IL-2 mutant and application thereof

Abstract

The invention relates to an IL-2 mutant protein and application thereof. The invention specifically provides an IL-2 mutant protein. Compared with an IL-2 wild type, the mutant protein at least comprises one mutation site. The mutation can regulate the affinity between IL-2 and IL-2R alpha or IL-2R beta gamma, so as to regulate the activation of immune cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an IL-2 mutant and application thereof. Background Art

[0002] IL-2 plays a vital role in the activation of immune effector cells. The precursor of human IL-2 has 153 amino acids, and mature secretory IL-2 is formed after the 20 amino acids at the N-terminus are removed. The activation and proliferation of T cells in vivo and in vitro depend on the binding of IL-2 to its corresponding cell surface receptors and further activation of downstream signaling pathways. The receptors of IL-2 include three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). IL-2Rα alone has a low affinity for IL-2, IL-2Rβγ dimers have a moderate affinity for IL-2, and IL-2Rαβγ trimers have the highest affinity for IL-2. CD25 is usually highly expressed on the surface of immunosuppressive regulatory T cells (Treg). Therefore, under low concentration conditions, IL-2 not only has less function in activating lymphocytes, but also preferentially amplifies suppressive Treg cells due to its preferential binding to the highly expressed receptor IL-2Rα on the surface of Treg. In order to overcome this problem, patients are usually given higher concentrations of IL-2 in clinical practice to activate immune effector cells. However, high concentrations of IL-2 usually cause pain, high fever, nausea, vomiting, pulmonary edema, and even death in severe cases.

[0003] One way to solve the above problems is to reduce or even block the binding of IL-2 to IL-2Rα, reduce IL-2 stimulation of regulatory T cells, and further increase IL-2's affinity for the IL-2Rβ subunit, promote IL-2 activation of cytotoxic lymphocytes such as T cells and promote their proliferation. To achieve this goal, some mutants have been designed for the IL-2 sequence to give IL-2 selective affinity, reduce or even eliminate its binding to IL-2Rα, and preferentially bind to the medium-affinity receptor IL-2Rβγ to preferentially expand and activate immune effector cells and reduce the expansion and activation of Treg cells. However, more IL-2 mutants that can selectively activate immune effector cells are still needed. Summary of the invention

[0004] In order to overcome the defects of the prior art, the present invention provides an IL-2 mutant, which enables IL-2 to have selective affinity, thereby preferentially amplifying and activating immune effector cells and reducing the amplification and activation of Treg cells.

[0005] The present invention provides a mutant protein of IL-2, which has an altered affinity for IL-2R compared to a control, and the mutant protein has at least one amino acid mutation compared to the sequence of the control.

[0006] In one or more embodiments, the sequence of the control is as shown in SEQ ID NO:55 or a sequence having at least 90% sequence identity thereto; alternatively, the sequence of the control is as shown in SEQ ID NO:55 and comprises a C125S mutation, or the sequence of the control is a sequence having at least 90% sequence identity thereto but comprises a C125S mutation.

[0007] In one or more embodiments, the mutation sites include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 selected from the following: E15, L19, R38, F42, P65, H79, R81, P82, R83, S87, E95, D109.

[0008] The IL-2 mutant protein provided in the first aspect of the present invention has reduced or eliminated affinity for IL-2Rα compared to the control, and the mutation comprises a mutation of 1, 2, 3 or 4 sites selected from the following: R38, F42, P65, D109. In one or more embodiments, the mutation comprises a mutation of a site selected from any one of the following groups:

[0009] (1) R38,

[0010] (2) R38 and P65,

[0011] (3) F42 and P65,

[0012] (4) P65 and D109,

[0013] (5) R38, F42 and P65.

[0014] In one or more embodiments, the mutation comprises a mutation at R38; the mutation optionally further comprises mutations at 1, 2 or 3 sites selected from the following: F42, P65, D109.

[0015] In one or more embodiments, the mutation comprises mutations at R38 and P65; the mutation optionally further comprises mutations at 1 or 2 sites selected from the following: F42, D109.

[0016] In one or more embodiments, the mutation comprises mutations at F42 and P65; the mutation optionally further comprises mutations at 1 or 2 sites selected from the following: R38, D109.

[0017] In one or more embodiments, the mutation comprises mutations at P65 and D109; the mutation optionally further comprises mutations at 1 or 2 sites selected from the following: R38, F42.

[0018] In one or more embodiments, the mutations comprise mutations at R38, F42 and P65; the mutations optionally further comprise mutations at D109.

[0019] In one or more embodiments, the mutant protein has one or more characteristics selected from the following:

[0020] The mutation of R38 is selected from the group consisting of: R38D, R38E, R38M, R38T, R38P, R38N, R38I, R38S, R38W, R38K, R38L, and R38F;

[0021] The mutation of F42 is selected from: F42K, F42A;

[0022] The mutation of P65 is selected from: P65R, P65W, P65I, P65H, P65V;

[0023] The mutation of D109 is selected from: D109K, D109T, and D109S.

[0024] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups:

[0025] (1)R38E;

[0026] (2) a mutation of R38 and P65W, wherein the mutation of R38 is selected from the group consisting of R38M, R38D, R38T, R38P, R38N, R38I, R38S, R38K, R38E, and R38F; and optionally further comprising: (a) any mutation of F42K and F42A, and / or, (b) any mutation of P65R, P65W, P65I, P65H, and P65V,

[0027] (3) R38W and P65I; optionally further comprising: (a) any mutation of F42K and F42A, and / or, (b) any mutation of P65R, P65W, P65I, P65H and P65V,

[0028] (4) R38D and P65H; optionally further comprising: (a) any mutation of F42K and F42A, and / or, (b) any mutation of P65R, P65W, P65I, P65H and P65V,

[0029] (5) a mutation of F42 and P65R, wherein the mutation of F42 is selected from the group consisting of: F42K, F42A,

[0030] (6) mutations of P65W and D109, wherein the mutation of D109 is selected from the group consisting of: D109K, D109T, and D109S,

[0031] (7) R38D, F42K and P65R; optionally including any mutation among D109K, D109T and D109S,

[0032] (8) R38L, F42K and P65W; optionally including any mutation among D109K, D109T and D109S,

[0033] (9) R38W, F42K and P65V; optionally, further comprising any one of the mutations D109K, D109T and D109S.

[0034] In one or more embodiments, the mutant protein of IL-2 further comprises mutations at one or more sites selected from the group consisting of: K35, T37, T41, K43, Y45, and L72. Preferably, the K35 point mutation is K35D; the T37 point mutation is T37E; the T41 point mutation is T41E; the K43 point mutation is K43E; the Y45 point mutation is Y45K; and the L72 point mutation is L72F. In one or more embodiments, the mutant protein of IL-2 further comprises the following mutations: K35D, T37E, T41E, K43E, Y45K, and L72F.

[0035] The IL-2 mutant protein provided in the second aspect of the present invention has an improved affinity for IL-2Rβγ compared to the control, and the mutation comprises at least 1, 2, 3, 4, 5, 6, 7 or 8 mutations selected from the following sites: E15, L19, H79, R81, P82, R83, S87, E95.

[0036] In one or more embodiments, the mutation comprises mutations at L19 and S87; the mutation optionally further comprises mutations at 1, 2, 3, 4, 5 or 6 sites selected from the following: E15, H79, R81, P82, R83, E95.

[0037] In one or more embodiments, the mutations comprise mutations at L19, R81 and S87; the mutations optionally further comprise mutations at 1, 2, 3, 4 or 5 sites selected from the group consisting of: E15, H79, P82, R83, E95.

[0038] In one or more embodiments, the mutations comprise mutations at H79, R81, P82 and R83; the mutations optionally further comprise mutations at 1, 2, 3 or 4 sites selected from the group consisting of: E15, L19, S87, E95.

[0039] In one or more embodiments, the mutation comprises a mutation at a site selected from any one of the following groups:

[0040] (1) L19,

[0041] (2) S87,

[0042] (3) E15 and L19,

[0043] (4) L19 and S87,

[0044] (5) L19, R81 and S87,

[0045] (6) H79, R81, P82, R83 and S87,

[0046] (7) L19, H79, R81, P82, R83 and S87,

[0047] (8) E15, L19, H79, R81, P82 and R83,

[0048] (9) H79, R81, P82, R83, S87 and E95.

[0049] In one or more embodiments, the mutation has one or more characteristics selected from the following: the E15 point mutation is selected from: E15R, E15D, E15A;

[0050] The L19 point mutation is selected from: L19W, L19F, L19D, L19Y;

[0051] The H79 point mutation is selected from: H79E, H79D, H79Q;

[0052] The R81 point mutation is selected from: R81D, R81N;

[0053] The P82 point mutation is selected from: P82T, P82A, P82I;

[0054] The R83 point mutation is selected from: R83E, R83D, R83A;

[0055] The S87 point mutation is selected from: S87D, S87Y, S87E, S87M;

[0056] The E95 point mutation is selected from: E95F, E95A, E95Y;

[0057] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups:

[0058] (1) L19W,

[0059] (2) S87Y,

[0060] (3) mutations at L19W and S87, wherein the mutation at S87 is selected from S87Y and S87M,

[0061] (4) L19F and S87D,

[0062] (5) L19Y and S87F,

[0063] (6) L19W and S87D,

[0064] (7) E15R and L19Y,

[0065] (8) L19W, R81D and S87E,

[0066] (9) H79E, R81D, P82I, R83E and S87D,

[0067] (10) H79E, R81W, P82I, R83E and S87Y,

[0068] (11) mutations at L19W, H79E, R81D, P82I, R83E and S87, wherein the mutation at S87 is selected from S87Y, S87F, S87M and S87D,

[0069] (12) L19F, H79E, R81D, P82I, R83E and S87D,

[0070] (13) E15R, L19Y, H79E, R81D, P82I and R83E,

[0071] (14) H79E, R81D, P82I, R83E, S87Y and E95F.

[0072] In one or more embodiments, the mutant protein of IL-2 further comprises mutations at one or more sites selected from the group consisting of: K35, T37, T41, K43, Y45, and L72. Preferably, the K35 point mutation is K35D; the T37 point mutation is T37E; the T41 point mutation is T41E; the K43 point mutation is K43E; the Y45 point mutation is Y45K; and the L72 point mutation is L72F. In one or more embodiments, the mutant protein of IL-2 further comprises the following mutations: K35D, T37E, T41E, K43E, Y45K, and L72F.

[0073] The IL-2 mutant protein provided in the third aspect of the present invention comprises:

[0074] (1) a first mutation that has reduced or eliminated affinity for IL-2Rα compared to the control, the first mutation being a mutation at 1, 2, 3 or 4 sites selected from the group consisting of R38, F42, P65, D109, and

[0075] (2) a second mutation having improved affinity for IL-2Rβγ compared to the control, wherein the second mutation is a mutation at 1, 2, 3, 4, 5, 6, 7 or 8 sites selected from the following: E15, L19, H79, R81, P82, R83, S87, E95.

[0076] In one or more embodiments, the first mutation is as described in the first aspect of the present invention, and the second mutation is as described in the second aspect of the present invention.

[0077] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups:

[0078] (1) R38D, F42K and P65R,

[0079] (2) L19W, R38D, F42K and P65R,

[0080] (3) R38D, F42K, P65R and S87Y,

[0081] (4) L19W, R38D, F42K, P65R and S87Y,

[0082] (5) R38D, F42K, P65R, H79E, R81D, P82I, R83E and S87D,

[0083] (6) K35D, T37E, R38E, T41E, K43E, Y45K, L72F, H79E, R81D, P82I, R83E and S87D,

[0084] (7) F42A and P65R,

[0085] (8) L19W, F42K, P65R and S87Y,

[0086] (9) L19W, P65W, D109K, H79E, R81D, P82I, R83E and S87Y,

[0087] (10) L19W, R38M, P65W and S87F,

[0088] (11) L19W, R38D, P65W, H79E, R81D, P82I, R83E and S87F,

[0089] (12) L19F, R38T, P65W and S87D,

[0090] (13) L19F, R38P, P65W, H79E, R81D, P82I, R83E and S87D,

[0091] (14) L19W, R38N, P65W and S87M,

[0092] (15) L19W, R38I, P65W, H79E, R81D, P82I, R83E and S87M,

[0093] (16) L19Y, R38S, P65W and S87F,

[0094] (17) E15R, L19Y, R38W and P65I,

[0095] (18) E15R, L19Y, R38D, P65H, H79E, R81D, P82I and R83E,

[0096] (19) R38K, P65W, H79E, R81D, P82I, R83E, S87Y and E95F,

[0097] (20) L19W, P65W, S87D and D109T,

[0098] (21) L19F, P65W, H79E, R81D, P82I, R83E, S87D and D109S,

[0099] (22) L19W, R38E, P65W and S87M,

[0100] (23) R38L, F42K, P65W, H79E, R81W, P82I, R83E and S87Y,

[0101] (24) L19W, R38F, P65W, R81D and S87E,

[0102] (25) L19W, R38W, F42K, P65V, H79E, R81D, P82I, R83E and S87D.

[0103] In one or more embodiments, the mutant protein of IL-2 further comprises mutations at one or more sites selected from the group consisting of: K35, T37, T41, K43, Y45, and L72. Preferably, the K35 point mutation is K35D; the T37 point mutation is T37E; the T41 point mutation is T41E; the K43 point mutation is K43E; the Y45 point mutation is Y45K; and the L72 point mutation is L72F. In one or more embodiments, the mutant protein of IL-2 further comprises the following mutations: K35D, T37E, T41E, K43E, Y45K, and L72F.

[0104] The present invention also provides a fusion protein comprising immunoglobulin Fc and

[0105] (1) the sequence as shown in SEO ID NO:55 and comprising the C125S mutation, or a sequence having at least 90% sequence identity thereto but comprising the C125S mutation, or

[0106] (2) The IL-2 mutant protein according to any one of the embodiments herein.

[0107] In one or more embodiments, the immunoglobulin Fc is located at the C-terminus of (1) or (2).

[0108] In one or more embodiments, the immunoglobulin Fc is a human immunoglobulin Fc.

[0109] In one or more embodiments, the immunoglobulin Fc is IgG1 Fc or IgG4 Fc.

[0110] In one or more embodiments, the immunoglobulin Fc is IgG4Fc, and the amino acid sequence of the IgG4Fc has the sequence shown in SEQ ID NO: 53, or a sequence having at least 70% sequence identity thereto.

[0111] In one or more embodiments, the fusion protein has a sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51, or a sequence having at least 70% sequence identity thereto.

[0112] The third aspect of the present invention provides a nucleic acid molecule comprising a sequence selected from the following:

[0113] (1) a nucleic acid sequence encoding an IL-2 mutant protein or fusion protein as described in any embodiment herein,

[0114] (2) a variant having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with (1),

[0115] (3) A complementary sequence of (1) or (2).

[0116] In one or more embodiments, the nucleic acid sequence has a sequence as shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52, or a variant thereof having at least 80% sequence identity thereto.

[0117] Another aspect of the present invention provides a nucleic acid construct, wherein the nucleic acid construct comprises the nucleic acid molecule described in any embodiment of the present invention.

[0118] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

[0119] The present invention also provides a host cell, wherein:

[0120] (1) comprising, expressing and / or secreting the IL-2 mutant protein and / or fusion protein described in any embodiment of the present invention, and / or

[0121] (2) comprising a nucleic acid molecule and / or a nucleic acid construct as described in any of the embodiments herein.

[0122] In one or more embodiments, the host cell is a bacterial cell or a mammalian cell.

[0123] In one or more embodiments, the bacteria are E. coli or CHO cells.

[0124] The present invention also provides a culture medium comprising the IL-2 mutant protein and / or fusion protein described in any embodiment herein; preferably, the culture medium may also comprise cells that express and / or secrete the IL-2 mutant protein and / or fusion protein.

[0125] In one or more embodiments, the culture medium is a culture medium for culturing immune effector cells.

[0126] In one or more embodiments, the immune effector cells are selected from one or more of the following: T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T, CIK, TIL, LAK, DC, DC-CIK, preferably TIL.

[0127] In one or more embodiments, the culture medium further comprises one or more substances selected from the group consisting of IL-1β, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-β, IFN-γ, G-CSF, M-CSF, GM-CSF, TNF-α, serum, antibodies or antigen-binding fragments thereof, and antibiotics.

[0128] In one or more embodiments, the antibody is an immune checkpoint antibody. In one or more embodiments, the immune checkpoint antibody is selected from any one or more of PD-1 antibody, CTLA-4 antibody, LAG3 antibody, TIM3 antibody, TIGIT antibody and BTLA antibody.

[0129] In one or more embodiments, the antibody is selected from any one or more of CD3 antibody, CD28 antibody, CD137 antibody, OX40 antibody, GITR antibody, ICOS, CD206 antibody and CD40 antibody. In one or more embodiments, the antibody is an activating antibody.

[0130] In one or more embodiments, the culture medium is a seed cell culture medium; preferably, it is a seed cell culture medium for TIL, which includes the following components: a basal culture medium, cytokines, and immune checkpoint antibodies or their antigen-binding fragments, wherein the cytokines include the IL-2 mutant protein and / or fusion protein described in any embodiment of the present invention, and the immune checkpoints include: PD-1, LAG-3, TIGIT and / or CTLA-4.

[0131] In one or more embodiments, the basal culture medium is selected from AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizerTM, FUJIFILM Irvin MHM-C.

[0132] The present application also provides seed cells of tumor infiltrating lymphocytes or a cell population thereof obtained by culturing with the seed cell culture medium described in the present application.

[0133] The present application also provides a pharmaceutical composition, which includes the seed cells of the tumor infiltrating lymphocytes described in the present application or a cell population thereof and a pharmaceutically acceptable excipient.

[0134] The present application also provides the use of the seed cells of tumor infiltrating lymphocytes described herein or a cell population thereof in the preparation of a drug for treating cancer. In one or more embodiments, the cancer is selected from the group consisting of gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, and pelvic poorly differentiated adenocarcinoma.

[0135] The present application also provides a method for culturing seed cells of tumor-infiltrating lymphocytes, which comprises the following steps: culturing isolated tumor-infiltrating lymphocytes in the seed cell culture medium described in the present application.

[0136] The present application also provides a method for amplifying tumor-infiltrating lymphocytes, which comprises the following steps: culturing isolated tumor-infiltrating lymphocytes in the seed cell culture medium described in the present application to obtain seed cells of the tumor-infiltrating lymphocytes.

[0137] In one or more embodiments, the isolated tumor infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, surgically resected primary lesion samples, synchronous and metachronous surgically resected metastatic lesion samples, puncture samples, and body fluids of a subject in need thereof.

[0138] In one or more embodiments, the body fluid comprises blood, interstitial fluid, lymphatic fluid and / or body cavity fluid.

[0139] In one or more embodiments, the isolated tumor infiltrating lymphocytes are derived from a tumor selected from the group consisting of gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, and pelvic poorly differentiated adenocarcinoma.

[0140] In one or more embodiments, the isolated tumor infiltrating lymphocytes are from a dissected tumor tissue having a diameter of about 1 mm to about 10 mm.

[0141] In one or more embodiments, the culturing period is about 3-20 days.

[0142] In one or more embodiments, the culturing is at a temperature of about 30-42°C.

[0143] The present invention also provides the use of the IL-2 mutant protein and / or fusion protein described in any embodiment of the present invention in the preparation of an immune effector cell culture medium or a medicament containing activated immune effector cells. In one or more embodiments, the medicament is used to treat cancer.

[0144] In one or more embodiments, the cancer is selected from: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, bile duct cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma.

[0145] In one or more embodiments, the immune effector cells are selected from one or more of the following: T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T, CIK, TIL, LAK, DC, DC-CIK. In one or more embodiments, the immune effector cells are Treg cells or CD8+T cells.

[0146] The present invention also provides a method for culturing or expanding immune effector cells, comprising incubating the immune effector cells with the culture medium described herein.

[0147] In one or more embodiments, the immune effector cells are selected from one or more of the following: T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T, CIK, TIL, LAK, DC, DC-CIK, preferably TIL.

[0148] In one or more embodiments, the incubation lasts at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days.

[0149] In one or more embodiments, the incubation lasts at most 20 days, at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days.

[0150] The present invention also provides a method for preparing a TIL cell population, comprising:

[0151] (1) incubating a tissue containing tumor cells with the seed cell culture medium described in any embodiment of the present invention to obtain a first TIL cell population,

[0152] (2) Incubating the first TIL cell population with expansion culture medium to obtain a second TIL cell population.

[0153] In one or more embodiments, the tumor tissue is obtained from a subject with cancer.

[0154] In one or more embodiments, the tumor tissue is pretreated. In one or more embodiments, the pretreatment comprises fragmenting and / or dissociating the tumor tissue.

[0155] In one or more embodiments, the tumor tissue is formed into fragmented tissue pieces by mechanical cutting.

[0156] In one or more embodiments, the size of the fragmented tissue block is 2×2×2 mm 3 ,3×3×3mm 3 ,4×4×4mm 3 , 5×5×5mm 3 or 6×6×6mm 3 .

[0157] In one or more embodiments, the incubation of step (1) lasts at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days.

[0158] In one or more embodiments, the incubation of step (1) lasts at most 20 days, at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days.

[0159] In one or more embodiments, the incubation of step (2) lasts at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days.

[0160] In one or more embodiments, the incubation of step (2) lasts at most 20 days, at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days.

[0161] In one or more embodiments, the initial seeding density of the first TIL cell population in step (2) is 2.0×10 5 / cm 2 ~5.0×10 5 / cm 2 , preferably, 2.5×10 5 / cm 2 ~4.5×10 5 / cm 2 , more preferably, 2.5×10 5 / cm 2 ~3.0×10 5 / cm2 .

[0162] In one or more embodiments, the tissue containing tumor cells is a tumor tissue or body fluid of a subject suffering from gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, bile duct cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, and the like.

[0163] In one or more embodiments, the body fluid comprises blood, pleural fluid, interstitial fluid, lymphatic fluid, and / or ascites.

[0164] In one or more embodiments, the expansion medium comprises IL-2, IL-7, IL-15 and immune checkpoint antibodies or antigen-binding fragments thereof, preferably, the immune checkpoint antibodies include PD-1 antibodies. In one or more embodiments, the expansion medium further comprises one or more selected from the following: serum, platelets, basal medium. In one or more embodiments, the basal medium is selected from AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizerTM, FUJIFILMIrvin MHM-C. In one or more embodiments, the serum is selected from human AB serum, subject autologous serum or animal serum. In one or more embodiments, the concentration of serum is 1-10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the concentration of serum is 1.5-9.5%, 2.5-8.5%, 3.5-7.5%, 4.5-6.5%. In one or more embodiments, the density of the platelets is 0.5×10 8 / mL-3×10 8 / mL, for example 0.5×10 8 / mL, 1×10 8 / mL, 1.5×10 8 / mL, 2×10 8 / mL, 2.5×10 8 / mL, 3×10 8 / mL. In one or more embodiments, the platelets are platelets derived from the subject's autologous blood or platelets derived from allogeneic blood. In one or more embodiments, the culture medium further comprises an antibiotic. Preferably, the antibiotic is selected from one or more of penicillin and streptomycin. In one or more embodiments, the concentration of penicillin in the culture medium is 10-1000IU / mL, 20-800IU / mL, 50-100IU / mL. In one or more embodiments, the concentration of penicillin in the culture medium is 10-1000IU / mL, 20-800IU / mL, 50-100IU / mL.

[0165] In one or more embodiments, the expansion medium is as described in WO2022 / 111571A1, which is incorporated herein by reference in its entirety.

[0166] In one or more embodiments, the incubation temperature in step (1) is 30-42°C, preferably 37°C.

[0167] In one or more embodiments, the incubation temperature in step (2) is 30-42°C, preferably 37°C.

[0168] In one or more embodiments, the CO2 concentration of the incubation in step (1) and step (2) is 5%.

[0169] The present invention provides the use of the IL-2 mutant protein and / or fusion protein described in any embodiment of the present invention in the culture of immune effector cells, and the immune effector cells include: T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DNT, CIK, TIL, LAK, DC, DC-CIK.

[0170] The present invention also provides a TIL cell population obtained by the method described in any embodiment of the present invention.

[0171] The present invention also provides use of a TIL cell population obtained by the method described in any embodiment of the present invention in the preparation of a drug for treating cancer.

[0172] In one or more embodiments, the cancer is selected from: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, bile duct cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma.

[0173] The present invention also provides a pharmaceutical composition, comprising the TIL cell population obtained by the method described in any embodiment of the present invention, and a pharmaceutically acceptable excipient.

[0174] Another aspect of the present invention provides a kit for activating immune effector cells, wherein the kit comprises the IL-2 mutant protein and / or fusion protein described in any embodiment of the present invention.

[0175] In one or more embodiments, the kit further comprises reagents required for culturing PBMC cells, for example, including but not limited to a culture medium suitable for culturing PBMC cells.

[0176] Beneficial effects of the present invention:

[0177] (1) The point mutations included in the present invention reduce or eliminate the affinity of IL-2 to IL-2Rα, greatly reduce the stimulation of Treg proliferation, and reduce the proportion of Treg in immune effector cells. It also further includes mutation sites that enhance the affinity of IL-2Rβγ, which enhances the stimulation of immune effector cells and thus increases their activation level.

[0178] (2) NK cells are non-specific immune effector cells of the innate immune system. They can quickly respond to tumor immunogens in the early stages of the immune response, but their immune killing effect on tumor cells is limited over a longer period of time. T cells targeting tumor antigens are an important anti-tumor component of the acquired immune system. As the main component of TIL, T cells specifically kill tumors by recognizing MHC-tumor antigen peptide complexes. This reaction lasts for a long time and is highly specific, making a major contribution to the killing and even eradication of tumors. If the proportion of NK cells in TIL is too high, it will have a certain impact on the tumor killing effect of TIL.

[0179] When the IL-2 mutant of the present invention is used in an in vitro TIL culture system, it can stimulate the proliferation of CD3+T cells more preferentially when culturing tumor tissues with a high level of NK cell infiltration, reduce the stimulation and proliferation of NK cells, and thus increase the proportion of CD3+T cells in the TIL finally obtained, and control the proportion of NK cells at a relatively low and reasonable level, which is of great significance for improving the tumor killing effect of TIL. BRIEF DESCRIPTION OF THE DRAWINGS

[0180] Figure 1 In Treg cell subsets, the activation effects of IL-2mut1 to IL-2mut7 on Treg were significantly reduced compared with IL-2mut0, indicating that the affinity of IL-2mut1 to IL-2mut7 to IL-2Rα was significantly reduced compared with IL-2mut0. Among them, the activation effects of IL-2mut3, IL-2mut4 and IL-2mut5 on Treg decreased the most.

[0181] Figure 2In the CD8+T cell subsets, the activation effects of IL-2mut3 to IL-2mut6 on CD8+T cells were significantly increased compared with IL-2mut0, indicating that compared with IL-2mut0, the affinity of IL-2mut3 to IL-2mut6 for IL-2Rβγ was significantly improved. DETAILED DESCRIPTION

[0182] Unless otherwise defined, all technical terms, symbols and other technical and scientific terms or specialized words used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the invention belongs. In some cases, terms with commonly understood meanings are defined herein for the purpose of clarification and / or ease of reference, and the inclusion of such definitions herein should not be construed as representing a significant difference from what is commonly understood in the art. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0183] All publications, including patent documents, academic papers and databases, mentioned in the present invention are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were independently incorporated by reference. If the definitions set forth herein are different from or otherwise inconsistent with the definitions set forth in the patents, published applications and other publications incorporated herein by reference, the definitions set forth herein shall prevail relative to the definitions in the documents incorporated herein by reference.

[0184] The methods and techniques of the present invention are generally performed according to conventional methods known in the art and described in various general and more specialized reference books cited and discussed in this specification, unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) and Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates (1992)), and Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990)), the contents of which have been incorporated herein by reference. Enzyme reactions and purification techniques are performed according to manufacturer's specifications and are generally performed according to methods known in the art or as described herein. Nomenclature, and experimental methods and techniques related to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery of the drug, and treatment of patients.

[0185] Definition of terms

[0186] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, "a" or "an" means "at least one or one" or "one (one) or more (ones)".

[0187] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other stated or intermediate value in the stated range is included in the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the stated smaller ranges and are also within the scope of the claimed subject matter unless the upper and lower limits of the stated range are explicitly excluded.

[0188] The term "about" as used herein refers to the usual error range for each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter per se.

[0189] As used herein, a subject includes any living organism, such as humans and other mammals. Mammals include, but are not limited to, humans and non-human animals, including farm animals, sports animals, rodents, and pets.

[0190] As used herein, a composition refers to any mixture of two or more products, substances or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0191] In the present invention, the term "IL-2" generally refers to a protein derived from lymphokines produced from normal peripheral blood lymphocytes and present in vivo at low concentrations. The term also includes proteins modified after the expression of IL-2, such as glycosylation, acetylation, phosphorylation, etc.

[0192] The "affinity" described herein refers to the binding ability between two molecules, which can usually be measured by KD. "KD" refers to the equilibrium dissociation constant of the interaction between two molecules (eg, a specific antibody and antigen or a ligand and receptor).

[0193] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0194] The term "coding sequence" refers to the portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product. The boundaries of the coding sequence are usually determined by the ribosome binding site (for prokaryotes) immediately upstream of the 5' open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. Coding sequences can include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0195] The term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0196] The term "disease and / or condition" refers to a physical condition of the subject, which physical condition is associated with the diseases and / or conditions described in the present invention.

[0197] The term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, alleviate and / or alleviate the disease or condition described in the present invention.

[0198] As used herein, the terms "treat," "therapeutic," and "therapeutic" refer to the complete or partial alleviation or reduction of a disease or condition or disorder, or a symptom, adverse effect or consequence, or phenotype associated therewith. In certain embodiments, the effect is therapeutic, such that it partially or completely cures a disease or condition or an adverse symptom associated therewith.

[0199] As used herein, a "therapeutically effective amount" of a compound or composition or combination refers to an amount that is effective, at dosages and for the necessary time periods, to achieve a desired therapeutic outcome, such as treatment of a disease, condition or disorder, and / or the pharmacodynamic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to a variety of factors, such as the disease state, age, sex, and weight of the subject, as well as the cell population being administered.

[0200] This document also includes all the terms described in the specific embodiments section of WO2022 / 111571A1. This patent application is incorporated into this application by reference in its entirety.

[0201] IL-2 mutants and their fusion proteins

[0202] The inventors have discovered that site-directed mutagenesis of IL-2 can regulate the affinity of IL-2 to IL-2Rα or IL-2Rβγ, thereby selectively activating immune effector cells.

[0203] The present invention first provides an IL-2 mutant having an altered affinity for IL-2R compared to a control, and the mutant protein has at least one amino acid mutation compared to the sequence of the control. In one or more embodiments, the mutation site comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 selected from the following: E15, L19, R38, F42, P65, H79, R81, P82, R83, S87, E95, D109. Herein, when referring to "IL-2 mutant", the control includes the wild-type sequence shown in SEQ ID NO: 55 or the sequence shown in SEQ ID NO: 55 and comprising the C125S mutation.

[0204] In some aspects, the IL-2 mutant protein has reduced or eliminated affinity for IL-2Rα compared to the control, and the mutation comprises mutations at 1, 2, 3 or 4 sites selected from the following: R38, F42, P65, D109. In an exemplary embodiment, the mutation of R38 is selected from: R38D, R38E, R38M, R38T, R38P, R38N, R38I, R38S, R38W, R38K, R38L, R38F; the mutation of F42 is selected from: F42K, F42A; the mutation of P65 is selected from: P65R, P65W, P65I, P65H, P65V; the mutation of D109 is selected from: D109K, D109T, D109S. The above mutations can be combined in any form, including any combination of the listed mutation sites, mutation numbers, and mutated amino acids, as long as the resulting IL-2 mutant protein shows reduced or eliminated affinity for IL-2Rα. In some embodiments, as measured by the SPR method, the Kd value of the affinity of the IL-2 mutant protein for IL-2Rα is greater than 1E-13, 5E-13, 1E-12, 5E-12, 1E-11, 5E-11, 1E-10, 5E-10, 9.65E-10, 1E-9, 5E-9, 1E-8, 5E-8.

[0205] In one or more embodiments, the mutation of IL-2 that reduces or eliminates affinity for IL-2Rα comprises a mutation at R38, optionally further comprising a mutation at 1, 2 or 3 sites selected from the group consisting of: F42, P65, D109; or, the mutation comprises a mutation at R38 and P65, optionally further comprising a mutation at 1 or 2 sites selected from the group consisting of: F42, D109; or, the mutation comprises a mutation at F42 and P65, optionally further comprising a mutation at 1 or 2 sites selected from the group consisting of: R38, D109; or, the mutation comprises a mutation at P65 and D109, optionally further comprising a mutation at 1 or 2 sites selected from the group consisting of: R38, F42; or, the mutation comprises a mutation at R38, F42 and P65, optionally further comprising a mutation at D109.

[0206] In one or more embodiments, the IL-2 mutation that reduces or eliminates affinity for IL-2Rα comprises a mutation selected from any of the following groups: (1) R38E; (2) a mutation of R38 and P65W, wherein the mutation of R38 is selected from: R38M, R38D, R38T, R38P, R38N, R38I, R38S, R38K, R38E, R38F, and optionally further comprises: (a) any mutation of F42K and F42A, and / or, (b) any mutation of P65R, P65W, P65I, P65H and P65V; (3) R38W and P65I, optionally further comprises: (a) any mutation of F42K and F42A, and / or, (b) any mutation of P65R, P65W, P65I, P65H and P65V; (4) R38D and P65H; Optionally, the invention further comprises: (a) any mutation of F42K and F42A, and / or (b) any mutation of P65R, P65W, P65I, P65H and P65V; (5) mutation of F42 and P65R, wherein the mutation of F42 is selected from the group consisting of F42K and F42A; (6) mutation of P65W and D109, wherein the mutation of D109 is selected from the group consisting of D109K, D109T, D109V and D109K. 9S; (7) R38D, F42K and P65R, optionally including any mutation among D109K, D109T and D109S; (8) R38L, F42K and P65W, optionally including any mutation among D109K, D109T and D109S; (9) R38W, F42K and P65V, optionally including any mutation among D109K, D109T and D109S.

[0207] In other aspects, the IL-2 mutant protein has an improved affinity for IL-2Rβγ compared to the control, and the mutation comprises at least 1, 2, 3, 4, 5, 6, 7 or 8 mutations selected from the following sites: E15, L19, H79, R81, P82, R83, S87, E95. In an exemplary embodiment, the E15 point mutation is selected from: E15R, E15D, E15A; the L19 point mutation is selected from: L19W, L19F, L19D, L19Y; the H79 point mutation is selected from: H79E, H79D, H79Q; the R81 point mutation is selected from: R81D, R81N; the P82 point mutation is selected from: P82T, P82A, P82I; the R83 point mutation is selected from: R83E, R83D, R83A; the S87 point mutation is selected from: S87D, S87Y, S87E, S87M; the E95 point mutation is selected from: E95F, E95A, E95Y. The above mutations can be combined in any form, including any combination of the listed mutation sites, mutation numbers, and mutated amino acids, as long as the resulting IL-2 mutant protein shows improved affinity for IL-2Rβγ. In some embodiments, as measured by SPR method, the Kd value of the affinity of the IL-2 mutant protein for IL-2Rβγ is less than 5E-10, 1.03E-10, 1E-10, 5E-11, 1E-11, 5E-12, 1E-12, 5E-13, 1E-13, 5E-14, 1E-14.

[0208] In one or more embodiments, the mutations of IL-2 that improve affinity for IL-2Rβγ include mutations at L19 and S87, and optionally also include mutations at 1, 2, 3, 4, 5 or 6 sites selected from the following: E15, H79, R81, P82, R83, E95; or, the mutations include mutations at L19, R81 and S87, and optionally also include mutations at 1, 2, 3, 4 or 5 sites selected from the following: E15, H79, P82, R83, E95; or, the mutations include mutations at H79, R81, P82 and R83, and optionally also include mutations at 1, 2, 3 or 4 sites selected from the following: E15, L19, S87, E95.

[0209] In one or more embodiments, the mutation of IL-2 that improves affinity for IL-2Rβγ comprises a mutation selected from any one of the following groups: (1) L19W, (2) S87Y, (3) L19W and a mutation at S87, wherein the mutation at S87 is selected from S87Y and S87M, (4) L19F and S87D, (5) L19Y and S87F, (6) L19W and S87D, (7) E15R and L19Y, (8) L19W, R81D and S87E, (9) H79E, R81D, P82I, R83E and S87D, (10) H79E, R81W, P82I, R83E and S87Y, (11) L19W, H79E, R81D, P82I, R83E and mutations at S87, wherein the mutation at S87 is selected from S87Y, S87F, S87M, S87D, (12) L19F, H79E, R81D, P82I, R83E and S87D, (13) E15R, L19Y, H79E, R81D, P82I and R83E, (14) H79E, R81D, P82I, R83E, S87Y and E95F.

[0210] In a further aspect, the IL-2 mutant protein comprises both the above mutations that reduce or eliminate affinity with IL-2Rα (first mutation) and the above mutations that increase affinity with IL-2Rβγ (second mutation). The first mutation and the second mutation can be combined in any form, including any combination of the listed mutation sites, mutation numbers, and mutated amino acids, as long as the resulting IL-2 mutant protein shows reduced or eliminated affinity for IL-2Rα and improved affinity for IL-2Rβγ.

[0211] In some embodiments, as measured by the SPR method, the Kd value of the affinity of the IL-2 mutant protein comprising the first mutation and the second mutation for IL-2Rα is greater than 1E-13, 5E-13, 1E-12, 5E-12, 1E-11, 5E-11, 1E-10, 5E-10, 9.65E-10, 1E-9, 5E-9, 1E-8, 5E-8. ​​In some embodiments, as measured by the SPR method, the Kd value of the affinity of the IL-2 mutant protein for IL-2Rβγ is less than 5E-10, 1.03E-10, 1E-10, 5E-11, 1E-11, 5E-12, 1E-12, 5E-13, 1E-13, 5E-14, 1E-14.

[0212] In a specific embodiment, the IL-2 mutant comprises a mutation selected from any one of the following groups: (1) R38D, F42K and P65R, (2) L19W, R38D, F42K and P65R, (3) R38D, F42K, P65R and S87Y, (4) L19W, R38D, F42K, P65R and S87Y, (5) R38D, F42K, P65R, H79E, R81D, P82I, R83E and S87D, (6) K35D, T37E, R38E, T41E, K43E, Y45K, L72F, H79E, R81D, P82I, I, R83E and S87D, (7) F42A and P65R, (8) L19W, F42K, P65R and S87Y, (9) L19W, P65W, D109K, H79E, R81D, P82I, R83E and S87Y, (10) L19W, R38M, P65W and S87F, (11) L19W, R38D, P65W, H79E, R81D, P82I, R83E and S87F, (12) L19F, R38T, P65W and S87D, (13) L19F, R38P, P65W, H79E, R81D, P82 I, R83E and S87D, (14) L19W, R38N, P65W and S87M, (15) L19W, R38I, P65W, H79E, R81D, P82I, R83E and S87M, (16) L19Y, R38S, P65W and S87F, (17) E15R, L19Y, R38W and P65I, (18) E15R, L19Y, R38D, P65H, H79E, R81D, P82I and R83E, (19) R38K, P65W, H79E, R81D, P82I, R83E, S87Y and E95F, (20) L19W, P65W, S87D and D109T, (21) L19F, P65W, H79E, R81D, P82I, R83E, S87D and D109S, (22) L19W, R38E, P65W and S87M, (23) R38L, F42K, P65W, H79E, R81W, P82I, R83E and S87Y, (24) L19W, R38F, P65W, R81D and S87E, (25) L19W, R38W, F42K, P65V, H79E, R81D, P82I, R83E and S87D.

[0213] In addition to the above mutations, the mutant protein of IL-2 described herein may also include mutations at one or more sites selected from the following: K35, T37, T41, K43, Y45, L72. By way of example only, the K35 point mutation may be K35D; the T37 point mutation may be T37E; the T41 point mutation may be T41E; the K43 point mutation may be K43E; the Y45 point mutation may be Y45K; the L72 point mutation may be L72F. In some embodiments, the mutant protein of IL-2 further includes the following mutations: K35D, T37E, T41E, K43E, Y45K and L72F.

[0214] The present invention also provides a fusion protein comprising an immunoglobulin Fc or a fragment thereof and (1) a sequence as shown in SEO ID NO: 55 and comprising a C125S mutation, or (2) an IL-2 mutant protein as described in any embodiment herein. The immunoglobulin Fc can be covalently fused to the N-terminus and / or C-terminus of the IL-2 protein or its mutant.

[0215] "Fc region" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. Thus, the Fc region is a polypeptide that constitutes the portion of the constant region of an antibody heavy chain other than the first constant region immunoglobulin domain (CH1 domain). In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the second (CH2 domain) and third (CH3 domain) constant domains of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH2-CH3-CH4 domains) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and a hinge region between CH1 and CH2. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain of the Fc region, that is, it extends from about amino acid 341 of IgG to about amino acid 447. "Fc fragment" refers to a fragment contained in the Fc region, such as an Fc CH2 domain or an Fc CH3 domain or other Fc fragments containing them. In one or more embodiments, the immunoglobulin Fc is IgG1Fc or IgG4Fc. In one or more embodiments, the immunoglobulin Fc is IgG4Fc, which has the sequence shown in SEQ ID NO: 53.

[0216] As used herein, "Fc region" or "Fc fragment" can be a natural Fc or a remodeled Fc. In addition, "Fc region" or "Fc fragment" can also refer to this region in an isolated state, or in a protein polypeptide containing Fc.

[0217] In some embodiments, the fusion protein comprising an immunoglobulin Fc or a fragment thereof and an IL-2 protein or a mutant thereof has a sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51, or a sequence having at least 70% sequence identity therewith.

[0218] The mutations described herein can be introduced by any method known in the art, such as homologous recombination, PCR, and the like.

[0219] The present invention also includes variants of the IL-2 mutant protein or fusion protein described above, such as mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity with the IL-2 mutant protein or fusion protein. More specifically, the present invention includes mutants having one or more (such as within 20, within 15, within 10, within 8, within 5 or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted compared to the signal transduction receptor described above. Such mutants retain the biological function of the IL-2 mutant protein or fusion protein described in the present invention.

[0220] The "sequence" of the present invention should be understood to include sequences that are substantially identical to the sequences of the present invention. The term "substantially identical sequences" refers to sequences that have at least 70, 75 or 80% sequence identity, preferably at least 90 or 95% sequence identity, and more preferably at least 97, 98 or 99% sequence identity between two peptide sequences after optimal alignment, such as using the GAP or BESTFIT program and using the default gap value. Preferably, the difference in the residue positions that are not identical is preferably a conservative amino acid substitution. "Conservative amino acid substitution" refers to an amino acid residue in which an amino acid residue is replaced by another amino acid residue with a side chain R group that has similar chemical properties (e.g., charge or hydrophilicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the protein. If the difference between two or more amino acid sequences is a conservative substitution, the sequence identity percentage or similarity can be adjusted upward to correct for the conservative nature of the substitution. See, for example, Pearson, Methods Mol. Biol. 243: 307-31 (1994). Examples of amino acid groups with side chains of similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. As indicated above, the IL-2 mutant protein or fusion protein according to the present invention may further contain other possible modifications not disclosed in the present invention in addition to the amino acid mutations provided by the present invention, as long as the substantially identical sequence requirements described above are met.

[0221] The mutants of the fusion protein described herein also include fragments, derivatives and analogs of the mutants, provided that they have the mutations described herein and have reduced affinity for IL-2Rα or increased affinity for IL-2Rβγ. Thus, the "fragments", "derivatives" and "analogs" substantially retain the same biological function or activity as the mutants. The fragments, derivatives or analogs may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted at mutation sites other than those described herein, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having substitution groups in one or more amino acid residues at mutation sites other than those described herein, or (iii) polypeptides formed by fusion of a mature mutant polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives and analogs are well within the scope known to those skilled in the art.

[0222] The polypeptide mutants described herein may be modified polypeptide mutants. Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0223] Nucleic Acids

[0224] The nucleic acid molecules of the present invention may be coding sequences of polypeptides or proteins, or nucleic acid constructs comprising polypeptides or proteins. The nucleic acid molecules may be in the form of DNA or RNA. The nucleic acid molecules described herein include sequences encoding IL-2 mutants or fusion proteins described in any embodiment of the present invention, as long as the amino acid sequence encoded by the nucleic acid molecules remains unchanged. The present invention also provides complementary sequences of these coding sequences.

[0225] Nucleic acid molecules encoding mature mutant polypeptides include: coding sequences encoding only mature mutant polypeptides; coding sequences of mature mutant polypeptides and various additional coding sequences; coding sequences of mature mutant polypeptides (and optional additional coding sequences) and non-coding sequences. The term "nucleic acid molecules encoding mutant polypeptides" may include nucleic acid molecules encoding the polypeptides, or may include nucleic acid molecules further including additional coding and / or non-coding sequences.

[0226] The present invention also relates to variants of the above-mentioned nucleic acid molecules, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. The variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it.

[0227] The present invention also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, 85%, 90%, 95% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably 95%.

[0228] The present invention also relates to complementary sequences of the above sequences or nucleic acid fragments that hybridize with the above sequences. As used herein, the length of "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used for nucleic acid amplification techniques (such as PCR) to determine and / or separate polynucleotides encoding desired polypeptides or domains. "Hybridization" as described herein mainly refers to nucleic acid sequence pairing under stringent conditions. Exemplary stringent conditions are hybridization and membrane washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS.

[0229] In certain embodiments, the nucleic acid molecule is a nucleic acid construct containing the coding sequence of a mutant described herein, and one or more regulatory sequences operably connected to these sequences. The regulatory sequence may be a suitable promoter sequence. The promoter sequence is usually operably connected to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence showing transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleotide sequence encoding the polypeptide. Any promoter and terminator that is functional in the selected host cell may be used herein. A typical example of a nucleic acid construct is an expression cassette that contains the complete elements required for expressing a gene of interest, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.

[0230] In certain embodiments, the nucleic acid construct is a vector. The vector may be a plasmid, a cosmid, a virus, and a phage. The vector may be a cloning vector, an expression vector, or an integration vector. Specifically, the coding sequence of the mutant herein may be cloned into many types of vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors may be used to provide the coding sequence of the mutant of the present invention. The expression vector may be provided to the cell in the form of a viral vector. Integration vectors are used to integrate the expression cassette described herein into the host genome. The present invention includes any vector known in the art for use in engineered bacteria (e.g., E. coli or yeast) for the production of polypeptides or proteins. The expression vector may be a transposon vector. In certain embodiments, the expression vector is one or more selected from the following transposon vectors: piggybac, sleeping beauty, frog prince, Tn5, and Ty. In addition to the polynucleotide molecules described in the present invention, the expression vector generally contains other elements commonly contained in the vector, such as a multiple cloning site, a resistance gene, a replication initiation site, and the like.

[0231] In certain embodiments, the expression vector uses pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 series vector as a backbone. In other embodiments, the recombinant expression vector uses pCDNA3 series vector, pCDNA4 series vector, pCDNA5 series vector, pCDNA6 series vector, pRL series vector, pUC57 vector, pMAX vector or pDC315 series vector as a backbone.

[0232] In the case where the polynucleotide sequence is known, each polynucleotide molecule can be prepared using a conventional method in the art, and a corresponding vector is constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley) or other standard textbooks described in the technology. Alternatively, the nucleic acid molecule and the vector can be reconstructed into a liposome for delivery to a target cell. The vector containing the nucleic acid molecule of the present invention can be transferred to a host cell by a well-known method, which varies according to the type of the cell host, including microinjection, gene gun method, electroporation, virus-mediated transformation method, electron bombardment method, calcium phosphate precipitation method, etc. For example, calcium chloride transfection is commonly used for prokaryotic cells, and calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0233] Host cells

[0234] Also provided herein is a host cell that contains, expresses and / or secretes a mutant described herein, or contains a nucleic acid molecule described herein. Herein, when expressing a heterologous nucleic acid sequence, "host cell" refers to a eukaryotic cell that is capable of replicating a vector and / or expressing a heterologous gene encoded by a vector. A host cell can be used as a recipient of a vector. A host cell can be "transfected" or "transformed," which refers to the process of transfection or transduction of an exogenous nucleic acid into a host cell. Transformed cells include primary subject cells and their progeny. The terms "engineered" and "recombinant" cells or host cells used herein often refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Therefore, a recombinant cell can be distinguished from a naturally occurring cell that does not contain an introduced recombinant nucleic acid. Host cells include both engineered bacteria that are ultimately used to produce polypeptides or proteins, and various cells used in the process of producing engineered bacteria.

[0235] Herein, host cells include cells carrying polynucleotide molecules and / or polypeptides described herein. Specifically, the present invention provides cells carrying polypeptides or proteins described herein or their coding sequences. The cells of the present invention may be immune effector cells.

[0236] As used herein, cells may be autologous, syngeneic, allogeneic, and in some cases even xenogeneic, relative to the individual receiving them.

[0237] In addition to carrying the signal transduction receptors and / or their coding sequences described herein, the cells of the present invention may also have one or more other properties that can be used for cellular immunotherapy (such as adoptive cell therapy for tumors). Such other properties may be inherent to the cells or may be part of the cells after genetic manipulation by humans. For example, the cells of the present invention may carry chimeric antigen receptors, αβT cell receptors, and / or antigen-specific receptors, such as tumor-specific receptors, or their coding sequences.

[0238] Culture medium

[0239] The IL-2 mutant protein, fusion protein and / or cell expressing the IL-2 mutant protein or fusion protein described herein can be used in the culture medium to replace or supplement the IL-2 used in the culture medium of the prior art. The mutant protein, fusion protein and / or cell can be used in any culture medium that requires a reduced affinity for IL-2Rα and / or an increased affinity for IL-2Rβγ. In some embodiments, the culture medium comprises a basal medium, and one or more selected from the following: (1) the IL-2 mutant protein described herein, (2) the fusion protein described herein and / or (3) a cell expressing the IL-2 mutant protein or fusion protein, wherein the concentration of the IL-2 mutant protein and / or fusion protein is about 3000 IU / mL or less, or the cell concentration contained is such that the concentration of the IL-2 mutant protein and / or fusion protein in the culture medium is about 3000 IU / mL or less.

[0240] In addition to using the IL-2 mutant protein and / or fusion protein to replace or supplement IL-2, the culture medium described herein can be the same as the culture medium in the prior art. For example, the culture medium may also include one or more substances selected from the following: IL-1β, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-β, IFN-γ, G-CSF, M-CSF, GM-CSF, TNF-α, serum, antibody or its antigen binding fragment, antibiotics. The culture medium may also include IL-2.

[0241] When referring to culture media, "antibodies" are generally used to inhibit, maintain or stimulate certain activities of cells, such as immune checkpoint antibodies, CD3 antibodies, CD28 antibodies, CD137 antibodies, OX40 antibodies, GITR antibodies, ICOS, CD206 antibodies and CD40 antibodies. Immune checkpoint antibodies include but are not limited to PD-1 antibodies, CTLA-4 antibodies, LAG3 antibodies, TIM3 antibodies, TIGIT antibodies and BTLA antibodies.

[0242] The culture medium described in this application can be used to cultivate activated immune effector cells.Herein, immune effector cells include T cells, such as cytotoxic T cells (also referred to as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+T cells or killer T cells), NK cells, NKT cells, CAR-T, CAR-NK, TCR-T, CIK, TIL; and other immune cells that can induce effector functions. Therefore, the IL-2 mutant protein and / or fusion protein described herein can be used to prepare drugs containing activated immune effector cells. The activated immune effector cells obtained by culturing the culture medium containing the IL-2 mutant protein and / or fusion protein described herein are also within the scope of the present invention.

[0243] When the culture medium described herein is an immune effector cell culture medium, the basal culture medium may be AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizer TM 、FUJIFILM Irvin MHM-C.

[0244] The culture medium may further include antibiotics, such as one or more selected from penicillin and streptomycin, at a concentration of about 1 to about 200 U / mL.

[0245] The culture medium may or may not include serum, such as human AB serum.

[0246] TIL seeding cell culture medium

[0247] In one or more embodiments, the culture medium is a seed cell culture medium for TIL, which includes: a basal culture medium, a cytokine, and an immune checkpoint antibody or an antigen-binding fragment thereof, wherein the cytokine includes an IL-2 mutant protein and / or a fusion protein as described in any embodiment herein. The culture medium also has any one or more characteristics of the aforementioned culture medium.

[0248] In one or more embodiments, the immune checkpoints include: PD-1, LAG-3, TIGIT and / or CTLA-4. The seed cell culture medium can be used to culture isolated tumor infiltrating lymphocytes to obtain seed cells of tumor infiltrating lymphocytes.

[0249] In the seed cell culture medium, the concentration of the IL-2 mutant protein and / or fusion protein is about 3000 IU / mL or less (for example, about 2900 IU / mL or less, about 2800 IU / mL or less, about 2700 IU / mL or less, about 2600 IU / mL or less, about 2500 IU / mL or less, about 2400 IU / mL or less, about 2300 IU / mL or less, about 2200 IU / mL or less, about 2100 IU / mL or less, about 2000 IU / mL or less, about 1900 IU / mL or less, about 1800 IU / mL or less). About 100 IU / mL or less, about 1500 IU / mL or less, about 1600 IU / mL or less, about 1500 IU / mL or less, about 1400 IU / mL or less, about 1300 IU / mL or less, about 1100 IU / mL or less, about 1100 IU / mL or less, about 1000 IU / mL or less, about 900 IU / mL or less, about 800 IU / mL or less, about 700 IU / mL or less, about 600 IU / mL or less, about 500 IU / mL or less, about 400 IU / mL or less, about 300 IU / mL or less, or less).

[0250] In one or more embodiments, the cytokine comprises IL-7, preferably at a concentration of about 200-about 1000U / mL. In one or more embodiments, the cytokine comprises IL-15, preferably at a concentration of about 200-about 500U / mL. In one or more embodiments, the cytokine comprises tumor necrosis factor TNF. In one or more embodiments, the cytokine comprises TNFα, preferably at a concentration of about 10-about 100pg / mL. In one or more embodiments, the cytokine comprises colony stimulating factor. In one or more embodiments, the cytokine comprises GM-CSF, G-CSF and / or M-CSF, preferably, the concentration of GM-CSF is about 200-about 5000U / mL, the concentration of G-CSF is about 300U / mL-about 1000U / mL, and the concentration of M-CSF is about 300U / mL-about 800U / mL. In one or more embodiments, the cytokine comprises interferon. In one or more embodiments, the cytokine includes IFN-γ, IFN-α and / or IFN-β, preferably, the concentration of the IFN-γ is about 300-about 1000U / mL, the concentration of the IFN-α is about 500U / mL-about 1000U / mL, and the concentration of the IFN-β is about 200U / mL-about 500U / mL. In one or more embodiments, the cytokine also includes: IL-4, IL-1α, IL-1β, IL-6, IL-9, IL-18, IL-12, IL-21 and / or IL-10. When contained, the concentration of IL-4 is about 200-about 500 U / mL, the concentration of IL-1α is about 200-about 500 U / mL, the concentration of IL-1β is about 200-about 500 U / mL, the concentration of IL-6 is about 200-about 500 U / mL, the concentration of IL-9 is about 200-about 500 U / mL, the concentration of IL-18 is about 200-about 500 U / mL, the concentration of IL-12 is about 200-about 1000 U / mL, the concentration of IL-21 is about 200-about 500 U / mL, and the concentration of IL-10 is about 200-about 500 U / mL.

[0251] In one or more embodiments, the immune checkpoint antibody or antigen binding fragment thereof is a human PD-1 antibody or antigen binding fragment thereof, preferably at a concentration of about 1 to about 100 μg / mL. In one or more embodiments, the immune checkpoint antibody or antigen binding fragment thereof is a LAG-3 antibody or antigen binding fragment thereof, preferably at a concentration of about 3 to about 10 μg / mL. In one or more embodiments, the immune checkpoint antibody or antigen binding fragment thereof is a TIGIT antibody or antigen binding fragment thereof, preferably at a concentration of about 1 to about 25 μg / mL. In one or more embodiments, the immune checkpoint antibody or antigen binding fragment thereof is a human CTLA-4 antibody or antigen binding fragment thereof, preferably at a concentration of about 1 to about 100 μg / mL. In one or more embodiments, the immune checkpoint antibody or antigen binding fragment thereof includes a CD137 antibody or antigen binding fragment thereof, preferably at a concentration of about 1 to about 100 μg / mL. In one or more embodiments, the immune checkpoint antibody or antigen binding fragment thereof includes a CD28 antibody or antigen binding fragment thereof, preferably at a concentration of about 1 to about 10 μg / mL.

[0252] In one or more embodiments, the seed cell culture medium further comprises a co-stimulatory receptor antibody or an antigen binding fragment thereof, wherein the co-stimulatory receptor or an antigen binding fragment thereof comprises: a CD40 antibody or an antigen binding fragment thereof, an OX-40 antibody or an antigen binding fragment thereof, a CD137 antibody or an antigen binding fragment thereof and / or a CD28 antibody or an antigen binding fragment thereof. In one or more embodiments, the seed cell culture medium further comprises a CD40 antibody or an antigen binding fragment thereof, preferably at a concentration of about 5 to about 10 μg / mL. In one or more embodiments, the seed cell culture medium further comprises an OX-40 antibody or an antigen binding fragment thereof, preferably at a concentration of about 3 to about 10 μg / mL.

[0253] In one or more embodiments, the seed cell culture medium further comprises serum at a concentration of about 1 to about 10% (v / v).

[0254] In one or more embodiments, the seed cell culture medium further comprises an M2 macrophage inhibitor, wherein the M2 macrophage inhibitor comprises RRx001 and / or CNI-1493. Preferably, the concentration of the M2 macrophage inhibitor is about 0.1 to about 100 μM.

[0255] In one or more embodiments, the seed cell culture medium further comprises a regulatory T cell (Treg) inhibitor, wherein the regulatory T cell inhibitor comprises CAL-101, dasatinib, imatinib and / or Panobinostat. Preferably, the concentration of the regulatory T cell inhibitor is about 0.1-about 100 μM.

[0256] In one or more embodiments, the seed cell culture medium further comprises a myeloid-derived suppressor cell (MDSC) inhibitor, wherein the myeloid-derived suppressor cell inhibitor comprises AG490, decitabine, sunitinib and / or BBI608. Preferably, the concentration of the myeloid-derived suppressor cell inhibitor is about 0.1-about 100 μg / mL.

[0257] In one or more embodiments, the seed cell culture medium further comprises a T cell activator, wherein the T cell activator comprises LYC-55716, GNE-1858 and / or methylene blue. Preferably, the concentration of the T cell activator is about 1 to about 10 μM.

[0258] In one or more embodiments, the seed cell culture medium further comprises a T cell differentiation inhibitor, and the T cell differentiation inhibitor comprises TWS119. Preferably, the concentration of the T cell differentiation inhibitor is about 1 to about 10 μM.

[0259] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0260] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 1000 U / mL IL-7, at least about 100 μg / mL PD-1 antibody or its antigen-binding fragment, at least about 10 pg / mL TNFα, at least about 5% human serum, serum-free culture medium X-VIVO 15 and 100 U / mL penicillin-streptomycin mixture PS dual antibody.

[0261] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-9, IL-15, PD-1 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0262] For example, the seed cell culture medium may include at least about 2000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500 U / mL IL-9, at least about 500 U / mL IL-15, at least about 50 μg / mL CTLA-4 antibody or its antigen-binding fragment, at least about 50 μg / mL PD-1 antibody or its antigen-binding fragment, at least about 3% human serum, serum-free culture medium AIM-V and 100 U / mL penicillin-streptomycin mixture PS dual antibody.

[0263] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, G-CSF, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO15 and penicillin-streptomycin mixture PS dual antibody.

[0264] For example, the seed cell culture medium may include at least about 2000U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500U / mL IL-7, at least about 1000U / mL G-CSF, at least about 36μg / mL PD-1 antibody or its antigen-binding fragment, at least about 20pg / mL TNFα, at least about 3% human serum, serum-free culture medium AIM-V and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0265] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-12, IL-21, GM-CSF, LAG3 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0266] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-12, at least about 500 U / mL IL-21, at least about 800 U / mL GM-CSF, at least about 10 μg / mL LAG-3 antibody or an antigen-binding fragment thereof, at least about 10 μg / mL CTLA-4 antibody or an antigen-binding fragment thereof, at least about 5% human serum, and serum-free medium CTS. TM OpTmizer TM and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0267] In the present application, the seed cell culture medium may include IL-1β, the IL-2 mutant protein and / or fusion protein described herein, IL-6, IL-21, IFN-γ, TIGIT antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0268] For example, the seed cell culture medium may include at least about 500U / mL IL-1β, at least about 2500U / mL IL-2 mutant protein and / or fusion protein described herein, at least about 200U / mL IL-6, at least about 200U / mL IL-21, at least about 1000U / mL IFN-γ, at least about 20μg / mL TIGIT antibody or its antigen-binding fragment, at least about 50μg / mL PD-1 antibody or its antigen-binding fragment, at least about 25pg / mL TNFα, at least about 5% human serum, serum-free culture medium X-VIVO15 and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0269] In the present application, the seed cell culture medium may include IL-1α, the IL-2 mutant protein and / or fusion protein described herein, IL-9, IL-18, IFN-α, IFN-β, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0270] For example, the seed cell culture medium may include at least about 500U / mL IL-1α, at least about 2000U / mL IL-2 mutant protein and / or fusion protein described herein, at least about 500U / mL IL-9, at least about 200U / mL IL-18, at least about 500U / mL IFN-α, at least about 500U / mL IFN-β, at least about 5μg / mL LAG-3 antibody or its antigen-binding fragment, at least about 25μg / mL PD-1 antibody or its antigen-binding fragment, at least about 25pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0271] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-4, IL-10, IL-21, CD137 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0272] For example, the seed cell culture medium can include at least about 3000U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200U / mL IL-4, at least about 200U / mL IL-10, at least about 250U / mL IL-21, at least about 10μg / mL CD137 antibody or its antigen-binding fragment, at least about 3μg / mL LAG-3 antibody or its antigen-binding fragment, at least about 20μg / mL PD-1 antibody or its antigen-binding fragment, at least about 10pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0273] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-6, IL-12, IL-21, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0274] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500 U / mL IL-6, at least about 500 U / mL IL-12, at least about 200 U / mL IL-21, at least about 20 μg / mL CD137 antibody or an antigen-binding fragment thereof, at least about 10 μg / mL CD28 antibody or an antigen-binding fragment thereof, at least about 10 μg / mL PD-1 antibody or an antigen-binding fragment thereof, at least about 10 pg / mL TNFα, at least about 3% human serum, and serum-free medium CTS. TM OpTmizer TM and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0275] In the present application, the seed cell culture medium may include IL-1β, the IL-2 mutant protein and / or fusion protein described herein, IL-7, IL-21, G-CSF, GM-CSF, IFN-γ, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0276] For example, the seed cell culture medium may include at least about 200 U / mL IL-1β, at least about 3000 U / mL IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-7, at least about 200 U / mL IL-21, at least about 500 U / mL G-CSF, at least about 500 U / mL GM-CSF, at least about 1000 U / mL IFN-γ, at least about 5 μg / mL LAG-3 antibody or an antigen-binding fragment thereof, at least about 3 μg / mL PD-1 antibody or an antigen-binding fragment thereof, at least about 15 pg / mL TNFα, at least about 5% human serum, serum-free medium CTS. TM OpTmizer TM and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0277] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0278] For example, the seed cell culture medium can include at least about 2500U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500U / mL IL-4, at least about 500U / mL IL-12, at least about 500U / mL GM-CSF, at least about 300U / mL M-CSF, at least about 200U / mL IFN-β, at least about 800U / mL IFN-γ, at least about 5μg / mL TIGIT antibody or its antigen-binding fragment, at least about 10μg / mL CTLA-4 antibody or its antigen-binding fragment, at least about 5% human serum, serum-free culture medium X-VIVO 15 and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0279] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, IL-15, IL-7, IL-15, GM-CSF, CD137 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.

[0280] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-7, at least about 200 U / mL IL-15, at least about 800 U / mL GM-CSF, at least about 10 μg / mL CD137 antibody or its antigen-binding fragment, at least about 20 μg / mL PD-1 antibody or its antigen-binding fragment, at least about 20 pg / mL TNFα, at least about 5% human serum, serum-free culture medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0281] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-4, IL-10, IL-10, G-CSF, M-CSF, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, OX-40 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.

[0282] For example, the seed cell culture medium can include at least about 2500U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500U / mL IL-4, at least about 500U / mL IL-10, at least about 250U / mL IL-10, at least about 500U / mL G-CSF, at least about 300U / mL M-CSF, at least about 5μg / mL CD137 antibody or its antigen-binding fragment, at least about 3μg / mL CD28 antibody or its antigen-binding fragment, at least about 10μg / mL OX-40 antibody or its antigen-binding fragment, at least about 10μg / mL PD-1 antibody or its antigen-binding fragment, at least about 5% human serum, serum-free medium AIM-V and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0283] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, IL-21, IFN-α, IFN-γ, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, OX-40 antibody or its antigen-binding fragment, at least about LAG-3 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.

[0284] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-7, at least about 500 U / mL IL-21, at least about 500 U / mL IFN-α, at least about 800 U / mL IFN-γ, at least about 5 μg / mL CD137 antibody or an antigen-binding fragment thereof, at least about 3 μg / mL CD28 antibody or an antigen-binding fragment thereof, at least about 5 μg / mL OX-40 antibody or an antigen-binding fragment thereof, at least about 3 μg / mL LAG-3 antibody or an antigen-binding fragment thereof, at least about 5 μg / mL CTLA-4 antibody or an antigen-binding fragment thereof, at least about 10 pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0285] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, IL-15, IFN-β, IFN-γ, CD137 antibody or its antigen-binding fragment, CD40 antibody or its antigen-binding fragment, OX-40 antibody or its antigen-binding fragment, TIGIT antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.

[0286] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500 U / mL IL-7, at least about 500 U / mL IL-15, at least about 250 U / mL IFN-β, at least about 1000 U / mL IFN-γ, at least about 5 μg / mL CD137 antibody or an antigen-binding fragment thereof, at least about 5 μg / mL CD40 antibody or an antigen-binding fragment thereof, at least about 5 μg / mL OX-40 antibody or an antigen-binding fragment thereof, at least about 5 μg / mL TIGIT antibody or an antigen-binding fragment thereof, at least about 15 μg / mL PD-1 antibody or an antigen-binding fragment thereof, at least about 5% human serum, serum-free culture medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0287] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, at least about 5% human serum, serum-free medium CTS TM OpTmizer TM And penicillin-streptomycin mixture PS double antibody.

[0288] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-7, at least about 200 U / mL IL-15, at least about 500 U / mL GM-CSF, at least about 1000 U / mL IFN-γ, at least about 3 μg / mL CD137 antibody or antigen-binding fragment thereof, at least about 3 μg / mL LCD28 antibody or antigen-binding fragment thereof, at least about 3 μg / mL PD-1 antibody or antigen-binding fragment thereof, at least about 10 pg / mL TNFα, at least about 5% human serum, serum-free medium CTS. TM OpTmizer TM and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0289] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, IL-12, G-CSF, GM-CSF, IFN-α, IFN-γ, CD28 antibody or its antigen-binding fragment, CD40 antibody or its antigen-binding fragment, TIGIT antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, TNFα, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS double antibody.

[0290] For example, the seed cell culture medium can include at least about 3000U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200U / mL IL-7, at least about 300U / mL IL-12, at least about 300U / mL G-CSF, at least about 200U / mL GM-CSF, at least about 500U / mL IFN-α, at least about 500U / mL IFN-γ, at least about 3μg / mL CD28 antibody or an antigen-binding fragment thereof, at least about 5μg / mL CD40 antibody or an antigen-binding fragment thereof, at least about 2.5μg / mL TIGIT antibody or an antigen-binding fragment thereof, at least about 3μg / mL PD-1 antibody or an antigen-binding fragment thereof, at least about 10pg / mL TNFα, at least about 5% human serum, serum-free medium X-VIVO 15, and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0291] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein, IL-7, PD-1 antibody or its antigen-binding fragment, RRx-001, CAL-101, human serum, serum-free culture medium CTS described herein. TM OpTmizer TM And penicillin-streptomycin mixture PS double antibody.

[0292] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 1000 U / mL of IL-7, at least about 50 μg / mL of PD-1 antibody or its antigen-binding fragment, at least about 3 μM RRx-001, at least about 1.5 μM CAL-101, at least about 5% human serum, serum-free medium CTS TM OpTmizer TM and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0293] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-10, IL-18, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, Sunitinib, Imatinib, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0294] For example, the seed cell culture medium can include at least about 2500 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-10, at least about 200 U / mL IL-18, at least about 10 μg / mL LAG-3 antibody or its antigen-binding fragment, at least about 5 μg / mL PD-1 antibody or its antigen-binding fragment, at least about 3 μM Sunitinib, at least about 5 μM Imatinib, at least about 5% human serum, serum-free culture medium X-VIVO 15 and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0295] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-1β, IL-6, GM-CSF, M-CSF, CNI-1493, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0296] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-1β, at least about 500 U / mL IL-6, at least about 500 U / mL GM-CSF, at least about 500 U / mL M-CSF, at least about 5 μM CNI-1493, at least about 5% human serum, serum-free culture medium X-VIVO 15, and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0297] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-9, IL-10, the IL-2 mutant protein and / or fusion protein 1 described herein, GM-CSF, OX40 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, Sunitinib, human serum, serum-free culture medium X-VIVO15 and penicillin-streptomycin mixture PS dual antibody.

[0298] For example, the seed cell culture medium may include at least about 2000U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200U / mL IL-9, at least about 200U / mL IL-10, at least about 500U / mL IL-21, at least about 500U / mL GM-CSF, at least about 5μg / mL OX40 antibody or its antigen-binding fragment, at least about 36μg / mL PD-1 antibody or its antigen-binding fragment, at least about 1.5μM Sunitinib, at least about 5% human serum, serum-free culture medium X-VIVO15 and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0299] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-6, IL-12, CD28 antibody or its antigen-binding fragment, OX-40 antibody or its antigen-binding fragment, CTLA-4 antibody or its antigen-binding fragment, TNFα, Imatinib, human serum, serum-free culture medium CTS TM OpTmizer TM And penicillin-streptomycin mixture PS double antibody.

[0300] For example, the seed cell culture medium may include at least about 2500 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500 U / mL of IL-6, at least about 1000 U / mL of IL-12, at least about 10 μg / mL of CD28 antibody or antigen-binding fragment thereof, at least about 3 μg / mL of OX-40 antibody or antigen-binding fragment thereof, at least about 5 μg / mL of CTLA-4 antibody or antigen-binding fragment thereof, at least about 15 pg / mL of TNFα, at least about 5 μM Imatinib, at least about 5% human serum, serum-free medium CTS. TM OpTmizer TM and 100U / mL penicillin-streptomycin mixture PS dual antibody.

[0301] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-7, IL-15, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, Dasatinib, human serum, serum-free culture medium X-VIVO 15 and penicillin-streptomycin mixture PS dual antibody.

[0302] For example, the seed cell culture medium may include at least about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 200 U / mL IL-7, at least about 300 U / mL IL-15, at least about 12 μg / mL CD137 antibody or its antigen-binding fragment, at least about 5 μg / mL CD28 antibody or its antigen-binding fragment, at least about 5 μg / mL LAG-3 antibody or its antigen-binding fragment, at least about 3 μg / mL PD-1 antibody or its antigen-binding fragment, at least about 3 μM Dasatinib, at least about 5% human serum, serum-free medium X-VIVO 15 and 100 U / mL penicillin-streptomycin mixture PS double antibody.

[0303] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-6, IL-12, G-CSF, M-CSF, IFN-β, IFN-γ, CTLA-4 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, Dasatinib, LYC-55716, GNE-1858, human serum, serum-free culture medium X-VIVO15 and penicillin-streptomycin mixture PS dual antibody.

[0304] For example, the seed cell culture medium can include at least about 3000U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500U / mL IL-6, at least about 500U / mL IL-12, at least about 1000U / mL G-CSF, at least about 500U / mL M-CSF, at least about 500U / mL IFN-β, at least about 800U / mL IFN-γ, at least about 3μg / mL CTLA-4 antibody or its antigen-binding fragment, at least about 5μg / mL PD-1 antibody or its antigen-binding fragment, at least about 2.5μM Dasatinib, at least about 10μM LYC-55716, at least about 4.5nM GNE-1858, at least about 5% human serum, serum-free medium X-VIVO 15 and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0305] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-1α, IL-9, GM-CSF, CD137 antibody or its antigen-binding fragment, CD28 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, TIGIT antibody or its antigen-binding fragment, CNI-1493, methylene blue, TWS119, human serum, serum-free culture medium AIM-V and penicillin-streptomycin mixture PS double antibody.

[0306] For example, the seed cell culture medium can include at least about 2500U / mL of the IL-2 mutant protein and / or fusion protein described herein, at least about 500U / mL IL-1α, at least about 500U / mL IL-9, at least about 800U / mL GM-CSF, at least about 3μg / mL CD137 antibody or its antigen-binding fragment, at least about 3μg / mL CD28 antibody or its antigen-binding fragment, at least about 5μg / mL LAG-3 antibody or its antigen-binding fragment, at least about 2.5μg / mL TIGIT antibody or its antigen-binding fragment, at least about 2.5μM CNI-1493, at least about 1μM methylene blue, at least about 5μM TWS119, at least about 5% human serum, serum-free medium AIM-V and 100U / mL penicillin-streptomycin mixture PS double antibody.

[0307] In the present application, the seed cell culture medium may include the IL-2 mutant protein and / or fusion protein described herein, IL-1α, IL-12, IL-21, G-CSF, M-CSF, IFN-α, IFN-β, IFN-γ, CD28 antibody or its antigen-binding fragment, CD40 antibody or its antigen-binding fragment, LAG-3 antibody or its antigen-binding fragment, PD-1 antibody or its antigen-binding fragment, CNI-1493, Dasatinib, GNE-1858, methylene blue, human serum, serum-free culture medium AIM-V and penicillin-streptomycin mixture PS double antibody.

[0308] For example, the seed cell culture medium may include about 3000 U / mL of the IL-2 mutant protein and / or fusion protein described herein, about 200 U / mL IL-1α, about 500 U / mL IL-12, about 500 U / mL IL-21, about 300 U / mL G-CSF, about 800 U / mL M-CSF, about 500 U / mL IFN-α, about 300 U / mL IFN-β, about 300 U / mL IFN-γ, about 3 μg / mL LCD28 antibody or an antigen-binding fragment thereof, about 5 μg / mL CD40 antibody or an antigen-binding fragment thereof, about 5 μg / mL LAG-3 antibody or an antigen-binding fragment thereof, about 3 μg / mL PD-1 antibody or an antigen-binding fragment thereof, about 3 μM CNI-1493, about 2 μM Dasatinib, about 5 nM GNE-1858, about 1.5 μM methylene blue, about 5% human serum, serum-free culture medium AIM-V and 100 U / mL penicillin-streptomycin mixture PS dual antibody.

[0309] In one or more embodiments, the seed cell culture medium described herein is as described in WO2022 / 111571A1, except that IL-2 is replaced with an IL-2 mutant or fusion protein at the same concentration. This patent application is incorporated herein by reference in its entirety.

[0310] The present application provides seed cells or cell groups of tumor infiltrating lymphocytes obtained by culturing the seed cell culture medium described in the present application. The term "tumor infiltrating lymphocytes" generally refers to infiltrating lymphocytes separated from tumor tissues. Immunohistochemical staining shows that tumor infiltrating lymphocytes may include CD3+T cells, CD20+, CD79α+B cells, plasma cells and CD56+NK cells; they may also include T cells, a small amount of B cells, NK cells, macrophages and dendritic cells. The tumor infiltrating lymphocytes can produce highly efficient and specific anti-tumor killing effects through direct cytotoxic T lymphocytes and secreted cytokines. In the present application, the tumor infiltrating lymphocytes may include mononuclear leukocytes, which leave the bloodstream and migrate to a tumor. The tumor infiltrating lymphocytes may be selected from the group consisting of T cells, B cells, natural killer cells, and natural killer T cells. "Isolated tumor infiltrating lymphocytes" can be derived from samples: ascites, surgical resection of primary lesions, synchronous and metachronous surgical resection of metastatic lesions, puncture samples and body fluids (such as blood, tissue fluid, lymph and / or body cavity effusion) of subjects in need. "Isolated tumor infiltrating lymphocytes" also come from tumors: gastric cancer, thyroid tumors, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma.

[0311] In the present invention, the term "seed cells" generally refers to tumor infiltrating lymphocytes cultured from tumor tissue using any suitable culture medium including the culture medium of the present application. These cells can be used as a starting cell population for further expansion of the culture, or as a cell population for terminal use, such as a cell population for reinfusion therapy of an individual subject. The seed cells can be obtained from tumor tissue after any different culture durations, and the culture duration of the seed cells can be determined based on the subsequent use of the seed cells, and is not limited to a specific culture duration. An exemplary culture time is 1-100 days. The scope of the present invention includes TIL or seed cells of TIL obtained by culture in the culture medium described herein.

[0312] Herein, "incubation" or "culture" refers to placing cells in the culture medium for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, or for up to 20 days, up to 15 days, up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days. Incubation can be carried out in general or special culture containers known in the art. Incubation can be static incubation or agitation incubation as required.

[0313] Preparation of TIL cell population

[0314] The present invention also provides a method for preparing a TIL cell population and a TIL cell population obtained thereby. The method comprises: (1) incubating a tissue containing tumor cells with a TIL seed cell culture medium as described in any embodiment of the present invention to obtain a first TIL cell population, and (2) incubating the first TIL cell population with an expansion culture medium to obtain a second TIL cell population.

[0315] The tissue containing tumor cells can be tumor tissue or body fluid (including blood, pleural effusion, tissue fluid, lymph fluid and / or ascites) of subjects suffering from cancers such as gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, etc. The tumor tissue is pretreated, including fragmentation and / or dissociation of the tumor tissue, for example, by mechanical cutting to form fragmented tissue blocks. The size of the tissue block is, for example, 2×2×2 mm 3 ,3×3×3mm 3 ,4×4×4mm 3 , 5×5×5mm 3 or 6×6×6mm 3 .

[0316] In one or more embodiments, the incubation of step (1) lasts for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days. In one or more embodiments, the incubation of step (1) lasts for at most 20 days, at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days. In one or more embodiments, the incubation of step (2) lasts for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days. In one or more embodiments, the incubation of step (2) lasts at most 20 days, at most 15 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days.

[0317] In one or more embodiments, the incubation temperature of step (1) is 30-42° C., preferably, 37° C. In one or more embodiments, the incubation temperature of step (2) is 30-42° C., preferably, 37° C. In one or more embodiments, the CO2 concentration of the incubation in step (1) and step (2) is 5%.

[0318] In one or more embodiments, the initial seeding density of the first TIL cell population in step (2) is 2.0×10 5 / cm 2 ~5.0×10 5 / cm 2 , preferably, 2.5×10 5 / cm 2 ~4.5×10 5 / cm 2 , more preferably, 2.5×10 5 / cm 2 ~3.0×10 5 / cm 2 .

[0319] In one or more embodiments, the expansion medium comprises IL-2, IL-7, IL-15 and immune checkpoint antibodies or antigen-binding fragments thereof, preferably, the immune checkpoint antibodies include PD-1 antibodies. In one or more embodiments, the expansion medium further comprises one or more selected from the following: serum, platelets, basal medium. In one or more embodiments, the basal medium is selected from AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizerTM, FUJIFILMIrvin MHM-C. In one or more embodiments, the serum is selected from human AB serum, subject autologous serum or animal serum. In one or more embodiments, the concentration of serum is 1-10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Preferably, the concentration of serum is 1.5-9.5%, 2.5-8.5%, 3.5-7.5%, 4.5-6.5%. In one or more embodiments, the density of the platelets is 0.5×10 8 / mL-3×10 8 / mL, for example 0.5×10 8 / mL, 1×10 8 / mL, 1.5×10 8 / mL, 2×10 8 / mL, 2.5×10 8 / mL, 3×10 8 / mL. In one or more embodiments, the platelets are platelets derived from the subject's autologous blood or platelets derived from allogeneic blood. In one or more embodiments, the culture medium also includes an antibiotic. Preferably, the antibiotic is selected from one or more of penicillin and streptomycin. In one or more embodiments, the concentration of penicillin in the culture medium is 10-1000IU / mL, 20-800IU / mL, 50-100IU / mL. In one or more embodiments, the concentration of penicillin in the culture medium is 10-1000IU / mL, 20-800IU / mL, 50-100IU / mL. In one or more embodiments, the expansion culture medium is as described in WO2022 / 111571A1, which is incorporated herein by reference in its entirety.

[0320] Pharmaceutical compositions and kits

[0321] The present application also provides a pharmaceutical composition, which includes a seed cell of the tumor infiltrating lymphocyte described in the present application or a cell group thereof, or a TIL cell group obtained by the method described in any embodiment of the present invention, and a pharmaceutically acceptable excipient. The pharmaceutical composition can be used to treat cancer, such as thyroid tumors, gastric cancer, gastrointestinal stromal tumors, intestinal cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma.

[0322] Typically, a pharmaceutical composition contains a therapeutically effective amount of TIL cells. A therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and / or relief of a disease or condition in a subject. The therapeutically effective amount can be determined based on factors such as the patient's age, gender, the disease and its severity, and other physical conditions of the patient. Herein, a subject or patient generally refers to a mammal, particularly a human.

[0323] The present invention also provides a kit for activating immune effector cells, the kit comprising the IL-2 mutant protein and / or fusion protein described in any embodiment of the present invention. The kit may also comprise reagents required for culturing PBMC cells, for example, including but not limited to a culture medium suitable for culturing PBMC cells.

[0324] The present invention is described below by specific embodiments. It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution.

[0325] Example

[0326] Example 1, Synthesis of IL-2 mutants

[0327] The coding sequences of synthetic IL-2mut0, IL-2mut1 to IL-2mut25 are SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52. The above IL-2mut0 and IL-2mut1 to IL-2mut25 sequences all contain IgG4Fc to increase the stability of the IL-2 molecule, and IgG4Fc is located at the C-terminus of the IL-2 part, and its amino acid sequence is shown in SEQ ID NO: 53, and the coding sequence is shown in SEQ ID NO: 54. The amino acid sequence of wild-type IL-2 without IgG4Fc is shown in SEQ ID NO: 55, and the coding sequence is shown in SEQ ID NO: 56. Linkers with EcoRI and XhoI restriction sites were connected to the 5' and 3' ends of each sequence, respectively. After double digestion with EcoRI and XhoI, the sequences were connected to the pCDNA3.1 expression vector to form expression vectors pCDNA3.1-IL-2mut0, pCDNA3.1-IL-2mut1 to pCDNA3.1-IL-2mut25 of IL-2 mutants, which were introduced into the CHO cell line and screened and cultured in 500 μg / mL G418, 5% v / v FBSDMEM culture medium. The medium was changed every 3 days. After culturing for 2-3 weeks, the limiting dilution method was used to screen high-expressing cell strains in 96-well plates. After ELISA detection and comparison, stable monoclonal CHO cell strains with high expression of wild-type IL-2 and its mutants were obtained. The CHO cell lines stably expressing the above-mentioned wild-type and mutant IL-2 were amplified and cultured, and then purified according to the method described in Example 2 of Chinese Patent CN102174111B (CN102174111B is incorporated herein by reference in its entirety) to obtain the above-mentioned mutant IL-2 proteins, whose amino acid sequences are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51, respectively. The IL-2mut0 sequence described in this article contains the point mutation C125S in the IL-2 clinical drug Proleukin, that is, the natural IL-2 amino acid position 125 is point mutated from cysteine ​​to serine (C125S), and the amino acid sequence is shown in SEQ ID NO: 1 (including IgG1Fc), which is intended to eliminate the original disulfide bond between cysteine ​​at position 125 and cysteine ​​at position 58 to reduce the excessive stability of the IL-2 structure and reduce in vivo toxicity, while reducing potential other disulfide bond mismatches and thereby reducing the toxic side effects of the drug itself.IL-2mut6 contains a point mutation of the IL-2 mutant Y01 described in Example 3 of WO2020057646A1, in which its affinity for IL-2Rα is recorded as undetectable, and its affinity for IL-2Rβ is significantly improved compared with IL-2mut0 (detection method: biofilm layer interferometry (BLI)). IL-2mut7 contains a point mutation of the IL-2 mutant P-0705 described in Table 11 of Example 6 of WO2020252418A2, in which the ratio of the EC50 value of mutant P-0705 binding to IL-2Rα to the EC50 value of wild-type IL-2 is recorded to be >500. The purified IL-2mut0 and IL-2mut1 to IL-2mut25 protein solutions were adjusted to the same final volume and the concentration was determined. The mutation sites of IL-2mut0 and IL-2mut1 to IL-2mut25 and the concentration of purified proteins obtained are shown in Table 1.

[0328] Table 1: IL-2 mutant mutation sites (based on SEQ ID NO: 1 sequence) and purified protein concentrations

[0329]

[0330]

[0331]

[0332] Example 2, Detection of affinity between IL-2 mutants and receptors

[0333] The SPR method was used to detect the affinity of IL-2mut0 and IL-2mut1 to IL-2mut25 obtained in Example 1 to IL-2Rα and IL-2Rβγ, respectively. All SPR detection experiments were performed using the Biacore 8K (Cytiva) system. IL-2Rα (acrobiosystems Cat. No.: ILA-H52H9) and IL-2Rβγ (acrobiosystems Cat. No.: ILG-H5283) were respectively immobilized on the surface of a CM5 sensor chip (Cytiva) to achieve a capture level of 600 response units (RU). The gradient dilutions of IL-2mut0 and each mutant protein flowed through the sensor chip surface where each IL-2 molecule was immobilized at a flow rate of 30 μL / min to determine the kinetic parameters. The buffer in the flowing dilution contained 10 mM HEPES pH 7.4, 150 mM NaCl and 0.05% v / v Tween 20. The generated data were analyzed using Biacore insight evaluationsoftware to obtain the affinity of the above IL-2 wild-type and mutant proteins to IL-2Rα and IL-2Rβγ, respectively.

[0334] The results are shown in Table 2. According to the results in Table 3, compared with IL-2mut0, the affinity of IL-2mut6 and IL-2mut7 to IL-2Rα has decreased significantly, which is consistent with the trend of the published reports (see Example 3 of WO2020057646A1 and Table 11 of Example 6 of WO2020252418A2, respectively). IL-2mut1 to IL-2mut5, IL-2mut23 and IL-2mut25 were all undetectable for binding to IL-2Rα, indicating that the combination of mutation sites introduced in IL-2mut1 to IL-2mut5, IL-2mut23 and IL-2mut25 greatly reduced the affinity of these mutants to IL-2Rα. The affinity K of IL-2mut6 to IL-2mut22 and IL-2mut24 for IL-2Rα DThe values ​​were also significantly increased compared with IL-2mut0, indicating that the affinity of IL-2mut6 to IL-2mut22 and IL-2mut24 to IL-2Rα decreased significantly compared with IL-2mut0. At the same time, the affinity of IL-2mut6 to IL-2Rβγ increased significantly compared with IL-2mut0, which is consistent with the trend of published reports; similarly, the affinity of mutants IL-2mut5 and IL-2mut25 containing some mutations in IL-2mut6 (H79E / R81D / P82I / R83E / S87D) to IL-2Rβγ also increased significantly. The affinity of IL-2mut1 and IL-2mut7 to IL-2Rβγ remained basically unchanged, indicating that the point mutations in IL-2mut1 and IL-2mut7 had little effect on the affinity of IL-2 to IL-2Rβγ. The affinity K of IL-2mut2 to IL-2mut4 and IL-2mut8 to IL-2mut24 for IL-2Rβγ D The values ​​were also significantly decreased compared with IL-2mut0, indicating that the affinity of IL-2mut2 to IL-2mut4 and IL-2mut8 to IL-2mut24 to IL-2Rβγ was also improved compared with IL-2mut0.

[0335] Table 2: Affinity K of each IL-2 mutant for IL-2Rα / IL-2Rβγ D value

[0336] IL-2 mutant ID IL-2Rα affinity (M) IL-2Rβγ affinity (M) IL-2mut0 9.65E-10 1.03E-10 IL-2mut1 NA 0.86E-10 IL-2mut2 NA 0.57E-10 IL-2mut3 NA 0.49E-10 IL-2mut4 NA 0.42E-10 IL-2mut5 NA 2.09E-11 IL-2mut6 1.49E-8 2.19E-11 IL-2mut7 1.33E-8 1.22E-10 IL-2mut 8 1.74E-8 0.33E-10 IL-2mut 9 1.49E-8 0.48E-10 IL-2mut 10 2.45E-8 2.53E-11 IL-2mut 11 2.07E-8 3.47E-11 IL-2mut 12 3.94E-8 0.49E-10 IL-2mut 13 2.32E-8 3.31E-11 IL-2mut 14 2.62E-8 0.52E-10 IL-2mut 15 2.69E-8 3.15E-11 IL-2mut 16 3.24E-8 0.51E-10 IL-2mut 17 2.52E-8 0.64E-10 IL-2mut 18 3.31E-8 2.94E-11 IL-2mut 19 4.02E-8 2.21E-11 IL-2mut 20 4.41E-8 0.33E-10 IL-2mut 21 9.92E-9 2.28E-11 IL-2mut 22 2.21E-8 0.52E-10 IL-2mut 23 NA 2.27E-11 IL-2mut 24 5.37E-8 0.65E-10 IL-2mut 25 NA 2.17E-11

[0337] Note: NA means no affinity detected

[0338] Example 3, Determination of the Stimulating Effect of IL-2 Mutants on T Cells

[0339] IL-2mut0 and mutants IL-2mut1 to IL-2mut7 in Example 1 were selected to treat PBMCs, and the activation levels of cell clusters of different phenotypes in the treated PBMCs were analyzed. Specifically, STAT5 is a known molecule that is involved in the activation of downstream signaling pathways after IL-2 binds to its receptor, and the level of phosphorylated STAT5 (pSTAT5) reflects the activation level of IL-2-mediated downstream cell signaling pathways. Therefore, by detecting the phosphorylation level of STAT5 in different T cell populations, the preference of each IL-2 mutant for the activation effect of different T cell subsets can be reflected. The specific detection method is as follows:

[0340] 1) Take out the frozen PBMC cells from liquid nitrogen, place them in a 37°C water bath for rapid recovery, add them to 10 mL of preheated X-VIVO 15 medium containing 5% human AB serum, centrifuge at 800 g for 5 minutes, and wash;

[0341] 2) Add 20 mL of X-VIVO 15 medium containing 5% human AB serum to the cell pellet and culture overnight at 37°C and 5% CO2;

[0342] 3) Centrifuge the cells cultured overnight in 2) at 800 g for 5 minutes, and wash the cell pellet twice with PBS;

[0343] 4) The PBMC cell pellet was resuspended in serum-free PBS and treated at 4°C for 1 hour. Then, 2×10 cells were treated with IL-2mut0 and mutants IL-2mut1 to IL-2mut7 in Table 2 of Example 2 diluted in a gradient at 37°C. 5 PBMC1h;

[0344] 5) After treatment with IL-2mut0 and its mutants in 4), fix them with Cytofix (BD Biosciences, Cat. NO.: 554655) at room temperature for 30 minutes according to the instructions, and then permeabilize with permeabilization buffer (BD Phosflow TM PermBuffer III, bdbiosciences, Cat.NO.: 558050) was treated at room temperature for 30 minutes as recommended by the instructions, and then washed with PBS;

[0345] 6) Treat the cells with Fc receptor blocking solution TruStain FcX (Biolegend, Cat. NO.: 422301) according to the instructions to block cell surface FcR.

[0346] 7) Each cell that has been fixed, permeabilized and FcR blocked is stained with a fluorescent flow cytometry antibody mixture containing anti-CD25-PE (Biolegend, Cat.NO.: 985802), anti-Foxp3-APC (R&D systems, Cat.NO.: IC8214A), anti-pSTAT5-FITC (ThermoFisher, Cat.NO.: 11-9010-42) and anti-CD4-PerCP Cy5.5 (Biolegend, Cat.NO.: 260180). The flow cytometry results are gated based on the CD4+CD25+Foxp3+ combination to divide the Treg subpopulation. The proportion of pSTAT5-positive cells in the Treg subpopulation is then divided separately, and the results are presented as the percentage of pSTAT5-positive cells in the Treg subpopulation, such as Figure 1 shown.

[0347] 8) Select samples treated with IL-2mut0, IL-2mut3, IL-2mut4, IL-2mut5 and IL-2mut6 from each of the cells obtained in 7) that have been fixed, permeabilized and FcR blocked, and stain the cells with a fluorescent flow cytometry antibody mixture containing anti-CD3-PE (Biolegend, Cat.NO.: 980008), anti-CD8-APC (Biolegend, Cat.NO.: 980904) and anti-pSTAT5-FITC (ThermoFisher, Cat.NO.: 11-9010-42). The flow cytometry results were gated by CD3+CD8+ combination to divide the CD8+T cell subpopulation. The proportion of pSTAT5-positive cells in the CD8+T cell subpopulation was then divided, and the results were presented as the percentage of pSTAT5-positive cells in the subpopulation, as shown in Figure 1. Figure 2 shown.

[0348] Example 4, Effects of IL-2 mutants on the proliferation and phenotype of tumor infiltrating lymphocytes (TIL) cultured in vitro

[0349] Collect freshly excised different tumor tissue samples and process them immediately under sterile conditions. According to the culture medium described in Implementation 1 of WO2022111571A1 and the tumor sample processing method and TIL culture method described in Example 2, each tumor tissue of this example is processed and cultured to obtain TIL, and WO2022111571A1 is incorporated herein by reference in its entirety.

[0350] The details are as follows:

[0351] 1) Prepare physiological saline containing a final concentration of 100 U / mL penicillin, 100 μg / mL streptomycin and 50 μg / mL gentamicin for later use;

[0352] 2) In a sterile environment in a Class II biosafety cabinet, the freshly isolated tumor tissue sample from the tumor patient was placed in a 10 cm dish to which 30 mL of the physiological saline prepared in step 1) had been added for washing, and then transferred to a new 10 cm dish to which 30 mL of the physiological saline prepared in step 1) had been added for washing, and the washing was repeated 3 times in total;

[0353] 3) Use a sterile surgical blade to remove fat tissue and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm 32 G-REX100 culture tanks (purchased from Wilsonwolf) were taken, 42 randomly selected tumor tissue blocks were placed in each G-REX100 culture tank, and seed cell culture medium was added to the culture tank, the components of the seed cell culture medium included: 3000IU / mL IL-2, 20ng / mL IL-7, 20ng / mL IL-15, 500U / mL GM-CSF, 1000IU / mL IFN-γ, 3μg / mL anti-CD137 mAb, 3μg / mL anti-CD28 mAb, 3μg / mL anti-PD-1 mAb, 10ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody and X-VIVO 15 basal culture medium supplemented to the final volume; the excess tumor tissue blocks were frozen with CryoStor10 (purchased from BioLifeSolutions) freezing solution through liquid nitrogen in a program cooling instrument;

[0354] 4) After adding 1 L of the above seed cell culture medium to the G-REX100 culture tank containing the tumor tissue block in 3), the tumor tissue block was cultured at 37° C. and 5% CO2. Half of the volume of the old seed cell culture medium was removed every 4 days, and half of the volume of fresh seed cell culture medium was added. After the TIL seed cells were harvested by centrifugation on the 12th day, the total number of cells and the viability were counted;

[0355] 5) Take the seed cells harvested in 4) and resuspend them to 5.0×10 in an expansion medium containing 500 IU / mL IL-2, 7 ng / mL IL-7, 30 ng / mL IL-15, 5% v / v human AB serum, 1× PS dual antibody and X-VIVO 15 basal medium supplemented to the final volume. 5 / mL, added to the cell culture vessels pre-treated with anti-CD3 mAb, anti-CD28 mAb and anti-CD137 mAb, activated at 37℃5% CO2 for 2 days, then the activated cells were collected by centrifugation and inoculated into G-REX500M culture tanks with pre-heated expansion medium. The expansion medium in the G-REX500M culture tank was the same as the above expansion medium. The volume of expansion medium in each G-REX500M was 5L. The activated seed cells were cultured at 2.5×10 5 / cm 2 The cells were inoculated at a density of 1.0 × 10 and cultured at 37 °C with 5% CO2. After counting the cells every 4 days, half of the old expansion medium was removed and half of the fresh expansion medium was added. The total number of cells in each G-REX500M tank reached 1.0 × 10 10 After that, the cells were divided into bottles at a ratio of 1:2, and each bottle was supplemented with fresh expansion medium to 5 L before continuing to culture. After a total of 12 days of culture in the expansion medium of the G-REX500M culture tank, the cells were harvested to obtain TIL.

[0356] The types of tumor tissues used and the sample numbers are shown in Table 3:

[0357] Table 3 Tumor sample numbers

[0358] Organization Number Cancer T01 Melanoma T02 Gastric cancer T03 Ovarian cancer T04 Cervical cancer T05 Cholangiocarcinoma T06 Lung cancer T07 Colorectal cancer T08 Osteosarcoma

[0359] Step 3) of the above-mentioned TIL culture process is configured with 8 groups of seed cell culture medium, and IL-2 in each group is IL-2mut0, IL-2mut5, IL-2mut6, IL-2mut11, IL-2mut14, IL-2mut19, IL-2mut22 and IL-2mut25 prepared in Example 1 of the present invention respectively. IL-2mut5, IL-2mut6, IL-2mut11, IL-2mut14, IL-2mut19, IL-2mut22 and IL-2mut25 are equimolar in culture medium with IL-2mut0 of final concentration 3000IU / mL, and other culture conditions and culture steps are the same. Cell counting is performed at different time points in the seed cell culture stage, and the proliferation curve of each sample seed cell is recorded. The TIL obtained after the final expansion culture of each sample under different seed cell culture medium culture is finally harvested, and the CD45+, CD3+, CD8+, CD4+ and CD56+ cell ratios are detected by flow cytometry.

[0360] The culture results showed that in all tissue samples, the proliferation rate of TIL seed cells in the seed culture medium containing IL-2mut5, IL-2mut6, IL-2mut11, IL-2mut14, IL-2mut19, IL-2mut22 and IL-2mut25 was significantly higher than that of TIL seed cells in the seed culture medium containing IL-2mut0. The phenotype of TIL cells finally harvested was detected by flow cytometry, and the results are shown in Table 4. In T01, T03, T04, T6 and T7, after culturing with seed culture medium containing IL-2mut0 and IL-2mut6, the proportion of NK cells (CD3-CD56+) in the finally harvested TIL was high, while after culturing with IL-2mut5, IL-2mut11, IL-2mut14, IL-2mut19, IL-2mut22 and IL-2mut25 seed culture medium, the proportion of NK cells in the finally harvested TIL from the above four tissues was significantly lower than that of the first two. In T02, T05 and T08, the proportion of NK cells in TIL obtained after culture in various daughter cell culture media was low, among which the proportion of NK cells in TIL obtained after culture in seed cell culture media containing IL-2mut0 and IL-2mut6 was still relatively high compared with other groups. The above results show that for tumor tissues with NK cell infiltration, using IL-2mut5, IL-2mut11, IL-2mut14, IL-2mut22 and IL-2mut25 to replace wild-type cytokine IL-2 in the TIL culture system can selectively reduce the proportion of nonspecific killing NK cells, increase the proportion of CD3+T cells, and thus enhance the potential tumor killing effect of TIL.

[0361] Table 4 Proportion of CD3-CD56+ cells (NK) in TILs obtained from different tissue cultures (%)

[0362]

[0363] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

[0364] Part of this article

[0365] SEQ ID NO:1

[0366] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Claims

1. A mutant protein of IL-2, which has an altered affinity for IL-2R compared to a control, and which has at least one amino acid mutation compared to the sequence of the control, Preferably, The control sequence is as shown in SEQ ID NO:55 or a sequence having at least 90% sequence identity thereto; or, the control sequence is as shown in SEQ ID NO:55 and comprises a C125S mutation, or the control sequence is a sequence having at least 90% sequence identity thereto but comprises a C125S mutation, and / or The mutation sites include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 selected from the following: E15, L19, R38, F42, P65, H79, R81, P82, R83, S87, E95, D109.

2. The mutant protein according to claim 1, characterized in that The IL-2 mutant protein has reduced or eliminated affinity for IL-2Rα compared to the control, and the mutation comprises mutations at 1, 2, 3 or 4 sites selected from the following: R38, F42, P65, D109, Preferably, the mutation comprises a mutation at a site selected from any one of the following groups: (1)R38， (2) R38 and P65, (3) F42 and P65, (4) P65 and D109, (5) R38, F42 and P65, More preferably, the mutant protein has one or more characteristics selected from the following: The mutation of R38 is selected from the group consisting of: R38D, R38E, R38M, R38T, R38P, R38N, R38I, R38S, R38W, R38K, R38L, and R38F; The mutation of F42 is selected from: F42K, F42A; The mutation of P65 is selected from: P65R, P65W, P65I, P65H, P65V; The mutation of D109 is selected from: D109K, D109T, and D109S.

3. The mutant protein according to claim 1, characterized in that The IL-2 mutant protein has an improved affinity for IL-2Rβγ compared to the control, and the mutation comprises at least one, two, three, four, five, six, seven or eight mutations selected from the following sites: E15, L19, H79, R81, P82, R83, S87, E95, Preferably, the mutation comprises a mutation at a site selected from any one of the following groups: (1)L19， (2)S87， (3) E15 and L19, (4) L19 and S87, (5) L19, R81 and S87, (6) H79, R81, P82, R83 and S87, (7) L19, H79, R81, P82, R83 and S87, (8) E15, L19, H79, R81, P82 and R83, (9) H79, R81, P82, R83, S87 and E95; More preferably, the mutation has one or more characteristics selected from the following: The E15 point mutation is selected from: E15R, E15D, E15A; The L19 point mutation is selected from: L19W, L19F, L19D, L19Y; The H79 point mutation is selected from: H79E, H79D, H79Q; The R81 point mutation is selected from: R81D, R81N; The P82 point mutation is selected from: P82T, P82A, P82I; The R83 point mutation is selected from: R83E, R83D, R83A; The S87 point mutation is selected from: S87D, S87Y, S87E, S87M; The E95 point mutation is selected from: E95F, E95A, and E95Y.

4. The mutant protein according to claim 1, characterized in that The IL-2 mutant protein comprises: (1) a first mutation having reduced or eliminated affinity for IL-2Rα compared to the control, the first mutation being a mutation at 1, 2, 3 or 4 sites selected from the group consisting of R38, F42, P65, D109, and (2) a second mutation having improved affinity for IL-2Rβγ compared to the control, the second mutation being a mutation at 1, 2, 3, 4, 5, 6, 7 or 8 sites selected from the group consisting of E15, L19, H79, R81, P82, R83, S87, E95, Preferably, the first mutation is the mutation described in claim 2, and / or the second mutation is the mutation described in claim 3.

5. The mutant protein according to any one of claims 1 to 4, characterized in that The mutant protein of IL-2 further comprises mutations at one or more sites selected from the following: K35, T37, T41, K43, Y45, L72, Preferably, the K35 point mutation is K35D; the T37 point mutation is T37E; the T41 point mutation is T41E; the K43 point mutation is K43E; the Y45 point mutation is Y45K; and the L72 point mutation is L72F.

6. A fusion protein comprising immunoglobulin Fc and (1) the sequence as shown in SEO ID NO:55 and comprising the C125S mutation, or a sequence having at least 90% sequence identity thereto but comprising the C125S mutation, or (2) The IL-2 mutant protein according to any one of claims 1 to 5, Preferably, the immunoglobulin Fc is IgG1Fc or IgG4Fc, More preferably, the fusion protein has the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51, or a sequence having at least 70% sequence identity therewith.

7. A nucleic acid molecule comprising a sequence selected from the group consisting of: (1) a nucleic acid sequence encoding the IL-2 mutant protein according to any one of claims 1 to 5 or the fusion protein according to claim 6, (2) a variant having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with (1), (3) a complementary sequence of (1) or (2), Preferably, the nucleic acid sequence has a sequence as shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52, or a variant thereof having at least 80% sequence identity thereto.

8. A nucleic acid construct comprising the nucleic acid molecule described in any embodiment of the present invention, Preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

9. A host cell, wherein: (1) comprising, expressing and / or secreting the IL-2 mutant protein according to any one of claims 1 to 5 or the fusion protein according to claim 6, and / or (2) comprising the nucleic acid molecule according to claim 7 and / or the nucleic acid construct according to claim 8, Preferably, the host cell is a bacterial cell or a mammalian cell.

10. A culture medium comprising the IL-2 mutant protein according to any one of claims 1 to 5 or the fusion protein according to claim 6, and / or cells expressing and / or secreting the IL-2 mutant protein and / or fusion protein, Preferably, the culture medium further comprises one or more substances selected from the following: IL-1β, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-β, IFN-γ, G-CSF, M-CSF, GM-CSF, TNF-α, serum, antibodies or their antigen-binding fragments, and antibiotics.

11. The culture medium according to claim 10, characterized in that The culture medium is a seed cell culture medium; preferably, it is a seed cell culture medium for TIL, comprising the following components: a basal culture medium, cytokines, and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include the IL-2 mutant protein of any one of claims 1 to 5 and / or the fusion protein of claim 6, and the immune checkpoints include: PD-1, LAG-3, TIGIT, and / or CTLA-4, Preferably, the basal culture medium is selected from one or more of AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizerTM, and FUJIFILM IrvinMHM-C.

12. A method for preparing seed cells of tumor infiltrating lymphocytes, comprising the following steps: culturing the separated tumor infiltrating lymphocytes in the culture medium of claim 10, Preferably, The isolated tumor infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, surgically removed primary lesion samples, synchronous and metachronous surgically removed metastatic lesion samples, puncture samples and body fluids, and / or The isolated tumor infiltrating lymphocytes are derived from a tumor selected from the group consisting of gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, and pelvic poorly differentiated adenocarcinoma.

13. Seed cells of tumor infiltrating lymphocytes or a cell population thereof prepared by the method according to claim 12.

14. Use of the IL-2 mutant protein according to any one of claims 1 to 5 and / or the fusion protein according to claim 6 in preparing an immune effector cell culture medium or a medicament containing activated immune effector cells, Preferably, the drug is used to treat cancer; more preferably, the cancer is selected from: gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, Preferably, the immune effector cells are selected from one or more of the following: T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T, CIK, TIL, LAK, DC, DC-CIK; in one or more embodiments, the immune effector cells are Treg cells or CD8+T cells.

15. A method for culturing or expanding immune effector cells, comprising incubating the immune effector cells with the culture medium according to claim 10 or 11; Preferably, the immune effector cells are selected from one or more of the following: T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T, CIK, TIL, LAK, DC, DC-CIK, more preferably TIL.

16. A method for preparing a TIL cell population, comprising: (1) incubating a tissue containing tumor cells with the culture medium according to claim 10 or 11 to obtain a first TIL cell population, (2) incubating the first TIL cell population with an expansion medium to obtain a second TIL cell population, Preferably, the tumor cell-containing tissue is a tumor tissue or body fluid of a subject suffering from cancers such as gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, bile duct cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, etc. Preferably, the expansion medium comprises IL-2, IL-7, IL-15 and an immune checkpoint antibody or an antigen-binding fragment thereof. Optionally, the expansion medium further comprises one or more selected from the following: serum, platelets, and basal culture medium.

17. A TIL cell population prepared by the method according to claim 15.

18. Use of the IL-2 mutant protein according to any one of claims 1 to 5 and / or the fusion protein according to claim 6 in culturing immune effector cells, Preferably, the immune effector cells include: T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T, CIK, TIL, LAK, DC, DC-CIK.

19. Use of (1) the seed cell of tumor infiltrating lymphocytes or a cell population thereof according to claim 13, and / or (2) the TIL cell population according to claim 17 in the preparation of a drug for treating cancer, Preferably, the cancer is selected from the group consisting of gastric cancer, thyroid tumor, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, bile duct cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, gastrointestinal stromal tumor, endometrial stromal sarcoma, and pelvic poorly differentiated adenocarcinoma.

20. A pharmaceutical composition comprising (1) the tumor infiltrating lymphocyte seed cells or cell populations thereof according to claim 13, and / or (2) the TIL cell population according to claim 17, and pharmaceutically acceptable excipients.

21. A kit for activating immune effector cells, the kit comprising the IL-2 mutant protein according to any one of claims 1 to 5 and / or the fusion protein according to claim 6, Preferably, the kit further comprises reagents required for culturing PBMC cells.

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