WO2023138598A1 - 肿瘤浸润淋巴细胞在疾病治疗中的用途 - Google Patents

肿瘤浸润淋巴细胞在疾病治疗中的用途 Download PDF

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WO2023138598A1
WO2023138598A1 PCT/CN2023/072784 CN2023072784W WO2023138598A1 WO 2023138598 A1 WO2023138598 A1 WO 2023138598A1 CN 2023072784 W CN2023072784 W CN 2023072784W WO 2023138598 A1 WO2023138598 A1 WO 2023138598A1
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til
cell
use according
cells
tils
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PCT/CN2023/072784
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English (en)
French (fr)
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刘雅容
种孟阳
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苏州沙砾生物科技有限公司
上海沙砾生物科技有限公司
珠海拓域生物科技有限公司
珠海沙砾生物科技有限公司
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Publication of WO2023138598A1 publication Critical patent/WO2023138598A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present application relates to the field of biomedicine, in particular to the use of tumor-infiltrating lymphocytes in disease treatment.
  • adoptive autologous transfer of tumor-infiltrating lymphocytes is an effective approach for patients with poor prognosis.
  • adoptive autologous transfer of tumor infiltrating lymphocytes to treat tumors requires a large number of tumor infiltrating lymphocytes, and the expansion ability of tumor infiltrating lymphocytes from patient tumors is weak, and the cell function is not strong.
  • the present application provides a use of tumor infiltrating lymphocytes in disease treatment.
  • the tumor infiltrating lymphocytes obtained by using the culture method described in this application can demonstrate effectiveness and safety in tumor treatment.
  • the present application provides a use of tumor-infiltrating lymphocytes (TIL) in the preparation of a medicament for preventing and/or treating tumors
  • TIL tumor-infiltrating lymphocytes
  • the TIL is obtained by the following culture method: TIL derived from tumor tissue undergoes at least one stage of in vitro expansion, wherein in a single in vitro expansion stage, TILs that have been expanded in vitro and/or have not been expanded in vitro are co-cultured with feeder cells after being contacted with T cell activators and/or T cell growth factors for a certain period of time.
  • the present application provides an application of tumor-infiltrating lymphocytes (TIL) in the preparation of medicines for preventing and/or treating tumors, and the TILs are obtained by the following culture method:
  • step (A) contacting the first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • the present application provides an application of tumor-infiltrating lymphocytes (TIL) in the preparation of medicines for preventing and/or treating tumors, and the TILs are obtained by the following culture method:
  • step (B) co-culture the second TIL population with feeder cells after contacting with T cell activator and/or T cell growth factor for a certain period of time, wherein the third TIL population is obtained through the step (B).
  • the present application provides an application of tumor-infiltrating lymphocytes (TIL) in the preparation of medicines for preventing and/or treating tumors, and the TILs are obtained by the following culture method:
  • step (A) contacting the first TIL population derived from tumor tissue and not expanded in vitro with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • step (B) contacting said second population of TILs with a T cell activator and/or a T cell growth factor, wherein said step (B) results in a third population of TILs;
  • the present application provides an application of tumor-infiltrating lymphocytes (TIL) in the preparation of medicines for preventing and/or treating tumors, and the TILs are obtained by the following culture method:
  • step (B) contacting said second population of TILs with a T cell activator and/or a T cell growth factor, wherein said step (B) results in a third population of TILs;
  • the present application provides a pharmaceutical composition, comprising TIL obtained according to the culture method described in the application of the present application, and a substance that reduces the number of lymphocytes and/or a substance that maintains the proliferative ability of the TIL.
  • the present application provides a kit comprising the pharmaceutical composition according to the present application.
  • Figure 1 shows the comparison of TIL proliferation ability of donors A-1, A-2, A-3 and A-4, when feeder cells were added after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 2 shows the ratio of CD45RA - CCR7 + central memory T cells (Tcm) in CD8 + or CD4 + CD45RA - CCR7 + central memory T cells (Tcm) in TILs cultured with feeder cells after 0 h, 24 h or 48 h after the addition of OKT3 and IL-2 for donors B-1 and B-2 Example comparison.
  • Figure 3 shows the ratio of CD45RA - CCR7 + central memory T cells (Tcm) in CD8 + or CD4 + CD45RA - CCR7 + central memory T cells in TILs cultured with feeder cells after adding OKT3 and IL-2 for 0 hours, 24 hours or 48 hours for donors B-3, B-4 and B-5.
  • Figure 4 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cultured with feeder cells after adding OKT3 and IL-2 for 0 hours, 24 hours or 48 hours for donors C-1 and C-2.
  • Figure 5 shows the ratio of PD1 + activated T cells and LAG3 + activated T cells in CD8 + or CD4 + in TIL cultured with feeder cells after adding OKT3 and IL-2 for 0 hours, 24 hours or 48 hours for donors D-1 and D-2.
  • Figure 6 shows the ratio of CD28 + activated T cells in CD8 + among TILs cultured with feeder cells after adding OKT3 and IL-2 for 0 hours, 24 hours or 48 hours for donors D-3, D-4 and D-5.
  • Figure 7 shows the ratio of CD103 + CD39 + tumor - specific T cells in CD8 + or CD4 + in TILs cultured with feeder cells after adding OKT3 and IL-2 for 0 hours, 24 hours or 48 hours for donors E-1 and E-2.
  • Figure 8 shows the comparison of the ratio of TCF1 + stem cell-like T cells in TIL cultured with feeder cells after adding OKT3 and IL-2 for donors F-1 and F-2 at 0 hours, 24 hours or 48 hours.
  • Figure 9 shows the results of cell proliferation ability of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 10 shows the results of CD45RA - CCR7 + central memory T cell (Tcm) ratio of TIL cells cultured after adding feeder cells for 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 11 shows the ratio of TCF1 + stem cell-like T cells in TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 12 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cells.
  • Figure 13 shows the ratio of activated T cells (PD1 + ) in TIL cells obtained by adding feeder cells after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 14 shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours after adding feeder cells.
  • Fig. 15 shows the ratio of activated T cells (CD28 + ) of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 16 shows the addition of OKT3 and IL-2 0 hours, 24 hours or 48 hours after the addition of feeder cell culture TIL, the proportion of activated T cells (41BB + ) in cultured TIL cells.
  • Fig. 17 shows the proportion of activated T cells (CD25 + ) in TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Fig. 18 shows the detection results of intracellular factor expression of TIL cells cultured by adding feeder cells after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2.
  • Figure 19 shows the cytokine secretion test results of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Figure 20 shows the results of cell proliferation ability of TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells.
  • Figure 21 shows the ratio of CD8 + T cells in TIL cells cultured after adding OKT3 and IL-2 at 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells.
  • Figure 22 shows the ratio of CD45RO + CD62L + T cells in TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells.
  • Figure 23 shows the NKT cell ratio of TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days.
  • Figure 24 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained after adding OKT3 and IL-2 for 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells to culture TILs.
  • Figure 25 shows the results of the cell killing ability of TIL cells cultured by adding feeder cells 48 hours after adding OKT3 and IL-2.
  • Figures 26A and 26B show the clinical imaging results of the treatment method of the present application.
  • cyclophosphamide generally refers to an agent that treats cells.
  • a mustard alkylating agent which can add an alkyl group (C n H 2n+1 ) to DNA.
  • cyclophosphamide could refer to the formula C 7 H 15 Cl 2 N 2 O 2 P ⁇ H 2 O, chemical name 2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphine 2-oxide monohydrate.
  • cyclophosphamide may have a CAS Registry No. 50-18-0 formulation. Cyclophosphamide is available commercially.
  • fludarabine generally refers to an agent that treats cells.
  • a fluorinated nucleotide analogue for example, fludarabine may refer to the preparation with molecular formula C 10 H 12 FN 5 O 4 and CAS accession number 21679-14-1.
  • the term "expression” generally refers to the process of transcription and/or translation of a gene encoding a target polypeptide within a cell.
  • the level of transcription of a gene encoding a polypeptide of interest in a host cell can be determined by measuring the amount of corresponding mRNA present in the cell. For example, mRNA transcribed from a gene encoding a polypeptide of interest can be quantitatively measured by PCR or by RNA hybridization.
  • the level of translation of a gene encoding a polypeptide of interest can be measured by various methods, such as by ELISA, by a polypeptide bioactivity assay, or by Western blot or radioimmunoassay.
  • stage in the term “one stage of in vitro expansion”, “single stage of in vitro expansion”, or “first stage of in vitro expansion” generally refers to a period of expansion process that TIL goes through in vitro.
  • each stage can be divided by changes in the number of TIL cells.
  • an increase in the number of TIL cells by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold is considered to be TIL cells entered the next stage of in vitro expansion.
  • each stage can also be divided by the conditions of TIL cell culture.
  • T cell co-stimulatory molecules and/or T cell growth factors when added or supplemented to the cell culture medium, it can be considered that the TIL cells enter the next stage of in vitro expansion. In one embodiment, after the TIL cells are centrifuged and/or washed, the TIL cells can be considered to enter the next stage of in vitro expansion. In one embodiment, each stage can also be divided by the days of TIL cell culture.
  • TIL cells when the TIL cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days or about 100 days Days later, TIL cells can be considered to have entered the next stage of in vitro expansion.
  • the term "the first stage of in vitro expansion” usually refers to the stage of using T cell growth factors to expand after primary TILs are obtained from tissues.
  • the tissue may be tumor tissue.
  • the amplification may be autologous or allogeneic in vivo amplification, or may be in vitro amplification.
  • the first stage of amplification may also be referred to as the preREP (Pre-Rapid Amplification) stage.
  • the term "the second stage of in vitro expansion” generally refers to the stage of expanding again after the tissue taken from the subject is expanded.
  • the number of TIL cells expanded in the second stage is increased, for example, may be at least about 10-fold (or at least about 20, 30, 40, 50, 60, 70, 80, or 90-fold), or in one embodiment the number of cells may be increased by at least about 100-fold, compared to TILs expanded in the first stage.
  • the culture conditions of the second-stage amplification may be different from those of the first-stage amplification, for example, the culture substances added may be different.
  • the second stage of amplification may also be referred to as the REP (Rapid Amplification) stage.
  • in vivo generally refers to an event that occurs in the body of a subject.
  • in vitro generally refers to events that occur outside the body of a subject.
  • ex vivo generally refers to an event involving treatment or surgery on cells, tissues and/or organs that have been removed from a subject.
  • the cells, tissues and/or organs can be returned to the body of the subject by surgery or therapy.
  • secretion generally refers to the transfer of an expressed polypeptide or protein by a cell to the extracellular environment.
  • secretion capacity generally refers to the ability of a cell to express a polypeptide or protein and to transfer said polypeptide or protein to the extracellular environment.
  • irradiation generally refers to the treatment of matter by radiation.
  • irradiating may refer to irradiating a substance with X-rays, ⁇ -rays, ⁇ -rays or ⁇ -rays.
  • engineered cell generally refers to a cell that has been genetically modified by adding additional genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • engineered cells can be genetically modified to express TILs of T cell co-stimulatory molecules and/or T cell growth factors according to the present application.
  • co-culture generally refers to the cultivation of two or more different populations of cells with some degree of contact between them.
  • the "contact" of the two or more different populations of cells may, in one embodiment, be by direct contact, ie, wherein cells of one population are in direct physical contact with cells of the other population. Or in one embodiment it may be through indirect contact mediated by a common culture medium.
  • the common medium may contain metabolites produced and released by at least one population of co-cultured cells and be used to culture cells of another population.
  • the term "contacting" generally means that two or more substances of different types are brought into contact together in any order, in any manner, and for any length of time.
  • it can be through direct contact, for example, a feeder cell, T cell co-stimulatory molecule and/or T cell growth factor can be added to the culture medium of TIL cells; in one embodiment, it can be through indirect contact, for example, the metabolites produced and released by feeder cells can be used to cultivate TIL cells.
  • mixture generally refers to a combination of two or more different substances.
  • the terms “concurrent contact”, “co-contact”, “simultaneously with”, “simultaneously” and “commonly” generally refer to administering two or more substances to a subject such that the substances are simultaneously present in the subject and/or in the environment in which the subject is cultivated.
  • Simultaneous contacting can include simultaneous administration in different compositions, administration in different compositions at different times, or administration in a composition in which two or more active pharmaceutical ingredients are present.
  • the term “expansion” generally refers to a several-fold increase in the number of cells over a period of time. In one embodiment the number of cells can be increased by at least about 3 fold (or 4, 5, 6, 7, 8 or 9 fold), in one embodiment the number of cells can be increased by at least about 10 fold (or 20, 30, 40, 50, 60, 70, 80 or 90 fold), or in one embodiment the number of cells can be increased by at least about 100 fold. In this application, the term “expanded” generally means that the cells have undergone one or more expansions as described above.
  • polymer generally refers to a molecule consisting of individual chemical moieties linked together, which may be the same or different.
  • the term “polymer” may refer to individual chemical moieties joined end to end to form linear molecules, as well as individual chemical moieties linked together in branched (eg, "multi-armed” or "star") structures.
  • the polymer may include, for example, a hydrogel, polyethylene glycol, or a poloxamer.
  • Poloxamers are nonionic triblock copolymers having a polyoxypropylene (poly(propylene oxide)) central hydrophobic chain flanked by two polyoxyethylene (poly(ethylene oxide)) hydrophilic chains.
  • the materials encompassed herein may be formulated with, or administered with, any polymer described herein or known in the art.
  • antibody generally refers to an immunoglobulin reactive with a specified protein or peptide or a fragment thereof.
  • Such antibodies include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, monoclonal antibodies, monospecific antibodies, polyclonal antibodies, polyspecific antibodies, nonspecific antibodies, bispecific antibodies, multispecific antibodies, humanized antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, graft-conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), and antibodies produced in vitro.
  • Antibodies can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (eg, IgGl, IgG2, IgG3, and IgG4).
  • the antibody can have a heavy chain constant region selected from, for example, IgGl, IgG2, IgG3, or IgG4.
  • Antibodies may also have light chains selected from eg kappa ( ⁇ ) or lambda ( ⁇ ).
  • the antibodies may be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, chicken, and cow.
  • the constant region of an antibody can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function).
  • an antibody specifically binds a predetermined antigen, eg, an antigen associated with a disorder, such as an inflammatory, immune, autoimmune, neurodegenerative, metabolic, and/or malignant disorder.
  • anti-CD3 antibody generally refers to CD3-targeting antibodies or variants thereof, such as monoclonal antibodies, including human, humanized, chimeric or murine antibodies, which target the CD3 receptor in the T cell antigen receptor of mature T cells.
  • Anti-CD3 antibodies can include OKT-3.
  • Anti-CD3 antibodies can also include other anti-CD3 antibodies including, for example, otelixizumab, teplizumab, and visilizumab in one embodiment.
  • IL-2 or "IL2” generally refers to the T-cell growth factor known as interleukin 2, and includes all forms of IL-2, which may include in one embodiment human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, or active fragments thereof.
  • the GeneID of the gene encoding IL2 may be 3558.
  • the terms “antigen presenting cell”, “antigen presenting cell”, or “APC” generally refer to cells of the immune system, such as helper cells (e.g., B cells, dendritic cells, etc.), that display exogenous antigens complexed with major histocompatibility complex (MHC) on their surface. T cells can recognize these complexes using their T cell receptor (TCR). APCs can process antigens and present them to T cells.
  • the antigen presenting cells may comprise a group selected from peripheral mononuclear cells, dendritic cells, and artificial antigen presenting cells.
  • TIL characteristics generally refers to the characteristics of TIL cells obtained through the cultivation method of this application. Changes in TIL properties may comprise: increased numbers of TIL cells, increased proportion of viable cells, increased viability, improved proportion of T cell subsets, increased cytokine secretion capacity, increased tumor cell killing capacity, increased T cell receptor (TCR) clonal diversity and increased numbers of TIL cells in tissues and/or tumors, or any combination thereof.
  • the changes in this application can be increased or decreased.
  • the term “amplification effect” generally refers to the effect that occurs after cells are expanded. Changes in amplification effects may include changes in the number and/or ratio of cells, changes in secretion capacity, changes in killing capacity or changes in expression capacity, or any combination thereof. The change may be an increase or a decrease.
  • expanded generally refers to being cultured to produce changes in the number of cells
  • expanded cells may also produce changes in the number and/or ratio of cells, changes in secretion capacity, changes in killing capacity or changes in expression capacity, or any combination thereof.
  • the change may be an increase or a decrease.
  • nanoparticle generally refers to microscopic particles having at least one dimension smaller than 100 nm.
  • nanoparticles typically have a diameter in the range of 50 nm to 500 nm (i.e., 0.05 ⁇ m to 0.5 ⁇ m); are structurally stable in physiological environments; and can accommodate smaller molecules, such as drugs or other bioactive agents, which can then be delivered to desired sites.
  • artificial antigen-presenting cell generally refers to an immune system cell artificially constructed to display exogenous antigen complexed with major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • isolated artificial antigen-presenting cells can be included, which can comprise expression HLA-A/B/C (the gene GeneID encoding it can be 3105, 3106 or 3107), CD64 (the gene GeneID encoding it can be 2209), CD80 (the gene GeneID encoding it can be 2209), CD80 (the gene GeneID encoding it can be 941), ICOS-L (the gene GeneID encoding it can be 23308) and CD58 (the gene GeneID encoding it can be 965), and can be modified to express more than one co-stimulatory molecule, and the above can include this number.
  • HLA-A/B/C the gene GeneID encoding it can be 3105, 3106 or 3107
  • CD64 the gene GeneID encoding it can be 2209
  • CD80 the
  • fusion protein generally refers to a polypeptide or protein comprising the amino acid sequence of a first polypeptide or protein or a fragment, analog or derivative thereof and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein or fragment, analog or derivative thereof that is different from the first polypeptide or protein or fragment, analog or derivative thereof, or generally not a part of the first polypeptide or protein or fragment, analog or derivative thereof).
  • a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide.
  • the heterologous protein, polypeptide or peptide may or may not be different types of preventive or therapeutic drugs.
  • two different proteins, polypeptides or peptides with immunomodulatory activity can be fused together to form a fusion protein.
  • the fusion protein may retain or increase activity compared to the activity of the original polypeptide or protein prior to fusion of the heterologous protein, polypeptide or protein.
  • the term "killing ability” generally refers to killing target cells by contacting the cells with an effective amount of substances.
  • the substance may be TIL cells.
  • the killing may include killing cells by themselves or promoting CDC, apoptosis, ADCC, and/or phagocytosis of other cells or substances, or by a combination of two or more of these mechanisms.
  • administering generally refers to delivering a substance to a subject in need thereof by any route known in the art.
  • Pharmaceutically acceptable carriers and formulations or compositions are also well known in the art. Routes of administration may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral and/or mucosal.
  • kit generally refers to two or more components packaged together in a container, receptacle or other container, one of which corresponds to the substance of the present application.
  • TIL cells of the present application are included.
  • the term "subject” generally refers to a cell or an animal, which can be a mammal, such as a human, a non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dog and cat), farm animal (poultry such as chicken and duck, horse, cow, goat, sheep, pig) and experimental animal (mice, rat, rabbit, guinea pig).
  • Human subjects include fetal, neonatal, infant, adolescent and adult subjects.
  • Subjects include animal disease models, such as tumor animal models, and other animal models known to those skilled in the art.
  • kit generally refers to two or more components packaged together in a container, receptacle or other container, one of which corresponds to the substance of the present application.
  • TIL cells of the present application are included.
  • feeder generally refers to a cultured cell that grows in vitro and secretes at least one factor into the culture medium and can be used to support the growth of another cell of interest in culture.
  • feeder cells may include antigen presenting cells.
  • the term "specifically binds” generally refers to an antibody that recognizes a specific antigen, but does not substantially recognize or bind to other molecules in a sample. For example, if an antibody can specifically bind said specific antigen from one species, said antibody can also specifically bind said antigen or a cognate antigen from one or more other species. This cross-species reactivity by itself may not alter the classification of the antibody as specific. In certain instances, an antibody that specifically binds to an antigen may also bind to a different allelic form of the antigen.
  • complete culture process generally refers to the complete process of separating cells from tumor tissue isolated from a patient, undergoing one or more expansions, and finally obtaining cells that can be administered to a subject.
  • cell culture medium generally refers to a nutrient solution in which cells, such as mammalian cells, are grown.
  • the formulation of cell culture media is well known in the art.
  • cell culture media include buffers, salts, carbohydrates, amino acids, vitamins and necessary trace elements.
  • Cell culture media may or may not contain serum, peptone, and/or protein.
  • the cell culture medium may be supplemented with additional components or components of increased concentration, such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc., depending on the requirements of the cells to be cultured and/or the desired cell culture parameters.
  • the term "pharmaceutical formulation” or “pharmaceutical composition” generally refers to a preparation that allows the biological activity of the active ingredient to be effective and that may contain no additional components that are unacceptably toxic to the subject to which the formulation will be administered. Such preparations are sterile. "Pharmaceutically acceptable” excipients (carriers, additives) are those which can reasonably be administered to a subject mammal to provide an effective dosage of the active ingredient employed.
  • TIL tumor infiltrating lymphocytes
  • TILs may include, but are not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages.
  • TILs can include primary TILs and secondary TILs.
  • Primary TILs can be those TIL cells obtained from a tissue sample of a subject, and "secondary TILs" can be any population of TIL cells in this application that has been or has been expanded.
  • the tumor-infiltrating lymphocytes may not be isolated and purified, or may infiltrate with tumor cells.
  • TIL in the present application may refer to TIL cell population.
  • CD4 + cells generally refers to CD4 positive cells, such as T cells.
  • the terms “CD4 + cells”, “CD4 positive cells” may be used synonymously.
  • These cells can be identified by methods known in the art, such as by staining the cells with a fluorescently labeled antibody directed against CD4 and using fluorescence activated cell sorting.
  • existing data can prove that an increase in the proportion of CD4 + cells can increase the ability of the cell population to secrete IFN- ⁇ and/or TNF, and can improve the effect of promoting tumor suppression of the T cell population. See, for example, Tay, RE, Richardson, EK et al. (2020). Cancer Gene Therapy, 1-13.
  • this application can provide a method to affect the ratio of CD4 + cells.
  • CD8 + cells generally refers to CD8 positive cells, such as T cells.
  • CD8 + cells CD8 positive cells
  • CD8 positive cells may be used synonymously. These cells can be identified by methods known in the art, such as by staining the cells with a fluorescently labeled antibody directed against CD8 and using fluorescence activated cell sorting.
  • central memory T cells generally refers to T cells that have long-term memory and are capable of receiving antigen restimulation.
  • Central memory T cells may have a CD45RA ⁇ CCR7 + phenotype, for example, central memory T cells may be identified by CD45RA ⁇ and CCR7 + .
  • central memory T cells may have a phenotype of CD45RO + CD62L + , for example, central memory T cells may be identified by CD45RO + and CD62L + .
  • Central memory T cells can have a stronger ability to resist tumor growth than ordinary T cells.
  • regulatory T cells generally refers to a subset of T cells that control autoimmune reactivity in vivo. Regulatory T cells may have a phenotype of CD4 + CD25 + Foxp3 + , for example, regulatory T cells may be identified by CD4 + , CD25 + and Foxp3 + . Regulatory T cells may have the ability to suppress the anti-tumor growth of T cells.
  • activated T cells generally refers to T cells that have been activated to have the ability to resist tumor growth.
  • Activated T cells may have a phenotype of PD1 + , LAG3 + or CD28 + , for example, activated T cells may be identified by PD1 + , LAG3 + or CD28 + .
  • Activated T cells may have the ability to fight tumor growth.
  • tumor-specific T cells generally refers to T cells that can specifically fight tumor growth.
  • Tumor-specific T cells may have a phenotype of CD103 + CD39 + , for example, tumor-specific T cells may be identified by CD103 + and CD39 + .
  • Tumor-specific T cells can have a more specific ability to resist tumor growth than ordinary T cells.
  • stem cell-like T cell generally refers to a type of T cell that may have the potential of self-proliferation and/or differentiation.
  • Stem cell-like T cells may have a TCF1 + phenotype, for example, stem cell-like T cells may be identified by TCF1 + .
  • Tumor-specific T cells may have stronger and/or longer-term anti-tumor growth capabilities than ordinary T cells.
  • NK cell is also called “natural killer cell”, and generally refers to a cell with large granules in the cytoplasm.
  • NK cells are developed from bone marrow lymphoid stem cells and can differentiate and develop depending on the bone marrow or thymus microenvironment.
  • the proportion of NK cells in TIL cells can be changed by the method of this application.
  • tumor fragments generally refers to tumor fragments that can be formed by crushing after the tumor tissue is removed from the subject.
  • composition or “pharmaceutical composition” generally refers to a mixture of at least one cell and at least one and optionally more than one other pharmaceutically acceptable chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients.
  • the term "pharmaceutically acceptable carrier” generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for human administration.
  • Other contemplated carriers, excipients, and/or additives that may be used in the formulations described herein may include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), and the like.
  • T cell co-stimulatory molecule generally refers to a ligand that binds to a corresponding binding receptor on a T cell and mediates a T cell co-stimulatory response.
  • Costimulatory molecules may be cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response.
  • Co-stimulatory molecules may include but not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activation receptors, BTLA (the gene GeneID encoding it can be 151888), Toll ligand receptor, OX40 (the gene GeneID encoding it can be 7293), CD2 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 914), CD7 (the gene GeneID encoding it can be 151888), 924), CD27 (the gene GeneID encoding it can be 939), CD28 (the gene GeneID encoding it can be 940), CD30 (the gene GeneID encoding it can be 943), CD40 (the gene GeneID encoding it can be 958), CDS, ICAM-1 (the gene GeneID encoding it can be 3383), LFA-1 (CD11a/CD18) (
  • Costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.
  • the intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain or a functional fragment thereof.
  • the term "T cell growth factor” generally refers to a biologically active polypeptide or small molecule compound that causes cell proliferation.
  • the T cell growth factor can be selected from one or more of the following groups: IL-2 (the gene GeneID encoding it can be 3558), IL-4 (the gene GeneID encoding it can be 3565), IL-7 (the gene GeneID encoding it can be 3574), IL-10 (the gene GeneID encoding it can be 3586), IL-12 (the gene GeneID encoding it can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3592 or 3593), IL-15 (the gene GeneID encoding it can be 3592 or 3593), Its gene GeneID may be 3600), and gamma interferon (the gene GeneID encoding it may be 3458).
  • substantially simultaneously usually means that TIL can be contacted with two or more substances simultaneously during a period of time during the contact process, but it is not limited to that TIL is always in contact with two or more substances simultaneously during the entire contact process.
  • substantially simultaneously can mean that the TIL can be in contact with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of each of the two or more substances simultaneously over a period of time.
  • the term "neoplastic” generally refers to any new pathological growth of tissue.
  • the tumors of this application may be benign or malignant.
  • the tumors of this application may be solid or hematological.
  • the term “tumor” may be selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.
  • tumor tissue generally refers to a sample of any tissue from a tumor in a subject, including any solid tumor and/or non-solid tumor in a subject.
  • the terms “about” and “approximately” generally mean within a statistically meaningful range of values. Such ranges may be within an order of magnitude of a given value or range, may be within 50%, may be within 20%, may be within 10%, may be within 5%.
  • the permissible variations encompassed by the term “about” or “approximately” depend on the particular system under study and are readily understood by those of ordinary skill in the art.
  • the terms “above”, “below”, “at most” and “at least” may be inclusive of numbers.
  • the present application relates to a use of tumor-infiltrating lymphocytes (TIL) in disease treatment, and the drug is used to prevent and/or treat tumors, wherein the tumor-infiltrating lymphocytes are cultured by the following method.
  • TILs can be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors.
  • the present application provides a method for culturing tumor-infiltrating lymphocytes (TILs), which includes: co-culturing the expanded TILs with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors for a period of time.
  • the expanded TIL may be an in vitro expanded TIL.
  • the lymphatic system and/or lymphatic system of the subject can be Cells are processed.
  • the subject can be depleted or substantially depleted of lymphocytes, the subject can be depleted or substantially depleted of lymphocytes, the subject can be depleted or substantially depleted of lymphocytes, or the subject can be depleted or substantially depleted of lymphocytes.
  • the treatment of the present application can reduce the number of lymphocytes in the subject by at least about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, relative to before the treatment.
  • reducing the number of lymphocytes in the subject of the present application comprises a lymphodepleting regimen.
  • the depletion regimen of the present application may comprise administering cyclophosphamide and/or fludarabine to the subject before administering the TIL obtained by the culturing method to the subject.
  • it may further comprise administering cyclophosphamide and/or fludarabine to the subject from day 4 to day 2 before administering the TIL obtained by the culturing method to the subject.
  • cyclophosphamide can be administered to the subject at a dose of about 300 to about 500 mg/ m2 /day. For example, about 50 mg/m 2 /day, about 100 mg/m 2 /day, about 150 mg/m 2 /day, about 200 mg/m 2 /day, about 250 mg/m 2 /day, about 300 mg/m 2 /day, about 350 mg/ m 2 / day, about 400 mg/m 2 /day, about 450 mg/m 2 /day, or about 500 mg/m 2 /day
  • the subject is administered cyclophosphamide at a daily dose.
  • cyclophosphamide can be administered to the subject for 3 days.
  • cyclophosphamide can be administered to the subject from day 4 to day 2 prior to administration of the TIL.
  • fludarabine can be administered to the subject at a dose of about 20 to about 30 mg/ m2 /day.
  • fludarabine can be administered to the subject at a dose of about 5 mg/ m2 /day, about 10 mg/ m2 /day, about 15 mg/ m2 /day, about 20 mg/ m2 /day, about 25 mg/ m2 /day, or about 30 mg/ m2 /day.
  • fludarabine can be administered to the subject for 3 days.
  • fludarabine may be administered to the subject from day 4 to day 2 prior to administration of the TIL.
  • the amount of TIL provided in the compositions of the present application can range from about 5 ⁇ 10 9 to 1 ⁇ 10 10 , from about 1 ⁇ 10 10 to 5 ⁇ 10 10 , or from about 5 ⁇ 10 10 to 1 ⁇ 10 11 . In some embodiments, the amount of TIL provided in the compositions of the present application can range from about 5 ⁇ 10 9 , about 1 ⁇ 10 10 , about 5 ⁇ 10 10 , or about 1 ⁇ 10 11 .
  • it may comprise administering the TIL obtained by the culture method to the subject as a one-time intravenous infusion.
  • multiple intravenous infusions of the TIL obtained by the culturing method to the subject may be included.
  • the TIL after administering the TIL obtained by the culturing method to the subject, the TIL can be maintained with the ability to proliferate.
  • it may further comprise administering interleukin-2 (IL-2) or a variant thereof to the subject after administering the TIL obtained by the culturing method to the subject.
  • IL-2 interleukin-2
  • IL-2 can be administered to the subject at a dose of about 200,000 to about 600,000 IU/kg/time.
  • a dose of about 200,000 to about 600,000 IU/kg/time For example, about 100,000IU/kg/time, about 200,000IU/kg/time, about 300,000IU/kg/time, about 400,000IU/kg/time, about IL-2 is administered to the subject at a dose of 500,000 IU/kg/time, or about 600,000 IU/kg/time.
  • IL-2 may be first administered to the subject 8 to 16 hours after the TIL obtained by the culturing method is administered to the subject.
  • IL-2 can be administered to the subject every 12 hours after the first administration of IL-2 to the subject.
  • the administration of IL-2 can be suspended once; for example, whether to continue the administration of IL-2 can be determined according to the patient's tolerance.
  • the IL-2 can be administered for the second time according to the tolerance of the subject.
  • whether to continue administering IL-2 can be determined according to the adverse reaction of the subject. For example, 10 or fewer doses of IL-2 can be administered to the subject.
  • the expanded TILs are TILs obtained by subjecting TILs derived from tumor tissue and not expanded in vitro to at least one stage of in vitro expansion.
  • at least 2 stages of in vitro expansion can be performed, at least 3 stages of in vitro expansion can be performed, at least 4 stages of in vitro expansion can be performed, at least 5 stages of in vitro expansion can be performed, at least 6 stages of in vitro expansion can be performed, at least 7 stages of in vitro expansion can be performed, at least 8 stages of in vitro expansion can be performed, at least 9 stages of in vitro expansion can be performed, or at least 10 stages of in vitro expansion can be performed.
  • each stage of in vitro expansion can be divided by the change in the number of TIL cells.
  • the number of TIL cells increases by at least about 1-fold, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • the number of TIL cells is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 10 fold 0 times, at least about 200 times, at least about 500 times, or at least about 1000 times, it can be considered that the TIL cells have entered the next stage of in vitro expansion.
  • each stage of in vitro expansion can also be divided by changing the conditions of TIL cell culture.
  • TIL cells when T cell activators and/or T cell growth factors are added or supplemented to the cell culture medium, TIL cells can be considered to enter the next stage of in vitro expansion.
  • T cell activators and T cell co-stimulatory molecules can be used interchangeably in this application.
  • IL-2 when added or supplemented to the cell culture medium, TIL cells can be considered to enter the next stage of in vitro expansion.
  • feeder cells are added or supplemented to the cell culture medium, TIL cells can be considered to enter the next stage of in vitro expansion.
  • each stage can also be divided by the days of TIL cell culture.
  • TIL cells when TIL cells are cultured in vitro for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about After 17 days, about 18 days, about 19 days, about 20 days, about 30 days, about 40 days, about 50 days or about 100 days, TIL cells can be considered to enter the next stage of in vitro expansion.
  • the second stage of in vitro expansion can be performed for at least about 7 days.
  • the second stage of in vitro expansion can be performed for at least about 9 days.
  • the second stage of in vitro expansion can be performed for up to about 14 days.
  • the second stage of in vitro expansion can be performed for up to about 13 days.
  • the second stage of in vitro expansion can be performed for about 7 days to about 14 days, about 9 days to about 14 days, about 7 days to about 13 days, or about 9 days to about 13 days.
  • the second stage in vitro expansion of the present application can be performed for at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the second stage of in vitro amplification of the present application can be carried out for about 9 days to about 14 days to about 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the second stage of in vitro expansion in the present application can be considered as the REP (rapid expansion protocol) stage.
  • the first stage of in vitro expansion can be performed for at least about 7 days.
  • the first stage of in vitro expansion can be performed for about 7 days to about 14 days.
  • the first stage in vitro expansion of the present application can be performed for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the first stage of in vitro amplification of the present application can be performed for about 7 days to about 14 days, about 8 days to about 14 days, about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, about 13 days to about 14 days, about 9 days to about 13 days, about 10 days to about 13 days, about 11 days to about 13 days, about 12 days to about 13 days, about 9 days to about 13 days 12 days, about 10 days to about 12 days, about 11 days to about 12 days, or about 10 days to about 11 days.
  • the first stage of in vitro expansion of the present application can be considered the preREP stage.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated from the start of the second stage of in vitro expansion. For example, when the second-stage in vitro expansion starts, it can be considered that the second-stage in vitro expansion has been performed for about 0 days. For example, if the second-stage in vitro expansion proceeds for about 24 hours, it can be considered that the second-stage in vitro expansion has been performed for about one day. For example, the day when the second-stage in vitro expansion starts can be considered as about 0 days after the second-stage in vitro expansion.
  • the number of days for the second stage of in vitro expansion in the present application can be calculated based on the number of days for the second stage of in vitro expansion. For example, on the second day after the start of the second-stage in vitro expansion, it can be considered that the second-stage in vitro expansion has been performed for about one day.
  • the method for cultivating TILs used in this application can be divided into two steps.
  • the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • the second TIL population can be contacted with a T cell activator and/or a T cell growth factor
  • co-cultivate with feeder cells wherein the third TIL population is obtained through the step (B).
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) may be performed for about 7 days to about 14 days.
  • the method for cultivating TILs used in this application can be divided into three steps.
  • (A) the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • (B) the second TIL population can be contacted with a T cell activator and/or a T cell growth factor, wherein a third TIL population is obtained through the step (B);
  • the third TIL population can be co-cultured with a feeder cell, wherein a fourth TIL population is obtained through the step (C).
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) can be performed for about 0 days to about 8 days.
  • step (C) may be performed for about 5 days to about 14 days.
  • the cultivation method of TIL used in this application can be divided according to four steps.
  • the first TIL population derived from tumor tissue and not expanded in vitro can be contacted with a T cell growth factor, wherein the second TIL population is obtained through the step (A);
  • the second TIL population can be contacted with a T cell activator and/or a T cell growth factor, wherein a third TIL population is obtained through the step (B);
  • the expression of optional genes of the third TIL population can be increased or decreased and/or the activity is enhanced or weakened, wherein the fourth TIL population is obtained through the step (C);
  • the fourth TIL population can be co-cultured with feeder cells, wherein the fifth TIL population is obtained through the step (D).
  • step (A) may be performed for about 7 days to about 14 days.
  • step (B) can be performed for about 0 days to about 4 days.
  • step (C) can be performed for about 0 days to about 4 days.
  • step (D) may be performed for about 5 days to about 14 days.
  • the improved TIL characteristics of the present application include one or more selected from the following group: increased TIL cell number, increased live cell ratio, increased viability, improved T cell subset ratio, improved cytokine secretion ability, improved tumor cell killing ability, increased T cell receptor (TCR) clonal diversity and increased TIL cell number in tissues and/or tumors.
  • the expanded TILs can be co-cultured with feeder cells after being contacted with a T cell co-stimulatory molecule. In one embodiment, the expanded TILs can be co-cultured with feeder cells after exposure to T cell growth factors. In one embodiment, the expanded TILs can be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and T cell growth factors. In one embodiment, the expanded TILs can be co-cultured with at least a portion of the feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors.
  • a portion of the feeder cells can be co-cultured with the expanded TILs while the expanded TILs are in contact with T cell co-stimulatory molecules and/or T cell growth factors, and at least another portion of the feeder cells can be co-cultured with the expanded TILs after the expanded TILs are contacted with T cell costimulatory molecules and/or T cell growth factors.
  • At least another portion of the feeder cells may contain all With about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, About 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.
  • the expanded TIL cells may be expanded in vitro. In one embodiment, the expanded TIL cells may be expanded autologously. In one embodiment, the expanded TIL cells may be expanded in vivo. In one embodiment, the expanded TIL cells may be expanded ex vivo.
  • the amount of the expanded TIL can be increased by at least 1-fold.
  • the amount of the expanded TIL can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 4-fold, at least about 4-fold 0 times, or at least about 50 times.
  • the number of expanded TIL cells can be increased by more than 50 times compared with TILs obtained from tumor tissue and not expanded in vitro.
  • the number of the expanded TIL cells may be increased by about 50 times or more, about 60 times or more, about 70 times or more, about 80 times or more, about 90 times or more, about 100 times or more, about 200 times or more, about 300 times or more, about 400 times or more, about 500 times or more, about 600 times or more, about 700 times or more, about 800 times or more, about 900 times or more, about 2000 times or more , about 3000 times or more, about 4000 times or more, about 5000 times or more, about 6000 times or more, about 7000 times or more, about 8000 times or more, about 9000 times or more, about 10000 times or more, about 15000 times or more, or about 20000 times or more. .
  • the present application provides a method for culturing the TIL used.
  • the first-stage expanded TILs are subjected to a second-stage expansion, wherein the TILs may be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors during the second stage expansion.
  • said TILs may be co-cultured with feeder cells after being contacted with a T cell co-stimulatory molecule. In one embodiment, in said second stage expansion, said TILs may be co-cultured with feeder cells after being contacted with T cell growth factors. In one embodiment, in said second stage expansion, said TILs may be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and T cell growth factors. In one embodiment, in said second stage expansion, said TILs may be co-cultured with at least a portion of feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors.
  • a Part of the feeder cells can be co-cultured with TILs while the TILs are in contact with T cell co-stimulatory molecules and/or T cell growth factors, and at least another part of the feeder cells is co-cultured with TILs after the TILs are in contact with T cell co-stimulatory molecules and/or T cell growth factors.
  • At least another portion of the feeder cells may comprise about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.
  • the present application can add the feeder cells of the present application to the cell culture medium of the TIL of the present application at a ratio of about 40:1 to about 400:1 of the feeder cells of the present application to the TIL of the present application.
  • the present application can be about 40:1 to about 400:1, about 40:1 to about 300:1, about 40:1 to about 200:1, about 40:1 to about 100:1, about 40:1 to about 90:1, about 40:1 to about 80:1, about 40:1 to about 70:1, about 40:1 to about 60:1, about 40:1 to about 5 0:1, about 50:1 to about 400:1, about 60:1 to about 400:1, about 70:1 to about 400:1, about 80:1 to about 400:1, about 90:1 to about 400:1, about 100:1 to about 400:1, about 200:1 to about 400:1, or about 300:1 to about 400:1
  • the ratio of the feeder cells to the TIL of the present application, the feeder cells of the present application are added to the cell culture medium of the T
  • the number of TIL cells expanded in the second stage can be increased by more than about 50 times.
  • the number of TIL cells expanded by the second stage can be increased by about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times More than, about 2000 times or more, about 3000 times or more, about 4000 times or more, about 5000 times or more, about 6000 times or more, about 7000 times or more, about 8000 times or more, about 9000 times or more, about 10000 times or more, about 15000 times or more, or about 20000 times or more.
  • the increase in the number of TIL cells can be represented by an expansion factor, and the expansion factor can be the factor to which the number of TIL cells is expanded after the second stage of expansion is completed compared to before the second stage of expansion begins. For example, if the number of TIL cells is 1 ⁇ 10 8 before the second stage of expansion starts, and the number of TIL cells is 1 ⁇ 10 9 after the second stage of expansion, it can be considered that the expansion factor of TIL cells is 10.
  • the present application provides a method for culturing TILs used, which includes: allowing TILs derived from tumor tissue and not expanded in vitro to undergo at least one stage of in vitro expansion, wherein in a single in vitro expansion stage, TILs that have been expanded in vitro and/or that have not been expanded in vitro can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • TILs derived from tumor tissue and not expanded in vitro can be subjected to at least two stages of In vitro expansion, wherein in the second stage of in vitro expansion and/or in a subsequent single in vitro expansion stage, the in vitro expanded and/or non-in vitro expanded TILs may be co-cultured with feeder cells after contact with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • TILs derived from tumor tissue and not expanded in vitro can be subjected to one stage of in vitro expansion, wherein in the first stage of in vitro expansion, TILs that have not been expanded in vitro can be co-cultured with feeder cells after being contacted with T cell costimulatory molecules and/or T cell growth factors for a certain period of time.
  • TILs derived from tumor tissue and not expanded in vitro can be subjected to two stages of in vitro expansion, wherein in the first stage of in vitro expansion, TILs that have not been expanded in vitro can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time. It is also possible to subject the TIL derived from tumor tissue and not expanded in vitro to two stages of in vitro expansion, wherein in the second stage of in vitro expansion, the TIL expanded in vitro can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • TIL derived from tumor tissue and not expanded in vitro can also undergo two stages of in vitro expansion.
  • the TIL that has not been expanded in vitro can be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time
  • TILs that have been expanded in vitro can be co-cultured with feeder cells after being in contact with T cell costimulatory molecules and/or T cell growth factors for a certain period of time.
  • TILs derived from tumor tissue and not expanded in vitro can be subjected to three stages of in vitro expansion, wherein in the first stage of in vitro expansion, TILs that have not been expanded in vitro can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time. It is also possible to subject the TILs derived from tumor tissue and not expanded in vitro to three stages of in vitro expansion, wherein in the second stage of in vitro expansion, the TILs expanded in vitro can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • TIL derived from tumor tissue and not expanded in vitro can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • TILs derived from tumor tissue and not expanded in vitro can also undergo three stages of in vitro expansion.
  • TILs that have not been expanded in vitro can be co-cultured with feeder cells after contacting T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time
  • TILs that have been expanded in vitro can be co-cultured with feeder cells after contacting T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • TILs derived from tumor tissue and not expanded in vitro can also undergo three stages of in vitro expansion, wherein in the first stage of in vitro expansion, TILs that have not been expanded in vitro can be combined with T cell co-stimulatory molecules and And/or co-culture with feeder cells after contacting with T cell growth factors for a certain period of time, and wherein in the third stage of in vitro expansion, the TILs expanded in vitro can be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • TIL derived from tumor tissue and not expanded in vitro can also undergo three stages of in vitro expansion.
  • the TIL that has not been expanded in vitro can be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time
  • TILs that have been expanded in vitro can be co-cultured with feeder cells after being in contact with T cell costimulatory molecules and/or T cell growth factors for a certain period of time.
  • TIL derived from tumor tissue and not expanded in vitro can also be subjected to three stages of in vitro expansion.
  • TILs that have not been expanded in vitro can be co-cultured with feeder cells after being in contact with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • the in vitro expanded TILs are co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors for a certain period of time.
  • the present application provides a method for culturing the TIL used.
  • the TIL can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors, and the TIL can go through two or more stages of expansion during the complete culture process, and the co-cultivation of the TIL with T cell costimulatory molecules and/or T cell growth factors and the TIL and feeder cells can occur in the same stage of expansion.
  • the TILs can undergo more than two stages of expansion during the entire culture process, and the TILs can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules. In one embodiment, the TILs can undergo more than two stages of expansion during the entire culture process, and the TILs can be co-cultured with feeder cells after being contacted with T cell growth factors. In one embodiment, the TILs can undergo more than two stages of expansion during the complete culture process, and the TILs can be co-cultured with feeder cells after being contacted with T cell co-stimulatory molecules and T cell growth factors.
  • the TILs can undergo two or more stages of expansion during the entire culture process, and the TILs can be co-cultured with at least a part of the feeder cells after being contacted with T cell co-stimulatory molecules and/or T cell growth factors.
  • the TILs can undergo two or more stages of expansion during the complete culture process.
  • a part of the feeder cells can be co-cultured with the TILs while the TILs are in contact with T cell co-stimulatory molecules and/or T cell growth factors, and at least another part of the feeder cells can be co-cultured with TILs after the TILs are in contact with T cell co-stimulatory molecules and/or T cell growth factors.
  • At least another portion of the feeder cells may comprise about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.
  • the TIL can undergo more than two stages of expansion during the entire culture process.
  • the TILs can be expanded through more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 stages during a complete culture process.
  • the contacting of TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivation of said TILs and feeder cells can occur during the first phase expansion and/or during the second phase expansion. In one embodiment, the contacting of TILs with T cell co-stimulatory molecules and/or T cell growth factors with co-cultivation of said TILs and feeder cells can occur in a first stage expansion, in a second stage expansion and/or in a third stage expansion. For example, contacting TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivation of said TILs and feeder cells can occur in the first stage of expansion.
  • contacting TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivation of the TILs and feeder cells can occur in a second stage of expansion.
  • contacting TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivation of said TILs and feeder cells can occur in a third stage of expansion.
  • contacting TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivation of said TILs and feeder cells can occur in a first stage expansion and in a second stage expansion.
  • contacting TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivation of the TILs and feeder cells can occur in the first stage of expansion and in the third stage of expansion.
  • contacting TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivation of the TILs and feeder cells can occur in the second stage of expansion and in the third stage of expansion.
  • contacting TILs with T cell co-stimulatory molecules and/or T cell growth factors and co-cultivating the TILs and feeder cells can occur in a first stage expansion, in a second stage expansion and in a third stage expansion.
  • co-cultivating the TIL and feeder cells after contacting the TIL with T-cell co-stimulatory molecules and/or T-cell growth factors for a certain time interval can increase the expansion effect of the TILs compared to co-cultivating the TILs and feeder cells while the TILs are in contact with T-cell costimulatory molecules and T-cell growth factors.
  • said enhancing the expansion effect of TIL may include being selected from the group consisting of increasing the number of TIL cells, changing the ratio of TIL cells, increasing the secretory ability of TIL cells, and increasing the killing ability of TIL cells.
  • the number of TIL cells can be increased by co-culturing the TIL with feeder cells after a time interval after contacting the TIL with a T cell co-stimulatory molecule and/or a T cell growth factor.
  • co-cultivating the TILs with feeder cells after a certain time interval after contacting the TILs with T cell co-stimulatory molecules and/or T cell growth factors can increase the secretion capacity of TIL cells.
  • co-cultivating the TIL and feeder cells after the TIL is contacted with T cell co-stimulatory molecules and/or T cell growth factors for a certain time interval can improve the killing ability of TIL cells.
  • co-cultivating said TILs with feeder cells after a time interval of contacting said TILs with T cell co-stimulatory molecules and/or T cell growth factors alters the proportion of TIL cells.
  • the TIL can be changed by The proportion of cells may comprise a proportion selected from the group consisting of: the proportion of central memory T cells (Tcm) in TIL may be increased, the proportion of TIL cells other than regulatory T cells (Treg) may be increased, the proportion of regulatory T cells (Treg) may be decreased, the proportion of activated T cells may be increased, the proportion of tumor-specific T cells may be increased, and the proportion of stem cell-like T cells may be increased.
  • the ratio of TIL cells that can be changed includes being selected from the following group: the ratio of CD45RA - CCR7 + central memory T cells (Tcm) in TIL can be increased, the ratio of TIL cells other than CD4 + CD25 + Foxp3 + regulatory T cells (Treg) can be increased, the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) can be reduced, the ratio of activated T cells can be increased, the ratio of CD103 + CD39 + tumor-specific T cells can be increased, and Can increase the proportion of TCF1 + stem-like T cells.
  • changing the proportion of TIL cells in the present application may include increasing the proportion of CD45RO + CD62L + central memory T cells (Tcm) in TIL.
  • said increasing the proportion of activated T cells includes being selected from the group consisting of increasing the proportion of PD1 + cells, increasing the proportion of LAG3 + cells and increasing the proportion of CD28 + cells.
  • Said changing the proportion of TIL cells may include increasing the proportion of central memory T cells, the proportion of activated T cells, the proportion of tumor-specific T cells, and/or stem cell-like T cells in the TIL cells cultured by the method of the present application, compared with co-cultivating said TIL and feeder cells while said TIL is in contact with T cell co-stimulatory molecules and T cell growth factors, increasing by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least About 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at
  • Said changing the ratio of TIL cells may comprise reducing regulatory T cells (Treg) in TIL cells cultured by the method of the present application by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99%.
  • Treg regulatory T cells
  • the TILs may be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors.
  • the later may refer to more than 2 hours later.
  • the TILs can be co-cultured with feeder cells 6 to 72 hours, or 12 to 48 hours after contact with T cell co-stimulatory molecules and/or T cell growth factors.
  • the TILs can be contacted with a T cell co-stimulatory molecule and/or a T cell growth factor for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours , about 23 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours later, with feeding Cell co-culture.
  • a T cell co-stimulatory molecule and/or a T cell growth factor for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,
  • the TILs can be co-cultured with feeder cells about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days after contacting with T cell costimulatory molecules and/or T cell growth factors.
  • the TILs may be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors.
  • the T cell costimulatory molecule can be selected from one or more of the following groups: CD80, CD86, B7-H3, 4-1BBL, CD27, CD30, CD134, B7h, CD40, LIGHT, antibodies specifically binding to CD3, antibodies specifically binding to CD28, antibodies specifically binding to HVEM, antibodies specifically binding to CD40L, antibodies specifically binding to OX40, and antibodies specifically binding to 4-1BB.
  • the contacting of TILs with T cell co-stimulatory molecules may comprise contacting one or more of said T cell co-stimulatory molecules with said TILs alone and/or contacting a plurality of said T cell co-stimulatory molecules with said TILs simultaneously.
  • one or more of said T cell co-stimulatory molecules may be included in contact with said TILs alone.
  • a plurality of said T cell co-stimulatory molecules may be included in simultaneous contact with said TIL.
  • one or more of the T cell co-stimulatory molecules can be added separately to the TIL cell culture medium, for example, multiple T cell costimulatory molecules can be added to the TIL cell culture medium at the same time.
  • one of the T cell costimulatory molecules can be added to the TIL cell culture medium in the form of one or more of the following group: engineered cells expressing the T cell costimulatory molecules, nanoparticles chimerized with the T cell costimulatory molecules, and polymers chimerized with the T cell costimulatory molecules.
  • multiple T cell costimulatory molecules can be added to the TIL cell culture medium in a form selected from the group consisting of mixtures, fusion proteins, engineered cells expressing multiple T cell costimulatory molecules, nanoparticles chimerized with multiple T cell costimulatory molecules, and polymers chimerized with multiple T cell costimulatory molecules.
  • the T cell co-stimulatory molecule can be an antibody that specifically binds to CD3, such as OKT3 from Miltenyi Biotech.
  • the TILs may be co-cultured with feeder cells after contacting with T cell co-stimulatory molecules and/or T cell growth factors.
  • the T cell growth factor may be selected from one or more of the following group: IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, and interferon- ⁇ .
  • the T cell growth factor can be IL-2.
  • the initial concentration of IL-2 in the TIL cell culture medium may be above about 1000 IU/mL.
  • the initial concentration of the IL-2 in the cell culture medium of the TIL can be about 1500IU/mL, about 2000IU/mL, about 2500IU/mL, about 2600IU/mL, about 2700IU/mL, about 2800IU/mL, about 2900IU/mL, about 3000IU/mL, about 3100IU/mL, about 3200IU/mL , about 3300IU/mL, about 3400IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, about 8000IU/mL, about 8 500IU/mL, or about 9000IU/mL or more.
  • the contacting of the TIL with T cell growth factors may comprise contacting one or more of the T cell growth factors with the TIL alone and/or contacting a plurality of the T cell growth factors with the TIL simultaneously. In one embodiment, one or more of said T cell growth factors may be included in contact with said TIL alone. In one embodiment, a plurality of said T cell growth factors may be included in simultaneous contact with said TIL. For example, one or more of the T cell growth factors can be added separately to the TIL cell culture medium, for example, multiple T cell growth factors can be added to the TIL cell culture medium at the same time.
  • one of the T cell growth factors can be added to the TIL cell culture medium in one or more of the following group: engineered cells expressing the T cell growth factor, nanoparticles chimerized with the T cell growth factor, and polymers chimerized with the T cell growth factor.
  • multiple T cell growth factors may be added to the TIL cell culture medium in a form selected from the group consisting of mixtures, fusion proteins, engineered cells expressing multiple T cell growth factors, nanoparticles chimerized with multiple T cell growth factors, and polymers chimeric with multiple T cell growth factors.
  • the TILs may be selected from the group consisting of TILs derived from fragments of tumor tissue, TILs derived from fragments of lymphoid metastases, TILs derived from pleural effusion, TILs derived from peritoneal effusion, TILs derived from tumor resection, TILs derived from biopsy and TILs derived from resuscitation after cryopreservation.
  • the TILs can be obtained by processing tumor tissue into tumor fragments.
  • the tumor fragments have a volume of about 1-27 cubic millimeters.
  • the tumor fragments have a volume of about 1 mm3, about 2 mm3, about 3 mm3, about 4 mm3, about 5 mm3, about 6 mm3, about 7 mm3, about 8 mm3, about 9 mm3, about 10 mm3, about 11 mm3, about 12 mm3, about 13 mm3, about 15 mm3, about 17 mm3, about 19 mm3, about 20 mm3, About 21 cubic millimeters, about 23 cubic millimeters, about 24 cubic millimeters, about 25 cubic millimeters, about 26 cubic millimeters, or 27 cubic millimeters.
  • the feeder cells may comprise antigen presenting cells.
  • the feeder cells may include one or more selected from the group consisting of peripheral mononuclear cells, dendritic cells, and artificial antigen-presenting cells.
  • the feeder cells may be peripheral mononuclear cells.
  • the feeder cells may be dendritic cells.
  • the feeder cells can be artificial antigen presenting cells.
  • the feeder cells can be isolated artificial antigen presenting cells (aAPCs), which can comprise cells expressing HLA-A/B/C, CD64, CD80, ICOS-L, and CD58, and can be modified to express more than one co-stimulatory molecule.
  • the feeder cells may be irradiated. For example, it may be irradiated with gamma rays, or it may be irradiated with X-rays.
  • the TILs can be co-cultured with feeder cells.
  • the co-cultivation can be that the surface of the TIL and the feeder cells are in contact, for example, the feeder cells can be added to the cell culture medium of the TIL.
  • the co-culture can be that the TIL is in contact with the surface of the feeder cells.
  • the feeder cells can be immobilized on the device and added to the cell culture medium of the TIL.
  • the feeder cells can be separated from the cells of the TIL by a membrane, mesh, grid, but material exchange can take place or contact can take place to a partial degree.
  • the cell metabolites of the feeder cells can be added to the cell culture medium of the TIL.
  • the present application can add the feeder cells of the present application to the cell culture medium of the TIL of the present application at a ratio of about 40:1 to about 400:1 of the feeder cells of the present application to the TIL of the present application.
  • the present application can be about 40:1 to about 400:1, about 40:1 to about 300:1, about 40:1 to about 200:1, about 40:1 to about 100:1, about 40:1 to about 90:1, about 40:1 to about 80:1, about 40:1 to about 70:1, about 40:1 to about 60:1, about 40:1 to about 5 0:1, about 50:1 to about 400:1, about 60:1 to about 400:1, about 70:1 to about 400:1, about 80:1 to about 400:1, about 90:1 to about 400:1, about 100:1 to about 400:1, about 200:1 to about 400:1, or about 300:1 to about 400:1
  • the ratio of the feeder cells to the TIL of the present application, the feeder cells of the present application are added to the cell culture medium of the T
  • the present application provides a method for culturing the TIL used.
  • the method of obtaining TIL cells from a tissue sample of a subject can be obtained by surgically obtaining an orthotopic tumor sample or a metastatic tumor sample, the weight of which can be at least about 1 g, or multiple pieces of tissue can be combined. Tumor tissue is transported at about 2-8 degrees in basal medium and processed within 48 hours. The tissue pieces can be mechanically crushed to a size of about 1-27 cubic millimeters each, transferred into a gas-permeable culture bag or Grex, added with T cell serum-free medium and IL-2 at a concentration of 1000-9000IU/mL (for example, 6000IU/mL) and cultured for about 3-14 days.
  • the cells in the culture medium can be collected and transferred together with the tissue pieces into a gas-permeable culture bag, or Grex, or Xuri equipment.
  • the T cell serum-free medium can be supplemented with about 30 ng/mL of CD3 antibody and IL-2 (1000-9000 IU/mL).
  • irradiated PBMC TIL and PBMC at a ratio of 1:40-1:400
  • the expansion culture is about 3-14 days. Filter the tissue block, use the cell processing system to collect the cells in the culture medium, clean and freeze them, and detect them.
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 70%, and the T cells greater than 80% can be memory effector T cells and effector T cells. After being stimulated, it can secrete IFN ⁇ , which can have the characteristics of up-regulation of the proportion of activated T cells.
  • the present application provides a tumor infiltrating lymphocyte (TIL), and the TIL can be cultured according to the cultivation method involved in the present application.
  • TILs provided in the present application may comprise one or a batch of TILs cultured by the cultivation method of the present application.
  • the TIL provided in the present application may comprise multiple or multiple batches of TIL cultured by the cultivation method of the present application and combined in any ratio.
  • TILs expanded using the methods of the present application can be administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition may be a suspension of TIL in a sterile buffer.
  • TILs expanded using the PBMCs of the present application can be administered by any suitable route known in the art.
  • T cells can be administered as a single intra-arterial or intravenous infusion, which can last for about 30 to 60 minutes. Other suitable routes of administration may include intraperitoneal, intrathecal and intralymphatic administration.
  • any suitable dose of TIL can be administered.
  • about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 TILs may be administered.
  • about 1 ⁇ 10 9 to about 12 ⁇ 10 10 TILs may be administered.
  • about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 TILs may be administered.
  • about 3 ⁇ 10 10 to about 12 ⁇ 10 10 TILs may be administered.
  • about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs may be administered.
  • about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs may be administered.
  • about 6 ⁇ 10 10 to about 8 ⁇ 10 10 TILs may be administered.
  • the therapeutically effective dose may be from about 2.3 ⁇ 10 9 to about 13.7 ⁇ 10 10 . In some embodiments, the therapeutically effective dose may be from about 1 ⁇ 10 9 to about 12 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 3 ⁇ 10 10 to about 12 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 4 ⁇ 10 10 to about 10 ⁇ 10 10 TILs.
  • a therapeutically effective dose may be from about 5 ⁇ 10 10 to about 8 ⁇ 10 10 TILs. In some embodiments, a therapeutically effective dose may be from about 6 ⁇ 10 10 to about 8 ⁇ 10 10 TILs. In some embodiments, the therapeutically effective dose may be from about 7 ⁇ 10 10 to about 8 ⁇ 10 10 TILs.
  • the amount of TIL provided in the composition of the present application can be about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , about 6 ⁇ 10 6 , about 7 ⁇ 10 6 , about 8 ⁇ 10 6 , about 9 ⁇ 10 6 , about 1 ⁇ 10 7 , about 2 ⁇ 10 7 , about 3 ⁇ 10 7 , about 4 ⁇ 10 7 , about 5 ⁇ 10 7 , about 6 ⁇ 10 7 , about 7 ⁇ 10 7 , about 8 ⁇ 10 7 , about 9 ⁇ 10 7 , about 1 ⁇ 10 8 , about 2 ⁇ 10 8 , about 3 ⁇ 10 8 , about 4 ⁇ 10 8 , about 5 ⁇ 10 8 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 , about 8 ⁇ 10 8 , about 9 ⁇ 10 8 , about 1 ⁇ 10 9 , about 2 ⁇ 10 9 , about 3 ⁇ 10 9 , about 4 ⁇ 10 9 , about 5 ⁇ 10 9 , about 6 ⁇ 10 8 , about 7 ⁇ 10 8 , about
  • the range of the amount of TIL provided in the composition of the present application can be about 1 ⁇ 10 6 up to 5 ⁇ 10 6 , about 5 ⁇ 10 6 to 1 ⁇ 10 7 , about 1 ⁇ 10 7 up to 5 ⁇ 10 7 , about 5 ⁇ 10 7 to 1 ⁇ 10 8 , about 1 ⁇ 10 8 up to 5 ⁇ 10 8 , about 5 ⁇ 10 8 to 1 ⁇ 10 9 , about 1 ⁇ 10 9 up to 5 ⁇ 10 9 , about 5 ⁇ 10 9 to 1 ⁇ 10 10 , about 1 ⁇ 10 10 up to 5 ⁇ 10 10 , about 5 ⁇ 10 10 to 1 ⁇ 10 11 , about 5 ⁇ 10 11 to 1 ⁇ 10 12 , about 1 ⁇ 10 12 to 5 ⁇ 10 12 , or about 5 x 10 12 to 1 ⁇ 10 13 .
  • the concentration of TIL provided in the compositions of the present application may be less than, for example, about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9% of the composition.
  • the concentration of TIL provided in the composition of the present application can be greater than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19.75%, about 19.50%, about 19.25%, about 19%, about 18.75%, about 18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about 17.25%, about 17%, about 16.75%, about 16.50%, about 16.25%, about 16%, about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about 14.50%, about 14.25%, about 14%, about 13.75%, About 13.50%, about 13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%, about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75%, about 10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%, about 9%, about
  • the concentration of TIL provided in the compositions of the present application may range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25% of the composition , about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14 %, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v/v.
  • the concentration of TIL provided in the compositions of the present application can range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0% of the composition. .08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w/w, w/v or v/v.
  • the amount of TIL provided in the compositions of the present application may be equal to or less than about 10 g, about 9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g, about 6.5 g, about 6.0 g, about 5.5 g, about 5.0 g, about 4.5 g, about 4.0 g, about 3.5 g, about 3.0 g, about 2.5 g, about 2.0 g , about 1.5g, about 1.0g, about 0.95g, about 0.9g, about 0.85g, about 0.8g, about 0.75g, about 0.7g, about 0.65g, about 0.6g, about 0.55g, about 0.5g, about 0.45g, about 0.4g, about 0.35g, about 0.3g, about 0.25g, about 0.2g, about 0.15g , About 0.1g, About 0.09g, About 0.08g, About 0.07g, About 0.06g, About 0.05g, About 0.04g
  • the amount of TIL provided in the compositions of the present application may be greater than about 0.0001 g, about 0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006 g, about 0.0007 g, about 0.0008 g, about 0.0009 g, about 0.001 g, about 0.0015 g, about 0.0 02g, about 0.0025g, about 0.003g, about 0.0035g, about 0.004g, about 0.0045g, about 0.005g, about 0.0055g, about 0.006g, about 0.0065g, about 0.007g, about 0.0075g, about 0.008g, about 0.0085g, about 0.009g, About 0.0095g, about 0.01g, about 0.015g, about 0.02g, about 0.025g, about 0.03g, about 0.035g, about 0.04g, about 0.045g, about 0.05g, about 0.055g, about 0.06g
  • TILs can be administered in a single dose. Such administration may be by injection, eg intravenous injection. In some embodiments, TILs can be administered in multiple doses. Doses may be once, twice, three, four, five, six or more than six times per year. Dosage can be monthly, biweekly, weekly, or every 2 days. In some embodiments, the administration of TILs can be administered continuously.
  • the present application provides a method for culturing the TIL used.
  • the method of obtaining TIL cells from a tissue sample of a subject can be obtained by surgically obtaining an orthotopic tumor sample or a metastatic tumor sample, the weight of which can be at least about 1 g, or multiple pieces of tissue can be combined.
  • the tumor tissue is transported at about 2-8 degrees in the sample transport solution, for example, a commonly used commercial tumor tissue transport solution, tumor tissue preservation solution or tumor tissue transfer solution, and processed within 48 hours.
  • the tissue pieces can be mechanically crushed to a size of about 1-27 cubic millimeters each, transferred into a gas-permeable culture bag or Grex, added with T cell serum-free medium and IL-2 at a concentration of 300-9000IU/mL (for example, 1000-9000IU/mL, for example, 6000IU/mL) and cultured for about 3-14 days.
  • the harvested TIL cells can be frozen and resuscitated, or the cells in the culture medium can be directly collected and transferred into a gas-permeable culture bag, or Grex or Xuri equipment, T cell serum-free medium can be added with the CD3 antibody of the present application, IL-2 at a concentration of 300-9000IU/mL (for example, 1000-9000IU/mL, for example, 6000IU/mL), after activating the TIL of the present application for a certain period of time, add irradiated PBMCs (the ratio of TIL to PBMC is about 1:40-about 1:400), and expand and culture for about 3-14 days.
  • Cells in the culture medium can be collected using a cell processing system, washed and frozen, and detected.
  • the CD3 ratio of the final product can be greater than 80%, the cell viability can be greater than 50%, and the T cells greater than 80% can be memory effector T cells and effector T cells.
  • IFN- ⁇ can be secreted after stimulation, and/or can be characterized by an up-regulated proportion of activated T cells.
  • the present application provides a pharmaceutical preparation.
  • it may comprise the TIL described herein and/or the composition described herein, and a pharmaceutically acceptable carrier.
  • the present application provides a pharmaceutical composition, which may include TIL obtained according to the culture method described in the application of the present application, and a substance that can reduce the number of lymphocytes and/or a substance that can maintain the proliferation ability of the TIL.
  • the present application provides a pharmaceutical composition, which may contain cyclophosphamide and/or fludarabine.
  • the present application provides a pharmaceutical composition, which may comprise interleukin-2 (IL-2) or a variant thereof.
  • IL-2 interleukin-2
  • the cyclophosphamide may be included at a dose of about 300 to about 500 mg/ m2 /day.
  • the dose contains cyclophosphamide.
  • the fludarabine may be included at a dosage of about 20 to about 30 mg/ m2 /day.
  • fludarabine can be included at a dose of about 5 mg/m 2 /day, about 10 mg/m 2 /day, about 15 mg/m 2 /day, about 20 mg/m 2 /day, about 25 mg/m 2 /day, or about 30 mg/m 2 /day.
  • the IL-2 can be contained at a dose of about 200,000 to about 600,000 IU/kg/time.
  • IL-2 may be included in a dose of about 100,000 IU/kg/time, about 200,000 IU/kg/time, about 300,000 IU/kg/time, about 400,000 IU/kg/time, about 500,000 IU/kg/time, or about 600,000 IU/kg/time.
  • the present application provides a kit, which may comprise the pharmaceutical composition according to the present application.
  • the present application provides a kit, which may comprise the T cell co-stimulatory molecules, T cell growth factors and/or feeder cells of the method for culturing tumor infiltrating lymphocytes (TIL) described in the present application, and instructions for recording the steps of the method for culturing tumor infiltrating lymphocytes (TIL) in the present application.
  • the present application provides a kit, which may comprise the TIL described in the present application and/or the pharmaceutical preparation described in the present application.
  • the present application provides a method for affecting the growth of tumor cells, which may include administering the TIL described in the present application and/or the pharmaceutical preparation described in the present application to a subject.
  • affecting tumor growth can comprise reducing the volume of the tumor to, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, About 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.
  • the present application provides the application of the TIL described in the present application and/or the pharmaceutical preparation described in the present application in the preparation of medicines, and the medicines can be used to prevent and/or treat tumors.
  • the tumor is selected from solid tumors.
  • the tumor may be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.
  • a tumor in the present application may refer to an advanced solid tumor.
  • the tumor in the present application may refer to an unresectable solid tumor.
  • tumors in the present application may refer to recurrent and/or metastatic solid tumors.
  • the tumor in the present application may refer to an unresectable recurrent and/or metastatic advanced solid tumor.
  • the present application provides a method for preventing and/or treating tumors, which may include administering the TIL described in the present application and/or the pharmaceutical preparation described in the present application to a subject.
  • the tumor is selected from solid tumors.
  • the tumor may be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.
  • a tumor in the present application may refer to an advanced solid tumor.
  • the tumor in the present application may refer to an unresectable solid tumor.
  • tumors in the present application may refer to recurrent and/or metastatic solid tumors.
  • the tumor in the present application may refer to an unresectable recurrent and/or metastatic advanced solid tumor.
  • a subject of the present application may meet one, more or all of the inclusion criteria of the present application.
  • a subject of the present application may not have one, more, or all of the exclusion criteria of the present application.
  • the effect of the present application can be evaluated through the safety, tolerability assessment, curative effect assessment and/or pharmacodynamic pharmacokinetic assessment described in the present application.
  • the subjects of the present application can isolate tissue blocks with a volume ⁇ 0.5 cm 3 (which can be derived from a single lesion or combined from multiple lesions) for the preparation of autologous tumor infiltrating lymphocytes.
  • the present application provides a TIL described in the present application and/or the pharmaceutical preparation described in the present application, which can be used for preventing and/or treating tumors.
  • the tumor is selected from solid tumors.
  • the tumor may be selected from one or more of the following group: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.
  • a tumor in the present application may refer to an advanced solid tumor.
  • the tumor in the present application may refer to an unresectable solid tumor.
  • tumors in the present application may refer to recurrent and/or metastatic solid tumors.
  • the tumor in the present application may refer to an unresectable recurrent and/or metastatic advanced solid tumor.
  • Embodiment 1 The culture method of tumor infiltrating lymphocyte (TIL) cell
  • PBMC peripheral blood mononuclear cells
  • red blood cell lysate according to the ratio of cell sedimentation volume and red blood cell lysate 1:2 to 1:3, mix well, lyse at room temperature for 10 minutes, and gently mix the centrifuge tube 2-3 times in the middle to ensure the lysing effect. After lysing, add PBS or normal saline to wash the cells. After cleavage, the cells were washed twice, centrifuged at 400g for 6 minutes, and samples were taken and counted before the last centrifugation.
  • Discard the supernatant resuspend the cells in the basal medium, adjust the cell density to about 2-3 ⁇ 10 7 /mL, the liquid level may not exceed 1 cm, and the volume in each T225 culture flask may be less than 200 mL; in the flat state, X-ray irradiation 50Gy.
  • the supernatant was discarded by centrifugation, and the cells were frozen according to the counting results, about 1-2 ⁇ 10 8 /mL, 1-2mL/vessel; the cells were placed in a programmed cooling box and transferred to a -80°C refrigerator for freezing.
  • cpro separation kit Aseptically connect the tubing of the blood bag to the input end of the cpro separation kit (Cytiva). If the blood volume is greater than 120 mL, a pre-concentration step is performed to concentrate the blood volume to less than 120 mL.
  • the neatcell program can be used to separate and wash PBMCs.
  • the washing liquid is physiological saline, with an intermediate volume of 20 mL; the resuspension liquid is the basal medium, and 80 mL/batch is added.
  • each donor’s PBMC is a bag of 100mL. In the flat state, the height of the liquid level can not exceed 1 cm, and the X-ray irradiation is 50Gy.
  • PBMC cells in blood samples were separated and frozen according to the above PBMC manual separation and cryopreservation procedures.
  • a culture bottle and bag with a gas-permeable surface such as G-Rex100 culture bottle (Wilson Wolf Manufacturing), and add 300mL of rewarmed complete medium.
  • the complete medium can be freely selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added at a concentration of about 1000-9000IU/ mL of IL-2, eg 6000 IU/mL of IL-2.
  • T cell culture medium such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium
  • essential amino acids and antibiotics can be added at a concentration of about 1000-9000IU/ mL of IL-2, eg 6000 IU/mL of IL-2.
  • ophthalmic scissors and ophthalmic tweezers to make preliminary cuts to remove fat tissue and necrotic tissue, and continue to cut each tissue block to a size of about 27 cubic millimeters.
  • take the non-suspended tumor tissue block use a 20mL syringe to remove the internal piston, connect it to the culture bag, and use a pipette to transfer about 1g of the tissue block into the culture bag through the syringe. Put the culture bag into the carbon dioxide incubator for cultivation.
  • the scissors and tweezers were cleaned and initially sterilized with 75% alcohol, then ultrasonically cleaned and sterilized to obtain primary TILs.
  • the liquid should be replaced every 3-7 days or half of the liquid should be replaced to ensure the nutrition of the cells.
  • Use complete medium complete medium can choose X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium, and can add essential amino acids and antibiotics, and add IL-2 at a concentration of about 1000-9000IU/mL, such as 6000IU/mL IL-2.
  • 3-14 days of the first stage of amplification for example, samples can be taken and counted on the 3rd, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days. If the number of cells is between 5 ⁇ 10 5 and 5 ⁇ 10 8 , enter the harvesting step of the first stage of amplification described below.
  • Collect the cells at the end of the first stage of expansion centrifuge, discard the medium, wash the cells once with PBS or saline, Obtain the TILs amplified in the first stage, and take a sample and count about 5 ⁇ 10 5 to 2 ⁇ 10 8 cells to enter the following first-stage expansion step; take about 5 ⁇ 10 5 cells for quality control testing; add an equal amount of 2 times the amount of cells to cryopreservation.
  • the complete medium can be arbitrarily selected from X-vivo 15 medium or other commercial T cell medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell medium, and can add essential amino acids and antibiotics, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, in a suspension 24-well culture plate, 1mL/well, add CD3 antibody, such as OK T3 is about 30ng/mL, add IL-2 at a concentration of about 1000-9000IU/mL, such as 6000IU/mL IL-2.
  • T n can be taken as 0 hours to 14 days
  • T n can be taken as 0 hours to 14 days
  • resuscitate the feeder cells mixed with 1-5 donors transfer the activated TIL cells, tissue pieces and feeder cells into G-Rex100 culture flasks or air-permeable bags, supplement the complete medium, take samples and count every 1-3 days, and replace the liquid or half the volume according to the state of the cells until the total number of cells is greater than 1 ⁇ 10 9 or the second-stage expansion culture reaches 14 days, and the culture is terminated.
  • the resuscitated therapeutic tumor-infiltrating lymphocytes may be administered to the subject as an intravenous infusion.
  • Tn can range from 0 hour to 14 days
  • Tn can range from 0 hour to 14 days
  • the feeder cells were added to the tumor-infiltrating lymphocyte culture bag.
  • Tn is selected from 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 5 days, 7 days, and 9 days to obtain TILs cultured at different addition times of feeder cells, and a comparative test of cell counts is carried out.
  • the proliferative ability analysis of TILs cultured at different feeding times of feeder cells is shown in Figure 1.
  • the numerical values on the vertical axis in each group of graphs in which TIL was cultured with feeder cells at different addition times indicate the expansion factor of the number of TIL cells after the end of the second stage of expansion compared to before the start of the second stage of expansion.
  • the proliferation results of TILs from 4 donors showed that the TILs cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (that is, at the same time), the proliferation ability was weaker than that of 24 hours after the addition of OKT3 and IL-2 After 48 hours or 48 hours, feeder cell cultured TIL was added.
  • Tn can range from 0 hour to 14 days
  • the feeder cells were added to the tumor-infiltrating lymphocyte culture bag.
  • Tn was selected from 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 5 days, 7 days, and 9 days to obtain TILs cultured at different addition times of feeder cells, and a comparative test of flow cytometry was performed.
  • Transcription Factor Buffer Set manufacturer BD, product number 562574; V-bottom 96-well plate, manufacturer Corning, product number 3894; flow tube, manufacturer Corning, product number 352052.
  • the flow cytometry antibodies in this example were purchased from BD or Biolegend. Add 1 ⁇ 10 5 to 5 ⁇ 10 5 cell samples in each group into flow tubes or V-bottom 96-well plates. Centrifuge at 600g for 3 minutes and discard the supernatant. Wash once with PBS, flow tube 1mL/tube, 96-well plate 250 ⁇ L/well, discard supernatant. Add the prepared antibody working solution for cell surface staining, the antibody (BD or Biolegend) concentration is 1:100 to 1:200, and the activity detection dye is 1:10000. Flow tube 100 ⁇ L/tube, 96-well plate 50 ⁇ L/well for staining, incubate at 2-8°C for 30 minutes in the dark.
  • Cell fixation and membrane rupture Sufficiently resuspend the cells, add an appropriate amount (96-well plate 100 ⁇ L/well, flow tube 1mL/tube) 1 ⁇ working solution A to fix and rupture the membrane, and incubate at 2-8°C for 40-50 minutes in the dark. After fixation and membrane rupture, add 1 ⁇ working solution B to wash the cells (250 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B 1 ⁇ working solution B to wash the cells (250 ⁇ L/time for 96-well plate, 2 mL/time for flow tube), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice.
  • 1 ⁇ working solution B to prepare intracellular antibody the antibody concentration is 1:100 to 1:200, 50 ⁇ L/well of 96-well plate, 100 ⁇ L/tube of flow tube, and stain for 30 minutes at 2-8°C in the dark. After staining, add 1 ⁇ working solution B to wash the cells (96-well plate 250 ⁇ L/time, flow tube 2 mL/time), centrifuge at 2-8°C, 350g for 6 minutes, and wash twice. After surface staining, cells were washed once with PBS (250 ⁇ L/time for 96-well plate, 1 mL/time for flow tube), centrifuged at 600 g for 3 minutes at room temperature, and the supernatant was discarded after centrifugation. Use 100-500 ⁇ L PBS to resuspend the cells for flow cytometry detection.
  • FIGS 2 to 3 show the ratio of CD45RA - CCR7 + central memory T cells (Tcm) in TIL cells cultured after adding OKT3 and IL-2 0 hour, 24 hours or 48 hours after adding feeder cells. The results showed that TIL cultured with feeder cells after 24 hours or 48 hours had a higher proportion of central memory T cells than TILs cultured with feeder cells at the same time.
  • Tcm central memory T cells
  • FIG. 4 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cells after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • FIGS 5 to 6 show the proportion of activated T cells in TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • the results showed that TILs cultured with feeder cells added after 24 hours or 48 hours had a higher proportion of activated T cells, such as a higher proportion of PD1 + , LAG3 + and/or CD28 + cells, than TILs cultured with feeder cells added at the same time.
  • Figure 7 shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured after adding feeder cells for 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of tumor-specific T cells than TILs cultured with feeder cells at the same time.
  • Figure 8 shows the ratio of TCF1 + stem cell-like T cells in TIL cells cultured by adding feeder cells after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. The results showed that the TIL cultured with feeder cells after 24 hours or 48 hours had a higher proportion of stem cell-like T cells than the TILs cultured with feeder cells at the same time.
  • Example 4 The statistics of the results of TIL cultured with feeder cells at different addition times
  • TIL activation of the second-stage expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, and add CD3 antibody, for example, about 30 ng/mL of OKT3, and add IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL of IL-2 in a suspended 24-well culture plate.
  • feeder cells were added into the culture environment of tumor infiltrating lymphocytes.
  • TIL and feeder cells can be added at a ratio of 1:40-1:400, and all the cells are collected after about 9-14 days of expansion and culture in the second stage, and the results of TIL obtained from the culture are detected and counted.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) was used as benchmark 1, and the data of the test group at other time points in the same batch were standardized, and the relative proliferation ability of each test group in the second stage of amplification relative to the 0h group was calculated.
  • Fig. 9 shows the results of cell proliferation ability of TIL cells cultured by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. Compared with the TIL cultured with feeder cells added 0 h after the addition of OKT3 and IL-2 (i.e. at the same time), the feeder cells were added 24 h or 48 h after the addition of OKT3 and IL-2. The proliferative ability of TIL in cell culture was significantly enhanced.
  • Flow cytometric detection was performed on the TIL population obtained by culturing the above-mentioned feeder cells at different addition times.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of the test group at other time points in the same batch were standardized, and the cell composition ratio of each test group expanded in the second stage relative to the 0h group was calculated.
  • test procedure of flow detection refer to the content of Example 3 of the present application.
  • FIG 10 shows the results of CD45RA - CCR7 + central memory T cell (Tcm) ratio of TIL cells obtained by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • Tcm central memory T cell
  • Figure 11 shows the ratio of TCF1 + stem cell-like T cells in TIL cells obtained by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of stem cell-like T cells in CD8 + compared with TILs cultured with feeders at the same time.
  • Figure 12 shows the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in TIL cells obtained by adding feeder cell-cultured TIL after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • FIG. 13 shows the proportion of activated T cells (PD1 + ) in TIL cells obtained by adding feeder cells cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • the results showed that TILs cultured with feeder cells added after 24 hours or 48 hours had a higher proportion of activated T cells, such as a higher proportion of PD1 + cells in CD8 + and/or CD4 + cells, than TILs cultured with feeder cells added at the same time.
  • Figure 14 shows the ratio of CD103 + CD39 + tumor-specific T cells in TIL cells cultured by adding feeder cells to TIL after 0 hour, 24 hours or 48 hours after adding OKT3 and IL-2. The results showed that TILs cultured with feeder cells after 24 hours or 48 hours had a higher proportion of tumor-specific T cells in CD8 + and/or CD4 + than TILs cultured with feeder cells at the same time.
  • FIG. 15 shows the proportion of activated T cells (CD28 + ) in TIL cells cultured by adding feeder cells cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TIL cultured with feeder cells after 24 hours or 48 hours had a higher proportion of activated T cells than TILs cultured with feeder cells at the same time, For example a higher proportion of CD8 + CD28 + cells.
  • FIG. 16 shows the proportion of activated T cells (41BB + ) in TIL cells cultured by adding feeder cells cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TIL cultured with feeder cells added after 24 hours or 48 hours had a higher proportion of activated T cells, such as a higher proportion of 41BB + cells in CD8 + and/or CD4 + , than TILs cultured with feeder cells at the same time.
  • FIG. 17 shows the proportion of activated T cells (CD25 + ) in TIL cells obtained by adding feeder cells cultured TILs 0 hour, 24 hours or 48 hours after the addition of OKT3 and IL-2.
  • the results showed that TIL cultured with feeder cells added after 24 hours or 48 hours had a higher proportion of activated T cells, such as a higher proportion of CD25 + cells in CD8 + and/or CD4 + cells, than TILs cultured with feeder cells added at the same time.
  • Figure 18 shows the detection results of intracellular factor expression in TIL cells obtained by adding feeder cells cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours. The results showed that TILs cultured with feeder cells added after 24 hours or 48 hours had a higher ability to express intracellular factors than TILs cultured with feeders added at the same time. For example, higher CD107a expression capacity in CD3 + , CD8 + and/or CD4 + .
  • the cytokine secretion detection method can refer to the instructions of the cytokine detection kit (BD), reconstitute the human Th1/Th2/Th17 cytokine standard freeze-dried powder (BD) with 2mL Assay Diluent diluent (BD) (the concentration of each cytokine in the standard stock solution is 5000pg/mL) and follow the sequence: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:51 2, 1:1024 serial dilution, marked as "standard tube”. Take 1 tube containing Assay Diluent dilution only as a reference.
  • Fig. 19 shows the cytokine secretion detection results of TIL cells cultured by adding feeder cells cultured TILs after adding OKT3 and IL-2 for 0 hour, 24 hours or 48 hours.
  • the results showed that TIL cultured with feeder cells added after 24 hours or 48 hours had higher cytokine secretion ability than TILs cultured with feeder cells added at the same time. For example, higher TNF- ⁇ secretion capacity, or higher IFN- ⁇ secretion capacity.
  • Example 5 The statistics of the results of TIL cultured with feeder cells at different addition times
  • TIL activation of the second-stage expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, add CD3 antibody, such as OKT3 at about 30 ng/mL, and add IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL IL-2 in a suspension 24-well culture plate. After 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after the addition of OKT3 and IL-2, feeder cells were added to the culture environment of tumor infiltrating lymphocytes.
  • CD3 antibody such as OKT3 at about 30 ng/mL
  • IL-2 at a concentration of about 1000-9000 IU/mL, such as 3000 or 6000 IU/mL IL-2 in a suspension 24-well culture plate.
  • feeder cells were added to the culture environment of tumor infiltrating lymphocytes.
  • TIL and feeder cells can be added at a ratio of 1:40-1:400, such as 1:200, and all cells are collected after about 9-14 days of expansion and culture in the second stage, and the results of TIL obtained from culture are detected and counted.
  • Figure 20 shows the result graph of the cell proliferation ability of TIL cells cultured after adding OKT3 and IL-2 for 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cell-cultured TIL. Compared with the TILs cultured with feeder cells added 0 hours after the addition of OKT3 and IL-2 (that is, at the same time), the proliferation ability of TILs cultured with feeder cells 12 hours or more after the addition of OKT3 and IL-2 was significantly enhanced.
  • Flow cytometric detection was performed on the TIL population obtained by culturing the above-mentioned feeder cells at different addition times.
  • TILs derived from tumors from different donors were regarded as different batches; the data of each batch of the test group in which OKT3 and IL-2 were added to feeder cells at the same time (0h group) were used as benchmark 1, and the data of the test group at other time points in the same batch were standardized, and the cell composition ratio of each test group expanded in the second stage relative to the 0h group was calculated.
  • test procedure of flow detection refer to the content of Example 3 of the present application.
  • Figure 21 shows the ratio of CD8 + T cells in TIL cells cultured after adding OKT3 and IL-2 at 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells to culture TIL. The results showed that feeder-cultured TILs added 12 hours or more after the addition of OKT3 and IL-2 TILs cultured from feeder cells have a higher proportion of CD8 + T cells.
  • Figure 22 shows the ratio of CD45RO + CD62L + T cells in TIL cells cultured after adding OKT3 and IL-2 at 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding feeder cells to culture TIL.
  • the results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a higher proportion of memory T cells (Tcm, CD45RO + CD62L + ) than TILs cultured with feeder cells at the same time.
  • Figure 23 shows the NK T cell ratio of TIL cells cultured after adding feeder cells for 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days after adding OKT3 and IL-2.
  • the results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a higher proportion of NK T cells than TILs cultured with feeder cells at the same time.
  • Figure 24 shows that the ratio of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in the cultured TIL cells was obtained after adding OKT3 and IL-2 at 0 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or 5 days later.
  • the results showed that TILs cultured with feeder cells added 12 hours or more after the addition of OKT3 and IL-2 had a lower proportion of regulatory T cells than TILs cultured with feeder cells at the same time.
  • TIL activation in the second stage of expansion in 1.4 of Example 1 take the amount of cells expanded in the first stage, adjust the cell density to 5 ⁇ 10 5 to 2 ⁇ 10 6 /mL, add CD3 antibody, such as about 30 ng/mL of OKT3, and add IL-2 at a concentration of about 1000 to 9000 IU/mL, such as 3000 or 6000 IU/mL of IL-2 in a suspended 24-well culture plate. 12 hours to 14 days after the addition of the above OKT3 and IL-2, for example 48 hours later, the feeder cells are added to the culture environment of tumor infiltrating lymphocytes. Among them, TIL and feeder cells can be added at a ratio of 1:40-1:400, and after about 9-14 days of expansion and culture in the second stage, all cells are collected, and the cell killing ability of TIL obtained from culture is detected and counted.
  • CD3 antibody such as about 30 ng/mL of OKT3
  • IL-2 at a concentration of about 1000 to 9000 IU/mL
  • TILs obtained from each test group for detection and target cells for co-culture such as A375 melanoma cells and/or Hela cervical cancer cells.
  • CFSE (6(6)-Carboxyfluorescein diacetate N-succinimidyl ester, Sigma, 21888-25MG-F): Wash tumor cells with PBS, resuspend tumor cells in 500 ⁇ L of PBS; add CFSE to 500 ⁇ L of PBS, mix with 500 ⁇ L of tumor cell suspension in PBS, to a final concentration of CFSE of 0.5 ⁇ mol/L .
  • TIL groups of each test group were centrifuged at 600g for 5 minutes, according to the effect-to-target ratio (The ratio of TIL cells to tumor cells) Resuspend TIL cells at 3:1 (that is, the concentration of resuspended TIL cells is 3 ⁇ 10 6 cells/mL).
  • TIL and tumor cells were added 100 ⁇ L to a U-bottom 96-well plate (Corning), and set up three replicate wells for each group. At the same time, a group of control groups containing only tumor cells was set up. Centrifuge the plate at 200g for 1 minute, and incubate at 37°C for 4 hours to overnight.
  • substances that activate TIL cells can be omitted as the non-activation group, or transACT (Miltenyi, a nano-matrix material containing CD3 antibody and CD28 antibody) can be added as the activation group.
  • Killing rate% Dapi + CFSE + cell number/total CFSE + ⁇ 100%, or the killing rate can be expressed by Dapi + cell number/total tumor cell number.
  • Figure 25 shows the results of the cell killing ability of TIL cells cultured by adding feeder cells cultured TILs 48 hours after the addition of OKT3 and IL-2. The results showed that 48 hours after the addition of OKT3 and IL-2, the TIL cultured with feeder cells had significant tumor cell killing ability, such as melanoma and/or cervical tumor.
  • the immune system of the subject can be treated, for example, cyclophosphamide and/or fludarabine can be administered to the subject on the 4th to 2nd day before the TIL obtained by the culturing method is administered to the subject.
  • cyclophosphamide is administered at a dose of about 300 to 500 mg/ m2 /day
  • fludarabine is administered at a dose of 20 to 30 mg/ m2 /day.
  • the TIL of the present application can be intravenously administered to the subject at a time with a cell number of 5 ⁇ 10 9 to 1 ⁇ 10 11 .
  • IL-2 (or its variant) can be administered to the subject for the first time at a dose of 200,000 to 600,000 IU/kg/time, and the administration of IL-2 can be suspended once after the administration of IL-2, and IL-2 can be administered to the subject every 12 hours according to the tolerance of the subject.
  • IL-2 can be administered to the subject 10 times, and the number of IL-2 administrations can be reduced according to the tolerance of the subject.
  • TIL tumor infiltrating lymphocytes
  • ECOG score standard an indicator for assessing health status and tolerance to treatment

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Abstract

一种肿瘤浸润淋巴细胞在疾病治疗中的用途，具体地，一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：使源自肿瘤组织的TIL经过至少一个阶段的体外扩增，其中在单个体外扩增阶段中，使经体外扩增和/或未经体外扩增的TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。

Description

肿瘤浸润淋巴细胞在疾病治疗中的用途 技术领域
本申请涉及生物医药领域，具体的涉及一种肿瘤浸润淋巴细胞在疾病治疗中的用途。
背景技术
使用过继性自体转移肿瘤浸润淋巴细胞治疗肿瘤是一种治疗预后不良患者的有效方法。但是过继性自体转移肿瘤浸润淋巴细胞治疗肿瘤需要大量的肿瘤浸润淋巴细胞，而且目前来自患者肿瘤的肿瘤浸润淋巴细胞的扩增能力弱，细胞功能不强。
因此如何提供一种能够用于细胞治疗的稳健可靠的肿瘤浸润淋巴细胞是亟待解决的问题。
发明内容
本申请提供了一种肿瘤浸润淋巴细胞在疾病治疗中的用途。使用本申请描述的培养方法获得的肿瘤浸润淋巴细胞在肿瘤治疗中，可以体现出有效性和安全性。
一方面，本申请提供了一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：使源自肿瘤组织的TIL经过至少一个阶段的体外扩增，其中在单个体外扩增阶段中，使经体外扩增和/或未经体外扩增的TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
一方面，本申请提供了一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：
(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
(B)使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
一方面，本申请提供了一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：
(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
(B)使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
一方面，本申请提供了一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：
(A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
(B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
(C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
一方面，本申请提供了一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：
(A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
(B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
(C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
一方面，本申请提供了一种药物组合物，包含根据本申请应用中所述培养方法获得的TIL，以及使淋巴细胞数量减少的物质和/或使所述TIL维持增殖能力的物质。
一方面，本申请提供了一种试剂盒，包含根据本申请的药物组合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是针对供者A-1、A-2、A-3和A-4，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养时，TIL增殖能力对比。
图2显示的是针对供者B-1和B-2，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL中，CD8+中或CD4+中CD45RA-CCR7+中心记忆T细胞(Tcm)比 例对比。
图3显示的是针对供者B-3、B-4和B-5，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL中，CD8+中或CD4+中CD45RA-CCR7+中心记忆T细胞(Tcm)比例对比。
图4显示的是针对供者C-1和C-2，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL中，CD4+CD25+Foxp3+调节性T细胞(Treg)比例对比。
图5显示的是针对供者D-1和D-2，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL中，CD8+中或CD4+中PD1+活化T细胞和LAG3+活化T细胞比例对比。
图6显示的是针对供者D-3、D-4和D-5，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL中，CD8+中CD28+活化T细胞比例对比。
图7显示的是针对供者E-1和E-2，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL中，CD8+中或CD4+中CD103+CD39+肿瘤特异性T细胞比例对比。
图8显示的是针对供者F-1和F-2，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL中，TCF1+干细胞样T细胞比例对比。
图9显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。
图10显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA-CCR7+中心记忆T细胞(Tcm)比例结果图。
图11显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1+干细胞样T细胞比例。
图12显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4+CD25+Foxp3+调节性T细胞(Treg)比例。
图13显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(PD1+)比例。
图14显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103+CD39+肿瘤特异性T细胞比例。
图15显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD28+)比例。
图16显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的 TIL，培养所得的TIL细胞的活化T细胞(41BB+)比例。
图17显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD25+)比例。
图18显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的胞内因子表达检测结果。
图19显示的是加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞因子分泌检测结果。
图20显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。
图21显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD8+T细胞比例。
图22显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RO+CD62L+T细胞比例。
图23显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的NK T细胞比例。
图24显示的是加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4+CD25+Foxp3+调节性T细胞(Treg)比例。
图25显示的是加入OKT3和IL-2的48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞杀伤能力结果。
图26A和26B显示的，本申请的治疗方法在临床上体现的影像学结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“环磷酰胺”通常是指一种处理细胞的制剂。例如一种芥子气烷基化剂，其可以将烷基(CnH2n+1)添加到DNA。例如，环磷酰胺可以是指分子式 C7H15Cl2N2O2P·H2O，化学名称2-[双(2-氯乙基)氨基]四氢-2H-1,3,2-氧氮磷环2-氧化物一水合物。例如，环磷酰胺的CAS登录号可以为50-18-0的制剂。环磷酰胺可以是可商购的。
在本申请中，术语“氟达拉滨”通常是指一种处理细胞的制剂。例如一种氟化核苷酸类似物。例如，氟达拉滨可以是指分子式为C10H12FN5O4，CAS登录号为21679-14-1的制剂。
在本申请中，术语“表达”通常是指编码目标多肽的基因在细胞内发生的转录和/或翻译过程。可以通过测量存在于细胞中的相应mRNA的量来确定宿主细胞中编码目标多肽的基因的转录水平。例如，可通过PCR或通过RNA杂交对编码目标多肽的基因转录的mRNA进行定量测量。可以通过多种方法测量编码目标多肽的基因的翻译水平，例如通过ELISA，通过多肽生物活性测试，或通过蛋白质印迹或放射免疫测试法。
在本申请中，术语“一个阶段的体外扩增”、“单个体外扩增阶段”、或“第一个阶段的体外扩增”等中的“阶段”通常是指TIL在体外经过的一段扩增过程。在一种实施方式中，每一个阶段之间可以是通过TIL细胞数量的变化来划分的，在一种实施方式中，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的条件来划分的。在一种实施方式中，当细胞培养基中添加了或补充添加了T细胞共刺激分子和/或T细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，当TIL细胞进行了离心和/或细胞洗涤后，可以认为TIL细胞进入了下一个阶段的体外扩增。在一种实施方式中，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。在一种实施方式中，当TIL细胞体外培养约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
在本申请中，术语“第一个阶段的体外扩增”通常是指从组织中获得初级TIL后，使用T细胞生长因子进行扩增的阶段。在一种实施方式中，所述组织可以是肿瘤组织。在一种实施方式中，所述扩增可以是自体或者异体进行的体内扩增，或者可以是体外扩增。所述第一阶段扩增也可以称为preREP(快速扩增前)阶段。
在本申请中，术语“第二个阶段的体外扩增”通常是指从受试者体内取出的组织并进行扩增后，再次进行扩增的阶段。在一种实施方式中，与经第一阶段扩增的TIL相比，所述经第二阶段扩增的TIL细胞数量增加，例如可以增加至少约10倍(或至少约20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在一种实施方式中，第二阶段扩增可以与第一阶段扩增的培养条件不同，例如加入的培养物质可以不同。所述第二阶段扩增也可以称为REP(快速扩增)阶段。
在本申请中，术语“体内”通常是指发生在受试者体内的事件。
在本申请中，术语“体外”通常是指在受试者体外发生的事件。
在本申请中，术语“离体”通常是指涉及对已从受试者体内移除的细胞、组织和/或器官进行治疗或进行手术的事件。在一种实施方式中，该细胞、组织和/或器官可以通过手术或治疗方法返回到受试者的身体。
在本申请中，术语“分泌”通常是指细胞将表达的多肽或蛋白转移到细胞外环境。
在本申请中，术语“分泌能力”通常是指细胞表达多肽或蛋白并将所述多肽或蛋白转移到细胞外环境的能力。
在本申请中，术语“辐照”通常是指通过射线对物质进行的处理。例如，在一种实施方式中，辐照可以是指通过X射线、α射线、β射线或γ射线对物质进行辐照。
在本申请中，术语“工程化细胞”通常是指将DNA或RNA形式的额外遗传物质加入细胞的总遗传物质而被基因修饰的细胞。在一种实施方式中，工程化细胞可以经过基因修饰以表达根据本申请的T细胞共刺激分子和/或T细胞生长因子的TIL。
在本申请中，术语“共培养”通常是指将两个或更多个不同群体的细胞在它们之间有一定程度的接触的情况下培养。所述两个或更多个不同群体的细胞的“接触”，在一种实施方式中可以通过直接接触，即其中一个群体的细胞与另一个群体的细胞直接物理接触。或者在一种实施方式中可以通过共用培养基所介导的间接接触。所述共用的培养基可以含有由共培养细胞的至少一个群体所产生和释放的代谢产物，并用于培养另一个群体的细胞。
在本申请中，术语“接触”通常是指两个或更多个不同类型的物质以任何顺序、任何方式以及任何时长接触在一起。在一种实施方式中可以通过直接接触，例如可以将一种饲养细胞、T细胞共刺激分子和/或T细胞生长因子加入TIL细胞的培养基，在一种实施方式中可以通过间接接触，例如可以将饲养细胞产生和释放的代谢产物，用于培养TIL细胞。
在本申请中，术语“混合物”通常是指两个或更多个不同物质的组合。
在本申请中，术语“同时接触”、“共同接触”、“与...接触同时”、“同时”和“共同”通常是指向受试者施用两种以上物质，使得物质同时存在于受试者和/或受试者培养的环境中。同时接触可以包括以不同的组合物同时施用、以不同的组合物在不同时间施用，或以其中存在两种以上活性药物成分的组合物施用。
在本申请中，术语“扩增”通常是指在一段时间内细胞的数量增加若干倍。在一种实施方式中细胞的数量可以增加至少约3倍(或4、5、6、7、8或9倍)，在一种实施方式中细胞的数量可以增加至少约10倍(或20、30、40、50、60、70、80或90倍)，或者在一种实施方式中细胞的数量可以增加至少约100倍。在本申请中，术语“经扩增”通常是指所述细胞经过上述一种或多种扩增。
在本申请中，术语“聚合物”通常是指由连接在一起的单独化学部分组成的分子，所述部分可相同或不同。在一种实施方式中，术语“聚合物”可以指尾尾相连而形成线性分子的单独化学部分，以及以分支(如“多臂”或“星型”)结构形式连接在一起的单独化学部分。在一种实施方式中聚合物可以包括例如水凝胶、聚乙二醇、或泊洛沙姆。泊洛沙姆是非离子三嵌段共聚物，其具有聚氧丙烯(聚(环氧丙烷))中央疏水链，侧连两条聚氧乙烯(聚(环氧乙烷))亲水链。本申请包含的物质可以与本文所描述的或本领域已知的任何聚合物一起配制，或与它们一起给予。
在本申请中，术语“抗体”通常是指对指定蛋白质或肽或其片段有反应性的免疫球蛋白。此类抗体包括但不限于人抗体、灵长类化抗体、嵌合抗体、单克隆抗体、单特异性抗体、多克隆抗体、多特异性抗体、非特异性抗体、双特异性抗体、多特异性抗体、人源化抗体、合成抗体、重组抗体、杂合抗体、突变型抗体、嫁接偶联抗体(即偶联或融合至其它蛋白质、放射性标记物、细胞毒素的抗体)、和体外生成的抗体。抗体可来自任何类的抗体，包括但不限于IgG、IgA、IgM、IgD、和IgE，及来自任何亚类(例如IgG1、IgG2、IgG3、和IgG4)的抗体。抗体可具有选自例如IgG1、IgG2、IgG3、或IgG4的重链恒定区。抗体还可具有选自例如卡帕(κ)或拉姆达(λ)的轻链。所述抗体可衍生自任何物种，包括但不限于小鼠、人、骆驼、美洲驼、鱼、鲨鱼、山羊、家兔、鸡、和牛。抗体的恒定区可进行改变，例如突变，以修饰抗体的特性(例如以提高或降低下述一项或多项：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应器细胞功能、或补体功能)。通常，抗体特异性结合预定抗原，例如与病症有关的抗原，病症例如炎性的、免疫性的、自身免疫性的、神经变性性的、代谢的、和/或恶性的病症。
在本申请中，术语“抗CD3抗体”通常是指靶向CD3的抗体或其变体，例如单克隆抗体，包括人、人源化、嵌合或鼠抗体，其针对成熟T细胞的T细胞抗原受体中的CD3受体。抗CD3抗体可以包括OKT-3。抗CD3抗体还可以包括其他抗CD3抗体包括例如在一种实施方式中otelixizumab、teplizumab和visilizumab。
在本申请中，术语“IL-2”或“IL2”通常是指称为白细胞介素2的T细胞生长因子，并包括所有形式的IL-2，可以包括在一种实施方式中人和哺乳动物形式、保守性氨基酸取代、糖型修饰或变体，或其活性片段。编码IL2基因的GeneID可以为3558。
在本申请中，术语“抗原呈递细胞”、“抗原递呈细胞”、或“APC”通常是指，在其表面上展示与主要组织相容性复合物(MHC)复合的外源抗原的免疫系统细胞，如辅助细胞(例如，B细胞、树突细胞等)。T细胞可以使用其T细胞受体(TCR)识别这些复合物。APC可以加工抗原并将其递呈至T细胞。在一种实施方式中，抗原呈递细胞可以包括选自以下组：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。
在本申请中，术语“TIL特性”通常是指TIL细胞经过本申请培养方法获得的特性。TIL特性的变化可以包含：增加的TIL细胞数量，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的T细胞受体(TCR)克隆多样性和提高的组织和/或肿瘤中TIL细胞数量，或它们的任何组合。本申请的变化可以是提高或者降低。在本申请中，术语“扩增效果”通常是指细胞经过扩增后出现的效果。扩增效果的变化可以包括，细胞的数量和/或比例变化，分泌能力变化，杀伤能力变化或表达能力的变化，或它们的任何组合。所述变化可以是提高或者降低。
在本申请中，术语“经扩增”通常是指经过培养以产生细胞的数量的变化，经扩增的细胞也可以产生细胞的数量和/或比例变化，分泌能力变化，杀伤能力变化或表达能力的变化，或它们的任何组合。所述变化可以是提高或者降低。
在本申请中，术语“纳米颗粒”通常是指至少一个尺寸小于100nm的微观颗粒。通常，纳米颗粒具有50nm至500nm(即0.05μm至0.5μm)范围内的直径；在生理环境中结构稳定；且可以容纳更小的分子(如药物或其他生物活性剂)，然后可以将该分子递送至希望的部位。
在本申请中，术语“人工抗原呈递细胞”通常是指人工构建的用于展示与主要组织相容性复合物(MHC)复合的外源抗原的免疫系统细胞。在一个实施方案中，可以包括分离的人工抗原呈递细胞(aAPC)，其可以包含表达HLA-A/B/C(编码其的基因GeneID可以为3105、3106或3107)、CD64(编码其的基因GeneID可以为2209)、CD80(编码其的基因GeneID可以为 941)、ICOS-L(编码其的基因GeneID可以为23308)和CD58(编码其的基因GeneID可以为965)的细胞，并可以被修饰以表达一种以上共刺激分子，所述以上可以包含本数。
在本申请中，术语“融合蛋白”通常是指含有第一多肽或蛋白质或其片段、类似物或衍生物的氨基酸序列和异源多肽或蛋白质(即，不同于第一多肽或蛋白质或其片段、类似物或衍生物的第二多肽或蛋白质或其片段、类似物或衍生物，或者通常不是第一多肽或蛋白质或其片段、类似物或衍生物的一部分)的氨基酸序列的多肽或蛋白质。在某些情形中，融合蛋白可包含与异源蛋白、多肽或肽融合的预防性或治疗性药物。其中，所述异源蛋白、多肽或肽可以是或不是不同类型的预防性或治疗性药物。例如，可将具有免疫调节活性的两种不同蛋白质、多肽或肽融合到一起形成融合蛋白。在某些情形中，与异源蛋白、多肽或蛋白质融合前的初始多肽或蛋白质的活性相比，融合蛋白可以保留或提高了活性。
在本申请中，术语“杀伤能力”通常是指通过使所述细胞接触有效量的物质从而杀伤靶细胞来实现。在一个实施方案中，所述物质可以是TIL细胞。所述杀伤可以包括通过自身或者促进其它细胞或物质的CDC、凋亡、ADCC、和/或吞噬作用，或通过两种或更多种这些机制的组合以杀伤细胞。
在本申请中，术语“施用”通常是指通过本领域已知的任意途径，将物质递送给有此需要的受试者。药用载体和制剂或组合物也是本领域众所周知的。给药途径可以包括：静脉内的、肌肉内的、真皮内的、皮下的、透皮的、粘膜的、瘤内的和/或粘膜的。
在本申请中，术语“试剂盒”通常是指一起被包装在容器、接受器或其它容器中的两种或更多种组分，其中一种对应于本申请的物质。例如，包含本申请的TIL细胞。
在本申请中，术语“受试者”通常是指细胞或动物，可以是哺乳动物，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型，例如肿瘤动物模型，和本领域技术人员已知的其它动物模型。
在本申请中，术语“试剂盒”通常是指一起被包装在容器、接受器或其它容器中的两种或更多种组分，其中一种对应于本申请的物质。例如，包含本申请的TIL细胞。
在本申请中，术语“饲养细胞(feeder)”通常是指体外生长和分泌至少一种因子至培养基并且可以用于支持培养的另一种所关注的细胞的生长的培养细胞。在一种实施方式中，饲养细胞可以包括抗原呈递细胞。
在本申请中，术语“特异性结合”通常是指识别特异性抗原，但是基本不识别或结合样品中其它分子的抗体。例如，如果一种抗体可以特异性结合来自一个物种的所述特异性抗原，则所述抗体还可以特异性结合来自其它的一个或多个物种的所述抗原或同源抗原。这种种间反应性本身可以不会改变抗体作为特异性的分类。在某些情形中，特异性结合至抗原的抗体还可以结合至抗原的不同等位形式。
在本申请中，术语“完整的培养过程”通常是指将细胞从患者体内分离的肿瘤组织中分离开始，经过一次或一次以上的扩增，最终获得可以施用于受试者的细胞的完整过程。
在本申请中，术语“细胞培养基”通常是指细胞例如哺乳动物细胞在其中生长的营养液。细胞培养基的配制在本领域中是熟知的。典型地，细胞培养基包括缓冲液、盐、碳水化合物、氨基酸、维生素以及必要的微量元素。细胞培养基可以含有或不含有血清、蛋白胨、和/或蛋白质。细胞培养基可以补充有另外的组分或浓度增加的组分，如氨基酸、盐、糖、维生素、激素、生长因子、缓冲液、抗生素、脂质、微量元素等，这取决于有待培养的细胞的要求和/或所希望的细胞培养参数。
在本申请中，术语“药物制剂”或“药物组合物”通常是指一种制备物，所述制备物可以允许有效成分的生物活性有效，并且可以不含有对于将会施用该制剂的受试者不可接受地有毒的额外组分。这类制剂是无菌的。“可药用的”赋形剂(载体、添加物)是可以合理地施用至受试哺乳动物以提供有效剂量的所用有效成分的那些赋形剂。
在本申请中，术语“肿瘤浸润淋巴细胞”或“TIL”通常是指最初作为白细胞获得的细胞群，所述细胞已经离开受试者的血流并迁移到肿瘤中。TIL可以包括但不限于CD8+细胞毒性T细胞(淋巴细胞)、Th1和Th17CD4+T细胞、天然杀伤细胞、树突细胞和M1巨噬细胞。TIL可以包括初级TIL和次级TIL。“初级TIL”可以是从受试者组织样品获得的那些TIL细胞，“次级TIL”可以是本申请中已扩增或经扩增的任何TIL细胞群。在一些实施方式中，所述肿瘤浸润淋巴细胞可以是未经分离纯化的，或者可以是与肿瘤细胞相互浸润的。在一种实施方式中，本申请的TIL可以是指TIL细胞群。
在本申请中，“CD4+细胞”通常是指CD4阳性的细胞，例如可以是T细胞。术语“CD4+细胞”，“CD4阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD4的抗体对细胞染色和使用荧光激活细胞分选。例如，已有的数据可以证明，CD4+细胞比例的提高可以使得细胞群分泌IFN-γ和/或TNF的能力提高，并可以提高T细胞群的促进肿瘤抑制的效果。例如，请见Tay,R.E.,Richardson,E.K.等人(2020). Cancer Gene Therapy,1-13.但是，本领域缺少一种提高CD4+细胞比例的方法，本申请可以提供一种影响CD4+细胞比例的方法。
在本申请中，“CD8+细胞”通常是指CD8阳性的细胞，例如可以是T细胞。术语“CD8+细胞”，“CD8阳性细胞”可以同义使用。这些细胞可通过本领域知道的方法来鉴定，例如通过用荧光标记的针对CD8的抗体对细胞染色和使用荧光激活细胞分选。
在本申请中，术语“中心记忆T细胞”通常是指具有长期记忆性的，并能够接受抗原再刺激的T细胞。中心记忆T细胞可以具有CD45RA-CCR7+的表型，例如可以是通过CD45RA-和CCR7+来鉴定中心记忆T细胞。又例如，中心记忆T细胞可以具有CD45RO+CD62L+的表型，例如可以是通过CD45RO+和CD62L+来鉴定中心记忆T细胞。中心记忆T细胞可以相比普通T细胞具有更强的抗肿瘤生长的能力。
在本申请中，术语“调节性T细胞”通常是指一类控制体内自身免疫反应性的T细胞亚群。调节性T细胞可以具有CD4+CD25+Foxp3+的表型，例如可以是通过CD4+、CD25+和Foxp3+来鉴定调节性T细胞。调节性T细胞可以具有抑制T细胞的抗肿瘤生长的能力。
在本申请中，术语“活化T细胞”通常是指经过活化而可以具有抗肿瘤生长的能力的T细胞。活化T细胞可以具有PD1+、LAG3+或CD28+的表型，例如可以是通过PD1+、LAG3+或CD28+来鉴定活化T细胞。活化T细胞可以具有抗肿瘤生长的能力。
在本申请中，术语“肿瘤特异性T细胞”通常是指可以特异性抗肿瘤生长的T细胞。肿瘤特异性T细胞可以具有CD103+CD39+的表型，例如可以是通过CD103+和CD39+来鉴定肿瘤特异性T细胞。肿瘤特异性T细胞可以相比普通T细胞具有更特异性的抗肿瘤生长的能力。
在本申请中，术语“干细胞样T细胞”通常是指可以具有自我增殖和/或分化的潜能的一类T细胞。干细胞样T细胞可以具有TCF1+的表型，例如可以是通过TCF1+来鉴定干细胞样T细胞。肿瘤特异性T细胞可以相比普通T细胞具有更强和/或更长期的抗肿瘤生长的能力。
在本申请中，术语“NK细胞”也称为“自然杀伤细胞”，通常是指一种细胞质中具有大颗粒的细胞。NK细胞由骨髓淋巴样干细胞发育而成，可以依赖于骨髓或胸腺微环境分化、发育。在本申请中，TIL细胞中的NK细胞的比例可以通过本申请的方法加以改变。
在本申请中，术语“肿瘤碎片”通常是指从受试者体内取出肿瘤组织后，可以通过破碎的方法，形成的肿瘤碎片。
在本申请中，术语“组合物”或“药物组合物”通常是指至少一种细胞以及至少一种和任选多于一种的其他药学上可接受的化学组分如运载体、稳定剂、稀释剂、分散剂、助悬剂、增稠剂和/或赋形剂的混合物。
在本申请中，术语“药学上可接受的载体”通常是指不干扰活性成分的生物活性的有效性的一种或多种非毒性材料。这类制剂常规地可以含有盐、缓冲剂、防腐剂、相容的载体、以及任选地其他治疗剂。这类药学上可接受的制剂还可以含有适合于给予人的相容的固体或液体填料、稀释剂或包封物质。可以用于在此所描述的配制品中的其他设想的载体、赋形剂、和/或添加剂可以包括：例如，调味剂、抗微生物剂、增甜剂、抗氧化剂、抗静电剂、脂质、蛋白质赋形剂(如血清白蛋白、明胶、酪蛋白)、成盐平衡离子(如钠)等等。适合用于在此所描述的配制品中的这些和另外已知的药物载体、赋形剂和/或添加剂是本领域中已知的。
在本申请中，术语“T细胞共刺激分子”通常是指与T细胞上的相应结合受体结合的配体，并介导T细胞共刺激反应。共刺激分子可以是有效免疫应答所需的除抗原受体或其配体之外的细胞表面分子。共刺激分子可以包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号淋巴细胞活化分子(SLAM蛋白)、NK细胞活化受体、BTLA(编码其的基因GeneID可以为151888)、Toll配体受体、OX40(编码其的基因GeneID可以为7293)、CD2(编码其的基因GeneID可以为914)、CD7(编码其的基因GeneID可以为924)、CD27(编码其的基因GeneID可以为939)、CD28(编码其的基因GeneID可以为940)、CD30(编码其的基因GeneID可以为943)、CD40(编码其的基因GeneID可以为958)、CDS、ICAM-1(编码其的基因GeneID可以为3383)、LFA-1(CD11a/CD18)(编码其的基因GeneID可以为3689)、4-1BB(CD137)(编码其的基因GeneID可以为3604)、B7-H3(编码其的基因GeneID可以为80381)、ICOS(CD278)(编码其的基因GeneID可以为29851)、GITR(编码其的基因GeneID可以为8784)、BAFFR(编码其的基因GeneID可以为115650)、LIGHT(编码其的基因GeneID可以为8740)、HVEM(LIGHTR)(编码其的基因GeneID可以为8764)、KIRDS2、SLAMF7(编码其的基因GeneID可以为57823)、NKp80(KLRF1)(编码其的基因GeneID可以为51348)、NKp44(编码其的基因GeneID可以为9436)、NKp30(编码其的基因GeneID可以为259197)、NKp46(编码其的基因GeneID可以为9437)、CD19(编码其的基因GeneID可以为930)、CD4(编码其的基因GeneID可以为920)、CD8α(编码其的基因GeneID可以为925)、CD8β(编码其的基因GeneID可以为926)、IL-2Rβ、IL-2Rγ、IL7Rα(编码其的基因GeneID可以为)、ITGA4(编码其的基因GeneID可以为3676)、VLA1(编码其的 基因GeneID可以为3672)、CD49a(编码其的基因GeneID可以为3672)、IA4(编码其的基因GeneID可以为3732)、CD49D(编码其的基因GeneID可以为3676)、ITGA6(编码其的基因GeneID可以为3655)、VLA-6(编码其的基因GeneID可以为3655)、CD49f(编码其的基因GeneID可以为3655)、ITGAD(编码其的基因GeneID可以为3681)、CD11d(编码其的基因GeneID可以为3681)、ITGAE(编码其的基因GeneID可以为3682)、CD103(编码其的基因GeneID可以为3682)、ITGAL(编码其的基因GeneID可以为3683)、CD11a(编码其的基因GeneID可以为3683)、LFA-1(编码其的基因GeneID可以为3683)、ITGAM(编码其的基因GeneID可以为3684)、CD11b(编码其的基因GeneID可以为3684)、ITGAX(编码其的基因GeneID可以为3687)、CD11c(编码其的基因GeneID可以为3687)、ITGB1(编码其的基因GeneID可以为3688)、CD29(编码其的基因GeneID可以为3688)、ITGB2(编码其的基因GeneID可以为3689)、CD18(编码其的基因GeneID可以为3689)、LFA-1(编码其的基因GeneID可以为3689)、ITGB7(编码其的基因GeneID可以为3695)、NKG2D(编码其的基因GeneID可以为22914)、NKG2C(编码其的基因GeneID可以为3822)、TNFR2(编码其的基因GeneID可以为7133)、TRANCE/RANKL(编码其的基因GeneID可以为8600)、DNAM1(CD226)(编码其的基因GeneID可以为10666)、SLAMF4(CD244、2B4)(编码其的基因GeneID可以为51744)、CD84(编码其的基因GeneID可以为8832)、CD96(Tactile)(编码其的基因GeneID可以为10225)、CEACAM1(编码其的基因GeneID可以为634)、CRTAM(编码其的基因GeneID可以为56253)、Ly9(CD229)(编码其的基因GeneID可以为4063)、CD160(BY55)(编码其的基因GeneID可以为11126)、PSGL1(编码其的基因GeneID可以为6404)、CD100(SEMA4D)(编码其的基因GeneID可以为10507)、CD69(编码其的基因GeneID可以为969)、SLAMF6(NTB-A、Ly108)(编码其的基因GeneID可以为114836)、SLAM(SLAMF1、CD150、IPO-3)(编码其的基因GeneID可以为6504)、BLAME(SLAMF8)(编码其的基因GeneID可以为56833)、SELPLG(CD162)(编码其的基因GeneID可以为6404)、LTBR(编码其的基因GeneID可以为4055)、LAT(编码其的基因GeneID可以为27040)、GADS(编码其的基因GeneID可以为9402)、SLP-76(编码其的基因GeneID可以为3937)、PAG/Cbp(编码其的基因GeneID可以为55824)、CD19a、和特异性结合CD3的配体、特异性结合CD28的配体、特异性结合HVEM的配体、特异性结合CD40L的配体、特异性结合OX40的配体、和特异性结合4-1BB的配体。共刺激胞内信号传导结构域指共刺激分子的胞内部分。胞内信号传导结构域可以包含从中衍生它的分子的完整胞内部分或完整天然胞内信号传导结构域或其功能性片段。
在本申请中，术语“T细胞生长因子”通常是指引起细胞增殖的生物活性多肽或小分子化合物。在一种实施方式中，T细胞生长因子可以选自以下组的一种或多种：IL-2(编码其的基因GeneID可以为3558)、IL-4(编码其的基因GeneID可以为3565)、IL-7(编码其的基因GeneID可以为3574)、IL-10(编码其的基因GeneID可以为3586)、IL-12(编码其的基因GeneID可以为3592或3593)、IL-15(编码其的基因GeneID可以为3600)、和γ干扰素(编码其的基因GeneID可以为3458)。
在本申请中，术语“基本上同时”通常是指接触过程的一段时间内TIL可以与两种以上的物质同时接触，但是可以不限于在整个接触过程中TIL总是与两种以上的物质同时接触。例如，基本上同时可以是指一段时间内TIL可以与至少10％、20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％的两种以上的物质的每种物质同时接触。
在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。本申请的肿瘤可能是良性的，也可能是恶性的。本申请的肿瘤可能是实体的，也可能是血液的。术语“肿瘤”可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
在本申请中，术语“肿瘤组织”通常是指来自对象中的肿瘤，包括对象中的任何实体肿瘤和/或非实体肿瘤的任何组织的样品。
在本申请中，术语“约”和“大约”通常是指在统计上有意义的数值范围内。这样的范围可以在给定值或范围的一个数量级内，可以包括在50％内，可以包括在20％内，可以包括在10％内，可以包括在5％内。术语“约”或“大约”所包含的可允许变化取决于所研究的特定系统，并且本领域普通技术人员可以容易地理解。术语“以上”、“以下”、“至多”和“至少”可以包括本数。
发明详述
一方面，本申请涉及一种肿瘤浸润淋巴细胞(TIL)在疾病治疗中的用途，所述药物用于预防和/或治疗肿瘤，其中的肿瘤浸润淋巴细胞采用如下方法培养。例如，可以使经扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与饲养细胞共培养。一方面，本申请提供一种培养肿瘤浸润淋巴细胞(TIL)的方法，其包括：可以使经扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一段时间之后与饲养细胞共培养。在一种实施方式中，所述经扩增的TIL可以为经过体外扩增的TIL。
例如，还包含在向受试者施用所述培养方法获得的TIL之前，可以使所述受试者的淋巴细胞数量减少。例如，为了使本申请的TIL的疗效提高，可以对受试者的淋巴系统和/或淋巴 细胞进行处理。例如，可以使受试者的淋巴细胞清除或基本被清除、可以使受试者的淋巴细胞耗竭或基本被耗竭、可以使受试者的淋巴细胞耗尽或基本被耗尽、或可以使受试者的淋巴细胞消耗或基本被消耗。例如，相对于为处理前，本申请的处理可以使受试者的淋巴细胞数量减少至少约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、或约0.1％。
例如，本申请的使所述受试者的淋巴细胞数量减少包含清淋方案。例如，本申请的清淋方案可以包含在向受试者施用所述培养方法获得的TIL之前，向所述受试者施用环磷酰胺和/或氟达拉滨。例如，还可以包含在向受试者施用所述培养方法获得的TIL之前第4天至第2天，向所述受试者施用环磷酰胺和/或氟达拉滨。
例如，可以以约300至约500mg/m2/天的剂量向所述受试者施用环磷酰胺。例如，可以以约50mg/m2/天、约100mg/m2/天、约150mg/m2/天、约200mg/m2/天、约250mg/m2/天、约300mg/m2/天、约350mg/m2/天、约400mg/m2/天、约450mg/m2/天、或约500mg/m2/天的剂量向所述受试者施用环磷酰胺。例如，可以向所述受试者施用环磷酰胺持续3天。例如，施用TIL之前第4天至第2天，可以向所述受试者施用环磷酰胺。
例如，可以以约20至约30mg/m2/天的剂量向所述受试者施用氟达拉滨。例如，可以以约5mg/m2/天、约10mg/m2/天、约15mg/m2/天、约20mg/m2/天、约25mg/m2/天、或约30mg/m2/天的剂量向所述受试者施用氟达拉滨。例如，可以向所述受试者施用氟达拉滨持续3天。例如，施用TIL之前第4天至第2天，可以向所述受试者施用氟达拉滨。
在一些实施方式中，本申请的组合物中提供的TIL数量的范围可以为约5×109至1×1010、约1×1010至5×1010、或约5×1010至1×1011。在一些实施方式中，本申请的组合物中提供的TIL数量的范围可以为约5×109、约1×1010、约5×1010、或约1×1011。例如，可以包含向所述受试者一次性静脉输注施用所述培养方法获得的TIL。例如，可以包含向所述受试者多次静脉输注施用所述培养方法获得的TIL。
例如，还可以包含在向受试者施用所述培养方法获得的TIL之后，可以使所述TIL维持增殖能力。例如，还可以包含在向受试者施用所述培养方法获得的TIL之后，向所述受试者施用白介素-2(IL-2)或其变体。
例如，可以以约200,000至约600,000IU/kg/次的剂量向所述受试者施用IL-2。例如，可以以约100,000IU/kg/次、约200,000IU/kg/次、约300,000IU/kg/次、约400,000IU/kg/次、约 500,000IU/kg/次、或约600,000IU/kg/次的剂量向所述受试者施用IL-2。
例如，可以在向受试者施用所述培养方法获得的TIL之后8至16小时，向所述受试者第一次施用IL-2。
例如，在向所述受试者第一次施用IL-2后，可以每12小时向所述受试者施用一次IL-2。例如，在第一次施用IL-2后，可以暂停施用IL-2一次；例如可以根据患者的耐受程度，确定是否继续施用IL-2。例如，在向所述受试者第一次施用IL-2的24小时后，可以根据受试者的耐受情况第二次施用所述IL-2。例如，可以根据受试者的不良反应情况，确定是否继续施用IL-2。例如，可以向所述受试者施用10次或更少次的IL-2。
在一种实施方式中，所述经扩增的TIL为使源自肿瘤组织且未经体外扩增的TIL可以经过至少一个阶段的体外扩增后获得的TIL。例如，可以经过至少2个阶段的体外扩增、可以经过至少3个阶段的体外扩增、可以经过至少4个阶段的体外扩增、可以经过至少5个阶段的体外扩增、可以经过至少6个阶段的体外扩增、可以经过至少7个阶段的体外扩增、可以经过至少8个阶段的体外扩增、可以经过至少9个阶段的体外扩增、或者可以经过至少10个阶段的体外扩增。
例如，每一个阶段体外扩增之间可以是通过TIL细胞数量的变化来划分的，例如，当TIL细胞的数量增加至少约1倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。在一些实施方式中，当TIL细胞的数量增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、至少约50倍、至少约100倍、至少约200倍、至少约500倍、或者至少约1000倍时，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段的体外扩增之间也可以是通过TIL细胞培养的条件的变化来划分的。例如，当细胞培养基中添加了或补充添加了T细胞激活剂和/或T细胞生长因子后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，本申请中T细胞激活剂可以和T细胞共刺激分子相互替换使用。例如，当细胞培养基中添加了或补充添加了IL-2后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当细胞培养基中添加了或补充添加了饲养细胞后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，当TIL细胞进行了离心和/或细胞洗涤的操作后，可以认为TIL细胞进入了下一个阶段的体外扩增。例如，每一个阶段之间也可以是通过TIL细胞培养的天数来划分的。例如，当TIL细胞体外培养约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约 17天、约18天、约19天、约20天、约30天、约40天、约50天或约100天后，可以认为TIL细胞进入了下一个阶段的体外扩增。
例如，所述第二阶段体外扩增可以进行至少约7天。例如，所述第二阶段体外扩增可以进行至少约9天。例如，所述第二阶段体外扩增可以进行至多约14天。例如，所述第二阶段体外扩增可以进行至多约13天。例如，所述第二阶段体外扩增可以进行约7天至约14天、约9天至约14天、约7天至约13天或约9天至约13天。例如，本申请的第二阶段体外扩增可以进行至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第二阶段体外扩增可以进行约9天至约14天，例如，本申请的第二阶段体外扩增可以进行约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第二阶段体外扩增可以认为是REP(rapid expansion protocol)阶段。
例如，所述第一阶段体外扩增可以进行至少约7天。例如，所述第一阶段体外扩增可以进行约7天至约14天。例如，本申请的第一阶段体外扩增可以进行至少约7天、至少约8天、至少约9天、至少约10天、至少约11天、至少约12天、至少约13天、或至少约14天。例如，本申请的第一阶段体外扩增可以进行约7天至约14天、约8天至约14天、约9天至约14天、约10天至约14天、约11天至约14天、约12天至约14天、约13天至约14天、约9天至约13天、约10天至约13天、约11天至约13天、约12天至约13天、约9天至约12天、约10天至约12天、约11天至约12天、或约10天至约11天。例如，本申请的第一阶段体外扩增可以认为是preREP阶段。
例如，本申请第二阶段体外扩增进行的天数可以是从第二阶段体外扩增的开始时刻进行计算。例如，第二阶段体外扩增开始的当时，可以认为是第二阶段体外扩增进行了约0天。例如，第二阶段体外扩增开始后进行了约24小时，可以认为是第二阶段体外扩增进行了约1天。例如，第二阶段体外扩增开始的当天，可以认为是第二阶段体外扩增进行了约0天。例如，本申请第二阶段体外扩增进行的天数可以是通过第二阶段体外扩增进行的天数进行计算。例如，第二阶段体外扩增开始后的第二天，可以认为是第二阶段体外扩增进行了约1天。
例如，本申请用到的TIL的培养方法可以按照两步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子 接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约7天至约14天。
例如，本申请用到的TIL的培养方法可以按照三步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；(C)可以使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约0天至约8天。例如，所述步骤(C)可以进行约5天至约14天。
例如，本申请用到的TIL的培养方法可以按照四步骤划分方式进行划分。例如，(A)可以使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；(B)可以使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；(C)可以使所述第三TIL群的任选的基因的表达提高或降低和/或活性增强或减弱，其中，经所述步骤(C)得到第四TIL群；(D)可以使所述第四TIL群与饲养细胞共培养，其中，经所述步骤(D)得到第五TIL群。例如，所述步骤(A)可以进行约7天至约14天。例如，所述步骤(B)可以进行约0天至约4天。例如，所述步骤(C)可以进行约0天至约4天。例如，所述步骤(D)可以进行约5天至约14天。
例如，本申请的改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，提高的T细胞受体(TCR)克隆多样性和提高的组织和/或肿瘤中TIL细胞数量。
在一种实施方式中，可以使经扩增的TIL在与T细胞共刺激分子接触之后，与饲养细胞共培养。在一种实施方式中，可以使经扩增的TIL在与T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，可以使经扩增的TIL在与T细胞共刺激分子和T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，可以使经扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与至少一部分饲养细胞共培养。例如，一部分饲养细胞与经扩增的TIL共培养可以是在经扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触的同时，至少另一部分的饲养细胞与经扩增的TIL共培养是在经扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后。例如至少另一部分的饲养细胞可以包含全部所 用饲养细胞的约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、或约0.1％。
在一种实施方式中，所述经扩增的TIL细胞可以是在体外进行扩增。在一种实施方式中，所述经扩增的TIL细胞可以是在自体体内进行扩增。在一种实施方式中，所述经扩增的TIL细胞可以是在异体体内进行扩增。在一种实施方式中，所述经扩增的TIL细胞可以是在离体进行扩增。
在一种实施方式中，与所述源自肿瘤组织且未经体外扩增的TIL相比，所述经扩增的TIL的数量可以增加至少1倍。例如，与所述源自肿瘤组织且未经体外扩增的TIL相比，所述经扩增的TIL的数量可以增加至少约1倍、至少约2倍、至少约3倍、至少约4倍、至少约5倍、至少约6倍、至少约7倍、至少约8倍、至少约9倍、至少约10倍、至少约11倍、至少约12倍、至少约13倍、至少约14倍、至少约15倍、至少约20倍、至少约30倍、至少约40倍、或者至少约50倍。
在一种实施方式中，所述经扩增的TIL细胞，与从肿瘤组织获得且未经体外扩增的TIL相比，所述经扩增的TIL细胞数量可以增加50倍以上。例如，与从肿瘤组织获得的TIL相比，所述经扩增的TIL细胞数量可以增加约50倍以上、约60倍以上、约70倍以上、约80倍以上、约90倍以上、约100倍以上、约200倍以上、约300倍以上、约400倍以上、约500倍以上、约600倍以上、约700倍以上、约800倍以上、约900倍以上、约2000倍以上、约3000倍以上、约4000倍以上、约5000倍以上、约6000倍以上、约7000倍以上、约8000倍以上、约9000倍以上、约10000倍以上、约15000倍以上、或者约20000倍以上。。
一方面，本申请提供一种用到的TIL的培养方法。其中，使经第一阶段扩增的TIL经受第二阶段扩增，其中在所述第二阶段扩增中，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与饲养细胞共培养。
在一种实施方式中，在所述第二阶段扩增中，可以使所述TIL在与T细胞共刺激分子接触之后，与饲养细胞共培养。在一种实施方式中，在所述第二阶段扩增中，可以使所述TIL在与T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，在所述第二阶段扩增中，可以使所述TIL在与T细胞共刺激分子和T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，在所述第二阶段扩增中，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与至少一部分饲养细胞共培养。例如，在所述第二阶段扩增中，一 部分饲养细胞与TIL共培养可以是在TIL在与T细胞共刺激分子和/或T细胞生长因子接触的同时，至少另一部分的饲养细胞与TIL共培养是在TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后。例如至少另一部分的饲养细胞可以包含全部所用饲养细胞的约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、或约0.1％。
例如，本申请可以以约40:1-约400:1的本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。例如，本申请可以以约40:1-约400:1、以约40:1-约300:1、以约40:1-约200:1、以约40:1-约100:1、以约40:1-约90:1、以约40:1-约80:1、以约40:1-约70:1、以约40:1-约60:1、以约40:1-约50:1、以约50:1-约400:1、以约60:1-约400:1、以约70:1-约400:1、以约80:1-约400:1、以约90:1-约400:1、以约100:1-约400:1、以约200:1-约400:1、或以约300:1-约400:1的本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。
在一种实施方式中，与第一阶段扩增的TIL相比，所述经第二阶段扩增的TIL细胞数量可以增加约50倍以上。例如，与第一阶段扩增的TIL相比，所述经第二阶段扩增的TIL细胞数量可以增加约50倍以上、约60倍以上、约70倍以上、约80倍以上、约90倍以上、约100倍以上、约200倍以上、约300倍以上、约400倍以上、约500倍以上、约600倍以上、约700倍以上、约800倍以上、约900倍以上、约1000倍以上、约2000倍以上、约3000倍以上、约4000倍以上、约5000倍以上、约6000倍以上、约7000倍以上、约8000倍以上、约9000倍以上、约10000倍以上、约15000倍以上、或者约20000倍以上。在一种实施方式中，TIL细胞的数量增加可以用扩增倍数表示，所述扩增倍数可以是，第二阶段扩增结束后相比于第二阶段扩增开始前，TIL细胞数量扩增至的倍数。例如，如果第二阶段扩增开始前TIL细胞数量为1×108，第二阶段扩增结束后TIL细胞数量为1×109，可以认为TIL细胞的扩增倍数为10。
一方面，本申请提供一种用到的TIL的培养方法，其包括：可以使源自肿瘤组织且未经体外扩增的TIL经过至少一个阶段的体外扩增，其中在单个体外扩增阶段中，可以使经体外扩增和/或未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
在另一种实施方式中，可以使源自肿瘤组织且未经体外扩增的TIL经过至少两个阶段的 体外扩增，其中在第二个阶段的体外扩增和/或之后的单个体外扩增阶段中，可以使经体外扩增和/或未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
例如，可以是使源自肿瘤组织且未经体外扩增的TIL经过一个阶段的体外扩增，其中在第一个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
例如，可以是使源自肿瘤组织且未经体外扩增的TIL经过两个阶段的体外扩增，其中在第一个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。也可以是使源自肿瘤组织且未经体外扩增的TIL经过两个阶段的体外扩增，其中在第二个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
例如，也可以是使源自肿瘤组织且未经体外扩增的TIL经过两个阶段的体外扩增，其中在第一个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，且其中在第二个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
例如，可以是使源自肿瘤组织且未经体外扩增的TIL经过三个阶段的体外扩增，其中在第一个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。也可以是使源自肿瘤组织且未经体外扩增的TIL经过三个阶段的体外扩增，其中在第二个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。也可以是使源自肿瘤组织且未经体外扩增的TIL经过三个阶段的体外扩增，其中在第三个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
例如，也可以是使源自肿瘤组织且未经体外扩增的TIL经过三个阶段的体外扩增，其中在第一个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，且其中在第二个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。例如，也可以是使源自肿瘤组织且未经体外扩增的TIL经过三个阶段的体外扩增，其中在第一个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和 /或T细胞生长因子接触一定时间之后与饲养细胞共培养，且其中在第三个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。例如，也可以是使源自肿瘤组织且未经体外扩增的TIL经过三个阶段的体外扩增，其中在第二个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，且其中在第三个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。例如，也可以是使源自肿瘤组织且未经体外扩增的TIL经过三个阶段的体外扩增，其中在第一个阶段的体外扩增中，可以使未经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，且其中在第二个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，且其中在第三个阶段的体外扩增中，可以使经体外扩增的TIL在与T细胞共刺激分子和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
一方面，本申请提供一种用到的TIL的培养方法。其中，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与饲养细胞共培养，所述TIL在完整的培养过程中可以经过两个以上阶段的扩增，所述TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在同一个阶段的扩增中。
在一种实施方式中，所述TIL在完整的培养过程中可以经过两个以上阶段的扩增，可以使的TIL在与T细胞共刺激分子接触之后，与饲养细胞共培养。在一种实施方式中，所述TIL在完整的培养过程中可以经过两个以上阶段的扩增，可以使TIL在与T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，所述TIL在完整的培养过程中可以经过两个以上阶段的扩增，可以使TIL在与T细胞共刺激分子和T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，所述TIL在完整的培养过程中可以经过两个以上阶段的扩增，可以使TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与至少一部分饲养细胞共培养。例如，所述TIL在完整的培养过程中可以经过两个以上阶段的扩增，一部分饲养细胞与TIL共培养可以是在TIL在与T细胞共刺激分子和/或T细胞生长因子接触的同时，至少另一部分的饲养细胞与TIL共培养是在TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后。例如至少另一部分的饲养细胞可以包含全部所用饲养细胞的约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约 0.2％、或约0.1％。
在一种实施方式中，所述TIL在完整的培养过程中可以经过两个以上阶段的扩增。例如，所述TIL在完整的培养过程中可以经过2、3、4、5、6、7、8、9、10、20、50、100个以上阶段的扩增。
在一种实施方式中，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第一个阶段扩增中和/或第二个阶段扩增中。在一种实施方式中，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第一个阶段扩增中、第二个阶段扩增中和/或第三个阶段扩增中。例如，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第一个阶段扩增中。例如，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第二个阶段扩增中。例如，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第三个阶段扩增中。例如，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第一个阶段扩增中和第二个阶段扩增中。例如，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第一个阶段扩增中和第三个阶段扩增中。例如，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第二个阶段扩增中和第三个阶段扩增中。例如，TIL与T细胞共刺激分子和/或T细胞生长因子接触与所述TIL和饲养细胞共培养可以发生在第一个阶段扩增中、第二个阶段扩增中和第三个阶段扩增中。
在一种实施方式中，与在所述TIL与T细胞共刺激分子和T细胞生长因子接触同时使所述TIL和饲养细胞共培养相比，在所述TIL与T细胞共刺激分子和/或T细胞生长因子接触一定时间间隔后使所述TIL和饲养细胞共培养可以提高所述TIL的扩增效果。例如，所述提高TIL的扩增效果可以包括选自以下组：增加TIL细胞的数量，改变TIL细胞的比例，提高TIL细胞的分泌能力，和提高TIL细胞的杀伤能力。在一种实施方式中，在所述TIL与T细胞共刺激分子和/或T细胞生长因子接触一定时间间隔后使所述TIL和饲养细胞共培养可以增加TIL细胞的数量。在一种实施方式中，在所述TIL与T细胞共刺激分子和/或T细胞生长因子接触一定时间间隔后使所述TIL和饲养细胞共培养可以提高TIL细胞的分泌能力。在一种实施方式中，在所述TIL与T细胞共刺激分子和/或T细胞生长因子接触一定时间间隔后使所述TIL和饲养细胞共培养可以提高TIL细胞的杀伤能力。
在一种实施方式中，在所述TIL与T细胞共刺激分子和/或T细胞生长因子接触一定时间间隔后使所述TIL和饲养细胞共培养可以改变TIL细胞的比例。例如，所述可以改变TIL 细胞的比例可以包括选自以下组：可以增加TIL中中心记忆T细胞(Tcm)比例，可以增加调节性T细胞(Treg)以外的TIL细胞的比例，可以降低调节性T细胞(Treg)的比例，可以增加活化T细胞比例，可以增加肿瘤特异性T细胞比例，和可以增加干细胞样T细胞比例。例如，所述可以改变TIL细胞的比例包括选自以下组：可以增加TIL中CD45RA-CCR7+中心记忆T细胞(Tcm)比例，可以增加CD4+CD25+Foxp3+调节性T细胞(Treg)以外的TIL细胞的比例，可以降低CD4+CD25+Foxp3+调节性T细胞(Treg)的比例，可以增加活化T细胞比例，可以增加CD103+CD39+肿瘤特异性T细胞比例，和可以增加TCF1+干细胞样T细胞比例。又例如，本申请改变TIL细胞的比例可以包括增加TIL中CD45RO+CD62L+中心记忆T细胞(Tcm)比例。例如，所述可以增加活化T细胞比例包括选自以下组：增加PD1+细胞比例、增加LAG3+细胞比例和增加CD28+细胞比例。所述改变TIL细胞的比例可以包括本申请方法培养的TIL细胞中中心记忆T细胞比例，活化T细胞比例，肿瘤特异性T细胞比例，和/或干细胞样T细胞，与在所述TIL与T细胞共刺激分子和T细胞生长因子接触同时使所述TIL和饲养细胞共培养相比，增加至少约1％、至少约2％、至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约97％、至少约98％或者至少约99％。所述改变TIL细胞的比例可以包括本申请方法培养的TIL细胞中调节性T细胞(Treg)，与在所述TIL与T细胞共刺激分子和T细胞生长因子接触同时使所述TIL和饲养细胞共培养相比，减少至少约1％、至少约2％、至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约97％、至少约98％或者至少约99％。
在一种实施方式中，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，所述之后可以是指之后2小时以上。例如，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触6到72小时、或12到48小时之后，与饲养细胞共培养。例如，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触约1小时、约2小时、约3小时、约4小时、约5小时、约6小时、约7小时、约8小时、约9小时、约10小时、约11小时、约12小时、约13小时、约14小时、约15小时、约16小时、约17小时、约18小时、约19小时、约20小时、约21小时、约22小时、约23小时、约24小时、约36小时、约48小时、约60小时或约72小时之后，与饲养 细胞共培养。例如，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、或约14天之后，与饲养细胞共培养。
在一种实施方式中，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，所述T细胞共刺激分子可以选自以下组的一种或多种：CD80、CD86、B7-H3、4-1BBL、CD27、CD30、CD134、B7h、CD40、LIGHT、特异性结合CD3的抗体、特异性结合CD28的抗体、特异性结合HVEM的抗体、特异性结合CD40L的抗体、特异性结合OX40的抗体和特异性结合4-1BB的抗体。
在一种实施方式中，所述TIL与T细胞共刺激分子接触可以包括一种或多种所述T细胞共刺激分子与所述TIL单独接触和/或多种所述T细胞共刺激分子与所述TIL同时接触。在一种实施方式中，可以包括一种或多种所述T细胞共刺激分子与所述TIL单独接触。在一种实施方式中，可以包括多种所述T细胞共刺激分子与所述TIL同时接触。例如，一种或多种所述T细胞共刺激分子可以单独加入到所述TIL的细胞培养基中，例如，多种所述T细胞共刺激分子可以同时加入到所述TIL的细胞培养基中。例如，一种所述T细胞共刺激分子可以以以下组的形式的一种或多种加入到所述TIL的细胞培养基中：表达所述T细胞共刺激分子的工程化细胞，嵌合所述T细胞共刺激分子的纳米颗粒，和嵌合所述T细胞共刺激分子的聚合物。例如，多种所述T细胞共刺激分子可以以选自以下组的形式加入到所述TIL的细胞培养基中：混合物，融合蛋白，表达多种所述T细胞共刺激分子的工程化细胞，嵌合多种所述T细胞共刺激分子的纳米颗粒，和嵌合多种所述T细胞共刺激分子的聚合物。例如，T细胞共刺激分子可以为特异性结合CD3的抗体，例如可以是Miltenyi Biotech的OKT3。
在一种实施方式中，可以使所述TIL在与T细胞共刺激分子和/或T细胞生长因子接触之后，与饲养细胞共培养。在一种实施方式中，所述T细胞生长因子可以选自以下组的一种或多种：IL-2、IL-4、IL-7、IL-10、IL-12、IL-15、和γ干扰素。例如，T细胞生长因子可以为IL-2。在一种实施方式中，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为约1000IU/mL以上。在一种实施方式中，所述IL-2在所述TIL的细胞培养基中的初始浓度可以为约1500IU/mL、约2000IU/mL、约2500IU/mL、约2600IU/mL、约2700IU/mL、约2800IU/mL、约2900IU/mL、约3000IU/mL、约3100IU/mL、约3200IU/mL、约3300IU/mL、约3400IU/mL、约3500IU/mL、约4000IU/mL、约4500IU/mL、约5000IU/mL、约5500IU/mL、约6000IU/mL、约6500IU/mL、约7000IU/mL、约7500IU/mL、约8000IU/mL、约8500IU/mL、或约9000IU/mL以上。
在一种实施方式中，所述TIL与T细胞生长因子接触可以包括一种或多种所述T细胞生长因子与所述TIL单独接触和/或多种所述T细胞生长因子与所述TIL同时接触。在一种实施方式中，可以包括一种或多种所述T细胞生长因子与所述TIL单独接触。在一种实施方式中，可以包括多种所述T细胞生长因子与所述TIL同时接触。例如，一种或多种所述T细胞生长因子可以单独加入到所述TIL的细胞培养基中，例如，多种所述T细胞生长因子可以同时加入到所述TIL的细胞培养基中。例如，一种所述T细胞生长因子可以以下组的形式的一种或多种加入到所述TIL的细胞培养基中：表达所述T细胞生长因子的工程化细胞，嵌合所述T细胞生长因子的纳米颗粒，和嵌合所述T细胞生长因子的聚合物。例如，多种所述T细胞生长因子可以以选自以下组的形式加入到所述TIL的细胞培养基中：混合物，融合蛋白，表达多种所述T细胞生长因子的工程化细胞，嵌合多种所述T细胞生长因子的纳米颗粒，和嵌合多种所述T细胞生长因子的聚合物。
在一种实施方式中，所述TIL可以选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL、源自肿瘤切除术的TIL、源自活体组织检查的TIL和源自冷冻保存后复苏的TIL。在一种实施方式中，可以通过将肿瘤组织处理成肿瘤碎片获得所述TIL。在一种实施方式中，所述肿瘤碎片的体积约为1-27立方毫米。在一种实施方式中，所述肿瘤碎片的体积约为约1立方毫米、约2立方毫米、约3立方毫米、约4立方毫米、约5立方毫米、约6立方毫米、约7立方毫米、约8立方毫米、约9立方毫米、约10立方毫米、约11立方毫米、约12立方毫米、约13立方毫米、约15立方毫米、约17立方毫米、约19立方毫米、约20立方毫米、约21立方毫米、约23立方毫米、约24立方毫米、约25立方毫米、约26立方毫米或27立方毫米。
在一种实施方式中，所述饲养细胞可以包括抗原呈递细胞。在一种实施方式中，所述饲养细胞可以包括选自以下组的一种或多种：外周单个核细胞，树突状细胞，和人工抗原呈递细胞。例如，所述饲养细胞可以为外周单个核细胞。例如，所述饲养细胞可以为树突状细胞。例如，所述饲养细胞可以为人工抗原呈递细胞。例如，所述饲养细胞可以为分离的人工抗原呈递细胞(aAPC)，其可以包含表达HLA-A/B/C、CD64、CD80、ICOS-L和CD58的细胞，并可以被修饰以表达一种以上共刺激分子。在一种实施方式中，所述饲养细胞可以经过辐照。例如，可以经过伽马射线辐照，或可以经过X射线辐照。
在一种实施方式中，所述TIL可以和饲养细胞共培养。在一种实施方式中，所述共培养可以是TIL和饲养细胞的表面相接触，例如可以将所述饲养细胞加入所述TIL的细胞培养基中。在一种实施方式中，所述共培养可以是TIL和饲养细胞的表面相接触。在一种实施方式 中，可以将所述饲养细胞固定在装置上并加入所述TIL的细胞培养基中。在一种实施方式中，可以将所述饲养细胞与TIL的细胞通过膜、网、栅格分离，但是可以进行物质交换或可以进行部分程度的接触。在一种实施方式中，可以将所述饲养细胞的细胞代谢产物加入所述TIL的细胞培养基中。例如，本申请可以以约40:1-约400:1的上述本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。例如，本申请可以以约40:1-约400:1、以约40:1-约300:1、以约40:1-约200:1、以约40:1-约100:1、以约40:1-约90:1、以约40:1-约80:1、以约40:1-约70:1、以约40:1-约60:1、以约40:1-约50:1、以约50:1-约400:1、以约60:1-约400:1、以约70:1-约400:1、以约80:1-约400:1、以约90:1-约400:1、以约100:1-约400:1、以约200:1-约400:1、或以约300:1-约400:1的本申请饲养细胞与本申请TIL的比例，将本申请饲养细胞添加至本申请TIL的细胞培养基中。
一方面，本申请提供一种用到的TIL的培养方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织在基础培养基内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入T细胞无血清培养基和浓度为1000-9000IU/mL(例如可以是6000IU/mL)IL-2培养约3-14天。收集培养基中细胞，可以与组织块共同转移入透气培养袋、或Grex、或Xuri设备，T细胞无血清培养基可以添加CD3抗体约30ng/mL，IL-2(1000-9000IU/mL)，活化一定时间后添加辐照PBMC(TIL与PBMC按照比率1:40-1:400)，扩增培养约3-14天。过滤组织块，可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于70％，大于80％的T细胞可以为记忆效应T细胞和效应T细胞。经刺激后可以分泌IFNγ，可以具有活化T细胞比例上调的特征。
一方面，本申请提供一种肿瘤浸润淋巴细胞(TIL)，所述TIL可以根据本申请涉及的培养方法培养得到。在一种实施方式中，本申请提供的TIL可以包含一种或一个批次的本申请的培养方法培养得到TIL。在一种实施方式中，本申请提供的TIL可以包含多种或多个批次的本申请的培养方法培养得到并以任意比例组合的TIL。
在一些实施方式中，可以将使用本申请方法扩增的TIL作为药物组合物施用于患者。在一些实施方式中，药物组合物可以是TIL在无菌缓冲液中的悬液。使用本申请的PBMC扩增的TIL可以通过本领域已知的任何合适途径施用。在一些实施方式中，T细胞可以以单次动脉内或静脉内输注施用，输注可以持续约30至60分钟。其他合适的施用途径可以包括腹膜内、鞘内和淋巴管内施用。
可以施用任何合适剂量的TIL。在一些实施方式中，例如当治疗肿瘤时，可以施用约2.3×109至约13.7×1010个TIL。在一些实施方式中，可以施用约1×109至约12×1010个TIL。在一些实施方式中，可以施用约1.2×1010至约4.3×1010个TIL。在一些实施方式中，可以施用约3×1010至约12×1010个TIL。在一些实施方式中，可以施用约4×1010至约10×1010个TIL。在一些实施方式中，可以施用约5×1010至约8×1010个TIL。在一些实施方式中，可以施用约6×1010至约8×1010个TIL。在一些实施方式中，可以施用约7×1010至约8×1010个TIL。在一些实施方式中，治疗有效剂量可以为约2.3×109至约13.7×1010。在一些实施方式中，治疗有效剂量可以为约1×109至约12×1010个TIL。在一些实施方式中，治疗有效剂量可以为约1.2×1010至约4.3×1010个TIL。在一些实施方式中，治疗有效剂量可以为约3×1010至约12×1010个TIL。在一些实施方式中，治疗有效剂量可以为约4×1010至约10×1010个TIL。在一些实施方式中，治疗有效剂量可以为约5×1010至约8×1010个TIL。在一些实施方式中，治疗有效剂量可以为约6×1010至约8×1010个TIL。在一些实施方式中，治疗有效剂量可以为约7×1010至约8×1010个TIL。
在一些实施方式中，本申请的组合物中提供的TIL的数量可以为约1×106、约2×106、约3×106、约4×106、约5×106、约6×106、约7×106、约8×106、约9×106、约1×107、约2×107、约3×107、约4×107、约5×107、约6×107、约7×107、约8×107、约9×107、约1×108、约2×108、约3×108、约4×108、约5×108、约6×108、约7×108、约8×108、约9×108、约1×109、约2×109、约3×109、约4×109、约5×109、约6×109、约7×109、约8×109、约9×109、约1×1010、约2×1010、约3×1010、约4×1010、约5×1010、约6×1010、约7×1010、约8×1010、约9×1010、约1×1011、约2×1011、约3×1011、约4×1011、约5×1011、约6×1011、约7×1011、约8×1011、约9×1011、约1×1012、约2×1012、约3×1012、约4×1012、约5×1012、约6×1012、约7×1012、约8×1012、约9×1012、约1×1013、约2×1013、约3×1013、约4×1013、约5×1013、约6×1013、约7×1013、约8×1013，或约9×1013。在一些实施方式中，本申请的组合物中提供的TIL数量的范围可以为约1×106至5×106、约5×106至1×107、约1×107至5×107、约5×107至1×108、约1×108至5×108、约5×108至1×109、约1×109至5×109、约5×109至1×1010、约1×1010至5×1010、约5×1010至1×1011、约5×1011至1×1012、约1×1012至5×1012，或约5×1012至1×1013。
在一些实施方式中，本申请的组合物中提供的TIL的浓度可以小于组合物的例如约100％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约 0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约0.0002％，或约0.0001％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度可以大于组合物的约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19.75％、约19.50％、约19.25％、约19％、约18.75％、约18.50％、约18.25％、约18％、约17.75％、约17.50％、约17.25％、约17％、约16.75％、约16.50％、约16.25％、约16％、约15.75％、约15.50％、约15.25％、约15％、约14.75％、约14.50％、约14.25％、约14％、约13.75％、约13.50％、约13.25％、约13％、约12.75％、约12.50％、约12.25％、约12％、约11.75％、约11.50％、约11.25％、约11％、约10.75％、约10.50％、约10.25％、约10％、约9.75％、约9.50％、约9.25％、约9％、约8.75％、约8.50％、约8.25％、约8％、约7.75％、约7.50％、约7.25％、约7％、约6.75％、约6.50％、约6.25％、约6％、约5.75％、约5.50％、约5.25％、约5％、约4.75％、约4.50％、约4.25％、约4％、约3.75％、约3.50％、约3.25％、约3％、约2.75％、约2.50％、约2.25％、约2％、约1.75％、约1.50％、约125％、约1％、约0.5％、约0.4％、约0.3％、约0.2％、约0.1％、约0.09％、约0.08％、约0.07％、约0.06％、约0.05％、约0.04％、约0.03％、约0.02％、约0.01％、约0.009％、约0.008％、约0.007％、约0.006％、约0.005％、约0.004％、约0.003％、约0.002％、约0.001％、约0.0009％、约0.0008％、约0.0007％、约0.0006％、约0.0005％、约0.0004％、约0.0003％、约或0.0002％，或者约0.0001％w/w、w/v或v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度范围可以为组合物的约0.0001％至约50％、约0.001％至约40％、约0.01％至约30％、约0.02％至约29％、约0.03％至约28％、约0.04％至约27％、约0.05％至约26％、约0.06％至约25％、约0.07％至约24％、约0.08％至约23％、约0.09％至约22％、约0.1％至约21％、约0.2％至约20％、约0.3％至约19％、约0.4％至约18％、约0.5％至约17％、约0.6％至约16％、约0.7％至约15％、约0.8％至约14％、约0.9％至约12％，或约1％至约10％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的浓度范围可以为组合物的约0.001％至约10％、约0.01％至约5％、约0.02％至约4.5％、约0.03％至约4％、约0.04％至约3.5％、约0.05％至约3％、约0.06％至约2.5％、约0.07％至约2％、约0.08％至约1.5％、约0.09％ 至约1％、或约0.1％至约0.9％w/w、w/v或者v/v。
在一些实施方式中，本申请的组合物中提供的TIL的量可以等于或小于约10g、约9.5g、约9.0g、约8.5g、约8.0g、约7.5g、约7.0g、约6.5g、约6.0g、约5.5g、约5.0g、约4.5g、约4.0g、约3.5g、约3.0g、约2.5g、约2.0g、约1.5g、约1.0g、约0.95g、约0.9g、约0.85g、约0.8g、约0.75g、约0.7g、约0.65g、约0.6g、约0.55g、约0.5g、约0.45g、约0.4g、约0.35g、约0.3g、约0.25g、约0.2g、约0.15g、约0.1g、约0.09g、约0.08g、约0.07g、约0.06g、约0.05g、约0.04g、约0.03g、约0.02g、约0.01g、约0.009g、约0.008g、约0.007g、约0.006g、约0.005g、约0.004g、约0.003g、约0.002g、约0.001g、约0.0009g、约0.0008g、约0.0007g、约0.0006g、约0.0005g、约0.0004g、约0.0003g、约0.0002g，或者约0.0001g。
在一些实施方式中，本申请的组合物中提供的TIL的量可以大于约0.0001g、约0.0002g、约0.0003g、约0.0004g、约0.0005g、约0.0006g、约0.0007g、约0.0008g、约0.0009g、约0.001g、约0.0015g、约0.002g、约0.0025g、约0.003g、约0.0035g、约0.004g、约0.0045g、约0.005g、约0.0055g、约0.006g、约0.0065g、约0.007g、约0.0075g、约0.008g、约0.0085g、约0.009g、约0.0095g、约0.01g、约0.015g、约0.02g、约0.025g、约0.03g、约0.035g、约0.04g、约0.045g、约0.05g、约0.055g、约0.06g、约0.065g、约0.07g、约0.075g、约0.08g、约0.085g、约0.09g、约0.095g、约0.1g、约0.15g、约0.2g、约0.25g、约0.3g、约0.35g、约0.4g、约0.45g、约0.5g、约0.55g、约0.6g、约0.65g、约0.7g、约0.75g、约0.8g、约0.85g、约0.9g、约0.95g、约1g、约1.5g、约2g、约2.5g、约3g、约3.5g、约4g、约4.5g、约5g、约5.5g、约6g、约6.5g、约7g、约7.5g、约8g、约8.5g、约9g、约9.5g，或者约10g。
在一些实施方式中，TIL可以单剂量施用。此种施用可以通过注射，例如静脉内注射。在一些实施方式中，TIL可以多剂量施用。剂量可以是每年一次、两次、三次、四次、五次、六次或超过六次。剂量可以是每月一次、每两周一次、每周一次或每2天一次。在一些实施方式中，TIL的施用可以连续施用。
在另一方面，本申请提供一种用到的TIL的培养方法。从受试者组织样品获得的TIL细胞的方法可以是患者手术取得原位肿瘤样本或转移肿瘤样本，重量可以至少约1g，也可以多块组织合并。肿瘤组织在样本运输液，例如可以是商业常用的肿瘤组织运输液、肿瘤组织保存液或肿瘤组织转运液，内约2-8度运输，48小时内处理。组织块可以机械破碎至每块约1-27立方毫米大小，转移入透气培养袋或Grex中，加入T细胞无血清培养基和浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2培养约3-14天。可以将收获的TIL细胞冻存后再复苏，也可以直接收集培养基中细胞，转移入透气培养袋、或 Grex、或Xuri设备，T细胞无血清培养基可以添加本申请的CD3抗体、浓度为300-9000IU/mL(例如可以是1000-9000IU/mL，例如可以是6000IU/mL)的IL-2，活化本申请的TIL一定时间后，添加辐照PBMC(TIL与PBMC按照比率约1:40-约1:400)，扩增培养约3-14天。可以使用细胞处理系统收集培养基中细胞，清洗冻存，并检测。最终产品CD3比例可以大于80％，细胞活率可以大于50％，大于80％的T细胞可以为记忆效应T细胞和效应T细胞。经刺激后可以分泌IFN-γ，和/或可以具有活化T细胞比例上调的特征。
一方面，本申请提供一种药物制剂。在一些实施方式中，其可以包含本申请所述的TIL和/或本申请所述的组合物，与药学上可接受的载体。
一方面，本申请提供了一种药物组合物，可以包含根据本申请应用中所述培养方法获得的TIL，以及可以使淋巴细胞数量减少的物质和/或使所述TIL维持增殖能力的物质。
一方面，本申请提供了一种药物组合物，可以包含环磷酰胺和/或氟达拉滨。
一方面，本申请提供了一种药物组合物，可以包含白介素-2(IL-2)或其变体。
例如，可以以约300至约500mg/m2/天的剂量包含所述环磷酰胺。可以以约50mg/m2/天、约100mg/m2/天、约150mg/m2/天、约200mg/m2/天、约250mg/m2/天、约300mg/m2/天、约350mg/m2/天、约400mg/m2/天、约450mg/m2/天、或约500mg/m2/天的剂量包含环磷酰胺。
例如，可以以约20至约30mg/m2/天的剂量包含所述氟达拉滨。例如，可以以约5mg/m2/天、约10mg/m2/天、约15mg/m2/天、约20mg/m2/天、约25mg/m2/天、或约30mg/m2/天的剂量包含氟达拉滨。
例如，可以以约200,000至约600,000IU/kg/次的剂量包含所述IL-2。可以以约100,000IU/kg/次、约200,000IU/kg/次、约300,000IU/kg/次、约400,000IU/kg/次、约500,000IU/kg/次、或约600,000IU/kg/次的剂量包含IL-2。
一方面，本申请提供了一种试剂盒，可以包含根据本申请的药物组合物。
一方面，本申请提供一种试剂盒，所述试剂盒可以包含本申请所述培养肿瘤浸润淋巴细胞(TIL)方法的T细胞共刺激分子、T细胞生长因子和/或饲养细胞与记载本申请培养肿瘤浸润淋巴细胞(TIL)方法的步骤的说明书。一方面，本申请提供一种试剂盒，所述试剂盒可以包含本申请所述的TIL和/或本申请所述的药物制剂。
一方面，本申请提供一种影响肿瘤细胞生长的方法，可以包括向受试者施用本申请所述的TIL和/或本申请所述的药物制剂。在一些实施方式中，影响肿瘤生长可以包含肿瘤的体积减少到施用前的例如约99％、约95％、约90％、约80％、约70％、约60％、约50％、约40％、约30％、约20％、约19％、约18％、约17％、约16％、约15％、约14％、约13％、 约12％、约11％、约10％、约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％、约1％、约0.5％、约0.4％、约0.3％、约0.2％或约0.1％。
一方面，本申请提供本申请所述的TIL和/或本申请所述的药物制剂在制备药物中的应用，所述药物可以用于预防和/或治疗肿瘤。在一些实施方式中，所述肿瘤选自实体瘤。在一些实施方式中，所述肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。例如，本申请的肿瘤可以是指晚期实体瘤。例如，本申请的肿瘤可以是指不可根治性切除实体瘤。例如，本申请的肿瘤可以是指复发和/或转移性的实体瘤。例如，本申请的肿瘤可以是指不可根治性切除的复发和/或转移性的晚期实体瘤。
一方面，本申请提供一种预防和/或治疗肿瘤的方法，可以包括向受试者施用本申请所述的TIL和/或本申请所述的药物制剂。在一些实施方式中，所述肿瘤选自实体瘤。在一些实施方式中，所述肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。例如，本申请的肿瘤可以是指晚期实体瘤。例如，本申请的肿瘤可以是指不可根治性切除实体瘤。例如，本申请的肿瘤可以是指复发和/或转移性的实体瘤。例如，本申请的肿瘤可以是指不可根治性切除的复发和/或转移性的晚期实体瘤。例如，本申请的受试者可以满足本申请的入选标准的一种、更多种或者全部的入选标准。例如，本申请的受试者可以不具有本申请的排除标准的一种、更多种或者全部的排除标准。例如，本申请的效果可以通过本申请记载的安全性、耐受性评估、疗效评价和/或药效药代动力学评价进行评估。例如，本申请的受试者可以分离出体积≥0.5cm3的组织块(可单一病灶来源或多个病灶合并)用于制备自体肿瘤浸润淋巴细胞。
一方面，本申请提供一种本申请所述的TIL和/或本申请所述的药物制剂，其可以用于预防和/或治疗肿瘤。在一些实施方式中，所述肿瘤选自实体瘤。在一些实施方式中，所述肿瘤可以选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。例如，本申请的肿瘤可以是指晚期实体瘤。例如，本申请的肿瘤可以是指不可根治性切除实体瘤。例如，本申请的肿瘤可以是指复发和/或转移性的实体瘤。例如，本申请的肿瘤可以是指不可根治性切除的复发和/或转移性的晚期实体瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1肿瘤浸润淋巴细胞(TIL)细胞的培养方法
1.1饲养细胞接收及制备
1.1.1单采血接收
记录单采血信息，批号及体积，并复温至室温。
1.1.2PBMC(外周血单个核细胞)手动分离及冻存
使用75％酒精消毒血袋，转移至生物安全柜内。使用无菌剪刀剪开血袋后，将单采血转移至50mL离心管内，使用20mL注射器注入20mL PBS或生理盐水清洗血袋，将洗涤液一并转入50mL离心管内。每个50mL离心管内液体体积可以不超过30mL。将单采血3000g离心10分钟。离心过程中准备6-8支50mL离心管，加入已复温的淋巴细胞分离液(天津灏洋Ficoll)，20mL/支。离心结束后，弃掉上层血浆，使用PBS或生理盐水稀释细胞沉淀，将稀释后的血细胞混合液缓慢滴加上淋巴细胞分离液上层，可以不破坏界面，每管约加25mL样品，可以不超过28mL。
离心使用水平转子，500-600g离心15-30分钟，温度18-22℃，离心结束后得到的白膜层将处于生理盐水及Ficoll的分界面处。吸弃上层血浆及生理盐水，用移液管吸取中间白膜层至另一干净的50mL离心管内。使用PBS或生理盐水稀释收集到的白膜层，600g离心10分钟，室温。离心结束后弃上清，PBS或生理盐水清洗细胞一次，500g离心5分钟，室温。
如红细胞较多，离心结束后可以进行裂红，按照细胞沉淀体积与红细胞裂解液1:2至1:3加入红细胞裂解液，混匀，室温裂解10分钟中，中间轻柔混匀离心管2-3次，保证裂解效果，裂解完成后加入PBS或生理盐水清洗细胞。裂红后清洗细胞两次，400g离心6分钟，最后一次离心前取样计数。
弃上清，基础培养基重悬细胞，调整细胞密度约2-3×107/mL，液面高度可以不超过1厘米，每T225培养瓶中体积可以低于200mL；平铺状态下，X射线辐照50Gy。离心弃上清，根据计数结果冻存细胞，约1-2×108/mL，1-2mL/支；将细胞放入程序降温盒内转移至﹣80℃冰箱内冻存。
1.1.3 PBMC自动分离及冻存
将血袋的管路与cpro分离套件(Cytiva)输入端无菌接管。若血量大于120mL，进行预浓缩步骤，可以将血液体积浓缩至120mL以内。可以使用neatcell程序进行PBMC分离及洗涤，洗涤液为生理盐水，中间体积20mL；重悬液为基础培养基，添加80mL/批。分离后每供者PBMC为一袋100mL，在平铺状态下，液面高度可以不超过1厘米，X射线辐照50Gy。 辐照后取样计数，将3-5名供者PBMC悬液按照0.5:1至1:2的比例混合，使用culture wash程序收集细胞并洗涤三次，洗涤液为生理盐水；设置中间体积及终体积，使得每1×109个细胞不少于2mL；加入等量至2倍冻存液混匀。使用1倍冻存液调整细胞密度约为1×107/mL至2×108/mL，分装20mL/袋，程序降温仪内冻存，液氮保存。
1.2肿瘤组织接收及处理
1.2.1组织接收
接收供者的肿瘤组织及血样，核对样品信息并记录，打印相应样品标签。
1.2.2组织处理及培养
使用75％酒精消毒样品管及采血管，转移至生物安全柜内。根据上述PBMC手动分离及冻存操作程序分离血样中PBMC细胞并进行冻存。取一种具有透气表面的培养瓶和袋子，例如G-Rex100培养瓶(Wilson Wolf Manufacturing)，加入300mL已复温的完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度约为1000～9000IU/mL的IL-2，例如6000IU/mL的IL-2。取数个10厘米培养皿，加入适量培养基，使用无菌眼科镊从样品管中取出肿瘤组织于10厘米培养皿中，培养基量以刚没过肿瘤组织为准，观察组织形态并记录。洗涤组织并更换培养皿。使用眼科剪及眼科镊将进行初步剪切，去除脂肪组织及坏死组织，每块组织块继续剪碎至约27立方毫米大小。取非悬浮肿瘤组织块，使用20mL注射器去除内部活塞后，与培养袋连接，使用移液管将约1g组织块通过注射器转入培养袋内。将培养袋放入二氧化碳培养箱内进行培养。清理剪刀及镊子，并用75％酒精进行初步消毒后，超声清洗后进行灭菌，获得初级TIL。
1.3第一阶段扩增及收获
1.3.1第一阶段扩增
根据细胞生长状态，每3-7天补液或半量换液，保证细胞营养。使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，并添加浓度约为1000～9000IU/mL的IL-2，例如6000IU/mL的IL-2。第一阶段扩增的3-14天，例如可以第3、4、5、6、7、8、9、10、11、12、13或14天时取样计数，若细胞数目处于5×105至5×108之间时进入下述第一阶段扩增的收获步骤。
1.3.2第一阶段扩增的收获
收集第一阶段扩增结束细胞，离心，弃去培养基，使用PBS或生理盐水洗涤细胞一次， 获得经第一阶段扩增的TIL，并取样计数留取约5×105至2×108细胞量进入下述第一阶段扩增步骤；取约5×105细胞量可以进行质量控制检测；其余细胞量加入等量2倍冻存液冻存。
1.4第二阶段扩增
1.4.1第二阶段扩增的TIL活化
取5×105至2×108的第一阶段扩增的细胞量，使用完全培养基，完全培养基可以任意地选用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基，并可以添加必须氨基酸及抗生素，调整细胞密度为5×105至2×106/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如6000IU/mL的IL-2。
1.4.2第二阶段扩增的扩大培养
第二阶段扩增加入OKT3和IL-2后的若干时间Tn以后(Tn可以取0小时到14天)，复苏1-5名供者混合的饲养细胞；将活化的TIL细胞，组织块及饲养细胞转入G-Rex100培养瓶或者透气袋内，补充完全培养基，每1-3天取样计数，并根据细胞状态补液或半量换液直至细胞总数大于1×109或第二阶段扩增培养达14天，终止培养。
1.4.3肿瘤浸润淋巴细胞的收获
取第二阶段扩增的细胞，离心后弃去培养基上清，并使用PBS或生理盐水或复方电解质溶液清洗三次，获得经第二阶段扩增的TIL，第三次清洗时取样计数，根据计数结果，最后一次离心后弃上清，取3×106细胞送质量控制检测；其余全部细胞加入冻存液，调整细胞密度1-3×108/mL冻存。
1.5肿瘤浸润淋巴细胞的应用
可以将复苏后的治疗性肿瘤浸润淋巴细胞给予受试者静脉滴注。
实施例2饲养细胞不同添加时间培养的TIL增殖能力对比
在实施例1的1.4的第二阶段扩增的TIL活化中，第二阶段扩增加入OKT3和IL-2后的若干时间Tn以后(Tn可以取0小时到14天)，将饲养细胞加入肿瘤浸润淋巴细胞培养袋中。本实施例中Tn选取0小时、6小时、12小时、24小时、48小时、72小时、5天、7天、和9天获得饲养细胞不同添加时间培养的TIL，并进行细胞计数的对比试验。
饲养细胞不同添加时间培养的TIL的增殖能力分析如图1所示。饲养细胞不同添加时间培养TIL的各组图中纵坐标的数值表示，第二阶段扩增结束后相比于第二阶段扩增开始前，TIL细胞数量扩增至的扩增倍数。4名供者来源的TIL增殖结果显示，加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，增殖能力弱于加入OKT3和IL-2后的24小 时或48小时后加饲养细胞培养的TIL。
实施例3饲养细胞不同添加时间培养的TIL流式检测对比
在实施例1的1.4的第二阶段扩增的TIL活化中，第二阶段扩增加入OKT3和IL-2后的若干时间Tn以后(Tn可以取0小时到14天)，将饲养细胞加入肿瘤浸润淋巴细胞培养袋中。本实施例中Tn选取0小时、6小时、12小时、24小时、48小时、72小时、5天、7天、和9天获得饲养细胞不同添加时间培养的TIL，并进行流式检测的对比试验。
TIL流式检测试验材料的来源
转录因子缓冲组(Transcription Factor Buffer Set)，厂家BD，货号562574；V底96孔板，厂家Corning，货号3894；流式管，厂家Corning，货号352052。
本实施例流式抗体购自BD或Biolegend。将每组1×105至5×105个细胞样品，加入流式管或V底96孔板内。600g离心3分钟，弃上清。PBS清洗一次，流式管1mL/管，96孔板250μL/孔，弃上清。加入配制好的抗体工作液进行细胞表面染色，抗体(BD或Biolegend)浓度为1:100至1:200，含活性检测染料1:10000。流式管100μL/管，96孔板50μL/孔染色，2-8℃避光孵育30分钟。染色过程中配制转录因子染色所需试剂：使用转录因子缓冲组(BD，Transcription Factor Buffer Set)稀释4×固定破膜液(BD，Fixation/Permeabilization)为1×工作液A；使用双蒸水稀释5×通透清洗液(BD，Perm/Wash Buffer)为1×工作液B，四度预冷待用。染色结束后加入适量PBS清洗细胞2次(96孔板250μL/次，流式管1mL/次)，600g离心3分钟，离心后弃上清。细胞固定、破膜：充分重悬细胞，加入适量(96孔板100μL/孔，流式管1mL/管)1×工作液A进行固定破膜，2-8℃避光孵育40-50分钟。固定破膜结束，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。使用1×工作液B配制胞内抗体，抗体浓度为1:100至1:200，96孔板50μL/孔，流式管100μL/管，2-8℃避光染色30分钟。染色结束后，加入1×工作液B清洗细胞(96孔板250μL/次，流式管2mL/次)，2-8℃离心，350g离心6分钟，清洗两次。表面染色结束后，PBS清洗细胞一次(96孔板250μL/次，流式管1mL/次)，室温600g离心3分钟，离心后弃上清。使用100-500μL PBS重悬细胞，进行流式上机检测。
饲养细胞不同添加时间培养的TIL的流式结果分析如图2到图8所示。
图2到图3表示加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA-CCR7+中心记忆T细胞(Tcm)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高中心记忆T细胞的比例。
图4表示加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4+CD25+Foxp3+调节性T细胞(Treg)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
图5到图6表示加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如PD1+、LAG3+和/或CD28+细胞比例更高。
图7表示加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103+CD39+肿瘤特异性T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的肿瘤特异性T细胞的比例。
图8表示加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1+干细胞样T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的干细胞样T细胞的比例。
实施例4饲养细胞不同添加时间培养的TIL的结果统计
在实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×105至2×106/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的0小时、24小时、48小时以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的结果。
增殖能力检测
对于上述饲养细胞不同添加时间培养获得的TIL进行细胞计数。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的相对增殖能力。
图9显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。相比于加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，加入OKT3和IL-2后的24小时或48小时后加饲养细 胞培养的TIL增殖能力显著增强。
流式检测TIL细胞组成
对于上述饲养细胞不同添加时间培养获得的TIL群进行流式检测。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的细胞组成比例。
流式检测的试验流程可以参照本申请实施例3的内容。
图10显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RA-CCR7+中心记忆T细胞(Tcm)比例结果图。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高CD8+中和/或CD4+中的中心记忆T细胞的比例。
图11显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的TCF1+干细胞样T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高CD8+中的干细胞样T细胞的比例。
图12显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4+CD25+Foxp3+调节性T细胞(Treg)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
图13显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(PD1+)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8+中和/或CD4+中的PD1+细胞比例更高。
图14显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD103+CD39+肿瘤特异性T细胞比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的CD8+中和/或CD4+中的肿瘤特异性T细胞的比例。
图15显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD28+)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例， 例如CD8+CD28+细胞比例更高。
图16显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(41BB+)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8+中和/或CD4+中的41BB+细胞比例更高。
图17显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的活化T细胞(CD25+)比例。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的活化T细胞的比例，例如CD8+中和/或CD4+中的CD25+细胞比例更高。
胞内因子表达检测
试验准备
配制胞内因子表达检测所需培养基：取T细胞培养基，按照体积比1:500添加CD107a抗体(BD)。
检测步骤
取各个试验组的TIL离心后，使用600μL上述胞内因子表达检测所需培养基重悬为1×106个细胞/mL，加入96孔板内，100μL/孔，置于37℃培养箱孵育过夜。
孵育结束后，200μL/孔PBS洗涤一次，600g离心3分钟，弃上清。配制抗体混合工作液进行细胞表面染色CD3/CD4/CD8(BD)，抗体浓度为1:100，viability(1:10000)，50μL/组染色，2-8℃避光孵育30分钟。染色结束后清洗细胞，使用PBS重悬，进行流式上机检测。
图18显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的胞内因子表达检测结果。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的胞内因子表达能力。例如，更高的CD3+中、CD8+中和/或CD4+中的CD107a表达能力。
细胞因子分泌检测
细胞因子分泌检测方法可以参照细胞因子检测试剂盒(BD)的说明书，将人Th1/Th2/Th17细胞因子标准品冻干粉(BD)使用2mL Assay Diluent稀释液(BD)复溶(标准品原液各细胞因子浓度均为5000pg/mL)并按顺序：1:2，1:4，1:8，1:16，1:32，1:64，1:128，1:256，1:512，1:1024梯度稀释，标记为“标准品管”。取1管仅含有Assay Diluent稀释液作为参照。按照2μL/Beads/孔加入每种Capture Beads(BD)，然后按照10μL/孔加入PE Detection Reagent检测试剂(BD)并混合配制为混合物(mix)，按照22μL/孔加入V底96孔板内，随后按照 10μL/孔加入各标准品和试验组的上清并混合，室温下避光孵育3小时。
孵育结束，每孔加入200μL Wash Buffer(BD)，500g离心3分钟。离心结束，每孔加入100μL Wash Buffer(BD)重悬，进行流式分析。
图19显示的是，加入OKT3和IL-2的0小时、24小时或48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞因子分泌检测结果。结果显示，24小时或48小时后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的细胞因子分泌能力。例如，更高的TNF-α分泌能力、或更高的IFN-γ分泌能力。
实施例5饲养细胞不同添加时间培养的TIL的结果统计
在实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×105至2×106/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的0小时、6小时、12小时、24小时、48小时、72小时、或5天以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，例如1:200，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的结果。
增殖能力检测
对于上述饲养细胞不同添加时间培养获得的TIL进行细胞计数。
图20显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞增殖能力结果图。相比于加入OKT3和IL-2后的0小时后(即同时)添加饲养细胞培养的TIL，加入OKT3和IL-2后的12小时或更多时间后加饲养细胞培养的TIL增殖能力显著增强。
流式检测TIL细胞组成
对于上述饲养细胞不同添加时间培养获得的TIL群进行流式检测。
不同供者肿瘤来源的TIL作为各自不同的批次；将各个批次的加入OKT3和IL-2同时(0h组)加入饲养细胞的试验组的数据作为基准1，将同批次其它时间点试验组的数据进行标准化处理，统计各试验组在第二阶段扩增相对于0h组的细胞组成比例。
流式检测的试验流程可以参照本申请实施例3的内容。
图21显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD8+T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加 饲养细胞培养的TIL，具有更高的CD8+T细胞的比例。
图22显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD45RO+CD62L+T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的记忆T细胞(Tcm，CD45RO+CD62L+)的比例。
图23显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的NK T细胞比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更高的NK T细胞的比例。
图24显示的是，加入OKT3和IL-2的0小时、6小时、12小时、24小时、48小时、72小时、或5天后添加饲养细胞培养的TIL，培养所得的TIL细胞的CD4+CD25+Foxp3+调节性T细胞(Treg)比例。结果显示，加入OKT3和IL-2后的12小时或更多时间后添加饲养细胞培养的TIL，相比同时添加饲养细胞培养的TIL，具有更少的调节性T细胞的比例。
实施例6本申请培养的TIL的杀伤能力检测
对于实施例1的1.4的第二阶段扩增的TIL活化中，取第一阶段扩增的细胞量，调整细胞密度为5×105至2×106/mL，于悬浮24孔培养板内，1mL/孔，添加CD3抗体，例如OKT3约30ng/mL，添加浓度约为1000～9000IU/mL的IL-2，例如3000或6000IU/mL的IL-2。加入上述OKT3和IL-2后的12小时至14天后，例如48小时以后，将饲养细胞加入肿瘤浸润淋巴细胞的培养环境中。其中，TIL与饲养细胞可以按照比率1:40-1:400加入，第二阶段扩增培养约9-14天后收集全部细胞，检测和统计培养所得TIL的细胞杀伤能力检测。
细胞准备
准备用于检测的各个试验组获得的TIL和用于共培养的靶细胞(例如A375黑色素瘤细胞和/或Hela宫颈癌细胞)。
检测步骤
用CFSE(5(6)-Carboxyfluorescein diacetate N-succinimidyl ester，Sigma，21888-25MG-F)标记肿瘤细胞：用PBS清洗肿瘤细胞，重悬肿瘤细胞于500μL的PBS中；将CFSE加入500μL的PBS中，与500μL的肿瘤细胞PBS重悬液混合，至CFSE的终浓度为0.5μmol/L。37℃孵育6分钟后，加含10％FBS的培养基清洗，600g离心5分钟，用X-vivo 15培养基或其它商用的T细胞培养基，例如Stem Cell，Lonza，Thermo，美天旎等品牌的T细胞培养基重悬肿瘤细胞浓度为1×106个细胞/mL。对各个试验组的TIL群600g离心5分钟，按照效靶比 (TIL细胞与肿瘤细胞的比例)3:1重悬TIL细胞(即重悬TIL细胞浓度为3×106个细胞/mL)。于U底96孔板(Corning)中加入肿瘤细胞和TIL细胞各100μL，每组设置三个复孔。同时设置一组只包含肿瘤细胞的对照组。将孔板200g离心1分钟，置于37℃孵育4小时至过夜。其中，TIL与肿瘤细胞共培养时，可以不加激活TIL细胞的物质作为无激活组，或者加入transACT(Miltenyi，包含CD3抗体和CD28抗体的纳米基质材料)作为激活组。
孵育完成后，600g离心3分钟，弃上清，每孔加入20μL胰酶，37℃培养箱内孵育3-5分钟消化肿瘤细胞，消化完成后加入180μL含10％FBS的培养基终止消化。将Dapi(碧云天，C0060)用1：100稀释，然后每孔加入20μL稀释后的Dapi。进行流式上机检测。
杀伤率％＝Dapi+CFSE+细胞数/总CFSE+×100％，或杀伤率可以通过Dapi+细胞数/总肿瘤细胞数表示。
图25显示的是，加入OKT3和IL-2的48小时后添加饲养细胞培养的TIL，培养所得的TIL细胞的细胞杀伤能力结果。结果显示，加入OKT3和IL-2后的48小时后添加饲养细胞培养的TIL均具有显著的肿瘤细胞杀伤能力，例如黑色素瘤和/或宫颈肿瘤。
实施例7
本申请肿瘤浸润淋巴细胞的安全性和有效性
在向受试者施用TIL之前，可以对受试者的免疫系统进行处理，例如可以在向受试者施用所述培养方法获得的TIL之前第4天至第2天，向所述受试者施用环磷酰胺和/或氟达拉滨。例如，以约300至500mg/m2/天的剂量施用环磷酰胺，以20至30mg/m2/天的剂量施用氟达拉滨。例如，本申请的TIL可以以5×109至1×1011的细胞数向所述受试者一次性静脉施用。在施用TIL之后8至16小时，可以以200,000至600,000IU/kg/次的剂量向所述受试者第一次施用IL-2(或其变体)，施用IL-2后可以暂停一次施用IL-2，根据受试者的耐受情况可以每12小时向所述受试者施用一次IL-2。可以向受试者施用10次IL-2，可以根据受试者的耐受情况减少IL-2施用次数。对于本申请的肿瘤浸润淋巴细胞(TIL)的治疗方案，评估肿瘤患者的疗效和安全性。
入选标准


排除标准

临床评估方案：


临床数据结果显示，两名宫颈癌的患者疗效评估为部分缓解(PR)。
例如，对于一名晚期(复发和转移)宫颈癌患者，过往经过根治性切除术，4个周期的多西紫杉醇/顺铂，以及参与过双特异性抗体临床试验；其ECOG评分标准(评估健康状况和对治疗耐受能力的指标)为1分。
对于安全性，该患者经过本申请的治疗方法治疗后，未出现与本申请疗法相关的安全性事件，并且在治疗后均已康复。对于有效性，如图26A和26B显示的，影像学结果表明本申请的治疗方法在临床上体现了优异的治疗效果，两个病灶均达到了部分缓解(PR)的情况。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本文所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (146)

  1. 一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：使源自肿瘤组织的TIL经过至少一个阶段的体外扩增，其中在单个体外扩增阶段中，使经体外扩增和/或未经体外扩增的TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
  2. 根据权利要求1所述的应用，所述培养方法包含使源自肿瘤组织的TIL经过至少两个阶段的体外扩增，且在第二个阶段的体外扩增中，使经体外扩增的TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养。
  3. 根据权利要求1-2中任一项所述的应用，与在单个体外扩增阶段中使TIL在与T细胞激活剂和/或T细胞生长因子接触同时与饲养细胞共培养相比，在单个体外扩增阶段中使TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养获得的TIL显示出改善的TIL特性。
  4. 根据权利要求3所述的应用，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，和提高的T细胞受体
    (TCR)克隆多样性。
  5. 根据权利要求4所述的应用，所述改善的T细胞亚群比例包含选自以下组的一种或多种：增加的中心记忆T细胞比例，降低的调节性T细胞的比例，增加的活化T细胞比例，增加的肿瘤特异性T细胞比例，和增加的干细胞样T细胞比例。
  6. 根据权利要求1-5中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之前，使所述受试者的淋巴细胞数量减少。
  7. 根据权利要求1-6中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之前，向所述受试者施用环磷酰胺和/或氟达拉滨。
  8. 根据权利要求1-7中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之前第4天至第2天，向所述受试者施用环磷酰胺和/或氟达拉滨。
  9. 根据权利要求1-8中任一项所述的应用，以约300至约500mg/m2/天的剂量向受试者施用环磷酰胺。
  10. 根据权利要求1-9中任一项所述的应用，向受试者施用环磷酰胺持续3天。
  11. 根据权利要求1-10中任一项所述的应用，以约20至约30mg/m2/天的剂量向受试者施用氟达拉滨。
  12. 根据权利要求1-11中任一项所述的应用，向受试者施用氟达拉滨持续3天。
  13. 根据权利要求1-12中任一项所述的应用，还包含以约5×109至约1×1011的细胞数向受试者施用所述培养方法获得的TIL。
  14. 根据权利要求1-13中任一项所述的应用，还包含向受试者一次性静脉输注施用所述培养方法获得的TIL。
  15. 根据权利要求1-14中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之后，使所述TIL维持增殖能力。
  16. 根据权利要求1-15中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之后，向所述受试者施用白介素-2(IL-2)或其变体。
  17. 根据权利要求1-16中任一项所述的应用，以约200,000至约600,000IU/kg/次的剂量向所述受试者施用IL-2。
  18. 根据权利要求1-17中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之后8至16小时，向所述受试者第一次施用IL-2。
  19. 根据权利要求1-18中任一项所述的应用，在向受试者第一次施用IL-2后，每12小时向所述受试者施用一次IL-2。
  20. 根据权利要求1-19中任一项所述的应用，在向受试者第一次施用IL-2的24小时后，根据受试者的耐受情况第二次施用所述IL-2。
  21. 根据权利要求1-20中任一项所述的应用，向受试者施用10次或更少次的IL-2。
  22. 根据权利要求1-21中任一项所述的应用，所述培养方法包含：使所述源自肿瘤组织的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使所述TIL与所述饲养细胞共培养。
  23. 根据权利要求22所述的应用，所述第一阶段体外扩增进行至少约7天。
  24. 根据权利要求22-23中任一项所述的应用，所述第一阶段体外扩增进行约7天至约14天。
  25. 根据权利要求22-24中任一项所述的应用，所述第二阶段体外扩增进行至少约7天。
  26. 根据权利要求22-25中任一项所述的应用，所述第二阶段体外扩增进行约7天至约14天。
  27. 根据权利要求1-26中任一项所述的应用，使所述TIL在与T细胞激活剂和/或T细胞生长因子接触至少约2小时之后与所述饲养细胞共培养。
  28. 根据权利要求1-27中任一项所述的应用，使所述TIL在与所述T细胞激活剂和/或 所述T细胞生长因子接触约6小时至约72小时之后与所述饲养细胞共培养。
  29. 根据权利要求1-28中任一项所述的应用，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约12小时至约48小时之后与所述饲养细胞共培养。
  30. 根据权利要求1-29中任一项所述的应用，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
  31. 根据权利要求1-30中任一项所述的应用，所述饲养细胞包含抗原呈递细胞。
  32. 根据权利要求1-31中任一项所述的应用，所述饲养细胞包含选自以下组的一种或多种：外周单个核细胞，树突状细胞和人工抗原呈递细胞。
  33. 根据权利要求1-32中任一项所述的应用，所述饲养细胞为外周单个核细胞。
  34. 根据权利要求1-33中任一项所述的应用，所述饲养细胞为经过辐照的饲养细胞。
  35. 根据权利要求1-34中任一项所述的应用，所述TIL与所述饲养细胞共培养包含使所述饲养细胞的表面与所述TIL的表面相接触。
  36. 根据权利要求1-35中任一项所述的应用，所述TIL与所述饲养细胞共培养包含将所述饲养细胞添加至所述TIL的细胞培养基中。
  37. 根据权利要求1-36中任一项所述的应用，以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中。
  38. 根据权利要求1-37中任一项所述的应用，所述培养方法还包含：使源自肿瘤组织的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述TIL与所述T细胞激活剂接触。
  39. 根据权利要求38所述的应用，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞激活剂接触。
  40. 根据权利要求1-39中任一项所述的应用，使所述源自肿瘤组织的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第二阶段体外扩增中，使所述TIL与所述T细胞激活剂接触。
  41. 根据权利要求1-40中任一项所述的应用，所述T细胞激活剂包含选自以下组的一种或多种：分化簇80(CD80)、CD86、CD276、4-1BB配体(4-1BBL)、CD27、CD30、CD134、CD275、CD40、CD258、以及它们的功能活性片段。
  42. 根据权利要求1-41中任一项所述的应用，所述T细胞激活剂包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、疱疹病毒进入介质(HVEM)、CD40L、OX40和4- 1BB。
  43. 根据权利要求1-42中任一项所述的应用，所述T细胞激活剂包含CD3激动剂和/或CD28激动剂。
  44. 根据权利要求1-43中任一项所述的应用，所述T细胞激活剂包含CD3激动剂。
  45. 根据权利要求1-44中任一项所述的应用，所述T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
  46. 根据权利要求1-45中任一项所述的应用，所述T细胞激活剂包含CD28激动剂。
  47. 根据权利要求1-46中任一项所述的应用，所述T细胞激活剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段。
  48. 根据权利要求1-47中任一项所述的应用，所述使TIL与所述T细胞激活剂接触包含选自以下组的一种或多种方式：(1)将所述T细胞激活剂添加至所述TIL的细胞培养基中；(2)将表达所述T细胞激活剂的工程化细胞添加至所述TIL的细胞培养基中；和(3)将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  49. 根据权利要求48所述的应用，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为至少约30ng/mL。
  50. 根据权利要求48-49中任一项所述的应用，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为约30ng/mL-约300ng/mL。
  51. 根据权利要求48-50中任一项所述的应用，所述固相介质的直径为约500纳米至约10微米。
  52. 根据权利要求48-51中任一项所述的应用，所述固相介质的直径为约1纳米至约500纳米。
  53. 根据权利要求51-52中任一项所述的应用，所述固相介质的直径通过透射电子显微镜测量。
  54. 根据权利要求48-53中任一项所述的应用，所述固相介质包含聚合物。
  55. 根据权利要求48-54中任一项所述的应用，每mg所述固相介质中包含的每一种所述T细胞激活剂的量各自独立地至少为约25μg。
  56. 根据权利要求48-55中任一项所述的应用，以约2:1-约1:2的所述固相介质与所述TIL的比例，将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  57. 根据权利要求48-56中任一项所述的应用，以约1:100-约1:2000的所述固相介质与所述TIL的比例，将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  58. 根据权利要求1-57中任一项所述的应用，所述培养方法还包含：使源自肿瘤组织的TIL经过至少一个阶段的体外扩增，其中，在至少一个阶段的所述体外扩增中，使所述TIL与所述T细胞生长因子接触。
  59. 根据权利要求58所述的应用，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞生长因子接触。
  60. 根据权利要求58-59中任一项所述的应用，在单个阶段的所述体外扩增中，使所述TIL与所述T细胞激活剂以及所述T细胞生长因子接触。
  61. 根据权利要求1-60中任一项所述的应用，使所述源自肿瘤组织的TIL经过第一阶段体外扩增和第二阶段体外扩增，且在所述第一阶段体外扩增和第二阶段体外扩增中，使所述TIL与T细胞生长因子接触。
  62. 根据权利要求61所述的应用，在所述第二阶段体外扩增中，使所述TIL基本上同时与所述T细胞激活剂以及所述T细胞生长因子接触。
  63. 根据权利要求1-62中任一项所述的应用，所述T细胞生长因子选自以下组的一种或多种：IL-2、IL-7、IL-12、IL-15、IL-21、γ干扰素、以及它们的功能活性片段。
  64. 根据权利要求1-63中任一项所述的应用，所述T细胞生长因子包含IL-2和/或其功能活性片段。
  65. 根据权利要求1-64中任一项所述的应用，所述TIL与所述T细胞生长因子接触包含将所述T细胞生长因子添加至所述TIL的细胞培养基中。
  66. 根据权利要求1-65中任一项所述的应用，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
  67. 根据权利要求1-66中任一项所述的应用，所述源自肿瘤组织的TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL、源自肿瘤切除术的TIL、源自活体组织检查的TIL和源自冷冻保存后复苏的TIL。
  68. 根据权利要求67所述的应用，所述碎片的体积为约1立方毫米至约27立方毫米。
  69. 根据权利要求1-68中任一项所述的应用，其中，所述肿瘤为实体瘤。
  70. 根据权利要求1-69中任一项所述的应用，其中，所述肿瘤选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
  71. 一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗 肿瘤，通过以下培养方法获得所述TIL：
    (A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
    (B)使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
  72. 一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：
    (A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
    (B)使所述第二TIL群在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养，其中，经所述步骤(B)得到第三TIL群。
  73. 根据权利要求72所述的应用，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
  74. 根据权利要求72-73中任一项所述的应用，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
  75. 根据权利要求71-74中任一项所述的应用，所述步骤(A)进行约7天至约14天。
  76. 根据权利要求71-75中任一项所述的应用，所述步骤(B)进行约7天至约14天。
  77. 一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：
    (A)使源自肿瘤组织且未经体外扩增的第一TIL群与T细胞生长因子接触，其中，经所述步骤(A)得到第二TIL群；
    (B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步骤(B)得到第三TIL群；
    (C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
  78. 一种肿瘤浸润淋巴细胞(TIL)在制备药物中的应用，所述药物用于预防和/或治疗肿瘤，通过以下培养方法获得所述TIL：
    (A)复苏和/或继续培养体外TIL群得到第二TIL群，其中，所述体外TIL群包含由源自肿瘤组织且未经体外扩增的第一TIL群体外扩增获得的TIL群；
    (B)使所述第二TIL群与T细胞激活剂和/或T细胞生长因子接触，其中，经所述步 骤(B)得到第三TIL群；
    (C)使所述第三TIL群与饲养细胞共培养，其中，经所述步骤(C)得到第四TIL群。
  79. 根据权利要求78所述的应用，所述体外TIL群包含由所述第一TIL群接触T细胞生长因子获得的TIL群。
  80. 根据权利要求78-79中任一项所述的应用，所述体外TIL群包含由所述第一TIL群冷冻保存获得的TIL群。
  81. 根据权利要求77-80中任一项所述的应用，所述步骤(A)进行约7天至约14天。
  82. 根据权利要求77-81中任一项所述的应用，所述步骤(B)进行约0天至约8天。
  83. 根据权利要求77-82中任一项所述的应用，所述步骤(C)进行约5天至约14天。
  84. 根据权利要求71-83中任一项所述的应用，与在单个体外扩增阶段中使TIL在与T细胞激活剂和/或T细胞生长因子接触同时与饲养细胞共培养相比，在单个体外扩增阶段中使TIL在与T细胞激活剂和/或T细胞生长因子接触一定时间之后与饲养细胞共培养获得的TIL显示出改善的TIL特性。
  85. 根据权利要求84所述的应用，所述改善的TIL特性包含选自以下组的一种或多种：增加的TIL细胞数量和扩增能力，增加的活细胞比例，增加的存续能力，改善的T细胞亚群比例，提高的细胞因子分泌能力，提高的肿瘤细胞杀伤能力，和提高的T细胞受体(TCR)克隆多样性。
  86. 根据权利要求85所述的应用，所述改善的T细胞亚群比例包含选自以下组的一种或多种：增加的中心记忆T细胞比例，降低的调节性T细胞的比例，增加的活化T细胞比例，增加的肿瘤特异性T细胞比例，和增加的干细胞样T细胞比例。
  87. 根据权利要求71-86中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之前，使所述受试者的淋巴细胞数量减少。
  88. 根据权利要求71-87中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之前，向所述受试者施用环磷酰胺和/或氟达拉滨。
  89. 根据权利要求71-88中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之前第4天至第2天，向所述受试者施用环磷酰胺和/或氟达拉滨。
  90. 根据权利要求71-89中任一项所述的应用，以约300至约500mg/m2/天的剂量向受试者施用环磷酰胺。
  91. 根据权利要求71-90中任一项所述的应用，向受试者施用环磷酰胺持续3天。
  92. 根据权利要求71-91中任一项所述的应用，以约20至约30mg/m2/天的剂量向受试者施用氟达拉滨。
  93. 根据权利要求71-92中任一项所述的应用，向受试者施用氟达拉滨持续3天。
  94. 根据权利要求71-93中任一项所述的应用，还包含以约5×109至约1×1011的细胞数向受试者施用所述培养方法获得的TIL。
  95. 根据权利要求71-94中任一项所述的应用，还包含向受试者一次性静脉输注施用所述培养方法获得的TIL。
  96. 根据权利要求71-95中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之后，使所述TIL维持增殖能力。
  97. 根据权利要求71-96中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之后，向所述受试者施用白介素-2(IL-2)或其变体。
  98. 根据权利要求71-97中任一项所述的应用，以约200,000至约600,000IU/kg/次的剂量向所述受试者施用IL-2。
  99. 根据权利要求71-98中任一项所述的应用，还包含在向受试者施用所述培养方法获得的TIL之后8至16小时，向所述受试者第一次施用IL-2。
  100. 根据权利要求71-99中任一项所述的应用，在向受试者第一次施用IL-2后，每12小时向所述受试者施用一次IL-2。
  101. 根据权利要求71-100中任一项所述的应用，在向受试者第一次施用IL-2的24小时后，根据受试者的耐受情况第二次施用所述IL-2。
  102. 根据权利要求71-101中任一项所述的应用，向受试者施用10次或更少次的IL-2。
  103. 根据权利要求71-102中任一项所述的应用，使所述TIL在与T细胞激活剂和/或T细胞生长因子接触至少约2小时之后与所述饲养细胞共培养。
  104. 根据权利要求71-103中任一项所述的应用，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时至约72小时之后与所述饲养细胞共培养。
  105. 根据权利要求71-104中任一项所述的应用，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约12小时至约48小时之后与所述饲养细胞共培养。
  106. 根据权利要求71-105中任一项所述的应用，使所述TIL在与所述T细胞激活剂和/或所述T细胞生长因子接触约6小时、约12小时、约24小时、约48小时或约72小时之后与所述饲养细胞共培养。
  107. 根据权利要求71-106中任一项所述的应用，所述饲养细胞包含抗原呈递细胞。
  108. 根据权利要求71-107中任一项所述的应用，所述饲养细胞包含选自以下组的一种或多种：外周单个核细胞，树突状细胞和人工抗原呈递细胞。
  109. 根据权利要求71-108中任一项所述的应用，所述饲养细胞为外周单个核细胞。
  110. 根据权利要求71-109中任一项所述的应用，所述饲养细胞为经过辐照的饲养细胞。
  111. 根据权利要求71-110中任一项所述的应用，所述TIL与所述饲养细胞共培养包含使所述饲养细胞的表面与所述TIL的表面相接触。
  112. 根据权利要求71-111中任一项所述的应用，所述TIL与所述饲养细胞共培养包含将所述饲养细胞添加至所述TIL的细胞培养基中。
  113. 根据权利要求71-112中任一项所述的应用，以约40:1-约400:1的所述饲养细胞与所述TIL的比例，将所述饲养细胞添加至所述TIL的细胞培养基中。
  114. 根据权利要求71-113中任一项所述的应用，所述T细胞激活剂包含选自以下组的一种或多种：分化簇80(CD80)、CD86、CD276、4-1BB配体(4-1BBL)、CD27、CD30、CD134、CD275、CD40、CD258、以及它们的功能活性片段。
  115. 根据权利要求71-114中任一项所述的应用，所述T细胞激活剂包含选自以下组的一种或多种靶点的激动剂：CD3、CD28、疱疹病毒进入介质(HVEM)、CD40L、OX40和4-1BB。
  116. 根据权利要求71-115中任一项所述的应用，所述T细胞激活剂包含CD3激动剂和/或CD28激动剂。
  117. 根据权利要求71-116中任一项所述的应用，所述T细胞激活剂包含CD3激动剂。
  118. 根据权利要求71-117中任一项所述的应用，所述T细胞激活剂包含抗CD3的抗体和/或其抗原结合片段。
  119. 根据权利要求71-118中任一项所述的应用，所述T细胞激活剂包含CD28激动剂。
  120. 根据权利要求71-119中任一项所述的应用，所述T细胞激活剂包含抗CD28的抗体和/或其抗原结合片段、CD80和/或其功能活性片段和/或CD86和/或其功能活性片段。
  121. 根据权利要求71-120中任一项所述的应用，所述使TIL与所述T细胞激活剂接触包含选自以下组的一种或多种方式：(1)将所述T细胞激活剂添加至所述TIL的细胞培 养基中；(2)将表达所述T细胞激活剂的工程化细胞添加至所述TIL的细胞培养基中；和(3)将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  122. 根据权利要求121所述的应用，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为至少约30ng/mL。
  123. 根据权利要求121-122中任一项所述的应用，每一种所述T细胞激活剂在所述TIL的细胞培养基中的初始浓度各自独立地为约30ng/mL-约300ng/mL。
  124. 根据权利要求121-123中任一项所述的应用，所述固相介质的直径为约500纳米至约10微米。
  125. 根据权利要求121-124中任一项所述的应用，所述固相介质的直径为约1纳米至约500纳米。
  126. 根据权利要求124-125中任一项所述的应用，所述固相介质的直径通过透射电子显微镜测量。
  127. 根据权利要求121-126中任一项所述的应用，所述固相介质包含聚合物。
  128. 根据权利要求121-127中任一项所述的应用，每mg所述固相介质中包含的每一种所述T细胞激活剂的量各自独立地至少为约25μg。
  129. 根据权利要求121-128中任一项所述的应用，以约2:1-约1:2的所述固相介质与所述TIL的比例，将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  130. 根据权利要求121-129中任一项所述的应用，以约1:100-约1:2000的所述固相介质与所述TIL的比例，将包含所述T细胞激活剂的固相介质添加至所述TIL的细胞培养基中。
  131. 根据权利要求71-130中任一项所述的应用，使所述TIL基本上同时与所述T细胞激活剂以及所述T细胞生长因子接触。
  132. 根据权利要求71-131中任一项所述的应用，所述T细胞生长因子选自以下组的一种或多种：IL-2、IL-7、IL-12、IL-15、IL-21、γ干扰素、以及它们的功能活性片段。
  133. 根据权利要求71-132中任一项所述的应用，所述T细胞生长因子包含IL-2和/或其功能活性片段。
  134. 根据权利要求71-133中任一项所述的应用，所述TIL与所述T细胞生长因子接触包含将所述T细胞生长因子添加至所述TIL的细胞培养基中。
  135. 根据权利要求71-134中任一项所述的应用，每一种所述T细胞生长因子在所述TIL的细胞培养基中的初始浓度各自独立地为至少约300IU/mL。
  136. 根据权利要求71-135中任一项所述的应用，所述源自肿瘤组织的TIL选自以下组：源自肿瘤组织的碎片的TIL、源自淋巴转移灶的碎片的TIL、源自胸腔积液的TIL、源自腹腔积液的TIL、源自肿瘤切除术的TIL、源自活体组织检查的TIL和源自冷冻保存后复苏的TIL。
  137. 根据权利要求136所述的应用，所述碎片的体积为约1立方毫米至约27立方毫米。
  138. 根据权利要求71-137中任一项所述的应用，其中，所述肿瘤为实体瘤。
  139. 根据权利要求71-138中任一项所述的应用，其中，所述肿瘤选自以下组的一种或多种：黑色素瘤、卵巢癌、宫颈癌、肺癌、膀胱癌、乳腺癌、头颈癌、胰腺癌、肝癌、胃癌、结直肠癌、和肾癌。
  140. 一种药物组合物，包含根据权利要求1-139中任一项所述应用中所述培养方法获得的TIL，以及使淋巴细胞数量减少的物质和/或使所述TIL维持增殖能力的物质。
  141. 根据权利要求140所述的药物组合物，所述使淋巴细胞耗竭的物质包含环磷酰胺和/或氟达拉滨。
  142. 根据权利要求140-141中任一项所述的药物组合物，所述使所述TIL维持增殖能力的物质包含白介素-2(IL-2)或其变体。
  143. 根据权利要求140-142中任一项所述的药物组合物，以约300至约500mg/m2/天的剂量包含所述环磷酰胺。
  144. 根据权利要求140-143中任一项所述的药物组合物，以约20至约30mg/m2/天的剂量包含所述氟达拉滨。
  145. 根据权利要求140-144中任一项所述的药物组合物，以约200,000至约600,000IU/kg/次的剂量包含所述IL-2。
  146. 一种试剂盒，包含根据权利要求140-145中任一项所述的药物组合物。
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