Preparation method of genetically modified T cell

Abstract

The invention provides a preparation method for preparing genetically modified T cells by taking PBMC (peripheral blood mononuclear cells) as a raw material without T cell sorting, cells produced by the preparation method and application of the cells. According to the preparation method for preparing the modified T cell, the material cost and the manpower consumption can be reduced, and the virus dosage can be reduced.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a method for preparing genetically modified T cells. Background Art

[0002] At present, the preparation process of genetically modified T cells represented by CAR-T cells involves multiple steps, such as cell sorting, activation, viral transduction, amplification, harvesting, etc., which takes 7-11 days in total. These processes are labor-intensive and the cost of cell sorting is high. Generally, the reagents and consumables required for sorting T cells are about 100,000 yuan, which accounts for the main part of the overall preparation process cost.

[0003] If T cells are not sorted and are directly cultured using peripheral blood mononuclear cells (PBMC) as the starting material, literature reports that because the composition of PBMCs varies greatly among different patients and the monocytes in PBMCs have phagocytic function, the preparation process will be unstable, resulting in problems such as unstable amplification multiples after cell amplification, low T cell purity, or low transduction rate.

[0004] In addition, the current preparation process of genetically modified T cells represented by CAR-T mostly involves cell sorting and activation on Day 0, viral transduction on Day 1 or Day 2, and cell expansion on Day 3-Day 9. The entire process is discontinuous and requires manual cell sorting, which increases manpower consumption and preparation costs.

[0005] Therefore, there remains a need for a new method of generating modified T cells that is simpler and cheaper than current methods. Summary of the invention

[0006] In order to improve the above technical problems, the present invention provides a process for preparing modified T cells using PBMC as raw materials without T cell sorting.

[0007] In one aspect, the present invention provides a method for preparing genetically modified T cells, characterized in that it comprises the following steps:

[0008] (1) On Day 0, the starting density of PBMCs was adjusted to 1e6 / ml-5e6 / ml using culture medium;

[0009] (2) On Day 0, transfer the PBMC cells at the density described in step (1) into a container and add culture medium to the initial culture volume;

[0010] (3) On Day 0, add an activator to the container of step (2);

[0011] (4) Between Day 0 and Day 2, the viral vector is added to the container of step (3) for transduction, and the container to which the viral vector has been added is cultured;

[0012] (5) At least 0.5 days after adding the viral vector, the culture medium is added to the container to the harvest volume, and then the container is shaken and cultured until the harvest day;

[0013] (6) On the harvest day, remove the container and harvest the cells.

[0014] In one embodiment of the present invention, the starting density of PBMC in step (1) is 1e6 / ml-3.5e6 / ml; preferably, the starting density of PBMC is 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml or 3.2e6 / ml.

[0015] In one embodiment of the present invention, the initial culture volume in step (2) is 75 ml-250 ml; preferably, the initial culture volume is 75 ml, 100 ml, 150 ml, 175 ml or 200 ml.

[0016] In one embodiment of the present invention, the culture medium is selected from the group consisting of X VIVO, AIM V, Optmizer, Optivitro, TexMacs, NutriT and T VIVO.

[0017] In one embodiment of the present invention, the activator in step (3) is selected from Dynabeads, TransAct and CD3 / CD28 / CD2 soluble tetramer;

[0018] Preferably, the activator is Dynabeads or The ratio of the number of magnetic beads: the number of cells is 0.5:1, 1:1, 2:1, 3:1 or 4:1, more preferably 2:1;

[0019] Preferably, the activator is TransAct, and the ratio of culture volume: activator volume is 60:1-10:1; more preferably 50:1, 40:1, 30:1 or 20:1;

[0020] Preferably, the activator is a soluble CD3 / CD28 / CD2 tetramer, and the ratio of culture volume: activator volume is 200:1-10:1; more preferably, it is 100:1, 75:1, 50:1, 25:1 or 20:1.

[0021] In one embodiment of the present invention, the cell density in the container when the viral vector is added for transduction in step (4) is 1e6-3.5e6 / ml;

[0022] Preferably, the cell density is 1.2e6 / ml, 1.5e6 / ml, 1.8e6 / ml, 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml or 3.2e6 / ml.

[0023] In one embodiment of the present invention, the culture in step (4) is shaking culture, rocking shaking culture or stirring culture;

[0024] Preferably, the rotation speed of the shaking culture is 50-100 rpm; more preferably, the rotation speed of the shaking culture is 75 rpm;

[0025] Preferably, the rotation speed of the rocking shaking culture is 5-30 rpm, and the angle is 2-30°; more preferably, the rotation speed of the rocking shaking culture is 15 rpm, and the angle is 15°;

[0026] Preferably, the rotation speed of the stirring culture is 50-300 rpm; more preferably, the rotation speed of the rocking shaking culture is 15 rpm.

[0027] In one embodiment of the present invention, the medium is added to the container to the harvest volume 0.5, 1, 1.5 or 2 days after the addition of the viral vector in step (5);

[0028] Preferably, one day after adding the viral vector in step (5), the culture medium is added to the container to the harvest volume.

[0029] In one embodiment of the present invention, the harvest day in step (6) is Day 4, Day 5, Day 6, Day 7 or Day 8;

[0030] Preferably, the harvest day in step (6) is Day 6.

[0031] In one embodiment of the present invention, the container is selected from a cell culture plate, a cell culture deep well plate, a cell culture chamber, a multilayer culture dish, a rotating tube, a controlled bioreactor, a shaking flask, a conical flask or a gas permeable bag.

[0032] In another aspect, the present invention provides a genetically modified T cell produced by the aforementioned method;

[0033] Preferably, the genetically modified T cells are CAR-T cells or TCR-T cells.

[0034] In another aspect, the present invention provides use of the aforementioned cell in the preparation of a medicament for treating a disease in a subject.

[0035] In another aspect, the present invention provides a method for treating a disease in a subject, comprising administering an effective amount of the aforementioned cells to the subject.

[0036] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned cell and a pharmaceutically acceptable carrier.

[0037] Beneficial Effects

[0038] Shaking the cultured cells allows the preparation of genetically modified T cells without sorting PBMCs, while also increasing the chance of virus-cell contact.

[0039] The CAR-T cell preparation method provided by the present invention can reduce material costs, manpower consumption, and reduce the amount of virus used; while achieving the above effects, the proportion of CAR-T cells in the final product is ≥95%, the cell expansion multiple is 30-50 times, the cell viability is ≥95%, and the transduction rate is between 30% and 60%. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Three process schemes for preparing CAR-T using unsorted PBMCs are shown.

[0041] Figure 2 The final T cell expansion fold when CAR-T was prepared using PBMCs with different starting densities is shown.

[0042] Figure 3 Shown are the effects of activator Dynabeads at different ratios relative to T cells on expansion and transduction rates.

[0043] Figure 4 Show Activator Effects of different ratios of T cells on expansion and transduction rates.

[0044] Figure 5 Shown are the effects of the activator TransAct on expansion and transduction rates at different ratios relative to T cells.

[0045] Figure 6 Shown is the effect of activator CD3 / CD28 / CD2 soluble tetramers at different ratios relative to T cells on expansion and transduction rates.

[0046] Figure 7 The figure shows the effect of different cell densities on the cell viability and transduction rate on Day 7 during transduction.

[0047] Figure 8 The CD3 in the cells harvested from the process of preparing CAR-T using unsorted PBMCs is shown. + The proportion of cells.

[0048] Fig. 9 The figure shows the viral transduction rate in the process of preparing CAR-T using unsorted PBMCs.

[0049] Fig.10 The figure shows the proportion of various T cell phenotypes in the cells harvested from the process of preparing CAR-T using unsorted PBMCs.

[0050] Fig.11 The figure shows the cell viability of cells harvested from the process of preparing CAR-T using unsorted PBMCs.

[0051] Fig.12 The figure shows the expansion fold of cells harvested from the process of preparing CAR-T using unsorted PBMCs. DETAILED DESCRIPTION

[0052] definition

[0053] Unless otherwise indicated, the terms "a" or "an" should be understood to mean "at least one or more of."

[0054] As used herein, "PBMC" refers to peripheral blood mononuclear cells. In some embodiments, PBMCs can be collected or obtained from a subject by any suitable method known in the art. For example, in some embodiments, blood can be collected by venipuncture or any other blood collection method for collecting blood and / or PBMC samples.

[0055] In some embodiments, PBMC can be obtained from the circulating blood of the subject by apheresis. The apheresis product usually contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In certain embodiments, the cells collected by apheresis, particularly leukocyte removal, can be washed to remove the plasma portion and placed in an appropriate buffer or culture medium for subsequent processing to separate the fresh apheresis, or to recover and wash the PBMCs previously separated and frozen to obtain PBMC cells. In certain embodiments, PBMC cells are obtained by recovering and washing the PBMCs previously separated and frozen.

[0056] As used herein, "genetically modified T cells" include CAR-T cells and TCR-T cells. "CAR-T cells" refer to engineered T cells containing a chimeric antigen receptor (CAR); "TCR-T cells" refer to engineered T cells containing a specific receptor (TCR) on the surface of T cells.

[0057] As used herein, "T cell subtypes" include T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and terminally differentiated effector memory cells (TEMRA).

[0058] As used herein, "viral vector" and "retroviral vector" are used interchangeably herein to refer to any form of nucleic acid derived from a retrovirus and used to transfer genetic material into cells via transduction. The term encompasses viral vector nucleic acids, such as DNA and RNA; encapsulated forms of these nucleic acids; and viral particles in which viral vector nucleic acids have been packaged.

[0059] As used herein, the term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome, particularly including the self-inactivating lentiviral vector provided in Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, gene delivery technology from Oxford BioMedica, LENTIMAX™ vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0060] The term "infection multiplier" (hereinafter "MOI") refers to the ratio of an infection such as a virus particle to an infection target such as a cell in a culture medium of a program such as a transduction program. In some embodiments, MOI may be equal to the number of functional virus particles added to the total number of target cells during the transduction program. In some embodiments, the number of functional virus particles added to the transduction program is determined by determining the titer of the functional virus particles. In some embodiments, the titer of functional virus particles is determined by using qPCR to determine the number of nucleic acid virus copies integrated per cell in a standard cell line stably transduced using techniques known in the art. For example, see Paugh, BS et al., SciRep11, 389 (2021), which is incorporated herein by reference in its entirety. In some embodiments, the virus particles are retroviral particles. In some embodiments, the virus particles are slow virus particles.

[0061] Transduction

[0062] Provided herein is a method for genetically modifying immune cells, including PBMCs and T cells produced by the methods disclosed herein. In some embodiments of the methods and compositions disclosed herein, T cells are contacted ex vivo with a retroviral vector without replication ability to genetically modify T cells so that they express exogenous gene products. In some embodiments, the exogenous gene product is CAR.

[0063] In some embodiments, the exogenous gene product is an epitope that is specific to a monoclonal antibody (i.e., specifically recognized by a monoclonal antibody), a suicide polypeptide, an inducible "on" or "accelerator" switch, such as an inducible caspase-9 (U.S. Application 2011 / 0286980) or thymidine kinase or an "off" switch. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO2016 / 120216, which is incorporated herein by reference in its entirety. In some embodiments, the exogenous gene product is an R epitope, such as RQR8. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. When the R epitope is expressed on the surface of a CAR immune cell, rituximab can bind to the R epitope to dissolve the CAR immune cell. In some embodiments, the exogenous gene product is a control switch, such as a dimerization domain.

[0064] In some embodiments, transduction can be performed in the same container as the activation step, without removing any culture medium. For example, blood cells enriched and separated from the collected blood sample, such as PBMC, can be activated in a shake bottle and then contacted with retroviral particles in the same shake bottle. In illustrative embodiments, blood cells are separated, separated and / or purified from granulocytes including neutrophils before contacting with retroviral vectors. Retroviral vectors can be recombinant lentiviral particles without replication ability in other exemplary embodiments, which can be introduced into the same permeable bag containing activated PBMC to form a transduction reaction mixture. In some embodiments, retroviral vectors are added to the transduction reaction mixture during the activation step. In some embodiments, retroviral vectors are added to the transduction reaction mixture after the activation step. In some embodiments, the activation step is performed before or simultaneously with the transduction step for no more than 12 or 24 hours. Figure 1 Potential transduction time points in an exemplary CAR-T cell production protocol are shown. Culture medium is typically present during transduction, such as culture medium known in the art for ex vivo culture of T cells, including basal medium and supplements, including cytokines, such as disclosed in more detail herein.

[0065] The transduction reaction, which in some embodiments begins when the retroviral vector is added to the PBMC cells, can be incubated between 23 and 39° C., and in some exemplary embodiments at 37° C. In some embodiments, the transduction reaction can be performed at 37-39° C. In some embodiments, the transduction reaction is incubated at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0% CO2. The transduction reaction can be incubated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 24, 36, 48 hours. In illustrative embodiments, the transduction reaction can be incubated at a pH within the starting pH range for a time between 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 4 to 12, 4 to 14, 4 to 16, 4 to 18, 4 to 20, 4 to 24, 4 to 26, 4 to 28, 4 to 30, 4 to 32, 4 to 36, 4 to 38, 4 to 40, 4 to 42, 4 to 44, 4 to 46, 4 to 48, or 4 to 72 hours. In some embodiments, the transduction reaction pH is passively controlled, for example, by adjusting the media buffer (e.g., sodium bicarbonate and / or HEPES) and the incubator pCO2 at the start of the culture to allow for a desired pH (e.g., a pH above 7.0) to achieve a target pH, e.g., 7.0 or higher. In some embodiments, the transduction reaction pH is actively controlled, for example by using a bioreactor with online pH measurement and a pH feedback control loop that maintains the pH by continuously (actively) adjusting the culture pH by adding CO2 gas.

[0066] In some embodiments, PBMC cells can be transduced at different ratios of retroviral or lentiviral particles to cells, which is referred to as the multiplicity of infection (MOI). In some embodiments, PBMC cells are transduced using an MOI (plaque forming units / cell) between 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 100, 150, 200, 250, 300, 350, 400, 450 to 500. In some embodiments, PBMC cells are transduced at an MOI of between 0.25, 0.50, 1.0, 5, 10, 15, 20, 25, or 30 to 50, 75, 100, 125, 150, 175, or 200. In some embodiments, PBMC cells are transduced at an MOI of about 1 to 10, 15, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, PBMC cells are transduced at an MOI of 1 to 20.

[0067] In some embodiments, the cell is transduced by a viral vector comprising a cell exogenous nucleic acid. In some embodiments, the exogenous nucleic acid encodes CAR. In some embodiments, the viral vector is a retrovirus, a slow virus or an AAV vector. In some embodiments, the viral vector is a mouse retrovirus vector.

[0068] The cells to be transduced to express CAR can be derived from allogeneic or autologous sources. In one embodiment, in the absence of non-human animal derived products, such as fetal calf serum (fetal calf serum) / fetal bovine serum (fetal bovine serum) in vitro transduction, culture and / or expansion of T cells.

[0069] In some embodiments, the transduction reaction cell population is incubated with a retroviral vector encoding a CAR at an MOI of at least 5, 10, 20, 30, 50, 100, 150 or 200 for 1 hour to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24 or 48 hours within the starting pH range and at a pH equal to or below 7.8 or equal to or below 7.9, and wherein at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the T cells express the CAR at least day 3, day 4, day 5 or day 6 after the retroviral vector is first contacted with the cells during the transduction reaction.

[0070] Preparation and cryopreservation

[0071] In some embodiments, engineered immune cells are formulated in therapeutically effective amounts by first harvesting the cells from their medium, followed by washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically saline NormosolTMR (Abbott) or Plasma-LyteTMA (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0072] In another embodiment, engineered immune cells, such as CAR-expressing T cells, are harvested, washed and concentrated, and then stored in a suitable cryopreservation medium, such as or (BioLife Solutions) at a predetermined cell concentration. Cryopreservation of engineered immune cells, such as CAR-expressing T cells, is performed using standard procedures for storage and / or preparation for use in human subjects. If desired, cryopreserved engineered immune cells can be thawed, grown, and expanded to produce more such cells.

[0073] Chimeric Antigen Receptor

[0074] As used herein, a chimeric antigen receptor (CAR) is a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). When bound to a target antigen, CAR can activate immune cells to attack and destroy cells (e.g., cancer cells) carrying the antigen. CAR can also have a co-stimulatory or signaling domain to increase its effectiveness. See, for example, Finney et al., Journal of Immunology, 1998, 161: 2791-2797; Song et al., Blood 119: 696-706 (2012); Kala et al., Sci. Transl. Med. 3: 95 (2011); Porter et al., N. Engl. J. Med. 365: 725-33 (2011) and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56: 59-83 (2016); U.S. Patents No. 7,741,465 and No. 6,319,494.

[0075] The chimeric antigen receptor described herein comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises an antigen binding domain. In some embodiments, the antigen-specific CAR comprises the following elements from 5' to 3': a signal sequence, an antigen binding domain, a hinge and a transmembrane region and one or more continuous signaling domains.

[0076] In some embodiments, the CAR further comprises a safety switch and / or a monoclonal antibody specific epitope. See, e.g., WO2016 / 120216.

[0077] Antigen binding domain

[0078] As discussed above, CAR described herein includes an antigen binding domain. As used herein, "antigen binding domain" means any polypeptide that binds to a specified target antigen. In certain embodiments, the polypeptide structure of the antigen binding domain is based on an antibody. Antigen binding domains include, but are not limited to, antibody binding regions that are immunologically functional fragments. The term "immunologically functional fragment" (or "fragment") of an antigen binding domain is a species of an antigen binding domain that includes a portion of an antibody (however the portion is obtained or synthesized), the portion lacking at least some of the amino acids present in the full-length chain, but still capable of specific binding to the target antigen. Such fragments are biologically active because they are bound to the target antigen and can compete with other antigen binding domains, including intact antibodies, for binding to a given epitope. Immunologically functional fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity determining regions ("CDRs"), bifunctional antibodies (heavy chain variable domains on the same polypeptide as light chain variable domains, connected via a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen binding domains (comprising two antigen binding sites), multispecific antigen binding domains, and single-chain antibodies. These fragments may be derived from any mammal, including, but not limited to, humans, mice, rats, camelids, or lagomorphs.

[0079] In some embodiments, the antigen binding domain comprises one or more complementary binding regions (CDRs) present in the full-length light or heavy chains of an antibody, and in some embodiments comprises a single chain and / or a light chain or a portion thereof. These fragments may be produced by recombinant DNA techniques or may be produced by enzyme or chemical cleavage of an antigen binding domain (including a complete antibody).

[0080] In some embodiments, the antigen binding domain is an antibody of a fragment thereof, including one or more CDRs in its complementarity determining region (CDR). In some embodiments, the antigen binding domain is a single chain variable fragment (scFv), which comprises light chain CDRs CDR1, CDR2 and CDR3, and heavy chain CDRs CDR1, CDR2 and CDR3.

[0081] In general, the assignment of amino acids to each of the framework, CDRs, and variable domains generally conforms to the numbering scheme of Kabat numbering (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia and Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226), contact numbering or the AbM scheme (Antibody Modeling Program, Oxford Molecular).

[0082] Variants of antigen binding domains (e.g., variants of CDRs, VH and / or VL) are also within the scope of the present disclosure, such as variable light chains and / or variable heavy chains each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99% or more than 99% consistency with the amino acid sequence of the antigen binding domain sequence. In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, the variant form contains two variable light chains and two variable heavy chains (or sub-parts thereof). Those skilled in the art will be able to determine suitable variants of antigen binding domains as described herein using well-known techniques. In certain embodiments, those skilled in the art can identify suitable regions of the molecule, which can be changed by targeting regions that are not considered important for activity without destroying the activity.

[0083] In some embodiments, the antigen binding domain is a scFv. In some embodiments, the antigen selective CAR comprises a leader or signal peptide. As will be appreciated by those skilled in the art, the antigen binding domain may include non-protein components.

[0084] The antigen binding domains in the CARs suitable for the methods and compositions of the present disclosure may have a variety of antigen binding specificities. In some embodiments, the antigen binding domains are specific for the epitopes present on the antigens expressed (synthesized) by the target cells. In one example, the target cell is a cancer cell-associated antigen. The antigen associated with cancer cells may be an antigen associated with: for example, breast cancer cells, B cell lymphomas, Hodgkin (Hodgkin) lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin B cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, gliomas, glioblastomas, medulloblastomas, colorectal cancer cells, etc. Cancer cell-associated antigens can also be expressed by non-cancerous cells.

[0085] Non-limiting examples of antigens to which the chimeric binding antigen can bind include, for example, CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, Ax1, Ror2, BCMA, tight junction protein (Claudin) and its isoforms, etc.

[0086] Hinge domain

[0087] The extracellular domain of the CAR of the present disclosure may include a "hinge" domain (or hinge region). The term includes any polypeptide that is used to connect the transmembrane domain in CAR to the extracellular antigen binding domain in CAR. Specifically, the hinge domain can be used to provide greater flexibility and accessibility for the extracellular antigen binding domain.

[0088] The hinge domain may comprise up to 300 amino acids, in some embodiments 10 to 100 amino acids, or in some embodiments 25 to 50 amino acids. The hinge domain may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, CD28, 4-1BB or IgG (specifically, the hinge region of IgG; it should be understood that the hinge region may contain some or all of a member of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or a fragment thereof), or all or part of an antibody heavy chain constant region.

[0089] Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring sequence or may be a completely synthetic sequence. In some embodiments, the hinge domain is a portion of a human CD8a chain (e.g., NP_001139345.1). In another specific embodiment, the hinge and transmembrane domain include a portion of a human CD8a chain. In some embodiments, the hinge domain of CAR described herein includes a subsequence of CD8a, CD28, IgG1, IgG4, PD-1 or FcγRIIIa molecules, specifically, any one of CD8a, CD28, IgG1, IgG4, PD-1 or FcγRIIIa molecules. In some embodiments, the hinge domain includes a human CD8a hinge, a human IgG1 hinge, a human IgG4 hinge, a human PD-1 hinge or a human FcγRIIIa hinge. In some embodiments, CAR disclosed herein includes scFv, CD8a human hinge and a transmembrane domain.

[0090] Transmembrane domain

[0091] The CAR disclosed herein is designed to have a transmembrane domain fused with the extracellular domain of CAR. It can be similarly fused with the intracellular domain of CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex. In some embodiments, a short linker can form a key between any one or some of the extracellular, transmembrane and intracellular domains of CAR. The transmembrane domain suitable for CAR disclosed herein has the following capabilities: (a) expressed on the surface of immune cells, such as but not limited to lymphocytes, such as CD4+ cells such as T helper (Th) cells, CD8+ cells such as cytotoxic T (Tc) cells, T regulatory (Treg) cells or natural killer (NK) cells; and / or (b) interacting with extracellular antigen binding domains and intracellular signaling domains to guide immune cells to target cell cellular responses.

[0092] The transmembrane domain may be derived from a natural source or a synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Particularly suitable transmembrane regions for the present disclosure may be derived from (comprising or corresponding to) CD28, CD8, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-I (PD-1), inducible T cell co-stimulatory molecules (ICOS), lymphocyte function-associated antigen-I (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, lgα (CD79a), DAP-10, Fe γ receptors, MHC1 class molecules, TNF receptor proteins, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2 Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIld, ITGAE, CD103, ITGAL, CDIla, LFA-1, ITG AM, CDIlb, ITGAX, CDIle, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2 B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0093] As non-limiting examples, the transmembrane region can be derived from a T cell receptor, a polypeptide constituting a CD3 complex, an IL-2 receptor, p55 (a chain), p75 (beta chain) or a y chain, a subunit chain of an Fc receptor, particularly an Fy receptor III or a CD protein, or be a part thereof. Alternatively, the transmembrane domain can be synthetic and can comprise mainly hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from a human CD8a chain (e.g., NP_001139345.1).

[0094] Intracellular domain

[0095] The intracellular (cytoplasmic) domain of the CAR disclosed herein can provide activation of at least one normal effector function of the immune cell comprising the CAR, such as signal I / activation and / or signal II / co-stimulation. For example, the effector function of T cells may refer to cell lysis activity or helper cell activity, including the secretion of cytokines. In some embodiments, the activated intracellular signaling domain for use in CAR may be, for example, (but not limited to) T cell receptors and cytoplasmic sequences of co-receptors that coordinate after antigen receptor binding to initiate signal transduction, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities.

[0096] It will be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) CD3ζ, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T-cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, lgα(CD79a), DAP-10, Feγ receptor, MHC1 class molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, toluidine ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL -2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIld, ITGAE, CD103, ITGAL, CDIla, LFA -1.ITGAM, CDIlb, ITGAX, CDIle, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMl(CD226), SLAMF4(CD2 44, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0097] In addition to the above-mentioned activation domain, the intracellular domain can also incorporate a co-stimulatory signaling domain (interchangeably referred to as a co-stimulatory molecule herein) to increase its effectiveness. The co-stimulatory domain can provide a signal in addition to the primary signal provided by the activation molecule as described herein.

[0098] It will be appreciated that suitable costimulatory domains within the scope of the present disclosure may be derived from (or correspond to) for example CD28, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CDS, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-I (LFA-1 (CD11a / CD18), CD247, CD276 (B7-H3), LI GHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Feγ receptor, MHC class I molecules, TNFR, integrin, signaling lymphocyte activation molecules, BTLA, toluidine ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2 Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-ld, ITGAE, CD103, ITGAL, CD1-la, LFA-1, ITGAM, CD l-lb, ITGAX, CD1-lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, C D96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or a fragment or combination thereof. It should be understood that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0099] In some embodiments, the intracellular / cytoplasmic domain of CAR can be designed to contain the 4-1BB / CD137 domain itself or to be combined with any other desired intracellular domain suitable for the situation of CAR. The complete native amino acid sequence of 4-1BB / CD137 is described in NCBI reference sequence: NP_001552.2. The complete native 4-1BB / CD137 nucleic acid sequence is described in NCBI reference sequence: NM_001561.5.

[0100] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to contain the CD28 domain itself or in combination with any other desired intracellular domain suitable for the situation of the CAR. The complete native amino acid sequence of CD28 is described in NCBI reference sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI reference sequence: NM_006139.1.

[0101] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the CD3 zeta domain itself or in combination with any other desired intracellular domain applicable to the situation of the CAR. In some embodiments, the intracellular signaling domain of the CAR may include a CD31 signaling domain. For example, the intracellular domain of the CAR may include a portion of a CD3 zeta chain and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR may be connected to each other randomly or in a specified order.

[0102] Nucleic acid and expression vector preparation

[0103] Provided herein are methods for preparing nucleic acids encoding CAR and vectors comprising nucleic acids encoding CAR.

[0104] A variety of known techniques can be utilized in preparing polynucleotides and vectors according to the present disclosure. For example, certain methods for making constructs and engineered immune cells of the present disclosure are described in publication WO2015 / 120096, the entirety of which is incorporated herein by reference.

[0105] The nucleotide sequence encoding CAR may be present in an expression vector. In the case where CAR includes two separate polypeptides, the nucleotide sequence encoding the two polypeptides may be cloned in the same or separate vector. The expression vector may include a selectable marker, a replication origin, and other features that provide replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, etc.

[0106] In order to clone a polynucleotide, an expression vector can be introduced into a host cell (isolated host cell) to allow replication of the vector itself, and thereby amplify copies of the polynucleotide contained therein. The cloning vector may contain sequence components, generally including but not limited to a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, a replication origin may be selected to promote autonomous replication of the vector in a host cell.

[0107] In other embodiments, the present disclosure relates to isolated polynucleotides encoding any one of the antigen binding domains described herein. In some embodiments, the present disclosure relates to isolated polynucleotides encoding CAR. Also provided herein are vectors comprising polynucleotides and methods for preparing the same.

[0108] In certain embodiments, the disclosure provides isolated host cells containing expression vectors provided herein. Host cells containing vectors can be used for expression or cloning of polynucleotides contained in the vectors. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells, and more specifically human cells.

[0109] The vector can be introduced into the host cell using any suitable method known in the art, including but not limited to DEAE-dextran mediated delivery, calcium phosphate precipitation method, cationic lipid mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor mediated gene delivery, delivery mediated by polylysine, histone, chitosan and peptide. Standard methods for viral transfection and transformation of cells for expressing the vector of interest are well known in the art. In another embodiment, a mixture of different expression vectors can be used for a donor population of genetically modified immune effector cells, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, wherein a certain proportion of engineered cells express more than one different CAR.

[0110] Retroviral particle preparation

[0111] In the illustrative embodiments disclosed herein, the transduction method may include the following steps: transducing immune cells, such as T cells, with replication-defective recombinant retroviral particles comprising one or more nucleic acids to produce transduced, engineered immune cells, such as engineered T cells. In some embodiments, one or more nucleic acids may encode one or more proteins, which are then expressed in transduced T cells, such as chimeric antigen receptors (CARs). Retroviral particles for transducing T cells and / or NK cells in the methods provided herein can be prepared according to methods known in the art. As disclosed herein, retroviral particles are common tools for gene delivery (Miller, Nature (1992) 357: 455-460). In some embodiments, the replication-defective recombinant retroviral particles may be derived from alpha retrovirus genus, beta retrovirus genus, gamma retrovirus genus, delta retrovirus genus, epsilon retrovirus genus, slow virus genus or foam virus genus. There are many kinds of retroviruses suitable for the methods disclosed herein. A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses", 1997 Cold Spring Harbor Laboratory Press editor: JM Coffin, SM Hughes, HE Warmus, pp. 758-763). Details on the genomic structure of some retroviruses can be found in the art. For example, details about HIV can be found from NCBI Genbank (i.e., Genome Accession No. AF033819).

[0112] In illustrative embodiments, the retroviral particles can be derived from recombinant retroviruses from the genus Lentivirus and can be replication-defective recombinant lentiviral particles. In some embodiments, the recombinant retrovirus can be derived from HIV, SIV or FIV. In other illustrative embodiments, the recombinant retrovirus can be derived from human immunodeficiency virus (HIV) in the genus Lentivirus.

[0113] In some embodiments, the replication-defective recombinant retroviral particles can be grown in culture in a culture medium that is specific for the manufacture of the replication-defective recombinant retroviral particles. Any suitable growth medium and / or supplement for growing replication-defective recombinant retroviral particles can be used for replication-defective recombinant retroviral particle inocula according to the methods described herein. According to some aspects, the retroviral particles can then be added to the culture medium during transduction.

[0114] Replication-deficient recombinant retroviral particles can be produced using mammalian cell lines according to methods known in the art. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines may include human cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), etc. In some cases, the cells are not immortalized cell lines, but are cells obtained from an individual or ex vivo cells (e.g., primary cells). For example, in some embodiments, the cell is an immune cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.

[0115] Treatment

[0116] Provided is a method for treating a disease or condition including cancer. In some embodiments, the present disclosure relates to generating a T cell-mediated immune response in a subject, comprising administering an effective amount of an engineered immune cell of the present disclosure to the subject. In some embodiments, the T cell-mediated immune response is directed to a target cell or cell. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the present disclosure comprises a method for treating or preventing a malignant tumor, comprising administering an effective amount of at least one separated antigen binding domain as described herein to a subject in need. In some aspects, the present disclosure comprises a method for treating or preventing a malignant tumor, comprising administering an effective amount of at least one immune cell to a subject in need, wherein the immune cell comprises at least one chimeric antigen receptor and / or separated antigen binding domain as described herein.

[0117] In some embodiments, the subject suffers from a solid tumor or a hematological malignancy, such as a lymphoma or a leukemia. In some embodiments, the cancer is present in the subject's bone marrow. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are heterologous immune cells, such as heterologous T cells. In some embodiments, ex vivo transfection and / or transduction of engineered cells. As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. Therapeutic agents, for example, "therapeutically effective amounts", "effective doses", "effective amounts", or "therapeutically effective doses" of engineered CART cells are any amounts, when used alone or in combination with another therapeutic agent, protect the subject from the onset of the disease or promote disease regression, the disease regression is demonstrated by a reduction in the severity of the disease symptoms, an increase in the frequency and duration of the disease-free period, or prevention of the disorder or disability caused by the disease ailment. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner (e.g., physician or clinician), for example, in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by analyzing the activity of the agent in in vitro assays.

[0118] The terms "patient" and "subject" are used interchangeably and include human subjects as well as those with a formally diagnosed condition, those without a formally recognized condition, those under medical observation, those at risk of developing the condition, etc.

[0119] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and disclosures in which a subject is reduced risk that he or she will develop a condition or other risk factors. Treatment does not require a complete cure of the condition and encompasses embodiments in which symptoms or potential risk factors are reduced. The term "prevention" does not require 100% elimination of the likelihood of an event occurring. Rather, it means that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0120] The total number of cells required for treatment in the composition comprises at least 2 cells (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells or two CD4+ T cells) or more typically more than 10 2 cells and at most 10 6 up to and including 10 8 or 10 9 cells, and can be 10 10 or 10 12 The number of cells will depend on the intended use of the composition and the type of cells included therein. The desired cell density is generally greater than 10 6 cells / mL and usually more than 10 7 cells / mL, usually 10 8 Clinically relevant numbers of immune cells can be divided into multiple infusions, with the cumulative amount equal to or greater than 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some aspects of the present disclosure, particularly since all infused cells will be redirected to a specific target antigen, administration may be performed within 10 6 / kg (10 per patient 6 -10 11 CAR therapy can be administered multiple times at doses within these ranges.

[0121] The cells can be autologous, allogeneic or xenogeneic to the patient being treated.

[0122] In some embodiments, the therapeutically effective amount of CART cells is about 1×10 5 cells / kg, about 2×10 5 cells / kg, about 3×10 5 cells / kg, about 4×10 5 cells / kg, about 5×105 cells / kg, about 6×10 5 cells / kg, about 7×10 5 cells / kg, about 8×10 5 cells / kg, about 9×10 5 cells / kg, 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg or about 9×10 7 cells / kg.

[0123] In some embodiments, the target dose of CAR+ / CAR-T+ cells is about 1×10 6 About 1×10 10 cells / kg, for example, about 1×10 6 cells / kg, about 1×10 7 cells / kg, about 1×10 8 cells / kg, about 1×10 9 cells / kg or about 1×10 10 Cells / kg. It should be understood that dosing above and below this range may be suitable for certain subjects, and appropriate dosing levels may be determined by a healthcare provider as needed. In addition, multiple doses of cells may be provided according to the present disclosure.

[0124] In some aspects, the disclosure comprises a pharmaceutical composition comprising at least one antigen binding domain as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0125] The CAR expressing cell colonies disclosed herein can be administered alone or in the form of a pharmaceutical composition in combination with a diluent and / or with other components (such as IL-2 or other cytokines or cell colonies). The pharmaceutical composition disclosed herein may include a cell colony expressing CAR in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients, such as T cells as described herein. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The disclosed compositions are preferably formulated for intravenous administration.

[0126] Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides that can serve as solvents or suspension media; polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates or phosphates and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral formulations may be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For therapeutic disclosure, injectable pharmaceutical compositions are preferably sterile.

[0127] The method may further include administering one or more chemotherapeutic agents to the patient before administering the engineered cells provided herein. In certain embodiments, the chemotherapeutic agent is a lymphocyte depletion (pretreatment) chemotherapeutic agent. For example, the method for adjusting a patient in need of T cell therapy comprises administering to the patient a specified beneficial dose of cyclophosphamide (between 200 mg / m2 and 2000 mg / m2 per day, between about 100 mg / m2 and about 2000 mg / m2 per day; for example, about 100 mg / m2 per day, about 200 mg / m2 per day, about 300 mg / m2 per day, about 400 mg / m2 per day, about 500 mg / m2 per day, about 600 mg / m2 per day, about 700 mg / m2 per day, about 800 mg / m2 per day, about 900 mg / m2 per day, about 1000 mg / m2 per day, about 1500 mg / m2 per day, about 2000 mg / m2 per day, about 3000 mg / m2 per day, about 400 mg / m2 per day, about 500 mg / m2 per day, about 600 mg / m2 per day, about 700 mg / m2 per day, about 800 mg / m2 per day, about 900 mg / m2 per day, about 1000 mg / m2 per day, about 1500 mg / m2 per day, about 1000 mg / m2 per day, about 2000 mg / m2 per day, about 2000 mg / m2 per day, about 3000 mg / m2 per day, about 400 mg / m2 per day, about 500 mg / m2 per day, about 600 mg / m2 per day, about 700 mg / m2 per day, about 800 mg / m2 m2 or about 2000 mg / m2 per day) and a specified dose of fludarabine (between 20 mg / m2 per day and 900 mg / m2 per day, between about 10 mg / m2 per day and about 900 mg / m2 per day; for example, about 10 mg / m2 per day, about 20 mg / m2 per day, about 30 mg / m2 per day, about 40 mg / m2 per day, about 40 mg / m2 per day, about 50 mg / m2 per day, about 60 mg / m2 per day, about 70 mg / m2 per day, about 80 mg / m2 per day, about 9020 mg / m2 per day, about 100 mg / m2 per day, about 500 mg / m2 per day, or about 900 mg / m2 per day). An exemplary dosing regimen involves treating a patient comprising administering to the patient daily about 300 mg / m2 of cyclophosphamide three days prior to administering to the patient a therapeutically effective amount of engineered T cells, in combination, or prior to or following daily administration of about 30 mg / m2 of fludarabine.

[0128] In other embodiments, the antigen binding domain, transduced (or otherwise engineered) cells, and chemotherapeutic agent are each administered in an amount effective to treat the disease or condition in the subject.

[0129] In certain embodiments, the composition comprising the immune effector cells expressing CAR disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylene melamine, triethylenephosphamide, triethylenethiophosphamide and trihydroxymethylmelamine; nitrogen mustard, such as chlorambucil, naphthalene nitrogen mustard, chlorphosphocyanate amine, estramustine, ifosfamide, dichloromethane, dichloromethane oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, for example, carmustine, chlorozostatin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, dactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin in), 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quela mycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiabendazole, thio ... Guanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; antiadrenal, such as aminoglutethimide, mitotane, trilostane;Folic acid supplements, such as folinic acid, acetoglucuronide, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, and elliptinomycin. niumacetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophylloic acid; 2-acetylhydrazine; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and docetaxel ( Rhne-PoulencRorer; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CP T-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid derivatives, such as TargretinTM (bexarotene), PanretinTM (alitretinoin); ONTAKTM (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. This definition also includes anti-hormonal agents used to regulate or inhibit the hormonal effects on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibition 4 (5) -imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and any of the above pharmaceutically acceptable salts, acids or derivatives. Combinations of chemotherapeutic agents are also administered when appropriate, including but not limited to CHOP, i.e., cyclophosphamide doxorubicin (Doxorubicin) (hydroxydoxorubicin), vincristine and prednisone.

[0130] In some embodiments, chemotherapeutic agents are administered simultaneously or within a week after the engineered cell, polypeptide or nucleic acid is administered. In other embodiments, chemotherapeutic agents are administered about 1-7 days, about 1 to about 4 weeks, or about 1 week to about 1 month, about 1 week to about 2 months, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, or about 1 week to about 12 months after the engineered cell, polypeptide or nucleic acid is administered. In other embodiments, chemotherapeutic agents are administered at least 1 month before the cell, polypeptide or nucleic acid is administered. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0131] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab, pembrolizumab, pidilizumab, and atezolizumab. Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to: ibrutinib, ofatumumab, rituximab, bevacizumab, trastuzumab, trastuzumab, trastuzumab, bemtansine, imatinib, cetuximab, panitumumab, catumaxomab, ibritu momab), ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib nilotinib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurine, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib,Binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukindiftitox; mTOR inhibitors, such as everolimus and temsirolimus; hedgehog inhibitors, such as sonidegib and vismodegib; CDK inhibitors, such as CDK inhibitors (palbociclib).

[0132] In some embodiments, the composition comprising the immune cells expressing CAR can be applied together with the treatment regimen to prevent cytokine release syndrome (CRS) or neurotoxicity. The treatment regimen for preventing cytokine release syndrome (CRS) or neurotoxicity may include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (eg, L-NIL or 1400W). In additional embodiments, the composition comprising the immune cells containing CAR can be applied together with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, ethylprednisolone, prednisolone, prednisone, triamcinolone); nonsteroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors and sialates. Exemplary analgesics include acetamide phenol, oxycodone, tramadol of propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CDS, etc.), cytokine inhibitors (e.g., TNF antagonists (e.g., etanercept, adalimumab and infliximab)), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules.Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0133] In certain embodiments, the compositions described herein are administered in combination with cytokines. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet growth factor; transforming growth factor (TG F), such as TGF-α and TGF-β; insulin-like growth factor-I and II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, β and γ; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.

[0134] Kits and Products

[0135] The present disclosure provides a kit comprising CAR-T cells obtained using the methods provided herein, and pharmaceutical compositions thereof. In one embodiment of the kit, the engineered CAR cells are cultured in a suitable culture medium, such as or (BioLifeSolutions).

[0136] The present disclosure also provides an article comprising any of the therapeutic compositions or kits described herein. Examples of articles include vials (e.g., sealed vials comprising immune cells expressing CAR).

[0137] As used herein, "making up the volume with culture medium" refers to adding culture medium during the fermentation process, ie, adding fresh culture medium to the fermentation container so that there is a certain volume of liquid in the container.

[0138] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0139] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0140] Example 1: Preparation of CAR-T using unsorted PBMC

[0141] 1.1 There are three process schemes for preparing CAR-T cells using unsorted PBMCs:

[0142] 1.1.1 Solution 1

[0143] (1) On Day 0, fresh single blood is collected to separate PBMCs to obtain PBMC cells, or previously separated and frozen PBMCs are revived and washed to adjust the initial density of PBMCs;

[0144] (2) On Day 0, the PBMC cells from step (1) were transferred into a shake flask and the volume was replenished to the initial culture volume using culture medium;

[0145] (3) On Day 0, add the activator to the shake flask in step (2);

[0146] (4) On Day 0, add the viral vector to the shake flask in step (3) for transduction;

[0147] (5) On Day 0, place the shake flask in step (4) in a CO2 shaking incubator for shaking culture;

[0148] (6) On Day 1, add culture medium to the shake flask to the harvest volume; on Day 1, place the shake flask in a CO2 shaking incubator and culture until Day 6;

[0149] (7) On Day 6, the shake flask was removed for cell harvesting and cryopreservation.

[0150] 1.1.2 Option 2

[0151] (1) On Day 0, fresh single blood is collected to separate PBMCs to obtain PBMC cells, or previously separated and frozen PBMCs are revived and washed to adjust the initial density of PBMCs;

[0152] (2) On Day 0, the PBMC cells from step (1) were transferred into a shake flask and the volume was replenished to the initial culture volume using culture medium;

[0153] (3) On Day 0, add the activator to the shake flask in step (2);

[0154] (4) On Day 1, add the viral vector to the shake flask in step (3) for transduction;

[0155] (5) On Day 1, place the shake flask in step (4) in a CO2 shaking incubator for shaking culture;

[0156] (6) On Day 2, add culture medium to the shake flask to the harvest volume; on Day 1, place the shake flask in a CO2 shaking incubator and culture until Day 6;

[0157] (7) On Day 6, the shake flask was removed for cell harvesting and cryopreservation.

[0158] 1.1.3 Option 3

[0159] (1) On Day 0, fresh single blood is collected to separate PBMCs to obtain PBMC cells, or previously separated and frozen PBMCs are revived and washed to adjust the initial density of PBMCs;

[0160] (2) On Day 0, the PBMC cells from step (1) were transferred into a shake flask and the volume was replenished to the initial culture volume using culture medium;

[0161] (3) On Day 0, add the activator to the shake flask in step (2);

[0162] (4) On Day 2, add the viral vector to the shake flask in step (3) for transduction;

[0163] (5) On Day 2, place the shake flask in step (4) in a CO2 shaking incubator for shaking culture;

[0164] (6) On Day 3, add culture medium to the shake flask to the harvest volume; on Day 1, place the shake flask in a CO2 shaking incubator and culture until Day 6;

[0165] (7) On Day 6, the shake flask was removed for cell harvesting and cryopreservation.

[0166] 1.2 Parameter optimization of preparation scheme

[0167] 1.2.1 Starting density of PBMC

[0168] The starting density of PBMC cells in step (1) was tested from 1e6 / ml to 5e6 / ml. The results are as follows: Figure 2 As shown, the optimal starting density is between 1e6 / ml-3.5e6 / ml.

[0169] 1.2.2 Initial culture volume of PBMC

[0170] The initial culture volume of PBMC cells in step (2) was tested to be between 50 ml and 300 ml, and the optimal initial culture volume was between 75 ml and 250 ml.

[0171] 1.2.3 Culture medium screening

[0172] X VIVO, AIM V, Optmizer, Optivitro, TexMacs, NutriT, and T VIVO media were tested.

[0173] 1.2.4 Types of activators and their relative cell ratios

[0174] We tested commonly used activators on the market, such as Dynabeads (Thermo Fisher Scientific, 11456D), (Tongli Haiyuan, cat: GMP-TL603-1000), TransAct (Miltenyi Biotec, cat: 200-076-204) and CD3 / CD28 / CD2 soluble tetramer (Stemcell technologies, cat: 10970) and their different ratios relative to cells. The results are shown in Figure 3-6 shown.

[0175] According to the test results, for magnetic beads Dynabeads and Bead number: cell number ratios of 0.5:1, 1:1, 2:1, 3:1 and 4:1 are all applicable; for TransAct reagent, culture volume: reagent volume ratios of 60:1 to 10:1 are all applicable; for CD3 / CD28 / CD2 soluble tetramers, culture volume: reagent volume ratios of 200:1 to 10:1 are all applicable.

[0176] 1.2.5 Virus transduction condition testing

[0177] Different viral transduction times and cell densities when adding viral vectors for transduction were tested, where the transduction time can be between 0 and 72 hours. Cell densities between 1e6 and 5e6 / ml were tested, and the experimental results are shown in the figure. Figure 7As shown, the transduction rate is optimal when the cell density is between 1e6-3.5e6 / ml.

[0178] 1.2.6 Cytokine screening

[0179] The following cytokine combinations were tested: IL2 alone, the combination of IL2+IL7+IL15, IL7 alone, IL15 alone, the combination of IL7+IL15, and the combination of IL7+IL15+IL21 were all applicable.

[0180] 1.3 CAR-T cell production process effect verification

[0181] After parameter optimization, CAT cells were prepared using the following process and the corresponding indicators of the prepared CAR-T cells were verified.

[0182] (1) On Day 0, fresh single blood was collected to isolate PBMCs to obtain PBMC cells, or previously isolated and frozen PBMCs were resuscitated and washed to adjust the starting density of PBMCs to 3e6 / ml;

[0183] (2) On Day 0, the PBMC cells from step (1) were transferred into a shake flask and the volume was supplemented to the initial culture volume of 200 ml using culture medium;

[0184] (3) On Day 0, add Dynabeads CD3 / CD28 to the shake flask in step (2), with the ratio of beads to cells being 2:1;

[0185] (4) On Day 0, add the lentiviral vector to the shake flask in step (3). At this time, the cell density in the shake flask is 3.5e6 / ml;

[0186] (5) On Day 0, place the shake flask in step (4) in a CO2 shaking incubator and shake at 75 rpm;

[0187] (6) On Day 1, add culture medium to the shake flask to a harvest volume of 1 L; on Day 1, place the shake flask in a CO2 shaking incubator and culture until Day 6;

[0188] (7) On Day 6, the shake flask was removed for cell harvesting and cryopreservation.

[0189] The above process was verified using PBMC cells from 10 donors. After 6 days of culture, the cells harvested from the 10 donors had CD3 + The proportion of cells is greater than 95% (such as Figure 8 The transduction rate of the virus expressing the CAR structure was between 36% and 60% (as shown in Fig. 9The proportion of cells with the phenotype of Tscm+Tcm in the harvested cells was greater than 80% (as shown in Fig.10 The viability of the harvested cells was above 95% (as shown in Fig.11 The harvested cells were expanded 30-50 times (as shown in Fig.12 The above results show that the process has good stability.

[0190] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing genetically modified T cells, characterized in that: It includes the following steps: (1) On Day 0, the starting density of PBMCs was adjusted to 1e6 / ml-5e6 / ml using culture medium; (2) On Day 0, transfer the PBMC cells at the density described in step (1) into a container and add culture medium to the initial culture volume; (3) On Day 0, add an activator to the container of step (2); (4) Between Day 0 and Day 2, the viral vector is added to the container of step (3) for transduction, and the container to which the viral vector has been added is cultured; (5) At least 0.5 days after adding the viral vector, add culture medium to the container to the harvest volume, and then culture until the harvest day; (6) On the harvest day, remove the container and harvest the cells.

2. The method according to claim 1, wherein the starting density of PBMC in step (1) is 1e6 / ml-3.5e6 / ml; preferably, the starting density of PBMC is 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml or 3.2e6 / ml.

3. The method according to claim 1 or 2, wherein the initial culture volume in step (2) is 75 ml-250 ml; Preferably, the initial culture volume is 75 ml, 100 ml, 150 ml, 175 ml or 200 ml.

4. The method according to any one of claims 1 to 3, wherein the culture medium is selected from the group consisting of X VIVO, AIM V, Optmizer, Optivitro, TexMacs, NutriT and T VIVO.

5. The method according to any one of claims 1 to 4, wherein the activator in step (3) is selected from Dynabeads, CD3 / CD28, TransAct, and CD3 / CD28 / CD2 soluble tetramers; Preferably, the activator is Dynabeads or CD3 / CD28, the ratio of the number of magnetic beads: the number of cells is 0.5:1, 1:1, 2:1, 3:1 or 4:1, more preferably 2:1; Preferably, the activator is TransAct, and the ratio of culture volume: activator volume is 60:1-10:1; more preferably 50:1, 40:1, 30:1 or 20:1; Preferably, the activator is a soluble CD3 / CD28 / CD2 tetramer, and the ratio of culture volume: activator volume is 200:1-10:1; more preferably, it is 100:1, 75:1, 50:1, 25:1 or 20:

1.

6. The method according to any one of claims 1 to 5, wherein the cell density in the container when the viral vector is added for transduction in step (4) is 1e6-3.5e6 / ml; Preferably, the cell density is 1.2e6 / ml, 1.5e6 / ml, 1.8e6 / ml, 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml, 3.2e6 / ml.

7. The method according to any one of claims 1 to 6, wherein the culture in step (4) is shaking culture, rocking shaking culture or stirring culture; Preferably, the rotation speed of the shaking culture is 50-100 rpm; more preferably, the rotation speed of the shaking culture is 75 rpm; Preferably, the rotation speed of the rocking shaking culture is 5-30 rpm, and the angle is 2-30°; more preferably, the rotation speed of the rocking shaking culture is 15 rpm, and the angle is 15°; Preferably, the rotation speed of the stirring culture is 50-300 rpm; more preferably, the rotation speed of the rocking shaking culture is 15 rpm.

8. The method according to any one of claims 1 to 7, wherein the culture medium is added to the container to the harvest volume 0.5, 1, 1.5 or 2 days after the addition of the viral vector in step (5); Preferably, one day after adding the viral vector in step (5), the culture medium is added to the container to the harvest volume.

9. The method according to any one of claims 1 to 8, wherein the harvest day in step (6) is Day 4, Day 5, Day 6, Day 7 or Day 8; Preferably, the harvest day in step (6) is Day 6.

10. The method according to any one of claims 1 to 9, wherein the container is selected from a cell culture plate, a cell culture deep well plate, a cell culture chamber, a multilayer culture dish, a rotating tube, a controlled bioreactor, a shake flask, a conical flask or a gas permeable bag.

11. A genetically modified T cell produced by the method of any one of claims 1-10; Preferably, the genetically modified T cells are CAR-T cells or TCR-T cells.

12. Use of the cell according to claim 11 in the preparation of a medicament for treating a disease in a subject.

13. A pharmaceutical composition comprising the cell according to claim 11 and a pharmaceutically acceptable carrier.

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