Method for preparing chimeric antigen receptor T cells using a serum-free medium

Abstract

We provide a method for preparing chimeric antigen receptor T cells in a serum-free medium, comprising the steps of: (a) providing PBMC cells; (b) negatively selecting the PBMC cells to obtain selected PBMC cells; (c) activating the selected PBMC cells to obtain activated T cells; (d) transfecting the activated T cells with a viral vector that expresses a chimeric antigen receptor to obtain transfected T cells; (e) removing the virus from the transfected T cells to obtain virus-free T cells; and (f) expanding and culturing the virus-free T cells to obtain chimeric antigen receptor T cells.

Description

Technical Field

[0001] The present invention relates to the field of biological technologies, and particularly to a method for preparing chimeric antigen receptor T cells by serum-free culture.

Background Art

[0002] Immunotherapy with chimeric antigen receptor T cells is a new treatment technology that endows T cells with scFv fragments and intracellular signal domains, and specifically recognizes tumor-associated antigens by means of gene editing, enhancing the targeting, killing, and durability of T cells. In recent years, chimeric antigen receptor T cell immunotherapy has shown good performance in clinical tumor immunotherapy, bringing hope for the clinical cure of tumors.

[0003] The preparation process of chimeric antigen receptor T cells is relatively complex and involves many operations such as cell activation, screening, transfection, and proliferation culture. Any inadequacy in the connection, such as the process flow, equipment, and reagent selection, has a significant impact on the quality of cell preparation, which in turn affects the viability, yield, safety, and subsequent clinical effects of chimeric antigen receptor T cells.

[0004] Serum is an important component in the conventional cell culture process, but it contains substances that are toxic and have side effects on cells. Furthermore, the quality of serum varies greatly from batch to batch. In addition, there is a possibility that mycoplasma, viruses, and other risks are introduced during the process of serum collection, which greatly hinders the development of cell therapy.

[0005] Currently, there is no satisfactory and efficient process for the serum-free preparation of commercially available chimeric antigen receptor T cells, which has a profound impact on the further development, promotion, and application of chimeric antigen receptor T cell immunotherapy.

[0006] Therefore, it is urgently needed in the art to develop a safe and efficient method for preparing chimeric antigen receptor T cells based on serum-free cell culture.

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a safe and efficient method for preparing chimeric antigen receptor T cells based on serum-free cell culture.

Means for Solving the Problems

[0008] In a first aspect of the present invention, a serum-free method for preparing chimeric antigen receptor T cells is provided: (a) providing PBMC cells resuspended in a serum-free medium; (b) performing negative sorting treatment on the PBMC cells in step (a) to obtain sorted PBMC cells; (c) activating the sorted PBMC cells obtained in step (b) to obtain activated T cells; (d) performing gene transfection on the activated T cells obtained in step (c) using a viral vector expressing a chimeric antigen receptor to obtain transfected T cells; (e) removing the virus from the transfected T cells to obtain virus-removed T cells; and (f) resuspending the virus-removed T cells in a growth medium and performing growth culture to obtain chimeric antigen receptor T cells, and collecting the chimeric antigen receptor T cells when the chimeric antigen receptor T cells reach a predetermined amount, wherein the growth medium is a serum-free medium containing cytokines, this step.

[0009] In another preferred embodiment, the serum-free medium is used in all steps of the method.

[0010] In another preferred embodiment, in step (a), the PBMC cells are resuscitated PBMC cells.

[0011] In another preferred embodiment, the PBMC cells are autologous or allogeneic.

[0012] In another preferred aspect, the PBMC cells are of human origin.

[0013] In another preferred embodiment, in step (b), the negative selection process uses CliniMACS.

[0014] In another preferred embodiment, in step (c), the anti-CD3 / CD28 antibody for activation performs an activation process.

[0015] In another preferred embodiment, in step (c), the activation process is performed in an activation medium containing a basic medium and an additive.

[0016] In another preferred embodiment, the basic medium is selected from the following group: LONZA X-VIVO, LIFE CTS AIM V, LIFE OpTmizer SFM, RPMI 1640, ImmunoCult™-XF, and Stemlineae. In another preferred embodiment, the additive is selected from the following group: human serum albumin, recombinant human serum albumin, plant-derived recombinant albumin, or a combination thereof, preferably recombinant albumin.

[0017] In another preferred embodiment, the albumin concentration is 0.01% - 50% (W / V), preferably 0.05% - 30% (W / V), more preferably 0.1% - 20% (W / V).

[0018] In another preferred embodiment, the activation process is selected from the following group: antibody coating process, free antibody process, activated magnetic bead process, or a combination thereof.

[0019] In another preferred embodiment, in the activation process, the antibody concentration is 10 ng / ml - 100 ng / ml.

[0020] In another preferred embodiment, in the activation treatment, the ratio of the number of activation magnetic beads to the cells is 0.25 to 5:1, preferably 0.5 to 3:1.

[0021] In another preferred embodiment, the time of the activation treatment is 3 to 8 days.

[0022] In another preferred embodiment, in step (d), the viral vector is selected from the following group: lentivirus, adeno-associated virus (AAV), adenovirus, or a combination thereof.

[0023] In another preferred embodiment, the multiplicity of infection (MOI) of gene transfection is 0.5 to 30, preferably 1 to 20, more preferably 2 to 10.

[0024] In another preferred embodiment, in step (e), the virus removal treatment includes the steps of resuspending the transfected cells in a serum-free medium, centrifuging the cells, and aspirating the supernatant to obtain virus-removed T cells.

[0025] In another preferred embodiment, in step (f), the predetermined amount is 1×10 9 ~1×10 11 Preferably 2×10 9 ~2×10 10 .

[0026] In another preferred embodiment, the growth medium in step (f) further contains cytokines.

[0027] In another preferred embodiment, the cytokines are selected from the following group: IL-2, IL-15, IL-7, or a combination thereof.

[0028] In another preferred embodiment, the concentration of each cytokine is independently 1 to 100 ng / ml, preferably 2 to 80 ng / ml, more preferably 5 to 50 ng / ml.

[0029] In another preferred embodiment, in step (f), the expansion culture is carried out in a container selected from the following group: a culture flask, a culture bag, or a combination thereof.

[0030] In another preferred embodiment, the culture flask is selected from the following group: a G-Rex culture flask, a T75 culture flask, or a combination thereof.

[0031] In another preferred embodiment, the container is a G-Rex culture flask.

[0032] In another preferred embodiment, the time of the expansion culture is 8 to 14 days.

[0033] In another preferred embodiment, in step (f), in the expansion culture, the operation of replacing the container is not performed.

[0034] In another preferred embodiment, the chimeric antigen receptor targets a target selected from the following group: CD19, CD23, etc.

[0035] In a second aspect of the present invention, chimeric antigen receptor T cells are provided. The chimeric antigen receptor T cells are prepared by a serum-free method for preparing the chimeric antigen receptor T cells according to claim 1.

[0036] In a third aspect of the present invention, a cell preparation comprising (a) the chimeric antigen receptor T cells according to claim 2 and (b) a pharmaceutically acceptable carrier is provided.

[0037] In another preferred embodiment, the cell preparation is a liquid preparation (for example, for injection).

[0038] It should be understood that within the scope of the present invention, the aforementioned technical features of the present invention and the technical features (for example, embodiments) specifically described below can be combined with each other to form new or preferred technical solutions.

[0039] Due to space constraints in writing, they are not described one by one.

Brief Description of the Drawings

[0040]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0041] After extensive and detailed research, the inventors have developed for the first time a unique and innovative process for serum-free culture for preparing chimeric antigen receptor T cells. Based on the serum-free culture method of the present invention, not only can the success rate and yield of culturing chimeric antigen receptor T cells be increased, but also the toxic side effects of serum on CAR-T cells can be avoided, and the risks introduced due to serum can be significantly reduced or eliminated, helping to further develop and promote chimeric antigen receptor T cell immunotherapy. Thus, the present invention is completed.

[0042] <Definition> <Method> In the present invention, positive selection or negative selection can be used. A preferred selection method is a selection method based on MACS. For example, the CliniMACS technology can be used for selection.

[0043] MACS is a highly specific cell sorting technology. Its main components include MACS microspheres, MACS sorting columns, and MACS separators. MACS microspheres are superparamagnetic particles conjugated with highly specific monoclonal antibodies. The MACS sorting column is placed within the MACS separator with a permanent magnetic field. Negative selection is a method of removing magnetic markers in non-target cells from a cell mixture, that is, non-magnetically labeled cells are target cells.

[0044] Preferably, in the method of the present invention, to obtain the desired target cells, the CliniMACS or CliniMACSplus sorting technology (and devices) is employed for negative selection.

[0045] Typically, the sorted cells in the present invention are essentially composed of CD3-positive cells.

[0046] <Chimeric Antigen Receptor (CAR)> As used herein, a chimeric antigen receptor (CAR) includes an extracellular domain, any hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes any signal peptide and a target-specific binding element (also known as an antigen-binding domain). The intracellular domain includes a co-stimulatory molecule and a ζ-chain moiety. When a CAR is expressed in a T cell, after the extracellular segment recognizes a specific antigen, it can transmit a signal through the intracellular domain, causing cell activation and proliferation, cell lysis toxicity, secretion of cytokines such as IL-2 and IFN-γ, affecting tumor cells, preventing tumor cells from proliferating, or promoting them to be killed or affected in other ways, and reducing or eliminating the tumor mass in the patient. The antigen-binding domain is preferably fused with one or more intracellular domains derived from co-stimulatory molecules and the ζ-chain.

[0047] <Preparation> The present invention provides a cell preparation as specifically described in the above-mentioned third aspect. In one embodiment, the cell preparation comprises (a) the chimeric antigen receptor T cells described in the second aspect of the present invention, and (b) a pharmaceutically acceptable carrier. In one embodiment, the cell preparation is a liquid preparation (e.g., an injection).

[0048] The present invention has the following main advantages: (a) Compared with conventional cell culture, the culture method of the present invention adopts a culture system such as a serum-free medium and G-Rex, avoids the introduction of substances with toxic side effects on cells, and greatly improves the success rate and safety of culturing chimeric antigen receptor T cells.

[0049] (b) The culture method of the present invention adopts a specially optimized process flow, thereby significantly improving the effectiveness of culturing chimeric antigen receptor T cells, particularly the positive rate and yield of the collected CAR-T cells.

[0050] The present invention will be further described in connection with specific embodiments. It should be understood that these embodiments are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods generally follow conventional conditions (e.g., the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or the conditions recommended by the manufacturer without indicating specific conditions in the following embodiments. Unless otherwise specified, percentages and parts are by weight percentage and parts by weight.

Examples

[0051] <Reagents> Serum-free medium: Basal medium + albumin

Examples

[0052] <Medium selection> 1.1 Frozen or fresh PBMC A cell count of 300×10 6 ~1000×10 6 of frozen or fresh PBMC is collected and resuspended in serum-free medium.

[0053] 1.2 Sorting Using CD19 and CD14-labeled magnetic beads, PBMC are labeled for 30 minutes, incubated, and sorted on CliniMACS as needed to set up the necessary pipes. After completion of cell incubation, a negative sorting operation is performed to remove CD19 and CD14-labeled impurity cells, and sorted cells mainly composed of CD3-positive cells are obtained.

[0054] 1.3 Cell activation The cells obtained from sorting are activated using CD3 / CD28, and inoculated and cultured for 2 days at a cell concentration of 3×10 6 ~6×10 6 / ml using serum-free medium (supplemented with IL-2) containing 1.0% albumin at the final concentration.

[0055] 1.4 Gene transduction The cells are centrifuged, the supernatant is discarded, the required amount of lentivirus is added according to MOI 2-10, resuspended in serum-free medium (containing 1.0% albumin at the final concentration), then transferred to a culture flask, and cultured at 37°C and 5% CO2 (for 12 - 48 hours).

[0056] Here, the lentivirus is a viral vector expressing the target CAR gene.

[0057] 1.5 Virus removal The cell suspension is centrifuged at 200 - 300 g for 6 - 8 minutes. The supernatant is discarded, the cells are resuspended, then the cells are transferred to serum-free medium (containing 1.0% albumin at the final concentration), and IL-2 is added to a concentration of 50 - 500 IU / ml, thereby obtaining virus-removed T cells.

[0058] 1.6 Growth medium Transfer the virus-removed T cells to a G-Rex culture flask and perform proliferation culture at 37°C and 5% CO2.

[0059] 3 to 10 days after the proliferation culture, take out the G-Rex flask from the incubator, mix the cells well, collect a sample, count the cells, supplement IL-2 to a concentration of 50 - 500 IU / ml, and continue the culture and collect when the cell yield reaches 1×10 9 ~1×10 10 CAR-T cells.

[0060] 1.7 Results In the collected cells, it was confirmed that the CAR-T positive rate exceeded 20%.

Example

[0061] <Selection of experimental consumables> 2.1 PBMC Resuscitation, Selection, Cell Activation and Gene Transfer The same method as in Example 1 was used for the resuscitation, selection, cell activation, and gene transfer of cryopreserved PBMC.

[0062] 2.2 Virus Removal Centrifuge the cell suspension at 200 - 300 g for 6 - 8 minutes. Discard the supernatant, flick to resuspend the cells, and obtain virus-removed T cells.

[0063] 2.3 Proliferation Medium Transfer the virus-removed T cells to a medium (OpTmizer SFM + 1.0% albumin), add IL-2 (final concentration 25 IU / ml), transfer to a G-Rex culture flask, culture bag, or T75 culture flask, and continue the culture at 37°C and 5% CO2.

[0064] After culturing for 3 days, collect samples from the G-Rex culture flask, culture bag, and T75 culture flask respectively, measure the cell count, and record the cell density and cell viability. The samples are stored for inspection.

[0065] Supplement with IL-2 (final concentration 25 IU / ml), return to the incubator, and continue culturing.

[0066] After culturing for 1 day, samples are taken from the G-Rex culture flask, culture bag, and T75 culture flask respectively, the cell count is measured, and the cell density and cell viability are recorded. The samples are stored for inspection.

[0067] After culturing for 2 days, samples are taken from the G-Rex culture flask, culture bag, and T75 culture flask respectively, the cells are counted, and the cell density and cell viability are recorded. The samples are stored for inspection.

[0068] 2.4 Results As shown in Figure 2, the CAR-T cells had a faster cell growth rate from day 3 to day 5 in the G-Rex culture system than in the culture bag and culture flask. As shown in Figure 3, the CAR-T cells could maintain a high CAR positive rate in G-Rex and had a lower degree of descent than the cell culture bag and culture flask. That is, when a large number of CAR-T positive cells are required in a short time, G-Rex has the advantages of a faster growth rate and a higher positive rate than the cell culture bag and culture flask.

Example

[0069] Cell culture addition experiment 3.1 Method In this embodiment, different additives IL-2, IL-7, IL-15 or combinations thereof were added to the cell culture medium, and a total of 5 experimental groups were used.

Table 1

[0070] The same method as in Example 1 was used to perform the resuscitation, selection, magnetic bead labeling activation, gene introduction, magnetic bead removal, virus removal, and proliferation culture of cryopreserved PBMC.

[0071] 1) The medium used was OpTmizer SFM + albumin (final concentration was 1.0%).

[0072] 2) All steps of adding IL-2 were replaced with the cytokines corresponding to experimental groups 2 - 5 in Table 1 for culturing.

[0073] 3.2 Results As shown in Figure 4, the combination of cytokines IL-2, IL-7, and IL-15 in the culture system had the most excellent effect on increasing cell proliferation and helped increase the amount of CAR-T cells.

Example

[0074] <Comparison experiment of positive selection and negative selection> 4.1 Test object: Using the positive selection method and the negative selection method respectively, the effects of the same PBMC on cell culture were compared.

[0075] 4.2 Cell selection: The revived PBMC was resuspended in the selection buffer, divided into two equal parts, and negative selection CD19 + CD14 + and positive selection CD3 + were performed respectively.

[0076] 4.3 Cell activation: The selected cells were inoculated with the medium, activated magnetic beads were added, mixed well and cultured.

[0077] 4.4 Virus transfection: When the number of activated cells reached the second day of culture, the number of required lentiviral vectors was determined from the MOI (2 - 10), the corresponding lentiviral vectors were collected, resuspended evenly in the medium, centrifuged to remove the original cell supernatant, the lentiviral vector suspension was added and resuspended, and the culture was continued.

[0078] 4.5 Remove the virus and activate the magnetic beads: On the third day of culture, the virus is removed by centrifugation, the activated magnetic beads are removed by a magnetic lattice, and the culture medium is added to continue the culture.

[0079] The test was conducted on the eighth day of culture.

[0080] 4.6 Results The results are shown in Table 2 and Figure 5: TIFF0007717458000002.tifThe ratio of target cells collected from PBMC by the negative selection method was close to the ratio of target cells collected from PBMC by the positive selection method, but with respect to the cell growth process, the negative selection method showed a clear increase compared to the positive selection method. That is, the use of the negative selection method ultimately resulted in the recovery of more positive CD3 cells.

[0081] All documents mentioned in the present invention are hereby incorporated by reference as if each document was individually cited as a reference. Further, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and it should be understood that these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. A serum-free method for preparing chimeric antigen receptor T cells, the method comprising the following steps: (a) providing PBMC cells in a serum-free medium; (b) performing negative selection on the PBMC cells of step (a) to obtain selected PBMC cells containing more CD3-positive cells, the step of reducing CD19-positive cells and CD14-positive cells by the negative selection; (c) Activating the selected PBMC cells in step (b) at a cell concentration of 3×10 6 to 6×10 6 / ml for 3 to 8 days to obtain activated T cells; (d) transducing the activated T cells of step (c) using a viral vector expressing a chimeric antigen receptor to obtain transduced T cells; (e) removing the viral vector from the transduced T cells; and (f) proliferating the T cells obtained in step (e) in a proliferation medium, the proliferation medium being a serum-free medium.

2. The method according to claim 1, wherein the serum-free medium is used in all steps.

3. The method according to claim 1, wherein in step (c), the activation is performed in an activation medium containing a basic medium and an additive.

4. The method according to claim 3, wherein the basic medium is RPMI 1640.

5. The method according to claim 3, wherein the additive is selected from the group consisting of: recombinant albumin, human serum albumin, recombinant human serum albumin, plant-derived recombinant albumin, or combinations thereof.

6. The method according to claim 1, wherein the proliferation medium in step (f) further comprises a cytokine.

7. The method according to claim 6, wherein the cytokine is selected from the group consisting of: IL-2, IL-15, IL-7, or combinations thereof.

8. The method according to claim 1, wherein in step (f), the T cells are proliferated in a container selected from the group consisting of a culture flask, a culture bag, or combinations thereof.

Images