METHOD FOR PREPARING CHIMERIC ANTIGEN RECEPTOR T CELLS BY SERUM-FREE CULTURE

Abstract

Provided is a method of preparing chimeric antigen receptor T cells by serum-free culture. The method comprises steps of: (a) providing PBMC cells; (b) performing negative sorting treatment on the PBMC cells to obtain sorted PBMC cells; (c) activating the sorted PBMC cells to obtain activated T cells; (d) transfecting the activated T cells with a viral vector expressing a chimeric antigen receptor, so as to obtain transfected T cells; (e) removing the viruses from the transfected T cells to obtain virus-removed T cells; and (f) subjecting the virus-removed T cells to expansion culture to obtain chimeric antigen receptor T cells.

Claims

1. A serum-free method of preparing chimeric antigen receptor T cells, wherein the method comprises steps of: (a) providing PBMC cells, which are resuspended in a serum-free medium; (b) performing negative sorting treatment on the PBMC cells at step (a) to obtain sorted PBMC cells; (c) activating the sorted PBMC cells obtained at step (b) to obtain activated T cells; (d) performing gene transfection on the activated T cells obtained at step (c) with a viral vector expressing a chimeric antigen receptor to obtain transfected T cells; (e) removing the viruses from the transfected T cells to obtain virus-removed T cells; and (f) resuspending the virus-removed T cells in an expansion medium and performing expansion culture to obtain chimeric antigen receptor T cells, and harvesting the chimeric antigen receptor T cells when the chimeric antigen receptor T cells reach a predetermined quantity, wherein the expansion medium is a serum-free medium containing a cell factor.

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

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

4. The method according to claim 1, wherein the basic medium is selected from the following group: LONZA X-VIVO, LIFE CTS AIM V, LIFE OpTmizer SFM, RPMI 1640, ImmunoCult-XF and Stemline.

5. The method according to claim 1, wherein 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.

6. The method according to claim 1, wherein the expansion medium at step (f) further contains a cell factor

7. The method according to claim 1, wherein the cell factor is selected from the following group: IL-2, IL-15, IL-7 or a combination thereof.

8. The method according to claim 1, wherein at step (f), the expansion culture is performed in a container, which is selected from the following group: a culture flask, a culture bag, or a combination thereof.

9. Chimeric antigen receptor T cells, wherein the chimeric antigen receptor T cells are prepared by the serum-free method of preparing chimeric antigen receptor T cells as described in claim 1.

10. A cell preparation, wherein the cell preparation contains (a) the chimeric antigen receptor T cells in claim 2 and (b) a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 shows changes in a growth curve of CAR-T cells in different media.

[0048] FIG. 2 shows changes in a growth curve of CAR-T cells in a G-Rex culture flask, a culture bag and a T75 culture flask.

[0049] FIG. 3 shows changes in a positive rate curve of CAR-T cells in a G-Rex culture flask, a culture bag and a T75 culture flask.

[0050] FIG. 4 shows effects of different cell culture additives on the proliferation of CAR-T cells.

[0051] FIG. 5 shows the proliferation rates of cells in different sorting methods.

DETAILED DESCRIPTION

[0052] After extensive and in-depth research, the inventors developed a unique and innovative process of serum-free culture for preparing chimeric antigen receptor T cells for the first time. Based on the serum-free culture method of the present invention, not only the success rate and yield of culture of chimeric antigen receptor T cells can be raised but also the toxic side effects of serum on CAR-T cells can be avoided and the risks introduced due to serum can be reduced significantly or eliminated, so as to provide help for the further development and promotion of chimeric antigen receptor T cell immunotherapy. On this basis, the present invention is completed.

Terminology

[0053] Method

[0054] In the present invention, positive sorting or negative sorting can be used. A preferred sorting method is a sorting method based on MACS. For example, CliniMACS technology can be used for sorting.

[0055] MACS is a highly specific cell sorting technology. The main components include MACS microspheres, an MACS sorting column and an MACS separator. The MACS microspheres are superparamagnetic particles coupled with highly specific monoclonal antibodies. The MACS sorting column is placed in an MACS separator of a permanent magnetic field. The negative sorting is a method of removing magnetic markers in non-target cells from a cell mixture, that is, non-magnetically labeled cells are target cells.

[0056] Preferably, the method of the present invention adopts CliniMACS or CliniMACSplus sorting technology (and device) for negative sorting, so as to obtain the desired target cells.

[0057] Typically, the sorted cells in the present invention are essentially composed of CD3 positive cells.

[0058] Chimeric Antigen Receptor (CAR)

[0059] As used herein, a chimeric antigen receptor (CAR) comprises an extracellular domain, an optional hinge region, a transmembrane domain and an intracellular domain. The extracellular domain comprises optional signal peptides and target-specific binding elements (also known as antigen binding domains). The intracellular domain comprises costimulatory molecules and chain parts. When CAR is expressed in T cells, the extracellular segment can recognize a specific antigen and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity, and secretion of cell factors such as IL-2 and IFN-, affecting tumor cells, causing the tumor cells to not grow, or to be promoted to die or affected in other ways, and leading to a reduced or eliminated tumor burden on the patient. The antigen binding domain is preferably fused with one or more intracellular domains from the costimulatory molecule and the chain.

[0060] Preparation

[0061] The present invention provides a cell preparation, specifically as described in the third aspect of the present invention. In an implementation manner, the cell preparation contains

[0062] (a) the chimeric antigen receptor T cells described in the second aspect of the present invention and (b) a pharmaceutically acceptable carrier. In an implementation manner, the cell preparation is a liquid preparation (e.g., an injection).

[0063] The present invention has the following main advantages:

[0064] (a) Compared with conventional cell culture, the culture method of the present invention adopts culture systems such as serum-free medium and G-Rex to avoid introducing substances that have toxic side effects on cells and to greatly improve the success rate and safety of the culture of chimeric antigen receptor T cells.

[0065] (b) The culture method of the present invention adopts a specially optimized process flow, thereby significantly improving the effectiveness of the culture of chimeric antigen receptor T cells, particularly the positive rate and yield of harvested CAR-T cells.

[0066] The present invention will be further described in conjunction with specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention only and not to limit the scope of the present invention. The experimental methods, without indicating specific conditions in the following embodiments, normally follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight.

[0067] Reagent

[0068] Serum-free medium: basic medium+albumin

EMBODIMENT 1

Medium Selection

[0069] 1.1 Cryopreserved or Fresh PBMC

[0070] Take cryopreserved or fresh PBMC with a cell count of 30010.sup.6 to 100010.sup.6 and resuspend the PBMC in a serum-free medium.

[0071] 1.2 Sorting

[0072] Use CD19 and CD14 labeling magnetic beads to label and incubate the PBMC for 30 minutes, install the pipes needed by sorting on CliniMACS according to the requirements, and after completion of the cell incubation, perform the operation of negative sorting to remove CD19- and CD14-labeled impurity cells and obtain sorted cells with CD3 positive cells as the main component.

[0073] 1.3 Cell Activation

[0074] Use CD3/CD28 to activate the cells obtained from sorting and then use a serum-free medium (supplemented with IL-2) containing albumin at a final concentration of 1.0% to perform inoculated culture at a cell density of 310.sup.6 to 610.sup.6/ml for 2 days.

[0075] 1.4 Gene Transduction

[0076] Centrifuge the cells, discard the supernatant, then add the required volume of lentivirus according to MOI 2-10, resuspend in a serum-free medium (containing albumin at a final concentration of 1.0%) and then transfer the liquid to a culture flask and culture it at 37 C., 5% CO.sub.2 (12 to 48 hours).

[0077] Here, the lentivirus is a viral vector expressing the target CAR gene.

[0078] 1.5 Virus Removal

[0079] Centrifuge the cell suspension at 200 to 300 g for 6 to 8 minutes. Suck and discard the supernatant and resuspend the cells, then transfer the cells to a serum-free medium (containing albumin at a final concentration of 1.0%) and add IL-2 to a concentration of 50 to 500 IU/ml, thereby obtaining virus-removed T cells.

[0080] 1.6 Expansion Culture

[0081] Transfer the virus-removed T cells to a G-Rex culture flask and perform expansion culture at 37 C., 5% CO.sub.2.

[0082] After 3 to 10 days of expansion culture, take the G-Rex bottle out of the incubator, mix the cells well, take a sample and count the cells, supplement IL-2 to a concentration of 50 to 500 IU/ml, continue the culture and harvest when the cell yield reaches 110.sup.9 to 110.sup.10 CAR-T cells.

[0083] 1.7 Results

[0084] It was determined that in the harvested cells, the CAR-T positive rate was greater than 20%.

EMBODIMENT 2

Selection of Experimental Consumables

[0085] 2.1 PBMC Resuscitation, Sorting, Cell Activation and Gene Transduction

[0086] Use the same method as in Embodiment 1 for resuscitation, sorting, cell activation, gene transduction of cryopreserved PBMC.

[0087] 2.2 Virus Removal

[0088] Centrifuge the foregoing cell suspension at 200 to 300 g for 6 to 8 minutes. Suck and discard the supernatant, and flick to resuspend the cells, so as to obtain virus-removed T cells.

[0089] 2.3 Expansion Culture

[0090] Transfer the virus-removed T cells to a medium (OpTmizer SFM+1.0% albumin), add IL-2 (the final concentration is 25 IU/ml), then transfer to a G-Rex culture flask, a culture bag and a T75 culture flask respectively and then continue the culture at 37 C., 5% CO.sub.2.

[0091] After continuing the culture for three days, take samples from the G-Rex culture flask, the culture bag and the T75 culture flask respectively, count the cells and record cell density and cell viability. Reserve samples for testing.

[0092] Supplement IL-2 (the final concentration is 25 IU/ml) and then put back into the incubator for continued culture.

[0093] After continuing the culture for one day, take samples from the G-Rex culture flask, the culture bag and the T75 culture flask respectively, count the cells and record cell density and cell viability. Reserve samples for testing.

[0094] After continuing the culture for two days, take samples from the G-Rex culture flask, the culture bag and the T75 culture flask respectively, count the cells and record cell density and cell viability. Reserve samples for testing.

[0095] 2.4 Results

[0096] As shown in FIG. 2, the CAR-T cells showed a faster cell proliferation rate from Day 3 to Day 5 in the G-Rex culture system than in the culture bag and the culture flask; as shown in FIG. 3, the CAR-T cells were able to maintain a high level of CAR positive rate in G-Rex and the decent degree was smaller than those in the cell culture bag and the culture flask; in summary, if a large number of CAR-T positive cells are needed in a short time, G-Rex has the advantage of a higher proliferation rate and a higher positive rate over the cell culture bag and culture flask.

EMBODIMENT 3

Cell Culture Additive Experiment

[0097] 3.1 Method

[0098] In this embodiment, different additives IL-2, IL-7, IL-15 or a combination thereof were added to a cell culture medium, and there were five experimental groups in total.

TABLE-US-00001 TABLE 1 Experi- Experi- Experi- Experi- Experi- mental mental mental mental mental group 1 group 2 group 3 group 4 group 5 IL-2 + + + + IL-7 + + + IL-15 + + +

[0099] Use the same method as in Embodiment 1 to perform resuscitation, sorting, magnetic bead-labeled activation, gene transduction, magnetic bead removal, virus removal and expansion culture of cryopreserved PBMC.

[0100] 1) The medium used was OpTmizer SFM+albumin (the final concentration was 1.0%).

[0101] 2) All the steps of adding IL-2 were replaced with the cell factors corresponding to experimental groups 2 to 5 in Table 1 for culture.

[0102] 3.2 Results

[0103] As shown in FIG. 4, combination of cell factors IL-2, IL-7 and IL-15 in a culture system had the best effect on increase of cell proliferation and helped to increase the quantity of CAR-T cells.

EMBODIMENT 4

Comparative Experiment of Positive Sorting and Negative Sorting

[0104] 4.1 Study Object:

[0105] Comparison of the effects of the same PBMC on cell culture using a positive sorting method and a negative sorting method respectively.

[0106] 4.2 Cell Sorting:

[0107] Resuspend the resuscitated PBMC in a sorting buffer and divide it into two equal parts and perform negative sorting CD19+CD14+ and positive sorting CD3+ respectively.

[0108] 4.3 Cell Activation:

[0109] Inoculate the sorted cells with a culture medium, add activating magnetic beads, mix them well and culture them.

[0110] 4.4 Virus Transfection:

[0111] Calculate the number of activated cells when the culture reaches the second day, calculate the required number of lentiviral vectors based on the MOI (210), take the corresponding lentiviral vector, evenly resuspend it in a culture medium, centrifuge to remove the original cell supernatant and add the lentiviral vector suspension for resuspension and continue the culture.

[0112] 4.5 Remove Virus and Activating Magnetic Beads:

[0113] On the third day of the culture, remove the virus by centrifugation and remove the activating magnetic beads by a magnetic grate and add a culture medium to continue the culture.

[0114] Conduct testing on the eighth day of the culture.

[0115] 4.6 Results

[0116] The results are as shown in Table 2 and FIG. 5:

TABLE-US-00002 TABLE 2 After sorting The 8.sup.th day of culture Red Red blood blood CD3+ CD19+ CD14+ cell Others CD3+ CD19+ CD14+ cell Others Negative 60% 2% 1% 34% 3% 98% 1% 0% 0% 1% sorting Positive 100% 0% 0% 0% 0% 100% 0% 0% 0% 0% sorting

[0117] The ratio of target cells collected from PBMC by the method of negative sorting was close to the ratio of target cells collected from PBMC by the method of positive sorting, but as for the process of cell proliferation, the method of negative sorting showed an obvious increase compared with the method of positive sorting. In summary, the use of the method of negative sorting eventually led to harvesting of more positive cells CD3.

[0118] All the documents mentioned in the present invention have been cited herein as references, as if each document is individually cited as a reference. Further, it should be understood that after reading the above-taught content of the present invention, those skilled in the art may make various changes or modifications to the present invention and these equivalent forms will also fall within the scope delimited by the claims of the present application.