T cell culture method

Abstract

The invention relates to a T cell culture method, engineered T cells are cultured by using micron-sized CD3 / 28 double diamagnetic beads and human platelet lysates, the viability of the engineered T cells can be effectively improved, and the T cell culture method has a huge application prospect in the field of immune cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of immune cell therapy, and in particular to a T cell culture method. Background Art

[0002] The use of engineered T cells to treat cancer began in the 1980s. Chimeric antigen receptor T cells (CAR-T) and T cell receptor engineered T cells (TCR-T) are T cells that have foreign genes introduced into them, enabling T cells to specifically kill target cells such as tumor cells. In recent years, adoptive cell immunotherapy has developed rapidly. Some CAR-T products have been approved in China, the United States, and Europe, and TCR-T products are also advancing rapidly. Although different CAR-T and TCR-T products differ in terms of targeted antigens, scFv acquisition, CAR or TCR molecule construction, and clinical strategies, better culture processes are crucial to the development of various CAR-T and TCR-T products. Summary of the invention

[0003] The applicant found during the experiment that the use of micron-sized CD3 / 28 dual anti-magnetic beads and CTS that have the function of sorting and activating T cells TM When T cells are cultured with immune cell serum substitutes (SR) or human ABS serum, the viability of T cells decreases significantly on the first and second days, especially the viability of T cells from tumor patients. However, when T cells are activated and cultured with nano-scale CD3 / 28 dual-antibody magnetic beads plus serum substitutes (SR) or human ABS serum, the problem of decreased T cell viability will not occur.

[0004] Based on the discovery of the above technical problems, the applicant used human platelet lysate to replace CTS TM Using immune cell serum replacement (SR) or human ABS serum to culture T cells can significantly increase T cell viability.

[0005] The present application provides a T cell culture method, characterized by comprising:

[0006] S1: CD3 / 28 dual anti-magnetic beads and human platelet lysate (hPLT) were added to the culture medium for T cell culture;

[0007] S2: Perform T cell gene modification operations to prepare engineered T cells;

[0008] S3: expanding and culturing the engineered T cells until harvest;

[0009] The CD3 / 28 dual anti-magnetic beads are in the micron range in diameter and have the functions of sorting and activating T cells.

[0010] Beneficial technical effects:

[0011] The T cell culture method of the present application can significantly improve the viability of the prepared engineered T cells, especially the viability of engineered T cells derived from tumor patients, and has great application prospects in the field of adoptive cell immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The corresponding CAR-T cell viability of hPLT, SR, and hPLT+SR groups.

[0013] Figure 2 CAR gene transduction rates on the sixth day in hPLT, SR, and hPLT+SR groups.

[0014] Figure 3 This is the memory phenotype of CAR-T cells in the hPLT, SR, and hPLT+SR groups on the sixth day.

[0015] Figure 4 Comparison of CAR-T cell viability in hPLT, SR, and ABS groups

[0016] Figure 5 is the CAR-T cell viability in different additive and culture medium groups.

[0017] Figure 6 The effect of different hPLT concentrations on the viability of CAR-T cells. DETAILED DESCRIPTION

[0018] In order to make the present invention more easily understood, certain technical and scientific terms of the present invention are explained before describing the embodiments.

[0019] Unless explicitly defined otherwise elsewhere in the present application document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0020] CD3 / CD28 dual anti-magnetic beads are superparamagnetic beads coated with anti-CD3 and anti-CD28 antibodies. There are micron-sized magnetic beads and nano-sized magnetic beads. CD3 / CD28CTS TM (Cat. No. 40203D) CTS (Cell Therapy System) Dynabeads CD3 / CD28 is a sterile, pyrogen-free suspension at a concentration of 4 x 10 in phosphate buffered saline (PBS). 8 / mL Dynabeads, diameter 4.5μm. CD3 / CD28 sorting activation magnetic beads, with a diameter of 4.5μm. Beijing Biopsies Biotechnology's Human T cell Activation / ExpansionCD3 / CD28 Beads, with a diameter of 5.5μm. The above three micron-sized magnetic beads have the dual functions of sorting and activating T cells, that is, they can be used for both sorting T cells and activating T cells. In addition, some commercial magnetic beads are nano-sized magnetic beads, such as Miltenyi's CD3 / CD28 magnetic beads.

[0021] CTS TM Immune cell serum replacement (SR) is designed to be added to basal cell culture media (such as CTS TM OpTmizer TM T cell expansion SFM or CTS TM AIM-V TM SFM) to support the in vitro culture and expansion of human immune cells (such as T cells). CTS TM ImmuneCell SR is a defined, xeno-free formulation that contains no bovine or other non-human animal derived components and is FDA registered by the manufacturer to support the expansion of a variety of polyclonal T cells (CD3 / CD28 bead activation and TILs), T cell phenotypes (CD4, CD8, and CD62L), and genetically modified CAR-T cells (lentiviral transduction).

[0022] Human platelet lysate refers to the contents released from platelets by various methods (such as freeze-thaw cycles that cause platelet lysis), including autologous or commercial. Specifically, human platelet lysate can be produced from buffy coat, platelet-rich plasma (PRP), or platelet concentrates derived from whole blood or apheresis. Platelets are usually lysed by a freeze-thaw process. Human platelet lysate can be further manufactured to inhibit coagulation factors (such as depleting fibrin). Autologous human platelet lysate is extracted from the T cell donor himself. Human platelet lysate can also be a commercial product of many manufacturers, including AventaCell BioMedical, MillCreek Life Sciences, Cook Regentec and Compass Biomedical, etc. They provide products in clinical or research grades. Clinical grade products have been filed with the FDA for drug master files, such as nLiven PR (Cook Regentec) and PLTGOLD (MILL CREEK).

[0023] The T cell culture method of the present application, in some embodiments, comprises:

[0024] S1: CD3 / 28 dual anti-magnetic beads and human platelet lysate (hPLT) were added to the culture medium for T cell culture;

[0025] S2: Perform T cell gene modification operations to prepare engineered T cells;

[0026] S3: expanding and culturing the engineered T cells until harvest;

[0027] The CD3 / 28 dual anti-magnetic beads are in the micron range in diameter and have the functions of sorting and activating T cells.

[0028] In some embodiments, the genetic modification operation is performed at the beginning of T cell culture. In some embodiments, the genetic modification operation is performed when the T cell culture does not exceed 6h, 12h or 24h. In some embodiments, the genetic modification operation is performed on the second, third or fourth day of T cell culture, preferably the genetic modification operation is performed on the second day of T cell culture.

[0029] In some embodiments, the expanded culture can be continued culture for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks after the engineering modification, or culture for no more than 3 weeks.

[0030] In some embodiments, the genetic engineering modification is selected from any one or a combination of gene introduction, gene knockout or gene knockdown. In some embodiments, the gene introduction is chimeric antigen receptor gene introduction, and / or T cell receptor gene introduction.

[0031] In some embodiments, the diameter of the micron dual-antimagnetic beads is 4.5 μm-5.5 μm, preferably 4.5 μm or 5.5 μm.

[0032] In some embodiments, step S1 further comprises adding IL2. In some embodiments, the concentration of IL2 is 20-2000 IU / mL, preferably 50-300 IU / mL, preferably 100 IU / mL.

[0033] In some embodiments, the hPLT concentration is 0.003%-20%. In some embodiments, the hPLT concentration is 1%-5%. In some embodiments, the hPLT concentration is 2-3%. In some embodiments, the hPLT concentration is 2.5. In some embodiments, the hPLT concentration is 3%. The above percentages are all volume ratios.

[0034] In some embodiments, step S1 may further add one or more fetal bovine serum substitutes other than human platelet lysate, including CTS immune cell serum substitute, human plasma, human AB serum, cord blood serum, whole blood extract, human platelet lysate, and the like.

[0035] In some embodiments, the T cells are derived from tumor patients or autoimmune disease patients. In some embodiments, the T cells are derived from tumor patients, in some embodiments, the T cells are derived from solid tumor patients, and in some embodiments, the T cells are derived from hematological tumor patients.

[0036] Example 1 Comparison of the difference between platelet lysate and CTS immune cell serum substitute in CAR-T culture

[0037] This experiment studied the effect of platelet lysate (hPLT) and CTS immune cell serum replacement (A2596101) (SR) on the viability of CTS (Cell Therapy System) Dynabeads CD3 / 28 magnetic beads (Thermo Fisher Scientific, Catalog No. 40203D) (hereinafter referred to as Dynabeads) when activating T cells.

[0038] Single blood samples were collected from patients with advanced tumors, and peripheral blood mononuclear cells (hereinafter referred to as PBMC) were separated using Sepax C-pro cell separator (Cytiva).

[0039] PBMCs were resuspended in X-VIVO15 medium (Lonza) or DPBS and counted using AO / PI fluorescent dye. Washed and added with magnetic beads at a ratio of 1:1 Dynabeads: T cells, mixed by hand or on a rotating mixer at 1–3 rpm, and incubated at room temperature for half an hour. Placed in DynaMag TM Place the cells on the magnet stand and remove the cell solution that has not bound to the magnetic beads.

[0040] The cells were removed from the magnetic rack and resuspended in X-VIVO15 medium supplemented with 100 IU / mL IL-2. The additives were divided into 2.5% hPLT group, 5% hPLT group, 2.5% hPLT plus 2.5% SR group and 5% SR group according to the grouping. The proportions of the above additives were all by volume.

[0041] AO / PI fluorescent dyes were used for counting, and each group was adjusted to 2*10 6 / mL and placed in a carbon dioxide cell culture incubator.

[0042] On the second day, AO / PI fluorescent dye was used for counting, and lentivirus carrying chimeric antigen receptor (hereinafter referred to as CAR) was added. For the construction and sequence of CAR, refer to patent CN113527491A.

[0043] On the fourth day, cells were counted using AO / PI fluorescent dye.

[0044] On the sixth day, AO / PI fluorescent dye was used for counting. Flow dye was used to detect the proportion of CAR-T. PE anti-CD197 (CCR7) Antibody (Biolegend) and Brilliant Violet 421 anti-human CD45RA (Biolegend) were used to detect cell memory phenotype.

[0045] Through flow cytometry detection, the cell population with positive CAR gene transduction rate of T cells was gated, and the CAR-T cell surface markers CCR7 and CD45RA were further cross-gated. CAR-T cells can be divided into four major subpopulations. They are naive cells (Tnaive) (CD45RA+CCR7+), central memory cells (Tcm) (CD45RA-CCR7+), effector memory cells (Tem) (CD45RA-CCR7-) and terminally differentiated effector memory cells (Temra) (CD45RA+CCR7-) that re-express CD45RA.

[0046] The results of the viability in the first six days of the experiment showed that the viability of the three groups with hPLT was significantly improved in all time periods compared with the 5% SR group (Table 1 and Figure 1 The results of the transduction on the sixth day showed that hPLT could improve the transduction rate (as shown in Table 2 and Figure 2 shown).

[0047] Table 1. T cell viability of the second test in the hPLT, SR, and hPLT+SR groups of the same donor

[0048] 2.5% hPL 2.5% hPL + 2.5% SR 5% hPL 5% SR Survival rate on day 2 37.95％ 46.51％ 62.64％ 29.07％ Survival rate on day 4 86.01％ 84.94％ 92.81％ 63.42％ Survival rate on day 6 93.37％ 90.29％ 93.20％ 75.00％

[0049] Table 2. CAR gene transduction rate of hPLT, SR, and hPLT+SR groups on the sixth day

[0050] 2.5% hPLT 5% hPLT 2.5% hPLT + 2.5% SR 5% SR Transduction rate 70.45％ 85.10％ 84.61％ 74.51％

[0051] The results of the memory phenotype detection of CAR-T on the sixth day of the first experiment are shown in Table 3 and Figure 3As shown. Among them, Tnaive of CAR-T is the initial T cell population in CAR-T (CD45RA+CCR7+), Tcm of CAR-T is the central memory T cell population in CAR-T (CD45RA-CCR7+), Tem of CAR-T is the effector memory T cell population in CAR-T (CD45RA-CCR7-), and Temra of CAR-T is the terminally differentiated effector memory T cell (CD45RA+CCR7-) in CAR-T that re-expresses CD45RA. The total proportion of the four groups is 100%. Since each group may have omissions in numerical rounding, the total may have errors.

[0052] Table 3. Memory phenotype of CAR-T cells in hPLT, SR, and hPLT+SR groups on day 6

[0053]

[0054] The results showed that compared with the 5% SR group, the three groups with the addition of hPLT increased the proportion of CAR-T central memory cell subsets and reduced the proportion of terminally differentiated effector memory cells in which CAR-T re-expressed CD45RA.

[0055] Example 2 Comparison of the effects of hPLT, SR and human AB serum on viability when Dynabeads activated T cells

[0056] This experiment was used to study the effects of hPLT, SR and human AB serum (abbreviated as ABS) on T cell viability when activated by Dynabeads.

[0057] Single blood samples were collected from patients with advanced tumors, and PBMCs were isolated using a Sepax C-pro cell separator.

[0058] PBMCs were resuspended in X-VIVO15 medium or DPBS and counted using AO / PI fluorescent dye. Washed and added with magnetic beads at a ratio of 1:1 Dynabeads:T cells, shaken manually or placed on a rotating mixer at 1–3 rpm, and incubated at room temperature for half an hour. Placed on DynaMag TM Place the cells on the magnet stand and remove the cell solution that has not bound to the magnetic beads.

[0059] The cells were removed from the magnetic rack and resuspended in X-VIVO15 medium with 100 IU / mL IL-2 added. The additives were added according to the grouping, namely 3% hPLT group, 5% SR group and 5% ABS group.

[0060] AO / PI fluorescent dyes were used for counting, and each group was adjusted to 2*10 6 / mL and placed in a carbon dioxide cell culture incubator.

[0061] On the next day, AO / PI fluorescent dye was used for counting and CAR lentivirus was added. For CAR construction and sequence, refer to patent CN113527491A.

[0062] On the fourth day, cells were counted using AO / PI fluorescent dye.

[0063] On the sixth day, cells were counted using AO / PI fluorescent dye.

[0064] Table 4. Comparison of T cell viability in hPLT, SR, and ABS groups

[0065] PL SR ABS Survival rate on day 2 66.36％ 32.76％ 45.46％ Survival rate on day 4 94.57％ 56.71％ 68.05％ Survival rate on day 6 94.66％ 74.28％ 81.35％

[0066] The results are shown in Table 4 and Figure 4 As shown, the survival rate of the hPLT-added group was significantly improved compared with the 5% SR group and the 5% ABS group.

[0067] Example 3 Comparison of the effects of different culture media and different additives on the viability when Dynabeads activated T cells

[0068] This experiment studied the effects of Dynabeads on T cell proliferation and viability when the basal medium was X-VIVO15 or RPMI1640, both supplemented with 100 IU / mL IL-2, and the additives were hPLT, SR, ABS or fetal bovine serum (hereinafter referred to as FBS).

[0069] Single blood samples were collected from patients with advanced tumors, and PBMCs were isolated using a Sepax C-pro cell separator. PBMCs were resuspended in X-VIVO15 medium or DPBS and counted using AO / PI fluorescent dye.

[0070] According to the instructions, CD3+ T cells were sorted using Miltenyi's CD3 sorting magnetic beads and magnetic rack.

[0071] The cells were resuspended by centrifugation. The solutions for each group are shown in Table 5 below.

[0072] Table 5. Composition of culture medium and supplements

[0073]

[0074] AO / PI fluorescent dyes were used for counting, and each group was adjusted to 2*10 6 / mL, wash and add magnetic beads at a ratio of 1:1 Dynabeads: T cells. Place in a carbon dioxide cell culture incubator.

[0075] On the second day, CAR lentivirus was added. For CAR construction and sequence, refer to patent CN113527491A.

[0076] On the third day, cells were counted using AO / PI fluorescent dye.

[0077] On the fifth day, cells were counted using AO / PI fluorescent dye.

[0078] Table 6. T cell viability in different supplement and culture medium groups

[0079]

[0080] The results showed that the survival rate of the group with hPLT was significantly higher than that of the group without hPLT, as shown in Table 6 and Figure 5 shown.

[0081] Example 4 Effects of different concentrations of hPLT and 5% SR on viability when Dynabeads activated T cells

[0082] This experiment studies the effect of hPLT gradient test at different concentrations on T cell proliferation and viability when Dynabeads is used to activate T cells. The basal medium is X-VIVO15, and the additives are hPLT, SR and a combination of the two at different concentrations.

[0083] PBMCs from healthy volunteers were used, resuspended in DPBS, and counted using AO / PI fluorescent dye.

[0084] According to the instructions, CD3+ T cells were sorted using Miltenyi's CD3 sorting magnetic beads and magnetic rack.

[0085] AO / PI fluorescent dyes were used for counting, and each group was adjusted to 2*10 6 / mL, wash and add magnetic beads at a ratio of 1:1 Dynabeads: T cells. Place in a carbon dioxide cell culture incubator.

[0086] The next day, add CAR lentivirus.

[0087] On the third day, cells were counted using AO / PI fluorescent dye.

[0088] On the fifth day, cells were counted using AO / PI fluorescent dye.

[0089] Table 7. Effects of different hPLT concentrations on T cell viability

[0090] Survival rate on day 3 Survival rate on day 5 hPLT 0.003% + SR 2.997% 71.05％ 91.90％ hPLT 0.015% + SR 2.985% 70.18％ 90.27％ hPLT 0.075%+SR 2.925% 72.05％ 90.88％ hPLT 0.750% 72.03％ 93.72％ hPLT 0.750% + SR 2.25% 70.03％ 94.38％ hPLT 3.000% 70.03％ 94.38％ hPLT 6.000% 82.49％ 96.44％ hPLT 9.000% 83.28％ 96.55％ hPLT 12.000% 83.04％ 96.07％ hPLT 20.000% 83.65％ 96.45％ hPLT 40.000% 83.29％ 93.39％ hPLT 60.000% 82.52％ 93.70％ hPLT 70.000% 83.58％ 87.89％ SR 5% 70.19％ 85.62％

[0091] The survival rate of the different groups with added hPLT was significantly higher than that of the 5% SR group. The test results are shown in Table 7 and Figure 6 shown.

[0092] In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A T cell culture method, characterized in that: include: S1: CD3 / 28 dual anti-magnetic beads and human platelet lysate (hPLT) were added to the culture medium for T cell culture; S2: Perform T cell gene modification operations to prepare engineered T cells; S3: expanding and culturing the engineered T cells until harvest; The CD3 / 28 dual anti-magnetic beads are in the micron range in diameter and have the functions of sorting and activating T cells.

2. The culture method according to claim 1, characterized in that The gene modification is selected from any one or a combination of gene introduction, gene knockout or gene knockdown.

3. The culture method according to claim 2, characterized in that The gene introduction includes chimeric antigen receptor gene introduction and / or T cell receptor gene introduction.

4. The culture method according to claim 1, characterized in that The diameter of the micron dual-resistance magnetic beads is 4.5 μm-5.5 μm, preferably 4.5 μm or 5.5 μm.

5. The culture method according to claim 1, characterized in that The step S1 further comprises adding IL2.

6. The culture method according to claim 5, characterized in that: The IL2 concentration is 20-2000 IU / mL; preferably, the IL2 concentration is 50-300 IU / mL; more preferably, the IL2 concentration is 100 IU / mL.

7. The culture method according to claim 1, characterized in that: The hPLT concentration is 0.003%-20%; preferably, the hPLT concentration is 1%-5%; more preferably, the hPLT concentration is 2-3%.

8. The culture method according to claim 1, characterized in that: The S1 may further be supplemented with one or more fetal bovine serum substitutes.

9. The culture method according to claim 8, characterized in that The fetal bovine serum substitute is selected from CTS immune cell serum substitute, human plasma, human AB serum, human umbilical cord blood serum, and whole blood extract.

10. The culture method according to claim 1, characterized in that: The T cells are derived from tumor patients or autoimmune disease patients.

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