CN111909899A

CN111909899A - Method for enriching T cells and application of method in adoptive T cell therapy

Info

Publication number: CN111909899A
Application number: CN201910375997.1A
Authority: CN
Inventors: 鲁薪安; 何霆; 齐菲菲
Original assignee: Beijing Yimiao Shenzhou Pharmaceutical Technology Co ltd
Current assignee: Beijing Yimiao Shenzhou Pharmaceutical Technology Co ltd
Priority date: 2019-05-07
Filing date: 2019-05-07
Publication date: 2020-11-10

Abstract

The invention relates to the technical field of adoptive T cell immunotherapy, in particular to a method for enriching T cells and application thereof in adoptive T cell therapy. The enrichment method comprises the steps of culturing Peripheral Blood Mononuclear Cells (PBMCs) for 1-4 hours, removing adherent cells, and then separating T cells from the rest cells by using magnetic beads. The method can effectively improve the purity of the T cells, and has high T cell yield and simple operation. The invention also improves the expression rate of the Chimeric Antigen Receptor (CAR) and reduces the operation risk by changing a virus infection method.

Description

Method for enriching T cells and application of method in adoptive T cell therapy

Technical Field

The invention relates to the technical field of adoptive T cell therapy, in particular to a method for enriching T cells and application of the method in preparing T cells for adoptive cell therapy.

Background

In recent years, there has been widespread interest and use of tumor immunotherapy, which controls and eliminates malignant cells by activating the body's anti-tumor immune response (Sanmamed MF, et al. cell.2019,176: 677). Cancer cells are transformed from normal cells by genetic mutations or epigenetic changes, which express many abnormal tumor antigens. Normally, the innate immune system of the human body specifically recognizes and kills cancer cells by recognizing tumor antigens. However, cancer tissues adopt various mechanisms to construct immunosuppressive microenvironments that allow immune cells such as T and B lymphocytes to develop immune tolerance against cancer cells, leading to malignant progression (Kalathil SG, et al cancer immunological impronother.2016, 65(7): 813). Compared with the traditional therapy, the tumor immunotherapy has the remarkable advantages of good curative effect and small toxic and side effect by breaking immune tolerance, restarting and maintaining tumor-immune circulation.

Adoptive T cell therapy is an emerging tumor immunotherapy that utilizes in vitro enrichment and engineered modification of human T cells to specifically target and kill cancer cells in patients (Yang JC, et al. adv immunol.2016,130: 279). The adoptive T cell therapy currently available includes tumor infiltrating lymphocytes, chimeric antigen receptor T cells (CAR-T), T cell receptor chimeric T cells (TCR-T), and the like. Various techniques have been developed at home and abroad to enrich naturally occurring T cells capable of targeting tumor antigens, or to specifically target known tumor antigens by genetically modifying T cells. Clinical studies have demonstrated that adoptive T cell therapy significantly inhibits cancer progression, prolongs patient survival, and in particular the emergence of CAR-T technology, has led to a milestone development in human control of cancer (June CH, et al science.2018,359: 1361). The application of this technology in tumor therapy started in 1989, and has achieved a response rate of 60-95% in hematological malignancies such as relapsed refractory leukemia, lymphoma, multiple myeloma (Park JH, et al. N Engl J Med 2018,378: 449; Neelapu SS, et al. N Engl J Med 2017,377: 2531; Cohen AD, et al. J Clin invest.2019,130: pii:126397), and also has shown potential therapeutic effects in some malignant solid tumors such as glioma, liver Cancer, melanoma (Migliorini D, et al. Clin Cancer Res.2018,24: 535; Gao H, et al. Clin Cancer Res.2014,20: 6418; Forsberg EMV, et al. Cancer Res.2019,79: 899).

The T cells currently used in CAR-T technology are mainly derived from the patient himself and are greatly affected by the condition of the patient. T cells were isolated by three methods: firstly, magnetic beads are directly separated from PBMCs; collecting cells from the PBMCs by a method of multiple adherence, and adding magnetic beads for activation; collecting cells from PBMCs by a method of multiple adherence, adding magnetic beads for activation, and separating T cells by using the magnetic beads after activation. The T cells separated by the first two methods have low purity, the activation efficiency, CAR expression rate and cell proliferation efficiency of the T cells are influenced, the sorting in the third method influences the activity of the cells after activation, the T cell yield is reduced, and the controllability on the number of the infectable T cells is low. In addition, the T cells separated by the prior art have the problems of low expression rate of target proteins and the like in the subsequent virus infection process. Therefore, methods for separating and purifying T cells and infecting viruses need to be improved, which is important for improving the success rate of clinical application of adoptive T cell therapy.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention relates to a method for enriching T cells, which comprises the steps of culturing PBMCs for 1-4 hours, removing adherent cells, and then separating the T cells from the rest cells by using magnetic beads.

T cells isolated from PBMCs have low purity and contain a part of adherent cells such as macrophages and dendritic cells, B cells and the like, which results in low T cell activation efficiency, low virus infection efficiency and unclear target cell components. The T cell enrichment method provided by the invention can effectively improve the purity of the T cells, and has high T cell yield and simple operation.

According to one aspect of the invention, the invention also relates to the use of a method as described above for the preparation of a medicament for T cell immunotherapy;

the T cell immunotherapy drug comprises one or more of the following cells: tumor infiltrating lymphocytes, cytotoxic T cells, chimeric antigen receptor T cells (CAR-T), T cell receptor chimeric T cells, natural killer T cells, and peripheral blood lymphocytes.

According to one aspect of the invention, the invention also relates to a method of making a CAR-T cell comprising:

i) enriching T cells using the methods described above;

ii) inoculating the virus carrying the CAR expression fragment 24-48 hours after T cell activation; and

iii) culturing the T cells.

According to the invention, the CAR expression rate is improved and the operation risk is reduced by changing a virus infection method.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing T cell proliferation isolated from PBMCs from different sources in one embodiment of the present invention;

FIG. 2 is a flow chart of monocyte depletion after PBMCs are attached in one embodiment of the invention; BNK: before adhering to the wall; 1-BNK: after adhering to the wall;

FIG. 3 is a graph of T cell sorting using different ratios of beads to T cells in one embodiment of the invention;

FIG. 4 is a graph of the effect of infection cofactors on CAR expression rate in one embodiment of the invention;

FIG. 5 is a graph showing the effect of different IL-2 concentrations on cell proliferation in one embodiment of the present invention;

FIG. 6 is a culture expansion of three patient-derived CAR-T cells in one embodiment of the invention.

Detailed Description

According to one aspect of the present invention, the present invention relates to a method for enriching T cells, which comprises culturing PBMCs for 1 to 4 hours, removing adherent cells, and separating T cells from the remaining cells using magnetic beads.

Lymphocytes are suspension cells or minimally adherent, and adherent treatment can remove mixed cells such as macrophages in the PBMCs.

The invention only carries out one-time treatment adherent culture, and the loss of T cells is less.

PBMCs refer to cells with a single nucleus in peripheral blood, including lymphocytes and monocytes.

In the present invention, unless otherwise specified, the cell culture conditions are all 30 ℃ to 45 ℃ and 1% to 10% CO₂Preferably 36-38 ℃ and 4-6% CO₂。

Specific subsets of the sorted T cells may be selected for example from CD28⁺、CD4⁺、CD8⁺、CD45RA⁺And CD45RO⁺Cells of any positive combination in T cells; in some embodiments, the T cell selected is CD3⁺CD28⁺T cells.

The above subpopulations of T cells may be further isolated by positive or negative selection techniques. For example CD3⁺CD28⁺T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., Dynabeads CD3/CD 28). In some embodiments, the magnetic beads conjugated with CD3/CD28 antibody are mixed with the remaining cells at a magnetic bead: t cells are incubated at a ratio of 1.5:1, 2:1, or 2.5:1, for example, and then sorted using the magnetic beads.

Wherein (1-3): 1 is the ratio of the number of magnetic beads to the number of T cells. The number of T cells and the estimation of the number of T cells by conventional methods such as morphological observation of the remaining cells.

In some embodiments, the PBMCs are cultured at a seeding density of (0.5-1.5). times.10⁸cells/30 mL; preferably 1X 10⁸cells/30mL。

In some embodiments, the culture area of cells available in the culture flask for said adherent culture is 175cm²In terms of volume, the addition amount of the culture medium used for adherent culture is 25-35 mL, and 30mL is preferably selected.

In some embodiments, the sorting is performed at a cell density of (0.15-0.6) x 10⁶cells/mL, preferably (0.25-0.5). times.10⁶cells/mL。

In some embodiments, the paramagnetic microparticles used in the present invention are of a size sufficient to be phagocytized by phagocytic monocytes, which are subsequently removed by magnetic separation. In certain embodiments, the paramagnetic microparticles are commercially available beads, for example, those manufactured by Dynal AS under the trade name Dynabeads^TMThe beads of (a). Dynabeads, as exemplified herein^TMIs M-280, M-450 and M-500. In one aspect, paramagnetic microparticles coated with "irrelevant" proteins (e.g., serum proteins or antibodies) remove other non-specific cells. Unrelated proteins and antibodies include those that do not specifically target the T cells to be expanded or fragments thereof. In certain embodiments, the unrelated beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.

In one aspect of the invention, enrichment of T cell populations by negative selection is accomplished with a combination of antibodies directed to cell surface unique markers of the negative selection. The preferred method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers on negatively selected cells. For example, enrichment of CD4 by negative selection⁺The cell, monoclonal antibody cocktail generally includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8.

T cells can be obtained from xenogeneic sources, e.g., mouse, rat, non-human primate, and pig.

In some embodiments, the PBMCs are from a subject in need of a T cell therapy.

In some embodiments, the PBMCs are from a single blood draw, peripheral blood whole blood, or peripheral blood stem cells.

At present, T cells used in the CAR-T culture process are all derived from patients, some patients are not suitable for single blood collection according to the condition of the patients, and some patients have low T cell activity and slow proliferation speed and cannot be cultured and expanded to the dose required by patient feedback, so that the patients cannot receive CAR-T treatment finally.

Preferably, the cells are isolated from the circulating blood of the individual by apheresis or leukapheresis. Apheresis typically contains lymphocytes (T cells, monocytes, granulocytes, B cells), other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis or leukapheresis may be washed to remove plasma components and placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate-buffered saline (PBS). In further embodiments, the wash solution may lack calcium, may lack magnesium, may lack all divalent ions, or lack many divalent ions. As those of ordinary skill in the art will readily appreciate, the washing step may be accomplished by methods well known in the art, such as by using semi-automated "extreme sedimentation" centrifugation according to the manufacturer's recommendations. After washing, the cells can be resuspended in various biocompatible buffers, e.g., Ca-free²⁺/Mg²⁺The PBS (1). In addition, unwanted components of the apheresis sample can be removed and the cells resuspended in culture medium.

In some embodiments, the PBMCs are freshly isolated cells.

In some embodiments, the PBMCs are cells that are thawed after cryopreservation.

the T cell immunotherapy drug comprises one or more of the following cells: tumor infiltrating lymphocytes, cytotoxic T cells, chimeric antigen receptor T cells, T cell receptor chimeric T cells, natural killer T cells and peripheral blood lymphocytes.

i) enriching T cells using the methods described above;

iii) culturing the T cells.

T cell activation can be performed by co-incubation with magnetic beads conjugated with CD3/CD28 antibodies as described above, or by the addition of certain well-known cytokine/cytokine antibodies that promote activation.

The term "inoculation" as used herein refers to the addition of virus to a host cell to initiate culture; or adding cells to the initial medium to start the culture.

Preferably, in step ii), the virus carrying the CAR-expressing fragment is inoculated 36-48 hours after T cell activation; alternatively, 40 hours, 43 hours, 44 hours or 46 hours, most preferably 48 hours, may be used.

In some embodiments, the virus carrying the CAR expression fragment is a retrovirus.

In some embodiments, the virus carrying the CAR expression fragment is a lentivirus.

In some embodiments, the virus carrying the CAR expression fragment is inoculated only once.

By "vaccinate once" is meant that the virus is added only once within 24 hours.

In some embodiments, no infection co-factor is added during the process of making the CAR-T cells.

The infection auxiliary factor is selected from, for example, Vectofusin-1 and Polybrene.

In some embodiments, in step iii), the cells are cultured using serum-free, antibiotic-free medium.

At present, CAR-T culture mostly adopts a culture medium containing serum and antibiotics, the serum source is AB serum or autologous serum, and great potential safety hazards exist. The serum-free and antibiotic-free culture medium used in the CAR-T culture process reduces the influence of serum and antibiotics on cells in the culture process and ensures that serum-free factors and antibiotics are not contained in the reinfusion preparation. The safety of the product is increased.

In some embodiments, the serum-free medium that can be used is selected from the group consisting of X-VIVO15 medium from Lonza.

In some embodiments, the medium contains 100-600 IU/mL Interleukin-2 (Interleukin 2, IL-2);

in some embodiments, the medium contains 500IU/mL IL-2.

IL-2 is also known as T Cell Growth Factor (TCGF) because it enables long-term, sustained T cell proliferation in vitro. Quiescent T cells do not express IL-2R and therefore cannot serve as target cells for IL-2; the surface of T cells activated by mitogen or other stimuli can express IL-2R and respond to IL-2 stimulation. IL-2 can enhance the killing activity of T cells, and can induce the generation of cytotoxic T cells (Tc or CTL) together with IL-4, IL-5 and IL-6 in vitro, so that the activity of the cytotoxic T cells is greatly enhanced; the T cells induced by IL-2 can generate obvious anti-tumor effect after being returned into the body of a patient, but the T cells are not easy to survive in the body, and if a small amount of IL-2 is simultaneously input, the survival time of the T cells in the body can be obviously prolonged, and the anti-tumor effect of the T cells can be enhanced.

In some embodiments, in step iii), the seeding density of T cells is 0.5-0.8 × 10⁶cells/mL；

In some embodiments, in step iii), the seeding density of T cells is 0.6-0.7 × 10⁶cells/mL。

In some embodiments, in step iii), the medium is changed every 12-36 hours (preferably 24 hours), and the ratio of the new medium to the old medium is (5-1): 1, or 4:1, 3:1 or 2: 1.

In some embodiments, in step iii), the incubation time is 7 to 8 days in total.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 isolation of autologous PBMCs from patients of different origins

The T cells used in the autologous CAR-T culture process are mainly derived from the patient, but in some cases, due to the condition of the patient or the condition of the patient, the PBMCs have low T cell content or insufficient T cell activity, which increases the difficulty of T cell separation and proliferation in the CAR-T production process. The CAR-T production can be carried out on the patients by using peripheral blood stem cells, PBMCs stored in the healthy state of the patients or high-activity PBMCs stored in the early onset stage of the patients, so that the culture failure caused by low activity of T cells can be effectively avoided.

Preparation of PBMCs from different sources

Scheme one is to separate PBMCs from autologous blood sample by using Ficoll separating medium.

The experimental procedure was as follows:

1. blood samples were collected as blood samples: the saline solution was diluted 1:4 by volume.

2. The diluted blood samples were separated using Ficoll medium (800g/20 min/liter 5 down 4).

3. The buffy coat layer was collected into a 50mL centrifuge tube and subjected to trim centrifugation (500g/6 min/L8 down 8).

4. The supernatant was discarded and washed twice with physiological saline (400g/6 min/L8, 8).

Scheme two PBMC is obtained by separating autologous peripheral blood with Ficoll separating medium.

The experimental procedure was as follows:

1. peripheral blood samples were as per blood sample: the saline solution is diluted in a volume ratio of 1: 1.

And (3) recovering the PBMCs frozen in the third scheme in a water bath, and washing out frozen stock solution to obtain the PBMCs.

The experimental procedure was as follows:

1. taking out the frozen PBMCs, rapidly resuscitating in water bath at 37 ℃, transferring into a 50mL centrifuge tube, balancing and centrifuging (400g/5 min/L8-step down 8)

2. The supernatant was discarded, and PBMCs were washed twice with physiological saline (400g/5 min/liter 8 down)

And recovering the peripheral blood stem cells stored in the fourth scheme in a water bath to obtain the low-purity PBMCs.

The experimental procedure was as follows:

1. the stored cells are taken out and quickly recovered in a water bath at 37 ℃, transferred into a 50mL centrifuge tube, and subjected to ingredient centrifugation (400g/5 min/L8-step-down 8)

T cells isolated using PBMCs from different sources proliferated well during CAR-T culture (FIG. 1). T cells sorted from the PBMCs from four sources can be cultured for CAR-T.

Example 2 obtaining autologous T cells from a patient

In the T cell sorting process, the high-purity T cells obtained by separation have promotion effects on improving the activation efficiency of the T cells, the proliferation multiple of the T cells and the CAR expression rate. However, in the CAR-T production process, PBMCs contain a large number of monocytes due to the patient himself, which results in a low T cell content, adding difficulties to the isolation of T cells to a high purity.

In the scheme, PBMCs are treated in an adherence way, monocytes in the PBMCs are removed, suspension cells are collected, and Dynabeads CD3/CD28 is added to sort T cells.

T cell isolation step:

1. the isolated PBMCs were dispensed in 30 mL/bottle (1X 10 total)⁸cells) were spread in a T175 flask and placed in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) And (5) culturing.

After 2.2 hours, the cells were removed, aspirated and centrifuged (400g/6 min/l 8 down 8).

3. After resuspension, the pellet was resuspended as beads: t-3: 1, 2:1, 1.5:1, 1:1 Dynabeads CD3/CD28 was added and incubated at room temperature for 20 min.

4. After incubation, the cell suspension was diluted to 0.5X 10⁶T cells/mL or 0.25X 10⁶Sorting was performed in T cells/mL.

5. And (4) spreading the sorted T cells into a culture flask for culture.

After PBMCs are attached to the wall, a monocyte removal flow chart is shown in figure 2, and as can be seen from the chart, the monocyte removal efficiency after the PBMCs are attached to the wall is high, and the proportion of lymphocytes is increased. PBMCs adherent treatment, monocyte removal efficiency and lymphocyte yield are shown in Table 1.

Sorting of different beads versus T cells is shown in FIG. 3. As can be seen from the figure, the ratio pairs of 3:1, 2:1, 1.5:1, 1:1 are all suitable for sorting T cells.

TABLE 1

Wherein, DP073, DP074, DP079, DP081, DP082, DP083, DP085, DP086, DP087 and DP088 are the apheresis group, and DP119 is the peripheral blood group.

Example 3

And (3) virus infection step:

1. collecting T cells after 24-48 hours of activation according to the proportion of 1.5 multiplied by 10⁶cells/mL were plated into culture flasks.

2. The amount of virus required was calculated as multiplicity of infection (MOI).

3. The desired virus volume was aspirated, added to the cell suspension, gently mixed, placed in a carbon dioxide incubator (37 ℃, 5% CO)₂) And (5) culturing.

In this protocol, the inventors found that the use of viral infection co-factors did not significantly contribute to CAR expression rates (table 2 and figure 4). Therefore, the invention does not add virus infection accessory factors.

Different infection times have more significant effects on CAR expression rate (Table 3), wherein the virus infection is better than 24 hours after activation, and the virus infection is carried out 24-48 hours after T activation, so as to meet the requirements of clinical production.

As can be seen from the effect of the number of infections on the CAR expression rate, the use of one infection was superior to the second (Table 4), and thus the present invention used a one infection protocol.

Through the adjustment, the operation process of virus infection is simplified, and the risk is reduced.

TABLE 2

TABLE 3

TABLE 4

Example 4 cell proliferation

In the cell proliferation process, the cell is cultured by adopting a serum-free and antibiotic-free X-VIVO15 culture medium, so that the dependence of the cell on serum factors and the antibiotic effect in the culture process are reduced, the finished product preparation is free of serum and antibiotic residues, and the safety is ensured. The culture time is shortened to 7-8 days, the clinical controllability is improved, the cells are in a high-speed proliferation stage during the preparation of finished products, and the activity of the cells is ensured. IL-2 (concentration 50IU/mL, 200IU/mL, 500IU/mL) is added in the cell proliferation process.

Cell expansion experiment procedure:

1. 24 hours after infection with virus, cell suspensions were collected and centrifuged (400g/5 min/L8 down 8).

2. The supernatant was discarded, and the medium was adjusted to 0.5X 10 using X-VIVO15⁶cells/mL,0.6×10⁶cells/mL,0.7×10⁶cells/mL,0.8×10⁶cells/mL weightAnd (5) suspension culture.

3. And (4) changing the culture medium every other day, wherein the ratio of the new culture medium to the old culture medium is 5:1, 4:1, 3:1, 2:1 or 1: 1.

4. And collecting cells when the cells are cultured to 7 days or 8 days for finished product preparation.

The effect of different concentrations of IL-2 on cell proliferation is shown in FIG. 5.

The number of viable cells of three patients cultured with CAR-T cells for 7-8 days is shown in FIG. 6.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The method for enriching the T cells is characterized by comprising the steps of culturing Peripheral Blood Mononuclear Cells (PBMCs) for 1-4 hours, removing adherent cells, and separating the T cells from the rest cells by using magnetic beads.

2. The method of claim 1, wherein the T cells selected are CD3⁺CD28⁺A T cell;

preferably, the magnetic beads are CD3/CD28 antibody conjugated magnetic beads which are bound to the remaining cells in a ratio of magnetic beads: incubating the T cells according to the proportion of (1-3) to 1, and separating the T cells by using the magnetic beads;

preferably, the seeding density of the PBMCs during culture is (0.5-1.5) × 10⁸cells/30mL；

Preferably, the sorting is performed at a cell density of (0.15 to 0.6). times.10⁶cells/mL；

Preferably, the PBMCs are from a subject in need of adoptive T cell therapy;

preferably, the PBMCs are from a single blood sample, peripheral blood whole blood or peripheral blood stem cells;

preferably, the PBMCs are freshly isolated cells or cells that are thawed after cryopreservation.

3. Use of the method of claim 1 or 2 for the manufacture of a medicament for T cell immunotherapy;

4. A method of making a CAR-T cell, comprising the steps of:

i) enriching T cells using the method of claim 1 or 2;

iii) culturing the T cells.

5. The method of claim 4, wherein the virus carrying the CAR-expressing fragment is a retrovirus;

preferably, the virus carrying the CAR expression fragment is a lentivirus;

preferably, the virus carrying the CAR expression fragment is inoculated only once;

preferably, no infection co-factors are added during the process of making the CAR-T cells.

6. The method according to claim 5, wherein in step iii) the cells are cultured using serum-free antibiotic-free medium.

7. The method according to claim 6, wherein the culture medium contains 100-600 IU/mL of interleukin-2.

8. The method according to claim 6 or 7, wherein in step iii) the seeding density of T-cells is 0.5-0.8 x 10⁶cells/mL。

9. The method as claimed in claim 8, wherein in step iii), the medium is changed every 12-36 hours, and the ratio of new medium to old medium is (5-1): 1.

10. The method according to claim 9, wherein in step iii), the cultivation time is 7 to 8 days in total.