WO2023164990A1

WO2023164990A1 - Preparation method and use of autologous tumor-draining lymph node lymphocytes

Info

Publication number: WO2023164990A1
Application number: PCT/CN2022/085870
Authority: WO
Inventors: 姚燕丹; 黄松音; 王瑞; 伍小华; 杨彦嘉; 鲍燕
Original assignee: 中山大学孙逸仙纪念医院深汕中心医院; 中山大学孙逸仙纪念医院
Priority date: 2022-03-03
Filing date: 2022-04-08
Publication date: 2023-09-07
Also published as: CN115433713A; CN115433713B

Abstract

The present invention provides a preparation method and the use of autologous tumor-draining lymph node lymphocytes. The preparation method comprises the following steps: A1, acquiring draining lymph node tissue from a patient, digesting same and separating same to prepare the draining lymph node mononuclear cells; and A2, culturing the cells obtained in the step A1, and activating and amplifying T cells contained in the cells to obtain the autologous tumor-draining lymph node lymphocytes. The preparation method of the present application can obtain the autologous tumor-draining lymph node lymphocytes (LNLs). Compared with tumor infiltrating lymphocytes, the LNLs as adoptive transfer cells facilitate in vitro culture amplification and increase the culture success rate. After adoptive transfer into a body, the LNLs can also provide a corresponding killing effect, and have excellent in-vivo amplification potential and in-vivo action durability and continuous activity, thus overcoming the defects of terminal differentiation, functional imbalance, poor durability and the like possibly caused by using tumor-infiltrating T cells as sources in the prior art.

Description

Preparation method and application of autologous tumor-draining lymph node lymphocytes

technical field

The invention relates to the technical field of cell culture, and more specifically, to a preparation method and application of autologous tumor-draining lymph node lymphocytes.

Background technique

Solid tumors Unlike hematological malignancies with lineage markers, solid tumors are highly heterogeneous, making it difficult to find ideal targets for all tumor cells in solid tumors. Adoptive cellular immunotherapy is one of the hot researches in the field of cancer treatment at present. Past studies have shown that it has the potential to be applied in both hematological malignancies and solid tumors to obtain significant curative effects. Among them, chimeric antigen receptor T, TIL and other therapies Diverse and remarkable advances have also been made in cancer therapy.

For solid tumors, chimeric antigen receptor T therapy targeting a single tumor antigen often produces antigen loss or leads to recurrence of more malignant tumor clones, and this type of therapy has some shortcomings. TIL therapy and chimeric antigen receptor T therapy belong to cellular immunotherapy, but TIL therapy has some significant advantages in the treatment of solid tumors. Adoptive cell therapy (ACT) was first proposed by Professor Steve A. Rosenberg and his colleagues at the National Institutes of Health (NIH). For the first time, TIL was isolated from multiple mouse tumor models. Adoptive cell therapy for systemic treatment of tumors. The therapy collects infiltrating lymphocytes from the patient's tumor, cultures and expands them in vitro, and finally returns them to the patient. Its principle of action is to use tumor-infiltrating lymphocytes to target multiple tumor-specific antigens, and it is MHC-restricted, with strong killing ability and few side effects. The effectiveness of TIL therapy has been demonstrated in many solid tumors, including breast cancer, ovarian cancer, melanoma renal cell carcinoma, non-small cell lung cancer, prostate cancer, bladder cancer, head and neck cancer, sarcoma, and pancreatic cancer. Compared with other adoptive cell therapies (such as chimeric antigen receptor T and TCR-T therapy), TIL is composed of T cells with diverse T cell receptor (TCR) clones, which can recognize a series of tumor antigens, superior tumor The homing ability and low off-target toxicity endow it with unique advantages in the treatment of solid tumors.

However, the acquisition of TIL is challenging. The success rate of culturing autologous tumors in vitro is low, and after long-term culture and massive expansion in vitro, the persistence of T cells in vivo is poor, resulting in terminal differentiation, dysfunction or failure of T cells. wait. At present, TILs are obtained by shredding or enzymatically digesting tumors obtained from surgical resection of patients, culturing cytotoxic T cells from tumor tissues, and expanding the cells through continuous passage until they reach the order of 10 ¹⁰ to 10 ¹¹ for reinfusion. However, the antitumor activity of TILs in vivo is highly dependent on the expansion, persistence, and continued activity of adoptively transferred cells. Although T cell products dominated by effector cells have stronger tumor cytotoxicity, these cells also tend to be terminal. differentiation and dysfunction. Although some researchers have optimized the above culture protocol, using a combination of

T cell activation

1, 2 and 3 signals (OKT3 antibody, agonistic CD137/4-1BB and IL-2) to culture TIL from tumor tissue has improved the success rate of culture. , but all TILs are obtained at the peripheral tissue level, which still cannot avoid the tendency of terminal differentiation and dysfunction after reinfusion, and the expansion, persistence and continuous activity of adoptively transferred cells still cannot be solved.

Therefore, the prior art urgently needs a kind of adoptively transferred cells that can be used as a source of T cells in ACT to overcome the tendency of terminal differentiation and dysfunction after reinfusion in the prior art, so as to improve the expansion, persistence, and persistence of cells after adoptive transfer. active effect.

Contents of the invention

The present invention aims at overcoming at least one deficiency of the above-mentioned prior art, and provides a method for preparing autologous tumor-draining lymph node lymphocytes and its application, through which the autologous tumor-draining lymph node lymphocytes that can be used as adoptively transferred cells can be obtained , to improve the culture success rate of adoptively transferred cells, as well as the curative effect and persistence of effect after reinfusion.

An object of the present invention is to provide a method for preparing autologous tumor-draining lymph node lymphocytes, comprising the steps of:

A1. Obtain draining lymph node tissue from the patient, digest and separate it, and prepare draining lymph node mononuclear cells;

A2. Culture the cells obtained in step A1, activate and expand the T cells contained therein, and obtain autologous tumor-draining lymph node lymphocytes.

The effect of adoptive T cell therapy is closely related to the degree of differentiation of T cells. For memory T cells with low degree of differentiation, they last longer in the body and have a stronger anti-tumor effect. However, CD8 ⁺ T cells exposed to different degrees of antigen stimulation directly affect cell survival, differentiation and therapeutic effect in vivo during cell expansion in vitro and after adoptive transfer therapy in vivo. Compared with effector memory T cells (T _EM ) and effector T cells (T _EFF ), T memory stem cells (T _SCM ) and central memory T cells (T _CM ) have better persistence and anti-tumor immunity, but if Frequently stimulated by antigenic and inflammatory signals, _TSCM will manifest as expression of multiple inhibitory receptors, such as PD-1, TIM-3, and LAG-3, as well as metabolic changes from oxidative phosphorylation to glycolysis, which will lead to T cell exhaustion. The adoptive transfer of T cells with and maintaining a less differentiated phenotype is crucial for antitumor efficacy and patient prognosis.

Lymph nodes are secondary lymphoid organs, and secondary lymphoid organs are the places where mature lymphocytes (T lymphocytes, B lymphocytes) settle. After the thymus matures, they circulate to the lymph nodes through blood circulation. In the lymph nodes of healthy people, T cells account for the majority of lymphocytes. 75%. Draining lymph nodes are the first lymph nodes that the primary tumor will inevitably pass through when lymph node metastasis occurs. T cells in lymph nodes can be divided into naive T cells (TN) that have not been exposed to antigens, and T memory stem cells (T _SCMs ) that have been stimulated to varying degrees by antigens. ), central memory T cells (T _CM ), effector memory T cells (T _EM ) and exhausted T cells (T _EX ). It is known that PD-1 is both a marker of activation and exhaustion, but the co-expression of multiple inhibitory receptors is the main feature of exhaustion, so the exhausted T cell marker can be set to CD3 ⁺ PD-1 ⁺ Tim3 ⁺ LAG3 ⁺ . Under chronic infection or long-term tumor antigen stimulation, T lymphocytes will expand and differentiate into effector cells and memory cells that clear pathogens. Memory cells can survive for a long time to ensure that the body responds quickly to protect the body when it is attacked again by the same antigen, and _TSCM cells have shown their strong potential for tumor treatment. However, most of the tumor-infiltrating T cells are effector T cells that tend to be terminally differentiated, have weak proliferative ability, tend to be exhausted, and have a low success rate of culture. On this basis, the inventors propose to obtain lymphocytes from draining lymph nodes, and As a source of T cells in ACT, it may be better than tumor tissue T cells. To this end, the inventors proposed a method for preparing autologous tumor-draining lymph node lymphocytes, and obtained corresponding autologous tumor-draining lymph node lymphocytes (LNL cells). The preparation method is simple and easy to operate, and the prepared LNL cells are easy to culture, which can improve the success rate of cell culture, and as adoptively transferred cells (including TIL) can provide effective tumor killing effect after adoptive reinfusion, compared with other sources Adoptive transfer of cells has significant curative effect and long-lasting effect. The preparation method can obtain T cells with a lower differentiation level and have been exposed to tumor antigens from lymph nodes, so as to increase the proportion of memory T cells in cell products, prolong the duration of in vivo treatment, and reduce T cell exhaustion. Moreover, the preparation method provided by the present application also exists as a culture method, which can improve the success rate of culture of adoptively transferred LNL cells, and overcome the defects in the prior art such as difficult culture of adoptively transferred cells.

Further, the autologous tumor-draining lymph node lymphocytes are DC-induced autologous tumor-draining lymph node lymphocytes, further comprising the steps of:

A3. Obtain DC cells from the peripheral blood of the same patient, pulse DC with tumor lysate, make DC present tumor antigen, and then co-culture with autologous tumor-draining lymph node lymphocytes in step A2 to obtain DC-induced autologous tumor-draining lymph node lymphocytes.

In addition to directly obtaining LNL cells and using their characteristics to overcome the defects of the prior art, this application can also use IL-2 and tumor lysates presented by DCs to further stimulate the specific proliferation of lymphocytes, increase the amplification factor, and increase the The proportion of T cell subtypes that are conducive to persistence and curative effect after adoptive reinfusion.

Further, step A1 specifically includes:

A11. Obtain draining lymph nodes by biopsy, remove excess fat, wash, shred, add to human lymphocyte serum-free medium containing collagenase I, collagenase III and DNase to incubate and digest;

A12. Dilute the tissue with normal saline after digestion, and use a cell strainer to collect the cells to obtain a single cell suspension;

A13. After obtaining the mononuclear suspension, use the human lymphocyte separation medium Ficoll density gradient centrifugation, absorb the buffy coat layer mainly composed of mononuclear cells after centrifugation, separate and prepare the mononuclear cell suspension, wash, plant in the orifice plate, and obtain drainage Lymph node mononuclear cells.

Further, step A2 specifically includes:

A21. Add the draining lymph node mononuclear cells obtained in step A1 to the initial medium for culture, the initial medium is a mixture of human lymphocyte serum-free medium, recombinant human IFN-γ and human AB serum or autologous plasma or serum substitute form;

A22. After initial culture, stimulate with CD3 monoclonal antibody and recombinant human IL-2 to activate and expand T cells;

A23. After the T cells are fully activated, regularly replace half of the fresh continuous expansion medium to maintain the proliferation of T cells, and obtain autologous tumor-draining lymph node lymphocytes. The continuous expansion medium is human lymphocyte serum-free medium, recombinant Human IL-2 and human AB serum or autologous plasma or serum substitutes are mixed to form.

Further, step A3 specifically includes:

A31. The peripheral blood of the same patient was extracted intravenously, centrifuged to remove the upper plasma, diluted with the same volume of normal saline, and then centrifuged with Ficoll density gradient of human lymphocyte separation medium to obtain peripheral blood mononuclear cells;

A32. After the peripheral blood mononuclear cells are obtained, culture them on the wall, and then replace the DC medium for culture to obtain DC cells. The DC medium is formed by mixing human lymphocyte serum-free medium and GM-CSF/IL-4;

A33. Use tumor lysate to pulse DC cells, and DCs are cultured and matured in the maturation medium and then co-cultured with autologous tumor-draining lymph node lymphocytes to obtain DC-induced autologous tumor-draining lymph node lymphocytes. The DC maturation medium is human lymphocyte-free Serum medium, TNFα/IL-1β/IL-6 are mixed to form.

Another object of the present invention is to provide the use of autologous tumor-draining lymph node lymphocytes obtained by the above preparation method in the preparation of drugs for preventing and treating tumors.

Further, drugs for the prevention and treatment of tumors include adoptive cellular immunotherapy drugs.

Another object of the present invention is to provide the use of the autologous tumor-draining lymph node lymphocytes obtained by the above preparation method in the preparation of ACT therapy cells. ACT therapy cells include TIL cells.

Another object of the present invention is to provide a cell preparation, which contains the above-mentioned autologous tumor-draining lymph node lymphocytes, and a pharmaceutically acceptable carrier or solvent.

Further, the solvent is PBS solution.

Another object of the present invention is to provide adoptive cells for tumor therapy, which are derived from autologous tumor-draining lymph node lymphocytes. In one embodiment of the present invention, adoptive reinfusion of LNL cells can achieve its tumor killing effect and inhibit tumor growth, and it has the function of adoptive infusion to treat tumors. Based on the source cells, it is expected to overcome the shortcomings of traditional TIL cells in the prior art.

Compared with the prior art, the beneficial effect of the present invention is: through the preparation method provided by the application, autologous tumor-draining lymph node lymphocytes (LNL) can be obtained. As far as cells are concerned, it is convenient to achieve in vitro culture expansion and improve the success rate of culture. When adoptively transferred into the body, it can also significantly enhance the effect of in vivo expansion and the persistence and continuous activity of the in vivo effect, overcoming the use of tumor infiltration in the prior art. Defects such as terminal differentiation, dysfunction, and poor persistence that may result from T cells as a source. The preparation method of the present application is simple to operate, has low requirements for experimental equipment, and is easy to be widely used in hospitals and other scientific research institutes. It is expected to become a new breakthrough in adoptive cellular immunotherapy programs including ACT therapy, and realize effective therapeutic preparations and treatment programs. So that more patients can be effectively treated.

Description of drawings

Figure 1 shows the results of flow cytometry: showing the proportion and type of T lymphocytes from different tissues, including: T _SCM : CD45RO ^- CD62L ⁺ CD95 ⁺ ; T _CM : CD45RO ⁺ CD62L ⁺ ; T _EM : CD45RO ⁺ CD62L ^- ; T _EX : CD3+PD-1 ⁺ Tim3 ⁺ LAG3 ⁺ ; Th1: CD4 ⁺ T-bet ⁺ ; NK: CD3 ^- CD56 ⁺ .

Figure 2 shows the results of cell culture: showing the culture morphology and growth curve of LNL cells.

Figure 3 shows the results of flow cytometry detection of cell subtypes on day 30 of LNL culture: T memory cells (T _SCM , T _CM , T _EM ), Th1, NK and T lymphocyte exhaustion (T _EX ).

Figure 4 shows the functional assessment of LNL at the cellular level: Tumor lysates or DCs stimulated with tumor lysates can stimulate the expression of CD134 and CD137, the markers of LNL activity in culture.

Figure 5 shows the curative effect of LNL on MDA-MB-231 tumor-bearing mice in Example 1: A, changes in body weight of mice during treatment; B, tumor growth curve; C, tumor suppression after treatment; D, tumor after treatment Infiltration of T cells in the tumor: IHC shows the infiltration of T cells in the tumor; E, the proportion of remaining T cells in the tumor cells after treatment.

Figure 6 shows the curative effect of LNL on PDX model mice in Example 1: A, changes in mouse body weight during treatment; B, tumor growth curve; C, tumor suppression after treatment (the red circle in the LNL treatment group represents complete regression of the tumor).

Figure 7 shows the main experimental process of Example 1.

Detailed ways

It should be pointed out that the following detailed description is exemplary and intended to provide further explanation to the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It should be noted that the terminology used here is only for describing specific embodiments, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof.

The present invention will now be further described in conjunction with specific examples. The following examples are only to explain the present invention, but not to limit the present invention. The test sample used in the following examples and the test process include the following (if the experimental specific conditions are not indicated in the examples, usually according to conventional conditions, or according to the conditions recommended by the reagent company; used in the following examples Reagents, consumables, etc., unless otherwise specified, can be obtained from commercial sources).

Example 1

In this embodiment, breast cancer is taken as an example to carry out the following experimental procedures.

In this example, the proportion and type of T lymphocytes in different tissue sources (tumor with the same volume and draining lymph node tissue with tumor metastasis) were detected by flow cytometry, as shown in Figure 1, the test results showed that T lymphocytes from different tissue sources There are significant differences in the proportion and type of cells. Compared with tumor-derived T cells, T cells in lymph nodes are more in a more immature memory cell state. On this basis, the applicant further carried out research on autologous tumor-draining lymph node lymphocytes and their use as a source of adoptively reinfused cells, hoping to overcome some of the shortcomings of other adoptively transferred cells in the prior art.

Specifically, the following experimental procedures are developed:

1. Mononuclear cell acquisition:

(1) Biopsy to obtain 2-3 draining lymph nodes;

(2) Use dissecting instruments to remove excess fat, place in normal saline, and repeat washing 3 times;

(3) Then use sterile tissue scissors to cut the tumor tissue or lymph nodes to about 1-2mm ³ size particles, add Super Culture ^TM L500 mixed with 1.5mg/ml collagenase I, III and 0.2mg/ml DNase Lymphocyte serum-free medium, incubate at 37°C for half an hour, take out and shake every 10 minutes, and digest the tissue pieces; further, an automatic tissue gentle processor can be used to realize this operation process;

(4) Dilute the tissue with normal saline after digestion, filter the cells with a sterile 70 μm cell filter to obtain a single cell suspension, and centrifuge for 5 min (2000 rpm);

(5) Use human lymphocyte separation medium Ficoll density gradient centrifugation, horizontal centrifugation for 20min (at room temperature, 800g, acceleration and deceleration speed adjusted to 1), absorb the buffy coat layer in the middle mainly mononuclear cells to separate and prepare mononuclear cell suspension, physiological After washing the cells twice with saline, count them, and seed them in a 24-well plate at 2-2.5× ¹⁰⁶ cells/mL;

(6) PBMC: extract peripheral blood from a vein, centrifuge to remove the upper layer of plasma, and use the same method as step (5) to separate and prepare peripheral blood mononuclear cells with human lymphocyte separation medium; the acquisition of DC is to adhere to the wall for 2 hours after obtaining PBMC, and replace Fresh DC medium (Super Culture ^TM L500 human lymphocyte serum-free medium + 2U/ml GM-CSF/IL-4) was obtained from culture.

2. Initial culture of T lymphocytes

Cultured in the initial medium: Super Culture ^TM L500 human lymphocyte serum-free medium + 10% human AB serum + 1000 U/mL recombinant human IFN-γ.

3. T lymphocyte activation and rapid expansion

(1) After culturing for 7 days, stimulate the cell suspension in step 2 with 100ng/mL CD3 monoclonal antibody and 1000IU/mL recombinant human IL-2 to activate and expand T cells; the T cells are stimulated by CD3 antibody After being activated, it is in the rapid expansion period.

(2) Replace half of the fresh medium every 2 to 3 days (the fresh medium is only added with recombinant human IL-2, without adding CD3 monoclonal antibody), and choose to expand the bottle to keep the cell count at 2-2.5× ¹⁰⁶ /mL.

4. DCs present tumor antigens to stimulate specific expansion of T cells

After T cells were stimulated and expanded by CD3 for 7 days, DCs loaded with tumor antigens were co-cultured with them to stimulate T cell-specific expansion. Specifically, the tumor tissue of the same patient was repeatedly frozen and thawed 5 times between liquid nitrogen and a 37°C water bath, centrifuged to remove cell debris, the lysate containing tumor antigens was placed in DC medium and pulsed for 24 hours, and DCs were stimulated by DC maturation factors. After 24 hours of maturation, DCs loaded with tumor antigens were co-cultured with T lymphocytes obtained after being stimulated and expanded by CD3 for 7 days;

Half of the fresh expansion medium (Super Culture ^TM L500 human lymphocyte serum-free medium + 10% human AB serum + 1000 IU/mL recombinant human IL-2) was replaced every 2 to 3 days to maintain the proliferation of T cells.

The culture status of the isolated LNL cells in step 3 and step 4 is shown in Figure 2, where Day is the morphology on the second day of culture, REP-1W, REP-2W, and REP-3W are the culture values of the first week and the second day, respectively. Representative pictures of cell culture morphology at 1 week and 3 weeks. The right of Figure 2 shows the corresponding growth curve of LNL cell culture, including CD3 monoclonal antibody and DC induction process. As shown in the right of Figure 2, LNL cells have been effectively cultured and expanded, and can continue to expand rapidly. That is, compared with other sources of adoptive cells in the prior art, LNL cells are easier to culture and expand, and can solve the problem that adoptive cells are difficult to cultivate and expand in large quantities in adoptive immune cell therapy in the prior art.

5. Detection of T cell memory subsets

(1) Continuously culture LNL cells until the 30th day and take samples, about ¹⁰⁵ /tube, centrifuge at 400g for 5min;

(2) Discard the supernatant, add 500μL PBS, resuspend the cells, and centrifuge at 400g for 5min;

(3) Discard the supernatant, add 100 μL PBS, label the test tubes with corresponding fluorescent CD3, CD4, CD8, CD45RO, CD62L, CD95, CD56, T-bet antibodies, incubate at room temperature in the dark for 20 minutes, and set up a negative control group;

(4) Add 500μL PBS, resuspend the cells, centrifuge at 400g for 5min, repeat twice;

(5) Discard the supernatant, add 100 μL PBS, and perform flow cytometry detection;

(6) Analyze with Flowjo software, set a gate on CD3 ⁺ cells, and analyze the proportion of each subgroup (T _SCM : CD45RO ^- CD62L ⁺ CD95 ⁺ ; T _CM : CD45RO ⁺ CD62L ⁺ ; T _EM : CD45RO ⁺ CD62L + ^- ;

(7) The proportion of LNL memory T cells cultured in the tumor draining lymph nodes was detected, and the detection results are shown in FIG. 3 . CD3 ⁺ T cells accounted for more than 95% of the cell preparations. Specifically, central memory T cells and effector memory T cells accounted for a relatively significant proportion, indicating that LNL cells still have corresponding differentiation and killing potential after cultured to day 30.

6. Detection of surface molecules associated with T cell exhaustion

(1) Sampling after culturing until the 30th day;

(2) Collect samples according to the collection method of T cell memory subpopulation detection process above, label fluorescent antibodies CD3, CD4, CD8, PD1, TIM3, LAG3, resuspend in 100 μL PBS after washing, and perform flow cytometry detection;

(3) Analyzed by Flowjo software, with CD3 ⁺ cells as the analysis object, and T cell exhaustion was expressed by the positive ratio of PD1, TIM3, and LAG3;

(4) As shown in Figure 3, it shows the expression of exhaustion-related surface molecules (T _EX ) of LNL T cells cultured in tumor-draining lymph nodes. After culturing until the 30th day, _TEX accounted for only a very small proportion, less than 3%, indicating that the LNL cells provided in the examples have long-term efficacy and can effectively overcome the condition of being prone to terminal differentiation and exhaustion.

7. Functional assessment of LNL at the cellular level

(1) On the 28th day of LNL culture, the tumor tissue of the same patient was repeatedly frozen and thawed 5 times between liquid nitrogen and a 37°C water bath according to the above method, and the cell debris was removed by centrifugation, and the lysate containing tumor antigen was placed in DC medium middle;

(2) After 24 hours, add DC maturation factors and culture for 24 hours;

(3) On the 30th day of LNL cell culture, replace with fresh medium (Super Culture ^TM L500 human lymphocyte serum-free medium), and place it in DC culture flask at the ratio of DC:LNL 1:10～1:100 , co-cultured with mature DC; or the same volume of tumor tissue lysate directly stimulated the same count of LNL at the same time; set up the control group at the same time: the negative control group was replaced with fresh medium for the same count of LNL; the positive control group was the same count of LNL Replace LNL with fresh medium and add CD3 antibody stimulation;

(4) After 24 hours, observe the changes in DC morphology and adherence, collect samples according to the method in step 5, and label fluorescent antibodies CD3, CD137, and CD134;

(5) Resuspend after washing, and perform flow cytometry detection. The results are shown in Figure 4, tumor lysates or DCs stimulated by tumor lysates can effectively stimulate the cultured LNL activity marker CD134, and the expression of CD137 is up-regulated.

8. In vivo evaluation of LNL cell efficacy and persistence in animal experiments

(1) Cell preparation: take the LNL cells (induced by DC) on the 14th day of culture, centrifuge at 750g for 5min, discard the supernatant, resuspend the cells with an appropriate volume of solvent (sterile PBS), count and adjust the cell concentration to 1×10 ⁷ cells/125 μL, live cells shall prevail when counting cells.

(2) Mouse model information

(a) Species & strains: NOD/SCID

Animal grade: SPF grade

Number of animals: 15

Gender: female

Age: 4 weeks old

Weight range: 17～22g

Modeling: MDA-MB-231 cells implanted subcutaneously to form tumors

Random grouping: PBS group, IL-2 group, LNL group (5 rats in each group)

(b) Species & strains: NOD/SCID

Animal grade: SPF grade

Number of animals: 12

Gender: female

Age: 4 weeks old

Weight range: 17～22g

Modeling: Orthotopic Tumor

Random grouping: PBS group, IL-2 group, LNL group (4 rats in each group)

(3) Treatment: In the cell therapy group, 1×10 ⁷ cells/rat were injected into the tail vein with a volume of 125 μL, and at the same time, 6000 IU/125 μL IL-2 was subcutaneously supplemented; the other control groups were injected with the same volume of sterile PBS and An equal volume of 6000IU IL-2 injection was injected subcutaneously.

(4) Safety and efficacy

a) Monitor the weight change of the mice and obtain the tumor growth curve;

b) monitoring tumor suppression in mice after 3-14 weeks of treatment;

c) Immunohistochemical detection of tumor lymphocyte infiltration (CD3) in PBS group, IL-2 group and LNL group.

d) The proportion of lymphocytes in PBS group, IL-2 group and LNL group was detected by flow cytometry after treatment;

(5) Safety and efficacy results

Regarding the safety and curative effect (non-specific) of LNL on MDA-MB-231 tumor-bearing mice, as shown in Figure 5A, the body weight of the mice in the treatment group was not significantly different from that of the IL-2 group and the PBS model group, indicating that LNL The cell preparation has no obvious effect on the body weight of the experimental mice, and has certain safety. Moreover, the tumors of the mice in the LNL treatment group cultured in the tumor-draining lymph nodes were significantly inhibited and gradually subsided (as shown in Figures 5B and 5C), indicating that LNL cells have effective tumor-killing effects. After the treatment, the results of immunohistochemistry showed the infiltration of lymphocytes in the tumor of the LNL group. As shown in Figure 5D, the LNL-derived cells were significantly infiltrated into the tumor; The proportions of different subtypes of T cells in the tumor are shown in Figure 5E. Compared with the CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ subtype cells that almost disappeared in other groups, the CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ subtype cells in the LNL group all had an equivalent A large proportion is retained, indicating its long-term action characteristics.

Regarding the safety and efficacy (specificity) of LNL to PDX model mice, as shown in Figure 6A, the body weight of the mice in the treatment group had no statistical difference compared with the IL-2 group and the PBS model group, indicating that the LNL cell preparation had no significant effect on the body weight of the experimental mice. No significant impact, with a certain degree of security. Moreover, the tumors of the mice in the LNL treatment group cultured in the tumor-draining lymph nodes were significantly inhibited and gradually subsided (as shown in Figures 6B and 6C), indicating that LNL cells have specific and effective tumor-killing effects.

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, rather than limiting the specific implementation manner of the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

A method for preparing autologous tumor-draining lymph node lymphocytes, comprising the steps of:

A1. Obtain draining lymph node tissue from the patient, digest and separate it, and prepare draining lymph node mononuclear cells;

A2. Culture the cells obtained in step A1, activate and expand the T cells contained therein, and obtain autologous tumor-draining lymph node lymphocytes.
The preparation method according to claim 1, wherein the autologous tumor-draining lymph node lymphocytes are DC-induced autologous tumor-draining lymph node lymphocytes, further comprising the steps of:

A3. Obtain DC cells from the peripheral blood of the same patient, pulse DC with tumor lysate, make DC present tumor antigen, and then co-culture with autologous tumor-draining lymph node lymphocytes in step A2 to obtain DC-induced autologous tumor-draining lymph node lymphocytes.
The preparation method according to claim 1, wherein step A1 specifically comprises:

A11. Obtain draining lymph nodes by biopsy, remove excess fat, wash, shred, add to human lymphocyte serum-free medium containing collagenase I, collagenase III and DNase to incubate and digest;

A12. Dilute the tissue with normal saline after digestion, and use a cell strainer to collect the cells to obtain a single cell suspension;

A13. After obtaining the mononuclear suspension, use the human lymphocyte separation medium Ficoll density gradient centrifugation, absorb the buffy coat layer mainly composed of mononuclear cells after centrifugation, separate and prepare the mononuclear cell suspension, wash, plant in the orifice plate, and obtain drainage Lymph node mononuclear cells.
The preparation method according to claim 1, wherein step A2 specifically comprises:

A21. Add the draining lymph node mononuclear cells obtained in step A1 to the initial medium for culture, the initial medium is a mixture of human lymphocyte serum-free medium, recombinant human IFN-γ and human AB serum or autologous plasma or serum substitute form;

A22. After initial culture, stimulate with CD3 monoclonal antibody and recombinant human IL-2;

A23. After the T cells are fully activated, regularly replace half of the fresh continuous expansion medium to maintain the proliferation of T cells, and obtain autologous tumor-draining lymph node lymphocytes. The continuous expansion medium is human lymphocyte serum-free medium, recombinant Human IL-2 and human AB serum or autologous plasma or serum substitutes are mixed to form.
The preparation method according to claim 2, wherein step A3 specifically comprises:

A31. The peripheral blood of the same patient was extracted intravenously, centrifuged to remove the upper plasma, diluted with the same volume of normal saline, and then centrifuged with Ficoll density gradient of human lymphocyte separation medium to obtain peripheral blood mononuclear cells;

A32. After the peripheral blood mononuclear cells are obtained, culture them on the wall, and then replace the DC medium for culture to obtain DC cells. The DC medium is formed by mixing human lymphocyte serum-free medium and GM-CSF/IL-4;

A33. Use tumor lysate to pulse DC cells, and DCs are cultured and matured in the maturation medium and then co-cultured with autologous tumor-draining lymph node lymphocytes to obtain DC-induced autologous tumor-draining lymph node lymphocytes. The DC maturation medium is human lymphocyte-free Serum medium, TNFα/IL-1β/IL-6 are mixed to form.
The use of autologous tumor draining lymph node lymphocytes obtained by the preparation method described in any one of claims 1 to 5 in the preparation of drugs for preventing and treating tumors.
The use according to claim 6, characterized in that the drugs for preventing and treating tumors include adoptive cellular immunotherapy drugs.
The use of autologous tumor draining lymph node lymphocytes obtained from the preparation method according to any one of claims 1 to 5 in the preparation of ACT therapy cells.
A cell preparation, characterized in that it contains autologous tumor-draining lymph node lymphocytes obtained by the preparation method according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier or solvent.
An adoptive cell for tumor treatment is characterized in that it is derived from autologous tumor draining lymph node lymphocytes.