CN103374548A - High-efficiency amplification culture solution for tumor infiltration lymphocytes - Google Patents
High-efficiency amplification culture solution for tumor infiltration lymphocytes Download PDFInfo
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- CN103374548A CN103374548A CN 201210127274 CN201210127274A CN103374548A CN 103374548 A CN103374548 A CN 103374548A CN 201210127274 CN201210127274 CN 201210127274 CN 201210127274 A CN201210127274 A CN 201210127274A CN 103374548 A CN103374548 A CN 103374548A
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Abstract
The invention provides a high-efficiency amplification culture solution for tumor infiltration lymphocytes. The culture solution contains IL-2, IL-12, anti-CD28 and anti-CTLA-4. Compared with the prior art, the high-efficiency amplification culture solution has the advantages that a combined serum-free culture medium containing IL-2, IL-12, anti-CD28, anti-CTLA-4 and other cell factors is adopted, the amplification efficiency of TIL cells is obviously improved, and the amplification efficiency is about 3-10 times higher than the amplification efficiency of a common amplification culture solution; moreover, an inhibitory function of Treg lymphocytes on cytotoxic T lymphocytes can be effectively achieved, and the amplified lymphocyte has an extremely strong tumor killing effect.
Description
Technical field
The present invention relates to a kind of knubble biological immunotherapy nutrient solution, particularly use the efficient amplification cultivation liquid that tumor infiltrating lymphocyte carries out the biological immune treatment.
Background technology
Tumor infiltrating lymphocyte (Tumor-infiltrating lymphocyte, TIL) is the CTL cell that a group of tumor locus infiltration has been activated by tumour antigen.1986 pioneering by Rosenberg etc., concrete grammar is to isolate til cell from specimens, ascites pleural fluid or peripheral blood, the nutrient solution that external use contains IL-2 and CD3 antibody feeds back to patient (Belldegrun A after being expanded to some amount, MuulL and Rosenberg S.Interleukin 2 Expanded Tumor-infiltratingLymphocytes in Human Renal Cell Cancer:Isolation, Characterization, and Antitumor Activity.Cancer Res 1988; 48:206-214.).Til cell after the amplification is take mature T cells as main, and the CD3+ cell can reach more than 95%, and the In Vitro Anti tumor activity than the high 50-100 of LAK doubly; Til cell is the T cell that tumour antigen activates, and the tumour target cell is had specificity, and the tumor cytotoxicity activity is strengthened greatly, feeds back in the body and can assemble in the tumor locus specificity.
The clinical study report is arranged, among the 62 routine nephrectomy MRCC patients, there are 55 routine patients to turn out TIL, 5 routine CR after TIL associating IL-2 treatment, 14 routine PR, MS is 22 months, serious toxicity (Figlin RA does not occur during the treatment, Pierce W, Bel ldegrun A, et al.Treatment ofmetastatic renal cell carcinoma with nephrectomy, interleukin-2andcytokine-primed or CD8 (+) selected tumor infi ltrating lymphocytes fromprimary tumor.J Urol.1997; 158:740-5.).In the other clinical study, 44 routine IV phase patients with gastric cancer after radical operations are divided at random: chemotherapy group (DDD unites 5-FU) and chemotherapy combined TIL group.The MS of chemotherapy combinatorial association TIL group is 8.3 and 11.5 months (P＜0.05), and multiplicity finds that the TIL treatment is relevant with prognosis with tumor size.Prompting TIL has anti-tumor activity (KonoK in cancer of the stomach, Takahashi A, Ichihara F, et al.Prognostic significance of adoptiveimmunotherapy with tumor-associated lymphocytes in patients withadvanced gastric cancer:a randomized trial.Clin Cancer Res, 2002,8:1767-1771.).In addition, research is found, the TIL treatment that 50 routine Hepatectomy patients adopt independent IL-2 or associating IL-2 to cultivate, and 1 year that unites the cultivation group is 64% and 92% without recurrence rate and survival rate, control group is 32% and 68%.And between two groups 3 years without recurrence rate and survival rate without significant difference (P＞0.05) (Zhang YX, Wang XY, Liu JB, et al.Effects of auto-tumor infiltratinglymphocytes induced by interleukin (IL)-12with IL-2on patients ofprimary hepatic carcinoma.Zhonghua Yi Xue Za Zi, 2008,88:973-976.).
Research and human clinical trial show that all TIL has the effect of target killing antigen positive tumour cell in the mouse model body, and prompting can be by sorting and these specificity TCRs V β subfamily T cell clone and be applied to the immunotherapy of tumour patient of increasing.But so far, the feasible program that really can be used for the tumour patient clinical treatment is but also few, and its reason is that the efficient of external evoked TIL is too low, and operating process is complicated, and proliferation time is long, and the amplification limited amount, does not therefore often reach the expection result for the treatment of.At first, the cytokine of the uses such as Rosenberg only has IL-2 (Belldegrun A, Muul L andRosenberg S.Interleukin 2Expanded Tumor-infiltrating Lymphocytes inHuman Renal Cell Cancer:Isolation, Characterization, and AntitumorActivity.Cancer Res 1988; 48:206-214.), the people such as Figlin continue to use the method for Rosenberg, also only adopt IL-2, the final cell quantity that obtains reaches 4 * 109～1 * 1010 (Figlin RA, Thompson JA, Bukowski RM, et al.Multicenter, Randomized, Phase IIITrial of CD81 Tumor-Infiltrating Lymphocytes in Combination WithRecombinant Interleukin-2in Metastatic Renal Cell Carcinoma.J ClinOncol 1999,17:2521-2529.); Afterwards, domestic employing IL-2 and IL-12 combination of cytokines contain serum free culture system liquid and cultivate, the result shows that the til cell of cultivation has the very high tumor activity (ZhangYX of killing, Wang XY, Liu JB, et al.Effects of auto-tumor infiltratinglymphocytes induced by interleukin (IL)-12with IL-2on patients ofprimary hepatic carcinoma.Zhonghua Yi Xue Za Zi, 2008,88:973-976.).The people such as the Ceng Yixin of DKFZ of Zhongshan University have adopted the blood serum medium that contains of the combination of cytokines that contains 1000IU/ml IL-2 and 30ng/mLanti-CD3, and use cultivating 14 days from the body feeder layer cells of 40Gy radioinactivation, 1000 times of average amplification efficiencies, final cell quantity reaches (1.2-20) * 109 (He J, Tang XF, Zeng YX, et al.Ex vivo expansion oftumor-infiltrating lymphocytes from nasopharyngeal carcinoma patientsfor adoptive immunotherapy.Chin J Cancer.2012Jan 17.doi:10.5732/cjc.011.10376.[Epub ahead of print]).
Tumor infiltrating lymphocyte not only comprises tumor-killing T lymphocyte (CTL), but also comprises the adjustment type T cell (Treg cell) that suppresses the CTL anti-tumor function and the medullary system inhibition cell (MDSC) of deriving.Immunity teaching and research room of Medical Center of Fudan University bear is thought east and waits the people by rejecting the CD4+CD25+Treg cell, significantly increased effect (the Xu L of TIL adoptive immunotherapy, Xu W, Xiong SD, et al.Depletionof CD4+CD25high regulatory T cells from tumor infiltrating lymphocytespredominantly induces Th1type immune response in vivo which inhibitstumor growth in adoptive immunotherapyCan Biol ﹠amp; Ther 2009,8:1,1-7.).
But above research all exists the amplification efficiency of til cell low and can not effectively suppress the Treg lymphocyte to the problem of the inhibit feature of cytotoxic T lymphocyte, thereby has limited its application aspect oncotherapy.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, and a kind of efficient amplification cultivation liquid of tumor infiltrating lymphocyte is provided.
The present invention realizes above-mentioned purpose by the following technical solutions:
A kind of efficient amplification cultivation liquid of tumor infiltrating lymphocyte contains IL-2 in the described nutrient solution, IL-12, anti-CD28 and anti-CTLA-4.
As the further improvement of the technical program, described IL-2, IL-12, the content of anti-CD28 and anti-CTLA-4 is respectively 1000U/ml, 7pg/ml, 2ug/ml and 3ug/ml.
As the further improvement of the technical program, also contain the serum-free medium of 2mM L-glutaminate and 0.5% human serum albumin in the described nutrient solution.
Compared with prior art, the present invention adopts and contains IL-2, IL-12, and the serum free medium of the combination of cytokines such as anti-CD28 and anti-CTLA-4, the amplification efficiency of til cell obviously improves, and is higher about 3～10 times than the amplification efficiency of amplification cultivation liquid commonly used; And can effectively suppress the Treg lymphocyte to the inhibit feature of cytotoxic T lymphocyte, the lymphocyte of amplification has the extremely strong knurl effect of killing.
Description of drawings
Fig. 1 is tumor infiltrating lymphocyte amplification of the present invention and testing process schema.
Embodiment
Further describe concrete technical scheme of the present invention below in conjunction with drawings and Examples, so that those skilled in the art understands the present invention, and do not consist of its Copyright law.
1. the tumour of excision is at first cleaned with HBSS, then under aseptic condition, remove immediately the tumor surface necrotic tissue, fatty tissue and obvious healthy tissues, be cut into the fragment of organizing about 0.5mmx0.5mmx0.5mm with the HBSS rinsing and with the knurl body, be suspended in the enzymic digestion liquid (without RPMI1640 nutrient solution AB serum, that contain the I type deoxyribonuclease of 0.01% V-type Unidasa, 0.1% IV Collagenase Type and 0.002%) 37 ℃ of water-baths concussions and stir digestion 4-5h, cross net, make single cell suspension.
2. be placed on first isopyknic 100% and 75% lymphocyte separation medium in the centrifuge tube, then cell suspension is slowly added to the superiors, carried out discontinuity density gradient centrifugation 300r/min centrifugal 20 minutes, and collected respectively the tumour cell of bed interface and the TIL of lower bed interface, use without Ca
2+And Mg
2+HBSS clean twice, carry out cell counting.
3. tumour cell is frozen: the tumour cell of fresh separated is placed the RPMI1640 nutrient solution that contains 30%AB serum and 10% dromisol, carry out packing and tight sealing with aseptic cryopreservation tube, and frozen for subsequent use in-80 ℃ of refrigerators.
4.TIL cultivation and propagation: place and contain IL-2 (1000U/ml), anti-CD28 (2ug/ml), (the perfect medium composition is as follows: the serum-free medium (Bo Tena company) that contains 2mM L-glutaminate and 0.5% human serum albumin) in cultivation in the perfect medium of IL-12 (7pg/ml) and anti-CTLA-4 (3ug/ml), inoculum density is 5 * 105 cells/ml, place 37 ℃, the 5%CO2 incubator, changed liquid 1 time in per 3 days, carried out bacterium every 3 days, the inspection such as fungi and intracellular toxin, 10～14d measures respectively the cell killing rate and collects TIL and counting, and cell proliferation is pressed proliferation index (EI)=cell count/ml/5x105/ml and calculated.
5.TIL Bacteria Detection: extract to cultivate first day and later on every 3 days TIL supernatant liquor 0.5ml, be transferred to and examine bacterium on the blood agar with transfer pipet, carry out mark, indicate cell numbering, operation item title, date etc.The blood agar that will add sample is just being put (lid is upper, and the glass dish of coated selective medium is lower) in the 37 degree constant temperature biochemical cultivation cases, and rear inversion overnight kept 48 hours.
6.TIL the cell killing rate is measured: the LDH method is measured cytotoxic activity, calculates as follows killing activity.
Calculation formula:
Killing activity (%)=(the effect target cell mixes release A value-Spontaneous release group A value)/(maximum release group A value-Spontaneous release group A value) x100%
7. detect the generation of cytokine with the ELISA method: get TIL at the supernatant liquor of each group nutrient solution, carry out IFN-γ, TNF-alpha content mensuration with the ELISA box.
8. infusion TIL and lymphocyte subgroup are measured: 15～20d til cell increases in a large number after operation, and killing activity reaches the peak, the tumour cell completely dissolve in the nutrient solution.5d patient all accepts intramuscular injection 100,000 U/ days IL-2 before feedback, and through the til cell 250ml of peripheral vein infusion 1～2x108-9/ml, 3～5d feeds back 1 time, totally 3～4 times at every turn.Before operation and after feeding back end, 1 week with flow cytometer two groups of patients are made Lymphocyte Subsets And Their Alternations respectively, observe the variation of human body immune function, observe simultaneously Clinical changes.
The present invention has adopted the substratum amplification tumor infiltrating lymphocyte that contains anti-CD28 and two kinds of antibody of anti-CTLA-4, and amplification efficiency is increased to 1000 times; Anti-CD28 has activated the lymphocytic activity of tumor-killing T effectively, the ability of the synthetic and release cells factor particularly, and such as INF-γ, TNF-α; Anti-CTLA-4 then can suppress the function of Treg cell effectively.
Above-described embodiment only is explanation technological thought of the present invention and characteristics; its purpose is to make those skilled in the art can understand content of the present invention and implements according to this; can not limit according to this protection scope of the present invention; the equalization of namely being done with disclosed spirit changes or derives, and must be encompassed in protection scope of the present invention.
Claims (3)
1. the efficient amplification cultivation liquid of a tumor infiltrating lymphocyte is characterized in that, contains IL-2 in the described nutrient solution, IL-12, anti-CD28 and anti-CTLA-4.
2. the efficient amplification cultivation liquid of a kind of tumor infiltrating lymphocyte according to claim 1 is characterized in that, described IL-2, and IL-12, the content of anti-CD28 and anti-CTLA-4 is respectively 1000U/ml, 7pg/ml, 2ug/ml and 3ug/ml.
3. the efficient amplification cultivation liquid of a kind of tumor infiltrating lymphocyte according to claim 1 is characterized in that, also contains the serum-free medium of 2mM L-glutaminate and 0.5% human serum albumin in the described nutrient solution.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103823068A (en) * | 2013-12-16 | 2014-05-28 | 周菊华 | Method for identifying anti-tumor lymphocytes |
| CN106085956A (en) * | 2016-06-12 | 2016-11-09 | 杨世成 | A kind of method reversing antitumor lymphocyte infiltration exhaustion and application thereof |
| CN106399239A (en) * | 2016-06-24 | 2017-02-15 | 安徽未名细胞治疗有限公司 | A kind of til cell culture medium and preparation method thereof |
| CN108707580A (en) * | 2018-06-20 | 2018-10-26 | 淮安诺康生物科技有限公司 | A kind of amplification in vitro method of tumor infiltrating lymphocyte TIL |
| CN108753718A (en) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | The amplification in vitro method of tumor infiltrating lymphocyte TIL |
| CN108753717A (en) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL |
| WO2021239083A1 (en) * | 2020-05-29 | 2021-12-02 | 上海君赛生物科技有限公司 | Seed cell medium of tumor-infiltrating lymphocyte and application thereof |
-
2012
- 2012-04-27 CN CN 201210127274 patent/CN103374548A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103823068A (en) * | 2013-12-16 | 2014-05-28 | 周菊华 | Method for identifying anti-tumor lymphocytes |
| CN103823068B (en) * | 2013-12-16 | 2016-05-25 | 周菊华 | A kind of antitumor lymphocytic authentication method |
| CN106085956A (en) * | 2016-06-12 | 2016-11-09 | 杨世成 | A kind of method reversing antitumor lymphocyte infiltration exhaustion and application thereof |
| CN106399239A (en) * | 2016-06-24 | 2017-02-15 | 安徽未名细胞治疗有限公司 | A kind of til cell culture medium and preparation method thereof |
| CN108707580A (en) * | 2018-06-20 | 2018-10-26 | 淮安诺康生物科技有限公司 | A kind of amplification in vitro method of tumor infiltrating lymphocyte TIL |
| CN108753718A (en) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | The amplification in vitro method of tumor infiltrating lymphocyte TIL |
| CN108753717A (en) * | 2018-06-20 | 2018-11-06 | 淮安诺康生物科技有限公司 | A kind of method of amplification in vitro tumor infiltrating lymphocyte TIL |
| WO2021239083A1 (en) * | 2020-05-29 | 2021-12-02 | 上海君赛生物科技有限公司 | Seed cell medium of tumor-infiltrating lymphocyte and application thereof |
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