免疫檢查點藥物研發--PBMC相關體外實驗

2019-05-05 14:50 嚴國樑
4536


【小編誤打誤撞進入此行業之后,經常聽很多研發人員講起用我們的PBMC做MLR, SEB, ADCC之類的實驗,但具體對于這類實驗的目的是什么,PBMC是如何用于此類實驗之類的問題往往是一知半解,為了給大家提供更好的服務,這段時間我們團隊也開始在學習這方面的內容,不過可能也是畢業有些年頭了,學習能力退化的比較厲害,因而也逼著自己寫寫公眾號,將看到的比較不錯的文獻,微信文章整理整理,分門別類一下,希望有一點點拋磚引玉的作用吧】


恰好前幾天查詢文獻的時候看到的一篇挺有意思的文章,相信讀者中很多人都看過,文章標題如下(所有的論文作者均來自BMS)。里面比較詳細的聊到了在check point藥物研發中使用PBMC做的相關體外實驗。


Mixed lymphocyte reaction (MLR).

Dendritic cells (DCs) were generated byculturing monocytes isolated from PBMCs using a monocyte purification kit invitro for 7 days with 500 U/mL IL- 4 and 250 U/mL GM-CSF.
CD4+ T cells (1 x105) and allogeneic DCs (1 x 104) were co-cultured with or without dosetitrations of nivolumab added at the initiation of the assay.
After 5 days, IFN secretion in culture supernatants was analyzed by ELISA, and cells were labeled with 3H-thymidine for an additional 18 hours to measure T-cell proliferation.


Staphylococcal enterotoxin B(SEB) stimulation of PBMC.

PBMCs from healthy human donors (N = 18) were cultured for 3 days with nivolumab or an isotype control antibody (20 μg/mL) at the initiation of the assay together with serial dilutions of SEB. Interleukin-2(IL-2) levels in culture supernatants were measured by ELISA analysis.


Antigen-specific recall response in vitro.

In a cytomegalovirus (CMV)-restimulationassay, 2 x 105 PBMCs from a CMV-positive donor were stimulated using lysate of CMV-infected cells, with serial dilutions of nivolumab added at the initiationof the assay. After 4 days, supernatants were assayed for IFNγ.


Suppression assay with regulatory T cells (Treg).

CD4+CD25+ Tregs and CD4+CD25- responder Tcells were purified from PBMC (CD4+CD25+ Treg isolation kit). In an allogeneicMLR assay, Tregs (5 x 104) were co-cultured with 1 x 105 responder T cells and2 x 104 monocyte-derived DCs, with 20 μg/mL nivolumab.
After 5 days, IFNγ production was assessed in supernatants,and cells were labeled with 3H-thymidine for an additional 18 hours for proliferation analysis.


Antibody-dependent cell-mediated cytotoxicity (ADCC).

PBMCs were incubated overnight with 50 U/mL IL-2 and used as effector cells.
Activated CD4+ T cells labeled with BATDA reagent were used as target cells at an effector-to-target cell ratio of 50:1. Serial dilutions of nivolumab or positive control (anti-MHC I antibody) were added; the cells were incubated for 3 hours at 37oC.
To measure cytotoxicity,supernatant was mixed with Europium-solution and read using a RUBYstar Model460 microplate reader (BMG LABTECH).

【Result】

Figure 2

In an allogeneic MLR, PD-1 blockade with nivolumab systematically resulted in a titratable enhancement of IFN release, and in some donor T cell/DC pairs, enhanced T-cell proliferation was observed(Fig. 2A).
Nivolumab also enhanced IL-2 secretion over isotype control inresponse to SEB using PBMCs (Fig. 2B).
Using a CMV- restimulation assay, nivolumab, compared with an isotype control, resulted in a concentration-dependent augmentation of IFNγ secretion from CMV-responsive donors (Fig. 2C).
While PD-1 expression can be observed in T cells prior to stimulation by allogeneic DCs, SEB or antigen,PD-1 expression is up regulated after T-cell activation in all of these assays (Figure S4A, 4B and data notshown).
In addition, PD-L1 expression and up regulation can be observed in multiple cell subsets in these assays (Figure S4B and data not shown).


As Tregs also express PD-1, nivolumab was assessed in an allogeneic MLR in which Tregs suppressed the proliferation and cytokine secretion of purified CD4+CD25- responder T cells, which were stimulated by allogeneic DCs.
In this assay, nivolumab completely restored proliferation and partially restored IFN release by the alloreactive T cells(Fig. 2D).


Taken together, these data show that nivolumab can, at very low concentrations (~1.5 ng/mL), enhance T-cellreactivity in the presence of a T-cell receptor stimulus. However, nivolumab had no effect in the absence of antigen or T-cell receptor stimulus. Specifically, there was no significant release of inflammatory cytokines, including IFNγ, TNF-α, IL-2, IL-4, IL-6, or IL-10, from unstimulated whole blood after co-incubation with nivolumab. In contrast, positive-control anti-CD3 antibody potently increased cytokine release (Table S2). These results demonstrate that nivolumab does not cause non- specific lymphocyte activation.


Figure 3

Lastly, the ability of nivolumab (tested from 0.003 μg/mL to 50 μg/mL) to mediate ADCC activity in vitro was tested. Using IL-2-activated PBMCs as a source of natural killer (NK) cells and activated human CD4+ T cells expressing high levels of cell-surface PD-1 as target cells, nivolumab (IgG4 [S228P]) did not mediate ADCC (Fig. 3). Limited ADCC activity was observed with the parental form of nivolumab, an IgG1 antibody purified from hybridoma supernatant at high antibody concentrations, whereas positive control anti-MHC-class I antibody was able to mediate ADCC of T cells at lowerantibody concentrations. In addition, nivolumab did not mediate complement-mediated cytotoxicity (CDC) of activated human CD4+ T cells in thepresence of human complement (data not shown).


【相關產品】

MLR: 我司有已經驗證過,配對好的PBMC個體,一組提取CD14分化DC,一組提取對應T cell,并可以持續穩定提供固定個體;

SEB:可推薦相應驗證過的個體滿足此實驗;

CMV positive donor PBMC;

Treg:可提供同一個體 Treg和剩余的T cell,可根據需要進行定制化分裝;

ADCC:可提供經過驗證的ADCC效應好的個體,且可大量穩定供應;

Isotype Control Antibody:Anti-HEL (Hen Egg Lysozyme) without anyknown cross-reactive to human.

Human:

huIgG1
huIgG1-N297A

huIgG1-LALA
huIgG2

huIgG3
huIgG4-S228P
huIgG4-L235E


Mouse:

MuIgG1
MuIgG2a


About 儒百】

儒百生物(Schbio)秉持質量優、速度快、服務好的理念,致力于支持治療性抗體研究。獨家代理全美最大的血液免疫學細胞供應商StemExpress?產品,并攜手重組蛋白服務商泰州百英生物推出陽性對照抗體、陰性對照抗體和重組靶點蛋白。

現階段主要產品線:

1.血液免疫學細胞——PBMC及亞型(CD3,CD4,CD8,CD56,CD14等),serum,plasma;Cord Blood;Bone Marrow;

2. 陽性對照抗體——Anti-PD1, Anti-PDL1,Anti-CD47,Anti-Tigit,Anti-Lag3,Anti-Tim3等;

3. 陰性對照抗體——Anti-HEL(與人源無交叉污染,Fc段可定制化);

4.重組靶點蛋白