CN104673897B - A kind of method of the biological activity of the measure pathway inhibitors of PD 1 - Google Patents
A kind of method of the biological activity of the measure pathway inhibitors of PD 1 Download PDFInfo
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Abstract
A kind of method for being used to determine the biological activity of the pathway inhibitors of PD 1, methods described includes：The effector cell of express alpha CD3TM and PD L1 target cell, expression NFAT reporter genes and PD 1 is in contact with the pathway inhibitors of PD 1, and measures the signal of NFAT reporter genes to determine the biological activity of the pathway inhibitors of PD 1.
Description
Technical field
A kind of method for being used to determine the biological activity of PD-1 pathway inhibitors, methods described includes：By express alpha
The effector cell of CD3TM and PD-L1 target cell, expression NFAT reporter genes and PD-1 is in contact with PD-1 pathway inhibitors,
Signal with measurement NFAT reporter genes is to determine the biological activity of PD-1 pathway inhibitors.
Background technology
Programmed death acceptor 1 (PD-1) be the inhibitive ability of immunity expressed first in the T cell and B cell of activation by
Body.The interaction of this receptor and its part shows the t cell response of decrease always in vitro and in vivo.PD-1 is blocked to match somebody with somebody with it
Interaction display between one of body PD-L1 improves the immunity of tumour-specific CD8+T cells, therefore, can help to be immunized
System removes tumour cell.
PD-1 (being encoded by gene Pdcd1) is the immunoglobulin superfamily member relevant with CTLA-4 with CD28.Already
Show that PD-1 can the transduction of negative regulator antigen receptor signal when being combined with its part (PD-L1 and/or PD-L2).Mouse is understood fully already
PD-1 structures and mouse PD-1 and human PD-L 1 cocrystallization structure (Zhang, X etc., Immunity 20：337-347
(2004)；Lin etc., Proc.Natl.Acad.Sci.USA 105：3011-6(2008)).PD-1 and similar family member are I
Type transmembrane glycoprotein, its Ig changeable types (v-shaped) domain for containing responsible ligand binding and responsible binding signal transduction molecule
Cytoplasmic tail.PD-1 cytoplasmic tails contain two signal transduction die body ITIM based on tyrosine, and (immunity receptor tyrosine suppresses
Act on die body) and ITSM (immunity receptor tyrosine transformation die body).
After T cell stimulation, PD-1 raises tyrosine phosphatidase SHP-2 to the ITSM die bodys in cytoplasmic tail, and this leads
The dephosphorylation of effector molecule is caused, effector molecule for example has CD3 ζ, PKC θ and ZAP70, these molecules participate in CD3T cells
Signal transduction is cascaded.The mechanism of PD-1 down-regulation t cell responses is similar to CTLA-4's, but different because this two
Plant molecule and all regulate and control overlapping a series of signal transducin (Parry etc., Mol.Cell Biol.25：9543-9553).
Bennett and partner show, only when activating and suppressing two kinds of signals all on same surface, and what PD-1 was mediated believes T cell
The inhibitory action of number transduction just can effectively, this show PD-1 signal transduction mechanisms determined by time and space (Bennett F. etc.,
J Immunol.170：711-8(2003)).
Research shows lymphocyte (periphery CD4+ and CD8+T cell, the B cell and monocyte) upper tables of PD-1 in activation
Reach, and expressed in CD4-CD8- (double-negative) T cells and NK-T cells during being additionally shown in thymus development.
PD-1 parts (PD-L1 and PD-L2) are constitutive expression, or can be induced in various kinds of cell type, and these are thin
Born of the same parents' type includes non-hematopoietic tissue and various tumor types.PD-L1 is in B cell, T cell, myeloid cell and BMDC
(DC) express, but also expressed in peripheral cells (such as CMEC) and non-lymphoid organ (as heart, lung) on.
In contrast, PD-L2 only exists in macrophage and DC.The expression pattern of PD-1 parts has pointed out PD-1 maintaining periphery resistance to
Effect by, and for adjusting T cell and the B cell response of self activation in the periphery.Two kinds of parts all be containing
The I type transmembrane receptors of IgV- samples and IgC- spline structures domain in extracellular regions.Two kinds of parts all contain containing unknown signaling transduction mould
The short cytoplasmic region of body.
There is substantial amounts of research to show that the interaction of PD-1 and its part causes suppression lymph thin in vitro and in vivo so far
Born of the same parents breed.Tamper indicating PD-1/PD-L1 interactions already can increase T cell propagation and cell factor is produced, and block cell
Cycle progress.Initially the analysis to Pdcd1-/- mouse does not have any obvious immunological phenotype of identification.However, aged mouse
It is spontaneous to have developed the autoimmune diseases different from Pdcd1 defects backcrossing strain.These diseases include lupoid acne proliferative joint
Inflammation (C57BL/6) (Nishimura H. etc., Int.Immunol.10：1563-1572 (1998)), lethal cardiomyopathy (BALB/
C) (Nishimura H. etc., Science 291：319-322 (2001)) and type i diabetes (NOD) (Wang J. etc.,
Proc.Natl.Acad.Sci.U.S.A 102：11823-11828(2005)).In a word, the analysis of knock-out animal is caused clear
PD-1 mainly plays a part of to induce and adjust peripheral tolerance.Therefore, Therapeutic blockade PD-1 approach can help to overcome immune
Tolerance.This selective exclusion can be used for treating cancer or infection and for being inoculated with (preventative or therapeutic) period reinforcement
Immunity.PD-1 pathway inhibitors prospect on clinical treatment is very tempting.In order to develop more PD-1 pathway inhibitors with
And the quality control in being produced for PD-1 pathway inhibitors (such as monoclonal antibody), it is necessary to exploitation suppresses to PD-1 paths
The method that the biological activity of agent is measured.
The biology of PD-1 pathway inhibitors (such as PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies) general at present
The assay method of activity, is the shadow for proving to block the PD-1 approach of lymphocyte effector cell using mixed lymphocyte reaction (MLP)
Ring.These methods carry out Determination of biological activity using the human peripheral blood mononuclear cell (PBMC) of people.However, this method variation
Property it is big, cost is high, and method durability and repeatability are poor.Its assay method is respectively：
(1) the anti-PD-1 or PD-L1 antibody of people cell proliferation and cell factor in mixed lymphocyte reaction (MLP) is produced
Influence：The influence of the PD-1 approach of blocking lymphocyte effector cell is proved using mixed lymphocyte reaction (MLP).In the assay,
In the presence or absence of anti-PD-1 or PD-L1 antibody, T cell test propagation, IFN-γ secretion and IL-2 are secreted.Make
Employment CD4+T cell enrichments post (R＆D Systems) purifies human T-cell by PBMC.Every part of culture is in 200 μ l cumulative volume
Include 105The T cell of individual purifying and 104The dendritic cells of individual allogene.Different antibodies concentration is added into every part of culture
Anti- PD-1 or PD-L1 monoclonal antibodies.It is used as negative control without antibody or Isotype control antibodies.By cell in 37 DEG C of cultures
5 days.After 5 days, 100 μ l culture mediums are taken out from every part of culture is used for cytokine measurements.Utilize OptEIA ELISA reagents
Box (BD Biosciences) measures the level of IFN-γ and other cell factors.Cell is used3H- thymidines are marked, and are further cultured for
18 hours, and analyze cell propagation.
(2) influence of the anti-PD-1 or PD-L1 antibody on human Blood Cytokines release of people：Anti- PD-1 or PD-L1 are resisted
Body mixed with human whole blood with determine single anti-PD-1 or PD-L1 antibody whether stimulate human blood cell discharge some cells because
Son.The human whole blood of 500 μ l test tube of hepari is added in each hole.Added into each hole 10 μ g or the 100 anti-PD-1 of μ g or
PD-L1 antibody.There is some holes to be incubated together with anti-cd 3 antibodies as positive control, or together with human IgG1 or the antibody of human IgG 4
Incubate the negative control matched as isotype.Cell is incubated 6 or 24 hours in 37 DEG C.Cell is centrifuged and blood is collected
Starch for cell factor IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-12 measurement, the measurement utilizes cell
Factor cytolytic dose pearl array determination method (BD Biosciences).The concentration (pg/ml) of every kind of cell factor is shown in following
Table 3a (incubating for 6 hours) and 3b (incubating for 24 hours).As a result the processing that the anti-PD-1 antibody 5C4 and 4H1 of independent employment is carried out is shown
Human blood cell is not stimulated to discharge any in cell factor IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-12
Kind.
The Determination of biological activity method of the traditional PD-1 pathway inhibitors of above two is required to gather, it is enough to separate
Human peripheral blood mononuclear cell (PBMC), it is thin in cell proliferative conditions and supernatant by being determined after culture in prolonged (2~5 days)
The expression of intracellular cytokine.Longer the time required to these traditional methods, cost is higher, and method is in itself easily by different people
The influence of PBMC activity differences between body, method variability is larger.
And present inventors have surprisingly discovered that, express alpha CD3TM and PD-L1 cell is used in combination (for example, to register
Number CGMCC No.10299 are deposited in the CHO-K1/ α of China Committee for Culture Collection of Microorganisms's common micro-organisms center
CD3TM/PD-L1 cells) (effectively it is connected so that right with detectable part with expression PD-1 and NFAT reporter genes
It, which is expressed, carries out quantitative NFAT genes) cell (for example, being deposited in China Microbiological bacterium with registration number CGMCC No.10298
Plant the JurKat/PD-1/NFAT cells of preservation administration committee common micro-organisms center) human PBMC can be replaced to simulate human body
Interior PD-1/PD-L1 mechanism of action.By using cross-film anti-cd 3 antibodies as molecules of immunization stimulus, co-suppression point is then studied
Sub- PD-L1 is realized to PD- by the stimulus signal with its acceptor PD-1 with reference to caused by suppressing cross-film CD 3-resisting monoclonal antibody
The measure of 1 or PD-L1 monoclonal antibody biological activities.This method does not need anyone blood-derived cells or other compositions,
Adding after detection liquid can be in different time detecting result, and the result that develops the color is stable, and quality is more controllable.The life that this method is determined
Thing activity is related to clinical efficacy, meets the related technical requirements of CFDA.
The content of the invention
In an aspect, the invention provides a kind of side for being used to determine the biological activity of PD-1 pathway inhibitors
Method, methods described includes：By the effector cell of express alpha CD3TM and PD-L1 target cell, expression NFAT reporter genes and PD-1
It is in contact with PD-1 pathway inhibitors, and the signal of measurement NFAT reporter genes is lived with the biology for determining PD-1 pathway inhibitors
Property.
In another aspect, the invention provides a kind of side for being used to determine the biological activity of PD-1 pathway inhibitors
Method, methods described includes：
(1) culture and expression NFAT reporter genes and PD-1 of express alpha CD3TM and PD-L1 target cell are provided respectively
Effector cell culture；
(2) PD-1 pathway inhibitors are added to the target cell culture；
(3) effector cell's culture is cultivated added to the target cell comprising the PD-1 pathway inhibitors
Thing；
(4) target cell culture, effector cell's culture and the PD-1 are included obtained by incubation step (3)
The mixture of pathway inhibitor；With
(5) signal of NFAT reporter genes is measured to determine the biological activity of PD-1 pathway inhibitors.
In some embodiments of the present invention, the PD-1 pathway inhibitors are selected from PD-1 monoclonal antibodies and PD-L1
Monoclonal antibody.
In some embodiments of the present invention, the target cell of the express alpha CD3TM and PD-L1 be selected from CHO-K1 cells,
Any of 293 cells, melanoma cells, breast cancer cell and lymphoma cell, preferably express alpha CD3TM and PD-L1
CHO-K1 cells.
In some embodiments of the present invention, the effector cell of the expression NFAT reporter genes and PD-1 is jurkat
Cell.
In some preferred embodiments, the target cell is with registration number CGMCC on December 30th, 2014
No.10299 is deposited in the CHO-K1/ α CD3TM/PD-L1 of China Committee for Culture Collection of Microorganisms's common micro-organisms center
Cell.
In some preferred embodiments, effector cell is with registration number CGMCC on December 30th, 2014
The JurKat/PD-1/NFAT that No.10298 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center is thin
Born of the same parents, NFAT reporter genes are NFAT- luciferases (NFAT-luciferase) in the cell.
In some embodiments of the present invention, the NFAT reporter genes are NFAT- luciferases.
In some embodiments of the present invention, the PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies are selected from
One or more in Nivolumab, pembrolizumab, CT-011, MPDL3280A and BMS-936559.
In some embodiments of the present invention, method of the invention is in-vitro method.
In some embodiments of the present invention, method of the invention is Non-diagnostic method.
In some embodiments of the present invention, the step of the inventive method (1) includes：
(1a) prepares express alpha CD3TM and PD-L1 target cell in the following manner：With the nucleic acid for including coding for alpha CD3TM
The carrier of sequence and carrier transfection CHO-K1 cells, 293 cells, melanoma cells, breast comprising the nucleotide sequence for encoding PD-L1
Adenocarcinoma cell or lymphoma cell.
In some embodiments of the present invention, the step of the inventive method (1) includes：
(1b) prepares the expression NFAT reporter genes and PD-1 effector cell in the following manner：With including coding
The carrier of the nucleotide sequence of NFAT reporter genes and the carrier transfection jurkat cells comprising the nucleotide sequence for encoding PD-1.
In another aspect, the invention provides method of the present invention in PD-1 monoclonal antibodies and PD-L1 Dan Ke
Application in the quality control of grand antibody producing.
In yet another aspect, it is applied to determine PD-1 monoclonal antibodies the invention provides one kind and PD-L1 monoclonals is anti-
The method of the biological activity of body.
The present invention it is other in terms of in, present invention also offers on December 30th, 2014 with registration number CGMCC
No.10299 is deposited in the CHO-K1/ α CD3TM/PD-L1 of China Committee for Culture Collection of Microorganisms's common micro-organisms center
Cell.
The present invention it is other in terms of in, present invention also offers on December 30th, 2014 with registration number CGMCC
The JurKat/PD-1/NFAT that No.10298 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center is thin
Born of the same parents.
In an additional aspect of the present invention, the invention provides a kind of recombinant anti human programmed death acceptor 1 (PD-1) list
The Determination of biological activity method of clonal antibody and recombinant anti human programmed death ligand 1 (PD-L1) monoclonal antibody, it is included
Following steps：
(1) target cell suspension is prepared, uniform bed board is incubated；
(2) the PD-1 monoclonal antibodies or PD-L1 monoclonal antibody solutions of various concentrations are prepared, the flat of step (1) is added to
In plate；
(3) effector cell's suspension is prepared, uniform bed board is incubated；
(4) relative chemical flat light emission value (RLU) is read on ELIASA, calculating PD-1 monoclonals by data processing resists
The biological activity of body or PD-L1 monoclonal antibodies.
In one embodiment, target cell can be express alpha CD3TM and PD-L1 cell.
In another embodiment, target cell can be express alpha CD3TM and PD-L1 cell, the express alpha CD3TM
With PD-L1 cell appointing in CHO-K1 cells, 293 cells, melanoma cells, breast cancer cell and lymphoma cell
It is a kind of.Preferably, target cell is express alpha CD3TM and PD-L1 CHO-K1 cells.
In some embodiments, the density of target cell suspension can be 1 × 104~5 × 104Individual/hole, preferably 2 ×
104~5 × 104Individual/hole, 2 × 104~4 × 104Individual/hole, 2 × 104~3 × 104Individual/hole, 3 × 104~5 × 104Individual/hole, 3 ×
104~4 × 104Individual/hole, 4 × 104~5 × 104Individual/hole, more preferably 1 × 104Individual/hole, 1.5 × 104Individual/hole, 2 × 104
Individual/hole, 2.5 × 104Individual/hole, 3 × 104Individual/hole, 3.5 × 104Individual/hole, 4 × 104Individual/hole, 4.5 × 104Individual/hole, 5 × 104
Individual/hole.It is particularly preferred that the density of target cell suspension is 2.5 × 104Individual/hole.
In preferred embodiments, effector cell can express NFAT reporter genes and PD-1 jurkat cells.
In some embodiments, the density of effector cell's suspension can be 2 × 104~10 × 104Individual/hole, preferably 2
×104~9 × 104Individual/hole, 2 × 104~8 × 104Individual/hole, 2 × 104~7 × 104Individual/hole, 2 × 104~6 × 104Individual/hole, 2
×104~5 × 104Individual/hole, 2 × 104~4 × 104Individual/hole, 2 × 104~3 × 104Individual/hole, 3 × 104~9 × 104Individual/hole, 3
×104~8 × 104Individual/hole, 3 × 104~7 × 104Individual/hole, 3 × 104~6 × 104Individual/hole, 3 × 104~5 × 104Individual/hole, 3
×104~4 × 104Individual/hole, 4 × 104~9 × 104Individual/hole, 4 × 104~8 × 104Individual/hole, 4 × 104~7 × 104Individual/hole, 4
×104~6 × 104Individual/hole, 4 × 104~5 × 104Individual/hole, 5 × 104~9 × 104Individual/hole, 5 × 104~8 × 104Individual/hole, 5
×104~7 × 104Individual/hole, 5 × 104~6 × 104Individual/hole, 6 × 104~9 × 104Individual/hole, 7 × 104~9 × 104Individual/hole, 7
×104~8 × 104Individual/hole, 8 × 104~9 × 104Individual/hole, more preferably 1 × 104Individual/hole, 2 × 104Individual/hole, 3 × 104
Individual/hole, 4 × 104Individual/hole, 5 × 104Individual/hole, 6 × 104Individual/hole, 7 × 104Individual/hole, 8 × 104Individual/hole, 9 × 104Individual/hole,
10×104Individual/hole.It is particularly preferred that the density of effector cell's suspension is 5 × 104Individual/hole.
According to the assay method of the present invention, PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies of the invention can be
Any one of Nivolumab, pembrolizumab, CT-011, MPDL3280A, BMS-936559.
In some embodiments, PD-1 monoclonal antibodies and PD-L1 monoclonal antibody solutions can be formulated as concentration 1
In the range of~100 μ g/mL, preferably 10 μ g/mL.
In some embodiments, data processing of the invention calculates reference material and half effect of sample according to four parametric regressions
Measure concentration (EC50)。
It is anti-in PD-1 monoclonal antibodies and PD-L1 monoclonals another aspect provides the assay method of the present invention
Application in the Quality Control of body.
Specifically, the present invention using various concentrations PD-1 monoclonal antibodies or PD-L1 monoclonal antibodies, by with effect
Cell (JurKat/PD-1/NFAT cells) surface PD-1 or target cell (CHO-K1/ α CD3TM/PD-L1 cells) surface PD-L1
With reference to the coinhibitory signals between blocking target cell and effector cell raise signal in effector cell, this method includes as follows
Step：
(1) effector cell is prepared；The cell membrane of the effector cell can express programmed death acceptor 1 (PD-1), cell
Luciferase (luciferase) can be expressed in matter；
(2) target cell is prepared；The cell membrane of the target cell can express cross-film anti-cd 3 antibodies (α CD3TM) and procedural dead
Die ligand 1 (PD-L1)；
(3) target cell suspension is prepared, and is uniformly spread according to 25000/hole into 96 orifice plates, 37 DEG C of overnight incubations；
(4) the PD-1 monoclonal antibodies of various concentrations or the solution of PD-L1 monoclonal antibodies are prepared, above-mentioned 96 orifice plate is added to
In；
(5) effector cell's suspension is prepared, and is uniformly spread according to 50000/hole into 96 orifice plates, 37 DEG C are incubated 6 hours；
(6) relative chemical flat light emission value (RLU) is read using chemiluminescence on ELIASA, is calculated by data processing
The biological activity of PD-1 monoclonal antibodies or PD-L1 monoclonal antibodies.
Chemical transfection or the method for electric shock transfection are used in the step (1) by pcDNA3.1 (+)/PD-1 and pGL4.30
[Luc2P/NFAT-RE] plasmid co-transfection then passes through screening (puromycin and the heredity of pressurizeing into jurkat cells (ATCC)
Mycin) mode screen acquisition stable cell line JurKat/PD-1/NFAT.
In the step (2) using chemical transfection or the method for electric shock transfection by pcDNA3.1 (+)/α CD3TM and
PcDNA3.1 (+)/PD-L1 plasmid co-transfections are to CHO-K1 cells (ATCC), 293 cells, melanoma cells, breast cancer cell
And in lymphoma cell, then screen acquisition stable cell line by way of screening (puromycin and hygromycin B) of pressurizeing
CHO-K1/αCD3TM/PD-L1。
Target cell is CHO-K1/ α CD3TM/PD-L1 cells or passes through artificial express alpha CD3TM, PD- in the step (3)
L1 293 cells, melanoma cells, breast cancer cell and lymphoma cell, the density of the target cell suspension is 1 × 104
~5 × 104Individual/hole, preferred density is 2.5 × 104Individual/hole.
PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies in the step (4) be Nivolumab,
Any one of pembrolizumab, CT-011, MPDL3280A, BMS-936559, described PD-1 monoclonal antibodies and PD-
L1 monoclonal antibody solutions sample or standard concentration are diluted to 1~100 μ g/mL, and preferred concentration is 10 μ g/mL, and then downward 3
Times or 2 times of doubling dilutions, 7 dilution factors.
Effector cell is JurKat/PD-1/NFAT cells in the step (5), and the density of effector cell's suspension is 2
×104~10 × 104Individual/hole, preferred density is 5 × 104Individual/hole.
After T cell stimulation, the NFAT activation of φt cell receptor (TCR) induction, Ca2+Interior stream, interleukin 2 (IL-2)
Produced with interferon-γ (IFN-γ).In expression NFAT- luciferases and PD-1 effector cell, pierced by CD3 antibody
After swashing, NFAT activation, the luciferase transcription expression of NFAT mediations；If on the contrary, thering is CD3 antibody and PD-L1 to act on table simultaneously
Up to NFAT- luciferases and PD-1 effector cell when, i.e. target cell (express alpha CD3TM and PD-L1 cell) and effector cell
After (expression NFAT- luciferases and PD-1 cell) is incubated altogether, suppresses signal PD-L1/PD-1 and play an active part in, it is suppressed that be a large amount of
The luciferase transcription of NFAT mediations.And if adding PD-1 and PD-L1 monoclonal antibody, then can effectively block PD-
L1/PD-1 suppression signal, so as to reverse suppression signal so that the luciferase transcription and up-regulated expression of NFAT mediations.
Definition
Unless otherwise defined, what all scientific and technical terminologies used herein had that those of ordinary skill in the art are understood is identical
Implication.
Term " target cell " used herein refers to natively or artificially (such as by that will be compiled containing destination protein
The carrier of code nucleotide sequence is transfected into cell) express alpha CD3TM and PD-L1 cell.The effector cell of the present invention serves PD-
In 1/PD-L1 paths target cell effect or simulation PD-1/PD-L1 paths in target cell (i.e. antigen presenting cell).
Term " express alpha CD3TM and PD-L1 CHO-K1 cells " used herein refers to containing natively or manually
Ground (such as by the way that the carrier containing destination protein nucleic acid sequence encoding is transfected into cell) express alpha CD3TM and PD-L1 CHO-
K1 cells.
Term " effector cell " used herein refers to natively or artificially (such as by that will contain destination protein
The carrier of nucleic acid sequence encoding is transfected into cell) expression PD-1 and NFAT cell.The effector cell of the present invention serves PD-1/
In PD-L1 paths effector cell effect or simulation PD-1/PD-L1 paths in effector cell (i.e. T cell, B cell or its
His bone marrow cell).
Term used herein " expression NFAT- luciferases and PD-1 jurkat cells " refers to natively or people
Express PD-1 and NFAT- luciferins in building site (such as by the way that the carrier containing destination protein nucleic acid sequence encoding is transfected into cell)
The jurkat cells of enzyme.
Term " PD-1 pathway inhibitors " used herein refers to suppress PD-1/PD-L1 pathway signal transductions
Inhibitor, it with PD-1 or PD-L1 by being combined so as to block the interaction between PD-1 and PD-L1 so that PD-1/
The signal transduction of PD-L1 paths is suppressed.PD-1 pathway inhibitors used include but is not limited in the present invention：PD-1 monoclonals
Antibody, PD-L1 monoclonal antibodies, micromolecular inhibitor etc..
Term " PD-1 " used herein refers to (the Programmed cell death of apoptosis albumen 1
Protein 1), also referred to as CD279 (cluster ofdifferentiation 279), it is in people by PDCD1 genes institute
Coding.
Term " PD-L1 " used herein refers to (the Programmed death-ligand of programmed death ligand 1
1), also referred to as CD274 (cluster of differentiation 274) or B7 homologues 1 (B7-H1), it is in people
By CD274 coded by said gene.
Term " NFAT " used herein refers to nuclear factor of activated T cells (Nuclear factor of activated
T-cells), transcription factor very crucial in its signal transduction pathway mediated for φt cell receptor (TCR).
Term " NFAT reporter genes " used herein refer to detectable part (such as GFP, eGFP, luciferase,
FITC, quantum dot etc.) effectively connect so that expressing it quantitative NFAT genes of progress.
" α CD3TM " refer to cross-film mouse anti-CD3antibody (OKT3) to term used herein, and it is in OKT3 heavy chain carboxylics
Cardinal extremity adds cross-film sequence (AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR).
Brief description of the drawings
Other aspects of the present invention are disclosed in the following description book and appended accompanying drawing.Pass through accompanying drawing hereafter and implementation
Example is to the present invention have been described in detail, and it is for illustration purposes only and has no intention to be limited.Due to the description and implementation
Example, other embodiments being also included in the present invention will be apparent to those skilled in the art.
Fig. 1 is effector cell's PD-1 expression quantity FACs results of different clones；
Fig. 2 is effector cell's luciferase activity measurement result of different clones；
Fig. 3 is that 12# target cells clone PD-L1 expression quantity FACs results；
Fig. 4 is that 12# target cells clone α CD3TM expression quantity FACs results；
Fig. 5 is anti-human PD-1 monoclonal antibodies (Nivolumab) and anti human PD-L 1 monoclonal antibody (MPDL3280A)
Reporter gene Determination of biological activity result.
The preservation of biomaterial
Chinese hamster ovary cell cell line CHO-K1/ α CD3TM/PD-L1 are on December 30th, 2014 with registration number
CGMCC No.10299 are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address：Court of Beijing
The positive institute 3 of area's North Star West Road 1, Institute of Microorganism, Academia Sinica).
Human T lymphocyte's cell line JurKat/PD-1/NFAT is on December 30th, 2014 with registration number CGMCC
No.10298 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica).
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1：Prepare the effector cell of expression PD-1 and NFAT- luciferases
The method that electric shock is transfected is used first by pcDNA3.1 (+)/PD-1 (limited public affairs of Yi Qiao Divine Land, Beijing biotechnology
Department) and pGL4.30 [Luc2P/NFAT-RE] (Pu Luomaige (Beijing) Bioisystech Co., Ltd) plasmid co-transfections to jurkat
In cell (ATCC), then cultivate and expressed in the culture medium containing 1 μ g/ml puromycins and 1mg/ml Geneticins
The effector cell of PD-1 and NFAT- luciferases.Using jurkat cells as control, gained is analyzed using flow cytometer (FACs)
The PD-1 expression quantity of cell, is as a result shown in Fig. 1.Stimulated with mouse anti-CD3antibody (BDBiosciences, cat.555336),
Pass through Bright-GloTMKit (Promega, cat.E2610) determines the expression of luciferase (luciferase), wherein firefly
Light element enzyme assay result is shown in Fig. 2.So as to obtain the effector cell of the present invention.Selection wherein higher gram of expression luciferase
Grand acquisition stable cell line JurKat/PD-1/NFAT, the cell is on December 30th, 2014 with registration number CGMCC
No.10298 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Embodiment 2：Prepare expression express alpha CD3TM and PD-L1 target cell
Using chemical transfection or the method for electric shock transfection by pcDNA3.1 (+)/α CD3TM and pcDNA3.1 (+)/PD-L1 matter
Grain (Sino Biological Inc.) cotransfection is into CHO-K1 cells (ATCC), then fast containing 1 μ g/ml
The cell through transfection is cultivated in purine mycin and the culture medium of 200 μ g/ml hygromycin Bs.It is anti-using the anti-mouse of donkey using CHO-K1 as control
The α CD3TM and PD-L1 of cell expression, wherein α CD3TM and PD-L1 expression quantity obtained by body and PD-L1 antibody tests
FACs results are shown in Fig. 3 and Fig. 4.So as to obtain the target cell of the present invention.The higher clone of selection wherein expression quantity, which obtains, stablizes thin
Born of the same parents strain CHO-K1/ α CD3TM/PD-L1, the cell is deposited on December 30th, 2014 with registration number CGMCC No.10299
China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Embodiment 3：The method according to the invention determines PD-1 monoclonal antibodies and the biology of PD-L1 monoclonal antibodies is lived
Property
(1) prepare CHO-K1/ α CD3TM/PD-L1 target cell suspensions, using DMEM/F12+10%FBS culture mediums according to
2.5×104Individual/hole, the even density in 100 μ l/ holes is spread into 96 orifice plates (corning), 37 DEG C of 5%CO2It was incubated in incubator
Night；
(2) the PD-1 monoclonal antibodies (Nivolumab) or PD-L1 of various concentrations are prepared using 1640+10% culture mediums
Monoclonal antibody (MPDL3280A) solution is diluted to 10 μ g/mL, and then downward 3 times of doubling dilutions, 7 concentration points, are sucked above-mentioned
DMEM/F12+10%FBS culture mediums in 96 orifice plates, by the PD-1 monoclonal antibodies prepared or PD-L1 monoclonal antibodies
Monoclonal antibody solution is added in 96 orifice plates according to 50 μ l/ holes；
(3) JurKat/PD-1/NFAT effector cell's suspension is prepared using 1640+10% culture mediums, and according to 5 × 104
Individual/hole, the even density in 50 μ l/ holes is spread into above-mentioned 96 orifice plate, in 37 DEG C of 5%CO2It is incubated 6 hours in incubator；
(4) relative chemical flat light emission value (RLU) is read using chemiluminescence on M200 or Pherastar ELIASAs,
The biological activity of PD-1 monoclonal antibodies or PD-L1 monoclonal antibodies is calculated by data processing, according to four parametric regression meters
Calculate PD-1 monoclonal antibodies and PD-L1 monoclonal antibody median effective dose concentration EC50Respectively 0.17 μ g/ml, 0.42 μ g/ml, specifically
As a result visible Fig. 5.
Multiplicating measure is carried out, it is found that the Determination of biological activity method stability of the present invention is higher, detection liquid is added
Afterwards can be in different time detecting results, the result that develops the color is stable, and quality is more controllable.Meanwhile, this method not need anyone
Blood-derived cells or other compositions.Also, the biological activity that this method is determined is related to clinical efficacy, meet CFDA related
Technical requirements.
Consider from the practice of specification disclosed herein or disclosure, other embodiments pair of present disclosure
It will be apparent for those skilled in the art.This specification and embodiment are intended to be regarded only as being used as example, this public affairs
The true scope for opening content is shown by following claims.Reference is made to any bibliography content by drawing
With being hereby incorporated by reference in its entirety.
Claims (22)
1. a kind of method for being used to determine the biological activity of PD-1 pathway inhibitors, methods described includes：By express alpha CD3TM
The effector cell of target cell, expression NFAT reporter genes and PD-1 with PD-L1 is in contact with PD-1 pathway inhibitors, and measurement
The signal of NFAT reporter genes is to determine the biological activity of PD-1 pathway inhibitors.
2. a kind of method for being used to determine the biological activity of PD-1 pathway inhibitors, methods described includes：
Step (1)：The culture and expression NFAT reporter genes and PD-1 of express alpha CD3TM and PD-L1 target cell are provided respectively
Effector cell culture；
Step (2)：PD-1 pathway inhibitors are added to the target cell culture；
Step (3)：By effector cell's culture added to the target cell culture comprising the PD-1 pathway inhibitors
Thing；
Step (4)：The target cell culture, effector cell's culture and the PD- are included obtained by incubation step (3)
The mixture of 1 pathway inhibitor；With
Step (5)：The signal of NFAT reporter genes is measured to determine the biological activity of PD-1 pathway inhibitors.
3. method according to claim 1 or 2, wherein the PD-1 pathway inhibitors be selected from PD-1 monoclonal antibodies and
PD-L1 monoclonal antibodies；And/or
Wherein described NFAT reporter genes are NFAT- luciferases.
4. method according to claim 1 or 2, wherein the target cell of the express alpha CD3TM and PD-L1 is selected from CHO-K1
Any of cell, 293 cells, melanoma cells, breast cancer cell and lymphoma cell；Wherein described expression NFAT
Reporter gene and PD-1 effector cell are jurkat cells.
5. method according to claim 1 or 2, wherein the target cell of the express alpha CD3TM and PD-L1 is express alpha
CD3TM and PD-L1 CHO-K1 cells.
6. method according to claim 1 or 2, wherein the target cell of the express alpha CD3TM and PD-L1 is in 2014
The CHO- that December 30 was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center with registration number 10299
K1/ α CD3TM/PD-L1 cells.
7. method according to claim 1 or 2, wherein the expression NFAT reporter genes and PD-1 effector cell be in
On December 30th, 2014 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center with registration number 10298
JurKat/PD-1/NFAT cells.
8. method according to claim 3, wherein the PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies are selected from
One or more in Nivolumab, pembrolizumab, CT-011, MPDL3280A and BMS-936559.
9. method according to claim 1 or 2, wherein the step (1) includes：
Step (1a)：Express alpha CD3TM and PD-L1 target cell is prepared in the following manner：With the nucleic acid for including coding for alpha CD3TM
The carrier of sequence and carrier transfection CHO-K1 cells, 293 cells, melanoma cells, breast comprising the nucleotide sequence for encoding PD-L1
Adenocarcinoma cell or lymphoma cell.
10. method according to claim 1 or 2, wherein the step (1) includes：
Step (1b)：The expression NFAT reporter genes and PD-1 effector cell are prepared in the following manner：With including coding
The carrier of the nucleotide sequence of NFAT reporter genes and the carrier transfection jurkat cells comprising the nucleotide sequence for encoding PD-1.
What 11. the method any one of claim 1-10 was produced in PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies
Application in quality control.
12. a kind of Determination of biological activity method of PD-1 monoclonal antibodies and PD-L1 monoclonal antibodies, methods described includes：
Step (1) prepares target cell suspension, and according to 1 × 104~5 × 104The even density in individual/hole is spread into 96 orifice plates, 37
DEG C 5%CO2It is incubated overnight in incubator, the target cell is CHO-K1/ α CD3TM/PD-L1 cells；
Step (2) prepares the PD-1 monoclonal antibodies of various concentrations or the solution of PD-L1 monoclonal antibodies, is added to above-mentioned 96 orifice plate
In；
Step (3) prepares effector cell's suspension, and according to 2 × 104~10 × 104The even density in individual/hole is spread into 96 orifice plates,
In 37 DEG C of 5%CO2It is incubated 6 hours in incubator, wherein the effector cell is JurKat/PD-1/NFAT cells；With
Step (4) reads relative chemical flat light emission value on ELIASA using chemiluminescence, and PD-1 is calculated by data processing
The biological activity of monoclonal antibody or PD-L1 monoclonal antibodies.
13. the density described in method according to claim 12, wherein step (1) is 2 × 104~5 × 104Individual/hole.
14. the density described in method according to claim 12, wherein step (1) is 2 × 104~3 × 104Individual/hole.
15. the density described in method according to claim 12, wherein step (1) is 2.5 × 104Individual/hole.
16. the concentration described in method according to claim 12, wherein step (2) is 1~100 μ g/mL.
17. the concentration described in method according to claim 12, wherein step (2) is 10 μ g/mL.
18. the density described in method according to claim 12, wherein step (3) is 2 × 104~10 × 104Individual/hole.
19. the density described in method according to claim 12, wherein step (3) is 4 × 104~8 × 104Individual/hole.
20. the density described in method according to claim 12, wherein step (3) is 4 × 104~6 × 104Individual/hole.
21. the density described in method according to claim 12, wherein step (3) is 5 × 104Individual/hole.
22. a kind of cell, the cell is to be deposited in Chinese microorganism strain on December 30th, 2014 with registration number 10299 to protect
Hide the CHO-K1/ α CD3TM/PD-L1 of administration committee's common micro-organisms center.
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