CN106474143A - Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides are preparing for treating the application in terms of the medicine of B cell lymphoma - Google Patents
Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides are preparing for treating the application in terms of the medicine of B cell lymphoma Download PDFInfo
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Abstract
The present invention relates to Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides prepare for treat B cell lymphoma medicine in application, the B cell lymphoma has MYD88L265P mutation, the oligonucleotides includes CCT repetitive sequence [(CCT) n], wherein, C is cytimidine, and T is thymidine, and n is 8 to 12 integer, wherein, the sequence of the oligonucleotides is selected from：SEQ ID NO.1：5’-cctcctcctcctcctcctcctcct-3’；SEQ ID NO.2：5’-cctcctcctcctcctcctcctcctcct-3’；SEQ ID NO.3：5’-cctcctcctcctcctcctcctcctcctcct-3’；SEQ ID NO.4：5’-cctcctcctcctcctcctcctcctcctcctcct-3’；SEQ ID NO.5：5’-cctcctcctcctcctcctcctcctcctcctcctcct-3’；SEQ ID NO.6：5’-cctccTcctcctccTcctcctccTcctcctccTcct-3’；Wherein, lowercase t represents the base of thio-modification, and capital T represents unmodified base.The oligonucleotides of the present invention can block TLR7 and TLR9 and MYD88L265P protein combination in the B cell lymphoma being mutated with MYD88L265P, block the activation of NF- κ B signal path, so as to suppress the propagation of tumour cell.
Description
Technical field
Present invention relates in general to a kind of Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides and its should
With specifically, the present invention relates to TLR7 the and TLR9 inhibition oligonucleotides comprising CCT repetitive sequence
Preparing for treating the application in terms of the medicine of B cell lymphoma.
Background technology
B cell lymphoma is the solid tumor that B cell is produced, and which includes that Hodgkin lymphoma and non-Hodgkin's drench
Bar knurl, its parting are numerous, and Hodgkin lymphoma includes classical Hodgkin lymphoma and Nodular lymphocyte
Type Hodgkin lymphoma, Hodgkin lymphoma are presently considered to be initiated by the tumour of B cell.Non-Hodgkin's
Lymthoma mainly include diffusivity large B cell lymphoid tumor (diffuse large B-cell lymphoma,
DLBCL), follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), primary lymphedema are thin
Born of the same parents' lymthoma/chronic lymphocytic leukemia, lymphoma mantle cell (MCL), above 5 kinds of B cell are non-suddenly
Strange gold lymthoma is relatively conventional, accounts for the 3/4 of NHL.
Diffusivity large B cell lymphoid tumor is modal class lymthoma, accounts for NHL
30% or so.The great heterogeneity of the clinical manifestation of DLBCL, morphology, immunophenotype and genetics characteristics.
DLBCL is divided into by centrum germinativum source B cell sample (germinal centre according to gene expression profile
B-cell-like, GCB) and two kinds of hypotypes of activating B cell sample (activated B-cell-like, ABC).
Survival analysis shows that the Overall survival of GCB type is significantly better than ABC type.For 5 years survival rates, Qian Zhewei
76%, and the latter is only 16%.In recent years, increasing research shows CD20 molecular targeted agents profit
Appropriate former times monoclonal antibody Combination chemotherapy can significantly improve CD20 the positive DLBCL patient complete remission rate and
DFS rate, is reduced significantly the prognosis difference of GCB type and non-GCB type.
Waldenstrom's macroglobulinemia (Macroglobulinemia, WM) belong to rare
B cell lymphoma, is lymphoplasmacytic lymphoma again, and annual morbidity is 0.3/10 ten thousand, drenches in all of B system
1%～2% is accounted in bar knurl, which is mainly characterized by marrow lymph sample plasmocyte infiltrating, monoclonal igm mass formed by blood stasis.
WM is indolent lymphoma, and the patient's median survival time for having clinical symptoms is 5～6 years.Treon etc.
(Treon SP.N Engl Med 2012；367(9):About 90% Fahrenheit macroglobulinemia 826-833) is found
There is MYD88 L265P mutation in disease.MYD88 L265P mutation is probably to cause the early stage for promoting WM to occur
Cancer event.Recently research have indicated that, in multiple B cell tumours, find the DNA sequence dna of MYD88 because specific
The base of tagmeme sports C by T, causes the 265th amino acids missense mutation of protein-coding region, i.e. leucine
Mistake is changed into proline (L → P).This mutational site be exactly in TIR (Toll-interleukin 1receptor,
TIR) domain, MYD88 also can occur dimerization in the case of without TLR and IL-1R signal stimulus,
Cause interleukin 1 receptor associated kinase (interleukin-1receptor-associated kinase, IRAK)
The activation of the NF- κ B signal of mediation, promotes cell propagation.In the AB type for carrying MYD88 L265P more
After IRAK4 being knocked out in unrestrained property large B cell lymphoid tumor cell line, Malignant cellular growth is limited, and this growth
Suppression can be corrected by introducing wild type IRAK4, and invalid to inactivation type IRAK4, IRAK4 inhibitor can
Cause the ABC-DLBCL cell line for carrying MYD88 L265P mutation dead, to carrying wild type
The GCB-DLBCL cell line of MYD88 is nothing lethal effect.Suppression MYD88/IRAK signal path is permissible
Suppression NF- κ B signal, and receive the ABC-DLBCL growth of tumour cell with MYD88 L265P mutation
To suppression.(the Ngo VN.Nature 2011 such as Ngo；470(7332):115-119) disturbed by RNA,
High-flux sequence finds that 29%ABC-DLBCL has MYD88 L265P mutation, and other kinds of
Few or do not have in diffusivity large B cell lymphoid tumor, such as GCB-DLBCL and Burkitt lymphoma
MYD88 L265P is mutated.The sustained activation of NF- κ B signal approach is the feature of ABC-DLBCL.Base
In this discovery, this area be devoted to always find can targeted therapy have MYD88 L265P mutation
Diffusivity large B cell lymphoid tumor.
Content of the invention
Present invention relates in general to Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides are in treatment B cell
Application in lymthoma, specifically, the present invention relates to Toll-like receptor TLR7 and TLR9 inhibition widow's core
Thuja acid prepare for treat B cell lymphoma medicine in application, wherein, the B cell lymphoma
It is mutated with MYD88 L265P, it is preferable that the B cell lymphoma is diffusivity large B cell lymphoid tumor,
And the diffusivity large B cell lymphoid tumor is activating B cell sample hypotype.
The oligonucleotides of the present invention includes CCT repetitive sequence [(CCT) n], and wherein, C is cytimidine, and T is
Thymidine, n are 8 to 12 integers, and wherein, the sequence of the oligonucleotides is selected from：
SEQ ID NO.1：5’-cctcctcctcctcctcctcctcct-3’；
SEQ ID NO.2：5’-cctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.3：5’-cctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.4：5’-cctcctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.5：5’-cctcctcctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.6：5’-cctccTcctcctccTcctcctccTcctcctccTcct-3’；
Wherein, lowercase t represents the base of thio-modification, and capital T represents unmodified base.
Toll-like receptor TLR7 of the present invention and TLR9 inhibition oligonucleotides are to MYD88 L265P
The B cell lymphoma of mutation produces dose dependent inhibitory action.In general, the Toll-like receptor
TLR7 and TLR9 inhibition oligonucleotides is with the dosage of 1.25mg/kg body weight to 500mg/kg body weight to having
The B cell lymphoma of MYD88 L265P mutation produces inhibitory action.
In one embodiment, the B cell lymphoma to being mutated with MYD88 L265P produces suppression
The dosage of Toll-like receptor TLR7 of effect and TLR9 inhibition oligonucleotides be 1.25mg/kg body weight extremely
12.5mg/kg body weight, or 12.5mg/kg body weight is to 25mg/kg body weight, or 25mg/kg body weight is to 50mg/kg
Body weight, or 50mg/kg body weight is to 125mg/kg body weight, or 125mg/kg body weight is to 250mg/kg body weight, or
250mg/kg body weight is to 500mg/kg body weight.
In a preferred embodiment, the B cell lymphoma to being mutated with MYD88 L265P produces suppression
Toll-like receptor TLR7 for making and the dosage of TLR9 inhibition oligonucleotides are 5mg/kg body weight, or
12.5mg/kg body weight, or 25mg/kg body weight, or 50mg/kg body weight.
In one embodiment of the invention, the medicine also includes pharmaceutically acceptable carrier, its
It is selected from：Solution, diluent, solvent, dispersant, liposome, emulsion, sugar-coat, antiseptic, anti-true
Microbial inoculum, the reagent of isotonic and delayed absorption.
In a preferred embodiment of the invention, the medicine is formulated into oral administered dosage form, vein note
Penetrate formulation, intramuscular injection formulation, inhalation formulation, topical applications.
The oligonucleotides of the present invention shows Toll-like receptor TLR7 and TLR9 inhibition, and which is for treatment tool
ABC type DLBCL for having MYD88 L265P mutation is particularly effective, and the oligonucleotides of the present invention is acted on
ABC type DLBCL with MYD88 L265P mutation, which can block TLR7 and TLR9 and MYD88
L265P protein combination, so as to block the activation of NF- κ B signal path, suppression ABC-DLBCL cell life
Deposit, and then effectively suppression lymphoma cell propagation.Thus, the present invention with Toll-like receptor TLR7 and
The oligonucleotides of TLR9 inhibition can be used for the B cell lymph that targeted therapy has MYD88 L265P mutation
Knurl simultaneously can be used to prepare the medicine of B cell lymphoma of the treatment with MYD88 L265P mutation.
Description of the drawings
Figure 1A to Fig. 1 C shows not homotactic oligonucleotides (SEQ ID NO.1, the SEQ ID of the present invention
NO.5, SEQ ID NO.6) outside people that imiquimod (Imiquimod, IMQ) and CpG685 are induced
The inhibitory action that all blood mononuclear cell (PBMC) breeds.X-axis represents CFSE, and y-axis represents CD19+B
Cell, in the proliferation experiment of PBMC, CFSE is dyeed, and is cultivated 6 days, flow cytomery cell
Propagation.
Figure 1A shows the widow of culture medium group, SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO.6
Impact of the nucleotides group to the proliferative conditions of PBMC.Culture medium group cell proliferation rate 3.78%, SEQ ID
The cell proliferation rate of the oligonucleotides group of NO.1, SEQ ID NO.5 and SEQ ID NO.6 respectively 1.21%,
0.823% and 1.08%, the cell proliferation rate of oligonucleotides group and culture medium group does not have difference, right as testing
According to group.
Figure 1B show the present invention not homotactic oligonucleotides (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) to IMQ (5 μM) stimulation human peripheral blood mononuclearcell propagation suppression situation.This
Bright oligonucleotides and IMQ are according to 1:1、1:3、1:9、1:27 ratio is added in PBMC, and streaming is thin
Born of the same parents' instrument detection CD19+B cell propagation.The concentration of the oligonucleotides of the present invention increases to 27 μM from 0 μM,
The not homotactic oligonucleotides of the present invention can all suppress the B cell proliferation that IMQ is induced, SEQ ID NO.1
Oligonucleotides suppression CD19+B cell breed by 30.60% widow for being reduced to 0.031%, SEQ ID NO.5
Nucleotides suppression CD19+B cell is bred by 30.60% few nucleosides for being reduced to 0.059%, SEQ ID NO.6
Acid suppression CD19+B cell propagation is reduced to 0.159% by 30.60%.
Fig. 1 C show the present invention not homotactic oligonucleotides (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) to CpG685 (1 μM) stimulation human peripheral blood mononuclearcell propagation suppression situation.
The concentration of oligonucleotides increases to 27 μM from 0 μM, and not homotactic oligonucleotides can all suppress CpG685
The B cell proliferation of induction, the oligonucleotides suppression CD19+B cell of SEQ ID NO.1 are bred by 28.50%
The oligonucleotides suppression CD19+B cell propagation for being reduced to 1.03%, SEQ ID NO.5 is reduced by 28.50%
Oligonucleotides suppression CD19+B cell propagation to 0.027%, SEQ ID NO.6 is reduced to by 28.50%
1.08%.The experiment is repeated 3 times and all obtains similar result.
Fig. 2A to Fig. 2 D respectively illustrates the not homotactic oligonucleotides (SEQ that CBA method detects the present invention
ID NO.1, SEQ ID NO.5, SEQ ID NO.6) to the single core of IMQ or CpG685 stimulation human peripheral blood
Cell factor IL-6 and the suppression situation of IL-10 that cell (PBMC) is secreted.X-axis is represented through not existing together
Human PBMC's group of reason, y-axis represent the content of cell factor.Control group is culture medium group and NOG group.
Fig. 2A show the variable concentrations of the present invention oligonucleotides (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) stimulate PBMC to secrete the impact of IL-6 IMQ.IMQ (5 μM) and different sequence,
The oligonucleotides (respectively 0,0.3,1.5,7.5 and 37.5 μM) of variable concentrations is added in PBMC and trains
Foster 36h, CBA (BD) determines the content of IL-6；
Fig. 2 B show the variable concentrations of the present invention oligonucleotides (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) stimulate PBMC to secrete the impact of IL-10 IMQ.IMQ (5 μM) and different sequences
Oligonucleotides (concentration be respectively 0,0.1,0.5,2.5 and 12.5 μM) be added to culture 36h in PBMC,
CBA (BD) determines the content of IL-10；The oligonucleotides of Fig. 2A and Fig. 2 B explanation present invention is lured to IMQ
Lead human PBMC's secrete cytokines IL-6 and IL-10 is inhibited, and be in dosage correlation.
Fig. 2 C show the variable concentrations of the present invention oligonucleotides (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) stimulate PBMC to secrete the impact of IL-6 CpG685.CpG685 (1 μM) and sheet
The oligonucleotides (concentration is respectively 0,1,3,9 and 27 μM) of invention is added to culture 36h in PBMC,
CBA method determines the content of IL-6；
Fig. 2 D shows oligonucleotides (SEQ ID NO.1, SEQ ID NO.5, the SEQ ID NO. of the present invention
6) PBMC is stimulated to secrete the impact of IL-10 CpG685 under the conditions of variable concentrations.CpG685(1μM)
It is added in PBMC with the oligonucleotides (concentration is respectively 0,1,3,9 and 27 μM) of the present invention and cultivates
36h, CBA determine the content of IL-10.Fig. 2 C and Fig. 2 D show that the oligonucleotides of the present invention is lured to CpG685
Lead human PBMC's secrete cytokines IL-6 and IL-10 is inhibited, and be in dosage correlation.Above reality
Test to be repeated 3 times and similar result is all obtained, be expressed as mean value ± SD.
Fig. 3 shows the MYD88 of Sanger method DNA sequencing detection clone OCI-Ly19 and OCI-Ly3.3
Mutational site.Sanger method DNA sequencing result shows, the MYD88 PCR primer of OCI-Ly19 cell
Sequencing, the sequence are consistent with the MYD88 sequence in ncbi database, there is no mutational site, OCI-Ly19
Belong to the cell line of MYD88 wild type.The MYD88 PCR primer sequencing of OCI-Ly3.3 cell, the sequence
It is that T sports C that MYD88 sequence in row and ncbi database has the mutational site of a base, therefore
OCI-Ly3.3 belongs to the cell line of MYD88 L265P mutation.2 clones of every plant of raji cell assay Raji.
Fig. 4 A and Fig. 4 B show flow cytomery OCI-Ly19 cell and OCI-Ly3.3 intracellular
The expression of TLR7 and TLR9.X-axis represents TLR7-PE or TLR9-APC, and Y-axis represents cell number.Detection
The average fluorescent strength (MIF) of the TLR7-PE of OCI-Ly19 or OCI-Ly3.3 and TLR9-APC.From Fig. 4 A
In as can be seen that in OCI-Ly19 groups of cells, TLR7-PE and TLR9-APC is substantially moved to right compared with Isotype control,
Average fluorescent strength (MIF) illustrates OCI-Ly19 cell inner expression apparently higher than the MIF of Isotype control group
TLR7 and TLR9 albumen.As can be seen that in OCI-Ly3.3 groups of cells from Fig. 4 B, TLR7-PE and
TLR9-APC is substantially moved to right compared with Isotype control, and average fluorescent strength (MIF) is apparently higher than Isotype control group
MIF, illustrates OCI-Ly3.3 cell inner expression TLR7 and TLR9 albumen.
Fig. 5 shows that WST-1 determines not homotactic oligonucleotides (SEQ ID NO.1, the SEQ of the present invention
ID NO.5, SEQ ID NO.6) to the propagation unrestraint effect of OCI-Ly19 cell, but substantially suppress
OCI-Ly3.3 cell is bred.X-axis represents the concentration of oligonucleotides, and Y-axis represents Cell viability.SEQ ID
The oligonucleotides of NO.1, SEQ ID NO.5, SEQ ID NO.6 is dense with 0,5,10,20 and 40 μM respectively
Degree effect OCI-Ly19 or OCI-Ly3.3 cell 72 hours, determines cell viability, numerical tabular by WST-1
It is shown as mean value ± SD.Fig. 5 shows that, with the increase of oligonucleotides concentration, the cell viability of OCI-Ly19 exists
The cell viability of 100% or so, OCI-Ly3.3 is gradually reduced by 100%, but the oligonucleotides as the present invention
Concentration be 40 μM when, its cell viability be 20% or so.The experiment is repeated 3 times and all obtains similar result.
The increase of oligonucleotides concentration is affected on the cell viability nothing of OCI-Ly19；But suppression OCI-Ly3.3's is thin
Born of the same parents' vigor, this inhibitory action have concentration dependent.
Fig. 6 show CBA method detection the present invention not homotactic oligonucleotides (SEQ ID NO.1,
SEQ ID NO.5, SEQ ID NO.6) to the secretion IL-10 nothing suppression of GCB-DLBCL clone OCI-Ly19
Make and use, but ABC-DLBCL clone OCI-Ly3.3 cell secretion of cytokines IL-10 can be suppressed.
X-axis represents the concentration of not homotactic oligonucleotides, and Y-axis represents the content of IL-10.The detection of CBA method is thin
The content of intracellular cytokine IL-10.By the not homotactic oligonucleotides of the present invention with 0,2,4,8,10 and 20 μM
Concentration and OCI-Ly19 or OCI-Ly3.3 co-culture 36 hours, as a result shows what OCI-Ly19 cell was secreted
IL-10 is less, and with the increase of oligonucleotides concentration, the amount of OCI-Ly19 secretion IL-10 does not change.
For OCI-Ly3.3 cell, when the concentration of oligonucleotides is 10 μM, IL-10 is almost close to 0,.With
The increase of the concentration of oligonucleotides, the amount of OCI-Ly3.3 cell secretion IL-10 is gradually decreased, this suppression
Effect is in dosage correlation, and three kinds of not homotactic oligonucleotides show similar inhibitory action.Should
Experiment is repeated 3 times and all obtains similar result.
Fig. 7 A and Fig. 7 B show that PI staining for flow cytometry detects oligonucleotides (the SEQ ID of the present invention
NO.6) the impact to clone OCI-Ly19 and OCI-Ly3.3 cell cycle.X-axis represents oligonucleotides
Concentration, Y-axis represent cell percentages.In Fig. 7 A the oligonucleotides of SEQ ID NO.6 concentration be respectively 0,
72h is together cultivated with cell OCI-Ly19 under conditions of 15 and 30 μM, cell cycle result shows, different
The oligonucleotides of the SEQ ID NO.6 of concentration was not all affected on the cell cycle of OCI-Ly19 each phase.Figure
In 7B the oligonucleotides of SEQ ID NO.6 concentration be respectively 0,15 and 30 μM under conditions of with cell
OCI-Ly3.3 cultivates 72h, and cell cycle result shows, SEQ ID NO.6 when concentration is for 30 μM, with
Concentration is compared (compared with control group) when being 0 μM, and OCI-Ly3.3 G1 cell cycle, cell proportion phase is obvious
Increase, S phase and G2, M phase cell proportion are significantly reduced, the oligonucleotides of SEQ ID NO.6 prevents
OCI-Ly3.3 cell was changed from G1 phase to S phase and G2, M phase so that cellular retention causes cell in the G1 phase
Propagation slows down.
Specific embodiment
Many aspects according to the present invention are described in detail below with reference to embodiment, these embodiments
It is only used for illustrating.Those of ordinary skill in the art are not it will be appreciated that the present invention is by institute in embodiment
The restriction of the concrete operation step of description.
Unless otherwise stated, all technical terms used herein and scientific terminology generally have and the present invention
The implication identical implication that person of an ordinary skill in the technical field is generally understood that.
Present invention generally provides Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides are used in preparation
Application in the medicine for the treatment of B cell lymphoma, wherein, the B cell lymphoma has MYD88
L265P is mutated, and the oligonucleotides includes CCT repetitive sequence [(CCT) n], and wherein, C is cytimidine, T
It is thymidine, n is 8 to 12 integer.
Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides
On the one hand, the present invention provides a kind of oligonucleotides comprising CCT repetitive sequence [(CCT) n], wherein,
C is cytimidine, and T is thymidine, and n is 8 to 12 integer.
Term " oligonucleotides " herein is referred to by sugared (as deoxyribose), phosphate group and base
The molecule of composition, wherein glycan molecule and base connect into nucleosides, and nucleosides is connected by phosphate group and forms nucleosides
Acid, the base for forming nucleosides have pyrimidine and purine, and pyrimidine has thymidine (being abbreviated as T or t) and cytimidine
(being abbreviated as C or c), purine have adenine (adenine, be abbreviated as A or a) and guanine (guanine,
It is abbreviated as G or g).Oligonucleotides in the present invention refers to oligodeoxynucleotide, and which is closed by automatic nucleic acid
Instrument is become to synthesize, therefore these oligonucleotides are artificial synthesized oligonucleotides.The oligonucleotides of the present invention
Comprising CCT repetitive sequence [(CCT) n], wherein, C is cytimidine or derivatives thereof, T be thymidine or
Its derivative, n represent the number of recurring unit, and which can be the integer of 8-12.
In certain embodiments of the present invention, the phosphate backbones in the oligonucleotides of the present invention can be for not repairing
Decorations.
Phosphate backbones in the oligonucleotides of the present invention can be also part modification or all modifying.Described
Modification may include multiple chemical modifications, for example, is modified using phosphorothioate moieties or all modifies this
Base in bright oligonucleotides.The modification can be carried out in the building-up process of oligonucleotides or closed
Carry out after becoming, and the modification can occur between nucleosides di-phosphate ester bridged bond on, ribose unit
On upper and/or natural nucleobases (adenine, guanine, cytimidine and thymidine).When in synthesis
When being modified during oligonucleotides, adorned base can be incorporated into that oligonucleotides is internal or is located at
Oligonucleotides end.When being modified after synthetic oligonucleotide, the modification can be by using work
Property group carry out, for example, carried out by amido modified composition, carried out by 3 ' or 5 ' hydroxyls, or pass through phosphorus
Acid esters group is carried out.
Chemical modification of the present invention may include that the skeleton of the oligonucleotides to the present invention is modified, bag
Include but be not limited to, skeleton is modified with thiophosphate, phosphorothioate backbone is obtained, this bone
Frame is a kind of sugar phosphate skeleton of stable nucleic acid molecules, in the skeleton, at least one nucleotides it
Between key on, sulphur instead of the oxygen of the phosphate not bridged, or, at each or every a nucleotides
Between key on, sulphur instead of the oxygen of the phosphate not bridged.Also other can be carried out to oligonucleotide backbone
Modification, for example, using non-ionic DNA analog, for example, alkyl phosphate and aryl phosphate ester,
Skeleton is modified, wherein, the oxygen in charged phosphate is replaced by alkyl or aryl, or is made
Skeleton is modified with di-phosphate ester and alkyl phosphotriester, wherein, by charged o-alkylation.
In certain embodiments of the present invention, the sequence of the oligonucleotides of the present invention is selected from：
SEQ ID NO.1：5’-cctcctcctcctcctcctcctcct-3’；
SEQ ID NO.2：5’-cctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.3：5’-cctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.4：5’-cctcctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.5：5’-cctcctcctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.6：5’-cctccTcctcctccTcctcctccTcctcctccTcct-3’；
Wherein, lowercase t represents the base of thio-modification, and capital T represents unmodified base.
The oligonucleotides that the present invention is provided has Toll-like receptor TLR7 and TLR9 inhibition, also referred to as
Toll-like receptor TLR7 and TLR9 inhibition oligonucleotides.In the present invention, " Toll-like receptor (TLR) "
People TLR family member, the family member has 11 acceptors, be than more conservative one in Evolution of Mammals
Individual receptor family.The related molecular pattern (PAMP) of TLR energy specific recognition cause of disease, not only in activation
Play a significant role in the innate immunity, but also acquired immunity is adjusted, be the connection innate immunity and acquisition
Property immunity bridge.TLR is I type transmembrane protein, can be divided into extracellular region, intracellular region and three part of transmembrane region,
Wherein extracellular region can recognize that specific part can recognize the PAMP of microorganism, the structure of intracellular region with
I receptor of IL-1 is homologous, referred to as Toll/ interleukin 1 receptor homeodomain (Toll/IL-1R
Homology domain, TIR), mainly it is responsible for intracellular signal transduction.TLR is in panimmunity cell
There are expression, such as B cell, dendritic cells (DC), macrophage and certain types of T cell etc..TLR
Positioning in cell is different, and wherein TLR1, TLR2, TLR4, TLR5 and TLR6 are present in carefully
After birth surface, and TLR3, TLR7, TLR8 and TLR9 then exist only in intracellular endoplasmic reticulum, interior body
On lysosome.The specific PAMP of each self-identifying of TLR, wherein TLR1 and TLR6 recognize trigalloyl fat respectively
Peptide；TLR2 can recognize peptide glycan, lipoprotein and zymosan；The viral double-stranded RNA of TLR3 identification
And Poly I (dsRNA):C；The lipopolysaccharides of TLR4 identification bacteria cell wall；TLR5 recognizes flagellin；
The viral single stranded RNA (ssRNA) of TLR7 and TLR8 identification and the antiviral chemical combination of artificial synthesized small molecule
(Imiquimod (S26308), its are the parts of TLR7/8 in 1988 for thing, such as imiquimod class compound
Year is reported first by American scholar Man Chen etc..The similar compound of series is subsequently found that successively, such as
R837, R848 etc.；Imiquimod family is proved to rely on path by TLR7/8-MYD88 signal
IFN is produced, with antiviral and antitumor activity, can be used as bone-marrow-derived lymphocyte activator；TLR9 knows
Other bacterium and the unmethylated CpG DNA of virus.
TLR/IL1-R and corresponding pathogen associated molecular pattern (pathogen associated molecular
Pattern, PAMP) combine after, directly by TIR domain or indirectly by carry TIR domain rank
Connect albumen (TIR domain-containing adaptor protein, TIRAP) and Bruton tyrosine-kinase
Enzyme (Bruton's tyrosine kinase, BTK) activates MYD88, and after activation, MYD88 is tied by DD
Homotype interaction between structure domain recruits IRAK4, and the latter makes IRAK1 that phosphorylation to occur, and combines tumor necrosis factor
Sub- correlation factor 6 (tumor necrosis factor receptor-asssociatd factor 6, TRAF6), shape
Become TLR-MYD88-IRAK4-IRAK1-TRAF6 compound connected with after birth；Subsequently,
IRAK1-TRAF6 is separated with compound, bindin kinase complex TAK1-TAB1-TAB4, activation
TAK1 and IRAK1 dissociate, make compound TRAF6-TAK1-TAB1-TAB4 enter kytoplasm, general
Elementization connection enzyme effect under, TRAF6 ubiquitination and degrade, the TAK1-TAB1-TAB4 compound of activation
Respectively two bars transduction pathway are started by phosphorylation.1st article of approach：The heterologous trimerization of TAK1 phosphorylation
Body, i.e. IKK complex (NEMO/IKK γ-IKK α-IKK β), the latter occur after making I κ B α phosphorylation again
Ubiquitination is degraded, and discharges the NF- κ B being made up of P65 and P50 Liang Fen subunit and is indexed into karyon, and activation promotes
The NF- κ B dependent gene of cell growth.2nd article of approach：TAK1 believes MAPK
After number (mitogen-activated protein kinase, MAPK) pathway components phosphorylation activated transcription because
Sub- c-Jun and c-Fos, c-Jun and c-Fos are indexed into karyon, form transcription factor AP -1, activation MAPK according to
Bad property gene.
(the Kian-Huat Lim.Cancer Research such as Kian-Huat Lim:April 15,2013；Volume
73, Issue 8, Supplement 1) find that TLR7 or TLR9 can be tied after MYD88 L265P mutation
MYD88 L265P albumen is closed, is made MYD88 that dimerization to occur, causes the activation of downstream NF- κ B signal.
After TLR7 and TLR9 is knocked out in the clone of ABC-DLBCL can substantially suppress NF- kB activation,
Promote Apoptosis.In the ABC-DLBCL clone for having knocked out endogenous TLR7 or TLR9, cancer cell
Propagation is substantially suppressed.The huge ball egg of about 90% lymphoplasmacytic lymphoma/Fahrenheit in B cell lymphoma
White mass formed by blood stasis (WM), 29% activated form Diffuse Large B-Cell Lymphoma, 33% central nervous system drench
Bar knurl (all detection specimens pathological are Diffuse Large B-Cell Lymphoma), 9% MALT lymphoma
(MALT), all detect in 2.9% chronic lymphocytic leukemia that MYD88 L265P is mutated.This
Bright oligonucleotides has Toll-like receptor TLR7 and TLR9 inhibition, and which is being acted on MYD88
(for example, diffusivity large B cell lymphoid tumor, for example, ABC type is more for the B cell lymphoma of L265P mutation
Unrestrained property large B cell lymphoid tumor) in can be tied by blocking TLR7 and TLR9 and MYD88 L265P albumen
Close, the activation of NF- κ B signal path is blocked, so as to suppress the propagation of tumour cell.Thus, the present invention
Oligonucleotides with Toll-like receptor TLR7 and TLR9 inhibition can be used for targeted therapy and have MYD88
The B cell lymphoma of L265P mutation simultaneously can be used to prepare B cell of the treatment with MYD88 L265P mutation
Lymphadenomatous medicine.
Medical usage
On the other hand, the present invention provides a kind of pressing down with Toll-like receptor TLR7 and TLR9 for use present invention
The individual internal method of B cell lymphoma of the oligonucleotide treatment of property processed and having using the present invention
The oligonucleotides of Toll-like receptor TLR7 and TLR9 inhibition is preparing the medicine for treating B cell lymphoma
Application in thing, wherein, the B cell lymphoma has MYD88 L265P mutation, for example：Diffuse
Property large B cell lymphoid tumor, including activating B cell sample hypotype diffusivity large B cell lymphoid tumor
(ABC-DLBCL).
In some embodiments, the above-mentioned oligonucleotides (SEQ ID NO.1-SEQ ID NO.6) of the present invention
The medicine for treating diffusivity large B cell lymphoid tumor can be together configured to pharmaceutically acceptable carrier
Preparation, wherein, the pharmaceutical preparation can be configured to oral administered dosage form according to clinical needs, and vein is noted
Penetrate formulation, intramuscular injection formulation, inhalation formulation, topical applications, etc..
Furthermore, the pharmaceutical preparation can also include extra active component, and which is selected from：CHOP (ring
Phosphamide, adriamycin, vincristine, metacortandracin), Btk inhibitor (Ibrutinib), PI3K δ suppression
Agent (Idelalisib), mTOR inhibitors (Everolimus), STAT3 inhibitor (OPB-51602),
Anti-CD 20 monoclonal antibodies (Rituximab), SYK inhibitor (Fostamatinib), Bcl-2 suppression
Agent (Navitoclax), protease inhibitors (Bortezomib) and immunomodulator (Lenalidomide),
One or more in above-mentioned extra active component can with the present invention with Toll-like receptor TLR7 and
The oligonucleotides of TLR9 inhibition together therapeutic alliance diffusivity large B cell lymphoid tumor.
" pharmaceutically acceptable carrier " of the present invention includes that one or more solid or liquid are filled
Agent, diluent or encapsulating substance, the carrier are suitable for for the oligonucleotides in the present invention being applied to individual body
Interior.The carrier can be organic, inorganic, natural or synthesis.The carrier include various solution,
Diluent, solvent, dispersant, liposome, emulsion, sugar-coat, antiseptic, antifungal agent, isotonic
With the reagent of delayed absorption and the carrier of other oligonucleotides applications being suitable in the present invention.Materia medica
The selection of upper acceptable carrier is decided by oligonucleotides application mode in the present invention.The carrier of injectable
Including water, physiological saline, balanced salt solution, glucose solution, glycerine etc..For solid mixture (such as
Powder, ball, tablet, capsule form) for, conventional non-toxic solid carriers includes：With pharmacology
Learn mannitol, lactose, starch and magnesium stearate of purity etc..In addition, as biologically neutral load
Body, can contain atoxic auxiliary substance in the pharmacology component of application, including humidifying or emulsifying agent,
Preservative, PH buffer reagent, sodium acetate and monolaurate etc..Above-mentioned pharmaceutically acceptable carrier is
Well known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical
Sciences, the 18th edition, Mack Printing Company, 1990, the 1289-1329 page, this is with reference to text
Offer and be incorporated herein by).
For example, the example of filler is lactose monohydrate, Lactis Anhydrous and various starch.Bonding
The example of agent is that various celluloses and PVPP, microcrystalline cellulose and silicified microcrystalline are fine
Dimension element.The example of suitable lubricant, including acting on by the medicament of the mobility of compressing powder, for example,
Colloidal silica, stearic acid, magnesium stearate, calcium stearate and silica gel.The example of sweetener is natural
Or artificial sweetener, such as sucrose, xylitol, saccharin sodium, honey element and aspartame.Preservative
Example be potassium sorbate, methylparoban, propylparaben, benzoic acid and its salt, such as to hydroxyl
The alcohol of the ester of the P-hydroxybenzoic acid of yl benzoic acid butyl ester etc, such as ethanol or phenmethylol etc, such as
The phenolic compounds of phenol etc, or quaternary compound, for example, benzalkonium chloride.Suitable diluent
Example includes pharmaceutically acceptable inert filler, for example, microcrystalline cellulose, lactose, calcium monohydrogen phosphate and sugar
Class.The example of diluent includes microcrystalline cellulose, the breast of such as lactose monohydrate and Lactis Anhydrous etc
Sugar, calcium monohydrogen phosphate, mannitol, starch, D-sorbite, sucrose and glucose.Suitable disintegrant
Including lightly crosslinked polyvinylpyrrolidone, cornstarch, farina, cornstarch and modified shallow lake
Powder, PVPP, sodium starch glycollate and its mixture.
The pharmaceutical preparation of the present invention can be aqueous solution form, saline solution form, particle, aerosol, powder
End, tablet, sugar coated tablet, microcapsules, suppository, syrup, emulsion, suspending agent, creme, drops or
Other are suitable to the form of multi-medicament delivery system.
The pharmaceutical preparation of the present invention can be administered in the following way：Oral administration, parenteral, sublingual
Administration, across mucosa delivery, cutaneous penetration, rectally, intraperitoneal injection administration, subcutaneous administration, flesh
In meat administration, intravenous administration, intraarterial delivery, intrathecal drug delivery, local be administered (with powder, ointment,
Gel, drops or transdermal patch form local be administered), buccal administration, by catheter drug delivery, pass through
Implant is administered, etc..In the case of any form of medication, the pharmaceutical preparation of the present invention must be no
Bacterium and be stable under production and storage condition, and bacterium or microorganism pollution will not be subject to.
The pharmaceutical preparation of the present invention can be also configured to by injecting (for example, fast injection or continuous infusion)
The formulation of mode parenteral.Formulation for drug administration by injection can be provided in a unit, for example,
There is provided in the form of ampulla or multi-dose container.The pharmaceutical preparation of the present invention can using such as oiliness carrier or
The form of suspension, solution or emulsion in aqueous carrier etc and can be containing such as suspending agent, stable
The conditioning agent of agent and/or dispersant etc, and polyvinylpyrrolidone, the agar being for example crosslinked can be added
Or alginic acid or its salt (for example, sodium alginate).
The suspension of the pharmaceutical preparation of the present invention can be prepared into suitable oily injection suspensions or Aqueous inj ection
Suspension, wherein, suitable lipophilic solvent or carrier include fat oil (for example, sesame oil) or close
The fatty acid ester (for example, ethyl oleate or triglycerides) for becoming or liposome；Water injection suspension liquid can
Material comprising the viscosity that can increase suspension, for example, sodium carboxymethylcellulose, D-sorbite or Portugal
Glycan.Optionally, suspension can also containing can suitably increase the solubility of compound stabilizer or
Reagent, so as to preparing the solution of high enrichment.For injection, the pharmaceutical preparation of the present invention can quilt
Aqueous solution is configured to, is preferably configured to physiological compatibility buffer solution (for example, Hanks solution, Lin Ge
Family name's solution or normal saline buffer solution).
Preferably, the pharmaceutical preparation of the present invention can be using the powder type being front dissolved in suitable carrier,
The suitable carrier for example, aseptic apirogen water.
For through mucosa delivery, the bleeding agent of barrier to be penetrated is suitable to used in said preparation.
These bleeding agents are generally known in the art.
For oral administration, tablet that the pharmaceutical preparation of the present invention can be prepared by conventional methods or
The form administration of lozenge.
For inhalation, the pharmaceutical preparation of the present invention can be formulated into be easy to by using suitable
Propellant (for example, dicholorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or its
His suitable gas) delivered in the form of the spraying provided by compression wrap or sprayer.Spray in use pressurization
In the case of mist, dosage unit can be determined by providing the valve for delivering the amount that measures.In inhalator or blow
Enter the capsule used in device and cartridge case (for example, gelatine capsule and cartridge case) can be configured to containing the present invention
Pharmaceutical composition and suitable powdered base (for example, lactose or starch) mixture of powders formulation.
The pharmaceutical preparation of the present invention can also be comprising suitable solid or gel phase carriers or excipient.These carry
The example of body or excipient include calcium carbonate, calcium phosphate, various carbohydrates, starch, cellulose derivative,
The polymer of gelatin and such as polyethylene glycol etc.
The present invention preferred embodiment in, the pharmaceutical preparation of the present invention is for treating the big B of diffusivity
Can also be with other activating agent administering drug combinations during cell lymphoma, other activating agents described are selected from：
CHOP (endoxan, adriamycin, vincristine, metacortandracin), Btk inhibitor (Ibrutinib),
PI3K δ inhibitor (Idelalisib), mTOR inhibitors (Everolimus), STAT3 inhibitor
(OPB-51602), anti-CD 20 monoclonal antibodies (Rituximab), SYK inhibitor (Fostamatinib),
Bcl-2 inhibitor (Navitoclax), protease inhibitors (Bortezomib) and immunomodulator
(Lenalidomide).
B cell lymphoma of the present invention is diffusivity big B lymthoma (diffuse large B-cell
Lymphoma, DLBCL), it is modal class lymthoma, accounts for the 30% of NHL
Left and right.The great heterogeneity of the clinical manifestation of DLBCL, morphology, immunophenotype and genetics characteristics.
DLBCL can betide any age, but common with the elderly, and middle position age of onset is 60～65 years old, man
Property is slightly more than women.Clinically with the rapid Silent Neuritis lump for increasing as typical performance, about 1/3 patient companion
The symptom such as there are heating, night sweat, lose weight.DLBCL can be betided in lymph node or knot is outer, and about 40%
It is outer that DLBCL is primary in knot, mainly intestines and stomach, and the outer position of other common knots includes bone, testis, saliva
Gland, thyroid gland, skin etc..CDNA microarray technology is applied to by Alizadeh in 2000 etc. first
The research of DLBCL gene expression profile.DLBCL is divided into by Germinal center B cell sample according to gene expression profile
(germinal centre B-cell-like, GCB) and activating B cell sample (activated B-cell-like, ABC)
Two kinds of hypotypes, survival analysis show that the Overall survival of GCB type is significantly better than ABC type.5 years survival rates are come
Say, the former is 76%, and the latter is only 16%.In recent years, increasing research shows CD20 molecule
Targeted drug rituximab combination chemotherapy regimen can significantly improve CD20 positive DLBCL patient's
Complete remission rate and DFS rate, are reduced significantly the prognosis difference of GCB type and non-GCB type.Ngo
Find that 29%ABC type Diffuse Large B-Cell Lymphoma is present Deng by RNA interference, high-flux sequence
MYD88 L265P, and in other kinds of Diffuse Large B-Cell Lymphoma such as GCB type and Hugh Burkitt lymph
Few or do not have in knurl.MYD88 L265P mutation causes MYD88 that dimerization occurs, and causes NF- κ B
The sustained activation of signal path, 60% ABC type DLBCL have NF- κ B in core to express, and only little portion
(about 30%) GCB type DLBCL is divided to have NF- κ B expression in core, research confirms, the NF- κ B's of continuation
Activation is necessary to the ABC type DLBC cell survival of part.MYD88 as herein described is substantially one
Soluble adaptin in kytoplasm is planted, is the Molecular regulator of Toll-like receptor and IL-1 receptor signaling pathways,
Participate in human body inherent immunity.Relative molecular weight 35kDa, comprising 2 special domains.C-terminal carry with
TLR molecule intracellular section identical TIR domain, is connected with TLR/IL-1R by The homotype interaction, opens
Dynamic signal transduction.N-terminal is death domain (death domain, DD), with death domain
Signaling molecule, the related signal path of apoptosis involvement.
The oligonucleotides of the present invention shows Toll-like receptor TLR7 and TLR9 inhibition, and which is for treatment tool
ABC type DLBCL for having MYD88 L265P mutation is particularly effective, and the oligonucleotides of the present invention is acted on
ABC type DLBCL with MYD88 L265P mutation, which can block TLR7 and TLR9 and MYD88
L265P protein combination, so as to block the activation of NF- κ B signal path, suppression ABC-DLBCL cell life
Deposit, and then effectively suppression lymphoma cell propagation.In certain embodiments of the present invention, by the present invention
Oligonucleotides or the pharmaceutical preparation of oligonucleotides comprising the present invention deliver medicine to patient and can alleviate or treat
ABC-DLBCL, for the serious disease occurred on the ABC-DLBCL type individuality with MYD88 L265P
For reason phenomenon, by the pharmaceutical preparation administration of the oligonucleotides of the present invention or oligonucleotides comprising the present invention
In the NF- κ B that patient can mitigate the symptom of ABC-DLBCL disease, suppression is mediated by MYD88 L265P
Signal activation causes the propagation of tumour cell.
Patient of the present invention refers to apply human individual and the non-human vertebrate of oligonucleotides of the present invention
Animal individual.Non-human vertebrate includes non-human primate, farming animals and pet animals etc..
Therapeutically effective amount
It is used for preparing treatment B cell in Toll-like receptor TLR7 of the present invention and TLR9 inhibition oligonucleotides
In terms of lymphadenomatous medicine, the medicine for treating B cell lymphoma be made up of the oligonucleotides of the present invention
The oligonucleotides of the present invention comprising therapeutically effective amount in preparation, the therapeutically effective amount is effectively to be realized
Expect the amount of purpose.Will be (especially) depending on the illness in treatment to the effective actual amount of application-specific.
The determination of effective dose is just (especially in accordance with concrete public herein in the limit of power of those skilled in the art
The content that opens).
For oligonucleotides as herein described, therapeutically effective amount can pass through cell culture detection method at first
Determine.Target blood concentration is the concentration of the reactive compound for being capable of Cell growth inhibition or division.Excellent
In the embodiment of choosing, at least the 25% of cytoactive is suppressed.Can induce at least about 30%, 50%,
The target blood concentration of the reactive compound of 75% or even 90% or higher cytoactive suppression is mesh
Front preferred.The percentage of cytoactive suppression in the patient can be monitored, so as to assess reached blood
The appropriateness of concentration, and can raise or lower dosage to realize desired suppression percentage.
As known in the art, can also be determined by animal model for the therapeutically effective amount of human body.Example
Such as, the dosage for human body can be formulated into the Efficient Cycle concentration for realizing having found in animal body.Such as
Upper described, the dosage in human body can be adjusted by monitoring Carbazole alkaloid and rise or downward dosage.
Treatment effective dose can also be by the known animal body for showing the compound for being similar to pharmacological activity
Interior or human trial data are determining.The dosage for being used can be based on the institute being compared with known compound
The effect of the compound of administration is adjusting (for example, Lakshmi Bhagat et al., IMO-8400, a selective
antagonist of TLRs 7,8and 9,inhibits MYD88 L265P mutation-driven signaling
and cell survival:A potential novel approach for treatment of B-cell lymphomas
harboring MYD88 L265P mutation,CANCER RESEARCH,October 1,2014 74:
2574 and Chinese invention patent application CN102171235A is disclosed).
Toll-like receptor TLR7 of the present invention and TLR9 inhibition oligonucleotides are to MYD88 L265P
The B cell lymphoma of mutation produces dose dependent inhibitory action.In general, the Toll-like receptor
TLR7 and TLR9 inhibition oligonucleotides is with the dosage of 1.25mg/kg body weight to 500mg/kg body weight to having
The B cell lymphoma of MYD88 L265P mutation produces inhibitory action.
In one embodiment, the B cell lymphoma to being mutated with MYD88 L265P produces suppression
The dosage of Toll-like receptor TLR7 of effect and TLR9 inhibition oligonucleotides be 1.25mg/kg body weight extremely
12.5mg/kg body weight, or 12.5mg/kg body weight is to 25mg/kg body weight, or 25mg/kg body weight is to 50mg/kg
Body weight, or 50mg/kg body weight is to 125mg/kg body weight, or 125mg/kg body weight is to 250mg/kg body weight, or
250mg/kg body weight is to 500mg/kg body weight.
In a preferred embodiment, the B cell lymphoma to being mutated with MYD88 L265P produces suppression
Toll-like receptor TLR7 for making and the dosage of TLR9 inhibition oligonucleotides are 5mg/kg body weight, or
12.5mg/kg body weight, or 25mg/kg body weight, or 50mg/kg body weight.
Embodiment
TLR7 and TLR9 inhibition widow's core of the present invention is illustrated in conjunction with following nonlimiting examples
The ABC type that thuja acid can be used to alleviate and treat with MYD88 L265P mutation diffuses big B lymthoma.
In an embodiment of the present invention, application human PBMC, ABC-DLBCL cell line OCI-Ly3.3 cell,
GCB-DLBCL cell line OCI-Ly19 cell, because human PBMC contains TLR7 and TLR9 acceptor, therefore
Stimulated after human PBMC using the part CpG685 of the ligand i MQ and TLR9 of TLR7, immune cell propagation,
TLR7 the and TLR9 inhibition oligonucleotides of the present invention can substantially suppress the people that IMQ or CpG 685 stimulates
PBMC propagation and the secretion of inflammatory cytokine IL-6, IL-10.
For the research of DLBCL clone, OCI-Ly19 cell belongs to MYD88 wild type, used as control
Group, OCI-Ly3.3 cell belong to MYD88 L265P saltant type, used as experimental group.The TLR7 of the present invention
The ABC-DLBCL type with MYD88 L265P mutation can be suppressed with TLR9 inhibition oligonucleotides
OCI-Ly3.3 cell propagation and the secretion of cell factor IL-10, and for MYD88 wild type
GCB-DLBCL type OCI-Ly19 cell is nothing similar effect.
Imiquimod (Imiquimod (R-837), C in embodiment14H16N4, HCl) and it is purchased from Invivogen.
Oligonucleotides used in embodiments of the invention is synthesized by Changchun Huapu Biotechnology Co., Ltd., all
Through processing nothing thermal source.Endotoxin application king crab ameboid cell dissolution method detection (Associates of in oligonucleotides
Cape Cod,Inc).Shown below is the sequence of the oligonucleotides that applies in an embodiment and title：
NOG1；5’-cctcctcctcctcctcctcctcct-3’(SEQ ID NO.1)；
NOG2:5’-cctcctcctcctcctcctcctcctcctcctcctcct-3’(SEQ ID NO.5)；
NOG3:5'-cctccTcctcctccTcctcctccTcctcctccTcct-3’(SEQ ID NO.6)；
CpG 685：5’-tcgtcgacgtcgttcgttctc-3’(SEQ ID NO.7).
1. present invention of embodiment not homotactic NOG1-3 oligonucleotides (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) IMQ or 685 stimulation human peripheral blood mononuclearcell (PBMC) of CpG are bred
Impact
Mononuclearcell (Ficoll reagent is separated with Ficoll Hypaque density-gradient centrifugation method from human peripheral
Purchased from Pharmacia company, blood sample is provided by Jilin Province's Blood Center).True with platform phenol orchid decoration method
The survival rate for determining human peripheral blood single nucleus cell is 95%-99%.Entered using CFSE (Invivogen company)
Line flag, using LSR Fortessa (BD company) flow cytomery human peripheral blood single nucleus cell
Proliferative conditions.
“CFSE(carboxyfluorescein succinimidyl ester)”：It is a kind of chemical dye, entirely
Referred to as Fluoresceincarboxylic acid succinimide ester, be by Carboxyfluorescein diaccete succinimidyl ester freedom
By cell membrane enter intracellular after by nonspecific esterase cut off acetyl group after formed, can be with cell
There is nonspecific, irreversibility, stable covalent bond in interior peptide and protein.CFSE pair
Cell does not have toxicity, stable chemical nature, will not separate again after combining with protein, CFSE per se with
Green fluorescence group, can be by the laser excitation of 488nm, and fluorescence signal is typically by FITC channel reception.
When cell is bred, intracellular CFSE can just be averaged and be assigned in two daughter cells, and the second generation is careful
The CFSE concentration of born of the same parents only has 1/4 for the half of original concentration, third generation daughter cell, and forth generation daughter cell only has
1/8, by that analogy, so by analysis breed after cell fluorescence intensity come calculate cell propagation activity.
Human peripheral blood single nucleus cell is with 5 × 105The density in individual/hole is inoculated into 96 orifice plate of U-shaped bottom, adds
CFSE, final concentration of 5 μM.IMQ (5 μM) or CpG685 (1 μ nol/L) and not homotactic NOG1-3
Oligonucleotides is according to molar concentration ratio 1:1、1:3、1:9、1:27 add in PBMC, are incubated 6 days.
CD19-APC-H7 (BD company), CD3-APC (BD company), LSR Fortessa (BD company)
Flow cytometry FITC-CFSE, the index reflection cell proliferative conditions.The experiment is repeated 3 times and all obtains
To similar result.
Experimental result as shown in Figure 1A～Fig. 1 C, compared with control group, the concentration of oligonucleotides NOG1-3 from
0 μM increases to 27 μM, the CD19+B cell propagation of oligonucleotides NOG1-3 suppression IMQ induction, makes thin
Born of the same parents' survival rate is reduced to intimate 0% by 30.60%；Oligonucleotides NOG1-3 suppression CpG685 induction
CD19+B cell is bred, and makes cell survival rate be reduced to intimate 0% by 28.50%.These results suggest that this
The oligonucleotides containing CCT repetitive sequence [(CCT) n] of invention can substantially suppress IMQ or CpG685 to lure
The propagation of the human peripheral CD19+B cell that leads.
2. present invention of embodiment not homotactic oligonucleotides NOG1-3 (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) human peripheral blood single nucleus cell (PBMC) that IMQ and CpG685 stimulates is secreted
Cell factor IL6 and IL-10 inhibitory action
Human peripheral blood single nucleus cell is separated with Ficoll Hypaque density-gradient centrifugation method from human peripheral
(Ficoll reagent is purchased from Pharmacia company, and blood sample is provided by Jilin Province's Blood Center).With platform
Phenol orchid decoration method determines that the survival rate of human peripheral blood single nucleus cell is 95%-99%.Examined with CBA (BD)
Survey the situation of human peripheral blood single nucleus cell secretion.
“CBA(cytometric bead array)”：I.e. trace sample multiplexed protein quantification technology, is base
Multiplexed protein quantification detection method in FCM analysis system.Replace cell using artificial microballoon,
Coated cell factor antibody on the microballoon, when in sample to be tested containing corresponding cell factor, manually micro-
Antibody on ball can be combined with cell factor, along with the anti-cytokine antibodies that PE is coupled, form " three
Mingzhi is sandwich " structure, by exciting PE fluorescein, according to the power of fluorescence signal come quantitative cell factor.
Human peripheral blood single nucleus cell is with 5 × 105The density in individual/hole is inoculated into 96 orifice plate of U-shaped bottom.Determine few core
Thuja acid NOG1-3 suppression IMQ stimulates PBMC secretion IL-6 and IL-10：IMQ (5 μM) and NOG1,
NOG2 and NOG3 (concentration be respectively 0,0.3,1.5,7.5 and 37.5 μM) according to different proportion, with people
PBMC together cultivates 36h, collects supernatant, and CBA (BD) determines the content of IL-6；IMQ (5 μM) and
NOG1, NOG2 and NOG3 (concentration is respectively 0,0.1,0.5,2.5 and 12.5 μM) be not according on year-on-year basis
Example, together cultivates 36h with human PBMC, collects supernatant, and CBA determines the content of IL-10.
NOG1-3 suppression CpG685 stimulates human PBMC secretion IL-6 and IL-10：CpG685 (1 μM) and
NOG1, NOG2 and NOG3 (concentration be respectively 0,1,3,9 and 27 μM) according to different proportion, with
Human PBMC together cultivates 36h, collects supernatant, and CBA method determines the content of IL-6；CpG685(1μM)
With NOG1, NOG2 and NOG3 (concentration is respectively 0,1,3,9 and 27 μM) according to different proportion,
36h is together cultivated with human PBMC, supernatant is collected, CBA method determines the content of IL-10.Above experiment repeats
3 times, as a result it is shown as mean value ± SD.
As shown in Figure 2 A and 2 B, culture medium group and oligonucleotides group are compared, with oligonucleotides
The increase of the concentration of NOG1-3, not homotactic oligonucleotides NOG1-3 stimulate human PBMC's secretion to IMQ
The inhibitory action of IL-6 and IL-10 strengthens；As shown in Figure 2 C and 2 D shown in FIG., by culture medium group and oligonucleotides group
It is compared, with the increase of oligonucleotides NOG1-3 concentration, not homotactic oligonucleotides NOG1-3
The inhibitory action of human PBMC secretion IL-6 and IL-10 is stimulated to strengthen to CpG685.These results suggest that this
The bright oligonucleotides comprising CCT repetitive sequence [(CCT) n] can substantially suppress IMQ or CpG685 induction
Human peripheral blood single nucleus cell secretes IL-6 and IL-10, as the concentration of oligonucleotides increases, inhibition
Strengthen, show dose dependent.
Embodiment 3. is to GCB-DLBCL cell line OCI-Ly19's and ABC-DLBCL cell line OCI-Ly3.3
The measure in MYD88 mutational site in DNA sequence dna
In order to determine MYD88 L265P mutational site in OCI-Ly19 or OCI-Ly3.3 cell, adopt
The method of Sanger method DNA sequencing.At 37 DEG C, in 5% (volume fraction) CO2 incubator, with supplement
There is the IMDM culture medium of 10% hyclone (GIBCO) and 1% penicillin/streptomycin (Hyclone)
(GIBCO) OCI-Ly19 and OCI-Ly3.3 cell is cultivated.Surviving for cell is determined with platform phenol orchid decoration method
Rate is 95%-99%.OCI-Ly19 or OCI-Ly3.3 cell number is (2～3) × 106Extract genomic DNA,
DNA extraction kit (purchased from Quan Shi gold biological reagent company), PCR expands MYD88 fragment, DNA
Poly- enzyme (Takara), upstream primer are 5 '-gttgaagactgggcttgtcc-3 ', and downstream primer is
5’-aggaggcagggcagaagta-3’.2 clones of every plant of raji cell assay Raji, trust money only intelligence biotechnology (north
Capital) Co., Ltd's sequencing.
Sanger method DNA sequencing result such as Fig. 3 shows, the MYD88 sequence of cell OCI-Ly19 and NCBI
MYD88 sequence in database is consistent, there is no mutational site, belongs to MYD88 wild type.Cell
There is the prominent of a base in the MYD88 sequence in the MYD88 sequence of OCI-Ly3.3 and ncbi database
It is that T sports C to become site, belongs to MYD88 L265P mutation.
The expression of the TLR7 and TLR9 of 4. cell line OCI-Ly19 and OCI-Ly3.3 of embodiment
At 37 DEG C, in 5% (volume fraction) CO2 incubator, with being supplemented with 10% hyclone (GIBCO)
With the IMDM culture medium (GIBCO) of 1% penicillin/streptomycin (Hyclone) culture OCI-Ly19 and
OCI-Ly3.3 cell.Determine that the survival rate of cell is 95%-99% with platform phenol orchid decoration method.Every kind of cell is pressed
Cell density is 5 × 105Individual/hole is inoculated in the U-shaped plate in 96 holes, carries out intracellular dyeing.Cell inner dyeing reagent
Purchased from BD company, 200 μ l Fixation Perm Buffer suspension cells, 4 DEG C, 20min is incubated, uses 200ul
Perm Wash Buffer is washed 1 time, is centrifuged with 2700rpm, continues 5min, abandons supernatant,
TLR7-PE and TLR9-APC antibody staining is added, room temperature lucifuge is incubated 30min, uses Perm Wash
Buffer is washed 2 times, with 400 μ l Perm Wash Buffer re-suspended cells, LSR Fortessa (BD company)
The mean fluorecence of the TLR7-PE of flow cytomery OCI-Ly19 or OCI-Ly3.3 and TLR9-APC is strong
Degree (MIF).
As shown in Figure 4 A and 4 B shown in FIG., the TLR7-PE and TLR9-APC of OCI-Ly19 and OCI-Ly3.3
Apparently higher than Isotype control group, this shows that OCI-Ly19 or OCI-Ly3.3 is thin to average fluorescent strength (MIF)
Intracellular all expresses TLR7 and TLR9.
5. present invention of embodiment not homotactic oligonucleotides NOG1-3 (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) for the propagation unrestraint effect of GCB-DLBCL clone OCI-Ly19 cell, but suppression
The propagation of ABC-DLBCL clone OCI-Ly3.3 cell processed
At 37 DEG C, in 5% (volume fraction) CO2 incubator, 10% hyclone (GIBCO) is being supplemented with
Cultivate with PRIM-1640 (GIBCO) culture medium of 1% penicillin/streptomycin (Hyclone)
OCI-Ly19.It is being supplemented with 10% hyclone (GIBCO) and 1% penicillin/streptomycin (Hyclone)
IMDM (GIBCO) culture medium in cultivate OCI-Ly3.3 cell.Cell is determined with platform phenol orchid decoration method
Survival rate be 95%-99%, cell in exponential phase when can do cell viability experiment.OCI-Ly19
Cell is with 8 × 103The density in/hole is inoculated into flat 96 orifice plate, and OCI-Ly3.3 cell is with 3 × 103The density in/hole connects
Plant and flat 96 orifice plate is arrived, 100uL/ hole, concentration is respectively 0,5,10,20 and 40 μM of oligonucleotides
NOG1, NOG2 and NOG3 and OCI-Ly19 or OCI-Ly3.3 are co-cultured 72 hours, add WST-1 examination
Agent (Chinese green skies biotechnology research institute) 10ul, is incubated 3h, determines OD450, calculates cell viability,
5 multiple holes of the Setup Experiments, repeat 3 independent experiments, are as a result expressed as mean value ± SD.
Fig. 5 shows with the increase of oligonucleotides NOG1-3 concentration, and the cell viability of OCI-Ly19 is 100%
Left and right, and the cell viability of OCI-Ly3.3 is gradually reduced by 100%, but the concentration of oligonucleotides NOG1-3
At 40 μM, OCI-Ly3.3 cell viability is 20% or so.
Cell proliferation experiment illustrates the oligonucleotides pair comprising CCT repetitive sequence [(CCT) n] of the present invention
In the cell line OCI-Ly19 cell propagation unrestraint effect of the GCB-DLBCL of MYD88 wild type, but
The substantially increasing of ABC-DLBCL cell line OCI-Ly3.3 cell of the suppression with MYD88 L265P mutation
Grow, this inhibitory action is in dosage correlation.As can be seen here, the present invention comprising CCT repetitive sequence
The oligonucleotides of [(CCT) n] can be effectively used for treating the ABC type diffusivity with MYD88 L265P mutation
Large B cell lymphoid tumor.
6. present invention of embodiment not homotactic oligonucleotides NOG1-3 (SEQ ID NO.1, SEQ ID NO.5,
SEQ ID NO.6) impact to cell factor IL-10 that OCI-Ly19 and OCI-Ly3.3 secrete
At 37 DEG C, in 5% (volume fraction) CO2 incubator, with being supplemented with 10% hyclone (GIBCO)
With the IMDM culture medium (GIBCO) of 1% penicillin/streptomycin (Hyclone) culture OCI-Ly19 and
OCI-Ly3.3 cell.Determine that the survival rate of cell is 95%-99% with platform phenol orchid decoration method.OCI-Ly19 and
OCI-Ly3.3 cell is all with 2 × 105/ hole density is inoculated into 96 orifice plates, by concentration be respectively 0,2,4,8,
10 and 20 μM oligonucleotides NOG1, NOG2 and NOG3 and OCI-Ly19 or OCI-Ly3.3 are co-cultured
36 hours.CBA method detects cell factor IL-10.The experiment is repeated 3 times and all obtains similar result.
As shown in fig. 6, the IL-10 nothing suppression that the oligonucleotides NOG1-3 of the present invention is secreted to OCI-Ly19 cell
Make and use, but NOG1-3 can substantially suppress cell factor IL-10 that OCI-Ly3.3 cell is secreted, and
This inhibitory action is in dosage correlation.
Impact of embodiment 7.NOG3 (SEQ ID NO.6) to OCI-Ly19 the and OCI-Ly3.3 cell cycle
At 37 DEG C, in 5% (volume fraction) CO2 incubator, with being supplemented with 10% hyclone (GIBCO)
With the IMDM culture medium (GIBCO) of 1% penicillin/streptomycin (Hyclone) culture OCI-Ly19 and
OCI-Ly3.3 cell.Determine that the survival rate of cell is 95%-99% with platform phenol orchid decoration method.Cell is in right
Number can do cell cycle experimental during growth period.OCI-Ly19 and OCI-Ly3.3 cell is all with 1.25 × 105/ hole
Density is inoculated into 96 orifice plates, concentration be respectively 0,15 and 30 μM of oligonucleotides NOG3 respectively with OCI-Ly19
Or OCI-Ly3.3 is co-cultured 72 hours, propidium iodide PI (prosperity Bioisystech Co., Ltd of Beijing ancient cooking vessel state)
Dyeing, LSR Fortessa (BD company) the flow cytomery cell cycle.The experiment is repeated 3 times all
Obtain similar result.
As shown in figures 7 a and 7b, the oligonucleotides NOG3 of the present invention is for OCI-Ly19 cell cycle nothing
Influence；But the cell cycle of impact OCI-Ly3.3, compared with control group, the OCI-Ly3.3 cell cycle
G1 phase cell proportion substantially increases, and S phase and G2, M phase significantly reduce, the oligonucleotides of this explanation present invention
NOG3 prevents OCI-Ly3.3 cell from being changed from G1 phase to S phase and G2, M phase so that cellular retention is in G1
Phase, cell propagation is caused to slow down.
Above a preferred embodiment of the present invention is described, but, these embodiments are used only for
Illustrate.Those skilled in the art can be to these embodiments on the premise of the essence without departing substantially from the present invention
Make multiple changes, modifications and replacement.The scope of the present invention is limited to the appended claims, and
Composition and its application and their equivalent in scope of the invention as claimed is also by appended right
Requirement is covered.
Claims (10)
1.Toll sample acceptor TLR7 and TLR9 inhibition oligonucleotides are being prepared for treating B cell lymphoma
Medicine in application, the B cell lymphoma have MYD88 L265P mutation, the oligonucleotides
Comprising CCT repetitive sequence [(CCT) n], wherein, C is cytimidine, and T is thymidine, and n is 8 to 12
Integer, wherein, the sequence of the oligonucleotides is selected from：
SEQ ID NO.1：5’-cctcctcctcctcctcctcctcct-3’；
SEQ ID NO.2：5’-cctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.3：5’-cctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.4：5’-cctcctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.5：5’-cctcctcctcctcctcctcctcctcctcctcctcct-3’；
SEQ ID NO.6：5’-cctccTcctcctccTcctcctccTcctcctccTcct-3’；
Wherein, lowercase t represents the base of thio-modification, and capital T represents unmodified base.
2. application as claimed in claim 1, wherein, the B cell lymphoma is diffusivity large B cell
Lymthoma.
3. application as claimed in claim 2, wherein, the diffusivity large B cell lymphoid tumor is activated b
Cell sample hypotype.
4. application as claimed any one in claims 1 to 3, wherein, Toll-like receptor TLR7 and
TLR9 inhibition oligonucleotides produces dose-dependant to the B cell lymphoma being mutated with MYD88 L265P
Property inhibitory action.
5. application as claimed any one in claims 1 to 3, wherein, Toll-like receptor TLR7 and
TLR9 inhibition oligonucleotides is with the dosage of 1.25mg/kg body weight to 500mg/kg body weight to MYD88
The B cell lymphoma of L265P mutation produces inhibitory action.
6. application as claimed in claim 5, wherein, the B cell to being mutated with MYD88 L265P is drenched
Bar knurl produces Toll-like receptor TLR7 of inhibitory action and the dosage of TLR9 inhibition oligonucleotides is
1.25mg/kg body weight is to 12.5mg/kg body weight, or 12.5mg/kg body weight is to 25mg/kg body weight, or 25mg/kg
Body weight is to 50mg/kg body weight, or 50mg/kg body weight is to 125mg/kg body weight, or 125mg/kg body weight is extremely
250mg/kg body weight, or 250mg/kg body weight is to 500mg/kg body weight.
7. application as claimed in claim 6, wherein, the dosage is 5mg/kg body weight, or 12.5mg/kg
Body weight, or 25mg/kg body weight, or 50mg/kg body weight.
8. the application as any one of claim 1 to 7, wherein, the medicine is also included pharmaceutically
Acceptable carrier.
9. application as claimed in claim 8, wherein, the pharmaceutically acceptable carrier is selected from：Solution,
Diluent, solvent, dispersant, liposome, emulsion, sugar-coat, antiseptic, antifungal agent, isotonic
Reagent with delayed absorption.
10. application as claimed in claim 1, wherein, the medicine be formulated into oral administered dosage form,
Intravenous form, intramuscular injection formulation, inhalation formulation, topical applications.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10004746B2 (en) | 2010-06-03 | 2018-06-26 | Pharmacyclics Llc | Use of inhibitors of Bruton's tyrosine kinase (Btk) |
| US10004745B2 (en) | 2010-06-03 | 2018-06-26 | Pharmacyclics Llc | Use of inhibitors of Bruton'S tyrosine kinase (Btk) |
| US10016435B2 (en) | 2010-06-03 | 2018-07-10 | Pharmacyclics Llc | Use of inhibitors of Bruton's tyrosine kinase (Btk) |
| US10478439B2 (en) | 2010-06-03 | 2019-11-19 | Pharmacyclics Llc | Use of inhibitors of bruton's tyrosine kinase (Btk) |
| US10653696B2 (en) | 2010-06-03 | 2020-05-19 | Pharmacyclics Llc | Use of inhibitors of bruton's tyrosine kinase (BTK) |
| US10751342B2 (en) | 2010-06-03 | 2020-08-25 | Pharmacyclics Llc | Use of inhibitors of Bruton's tyrosine kinase (Btk) |
| US11672803B2 (en) | 2010-06-03 | 2023-06-13 | Pharmacyclics Llc | Use of inhibitors of Brutons tyrosine kinase (Btk) |
| US10954567B2 (en) | 2012-07-24 | 2021-03-23 | Pharmacyclics Llc | Mutations associated with resistance to inhibitors of Bruton's Tyrosine Kinase (BTK) |
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