阅读说明：本技术 程序性死亡配体1的肠表达 (The intestines of programmed death ligand 1 are expressed ) 是由 S·M·道芬内 C·D·A·麦卡锡 J·杜鲍尔-驰寇内 E·许 G·驰克 A·张 于 2017-11-09 设计创作，主要内容包括：本文提供了用于改善炎性病症的方法和组合物,所述方法和组合物包括程序性死亡配体1的肠表达。(There is provided herein the method and composition for improving inflammatory conditions, the method and composition include the intestines expression of programmed death ligand 1.)

1. a kind of method for the inflammatory conditions for treating patient in need, the method includes applying to the gastrointestinal tract of the patient Expression vector comprising programmed death ligand 1 (" PD-L1 ") nucleic acid.

2. according to the method described in claim 1, the wherein PD-L1 nucleic acid encode human PD-L 1 polypeptide.

3. according to the method described in claim 2, wherein the PD-L1 polypeptide is soluble human PD-L1 polypeptide, the solubility Human PD-L 1 polypeptide includes the IgV structural domain and IgC structural domain (the amino acid 1 9-239 of SEQ ID NO:1) or by it of human PD-L 1 Composition.

4. according to the method described in claim 2, wherein the PD-L1 polypeptide is soluble human PD-L1 polypeptide, the solubility Human PD-L 1 polypeptide includes signal transduction structural domain, IgV structural domain and the IgC structural domain (amino of SEQ ID NO:1 of human PD-L 1 Sour 1-239) or be made from it.

5. the method according to any one of claim 2-4, wherein the N-terminal area of the PD-L1 polypeptide and the area human IgG1 Fc Or part thereof fusion.

6. according to the method described in claim 5, wherein the PD-L1 polypeptide via (GGGGS) n (SEQ ID NO:5) ammonia Base acid sequence and the area human IgG1 Fc or part thereof are merged.

7. according to the method described in claim 5, wherein the human IgG1 Fc is by changing the following amino in Fc structural domain One or more of acid and be mutated to reduce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDCC): E233P, L234V, L235A, G236 missing, A327G, A330S and P331S.

8. according to the method described in claim 5, wherein the PD-L1 nucleic acid includes the sequence of SEQ ID NO.4.

9. the film combines according to the method described in claim 2, wherein the PD-L1 polypeptide is the PD-L1 polypeptide that film combines PD-L1 polypeptide include human PD-L 1 signal transduction sequence, IgV structural domain, IgC structural domain and transmembrane domain (SEQ ID The amino acid 1-259 of NO:1) or be made from it.

10. according to the method described in claim 9, wherein the PD-L1 polypeptide also includes the cytoplasmic domain of human PD-L 1.

11. according to the method described in claim 10, wherein the PD-L1 nucleic acid includes the sequence of SEQ ID NO.3.

12. method described in any one of -11 according to claim 1, wherein the inflammatory conditions are inflammatory bowel disease, exedens knot Enteritis or Crohn disease.

13. method described in any one of -11 according to claim 1, wherein the inflammatory conditions are graft versus host disease(GVH disease)s (GvHD)。

14. a kind of expression vector comprising PD-L1 nucleic acid, wherein the sequence of the PD-L1 nucleic acid is wrapped compared with SEQ ID NO:2 Containing at least one synonymous substitution for detecting after application, and preferably include multiple synonymous substitutions.

15. expression vector according to claim 14, wherein the PD-L1 nucleic acid sequence is through codon optimization for table It reaches.

16. expression vector according to claim 14, wherein the PD-L1 nucleic acid also includes heterologous sequence.

17. expression vector according to claim 16, wherein the heterologous sequence includes Fc structural domain, protein tag, sews The therapeutic agent or their combination of conjunction.

18. expression vector according to claim 17, wherein the Fc structural domain includes area human IgG1 Fc or part thereof.

19. expression vector according to claim 18, wherein the PD-L1 nucleic acid includes the sequence of SEQ ID NO.4.

20. expression vector according to claim 15, wherein the PD-L1 nucleic acid includes the sequence of SEQ ID NO.3.

21. expression vector described in any one of 4-20 is in the inflammatory conditions for treating patient in need according to claim 1 Purposes.

22. expression vector described in any one of 4-20 is preparing the inflammation for treating patient in need according to claim 1 Purposes in the drug of venereal disease disease.

23. the purposes according to claim 21 or 22, wherein the inflammatory conditions be inflammatory bowel disease, ulcerative colitis or Crohn disease.

24. the purposes according to claim 21 or 22, wherein the inflammatory conditions are graft versus host disease(GVH disease) (GvHD).

Invention field

This disclosure relates to improve the method and composition of inflammatory conditions using the gene expression of PD-L1.

Background of invention

Immune activation in general, specifically T cell activation state by by T cell costimulatory molecules (such as CD28, ICOS, OX40 etc.) and Co inhibitor (such as CTLA-4, death protein -1 (" PD-1 ") etc.) host be situated between The influence for the complicated overlapping adjustment mechanism led.Programmed death ligand-1 (" PD-L1 ", also referred to as B7H1) is by Dendritic Cells It is constitutively expressed with other antigen presenting cells, and seems that mediate T cell is lowered by the interaction with PD-1. Patsoukis et al., Selective effects of PD-1 on Akt and Ras pathways regulate molecular components of the cell cycle and inhibit T cell proliferation, Sci.Signal 5:ra46(2012)。

Unfortunately, influence of the people to the interaction of PD-L1 and CD80 is still not clear, to both approach Between interaction be also not clear.In some cases, block PD-1 that can effectively enhance T cell by soluble PD-L1 Reaction, and enhance graft versus host disease(GVH disease) (" GvHD ").Blazar et al., Blockade of Programmed Death-1 Engagement Accelerates Graft-Versus-Host Lethality by an IFN-γ-Dependent Mechanism J.Immunol.171:1272-77(2003).On the contrary, other researchers can use the PD- of soluble form L1 improves GvHD, but being only could be so in PD-L1 knockout background.Deng et al. B7H1/CD80Interaction Augments PD-1 Dependent T Cell Apoptosis and Ameliorates Graft-versus-Host Disease,J.Immunol.194:560-74(2105).In addition, periphery Dendritic Cells seems to compare to the effect of t cell responses What is had previously been thought that is more complicated.Although having been observed for example, immature periphery Dendritic Cells does not cause T cell differentiation usually To Dendritic Cells group infiltration central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) mouse model And epitope is promoted to spread.Spagnuolo et al., Involvement of Immune Regulation in Multiple Sclerosis,Immunology and Immunogenetics Insights 9:1-10。

Therefore, this field clearly requires the work for more fully understanding PD-L1 in immune protective environment in T cell activation With preferably to utilize treatment potentiality of the PD-L1 in inflammatory conditions.

Summary of the invention

The present invention is by successfully using PD-L1 polypeptide (including soluble PD-L1 polypeptide) in immune protective environment The expression of local intestines solve above-mentioned scientific uncertainty in the prior art, for treating a variety of inflammatory conditions, including for example Inflammatory bowel disease (such as ulcerative colitis, Crohn disease etc.) and the graft-versus-host induced by organ or bone-marrow transplantation Sick (GvHD).Therefore, in one aspect, the present invention provides a kind of method of inflammatory conditions for treating patient in need, institutes The method of stating includes to expression vector of the enteron aisle of the patient application comprising PD-L1 nucleic acid.

In one embodiment, PD-L1 polypeptide is the PD-L1 polypeptide that film combines.In preferred embodiments, PD- L1 polypeptide is human PD-L 1 or its splice variant.In such embodiment, PD-L1 nucleic acid encode PD-L1 polypeptide, institute State amino acid sequence that PD-L1 polypeptide includes SEQ ID NO:1 or with SEQ ID NO:1 at least about 80%, 85%, 90%, 95%, the identical amino acid sequence of 96%, 97%, 98% or 99%.

In alternative embodiment, PD-L1 polypeptide is soluble PD-L1 polypeptide, such as (and preferably comprising PD-L1 Human PD-L 1) signal sequence, IgV structural domain and IgC structural domain (i.e. the amino acid 1-239 of SEQ ID NO:1).In some realities It applies in scheme, soluble PD-L1 polypeptide can lack all or part of signal sequence (for example, it may include the ammonia of SEQ ID NO:1 Base acid 19-238).

In some embodiments, PD-L1 nucleic acid by expression vector of the capsule encapsulating in delivery of nucleic acids medium come Delivering.Preferred delivery of nucleic acids medium includes chitosan or chitosan derivative nano particle.At one In such embodiment, chitosan derivative nano particle includes the de- second being coupled with arginine and/or gluconic acid Acyl chitin.In embodiment as another, chitosan derivative nano particle includes and arginine and parent The chitosan of aqueous polyalcohol coupling.In one embodiment, hydrophilic polyol is glucose.

On the other hand, the inflammation of patient in need is treated the present invention provides the expression vector comprising PD-L1 nucleic acid Venereal disease disease, wherein the expression vector is administered to the intestines of the subject.In some embodiments, PD-L1 nucleic acid includes SEQ ID NO:2.Preferably, PD-L1 nucleic acid includes at least one same sense mutation in SEQ ID NO:2.It is highly preferred that PD-L1 nucleic acid includes that multiple such mutation are expressed with helping to break up and optionally improve.In an exemplary embodiment, PD- L1 nucleic acid includes SEQ ID NO:3.

In some embodiments, PD-L1 nucleic acid also includes heterologous sequence.Heterologous sequence may include Fc structural domain, albumen Label, the therapeutic agent of conjugation or their combination.In one embodiment, the N-terminal area of PD-L1 polypeptide and the area human IgG1 Fc Or part thereof fusion.PD-L1 polypeptide can pass through the amino acid sequence of (GGGGS) n (SEQ ID NO:5) and the area human IgG1 Fc Or part thereof fusion.In some embodiments, IgG1 Fc is by changing one in the following amino acid in Fc structural domain Or it is multiple and be mutated to reduce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDCC): E233P, L234V, L235A, G236 missing, A327G, A330S and P331S.In an exemplary embodiment, PD-L1 nucleic acid includes SEQ ID NO:4。

The inflammatory conditions that can obtain favourable therapeutic through the invention include chronic disease and acute illness, including with infection phase The inflammation (such as septic shock, septicemia or systemic inflammatory response syndrome (SIRS)) of pass, ischemia reperfusion injury, Endotoxin is lethal, inflammatory bowel disease, Crohn disease, colitis or as caused by cell factor (such as TNF or IL-1) excess generation Illness and GvHD.

It is herein incorporated by reference

The all publications, patents and patent applications referred in this specification are herein incorporated by reference, and degree is such as It is pointed out specifically and individually to be incorporated by reference with each individually publication, patent or patent application.

Detailed description of the invention

The disclosure is disclosed with reference to the accompanying drawings, in the accompanying drawings:

Fig. 1: pVax-hPD-L1 carrier (SEQ ID NO:6).Encode the plasmid of the carrier for the human PD-L 1 polypeptide that film combines The human PD-L 1 cDNA of sequence, optimization is highlighted.

Fig. 2: pVax-hPD-L1 Fc carrier (SEQ ID NO:7).The carrier of encoding soluble human PD-L 1 Fc polypeptide The human PD-L 1 cDNA of plasmid sequence, optimization is highlighted.

Fig. 3 A: human PD-L 1 plasmid and vivoexpression.(A) it is cloned in the hPD-L1 DNA sequence dna of the optimization in pVax skeleton Plasmid map, which contains Plasmid replication origins (pUC ori；), the Plasmid replication origins by human cytomegalovirus immediately Early gene promoter (CMV；) control and have kalamycin resistance gene.

Fig. 3 B: external PD-L1 and PD-L1-Fc expression: use Lipofectamine 2000 (Invitrogen) with 1 μ g Or 2.5 Fc controls (white) of μ g, PD-L1-Fc (black) or PD-L1 (grey) plasmid DNA transfection HEK293T cell.Transfection 48h quantifies the PD-L1 protein content in cell culture supernatant or cell pyrolysis liquid afterwards.

Fig. 4: the vivoexpression of the PD-L1-Fc from polymer composites transfection.(A-B) as noted, with containing The PD-L1-Fc polymer composites for increasing the pVAX-opt-hPD-L1-Fc DNA of concentration transfect HEK-293T.(A) after transfection 48 hours collection supernatants, and hPD-L1-Fc albumen is measured by ELISA.Data standard is turned into total protein of cell.(B) The table of EC50 and maximum protein expression.

Fig. 5: the purifying and expression of the construct of the Fc fusion generated in vitro.(A) pass through coomassie gel dyeing confirmation Protein expression: using Lipofectamine 2000 (Invitrogen), with Fc control or PD-L1-Fc plasmid DNA transfection HEK293T cell.Cell culture medium is changed to serum free medium for 24 hours after transfection.72h collects cells and supernatant after transfection Liquid, and Fc fusion protein is purified using Protein G HiTrap column (GE).Fluid exchange will be buffered using PD-10 desalting column (GE) For PBS, and protein are eluted with four equal fractions (eluent 1-4).(B) Lipofectamine 2000 is used (Invitrogen), with Fc control or PD-L1-Fc plasmid DNA transfection HEK293T cell.After transfection for 24 hours by cell culture medium more It is changed to serum free medium.72h collects cell culture supernatant after transfection, and is purified using Protein G HiTrap column (GE) Fc fusion protein.Buffer is changed to PBS using PD-10 desalting column (GE), and with four equal fraction (F1-4) elutions Protein.PD-L1 protein concentration is measured by ELISA.(C) Lipofectamine 2000 is used in 6 orifice plates (Invitrogen) NIH/3T3 cell is transfected with wild type PD-L1 Plasmid DNA or control (pVax).Wash cell and by cell Renewed vaccination for 24 hours is in 96 hole flat undersides after transfection.Second day (the 2nd day) and subsequent 3 days, pass through flow cytometry point Analyse the PD-L1 expression on cell surface.(D) in 6 orifice plates using Lipofectamine 2000 (Invitrogen) with wild Type PD-L1 Plasmid DNA or control (pVax) transfect NIH/3T3 cell.Wash cell and the renewed vaccination for 24 hours after transfection by cell In 96 hole flat undersides.Second day (the 2nd day) and subsequent 3 days, pass through the PD-L1 on flow cytometry cell surface Expression.

Fig. 6: people PD-1 expression of receptor PD-L1-Fc signal is passed through with dosage-dependent manner.Cell PD-L1-Fc is turned It contaminates and is purified from cell culture supernatant.(A) PathHunter PD-1 is stimulated with the PD-L1-Fc (40min) for increasing concentration Signal transduction measures cell, and passes through the (RLU that shines；Relative light units) measuring signal activation.(B) with containing and be free of anti-PD-1 It is (low: 0.1 μ g/mL；It is high: 1.0 μ g/mL) recombination IgG1-Fc or PD-L1-Fc (50 μ g/mL, 40min) be incubated for PathHunter PD-1 signal transduction measures cell, and passes through the (RLU that shines；Relative light units) measuring signal activation.Triplicate measurement is thin Born of the same parents' activation, and data are expressed as the RLU relative to the control not stimulated.Data are expressed as average value ± standard deviation, and Represent 4 independent experiments.Using student's t check analysis data, and asterisk indicate statistically significant difference (*, p≤ 0.05；*, p≤0.01；* *, p≤0.001).

Fig. 7: the PD-L1-Fc that Plasmid DNA generates inhibits T cell activation in vitro.(A) divide from three C57BL/6 mouse CD4+T cell fusion from purifying is simultaneously.Seed cells into 96 hole flat undersides, the plate be pre-coated with individually or containing 5 μ g/ml without The AntiCD3 McAb (0.2 μ g/ml) for the PD-L1-Fc that blood serum medium generates.It recombinates IgG1-Fc and recombination PD-L1-Fc is used as control. Stimulation cell 3 days, and cell activation is measured by the cell size in Flow cytometry FSC-SSC.(B) in 6 holes With wild type PD-L1 Plasmid DNA or control (pVax in plate；) transfection NIH/3T3 cell.48 hours after transfection, again by cell It is seeded in 96 hole v-shaped bottom plates, and is incubated for the recombined human PD-1 of various concentration.The anti-human PD- for washing cell and being conjugated with APC 1 or isotype controls dyeing, and by flow cytometry by evaluation transfection cell on APC test and analyze PD-1 combine.Also The combination of the anti human PD-L 1 or isotype controls of PE conjugation is evaluated.In view of recombination PD-1 with express on NIH/3T3 cell PD-L1 is combined, and the reduction that prediction anti human PD-L 1-PE is combined is the result for the steric hindrance that antibody combines.Fig. 7 A is disclosed " GGGGS " is used as SEQ ID NO:5, and " (GGGGS) 3 " is used as SEQ ID NO:8.

Fig. 8: disease model optimization.(A) to Naive T cells are injected in male Rag1-/- mouse peritoneum, it is defined as CD4+ CD25-CD45RB high or untreated.As by the way that observed by weight loss, Naive T cells transfer induces disease.(B) it is Induction acute graft versus host disease (GvHD) irradiates female BAl BIc/c mouse, and and allogeneic with 700 cGY C57Bl6/J marrow (10M cell) and 2.5M splenocyte shift together.Such as subtracted by the original body mass observed in first 10 days Observed by light, GvHD is successfully induced using these conditions.In addition, about in the case where the 20th day is initially observed secondary weight Drop.No graft group confirms successfully irradiation and BMT control confirms the successful transfer of allogeneic bone marrow.

Therapeutic efficiency of Fig. 9: the PD-L1 and PD-L1 Fc polymer composites in GvHD.(A) as previously opened up in the fig. 8b The induction GvHD shown.PVAX, PD-L1 or the PD-L1 for being c1000 by bowel lavage injection concentration without treatment or weekly to mouse Fc polymer composites continue 7 weeks.It is up to 75% animal euthanasia of its original body mass.It is poly- to apply PD-L1 and PD-L1 Fc Close the weight that compound has saved animal.(B) clinical sign of disease is reduced in the mouse that PD-L1 or PD-L1-Fc is treated, And the average clinical sign observed in the mouse that PD-L1 and PD-L1-Fc is treated at the 21st day is also reduced.(C) it induces The animal of the survival curve of mouse after GvHD, all untreated animals and pVAX treatment all dies of disease, about 40% PD- The animal (green) that the animal of L1 Fc treatment and 30% PD-L1 are treated survived to the 75th day.

Therapeutic efficiency of Figure 10: the PD-L1 Fc and PD-L1 polymer composites in GvHD.(A) such as previous institute in the fig. 8b The induction GvHD of displaying.PVAX the or PD-L1 Fc for being c1000 by bowel lavage injection concentration without treatment or weekly to mouse Polymer composites continue 7 weeks.It is up to 75% animal euthanasia of its original body mass.Apply PD-L1 Fc polymer composites The weight of animal is saved.(B) the induction GvHD as previously shown in the fig. 8b.Mouse is passed through without treatment or weekly Bowel lavage injection concentration is pVAX the or PD-L1 polymer composites of c1000, continues 7 weeks.It is up to the dynamic of the 75% of its original body mass Object euthanasia.Application PD-L1 polymer composites have saved the weight of animal.(C) induce GvHD after mouse survival curve, own The animal of pVAX treatment all dies of disease, the animal that about 40% animal of PD-L1 Fc treatment and 30% PD-L1 are treated Survival was to the 75th day.

Therapeutic effect of Figure 11: the PD-L1 and PD-L1 Fc in GvHD.(A) induction as previously shown in the fig. 8b GvHD.Mouse is polymerize without treatment or weekly by pVAX, PD-L1 or PD-L1 Fc that bowel lavage injection concentration is c1000 Compound continues 7 weeks.Compared with the animal without treatment and the pVAX started for the 18th day treatment, the animal of PD-L1 treatment and PD- The weight of the animal of L1 Fc treatment greatly improves.(B) survival curve of mouse after GvHD, the animal of all pVAX treatment are induced And untreated animal all dies of disease.On the other hand, 50% PD-L1 Fc treatment animal and 25% application PD- The animal of L1 survived to the 45th day.

Figure 12: the application of PD-L1 Fc is recombinated in colitis DSS model.(A) figure is from Song et al., Gut 2015.In 2%DSS is applied in Rag1-/- mouse drinking water, and intraperitoneal injection recombination PD-L1 Fc albumen or PBS on day 2.With note The animal for penetrating PBS is compared, and weight loss caused by injection recombination PD-L1 Fc is less serious.(B) in Rag1-/- mouse drink PD-L1 Fc or PBS are recombinated with the 6th day intraperitoneal injection with applying 4%DSS in water, and on day 3.In entire research, move The weight of object is equivalent.However, not observing therapeutic effect, therefore the knot of Song et al. using PD-L1-Fc soluble protein Fruit not reproduce.

Therapeutic efficiency of Figure 13: the PD-L1 and PD-L1 Fc in T cell colitis model.(A-B) T is induced according to Fig. 8 A Cell colitis.Since after transfer the 15th day, pVAX or PD-L1 is injected weekly to mouse by bowel lavage at next 6 weeks C1000 polymer composites, or without treatment.To the 75% fixed scoring that the animal for dying of disease is its original body mass. As by the way that observed by weight stable in entirely research, injection PD-L1 polymer composites inhibit progression of disease.(B) T is induced The survival curve of mouse shows that the animal of PD-L1 treatment does not die of disease after cell colitis.On the other hand, it is necessary to by 1 The animal of pVAX treatment and 2 untreated animal euthanasias.(C-E) according to Fig. 8 A inducing T cell colitis.Weekly two to Weight (C) and the clinical sign (D) of animal are monitored three times, and the 0th day weight is used as baseline weight (100%；0th day It is first day that colon instils, corresponds to after transfer the 19th day).Since after transfer the 19th day, animal received weekly colon Interior instillation continues 7 weeks.(E) after inducing T cell colitis mouse survival.When animal reaches the 75% of its original body mass, or Person is when active serious reduction, by animal euthanasia.For drawing purpose, for the remainder of experiment, by the mouse that is euthanized Weight be expressed as the 75% of its original body mass.Similarly, the clinical score for the mouse that is euthanized is expressed as maximum scores 8, directly Terminate to research.For (C-E), sucrose n=8, pVAX n=9, PD-L1-Fc n=9.

The expression of FoxP3 in the application induction regulatory T cells of Figure 14: PD-L1 polymer composites.In lymphonodi mesenterici With the terminal of spleen, the average fluorescent strength of FoxP3 is examined in regulatory T cells (being defined as CD4+CD25+FoxP3+ cell) (MFI).FoxP3 compared with pVax group, in (A) lymphonodi mesenterici (MLN) and (B) spleen, in the animal of PD-L1 treatment It expresses all higher.

Specific embodiment

Present invention contemplates the expression of the intestines of PD-L1 nucleic acid, for treating inflammatory conditions.

PD-L1 protein and polynucleotides

The PD-L1 polypeptide imagined herein includes full length sequence, soluble fragments and its variant.

Overall length PD-L1 albumen generally comprises signal sequence, IgV structural domain and IgC structural domain, transmembrane domain and cell Matter structural domain.The public can obtain human sequence at NM_014143.3 and NP_054862.1 in GenBank database.People PD- The sequence of L1 transcript variant 1 is canonical sequence, and all location informations about the description of known isotype are determined from the sequence. In the isotype, signal sequence is shown as about amino acid 1 to about 18, IgV structural domain and is shown as about 19 to about 134, IgC structure Domain is shown as about 135 to about 227, and transmembrane domain is shown as about 239 to about 259, and cytoplasmic domain be shown as about 260 to About 290.At least five kinds of transcript (i.e. montage) variants of coding different people PD-L1 isotype are known and are described in for example In U.S. Patent Publication number 2016/0122829, the disclosure is expressly incorporated herein.

The nucleic acid of PD-L1 and amino acid sequence information are well known in the art in a variety of species, and are easy Obtained in public database, these PD-L1 include such as monkey PD-L1 (NM 001083889.1 and NP_001077358.1), Chimpanzee PD-L1 (XM_0011401705.2 and XP_001140705.1), mouse PD-L1 (NM 021893.3 and NP_ 068693.1), P of Rats D-L1 (NM_001191954.1 and NP_001178883.1), chicken D-L1 (XM_424811.3 and XP_ 424811.3), ox PD-L1 (NM_001163412.1 and NP_001156884.1) and dog PD-L1 (XM_541302.3 and XP_ 541302.3。

PD-L1 nucleic acid of the invention will generally comprise the nucleic acid sequence of encoding human PD-L1 polypeptide, and wherein PD-L1 polypeptide is excellent Selection of land include SEQ ID NO:1 amino acid sequence or with SEQ ID NO:1 at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or its segment.In some embodiments, PD-L1 polypeptide is that film combines , that is, it include the overall length PD-L1 of transmembrane domain and cytoplasmic domain.In alternative embodiment, PD-L1 polypeptide is can Dissolubility PD-L1 polypeptide, the solubility PD-L1 polypeptide includes at least the IgV structural domain and IgC structural domain of human PD-L 1, and appoints Selection of land also includes signal sequence.In one embodiment, PD-L1 polypeptide includes the amino acid 1-239 of SEQ ID NO:1, this Nucleotide 1-717 corresponding to full length cDNA sequence.In other embodiments, soluble PD-L1 polypeptide can lack all or Part signal sequence (for example, it may include the amino acid 1 9-239 of SEQ ID NO:1).See, e.g. U.S. Patent Publication number 2017/0189476, the disclosure of which is expressly incorporated herein.

The amino acid sequence of table 1- human PD-L 1

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFV HGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVV DPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENH TAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ ID NO:1)

In some embodiments, PD-L1 nucleic acid and natural PD-L1 nucleic acid sequence (SEQ ID NO:2) or its segment phase Together.Preferably, at least one is carried out to replace with phosphorothioate odn to allow after applying theme nucleic acid from endogenous human PD-L 1 nucleosides Acid differentiation and detection gained transcript, and more preferably carry out multiple such same sense mutations.In particularly preferred embodiment party In case, PD-L1 nucleic acid sequence improves expression through codon optimization.In an exemplary embodiment, PD-L1 nucleic acid preferably wraps The nucleic acid sequence of the NO:3 of ID containing SEQ or with SEQ ID NO:3 at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical sequence.

In some embodiments, PD-L1 polypeptide of the invention can be merged with the area Fc or part thereof.In exemplary implementation In scheme, PD-L1 nucleic acid include SEQ ID NO:4 nucleic acid sequence or with SEQ ID NO:4 at least about 70%, 80%, 85%, 90%, the identical nucleic acid sequence of 95%, 96%, 97%, 98% or 99%.

PD-L1 nucleic acid of the invention can integrally include preceding 35 nucleotide of exons 1-4 and exon 5.It needs to infuse Meaning, the site ATG (initiation codon) is located at the 13rd of exon 2.Therefore, PD-L1 nucleic acid of the invention may include exon 2 Last 52 nucleotide, exon 3 and 4 whole and exon 5 preceding 35 nucleotide.

The nucleic acid sequence of table 2- human PD-L 1

ATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGT TCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTA GACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGAAGACC TGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACT TCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAG CGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTG AACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCT GAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAAC ACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCA TCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCT TGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGAT ACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAA(SEQ ID NO.2)

The nucleic acid sequence through codon optimization of table 3- film combination PD-L1

ATGAGAATCTTCGCGGTGTTCATCTTCATGACCTACTGGCACCTCCTGAACGCTTTCACTGTGACCGT GCCTAAGGACCTCTACGTCGTGGAATACGGCTCCAACATGACCATCGAGTGCAAATTCCCAGTGGAGAAGCAGCTG GACCTGGCTGCCCTGATCGTGTACTGGGAAATGGAGGACAAGAACATCATCCAATTCGTGCATGGGGAGGAGGACC TGAAGGTCCAGCATTCGTCATATCGGCAAAGAGCCAGGCTGCTGAAGGATCAGCTGTCCCTCGGCAATGCGGCACT GCAGATTACCGATGTGAAGCTGCAGGACGCCGGAGTCTACCGGTGCATGATTTCCTACGGCGGAGCAGACTACAAG CGCATTACCGTGAAGGTCAACGCTCCCTACAACAAGATCAACCAGCGGATTCTGGTGGTCGACCCTGTGACCTCCG AGCATGAGCTGACCTGTCAAGCCGAAGGTTACCCGAAAGCGGAAGTGATCTGGACGTCGAGCGACCACCAGGTCTT GAGCGGAAAGACGACCACTACTAACAGCAAGCGGGAAGAGAAACTGTTTAACGTGACCAGCACTCTTCGGATCAAC ACCACCACTAACGAGATTTTCTACTGTACCTTTCGCCGGCTTGACCCGGAAGAAAATCACACCGCCGAGCTCGTGA TCCCCGAGCTGCCCCTCGCCCACCCTCCTAACGAAAGAACCCACCTGGTCATCTTGGGGGCCATCCTGCTGTGCCT GGGAGTGGCCCTGACCTTCATTTTTAGGCTCCGAAAGGGCCGCATGATGGACGTGAAGAAATGCGGAATCCAGGAC ACTAACTCCAAGAAGCAGTCCGATACTCACCTGGAAGAAACCTAG(SEQ ID NO.3)

The nucleic acid sequence through codon optimization of the soluble PD-L1 of table 4- and Fc segment composition

ATGAGAATCTTCGCGGTGTTCATCTTCATGACCTACTGGCACCTCCTGAACGCTTTCACTGTGACCGT GCCTAAGGACCTCTACGTCGTGGAATACGGCTCCAACATGACCATCGAGTGCAAATTCCCAGTGGAGAAGCAGCTG GACCTGGCTGCCCTGATCGTGTACTGGGAAATGGAGGACAAGAACATCATCCAATTCGTGCATGGGGAGGAGGACC TGAAGGTCCAGCATTCGTCATATCGGCAAAGAGCCAGGCTGCTGAAGGATCAGCTGTCCCTCGGCAATGCGGCACT GCAGATTACCGATGTGAAGCTGCAGGACGCCGGAGTCTACCGGTGCATGATTTCCTACGGCGGAGCAGACTACAAG CGCATTACCGTGAAGGTCAACGCTCCCTACAACAAGATCAACCAGCGGATTCTGGTGGTCGACCCTGTGACCTCCG AGCATGAGCTGACCTGTCAAGCCGAAGGTTACCCGAAAGCGGAAGTGATCTGGACGTCGAGCGACCACCAGGTCTT GAGCGGAAAGACGACCACTACTAACAGCAAGCGGGAAGAGAAACTGTTTAACGTGACCAGCACTCTTCGGATCAAC ACCACCACTAACGAGATTTTCTACTGTACCTTTCGCCGGCTTGACCCGGAAGAAAATCACACCGCCGAGCTCGTGA TCCCCGAGCTGCCCCTCGCCCACCCTCCTAACGAAAGAACTCCCAAGTCTTGCGATAAGACCCACACATGCCCGCC ATGCCCAGCCCCGCCCGTGGCGGGCCCCTCCGTGTTTCTTTTCCCGCCGAAGCCTAAGGATACCCTGATGATCTCC CGCACCCCCGAAGTCACTTGTGTGGTGGTGGACGTCAGCCACGAAGATCCGGAAGTCAAGTTCAATTGGTACGTGG ACGGGGTCGAAGTGCACAACGCCAAGACCAAGCCCCGCGAGGAACAGTACAACTCAACGTACCGGGTGGTGTCCGT GCTGACCGTGCTGCATCAGGACTGGCTGAACGGAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGACTGCCGAGC TCGATCGAAAAGACCATTTCGAAGGCCAAGGGGCAGCCTAGGGAGCCACAGGTCTATACCCTCCCGCCCTCACGAG ATGAACTGACCAAGAACCAAGTGTCATTGACTTGCCTCGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAATG GGAATCCAACGGACAGCCGGAGAACAACTACAAGACTACTCCGCCCGTGCTTGACTCCGACGGTTCGTTCTTCCTG TACTCCAAGCTGACCGTGGATAAGTCCCGCTGGCAACAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCCC TGCACAACCACTACACCCAGAAGTCCCTCTCGTTGAGCCCTGGAAAATAG(SEQ ID NO.4)

As defined in genetic code, in the amino acid sequence of specific protein and the nucleotides sequence of the codified protein There is corresponding relationship that is known and determining between column.Similarly, as defined in genetic code, in the nucleotides sequence of specific nucleic acid It arranges and by there is corresponding relationship that is known and determining between the amino acid sequence of the nucleic acid encode.

Table 3

Genetic code

Alanine (Ala, A) GCA, GCC, GCG, GCT

Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

Asparagine (Asn, N) AAC, AAT

Aspartic acid (Asp, D) GAC, GAT

Cysteine (Cys, C) TGC, TGT

Glutamic acid (Glu, E) GAA, GAG

Glutamine (Gln, Q) CAA, CAG

Glycine (Gly, G) GGA, GGC, GGG, GGT

Histidine (His, H) CAC, CAT

Isoleucine (Ile, I) ATA, ATC, ATT

Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

Lysine (Lys, K) AAA, AAG

Methionine (Met, M) ATG

Phenylalanine (Phe, F) TTC, TTT

Proline (Pro, P) CCA, CCC, CCG, CCT

Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

Threonine (Thr, T) ACA, ACC, ACG, ACT

Tryptophan (Trp, W) TGG

Tyrosine (Tyr, Y) TAC, TAT

Valine (Val, V) GTA, GTC, GTG, GTT

One of genetic code it is important and well-known feature be its redundancy, thus for being used to prepare protein Most of amino acid can use more than one coding nucleotide triplet.Therefore, a variety of different nucleotide sequences can be compiled The given amino acid sequence of code.Such nucleotide sequence is considered as that function is equivalent, because they cause in all organisms It is middle to generate identical amino acid sequence (although certain organisms more effectively translate some sequences than other organisms).This Outside, the methylated variant of purine or pyrimidine can be found in given nucleotide sequence once in a while.Such methylation does not influence three Encoding relation between nucleotide codon and corresponding amino acid.

In view of the foregoing, the nucleosides of the DNA or RNA of fusion protein or polypeptide (or its any part) of the invention are encoded Acid sequence can be used for derivative fusion albumen or polypeptid acid sequence, and the DNA or RNA are translated into amino acid using genetic code Sequence.Similarly, for fusion protein or polypeptid acid sequence, (due to its redundancy, can be given from genetic code for any Multiple nucleic acid sequences will be generated by determining amino acid sequence) derive the corresponding nucleotide sequence that can encode the fusion protein or polypeptide Column.Therefore, herein to the description of the polynucleotide sequence of encoding fusion protein or polypeptide and/or openly should be understood as further include To the description by the nucleotide sequence coded amino acid sequence and/or openly.Similarly, herein to fusion protein or polypeptide ammonia The description of base acid sequence and/or openly should be understood as further including all possible nucleotide to the codified amino acid sequence The description of sequence and/or openly.

In some embodiments, PD-L1 albumen of the invention do not include signal sequence because this sequence usually from It is cut before cell secretory polypeptide.In other embodiments, PD-L1 albumen is soluble, i.e., by IgV structural domain and IgC Structural domain (that is, the extracellular part for the PD-L1 that the film of overall length combines) composition, and also may include heterologous sequence, such as Fc knot Structure domain, protein tag, the therapeutic agent of conjugation etc..Such solubility PD-L1 isotype can be by more known to technical staff Kind mode passes through alternative splicing and generates.

In preferred embodiments, as shown in this paper Fig. 2, soluble PD-L1 includes the PD- for eliminating the film of overall length and combining The part of the exon 5 of L1 cDNA and exon 6 and 7 whole.It can be by the way that the N-terminal area of PD-L1 be merged with human IgG1 Fc Soluble PD-L1 isotype is generated, and in some embodiments, amino acid sequence (GGGGS) n (SEQ ID can be passed through NO:5 it) connects.In preferred embodiments, IgG1 Fc can be mutated by changing the following amino acid in Fc structural domain To reduce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDCC): E233P, L234V, L235A, G236 missing, A327G, A330S and P331S.Fc structural domain can be source of people, and can also originate from the Fc of human IgG1.

Delivery of nucleic acids medium

This field has can be used comprising the expression vector of PD-L1 nucleic acid to realize that effective intestines express this purpose in application Any method for knowing is realized.In preferred embodiments, theme nucleic acid delivery vehicle is applied to the part of intestines (i.e. not It systemically applies).In one embodiment, delivery of nucleic acids medium includes viral vectors.In another embodiment, Delivery of nucleic acids medium includes cationic-liposome.In preferred embodiments, delivery of nucleic acids medium includes deacetylated shell Polysaccharide.In alternative embodiment, biological gene delivery vehicle (BGV), such as enterobacteria, bacteriophage, disease are used Malicious sample particle, biological liposome etc., by PD-L1 delivery of polynucleotides to enteric epithelium.About the comprehensive of the BGV currently studied It states, referring to Seow and Wood Biological Gene Delivery Vehicles Beyond Viral Vectors, Molecular Therapy 17:767-777 (2009), content are integrally incorporated herein.

In specific embodiments, delivery of nucleic acids medium includes chitosan derivative, such as in conjunction with other Functionalization, such as the chitosan of the ligand with attachment." chitosan " is interpreted as wrapping as used herein Include wide class N- acetyl group-d-glucosamine comprising covalent modification and/or d-glucosamine unit it is more based on deacetylated shell The polymer of sugar, and combine other units or be attached to the polymer based on chitosan of other parts.Derivative It is the modification based on hydroxyl or amido to aminoglucose in many cases, such as with the functionalized chitosan of arginine The modification of completion.The example of chitosan derivative includes but is not limited to tri-methylated chitosan, PEGylated de- second Acyl chitin, galactosylation chitosan, is alkylated chitosan, in conjunction with PEI at sulfhydrylation chitosan Chitosan, uronic acid modification chitosan, glycol chitosan etc..It is more about deacetylated shell The further introduction of sugar derivatives, see, e.g. K.Taira, K.Kataoka, T.Niidome (editor), Springer- Verlag Tokyo, 2005, ISBN 4-431-25122-7, the 63-74 pages of " Non-viral Gene Therapy "；Zhu Et al., Chinese Science Bulletin, in December, 2007, the 52nd (23) volume, the 3207-3215 pages；With Varma etc. People, Carbohydrate Polymers 55 (2004) 77-93

The chitosan with any deacetylation (DDA) greater than 50% is used in the present invention, functionalization exists Between 1% and 50%.(quantity relative to the free amino moieties on chitosan polymer determines functionalization percentage Than.) the functionalized specific charge density of chitosan derivative of deacetylated and functionalized degree imparting.Resulting electricity Lotus Effects of Density solubility, nucleic acid combines and subsequent release, and the interaction with mammalian cell membrane.Therefore, root According to the present invention, it is necessary to optimize these characteristics to obtain optimal efficacy.Illustrative chitosan derivative is described in Baker Et al. submitted on January 24th, 2007 11/657,382 in, the document is herein incorporated by reference.In an embodiment In, double derivative chitosan as described herein includes the chitosan that deacetylation is at least 50%.One In a embodiment, deacetylation is at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably extremely Few 90%, most preferably at least 95%.In preferred embodiments, double derivative chitosan packet as described herein The chitosan for being at least 98% containing deacetylation.

Chitosan derivative as described herein has the soluble average molecular weight range under neutral and physiological pH, It and for purposes of the present invention include the molecular weight ranges of 3-110kDa.The embodiment described herein is characterized in that derivative The average molecular weight of change chitosan is lower, and (to about 25kDa), these derivatizations are deacetylated by < 25kDa, for example, about 5kDa Chitin has desired delivering and transfection characteristics, and size is small and has advantageous solubility.Compared with the derivative of low average molecular weight It is usually more more soluble than the chitosan with higher molecular weight to change chitosan, therefore the former generates nucleic acid/de- Chitosan compound, the compound will more easily discharge nucleic acid and provide increased cell transfecting.

In preferred embodiments, use chitosan or chitosan derivative as delivery of nucleic acids matchmaker Jie's object completes the application of the expression vector comprising PD-L1 nucleic acid.Chitosan derivative as described herein is by with band Positive charge and/or hydrophilic part is functionalized gained free amine group and generates.International patent application no PCT/CA2013/ The deacetylated shell of derivatization described in 050218 and PCT/CA2014/050921 (its content is integrally incorporated herein by reference) The characteristic that there is polysaccharide many to be conducive to delivery of nucleic acids medium, comprising: they are effectively combined and compound negatively charged core Acid, they can form the controllable nano particle of size, they can be absorbed by cell, and they can be in the cell suitable Nucleic acid is discharged when the time.

In preferred embodiments, " double derivative chitosan " or " DD- chitosan " is used, Refer to that (" dual functionalisation's chitosan " or " the deacetylated shell of DF- is more by the chitosan of dual functionalisation Sugar "), such as be coupled with both Arg and hydrophilic polyol, both of which and chitosan are covalently attached.Arg can make It is covalently attached for single amino acid or as polypeptide and chitosan.Hydrophilic polyol can be sugared such as glucose. " DD- chitosan nucleic acid polymerization compound " or its grammatical equivalents mean comprising multiple DD- chitosan molecules With the nucleic acid molecules of multiple coding PD-L1 or the compound of its segment.In preferred embodiments, double derivativeization is deacetylated The nucleic acid of chitin and coding PD-L1 polypeptide is compound.

DD- chitosan PD-L1 nucleic acid polymerization compound includes PD-L1 nucleic acid component and DD- chitosan Component.Chitosan and DD- chitosan nucleic acid polymerization compound can pass through any method system known in the art It is standby.For example, adjustable functionalized chitosan and nucleotide material concentration are to adapt to various amine and phosphate ratio (N/ P), mixing ratio and target nucleotide concentration.The preferred method formed for polymer composites is disclosed in WO 2009/039657, It is whole by reference to be expressly incorporated herein.

Expression vector and expression control zone

The present invention relates to the expression vectors comprising PD-L1 nucleic acid, promoter and transcription and translation termination signal.It can incite somebody to action Various nucleotide and control sequence link together to generate expression vector, which may include one or more convenient Restriction site, with allow these sites be inserted into or replace coding PD-L1 nucleic acid.It alternatively, can be by that will include The polynucleotides or nucleic acid construct of PD-L1 nucleic acid are inserted into carrier appropriate and are expressed to express PD-L1 nucleic acid.It is being formed When expression vector, the coded sequence of PD-L1 is located in carrier, so that it is operably connected to carry out with control sequence appropriate Expression.

Expression vector, which can be, to be convenient to carry out recombinant DNA program and can cause any carrier (example of expression of nucleic acid Such as plasmid or virus).The selection of carrier will generally depend on the compatibility of carrier with the host cell that will introduce carrier.Carrier It can be linear or closed loop plasmid.Carrier can be the carrier independently replicated, i.e., as the existing load of extrachromosomal entity Body is replicated independently of chromosome replication, these carriers such as plasmid, extra-chromosomal element, minichromosome or artificially colored Body.Carrier may include any component for ensuring self-replacation.Alternatively, carrier may be such that thin when being introduced into host When in born of the same parents, it is integrated into genome and is replicated together with the one or more chromosomes being integrated into.In addition it is possible to use single carry Two or more carriers or matter of body or plasmid or the total nucleic acid jointly containing host cell gene group to be introduced or transposons Grain.

Carrier optionally includes one or more selected marker objects, and one or more of selected marker objects allow Cell that be readily selected conversion, transfection, transduction or similar.Selected marker object be its product provide biocide or Virus resistance, to the resistance of heavy metal, to the gene of auxotrophic prototrophy etc..For independently replicating, carrier can also be wrapped Containing the replication orgin for enabling carrier independently to replicate in the host cell discussed.Replication orgin can be mediation in cell In any plasmid replicon independently replicated that works.Term " replication orgin " or " plasmid replicon " mean that plasmid can be made Or the polynucleotides that carrier replicates in vivo.

In preferred embodiments, expression vector of the invention includes the nucleic acid molecules or its segment of coding PD-L1, institute Stating nucleic acid molecules includes the expression control zone being operably connected with the code area of PD-L1 polypeptide as described herein.In some realities It applies in scheme, nucleic acid is DNA or RNA, such as mRNA.In some embodiments, PD-L1 nucleic acid is artificial nucleic acid.Preferably Artificial nucleic acid includes but is not limited to peptide nucleic acid (PNA), phosphorodiamidate morpholino oligomer (phosphorodiamidate Morpholino oligo, PMO), lock nucleic acid (LNA), ethylene glycol nucleic acid (GNA) and threose nucleic acid (TNA).

In a specific embodiment, expression vector is such as pVAX_hPD-L1 (Fig. 1) exemplified here.Another In a specific embodiment, expression vector is pVax_hPD-L1 Fc (Fig. 2).

In some embodiments, expression control zone has constitutive activity.In multiple preferred embodiments, expression Control zone does not have constitutive activity.This provides the Expression of PD-L1 nucleic acid." dynamic " expression, which refers to, to be changed over time Expression.Expression may include several such low expressions or the period without expression, be separated with the period of detectable expression.In In multiple preferred embodiments, PD-L1 nucleic acid is operably connected with adjustable promoter.This provides nucleic acid molecules Regulatable expression.Expression control zone includes modulability polynucleotides (this for the expression for influencing the PD-L1 nucleic acid being operably connected Text is sometimes referred to as element), such as promoter and enhancer.

The expression control element for including herein can come from bacterium, yeast, plant or animal, and (mammal or non-lactation are dynamic Object).Expression control zone includes overall length promoter sequence, such as natural promoter and enhancer element, and retains whole or portion Point overall length or non-variant function (for example, retain a certain amount of nutrition adjustment or cell/tissue is specific expressed) subsequence Or polynucleotides variant.As used herein, term " functionality " and its grammatical variants, when for referring to nucleic acid sequence, subsequence Or when segment, it is intended that the sequence has one or more function of native sequence nucleic acid (such as non-variant or unmodified sequence) Energy.As used herein, term " variant " mean sequence replace, miss or add or other modifications (such as chemical derivative, it is all The form of modification such as resistant to nuclease).

As used herein, term " operable connection " refers to the physics juxtaposition of described component, with allow they with Its expected mode plays a role.In the example for the expression control element being operatively connected with nucleic acid, which is so that control The expression of element regulation nucleic acid processed.In general, the expression control zone juxtaposition of regulatory transcription near 5 ' ends of transcribed nucleic acid (i.e. " upstream ").Expression control zone may be located on the 3 ' ends (i.e. " downstream ") of transcription sequence or be located in transcript (for example, interior Containing in son).Expression control element can be located remotely from transcription sequence at a certain distance from (for example, apart from nucleic acid 100 to 500, 500 to 1000,2000 to 5000 or more nucleotide).The specific example of expression control element is promoter, usually Positioned at the 5 ' of transcription sequence.Another example of expression control element is enhancer, can be located at the 5 ' or 3 ' of transcription sequence, Or it is located in transcription sequence.

Some expression control zones assign the adjustable expression of PD-L1 nucleic acid being operably connected.Signal (sometimes referred to as pierces Swash) it can increase or decrease and this expression for expressing the PD-L1 nucleic acid that control zone is operably connected.Increase in response to signal Such expression control zone of expression is added to be commonly known as induction type.Such expression control zone of expression is reduced in response to signal Commonly known as suppressive.In general, the amount increased or decreased that these elements assign is proportional to existing semaphore；Signal Amount is bigger, and increasing or decreasing for expression is bigger.

A variety of adjustable promoters are well known in the art.Preferred inducible expression control zone includes containing useful The expression control zone of the inducible promoter of small molecule compound stimulation.In one embodiment, expression control zone in response to It is orally available to deliver but be generally not present in the chemical substance in food.Specific example is found in such as United States Patent (USP) 5,989, 910；5,935,934；6,015,709；With 6,004,941, all these patents are integrally incorporated herein by reference.

In one embodiment, expression vector also includes integration sequence.In one embodiment, expression vector includes Single integration sequence.In another embodiment, expression vector includes the first integration sequence and the second integration sequence, to be used for PD-L1 nucleic acid or part thereof is integrated into the genome of target cell.In preferred embodiments, one or more integrates sequence Column play a role with being selected from conjunction with the integration component of group being made up of: mariner, sleeping beauty, FLP, Cre, Ф C31, R, λ, and the integration component of the integration virus from such as AAV, retrovirus and slow virus.

In one embodiment, theme composition also includes non-therapeutic construct in addition to PD-L1 construct, wherein Non-therapeutic construct includes the nucleic acid sequence for integrating component that coding is operably connected with the second expression control zone.This second Expression control zone and the expression control zone being operably connected with PD-L1 nucleic acid can be identical or different.The integration component of coding It is preferably chosen from the group being made up of: mariner, sleeping beauty, FLP, Cre, Ф C31, R, λ, and from all Such as the integration component of the integration virus of AAV, retrovirus and slow virus.

It with the program for constructing recombinant expression carrier of the invention is that those skilled in the art are known for connecting said elements , see, for example, Sambrook et al., 1989, ibid).About further introduction, referring to WO2008020318, with reference Mode is integrally expressly incorporated herein.

Pharmaceutical preparation

The present invention also provides " the pharmacy comprising DD- chitosan nucleic acid polymerization composite compositions of the invention It is upper acceptable " or " physiologically acceptable " preparation.Such preparation can be applied to subject in vivo to implement to treat Method.

As used herein, term " pharmaceutically acceptable " and " physiologically acceptable " refer to can be applied to it is tested Person does not generate carrier, diluent, the excipient etc. of excessive adverse side effect (such as nausea, abdominal pain, headache etc.) preferably Deng.Such preparation for application includes sterile aqueous or non-aqueous solution, suspension and lotion.

Pharmaceutical preparation can be by applying compatible carrier, diluent, excipient, solvent, decentralized medium, packet with subject Clothing, antibacterial agent and antifungal agent, isotonic agent and absorption delaying agent etc. preparation.Such preparation may be embodied in tablet (cladding Or it is uncoated), capsule (hard or soft), microballon, lotion, powder, pellet, crystal, suspension, in syrup or elixir. Other than other additives, there may also be the reactive compound of supplement and preservative, for example (,) it is antimicrobial, anti-oxidant Agent, chelating agent and inert gas etc..

Excipient may include salt, isotonic agent, haemocyanin, buffer or other pH controlling agents, antioxidant, thickener, Uncharged polymer, preservative or Cryoprotectant.Excipient for the present composition may also include isotonic agent and Buffer or other pH controlling agents.These excipient can be added to reach preferred pH range (about 6.0-8.0) and held with seeping Rub (about 50-400mmol/L).The example of suitable buffer be acetate, borate, carbonate, citrate, phosphate and Sulfonation organic molecule buffer.These buffers can be present in composition with the concentration of 0.01% to 1.0% (w/v).Deng Penetration enhancer can be selected from any isotonic agent known in the art, such as mannitol, dextrose, glucose and sodium chloride or other electricity Xie Zhi.Preferably, isotonic agent is glucose or sodium chloride.Can be used makes composition have the biotic environment phase introduced with its The isotonic dosage of same or similar osmotic pressure.The concentration of composition isotonicity agent will depend on the property of specific isotonic agent used, And it can be in the range of about 0.1% to 10%.When using glucose, preferably with 1% to 5%w/v, more particularly 5% The concentration of w/v uses.When isotonic agent is sodium chloride, preferably to reach 1%w/v, particularly the amount of 0.9%w/v is used.The present invention Composition can also contain preservative.The example of preservative is that polyhexamethylene-biguanides, benzalkonium chloride, stabilized oxygen chlorine are multiple Object is closed (to be such as known asCompound), phenylmercuric acetate, chlorobutanol, sorbic acid, Chlorhexidine, benzylalcohol, para hydroxybenzene Formic acid esters and thimerosal.In general, the concentration of such preservative is about 0.001% to 1.0%.In addition, composition of the invention is also Cryoprotectant can be contained.Preferred Cryoprotectant be glucose, sucrose, mannitol, lactose, trehalose, D-sorbite, Colloidal silicon dioxide, molecular weight are preferably shorter than the glucan, glycerol and molecular weight of 100,000g/mol lower than 100,000g/mol Polyethylene glycol or their mixture.Most preferably glucose, trehalose and polyethylene glycol.In general, such freezen protective The concentration of agent is about 0.01% to 10%.

It can be by formulating medicinal preparations at compatible with expected administration method.For example, for being administered orally, it can be by composition With excipient blend and with tablet, pastille, capsule (such as gelatin) or coating (such as enteric coating (Or)) form use.The adhesive of pharmaceutically compatible and/or Adjuvanting material can be included in oral preparation In.Tablet, pill, capsule, pastille etc. contain following any ingredient or the compound with similarity: adhesive, all Such as microcrystalline cellulose, bassora gum or gelatin；Excipient, such as starch or lactose；Disintegrating agent, such as alginic acid, Primogel or Cornstarch；Lubricant, such as magnesium stearate or other stearate；Glidant, such as colloidal silicon dioxide；Sweetener, it is all Such as sucrose or saccharin；Or flavoring agent, such as peppermint, gaultherolin or seasoning.

Preparation can also protect composition to eliminate from fast degradation or from internal comprising carrier, such as controlled release preparation, Including implantation material and microencapsulated delivery systems.For example, can be used alone or time delay material is applied in combination such as with wax Glycerin monostearate or tristerin.

Also contemplate suppository and other can rectal administration preparation (such as can by bowel lavage apply preparation).About rectum The further information of delivering, see, e.g. Song et al., Mucosal drug delivery:membranes, methodologies,and applications,Crit.Rev.Ther.Drug.Carrier Syst.,21:195-256, 2004；Wearley,Recent progress in protein and peptide delivery by noninvasive routes,Crit.Rev.Ther.Drug.Carrier Syst.,8:331-394,1991。

Other drugs preparation suitable for application is well known in the art, and is suitable for method and composition of the invention (see, e.g. Remington's Pharmaceutical Sciences (1990) the 18th edition, Mack Publishing Co.,Easton,Pa.；The Merck Index (1996) the 12nd edition, Merck Publishing Group, Whitehouse, N.J.；With Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co.,Inc.,Lancaster,Pa.,(1993))。

Intestines application

Theme composition can be with oral administration.Oral administration may relate to swallow, so that compound enters gastrointestinal tract.This The composition of invention can also be applied directly to gastrointestinal tract.Syringe, endoscope, casing, intubation, conduit and other articles are available In this application.Preparation suitable for oral administration includes solid pharmaceutical preparation, such as tablet, capsule, the packet containing particle or coated particle Cover capsule, liquid or powder, pastille (pastille including filling liquid), chaw, more particles and nano particle, gel, film, Pearl shape agent (ovule) and spray.Liquid preparation includes suspension, solution, syrup and elixir.Liquid preparation can pass through reconstruct It is prepared by solid.

Tabules usually contain disintegrating agent.The example of disintegrating agent includes carboxyrnethyl starch sodium, sodium carboxymethylcellulose, carboxylic first Base cellulose calcium, croscarmellose sodium, Crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, Hydroxypropyl cellulose, starch, pregelatinized starch and the sodium alginate that low alkyl group replaces.In general, disintegrating agent accounts for the 1 of dosage form Weight % to 25 weight %, preferably 5 weight % are to 20 weight %.

Adhesive is commonly used in imparting tablet formulation adhesiveness.Suitable adhesive include microcrystalline cellulose, gelatin, sugar, Polyethylene glycol, natural and paragutta, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methyl are fine Dimension element.Tablet can also contain diluent, such as lactose (monohydrate, the monohydrate of spray drying, anhydride etc.), sweet dew Sugar alcohol, xylitol, dextrose, sucrose, D-sorbite, microcrystalline cellulose, starch and dicalcium phosphate dihydrate.

Tablet also optionally including surfactant (such as NaLS and polysorbate80) and helps stream Agent (such as silica and talcum).When it is present, surfactant can account for the 0.2 weight % to 5 weight % of tablet, and help Stream agent can account for the 0.2 weight % to 1 weight % of tablet.

Tablet usually also contains lubricant, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, with And the mixture of magnesium stearate and NaLS.Lubricant usually accounts for the 0.25 weight % to 10 weight % of tablet, preferably 0.5 weight % to 3 weight %.

Other possible ingredients include antioxidant, colorant, flavoring agent, preservative and odor mask.Tablet blend can Direct pressing is in blocks by roll pressing.It before tableting, can be by tablet blend or blend part wet granulation, dry method grain Change or melt granulation, melting and solidification or extrusion.Final preparation may include one or more layers and can be cladding or it is uncoated 's；It can even is that capsule encapsulating.

Pharmaceutical Dosage Forms of the tablet formulation in H.Lieberman and L.Lachman: Tablets has been discussed in volume 1 (Marcel Dekker, New York, 1980).

The oral film that consumes for people's purposes or veterinary purpose is usually flexible water solubility or water-swellable membrane agent Type, their rapidly dissolvable or mucoadhesives, and generally comprise film forming polymer, adhesive, solvent, moisturizer, plasticizer, Stabilizer or emulsifier, viscosity improver and solvent.Some components of preparation can execute more than one function.

The invention also includes more non-pareil beads containing the present composition.

Other possible ingredients include antioxidant, colorant, seasoning and flavoring agent, preservative, saliva stimulant, cool Refreshing agent, cosolvent (including oil), emollient, incremental agent, defoaming agent, surfactant and odor mask.

Film according to the present invention usually passes through the thin water that evaporation drying is coated on strippable backing support object or paper It is prepared by property film.This can be completed in drying oven or tunnel (usually combined type coating machine drier), or pass through freezing Drying vacuumizes to complete.Solid pharmaceutical preparation for oral administration, which can be formulated into, to be released immediately and/or modified release.Modification Delivery formulations include sustained release, sustained release, pulsed release, control release, Targeting delivery and sequencing release.

Other suitable release tech such as high energy dispersions and permeability and coated particle are known.

Rectum/intravaginal administration

The compound of the present invention can be with per rectum or vaginal application, such as is applied in the form of suppository, pessary or enema With.Cupu oil is traditional suppository base, but can take the circumstances into consideration to use various substitutes.

It can be formulated into and release immediately and/or modified release for rectum/vaginal application preparation.Modified release preparation packet Include sustained release, sustained release, pulsed release, control release, Targeting delivery and sequencing release.

Dosage or " effective quantity " for treating subject are preferably enough to improve disease with measurable or detectable degree A kind of, several or all symptoms of trouble, but preventing or inhibit the progress or deterioration of inflammatory conditions or illness or symptom is to make us full The result of meaning.Therefore, the amount of the PD-L1 albumen generated to improve the illness that can be treated by the method for the invention will depend on disease Suffer from and required as a result, and can be readily determined by technical staff.Amount appropriate will depend on treated illness, required Therapeutic effect and individual subjects (such as bioavilability, gender, age in subject etc.).It is relevant by measuring Physiological effect can determine effective quantity.

Present invention further contemplates veterinary applications.Therefore, in one embodiment, the present invention provides treat inhuman lactation The method of animal, it is in need for the treatment of non-the method includes the nano particle of the invention based on chitosan to be applied to People mammal.

Treatment method

The compositions and methods of the invention have advantageous purposes in regulation aspect of inflammation.For example, therapeutical peptide can be with Inhibit the proliferation and differentiation of the cell of participation inflammatory reaction.These molecules can be used for treating inflammatory condition (chronic disease and acute Illness), including inflammation relevant to infection (such as septic shock, septicemia or systemic inflammatory response syndrome (SIRS)), ischemia reperfusion injury, endotoxin be lethal, inflammatory bowel disease (IBD), Crohn disease, colitis or by cell factor Illness caused by (such as TNF or IL-1) excess generation.The inflammatory conditions packet of special interest for treatment of the invention It includes but is not limited to Crohn disease and inflammatory bowel disease.

Therapeutic composition of the invention can also be used to treating and/or preventing organ rejection or graft versus host disease(GVH disease) (GvHD).Host immune cell destroys the tissue of transplanting by immune response, thus organ rejection occurs.Similarly, in GvHD Also it is related to being immunoreacted, still, in this case, the immune cell destruction host tissue of xenotransplant.Application inhibits immune Reaction, the therapeutic composition of the invention of the especially proliferation of T cell, differentiation or chemotaxis can be prevention organ rejection or Effective therapy of GvHD.

Example set forth herein shows several embodiments of the disclosure, but is not necessarily to be construed as limiting in any way The scope of the present disclosure.