CN105968189A

CN105968189A - B and T lymphocyte attenuator immunogen polypeptide and application thereof

Info

Publication number: CN105968189A
Application number: CN201610438242.8A
Authority: CN
Inventors: 罗瑞雪
Original assignee: Suzhou Puluoda Biological Science and Technology Co Ltd
Current assignee: Suzhou Puluoda Biological Science and Technology Co Ltd
Priority date: 2016-06-19
Filing date: 2016-06-19
Publication date: 2016-09-28

Abstract

The invention discloses a B and T lymphocyte attenuator immunogen polypeptide, belongs to the field of anti-cancer vaccines, and particularly relates to optimized polypeptide of B and T lymphocyte attenuator immunogen serving as an anti-cancer vaccine, and T cell immune responses are enhanced. The amino acid sequence is a brand-new sequence. The invention relates to application of the polypeptide in preparation of drugs for treating tumors, in particular to application in preparation of drugs which are used for tumor immune cells and used for enhancing the T cell immune responses and application in preparation of drugs for CAR-T cell immunotherapy. The B and T lymphocyte attenuator immunogen polypeptide has the advantages that the immunogen polypeptide has the brand-new amino acid sequence, can be used in tumor immune cell technologies, can promote T cell proliferation, can promote specific binding of T cells and tumor surface antigens, can inhibit tumor growth in tumor-burdened mouse bodies and is applied to cell therapies of CAR-T and the like by serving as target molecules of the cell therapies.

Description

B/T lymphocyte weakens factor immunogen polypeptide and application thereof

Technical field

The present invention is relevant anti-cancer vaccine field.In particular it relates to the B/T being used as anti-cancer vaccine drenches The bar cell weakening factor immunogenic optimization polypeptide, strengthens T cell immunne response.

Background technology

Tumor cell immunization therapy be a kind of emerging, there is the tumor treatment model of significant curative effect, be a kind of from The novel method for the treatment of of body immunity anticancer.It uses biotechnology and biological preparation to gathering from the patient The method that immunocyte feeds back in patient body after carrying out In vitro culture and amplification, excites, enhancing body self Immunologic function, thus reach to treat the purpose of tumor.Tumor cell immunotherapy is continue operation, radiation and chemotherapy The fourth-largest oncotherapy technology afterwards.

B/T cell weakening molecule (BTLA) is that the B/T lymphocyte reduction factor family found recently presses down altogether Molecule processed, is CD28 family member, is to divide with Cytotoxic T lymphocyte-associated antigen-4 and programmed death The similar Inhibitory receptor of son-1.Mainly it is expressed in B cell, T cell and antigen presenting cell surface. The part of BTLA is TNF superfamily member Herpesvirus entry mediator (HVEM).HVEM-BTLA The activation of T, B cell is played down regulation by signal pathway, at regulation immune response, maintains self Play a very important role during immunity is stable, be one of the focus of current immunotherapy of tumors research.

Recently Gertner-Dardenne etc. think the signal pathway that lymphoma cell may be mediated by BTLA The propagation (retardance S phase) of suppression gamma delta T cells so that the generation of immunosurveillance escape.It addition, BTLA is high expressed on melanoma specific C D8+T cell, and by mediating with its part HVEM The function of signal pathway suppression tumour-specific CD8+T cell, can increase after closing BTLA signal pathway Strong its increment activity and generation of cytokine；After melanoma patients inoculation CpG vaccine, can lower BTLA is in tumour-specific CD8+T cell and the expression of B cell, and it declines degree and immunity The number of times of inoculation is proportionate.Hobo etc. use Allogeneic stem cell to adopt and treat grinding of malignant hematologic disease Study carefully middle discovery, BTLA secondary tissue compatibility antigen (Minor histocompatibility antigens, MiHA) high expressed weaken the increment of T cell and the generation of cytokine on specific C D8+T cell, Its function can be recovered after blocking its signal pathway.These researchs are the tumour immunity using BTLA as target spot Treatment provides clue.BTLA can be with multiple co-suppression molecule (such as PD-1, TIM-3 etc.) table simultaneously Reach at tumour-specific CD8+T cell and make T cell dysfunction.Radvanyi etc. use external expansion Increase melanoma patients autologous tumor lymphocyte infiltration to carry out finding during adoptive cellular treatment, BTLA+CD8 + TILs quantity and clinical treatment curative effect are high-positive correlation.Prompting BTLA adopts treatment at TILs cell Malignant melanoma is probably a new important biomarker.

Along with the further investigation to BTLA, it has been found that BTLA not only at T, bone-marrow-derived lymphocyte, huge bite The inherent immunity cell surface expressions such as cell, DC, NK, mononuclear cell, and newly discovered gamma delta T cells, Hematological system tumor Cells B Cells type malignant tumor, multiple myeloma cells (MM), acute myeloid are white Also expression is had on disorders of blood cell (AML).BTLA expression on tumor cell points out it to be probably one Tumor marker, detects its expression and may have important guiding effect to clinical diagnosis.

B/T lymphocyte weakens the factor can be as the specific immunogens of tumor vaccine.But, B/T drenches The bar cell weakening factor is by 414 amino acid whose albumen, and molecular weight is bigger, it is more difficult to obtain the B/T that purity is high Lymphocyte weakens the factor.So, bring difficulty not only can to the Specific T cell immunity in later stage, and The autoimmune of patient can be caused.Therefore, the invention provides a kind of high specificity, the little molecule that purity is higher Polypeptide is as immunogen.

Summary of the invention

Goal of the invention

The present invention provides a kind of B/T lymphocyte to weaken factor immunogen polypeptide, can be used for tumor cell immunity Immunogen, strengthens T cell immunne response, has the advantages that molecular weight is little, specific immunogenic is strong.

Technical scheme

Technical program of the present invention lies in providing a kind of B/T lymphocyte to weaken factor immunogen polypeptide, sequence is RRGDPWLLYSLLPSSLDFTTCFCLFCCLFFL (SEQ ID NO:1).This aminoacid sequence For brand-new sequence, the application in preparation is used for tumor.Especially thin for tumour immunity in preparation Born of the same parents, strengthen the application in T cell immunne response medicine, and in preparation for CAR-T cellular immunotherapy Application.

Beneficial effect

The B/T lymphocyte of the present invention weakens factor immunogen polypeptide, for brand-new sequence, can be used for tumor and exempts from Immunogen in epidemic disease cell technology.Having the beneficial effects that (1) promotes T cell propagation, (2) can promote that T is thin Born of the same parents are specific binding with TSA, and (3), in tumor-bearing mice body, can suppress the growth of tumor.Make For the target molecules of cell therapy, it is applied to the cell therapys such as CAR-T.

Detailed description of the invention

Polypeptide is by Shanghai raw work gill synthesis.

Embodiment 1

Employment diffusivity large B cell lymphoid tumor Transplanted tumor model detection B/T lymphocyte weakens factor immunogen polypeptide Internal vigor.

6-8w SCID mice in age, mice is randomly divided into 4 groups, male and female half and half, often group 10.(1) blank Group；(2) polypeptide low dose group；(3) dosage group in polypeptide；(4) polypeptide high dose group.Set up people's diffusivity Large B cell lymphoid tumor (DLBCL) Transplanted tumor model, the 3rd, 5,7 after inoculation DLBCL cell My god, carry out immunity respectively.Scheme is: blank group adds the solvent of same volume, and experimental group polypeptide sets 3 0.5,1.0,2.0mg/Kg dosage:, multi-point injection around tumor.After 21 days, observe mouse survival quantity, Calculate survival rate.Result shows, polypeptide can protect white mice effectively, dosage 0.5,1.0,2.0mg/Kg Time can improve the survival rate of tumor-bearing mice, survival rate is respectively 41.27,65.12,78.31%.

Embodiment 2

Employment small cell lung cancer tumor model inspection B/T lymphocyte weakens the internal vigor of factor immunogen polypeptide.

6-8w C57BL/6 in age nude mice, mice is randomly divided into 4 groups, male and female half and half, often group 10.(1) Blank group；(2) polypeptide low dose group；(3) dosage group in polypeptide；(4) polypeptide high dose group.Set up people little Cell lung cancer tumors model, the 3rd, 5,7 days after inoculated tumour cell, carry out immunity respectively.Scheme is: 1.0,2.0,4.0mg/Kg blank group adds the solvent of same volume, and experimental group polypeptide sets 3 dosage:, Multi-point injection around tumor.After 21 days, observe mouse survival quantity, calculate survival rate.Result shows, Polypeptide can protect white mice effectively, dosage 1.0,2.0,4.0mg/Kg time can improve tumor-bearing mice Survival rate, survival rate is respectively 36.89, and 57.12,69.21%.

Embodiment 3

The proliferation function of T lymphocyte: aseptic take mouse spleen, 1640 culture medium clean 3 times, and 5ml injects Device core grinds, and 200 eye mesh screens filter, and make single cell suspension, and centrifugal (1000r/min, 5min) abandons Clearly, Tris-NH₄CL cracks erythrocyte, and ice-water bath stands 3-5min, centrifugal (1000r/min, 5min), abandons Supernatant, with aseptic cold PBS washed cell twice.The RPMI 1640 being eventually adding 10% calf serum cultivates Liquid (5ml) suspension cell, cell counting, adjusting cell concentration is 5 × 10⁶Individual/ml, in 96 well culture plates Middle cultivation.

Experiment set blank group, model group (concanavalin A, Con A, sigma company buy), each dosage of polypeptide (5.0, 10.0,20.0mg/ml) group.After each group is separately added into spleen lymphocyte suspension 100 μ l/ hole, blank Group adds RPMI-1640 100 μ l, and model group adds ConA (final concentration of 5 μ g/ml), each dosage of polypeptide Group adds ConA (final concentration of 5 μ g/ml) on the basis of the polypeptide adding variable concentrations.37 DEG C of cell culture incubators Static gas wave refrigerator 48h, cultivates every hole after terminating and adds 20 μ l MTT, continue to cultivate 4h, finally discard every hole institute Solution, every hole is had to add 100 μ lDMSO, concussion, detect OD value at 570nm by microplate reader, every hole sets 5 parallel.

Table 1 polypeptide proliferation function to T lymphocyte

* P < 0.05, * * P < 0.01 is compared with model group.

Experimental result is shown in Table 1, compares with model group, and many Toplink promote the propagation of mouse spleen lymphocyte, Dosage be 5～20mg/ml scope present good dose-dependence.

Embodiment 4

B/T lymphocyte weakens the T cell binding tests of factor immunogen polypeptide: use rosette Evaluate the binding ability of T cell and people's diffusivity large B cell lymphoid tumor cell strain (SUDHL-4).Aseptic Taking Thymus of Guinea Pigs, 1640 culture medium are cleaned 3 times, and 5ml piston grinds, and 200 eye mesh screens filter, and make Single cell suspension, 10 minutes, abandons supernatant by centrifugal 1000 revs/min, adjusts cell concentration for 3 with Hank ' s liquid ×10⁶/ml.Cultivate in 96 well culture plates.

Experiment sets blank group, each dosage of polypeptide (5,10,20mg/ml) group.Each group is separately added into thymus Behind lymphocyte suspension 100 μ l/ hole, blank group adds RPMI-1640 500 μ l, and polypeptide each dosage group is adding Enter the polypeptide of variable concentrations.37 DEG C of cell culture incubators, cultivate 48h, cultivate and add 100 μ l/ in every hole after terminating (cell concentration is 1 × 10 to people from hole diffusivity large B cell lymphoid tumor cell SUDHL-4⁶/ ml, containing 10% tire cattle Serum), mixing, 500 revs/min, centrifugal 5 minutes.Abandon supernatant, add a small amount of glutaraldehyde, rock gently, make Cell suspension, adds violet stain, and 400 power microscopes are observed.The T lymph of all more than 3 tumor cells of combination is thin Born of the same parents are garland positive cell.100 T cell that count are formed the lymphocyte percentage of garland, is T cell Combination rate with SUDHL-4 cell.Every hole set 5 parallel.

Experimental result is shown in Table 2, compares with blank group, and B/T lymphocyte weakens factor immunogen polypeptide and can show Work property increases the combination rate (P < 0.01) of T cell and SUDHL-4 cell, the combination rate of low middle high dose group It is respectively 67.33,81.33 and 86.82%.Good dosage is presented in the scope that dosage is 5～20mg/ml Dependence.

The impact that T cell is combined by table 2 polypeptide

* P < 0.05, * * P < 0.01 is compared with blank group.

SEQUENCE LISTING

<110> Pu Luoda bio tech ltd, Suzhou

<120> B/T lymphocyte weakens factor immunogen polypeptide and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 31

<212> PRT

<213> Artificial sequence

<400> 1

Arg Arg Gly Asp Pro Trp Leu Leu Tyr Ser Leu Leu Pro Ser Ser Leu

1 5 10 15

Asp Phe Thr Thr Cys Phe Cys Leu Phe Cys Cys Leu Phe Phe Leu

20 25 30

Claims

1. a B/T lymphocyte weakens factor immunogen polypeptide, it is characterised in that: described peptide sequence is SEQ ID NO:1.

Polypeptide the most according to claim 1, it is characterised in that: the application in preparation is used for tumor.

Polypeptide the most according to claim 2, it is characterised in that: in preparation for tumor vaccine cells, strengthen the application in T cell immunne response medicine.

Polypeptide the most according to claim 2, it is characterised in that: the application of CAR-T cellular immunotherapy it is used in preparation.