CN108853144A

CN108853144A - The combination of toll-like receptor agonist and immune effector cell

Info

Publication number: CN108853144A
Application number: CN201810469904.7A
Authority: CN
Inventors: 李宗海; 狄升蒙
Original assignee: Keji Biomedical (shanghai) Co Ltd; Shanghai Cancer Institute
Current assignee: Clegg Medical Co ltd
Priority date: 2017-05-16
Filing date: 2018-05-16
Publication date: 2018-11-23
Also published as: WO2018210279A1

Abstract

Combine the present invention provides toll-like receptor agonist and immune effector cell and is preparing the purposes in anti-tumor drug or antipathogen drug or the low drug of anti-immunity, the effect of immune effector cell can be enhanced in toll-like receptor agonist, and the present invention also provides the compositions of toll-like receptor agonist and immune effector cell.

Description

The combination of toll-like receptor agonist and immune effector cell

This application claims the CN201710344558.5 (applyings date：2017.5.16) and CN201710561825.4 (applies Day：2017.7.11) the priority of two patents, is fully incorporated herein by way of reference.

Technical field

The invention belongs to immunotherapy fields；It is related to the immunological effect with targets identification tumour antigen or pathogen antigen The use in conjunction of cell and toll-like receptor agonist.

Background technique

In recent years, genetically engineered immunocyte is increasingly used in therapeutic field of tumor or infectious diseases Therapy field, the NK cell (CAR-NK that the T cell (CAR-T cell) modified such as Chimeric antigen receptor, Chimeric antigen receptor are modified Cell), the T cell (TCR-T) etc. of TCR modification.

But due to the complexity of tumor microenvironment, in oncotherapy especially treatment of solid tumors, immune effector cell exists Antitumor action generally can not be preferably played in vivo.

Summary of the invention

Mesh of the present invention be to provide a kind of toll-like receptor agonist and immune effector cell use in conjunction in tumour, infect Purposes in the treatment of disease and other immune related diseases.Toll-like receptor agonist can increase the function of immune effector cell Effect.

In the first aspect of the present invention, the group comprising immune effector cell and the immune effector cell synergist is provided Object is closed, the immune effector cell has the receptor of targets identification tumour antigen or pathogen antigen, the immune effector cell Synergist is toll-like receptor agonist.

Specifically, the present invention provides the composition comprising immune effector cell, the immune effector cell has targeting Identify the receptor of tumour antigen or pathogen antigen, the immune effector cell with have and tumour antigen or pathogen antigen The relevant disease of expression subject treatment in the agent of the effect of increasing the immune effector cell be applied in combination, In：

(i) receptor has extracellular region, transmembrane region and intracellular region, wherein extracellular region has antigen-binding domains, institute It states antigen-binding domains and is bound to tumour antigen relevant to the disease or pathogen antigen；

(ii) agent for increasing immune effector cell effect is toll-like receptor agonist.

In preferred embodiments, the intracellular region of the receptor has the structural domain for participating in causing activated immune cell.

In preferred embodiments, the toll-like receptor agonist is Toll-like receptor 3 (TLR3) agonist.

In preferred embodiments, the TLR3 agonist is selected from natural or synthetic double stranded RNA.

In a preferred example, the TLR3 agonist is poly I:poly C (poly (I:C)).

In a preferred example, the TLR3 agonist is the double stranded RNA poly (I of mispairing:C11-14U), example Property, such as poly (I:C12U),poly(I:C13U).

In preferred embodiments, the identification tumour antigen or pathogen antigen receptor include Chimeric antigen receptor (CAR), T cell (antigen) receptor (TCR), T cell fusion protein (TFP) or T cell antigen coupler (TAC).

In a preferred example, the Chimeric antigen receptor includes：

(i) antibody, the transmembrane region of CD28, the costimulatory signal structural domain of CD28 and the CD3 ζ of the antigen are specifically bound Endochylema signal transduction structural domain；Or

(ii) specifically bind the antibody of the antigen, the transmembrane region of CD28, the costimulatory signal structural domain of CD137 and CD3 ζ endochylema signal transduction structural domain；Or

(iii) specifically bind the antibody of the antigen, the transmembrane region of CD28, CD28 costimulatory signal structural domain, The costimulatory signal structural domain and CD3 ζ endochylema signal transduction structural domain of CD137.

In preferred embodiments, the TFP includes:(a) TCR subunit, the TCR subunit include the part TCR born of the same parents Extracellular portion, transmembrane domain and TCR intracellular domain, the intracellular domain include stimulus signal conducting structure domain；(b) Antigen-binding domains, the TCR subunit and the antigen-binding domains effectively connect, extracellular, the cross-film of the TCR subunit CD3 ε or CD3 γ are derived from intracellular signal structural domain, the TFP is integrated into the TCR expressed in T cell.

In preferred embodiments, the TAC includes：(a) extracellular domain：The extracellular domain includes having Antigen-binding domains and the single-chain antibody in conjunction with CD3；(b) transmembrane region；(c) intracellular domain, the intracellular domain connect Meet protein kinase LCK.

In preferred embodiments, the immune effector cell is selected from T cell, natural kill (NK) cell, nature Killer T (NKT) cell, mast cell or bone marrow derived phagocyte.

In a preferred example, the immunocyte is T cell, and the Intracellular domain of the receptor can cause the work of T cell Change.

In another preferred example, the immune effector cell also expresses other extrinsic proteins, it is preferred that the external source Property albumen is selected from the group：FLT3L, interferon, interleukin, interleukin-2-receptor, PD-1 inhibitor (antibody of such as PD-1), PD-L1 Inhibitor (antibody of such as PD-L1, soluble PD-1 or the albumen containing soluble PD-1), or combinations thereof.

In another preferred example, the composition further includes MDSC inhibitor, and/or immunologic test point inhibitor.

In another preferred example, the MDSC inhibitor is selected from the group：Anti- Gr1 antibody, Cyclooxygenase-2 Inhibitor, forefront Parathyrine class stem cell factor inhibitor, macrophage colony stimulating factor inhibitor, G-GSF inhibit Agent, vascular endothelial growth factor inhibitor, or combinations thereof.

In another preferred example, the immunologic test point inhibitor is selected from the group：PD-1 inhibitor (antibody of such as PD-1), PD-L1 inhibitor (antibody of such as PD-L1), CTLA-4 inhibitor (such as CTLA-4 antibody), or combinations thereof.

In another preferred example, the immune effector cell is the T cell that TCR is knocked out or PD-1 is knocked out.

In another preferred example, the expression of the tcr gene or PD-1 gene in the immune effector cell is to be silenced 's.

In another preferred example, described " expression of gene is silenced " refers to that being silenced gene does not express or low expression.

In another preferred example, described " low expression " refers to that the immune effector cell is silenced gene expression amount G1 and normal The ratio of T cell corresponding gene expression quantity G0, i.e. G1/G0≤0.5, preferably G1/G0≤0.3, more preferably≤0.2, more preferably ≤ 0.1, it is most preferably 0.

In the second aspect of the present invention, the combination for additionally providing immune effector cell and immune effector cell synergist is being made For the purposes in the low drug of anti-tumor drug or antipathogen drug or anti-immunity power, the immune effector cell has target To identification tumour antigen or the receptor of pathogen antigen, the immune effector cell synergist is toll-like receptor agonist.

Specifically, the combination that the present invention provides toll-like receptor agonist and immune effector cell is preparing anti-tumor drug Or the purposes in antipathogen drug or the low drug of anti-immunity power, the immune effector cell are anti-with targets identification tumour Former or pathogen antigen receptor, this receptor have extracellular region, transmembrane region and intracellular region, and the extracellular region has in conjunction with described Tumour antigen or pathogen antigen antigen-binding domains.

In preferred embodiments, the toll-like receptor agonist is Toll-like receptor 3 (TLR3) agonist, described TLR3 agonist preferably be selected from natural or synthetic double stranded RNA.

In a preferred example, the TLR3 agonist is poly I:poly C (poly (I:C)).

In a preferred example, the TLR3 agonist is mispairing double stranded RNA, illustratively, such as poly (I: C12U)、poly(I:C13U)。

In a preferred example, the amount of the mispairing double stranded RNA be enough with Toll-like receptor 3 (TLR3) combine and Reduce or eliminate the proliferation of the tumour or other transformed cells in the study subject.

In preferred embodiments, the toll-like receptor agonist there is collaboration to make the immune effector cell With.

In preferred embodiments, the administration mode of the toll-like receptor agonist and the immune effector cell is Parenteral administration, preferably intravenous injection and intratumor injection；To which the toll-like receptor agonist and the immunological effect are thin Born of the same parents can be prepared into parenteral administration, preferably be injected intravenously the drug with intratumor injection administration.

In preferred embodiments, the administration mode of the toll-like receptor agonist and the immune effector cell can With identical, can also be different.

In preferred embodiments, medication is to give the toll-like receptor agonist and the immune effect jointly Cell is answered, or first gives the immune effector cell, then gives the toll-like receptor agonist；Or it first gives described Toll-like receptor agonist, then give the immune effector cell.

In a preferred example, the medication is first to give the immune effector cell, then give the Toll-like Receptor stimulating agent.

In preferred embodiments, the tumour antigen be selected from EGFRvIII, GPC3, CLD18A2, CD19, CD20, CD22、CD30、WT1、CLDN6、MUC1、BCMA、IL-11Ra、IL-13Ra、FAP。

In preferred embodiments, the toll-like receptor agonist administration amount can be 1mg/kg~4mg/kg, excellent 2mg/kg~3mg/kg is selected, 2.08mg/kg-2.77mg/kg is more preferably selected from.

In preferred embodiments, the administration number of times of the toll-like receptor agonist is 2~4 times, preferably 2-3 times.

In preferred embodiments, the effect epidemic disease cell of exempting from is selected from T cell, natural kill (NK) cell, nature Killer T (NKT) cell, mast cell or bone marrow derived phagocyte, preferably T cell.

In a preferred example, the immune effector cell is T cell, and the Intracellular domain of the receptor can cause T cell Activation.

In preferred embodiments, it is thin to be selected from the T cell of expression CAR, the T of expression TCR for the immune effector cell Born of the same parents, the NK cell for expressing CAR, the T cell for expressing TFP, the NK cell for expressing TFP, the T cell for expressing TAC, the NK for expressing TAC Cell.

In a preferred example, the Chimeric antigen receptor (CAR) includes：

In preferred embodiments, the TFP includes：(a) TCR subunit, the TCR subunit include the part TCR born of the same parents Extracellular portion, transmembrane domain and TCR intracellular domain, the intracellular domain include stimulus signal conducting structure domain；(b) Antigen-binding domains, the TCR subunit and the antigen-binding domains effectively connect, extracellular, the cross-film of the TCR subunit CD3 ε or CD3 γ are derived from intracellular signal structural domain, the TFP is integrated into the TCR expressed in T cell.

In preferred embodiments, the immune effector cell, which is also expressed, other albumen, it is preferred that as PD1, Antibody, I type interferon of PDL1 etc..

In another preferred example, the drug further includes MDSC inhibitor or immunologic test point inhibitor.

In the third aspect of the present invention, immune effector cell, immune effector cell synergist and lymphocyte are additionally provided The combination of scavenger is preparing the purposes in antitumor or antipathogen or the low drug of anti-immunity power.

In a preferred embodiment, immune effector cell and immune effector cell synergist are being given to this needs Before object, lymphocyte scavenger is first given.

In a preferred embodiment, the immune effector cell synergist is toll-like receptor agonist.

Specifically, the present invention also provides immune effector cell, toll-like receptor agonist and lymphocyte scavengers It combines and is preparing the purposes in anti-tumor drug or antipathogen drug or the low drug of anti-immunity power, the immunological effect is thin Born of the same parents have the receptor of targets identification tumour antigen or pathogen antigen, and this receptor has extracellular region, transmembrane region and intracellular region, described Extracellular region have in conjunction with the tumour antigen or pathogen antigen antigen-binding domains.

In a preferred embodiment, it before giving toll-like receptor agonist and immune effector cell, first gives and drenches Bar cell clearance agent.

In a preferred embodiment, the lymphocyte scavenger includes the drug or progress spoke for removing lymphocyte Penetrate processing.In a preferred example, the drug for removing lymphocyte includes but is not limited to fludarabine and/or cyclophosphamide.

In a preferred embodiment, toll-like receptor agonist and immune effector cell are given to the object of this needs Including giving or sequentially giving simultaneously；For example, first give toll-like receptor agonist gives immune effector cell again, or first give It gives immune effector cell and gives toll-like receptor agonist again；It is highly preferred that first give immune effector cell gives Toll-like again Receptor stimulating agent.

In the fourth aspect of the present invention, the method for the treatment of tumour or pathogenic infection or hypoimmunity, institute are additionally provided The method of stating includes：The combination of immune effector cell and immune effector cell synergist is given to the object of this needs；Or

Give the combination of immune effector cell, immune effector cell synergist and lymphocyte scavenger to this needs Object, wherein before the object that immune effector cell and immune effector cell synergist are given to this needs, first give and drench Bar cell clearance agent.

Specifically, the present invention provides the method for the treatment of tumour or pathogenic infection or hypoimmunity, the method includes：

The combination of immune effector cell and toll-like receptor agonist is given to the object of this needs, the immunological effect Cell has the receptor of targets identification tumour antigen or pathogen antigen, and this receptor has extracellular region, transmembrane region and intracellular region, institute The extracellular region stated has the antigen-binding domains in conjunction with the tumour antigen or pathogen antigen；Or

The combination of immune effector cell, toll-like receptor agonist and lymphocyte scavenger is given to pair of this needs As.

In a preferred example, the TLR3 agonist is poly I:poly C (poly (I:C)).

Fifth aspect present invention provides a kind of composition product, which is characterized in that including：

(i) the first pharmaceutical composition, first pharmaceutical composition include at least one immune effector cell synergist, institute Stating immune effector cell synergist includes toll-like receptor agonist；And pharmaceutically acceptable carrier；

(ii) the second pharmaceutical composition, second pharmaceutical composition includes immune effector cell, and the immunological effect is thin Born of the same parents have the receptor of targets identification tumour antigen or pathogen antigen；And pharmaceutically acceptable carrier；With

(iii) third pharmaceutical composition, the third pharmaceutical composition include that MDSC inhibitor or immunologic test point inhibit Agent；And pharmaceutically acceptable carrier.

In another preferred example, first pharmaceutical composition, the second pharmaceutical composition, third pharmaceutical composition are mixing Or it is self-existent.

In another preferred example, first pharmaceutical composition, the second pharmaceutical composition and third pharmaceutical composition Total content is 70~100wt%, preferably 80~100wt%, is more preferably 90~100wt%, by the composition product Total weight.

In another preferred example, the dosage of the immune effector cell is 1 × 10⁶-1×10¹²Cell/kg, more preferably Ground, 1 × 10⁷-1×10¹⁰Cell/kg.

In another preferred example, the composition product is for treating tumour or pathogenic infection or hypoimmunity Pharmaceutical composition.

In another preferred example, the dosage form of the composition includes injection type, external drug dosage forms and peroral dosage form.

In another preferred example, the composition can be given by way of subcutaneous injection, intravenous injection, intramuscular injection Medicine.

In another preferred example, the peroral dosage form includes tablet, capsule, film and granule.

In another preferred example, the dosage form of the composition includes slow-release dosage form and non-time-release type dosage form.

Sixth aspect present invention provides a kind of purposes of composition product described in fifth aspect present invention, is used to prepare Anti-tumor drug or antipathogen drug or the low drug of anti-immunity.

In another preferred example, in the composition product, the dosage of the immune effector cell synergist be can be 1mg/kg~4mg/kg, preferably 2mg/kg~3mg/kg are more preferably selected from 2.08mg/kg-2.77mg/kg.

In another preferred example, in the composition product, the dosage of the immune effector cell is 1 × 10⁶-1× 10¹²Cell/kg, more preferably, 1 × 10⁷-1×10¹⁰Cell/kg.

In another preferred example, in the composition product, the dosage of the MDSC inhibitor is 2mg/kg-5mg/ Kg, preferably, 2mg/kg-4mg/kg.

In another preferred example, in the composition product, the administration number of times of the immune effector cell synergist is 2~ 4 times, preferably 2-3 times.

In another preferred example, in the composition product, the administration number of times of the immune effector cell is 2~4 times, excellent It selects 2-3 times.

In another preferred example, in the composition product, the administration number of times of the MDSC inhibitor is 2~4 times, excellent It selects 2-3 times.

Seventh aspect present invention provides a kind of method for improving immune effector cell vigor, which is characterized in that including step Suddenly：

(i) immune effector cell is applied to the object of needs；(ii) immune effector cell synergist；And/or (iii) is optional MDSC inhibitor or immunologic test point inhibitor, wherein the immune effector cell have targets identification tumour antigen or cause of disease The receptor of body antigen, the immune effector cell synergist are toll-like receptor agonist.

In another preferred example, the object includes people or non-human mammal.

In another preferred example, the non-human mammal includes rodent and primate, preferably mouse, big Mouse, rabbit, monkey.

In another preferred example, the component (i), component (ii), component (iii) are applied simultaneously or successively.

In another preferred example, compared with immune effector cell is administered alone, (i) immune effector cell is applied；(ii) exempt from Epidemic disease effector cell's synergist；The tumour inhibiting rate of (iii) optional MDSC inhibitor or immunologic test point inhibitor improves >= 20%, preferably, >=30%.

In another preferred example, compared with immune effector cell is administered alone, (i) immune effector cell is applied；(ii) exempt from Epidemic disease effector cell's synergist；The interferon emission levels of (iii) optional MDSC inhibitor or immunologic test point inhibitor mention High >=20%, preferably, >=30%, more preferably, >=50%.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 is recombinant retroviral vector EGFRvIII-28Z structural schematic diagram.

Fig. 2 is retroviral infection mouse T lymphocyte efficiency.

Fig. 3 is the detection of EGFRvIII-28Z CAR-T cells in vitro killing ability.

Fig. 4 A is showing EGFRvIII-28Z CAR-T cell and poly I in mouse breast cancer model:Resist in C body swollen Tumor effect；Fig. 4 B shows EGFRvIII-28Z CAR-T cell and poly I in mouse breast cancer model:Antitumor reality in C body Tumor weight after testing；Fig. 4 C shows EGFRvIII-28Z CAR-T cell and poly I in mouse breast cancer model:C The IFN γ of peripheral blood is horizontal after internal anti-tumor experiment；Fig. 4 D shows that anti-tumor experiment terminates mouse breast cancer model Spleen and tumour in CAR-T cell copy number.

Fig. 5 A is EGFRvIII-28Z CAR-T cell and poly I in mouse junction cancer model:The internal antitumor work of C With；Fig. 5 B shows EGFRvIII-28Z CAR-T cell and poly I in mouse junction cancer model:Anti-tumor experiment knot in C body Tumor weight after beam；Fig. 5 C shows EGFRvIII-28Z CAR-T cell and poly I in mouse junction cancer model:In C body The IFN γ of peripheral blood is horizontal after anti-tumor experiment；Fig. 5 D shows that anti-tumor experiment terminates the spleen of mouse junction cancer model CAR-T cell copy number in dirty and tumour.

Fig. 6 is the plasmid figure of MSCV-EGFRvIII-28Z-FLT3L.

Fig. 7 shows EGFRvIII-28Z-FLT3L CAR-T+poly I:Work of the C cell in mouse junction cancer model With.

Fig. 8 A shows that I type interferon is closed the growing state of rear intestinal cancer transplantable tumor；Fig. 8 B shows I type interferon quilt The tumor weight of intestinal cancer transplantable tumor after closing.

Fig. 9 A shows that I type interferon is closed the growing state of rear breast cancer transplantable tumor；Fig. 9 B shows I type interferon It is closed the tumor weight of rear breast cancer transplantable tumor.

Figure 10 is shown using MDSC inhibitor, CAR T cell and polyI:C is in mouse tumor model at any time to swollen Comparison diagram (Figure 10 A), tumour weight (Figure 10 B) and the tumour photo comparison that knurl product influences scheme (Figure 10 C).

Specific embodiment

In order to overcome the defects of the prior art, the present invention conducts in-depth research, provide (a) TLR agonist and (b) Chimeric antigen receptor and (c) optional MDSC inhibitor and/or immunologic test point inhibitor use in conjunction tumour, infection The treatment method of disease and other immune related diseases has significantly excellent raising immunity of organisms, kills tumour or cause of disease The ability of body.

Term

As used herein, term " agent for increasing immune effector cell effect ", " immune effector cell synergy Agent " is used interchangeably, and refers both to the drug or preparation of enhancing immune effector cell effect.

The scientific and technical terminology used herein has the same or similar meaning routinely understood with those skilled in the art.For just In understanding the present invention, some terms are defined as follows.

When being related to measurable magnitude such as dosage etc., including designated value ± 20%, or in some cases ± 10%, Or in some cases ± 5%, or in some cases ± 1%, or ± 0.1% variation in some cases, therefore in this way Variation be adapted for disclosed method.

Term, " immune effector cell (being also referred to as immunocyte herein) ", refers to participation immune response, example Such as, promote the cell of immune effector response.The example of immune effector cell includes that T cell, B cell, natural kill (NK) are thin Born of the same parents, natural killer T (NKT) cell, mast cell and bone marrow derived phagocyte.For example, T cell can be directly from periphery The T cell of blood, the hypotype for being also possible to T cell, such as the T cell of CD8+, the T cell of CD4+, T cell, the gamma/delta T cell of α/β Deng.

" Chimeric antigen receptor " used herein or " CAR " refer to that one group of polypeptide is given when it is in immune effector cell The cell provides the specificity for being directed to target cell (usually cancer cell), and there are Intracellular signals to generate.CAR is usual Including at least one extracellular antigen binding structural domain, transmembrane domain (also referred to as transmembrane region) and cytoplasm signal transduction structural domain (also referred herein as " intracellular region ") comprising from such as undefined irritation molecule and/or the function of costimulation molecule It can signal transduction structural domain.In some aspects, polypeptide group is adjacent to each other.Polypeptide group is included in that there are can make when dimerization chemoattractant molecule The dimerization Switching that polypeptide is coupled each other, for example, antigen-binding domains can be made to be coupled to intracellular signal transduction structural domain.? On one side, irritation molecule is the ζ chain in conjunction with T cell receptor complex.In one aspect, cytoplasm signal transduction structure Domain further comprises one or more functional signal conduction knots from least one such as undefined costimulation molecule Structure domain.In one aspect, costimulation molecule is selected from costimulation molecule described herein, such as 4-1BB (that is, CD137), CD27 And/or CD28.In one aspect, CAR includes chimeric fusion protein, the fusion protein include extracellular antigen binding structural domain, Transmembrane domain and include from irritation molecule functional signal conducting structure domain intracellular signal transduction structural domain.? On one side, CAR include chimeric fusion protein, the fusion protein include extracellular antigen binding structural domain, transmembrane domain and It is passed comprising the functional signal conducting structure domain from costimulation molecule and the functional signal from irritation molecule The intracellular signal transduction structural domain of transduction domain.In an aspect, CAR includes chimeric fusion protein, which includes Extracellular antigen binding structural domain, transmembrane domain and two functions comprising deriving from one or more costimulation molecules Property signal transduction.

Term, " the signal transduction structural domain " refer to the protein to work and transmitting information in the cell Functional portions, for playing effector functions via determining signal by generating second messenger or by responding such courier The activity of pathway adjusting cell.

" T cell (antigen) receptor (T the cell receptor, TCR) ", for the characteristic on all T cell surfaces Mark forms TCR-CD3 compound with non-covalent bond in conjunction with CD3.The effect of TCR is identification antigen.TCR be by two not With the heterodimer that peptide chain is constituted, it is made of two peptide chains of α, β, every peptide chain can be divided into variable region (area V), constant region (C again Area), several parts such as transmembrane region and cytoplasmic region；Its main feature is that cytoplasmic region is very short.TCR molecule contactin, Antigentic specificity is present in the area V；The area V (V α, V β) is again each, and there are three hypervariable region CDR1, CDR2, CDR3, wherein most with CDR3 variation Greatly, the antigen-binding specificity of TCR is directly determined.TCR identify MHC- antigenic peptide complexes when, CDR1, CDR2 identification and In conjunction with the side wall of MHC molecule antigen binding slot, and CDR3 is directly combined with Antigenic Peptide.TCR is divided to for two classes：TCR1 and TCR2； TCR1 is made of two chains of γ and δ, and TCR2 is made of two chains of α and β.Any T cell only expresses one of TCR2 and TCR1.

" T cell fusion protein (T CELL the FUSION PROTEIN, TFP) ", P include the various more of composition TCR Recombinant polypeptide derived from peptide, the surface antigen and ii that can i) be integrated on target cell) with complete TCR compound its He interacts at polypeptide, usually same to be located in T cell surface.TFP is by a TCR subunit and people or humanized antibody structural domain group At an antigen-binding domains composition, wherein TCR subunit include at least partly TCR extracellular domain, transmembrane domain, The stimulus structure domain of the intracellular signal structural domain of TCR intracellular domain；The TCR TCR subunit and the antibody domain effectively connect It connects, wherein the extracellular of TCR subunit, cross-film, intracellular signal structural domain derive from CD3 ε or CD3 γ, also, the TFP is integrated into T The TCR expressed on cell.

" valid link " refers to a functional cohesion between regulating and controlling sequence and heterologous nucleic acid sequence, can lead Cause the expression of heterologous nucleic acid sequence.For example, when the first nucleic acid sequence is located at second nucleotide sequence function controlling area, the first nucleic acid Sequence is operationally connect with second nucleotide sequence.For example, the transcript and expression of coded sequence is influenced if necessary to promoter, that This promoter can effectively be connect with the coded sequence.Linkable DNA sequence dna can be adjacent to each other, for example, can be with Two protein-coding regions are placed in the same reading frame.

" T cell antigen coupler (T CELL the ANTIGEN COUPLER, TAC) ", including three functional structures Domain：Cancer target structural domain, ankyrin repeat protein (the designed ankyrin repeat including single-chain antibody, design Protein, DARPin) or other targeting groups 2；For extracellular region structural domain, single-chain antibody in conjunction with CD3, so that TAC Receptor and other TCR receptors are close；The intracellular region of transmembrane region and CD4 co-receptor, wherein intracellular region connects protein kinase LCK, urges Change initial step of immunoreceptor tyrosine activating motif (ITAMs) phosphorylation of TCR compound as T cell activation.

" Toll-like receptor " (TLR), which refers to, to be bound to pathogen associated molecular pattern (PAMP) and promotes lactation The member of the receptor family of immune response in animal.Known 10 kinds of mammal TLR, such as TLR1-10.Term " toll sample Receptor stimulating agent " (TLR agonist) refers to the molecule for interacting with TLR and stimulating the receptor active.The TLR excitement of synthesis Agent is designed as that the compound of the receptor active is interacted and stimulated with TLR.The example of TLR agonist includes that TLR-7 swashs Dynamic agent, TLR-3 agonist or TLR-9 agonist.

It is described that " Toll-like receptor 3 (TLR3) is one of Toll-like receptor family member, it being capable of specific recognition virus Double-stranded RNA and the like Poly (I:C).DsRNA is with two complementary strands that can be formed during viral replication cycle RNA.In identification, TLR3 induces the activation of transcription factor such as NF- κ B and Interferon regulatory factor-3 (IRF3) to improve to it His cell issues signal to increase the generation of the I type interferon of its antiviral defense.Existing research shows that internal panimmunity is thin Born of the same parents include that TLR3 can be expressed in NK cell, macrophage, Dendritic Cells and T cell.And Poly (I:C) as TLR3's Agonist can promote the growing multiplication and antigen presentation of B cell.In addition, the agonist of TLR3 can also promote dendron Cell (DC) cell maturation, is dendritic cell maturation agent.

" poly I: C (Poly (the I:C)) " (in present specification, with Poly I:C has same Meaning, also in generation, refers to Poly (I to pIC in the accompanying drawings:C)), Chinese be poly- flesh liver, be synthesis dsRNA analog, for The up to double stranded rna molecule of the MW distribution of such as 3,600,000 dalton.Poly(I:C) as the agonist of TLR3, can promote Into the growing multiplication and antigen presentation of B cell.Also, in the case where no specific antigen stimulates, TLR3 agonist Poly(I:C B cell) can directly be induced to generate IgG1 κ antibody.Its antitumor action depends on CD8+T cell and NK is thin Born of the same parents, and the panimmunities cell such as energy coordinate induction DC cell, macrophage/neutrophil leucocyte, NK cell and T cell secretes I type Interferon collectively promotes anti-tumor effect.

Term " dendritic cells (Dendritic cell, DC) " is originated from the progenitor cells in marrow, moves as immature cell Peripheral tissues are moved on to, their endocytosis antigens and carry out complicated maturation there.Antigen is interior via many surface moleculars It gulps down, including complement receptors (for example, CD11c/CD18) and endocytic receptor (for example, DEC-205, DC-SIGN and Toll-like receptor). During antigen obtains, immature DC also receives the pathogen relevant molecule form of such as bacteria cell wall lipopolysaccharides (LPS) " danger signal ", or the inflammatory stimulus of the cell factor via such as IFN-γ.Then DC moves to secondary lymphatic organ, Maturation becomes competence APC.Receptor such as CD11c/CD18, DEC-205, DC-SIGN and Toll-like receptor are captured and are passed in Ag In playing the part of pivotal player in the process, and mainly express on DC.

Term " dendritic cell maturation agent " refers to the substance that induction immature DC cell maturation is competence APC, including choosing From the following group or combinations thereof：STING agonist；TLR agonist it is such as heat-inactivated or through formalin handle BCG vaccine (BCG), the preferably cell wall constituent of BCG, fat arabian mannan or BCG ingredient derived from BCG；From derived from Escherichia coli Lipopolysaccharides (LPS)；Picibanil (Picibanil) (OK432) or the Gram-positive or gram-negative micro-organism of inactivation； Imidazole quinoline compound, preferably imidazole quinoline -4- amine compounds, especially 4- amino -2- ethoxyl methyl-x- dimethyl -1H- Imidazoles [4,5-c] quinoline -1- ethyl alcohol or 1- (2- methyl-propyl) -1H- imidazoles [4,5-c] quinolin-4-amines, or derivatives thereof；It closes At double-strand polyribonucleotide, preferably poly I:C；Natural double-stranded RNA or RNA virus or RNA segment synthesize similar Object or synthesis comprising unmethylated CpG motif or natural nucleic acid molecules；Combination of cytokines, preferably neoplasm necrosis Factor-alpha (TNF-α), interferon gamma (IFN-γ), IL-1, IL-6, IL-12 or prostaglandin E 6, CD40L, preferably recombinate CD40L or fusion protein comprising the domain CD40L；Or the genetically engineered primary cell or cell line for being transformed into expression CD40L, or The activated T lymphocytes such as T lymphocyte of up-regulation CD40L expression in a physiologically；2-Hydroxypropyl-β- Cyclodextrin (HP- β-CD) [bibliography：Front Immunol.2016Oct20；7:435.eCollection 2016], [the bibliography J Immunol.2005Sep1 of galectin (Gal) -9；175(5):2974-81].

Term, " tumour " refer to a kind of disease characterized by the pathological proliferation of cell or tissue, and its Its hetero-organization or organ are invaded in subsequent migration.Tumour growth is usually uncontrolled and progressive, does not induce or presses down Normal cell proliferation processed.Tumour can influence various kinds of cell, tissue or organ, including but not limited to selected from bladder, bone, brain, mammary gland, Cartilage, Deiter's cells, oesophagus, fallopian tubal, gall-bladder, heart, intestines, kidney, liver, lung, lymph node, nerve fiber, ovary, pancreas Gland, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus gland, thyroid gland, tracheae, urethra, ureter, urethra, uterus, Vagina organ, or tissue or corresponding cell.Tumour includes cancer, such as sarcoma, and (the pernicious of thick liquid cell is swollen for cancer or plasmacytoma Tumor).Tumour of the present invention, it may include, but it is not limited to leukaemia (such as acute leukemia, acute lymphoblastic leukemia, urgency Property myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelo-monocytic leukemia, Acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, Polycythemia vera), lymthoma (Hodgkin's disease, non-Hodgkin lymphoma), primary macroglobulinaemia, heavy chain disease, entity Tumor such as sarcoma and cancer (such as fibrosarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, Lymphangioendothelial sarcoma, angiosarcoma, lymphangioendothelial sarcoma, synovial membrane vioma, celiothelioma, Ewing' s tumor, leiomyosarcoma, band Muscle tumor, colon cancer, cancer of pancreas, breast cancer, oophoroma, prostate cancer, squamous cell carcinoma, basal-cell carcinoma, gland cancer, sweat gland Cancer, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, cancer, bronchiolar carcinoma, cephaloma, clear-cell carcinoma, liver cancer, Nile pipe cancer, Suede cancer, seminoma, embryonal carcinoma, the nephroblastoma, cervical carcinoma, uterine cancer, carcinoma of testis, lung cancer, Small Cell Lung Cancer, bladder Cancer, epithelioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, Angioblast Tumor, acoustic neurinoma, mesoglioma, neurinoma, meningioma, melanoma, neuroblastoma, retinoblastoma), The cancer of the esophagus, gallbladder cancer, kidney, Huppert's disease.Preferably, " tumour " includes but is not limited to：Cancer of pancreas, liver cancer, Lung cancer, gastric cancer, the cancer of the esophagus, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymthoma, gallbladder cancer, kidney, Leukaemia, Huppert's disease, oophoroma, cervical carcinoma and glioma.

Term, " tumour antigen " refer to fully or with pieces (such as MHC/ peptide) in tumor cell surface On the molecule (usually protein, carbohydrate or lipid) that is expressed, and it is used for the preferential target of pharmacotoxicological effect agent To tumour cell.Tumour antigen can be the marker expressed by normal cell and cancer cell, such as lineage marker is for example in B CD19 on cell；Tumour antigen may be the cell surface molecule being overexpressed compared with normal cell in cancer cell, example Such as compared with normal cell 1 times be overexpressed, 2 times be overexpressed, 3 times or more be overexpressed；Tumour antigen may be in cancer cell The cell surface molecule inadequately synthesized, for example, compared with the molecule being expressed on normal cell containing missing, addition or The molecule of mutation；Tumour antigen can also be expressed in specific manner all or with pieces (for example, MHC/ peptide) in cancer cell Cell surface on, rather than on the surface of normal cell synthesize or express.In some embodiments, CAR of the invention Including the CAR comprising combining the antigen-binding domains (for example, antibody or antibody fragment) of MHC presentation peptide.In general, from interior The pocket of the I class molecule of peptide filling major histocompatibility complex (MHC) of source protein matter, and by CD8+T lymphocyte On T cell receptor (TCRs) identification.MHC I class complex is by whole karyocyte constitutive expressions.In tumour, virus is special Anisotropic and/or tumour-specific peptide/MHC complex represents the cell surface target spot of the unique types of immunotherapy.

Tumour antigen includes but is not limited to：Thyrotropin receptor (TSHR)；CD171；CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24)；C-type agglutinin molecule -1 (CLL-1)；Ganglioside, GD3 (aNeu5Ac (2- 8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer)；Tn antigen (Tn Ag)；CD19；CD20；CD 22；CD 30； CD 70；CD 123；CD 138；CD 33；CD 44；CD44v7/8；CD38；CD44v6；B7H3 (CD276), B7H6；KIT (CD117)；Interleukin-13 receptor subunit α (IL-13Ra)；Interleukin 11 receptor alpha (IL-11Ra)；Prostate stem cell antigen (PSCA)；Prostate-specific membrane antigen (PSMA)；Carcinomebryonic antigen (CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100； Tyrosinase；Mesothelin；EpCAM；Protease serine 21 (PRSS21)；Vascular endothelial growth factor receptor, blood vessel endothelium are raw Growth factor receptor body 2 (VEGFR2)；Louis (Y) antigen；CD24；Platelet derived growth factor receptor β (PDGFR- β)；Stage Specific embryonic antigen -4 (SSEA-4)；The relevant mucin 1 of cell surface (MUC1), MUC6；EGF-R ELISA man Race and its mutant (EGFR, EGFR2, ERBB3, ERBB4, EGFRvII)；N-CAM (NCAM)；Carbonic anhydrase IX(CAIX)；Proteasome (Prosome, Macropain) subunit, β type, 9 (LMP2)；Ephrins A receptor 2 (EphA2)； Fucosido GM1；Saliva acidic group Louis adhesion molecule (sLe)；Ganglioside GM3 (aNeu5Ac (2-3) bDGalp (1-4) bDGlcp(1-1)Cer；TGS5；High molecular weight melanoma associated antigen (HMWMAA)；Adjacent acetyl group GD2 gangliosides (OAcGD2)；Folacin receptor；Tumor vascular endothelium label 1 (TEM1/CD248)；Tumor vascular endothelium label 7 is relevant (TEM7R)；Claudin 6 (CLDN6), Claudin18.2, Claudin18.1；ASGPR1；CDH16；5T4；8H9；α v β 6 is whole Close element；B cell maturation antigen (BCMA)；CA9；κ light chain (kappa light chain)；CSPG4；EGP2, EGP40；FAP； FAR；FBP；Embryo type AchR；HLA-A1,HLA-A2；MAGE A1,MAGE3；KDR；Lambda；MCSP；NKG2D ligand； PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；Fibronectin；Tenascin；The cancer embryo variant in neoplasm necrosis area；G egg White 5 groups of coupled receptor C class, member D (GPRC5D)；X chromosome open reading frame 61 (CXORF61)；CD97；CD179a；Between become Property lymphom kinase (ALK)；Poly sialic acid；Placental-specificity 1 (PLAC1)；The hexose part of globoH glycoceramide (GloboH)；Mammary gland differentiation antigen (NY-BR-1)；uroplakin 2(UPK2)；Hepatitis A virus cell receptor 1 (HAVCR1)；Adrenocepter β 3 (ADRB3)；pannexin 3(PANX3)；G protein coupled receptor 20 (GPR20)；Lymph 6 compound of cellular antigens, locus K9 (LY6K)；Olfactory receptor 51E2 (OR51E2)；TCR γ replaces reading frame albumen (TARP)；Nephroblastoma albumen (WT1)；ETS transposition mutant gene 6 is located at chromosome 12p (ETV6-AML)；Sperm protein 17(SPA17)；X antigen family, member 1A (XAGE1)；Angiogenin combination cell surface receptor 2 (Tie2)；Melanoma cancer Testis antigen -1 (MAD-CT-1)；Melanoma cancer testis antigen -2 (MAD-CT-2)；Fos related antigen 1；P53 mutant；People end Human telomerase reverse transcriptase (hTERT)；Sarcoma translocation breakpoint；The melanoma inhibitory (ML-IAP) of Apoptosis；ERG (transmembrane protein Enzyme, (TMPRSS2) ETS of serine 2 fusion)；N-acetylglucosaminyltransferase V (NA17)；Match box protein Pax-3 (PAX3)；Androgen receptor；Cell periodic protein B 1；V-myc bird myelocytomatosis viral oncogene neuroblastoma is derivative Homologue (MYCN)；Ras homologue family member C (RhoC)；Cytochrome P450 1B1 (CYP1B1)；CCCTC combination because Sub (zinc finger protein) sample (BORIS)；The squamous cell carcinoma antigen 3 (SART3) identified by T cell；Match box protein Pax-5 (PAX5)；Proacrosin binding protein sp32 (OYTES1)；Lymphocyte-specific protein-tyrosine kinase (LCK)；A kinases Anchorin 4 (AKAP-4)；Synovial sarcoma, X breakpoint 2 (SSX2)；CD79a；CD79b；CD72；Leucocyte associated immunoglobulin Sample receptor 1 (LAIR1)；The Fc segment (FCAR) of IgA receptor；Leukocytic immunity globulin sample receptor subfamily member 2 (LILRA2)；CD300 molecule sample family member f (CD300LF)；12 member A (CLEC12A) of c-type Lectin domain family；Bone Marrow stromal cell antigen 2 (BST2)；Contain EGF egf block mucoprotein sample hormone receptor sample 2 (EMR2)；Lymphocyte antigen 75 (LY75)；Monophosphoinositideproteoglycans proteoglycans-3 (GPC3)；Fc receptor sample 5 (FCRL5)；Immunoglobulin λ sample polypeptide 1 (IGLL1). In specific embodiments, tumour antigen can be, but not limited to for EGFRvIII, GPC3, CLD18A2, CD19, CD20, CD22、CD30、WT1、CLDN6、MUC1、BCMA、IL-11Ra、IL-13Ra、FAP。

Term, " pathogen " be refer to cause the protozoan of disease, including：Virus, bacterium, fungi are posted It is infested." viral antigen " refers to expressing viral, can induce the polypeptide of immune response.

As used herein, term " antibody " refers to derived from specifically in conjunction with the immunoglobulin of antigen point The protein or polypeptide sequence of son.Antibody can be polyclonal or monoclonal, multichain or single-stranded or complete immune globulin It is white, and natural origin or recombinant sources can be derived from.Antibody can be the tetramer of immunoglobulin molecules.

Term " antibody fragment " interacts (for example, by combination, space with referring to the epitope specificity of reservation and antigen Steric hindrance, stabilisation/stabilization removal, spatial distribution) ability antibody at least part.The example of antibody fragment includes, but It is not limited to Fab, Fab', F (ab') 2, Fv segment, scFv antibody fragment, disulfide bond-connection Fvs (sdFv), is tied by VH and CH1 Structure domain composition Fd segment, linear antibodies, single domain antibody such as sdAb (VL or VH), camelid VHH structural domain, by antibody piece Section (bivalent fragment for example including two Fab segments connect in hinge area by disulfide bond) formed multi-specificity antibody with The isolated CDR or other epitope binding fragments of antibody.Antigen-binding fragment can also be impregnated in single domain antibody, maximum antibody, Mini-antibody, nano antibody, intracellular antibody, double antibody, three antibody, four antibody, v-NAR and double-scFv (see, e.g. Hollinger and Hudson, Nature Biotechnology 23:1126-1136,2005).Antigen-binding fragment can also be with The bracket based on polypeptide, such as type III fibronectin (Fn3) are grafted to (referring to U.S. Patent number:6,703,199, which depict Fibronectin polypeptide mini-antibody).

Term " scFv " refer to comprising at least one include light chain variable region antibody fragment and at least one include heavy chain Variable region antibody fragment fusion protein, wherein the light chain and heavy chain variable region are adjacent (such as connect via synthesis For example short flexible polypeptide connector of head), and can be expressed in the form of single chain polypeptide, and wherein the scFv retains its source Complete antibody specificity.Unless specified otherwise, as used herein, scFv can (example in any order Such as relative to the end N- of polypeptide and C-terminal) there is the variable region VL and VH, scFv may include VL- connector-VH or can To include VH- connector-VL.

Herein, term " binding structural domain " or " antibody molecule " refer to protein, for example, immunoglobulin chain or Its segment includes at least one immunoglobulin variable domain domain sequence.Term " binding structural domain " or " antibody molecule " include Antibody and antibody fragment.Antibody molecule can be multi-specificity antibody molecule, for example, it includes multiple immunoglobulin variable knots Structure domain sequence, wherein the first immunoglobulin variable domain domain sequence has binding specificity and multiple the to the first epitope Two immunoglobulin variable domain domain sequences have the binding specificity for the second epitope.Multi-specificity antibody molecule can also be with For bi-specific antibody molecule.Bispecific antibody has to the specificity for being no more than two antigen.Bi-specific antibody molecule It is characterized in that the first epitope is characterized in that having the first epitope with the special bi-specific antibody molecule of combination to combine First immunoglobulin variable domain domain sequence of specificity and second immune globulin to the second epitope with binding specificity White variable domain sequence.

Term " antigen " or " Ag " refer to the molecule for causing immune response.The immune response can be related to antibody and generate or have The activation of cell or both of specific immunity ability.It should be understood by those skilled in the art that include actually all proteins or Any macromolecular of peptide can serve as antigen.In addition, antigen can be from recombination or genomic DNA.Make when herein When with the term, it should be understood by those skilled in the art that including nucleotide sequence or the portion of the protein that coding causes immune response Any DNA of pyrene nucleotide sequence, therefore encode " antigen ".In addition, it should be understood by those skilled in the art that antigen is without only leading to Cross the full length nucleotide sequential coding of gene.It is readily apparent that the present invention includes but is not limited to use more than a gene Partial nucleotide sequence, and these nucleotide sequences are arranged with various combination to encode the polypeptide for causing expectation immune response. Moreover, it should be understood by those skilled in the art that antigen is not necessarily to be encoded by " gene " at all.It is readily apparent that antigen can synthesize It generates, can perhaps derive from biological sample or can be the macromolecular other than polypeptide.Such biological sample can be with Include, but are not limited to tissue sample, tumor sample, cell or liquid with other biological components.

Term " antitumaous effect " refers to the biological effect that can be showed by multiple means, including but not limited to such as tumour Volume reduces, cancer cell number is reduced, transfer quantity is reduced, life expectancy increases, cancer cell multiplication is reduced, cancer cell survival rate Reduction or the improvement of various pathophysiological conditions related with cancer disorder." antitumaous effect " can also be by peptide, polynucleotides, thin The ability that born of the same parents and the pre- anti-cancer of antibody first appear.Term " antitumaous effect " refers to the biology that can be showed by various means Effect, including but not limited to such as gross tumor volume reduce, tumour cell quantity is reduced, tumor cell proliferation reduces or tumour cell Survival rate reduces.

Such as term is it is used in the present context, " deriving from " indicates the relationship between the first and second molecules.It is usually Refer to structural similarity between the first molecule and the second molecule, and does not imply that or including to from the dimolecular firstth The process of molecule or the limitation in source.For example, in the case where deriving from the intracellular signal transduction structural domain of CD3 ζ molecule, it is intracellular It is required that signal transduction structural domain keeps enough CD3 ζ structures that it is had the function of, that is, generates signal under suitable condition Ability.It does not imply that or includes the limitation to the detailed process for generating intracellular signal transduction structural domain, for example, it is not intended to , in order to provide intracellular signal transduction structural domain, it is necessary to since CD3 ζ sequence and lack unwanted sequence, or apply prominent Become, to obtain intracellular signal transduction structural domain.

Phrase " disease relevant to the expression of tumour antigen as described herein " includes, but be not limited to such as this paper institute State the relevant disease of expression or situation relevant to the cell for expressing tumour antigen as described herein of tumour antigen, including example Such as, proliferative disease such as cancer or malignant tumour or precancerous condition, such as myelodysplasia, myelodysplastic syndrome or white Blood disease early period；Or the relevant idicatio of relevant to the cell for expressing tumour antigen as described herein non-cancer.A side In face, cancer relevant to the expression of tumour antigen as described herein is hematologic cancers.In an aspect, with it is as described herein The relevant cancer of the expression of tumour antigen is solid carcinoma.Other diseases packet relevant to the expression of tumour antigen as described herein Include, but be not limited to, for example, atypia and/or non-classical cancer, malignant tumour, precancerous condition or with tumour as described herein The relevant proliferative disease of the expression of antigen.The related idicatio packet of relevant to the expression of tumour antigen as described herein non-cancer It includes, but is not limited to, for example, autoimmune disease (for example, lupus), inflammatory conditions (allergy and asthma) and transplanting.Tumour Antigen presentation can be expressed with cell, or be expressed at any time, and the mRNA of the tumour antigen is encoded.Tumour antigen expression is thin Born of the same parents can produce tumor antigen protein (for example, wild type or mutant), and the tumor antigen protein can be in normal water Horizontal presence that is flat or reducing.Tumour antigen expression cell can generate the tumor antigen protein of detectable level in point, And then generate substantially undetectable tumor antigen protein.

Term " stimulation " refer to by irritation molecule (for example, TCR/CD3 complex or CAR) and its cognate ligand (or In the case where CAR be tumour antigen) combination, thus mediated signal transduction event (is such as but not limited to compound via TCR/CD3 The signal transduction of body or via suitable NK receptor or CAR signal transduction structural domain signal transduction) and induce for the first time answer It answers.Stimulation can mediate the expression of the change of certain molecules.

Term " irritation molecule " refers to by the molecule of the offer cytoplasm signal transduction sequence of immunocyte expression, the letter Number conduction sequence adjusts the immunocyte of at least some aspects for immunocyte signal transduction path in a manner of irritation Activation.In one aspect, signal is the first of the combination starting for the MHC molecule for having peptide for example, by TCR/CD3 complex and load Grade signal, and it leads to mediate T cell response, includes, but are not limited to proliferation, activation, differentiation etc..It is worked with stimulation mode Level-one cytoplasm signal transduction sequence (also referred to as " " one stage signal conducting structure domain ") can containing be referred to as based on it is immune by The activation motifs of body tyrosine or the signal transduction motif of ITAM.It is particularly used for the cytoplasm signal of the invention containing ITAM The example of conduction sequence includes, but are not limited to from those of following:CD3 ζ, common FcR γ (FCER1G), Fc γ RIIa, FcR β (FcEpsilon R1b), CD3 γ, CD3 δ, CD3 ε, CD79a, CD79b, DAP10 and DAP12.Of the invention In specific C AR, the intracellular signal transduction structural domain in any one or more CAR of the invention includes that Intracellular signals pass Sequence is led, such as the primary signal of CD3- ζ conducts sequence.In specific C AR of the invention, the primary signal of CD3- ζ is conducted Sequence is the equivalent ones from people or non-human type such as mouse, rodent, monkey, ape etc..Of the invention special Property CAR in, the primary signal of CD3- ζ conduction sequence be from people or non-human type for example mouse, rodent, monkey, The equivalent ones of ape etc..

Term " antigen presenting cell " or " APC " refer to be presented and major histocompatibility complex (MHC on its surface ) immune system cell of compound exotic antigen, such as auxiliary cell (for example, B- cell, dendritic cells etc.).T- cell can To use its T-cell receptors (TCR) to identify these complexs.APC process antigen and it is presented to T- cell.

Term " intracellular signal transduction structural domain " refers to the intracellular portion of molecule.Intracellular signal transduction structural domain generates rush Into the signal of the immunological effect subfunction of such as CART cell of the cell containing CAR.The immune effector in such as CART cell The example of function includes cell lysis activity and auxiliary activity, the secretion including cell factor.In one embodiment, intracellular Signal transduction structural domain may include level-one intracellular signal transduction structural domain.Illustrative level-one intracellular signal transduction structural domain packet It includes from responsible those of primary stimulus or the molecule of antigen dependent stimulation.In one embodiment, intracellular signal passes Transduction domain may include costimulation intracellular domain.Illustrative costimulation intracellular signal transduction structural domain includes deriving from It is responsible for those of the molecule of costimulatory signal or the independent stimulation of antigen.For example, level-one intracellular signal passes in the case where CART Transduction domain may include the cytoplasmic sequences of T cell receptor, and costimulation intracellular signal transduction structural domain may include and From coreceptor or the cytoplasmic sequences of costimulation molecule.

Level-one intracellular signal transduction structural domain may include activation motifs referred to as based on immunity receptor tyrosine or The signal transduction motif of ITAM.The example of level-one cytoplasm signal transduction sequence containing ITAM includes, but are not limited to derive from It is those of following:CD3 ζ, common FcR γ (FCER1G), Fc γ RIIa, FcR β (FcEpsilon R1b), CD3 γ, CD3 δ, CD3 ε, CD79a, CD79b, DAP10 and DAP12.

Term " ζ " or alternatively " ζ chain ", " CD3- ζ " or " TCR ζ " are defined as such as GenBan Acc.No.BAG36664.1 The protein of offer or the equivalent ones for coming from non-human type (such as mouse, rodent, monkey, ape etc.), and " ζ thorn Swash structural domain " or alternatively " CD3- ζ stimulus structure domain " or " TCR- ζ stimulus structure domain " is defined as the cytoplasm knot from ζ chain The amino acid residue in structure domain or its functional derivatives are enough initial signal needed for functionally transmitting T cell activation.? On one side, the cytoplasmic domain of ζ includes the residue 52 to 164 of GenBank Acc.No.BAG36664.1 or as its function The equivalent ones from non-human type (such as mouse, rodent, monkey, ape etc.) of energy property homologue.

Term " costimulation molecule " refers to the homologous binding partners in T cell, specifically matches in conjunction with costimulation Body is such as but not limited to be proliferated so that the costimulation of mediate T cell is reacted.Costimulation molecule be in addition to antigen receptor or its Cell surface molecule except ligand promotes effective immune response.Costimulation molecule includes but is not limited to MHC I class point Son, BTLA and Toll ligand receptor and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4-1BB (CD137).The further example of such costimulation molecule include CDS, ICAM-1, GITR, BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160、CD19、CD4、CD8α、 CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、 ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、 ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4 (CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、 CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and specifically combine The ligand of CD83.

Costimulation intracellular signal transduction structural domain can be the intracellular portion of costimulation molecule.Costimulation molecule It can be represented with following protein families:TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin egg White, signal transduction lymphatic anakmetomeres (SLAM protein) and NK cell receptor.The example of such molecule includes CD27, CD28,4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1 and lymphocyte The relevant antigen -1 (LFA-1) of function, CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and the ligand etc. for specifically combining CD83.

Intracellular signal transduction structural domain may include the whole intracellular portions or all natural intracellular signal transduction of molecule Structural domain or its function fragment or derivative.

Term " 4-1BB " refers to the TNFR with the amino acid sequence provided such as GenBank Acc.No.AAA62478.2 The member of superfamily, or the equivalent ones from non-human species such as mouse, rodent, monkey, ape etc.；And " 4- 1BB costimulation structural domain " is defined as the amino acid residue 214-255 of GenBank Acc.No..AAA62478.2, or comes from The equivalent ones of non-human species such as mouse, rodent, monkey, ape etc..In one aspect, " 4-1BB costimulation structure Domain " is the equivalent ones from people or from non-human species such as mouse, rodent, monkey, ape etc..

Term " coding " refers to the polynucleotides such as specific sequence of gene, cDNA or the nucleotide in mRNA in life Inherent characteristic during object as the template for synthesizing other polymer and macromolecular, the polymer and macromolecular have true Fixed nucleotide sequence (for example, rRNA, tRNA and mRNA) or the amino acid sequence and biological characteristics therefrom determined. Therefore, if the transcription and translation of mRNA corresponding with the gene generates protein in cell or other biosystems, Gene, cDNA or RNA coding protein.Its nucleotide sequence is identical as mRNA sequence and is normally provided in the volume in sequence table Code both chain and the noncoding strand of template as open gene or cDNA can be said to encode the gene or cDNA's Albumen or other products

Unless otherwise stated, " nucleotide sequence of encoding amino acid sequence " include as mutual degeneracy form and Encode all nucleotide sequences of same amino acid sequence.The nucleotide sequence of phrase coding protein or RNA may also comprise interior Containing son, the nucleotide sequence for reaching coding protein can be in some forms containing the degree of introne.

Term " effective quantity " or " therapeutically effective amount " use interchangeably herein, and refer to have as described herein Realize the amount of the compound of particular biological result, preparation, substance or composition in effect ground.

Term " expression " refers to the transcription and/or translation of the specific nucleotide sequence by promoter driving.

Term " transfer vector " refers to including isolated nucleic acid and can be used for separated delivery of nucleic acids to cell interior Composition of matter.A large amount of carriers be it is known in the art, include, but are not limited to linear polynucleotides, with ion or amphipathic The related polynucleotides of compound, plasmid and virus.Therefore, term " transfer vector " includes the plasmid independently replicated or virus. The term, which should also be as being interpreted, to be further comprised non-plasmid and nucleic acid is promoted to be transferred to non-viral compound in cell, such as Such as polylysin compounds, liposome etc..It is adjoint that the example of Baculovirus transfer vector includes, but are not limited to adenovirus vector, gland Viral vectors, retroviral vector, slow virus (lentiviral) carrier etc.

Term " expression vector " refers to the carrier comprising recombination of polynucleotide, and it includes effectively connect the nucleotide to be expressed The expression control sequence of sequence.Expression vector includes enough cis-acting elements for expression；Other members for expression Part can be by host cell offer or in vitro in expression system.Expression vector includes all that known in the art, including It is impregnated in the clay of recombination of polynucleotide, plasmid (for example, naked or be included in liposome) and viral (for example, slow virus, inverse Retroviral, adenovirus and adeno-associated virus).

In the context of the present invention, using the abbreviation of following usually existing nucleic acid bases." A " refers to that adenosine, " C " are Refer to that cytimidine, " G " refer to that guanosine, " T " refer to that thymidine and " U " refer to uridine.

" parenteral " application of the composition of term immunogenicity includes for example subcutaneous (s.c.), intravenous (i.v.), flesh In meat in (i.m.) or breastbone inner injection, tumor or infusion techniques.Term " nucleic acid " or " polynucleotides " refer to single-stranded or double-stranded shape The DNA (DNA) or ribonucleic acid (RNA) and its polymer of formula.Unless clearly limiting, otherwise the term includes The nucleic acid of known analog containing natural nucleotide, the known analog have the binding property similar with reference nucleic acid, And by with as naturally occurring ucleotides in a manner of be metabolized.Unless otherwise stated, specific nucleic acid sequence is also impliedly Variant (for example, degenerate codon substitution), allele, ortholog, SNP and complementary series including its conservative modification with And clearly specified sequence.Particularly, degenerate codon replace can by generate sequence realize, in the sequence one or Three positions of more selected (or all) codons are mixed base and/or deoxidation inosine residue replaces (Batzer etc., Nucleic Acid Res.19:5081(1991)；Ohtsuka etc., J.Biol.Chem.260:2605-2608 (1985)；With Rossolini etc., Mol.Cell.Probes 8:91-98(1994)).

Term " peptide ", " polypeptide " and " protein " is interchangeably used, and refers to the amino acid connected by covalent peptide bonds The compound of residue composition.Protein or peptide must contain at least two amino acid, and for may include protein or peptide Sequence amino acid maximum quantity do not limit.Polypeptide includes containing two or more amino acid each other by peptide linkage Any peptide or protein matter.As used herein, which refers to that (it is also commonly referred to as example short chain in this field Peptide, oligopeptides and oligomer) and longer chain (it is also commonly referred to as protein in this field, and there are multiple types)." polypeptide " Including such as biological active fragment, substantially homologous polypeptide, oligopeptides, homodimer, heterodimer, polypeptide variant, Polypeptide, derivative, analog, fusion protein of modification etc..Polypeptide includes native peptides, recombinant peptide or combinations thereof.

As used herein, term " treatment (treat, treatment, treating) " refers to due to application One or more therapies (such as one or more therapeutic agents such as of the invention CAR) slow down or improve proliferative disorders into Exhibition, severity and/or duration, or improving one or more symptoms of proliferative disorders (preferably, one or more can The symptom of discrimination).Term " treatment " is can also to refer to improve the measurable physical parameters of at least one of proliferative disorders such as Tumour growth, it is not necessary to be that patient is recognizable.Term " treatment " may also mean that for example, by stablizing recognizable symptom with object Reason mode, the progress for example, by stablizing physical parameter Inhibiting proliferation venereal disease disease in a manner of physiology or both.Term " treatment " It may also mean that reduction or stablize tumor size or cancer cell counting.

Term " signal transduction pathway ", which refers to, is transduceing signal to another part of cell from a part of cell In biochemical relationship between the multi-signal transduction molecule that works.Phrase " cell surface receptor " includes that can cross over cell membrane Receive signal and transmits the molecule and molecular complex of signal.

Term " subject " refers to the living organism including can wherein draw immune response (for example, mammal, people Class).

Term " substantially purifies " cell and refers to the cell substantially free of other cell types.What is substantially purified is thin Born of the same parents refer to its cell separated with related other cell types usual in its naturally occurring state.In some cases, The cell mass substantially purified refers to the homogenous population of cell.In other cases, which only refers under its native state The cell separated with its natural related cell.In certain aspects, the cell is in vitro culture.In other aspects, The cell is not in vitro culture.

As used herein, term " treatment " refers to treatment.Therapeutic effect is by mitigation, inhibition, alleviation Or eradicate what morbid state obtained.

As used herein, term " prevention " refers to preventative or protectiveness treatment disease or morbid state.

Term " transfection " or " conversion " or " transduction " refer to Exogenous Nucleic Acid by its transfer or are introduced into host Process in cell." transfection " or " conversion " or " transduction refers to Exogenous Nucleic Acid by its transfer or is introduced into host Process in cell." transfection " or " conversion " or " transduction " cell are to have been transfected, converted or turned with Exogenous Nucleic Acid The cell led.The cell includes primary subject cell and its offspring.

Term " specifically combining " refers to binding partners that identification and combining is present in sample, and (such as tumour is anti- It is former) antibody or ligand of protein, but the antibody or ligand will not substantially identify or in conjunction with other molecules in sample.

Range: in entire disclosure, various aspects of the invention can exist with range format.It should be appreciated that range For the sake of the description of form is only convenienct and succinct, and it should not be construed as to the unmodifiable limitation of the scope of the present invention. Therefore, the description of range should be considered particularly disclosing all possible subrange and the independent numerical value within the scope of this. For example, the description of range such as from 1 to 6 should just be considered specifically disclosing subrange such as 1 to 3,1 to 4,1 to 5,2 to 4,2 to 6,3 to 6 etc. and the independent numerical value within the scope of this, such as 1,2,2.7,3,4,5,5.3 and 6.As another reality Example, the identity of range such as 95-99% include the range with 95%, 96%, 97%, 98% or 99% identity, and Identity including subrange such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99%.Do not consider The width of range, this is applicable in.

Term, " FLT3L " can be with the FLT3L of people, the IV type transmembrane protein being made of 235 amino acid residues (NCBI Reference Sequence:NP_001191431.1), amino acid sequence such as SEQ ID NO：Shown in 8；It is also possible to With SEQ ID NO：8 at least 80% similitude polypeptides, preferably with SEQ ID NO：8 have at least 85% or at least 90% The polypeptide of similitude；" FLT3L " could also be from other kinds, including but not limited to mouse, monkey, pig.FLT3L can be It is naturally occurring, for example it can be by isolated or purified from mammal；It is also possible to manually to prepare, such as can be according to routine Genetic engineering recombinant technique produce each element of recombination or FLT3L.Preferably, the present invention can be used recombination each element or FLT3L。

Composition

The present invention provides the composition comprising immune effector cell, the composition also includes toll-like receptor agonist, The immune effector cell has the receptor of targets identification tumour antigen or pathogen antigen, and the toll-like receptor agonist exists The effect of to can increase the immune effector cell in the treatment of subject, wherein：The receptor have extracellular region, transmembrane region and Intracellular region, wherein the antigen-binding domains of tumour antigen or pathogen antigen that extracellular region has identification described, the antigen Binding structural domain is bound to tumour antigen relevant to disease or pathogen antigen.In a preferred embodiment, of the invention Composition may also include MDSC inhibitor or immunologic test point inhibitor.

" immune effector cell " can come from autogenous cell, can be from homogeneous variant cell.

The receptor of the identification tumour antigen or pathogen antigen includes but is not limited to Chimeric antigen receptor (CAR), T thin Born of the same parents (antigen) receptor (TCR), T cell fusion protein (TFP) or T cell antigen coupler (TAC).

" immune effector cell " can also express other extrinsic proteins, for example, the extrinsic protein can be with It is but not limited to FLT3L, interferon, interleukin, PD-1 or PD-L1 inhibitor.The presence of these extrinsic proteins can be enhanced Antitumor or resisting pathogenic microbes the ability of " immune effector cell ".

In a specific embodiment, the interferon includes but is not limited to I type interferon；The interleukin include but It is not limited to IL-2, IL-12, IL-21, IL18；The interleukin-2-receptor includes but is not limited to the receptor of IL-4；PD-1 or PD- The inhibitor of L1 include but is not limited to soluble PD-1 or containing soluble PD-1 fusogenic peptide (reference can be made to WO2017080377), the antibody of anti-PD-1 or anti-PD-L1.

The toll-like receptor agonist can be, but not limited to as Toll-like receptor 3 (TLR3) agonist.The TLR3 Agonist can be natural or synthetic double stranded RNA such as poly I:poly C (poly (I:C)).The TLR3 swashs Dynamic agent can also be the double stranded RNA poly (I of mispairing:C11-14U), illustratively, such as poly (I:C12U),poly(I: C13U)。

The Chimeric antigen receptor may include conventional all Chimeric antigen receptor structures, and transmembrane region usually can be optional Known albumen or protein fragments with cross-film function, intracellular region can also select conventional having to cause according to the prior art The structural domain of activated immune cell, including but not limited to CD3 ζ, FcR γ (FCER1G), Fc γ RIIa, FcR β (FcEpsilon R1b), the overall length or active tablet of CD3 γ, CD3 δ, CD3 ε, CD79a, CD79b, DAP10, DAP12, CD137, CD27, CD28 etc. Section (such as CN201580013987 description as described in Intracellular domain).For example, can also having for Chimeric antigen receptor is following several Structure：

The TFP includes：(a) TCR subunit, the TCR subunit include the part TCR extracellular domain, transmembrane domain, With TCR intracellular domain, the intracellular domain includes stimulus signal conducting structure domain；(b) antigen-binding domains, it is described TCR subunit and the antigen-binding domains effectively connect.In one embodiment, the extracellular of TCR subunit, cross-film and Intracellular signal structural domain derives from CD3 ε or CD3 γ, and the TFP is integrated into the TCR expressed in T cell.

The TAC includes：(a) extracellular domain：The extracellular domain include with antigen-binding domains and with The single-chain antibody that CD3 is combined；(b) transmembrane region；(c) intracellular domain, the intracellular domain connect protein kinase LCK.

In the present invention, it is thin to be selected from T cell, natural kill (NK) cell, natural killer T (NKT) for the immunocyte Born of the same parents, mast cell or bone marrow derived phagocyte.In a preferred example, the immunocyte is T cell, the receptor Intracellular domain can cause the activation of T cell.

The present invention also provides have the immune effector cell of Chimeric antigen receptor and pharmaceutically acceptable comprising expressing The composition of carrier or excipient, the composition also include TLR agonist and/or dendritic cell maturation promotor.

On the other hand, the present invention provides the compositions comprising immune effector cell, in addition to swashing comprising toll-like receptor Dynamic agent can also include the antibody of MDSC inhibitor, anti-PD-1 antibody or anti-PD-L1.

The present invention also provides the immune effector cells, TLR agonist and/or dendron that have Chimeric antigen receptor comprising expressing How cell maturation promotor and guidance give the kit of the specification of the cell to individual.

Medication

The present invention also provides the method for the treatment of, control or pre- preventing tumor or pathogenic infection, this method includes giving Give a effective amount of immune effector cell of subject and toll-like receptor agonist.There is the immune effector cell targeting to know The receptor of other tumour antigen or pathogen antigen, the toll-like receptor agonist can increase described in the treatment to subject The effect of immune effector cell, wherein：The receptor has extracellular region, transmembrane region and intracellular region, wherein extracellular region has identification The antigen-binding domains of the tumour antigen or pathogen antigen, the antigen-binding domains are bound to related to disease Tumour antigen or pathogen antigen.

In a specific embodiment, immune effector cell and toll-like receptor agonist can be administered simultaneously, can also be first Immune effector cell is given, then gives toll-like receptor agonist, can also first give toll-like receptor agonist, then give and exempt from Epidemic disease effector cell.

The toll-like receptor agonist can be, but not limited to as Toll-like receptor 3 (TLR3) agonist.The TLR3 Agonist can be natural or synthetic double stranded RNA such as poly I:poly C (poly (I:C)).The TLR3 swashs Dynamic agent can also be the double stranded RNA poly (I of mispairing:C_11-14U), illustratively, such as poly (I:C₁₂U)、poly(I: C₁₃U)。

The TFP includes:(a) TCR subunit, the TCR subunit include the part TCR extracellular domain, transmembrane domain, With TCR intracellular domain, the intracellular domain includes stimulus signal conducting structure domain；(b) antigen-binding domains, it is described TCR subunit and the antigen-binding domains effectively connect.In one embodiment, the extracellular of TCR subunit, cross-film and Intracellular signal structural domain derives from CD3 ε or CD3 γ, and the TFP is integrated into the TCR expressed in T cell.

In the present invention, the immunocyte includes but is not limited to T cell, natural kill (NK) cell, natural killer T (NKT) any or combinations thereof in cell, mast cell or bone marrow derived phagocyte.In a preferred example, described Immunocyte is T cell, and the Intracellular domain of the receptor can cause the activation of T cell.

On the other hand, the present invention also provides the method for the treatment of, control or pre- preventing tumor or pathogenic infection, This method includes also giving MDSC in addition to giving a effective amount of immune effector cell of subject, toll-like receptor agonist The antibody of inhibitor, anti-PD-1 antibody or anti-PD-L1.

Prepare the purposes in treatment/prevention/inhibition tumour or pathogenic infection or the drug of adjusting immune tolerance ability

The present invention also provides toll-like receptor agonist and immune effector cell use in conjunction tumour, infectious disease and other The treatment method of immune related diseases, the tumour include but is not limited to：Cancer of pancreas, liver cancer, lung cancer, gastric cancer, neck Portion's squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymthoma, gallbladder cancer, kidney, leukaemia, myeloma, oophoroma, Cervical carcinoma, oophoroma, cervical carcinoma or glioma；The pathogen includes but is not limited to：Virus, bacterium, fungi, protozoan Or helminth；Preferably, the virus includes：Cytomegalovirus, epstein-Barr virus, human immunodeficiency virus Or influenza virus.

In preferred embodiments, the toll-like receptor agonist is Toll-like receptor 3 (TLR3) agonist, described TLR3 agonist preferably be selected from natural or synthetic double stranded RNA.In a preferred example, the TLR3 agonist is poly- Inosinicacid cytidine monophosphate (poly (I:C)).The TLR3 agonist can also be mispairing double stranded RNA, illustratively, such as poly(I:C12U)、poly(I:C13U)。

Those skilled in the art can rely on professional knowledge and the prior art, according to the age of patient or object, weight, property Not, the factors such as the severity of disease to be treated determine the administration of the toll-like receptor agonist and the immune effector cell Mode and dosage.In specific embodiments, the toll-like receptor agonist and the immune effector cell give prescription Formula may be the same or different.For example, medication is to give the toll-like receptor agonist and the immune effect jointly Answer cell；Or the immune effector cell is first given, then give the toll-like receptor agonist；Or it first gives described Toll-like receptor agonist, then give the immune effector cell.In a preferred embodiment, the medication is first The immune effector cell is given, then gives the toll-like receptor agonist.

In preferred embodiments, the toll-like receptor agonist administration amount can be 1mg/kg~4mg/kg, excellent 2mg/kg~3mg/kg is selected, 2.08mg/kg-2.77mg/kg is more preferably selected from.In preferred embodiments, the Toll-like The administration number of times of receptor stimulating agent is 2~4 times, preferably 2-3 times.

In preferred embodiments, the immunocyte is selected from T cell, natural kill (NK) cell, natural killer T (NKT) cell, mast cell or bone marrow derived phagocyte, preferably T cell.In a preferred example, the immunocyte is T Cell, the Intracellular domain of the receptor can cause the activation of T cell.

In preferred embodiments, the identification tumour antigen or pathogen antigen receptor include Chimeric antigen receptor (CAR), T cell (antigen) receptor (TCR), T cell fusion protein (TFP) or T cell antigen coupler (TAC).Preferred In embodiment, the immunocyte can be the T cell, the T cell of expression TCR, the NK cell for expressing CAR of expression CAR, The T cell for expressing TFP, the NK cell for expressing TFP, the T cell for expressing TAC, the NK cell for expressing TAC.

The Chimeric antigen receptor (CAR) can be, but not limited to having structure：(i) the anti-of the antigen is specifically bound Body, the transmembrane region of CD28, CD28 costimulatory signal structural domain and CD3 ζ；Or (ii) specifically bind the antigen antibody, The transmembrane region of CD28, the costimulatory signal structural domain of CD137 and CD3 ζ；Or (iii) specifically bind the antigen antibody, The transmembrane region of CD28, the costimulatory signal structural domain of CD28, CD137 costimulatory signal structural domain and CD3 ζ.

In preferred embodiments, the TFP includes：(a) TCR subunit, the TCR subunit include the part TCR born of the same parents Extracellular portion, transmembrane domain and TCR intracellular domain, the intracellular domain include stimulus signal conducting structure domain；(b) Antigen-binding domains, the TCR subunit and the antigen-binding domains effectively connect.In a preferred example, the TCR is sub- The extracellular of base, cross-film and intracellular signal structural domain derive from CD3 ε or CD3 γ, and the TFP is integrated into the TCR expressed in T cell.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.

Illustrative antigen receptor of the invention, including CAR, and for it is engineered and will receptor import cell in Method, see, for example Chinese patent application publication number CN107058354A, CN107460201A, CN105194661A, CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、 CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication No. Disclosed in WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018018958A1 those.

The preparation of embodiment 1.CAR-T cell

The present embodiment selects the target spot of EGFRvIII effect CAR-T cell, intracorporal in mouse in order to more accurately verify Antitumous effect, therefore, signal peptide, transmembrane region, intracellular region for selecting etc. are source of mouse.The method of preparation is according to this field routine CAR-T cell preparation method operation.

1, retroviral vector construct

By mouse CD8 alpha signal peptide (SEQID NO：1), anti-EGFRvIII antibody mab (SEQID NO：2), CD8 α glue chain Area and transmembrane region (SEQID NO：3), CD28 Intracellular domain (SEQID NO：4), CD3 ζ Intracellular domain (SEQID NO：5) it is sequentially connected, EGFRvIII-28Z genetic fragment is obtained by outer-gene synthetic method, and inverse with Mlu I and the displacement of I double enzyme site of Sal IRES-GFP segment in transcription vector MSCV-IRES-GFP obtains recombinant vector MSCV-EGFRvIII-28Z.Each group It is as shown in Figure 1 at segment built-up sequence.

2, prepared by retrovirus

It operates as follows：

(1) with 4.5 × 106 density inoculation 293T cell in culture dish, CO2 overnight incubation, culture medium is addition tire The DMEM of cow's serum,；

(2) 9 μ g MSCV-EGFRvIII-28Z and 9 μ g pCL-Eco 800 μ L serum-free DMEM culture solutions are dissolved in obtain Plasmid mixed liquor；54 μ g PEI (1 μ g/ μ l) are dissolved in the serum-free DMEM culture solution of 800 μ l, PEI mixed liquor is obtained；It will Plasmid mixed liquor is added in PEI mixed liquor and is incubated at room temperature, and obtains transfection composite.

(4) transfection composite 1.6ml is added dropwise in the culture dish containing step (1) and is cultivated, collected supernatant, obtain.

3, retroviral infection mouse spleen T lymphocyte

Healthy Balb/c mouse is taken, de- neck takes out spleen after putting to death, and spleen cell is prepared into single cell suspension；Add 200ul erythrocyte cracked liquid splits red rear small using mouse CD3+T cell sorting kit (STEMCELL, #19851) sorting acquisition Mouse splenic T lymphocyte.

Mouse CD3+T lymphocyte is pressed 1 after purification:Dynabeads Mouse T-activator is added in 1 ratio CD3/CD28 is cultivated 24 hours in 1640 complete medium of RPMI.

Hole every in 48 orifice plates is added after 400 μ l retroNectin solution (5 μ g/mL) are incubated overnight and is discarded retroNectn；The mouse spleen T lymphocyte of activation for 24 hours is inoculated in plate, 1ml is added in every hole cell number 1 × 106 Retrovirus, supplementing culture medium to 2mL are cultivated and are passed on after centrifugation.

4, expression of the Chimeric antigen receptor in T lymphocyte

After infection 3 days, 4 × 105 T cell being taken, cell being resuspended, the EGFRvIII albumen of Biotin label is added to dense eventually Degree is to clean after 25ug/ml is incubated on ice；50 μ L PBS+2%FCS resuspension is added, 1 μ LPE label is added Streptavidin after being incubated for cleaning on ice, is added 400 μ L PBS+2%FCS and cell, flow cytometer detection, as a result such as Fig. 2 institute is resuspended Show, EGFRvIII-28Z CART cell infection efficiency is 76.0%.

Embodiment 2.EGFRvIII-28Z CAR T lymphocyte in vitro toxicity

By mouse colonic cell CT26 (being purchased from American Type Culture Collection (ATCC)) and mice pancreatic cancer cell E0771 (is obtained from Baylor College Medicine), and using this field conventional technology, the CT26- for being overexpressed EGFRvIII is prepared EGFRvIII and E0771-EGFRvIII.

It is measured using the cytotoxicity assay kits (Promega, Madison, USA) of 96 non-radioactive of cytoTox Vitro cytotoxicity of the EGFRvIII-28Z CAR T lymphocyte to target cell.Adjust target cell CT26, CT26- EGFRvIII, E0771, E0771-EGFRvIII concentration are 2 × 105/mL, and 50 μ L is taken to be inoculated into 96 orifice plates；By effect target than 0.3: 1,1:1 and 3:1 CAR-T cell and control T cell (50ul) are added into 96 orifice plates, and each control is arranged by kit specification Hole, each group are all provided with 5 multiple holes；It is incubated for 18 hours altogether.Time-and-motion study is carried out according to kit specification after incubation EGFRvIII-28Z CAR T lymphocyte is to the vitro cytotoxicity of target cell, as a result as shown in Figure 3.

The results show that the EGFRvIII-28Z CAR-T cell CT26 and E0771 cell negative to EGFRvIII expression without Overt toxicity, and CT26-EGFRvIII, E0771-EGFRvIII cells show of expression EGFR-vIII target antigen are gone out relatively strong Lethal effect.In effect target than 3:When 1, EGFRvIII-28Z CAR-T cell is respectively 71% to the killing of two kinds of cells With 67.8%；T cell is uninfected by then to target cell without apparent killing activity.And EGFRvIII-28Z CAR T be uninfected by There are significant differences for fragmentation effect of the T cell to EGFR-vIII target antigen positive cell.These are the result shows that we constructed EGFRvIII-28Z CAR T lymphocyte can express positive tumour cell in vitro with specific killing EGFRvIII.

Embodiment 3.EGFRvIII-28Z CAR-T combines poly I:Antitumor action of the C in mouse breast cancer model

Being inoculated with 1 × 106E0771-EGFRvIII cell, (weight 18g-24g is purchased from Shanghai in the female C57 mouse of 6 week old Ling Chang Biotechnology Co., Ltd) the 4th pair of mammary fat pad；Tumor inoculation 13 days, tumor-bearing mice is divided into 4 groups, is divided into UT Groups of cells, UT+poly I:C group, EGFRvIII-28Z CAR-T groups of cells and EGFRvIII-28Z CAR-T+poly I:C group, Every group of gross tumor volume average value is about 150mm³；After grouping, pass through tail vein infusion 5.0 × 10⁶EGFRvIII-28Z CAR-T is thin Born of the same parents' (injection day is the 0th day), while using U T cell group as control, the 3rd, 6 day intratumor injection 50ug after CAR-T cell is fed back polyI:C, UT groups of cells are the physiological saline of intratumor injection same dose.The growth of transplantable tumor is observed, as a result as shown in Figure 4 A.

The results show that feeding back although life that 5 × 106EGFRvIII-28Z is able to suppress E0771-EGFRvIII transplantable tumor It is long；And combine poly I:It is then more significant to the inhibitory effect of E0771-EGFRvIII growth of transplanted human after C, show poly I:C further enhances the anti-tumor capacity of CAR-T cell.Relative to UT group, the EGFRvIII-28Z CAR-T at the 16th day Groups of cells tumour inhibiting rate is 36.1%, and is combined poly I:C group then reaches 69.6%, and difference has conspicuousness (P<0.05), tumor suppression Rate is improved more than 30%.

Reach 2000mm to control group mice tumor size³When, i.e., experiment is put to death, after separating tumour, measures tumour weight As a result as shown in Figure 4 B compared with individually giving CAR-T, Poly is given in combination in amount:The tumor weight of IC group reduces by 77.8%

Mouse after execution takes periphery hematometry IFN γ horizontal, as a result as shown in Figure 4 C, takes spleen and tumor tissues, surveys Determine CAR-T cell copy number, as a result as shown in Figure 4 D.

Embodiment 4.EGFRvIII-28Z CAR-T combines poly I:Antitumor action of the C in mouse junction cancer model

6 week old female Balb/c mouse (being purchased from Shanghai Ling Chang Biotechnology Co., Ltd) use 3Gy gamma ray spoke After (i.e. progress lymphocyte removing), the same day inoculates 3 × 105CT26-EGFRvIII cell；It tumor inoculation 13 days, will Tumor-bearing mice is divided into 4 groups, is divided into UT groups of cells, UT+poly I:C group, EGFRvIII-28Z CAR-T groups of cells and EGFRvIII-28Z CAR-T+poly I:C group, every group of gross tumor volume average value is about 150mm3；After grouping, pass through tail vein 5.0 × 106EGFRvIII-28Z CAR-T cell is transfused, while using U T cell group as compareing, the after CAR-T cell feedback 3,6 days intratumor injection 50ug polyI:C.GFRvIII-28Z CAR-T combines poly I:C is to CT26-EGFRvIII transplantable tumor Therapeutic effect it is as shown in Figure 5.

CT26-EGFRvIII transplantable tumor can obviously be inhibited by individually feeding back 5 × 106CT26-EGFRvIII as the result is shown Growth；And combine poly I:It is then more significant to the inhibitory effect of CT26-EGFRvIII growth of transplanted human after C, show poly I:C further enhances the anti-tumor capacity of CAR-T cell.Relative to UT group, the EGFRvIII-28Z CAR-T at the 16th day Groups of cells tumour inhibiting rate is 36.1%, and is combined poly I:C group then reaches 74.3%, and difference has conspicuousness (P<0.05, P< 0.01), tumour inhibiting rate is improved more than 30%.

Reach 2000mm to control group mice tumor size³When, i.e., experiment is put to death, after separating tumour, measures tumour weight As a result as shown in Figure 5 B compared with individually giving CAR-T, Poly is given in combination in amount:The tumor weight of IC group reduces 48.5%.

Mouse after execution takes periphery hematometry IFN γ horizontal, as a result as shown in Figure 5 C, takes spleen and tumor tissues, surveys Determine CAR-T cell copy number, as a result as shown in Figure 5 D.

Embodiment 5 expresses FLT3L CAR-T cell and Poly I:The combination of C

Referring to conventional CAR-T cell preparation method, by mouse muFLT3L genetic fragment (SEQ ID No：6) and implement The gene order of the EGFRvIII-28Z constructed in example 1 is by passing through F2A (SEQ ID No：7) it connects, i.e., by F2A-FLT3L base After CD3 ζ segment in insertion EGFR-28Z gene, EGFRvIII-28Z-FLT3L retroviral vector MSCV- is constructed EGFRvIII-28Z-FLT3L, plasmid figure are shown in that expression EGFRvIII- is prepared in Fig. 6, infecting mouse splenic T lymphocyte 28Z-FLT3L CAR T cell.

Referring to the operation of embodiment 4, mouse junction cancer model is constructed, tumor-bearing mice is divided into EGFRvIII-28Z CAR-T Cell+poly I:C group and EGFRvIII-28Z-FLT3L CAR-T+poly I:C groups of cells

As a result as shown in fig. 7, in Balb/c mouse CT26-EGFRvIII Transplanted tumor model, EGFRvIII-28Z- FLT3L CAR-T+poly I:The tumor inhibitory effect of C groups of cells is substantially better than EGFRvIII-28Z CAR-T cell+poly I:C group, EGFRvIII-28Z-FLT3L CAR-T+poly I:The tumour inhibiting rate of C combination therapy group is the after CAR T cell infusion 7,47.8%, 50.1% and 54.0% is respectively reached within 10,14 days.

6 interferon inhibitor of embodiment is to CAR-T+poly I:The influence of the antitumor action of C

Referring to embodiment 3 and 4, mouse breast cancer model and model of colon cancer are constructed respectively.Using interferon receptors α chain Release of the antibody (clone MAR1-5A3 is purchased from Bioxcell) as blocking agent I type interferon.

Mouse intestinal cancer model is taken to be divided into UT group (injecting normal saline), EGFRvIII-28Z CAR-T cell+poly I:C Group and EGFRvIII-28Z CAR-T cell+poly I:C group+interferon receptors α chain sealer.After CAR-T cell is fed back 3rd, 6 day intratumor injection 50ug polyI:C is giving poly I:The 0th day and the 2nd day after C or physiological saline, intratumor injection is given Give 50ug interferon receptors α chain sealer.The growing state of tumour is measured, as a result as shown in Figure 8 A.To control group mice tumour Size reaches 2000mm³When, i.e., experiment is put to death, after separating tumour, measures tumor weight, as a result as shown in Figure 8 B.

Mouse breast cancer model is taken to be divided into UT group (injecting normal saline), EGFRvIII-28Z CAR-T cell+poly I: C group and EGFRvIII-28Z CAR-T cell+poly I:C group+interferon receptors α chain sealer.After CAR-T cell is fed back 3rd, 6 day intratumor injection 50ug polyI:C is giving poly I:The 0th day and the 2nd day after C or physiological saline, intratumor injection is given Give interferon receptors α chain sealer.The growing state of tumour is measured, as a result as shown in Figure 9 A.To control group mice tumor size Reach 2000mm³When, i.e., experiment is put to death, after separating tumour, measures tumor weight, as a result as shown in Figure 9 B.

The above results show that giving CAR-T cell and poly I after closing I type interferon:C causes anti-tumor activity to drop Low, i.e. the presence of I type interferon is beneficial to CAR-T cell and poly I:The antitumor action of C.

Embodiment 7MDSC inhibitor is to CAR-T+poly I:The influence of the antitumor action of C

Referring to embodiment 4, mouse junction cancer model is constructed.BioXell (is purchased from using MDSC scavenger anti-Gr1 antibody E, clone RB6-8C5), inhibit MDSC.

MDSC is the inhibitory cells of derived from bone marrow, positive in CD11b+Gr1+.Individually give anti-Gr1 antibody and UT Group is compared, without apparent anti-tumor activity.

Take mouse intestinal cancer model be divided into UT group (injecting normal saline), UT+anti-Gr1 group (give anti-Gr1 antibody and Physiological saline), CAR-T+poly I:C group (gives EGFRvIII-28Z-CAR-T and poly I:C), CAR-T+anti-Gr1 group (giving EGFRvIII-28Z-CAR-T and anti-Gr1 antibody), CAR-T+poly I:C+anti-Gr1 group (is given EGFRvIII-28Z-CAR-T,poly I:C and anti-Gr1 antibody).

CAR-T cell feed back after the 2nd day, inject anti-Gr1 antibody according to 10mg/kg mouse peritoneal, next weekly 3 Secondary injection amounts to two weeks.CAR-T cell injects 50ug polyI in tumor on the 3rd, 6 day after feeding back respectively:C.

The growing state of tumour is measured, as a result as shown in Figure 10 A.When control group mice tumor size reaches 2000mm3, Execution will be tested, after separating tumour, measures tumor weight, as a result as shown in Figure 10 B.It takes pictures after separation tumour, as a result As illustrated in figure 10 c.

As illustrative, the CAR-T cell of above-described embodiment selection targeting EGFR, it should be appreciated that selection index system is in other The CAR-T cell of target spot also has the same effect, such as GPC3, CLD18A2, CD19, BCMA.Used antibody can be Mouse is anti-to be also possible to humanization, and the transmembrane domain of use, Intracellular domain can also such as be adopted according to purpose difference using different genera Employment.

As illustrative, although above-described embodiment, using CAR-T cell, which can also express it He enhances the cell factor of CAR-T cell function, and CAR-T cell, CAR and the PD1 as CAR and I type interferon co-expresses are total to table CAR-T cell reached etc..

As illustrative, although above-described embodiment using CAR-T cell, it is also possible to select other immune thin Born of the same parents can also be specifically chosen the specific subtype of immunocyte, such as gamma/delta T cell such as NK cell, NK-T cell.

As illustrative, the CAR of above-described embodiment selection source of mouse, but its signal peptide, hinge area, transmembrane region etc. can roots Other kinds are selected according to the difference of purpose.Including but not limited to signal peptide, hinge area, transmembrane domain, the intracellular region of people.Antibody can also With according to different purposes, selection is for the mouse of different target spots is anti-or the antibody of humanization or the antibody of full people.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Section's Ji biological medicine（Shanghai）Co., Ltd

Shanghai Inst. of Tumor

<120>The combination of TOLL sample receptor stimulating agent and immune effector cell

<130> P2018-0911

<150> CN 201710561825.4

<151> 2017-07-11

<150> CN 201710344558.5

<151> 2017-05-16

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 81

<212> DNA

<213>Mouse (Mus musculus)

<400> 1

atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60

atcctgggga gtggagaagc t 81

<210> 2

<211> 720

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 2

gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc 60

atcacttgcc attcaagtca ggacattaac agtaatatag ggtggttgca gcagagacca 120

gggaaatcat ttaagggcct gatctatcat ggaaccaact tggacgatga agttccatca 180

aggttcagtg gcagtggatc tggagccgat tattctctca ccatcagcag cctggaatct 240

gaagattttg cagactatta ctgtgtacag tatgctcagt ttccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa acgtggtgga ggcggttcag gcggaggtgg ctctggcggt 360

ggcggatcgg ccgatgtgca gcttcaggag tcgggaccta gcctggtgaa accttctcag 420

tctctgtccc tcacctgcac tgtcactggc tactcaatca ccagtgattt tgcctggaac 480

tggatccggc agtttccagg aaacaagctg gagtggatgg gctacataag ttatagtggt 540

aacactaggt acaacccatc tctcaaaagt cgaatctcta tcactcgaga cacatccaag 600

aaccaattct tcctgcagtt gaattctgtg actattgagg acacagccac atattactgt 660

gtaacggcgg gacgcgggtt tccttattgg ggccaaggga ctctggtcac tgtctctgca 720

<210> 3

<211> 216

<212> DNA

<213>Mouse (Mus musculus)

<400> 3

actactacca agccagtgct gcgaactccc tcacctgtgc accctaccgg gacatctcag 60

ccccagagac cagaagattg tcggccccgt ggctcagtga aggggaccgg attggacttc 120

gcctgtgata tttacatctg ggcacccttg gccggaatct gcgtggccct tctgctgtcc 180

ttgatcatca ctctcatctg ctaccacagg agccga 216

<210> 4

<211> 123

<212> DNA

<213>Mouse (Mus musculus)

<400> 4

aatagtagaa ggaacagact ccttcaaagt gactacatga acatgactcc ccggaggcct 60

gggctcactc gaaagcctta ccagccctac gcccctgcca gagactttgc agcgtaccgc 120

ccc 123

<210> 5

<211> 321

<212> DNA

<213>Mouse (Mus musculus)

<400> 5

agcaggagtg cagagactgc tgccaacctg caggacccca accagctcta caatgagctc 60

aatctagggc gaagagagga atatgacgtc ttggagaaga agcgggctcg ggatccagag 120

atgggaggca aacagcagag gaggaggaac ccccaggaag gcgtatacaa tgcactgcag 180

aaagacaaga tggcagaagc ctacagtgag atcggcacaa aaggcgagag gcggagaggc 240

aaggggcacg atggccttta ccagggtctc agcactgcca ccaaggacac ctatgatgcc 300

ctgcatatgc agaccctggc c 321

<210> 6

<211> 696

<212> DNA

<213>Mouse (Mus musculus)

<400> 6

atgacagtgc tggcgccagc ctggagccca aattcctccc tgttgctgct gttgctgctg 60

ctgagtcctt gcctgcgggg gacacctgac tgttacttca gccacagtcc catctcctcc 120

aacttcaaag tgaagtttag agagttgact gaccacctgc ttaaagatta cccagtcact 180

gtggccgtca atcttcagga cgagaagcac tgcaaggcct tgtggagcct cttcctagcc 240

cagcgctgga tagagcaact gaagactgtg gcagggtcta agatgcaaac gcttctggag 300

gacgtcaaca ccgagataca ttttgtcacc tcatgtacct tccagcccct accagaatgt 360

ctgcgattcg tccagaccaa catctcccac ctcctgaagg acacctgcac acagctgctt 420

gctctgaagc cctgtatcgg gaaggcctgc cagaatttct ctcggtgcct ggaggtgcag 480

tgccagccgg actcctccac cctgctgccc ccaaggagtc ccatagccct agaagccacg 540

gagctcccag agcctcggcc caggcagctg ttgctcctgc tgctgctgct gctgcctctc 600

acactggtgc tgctggcagc cgcctggggc cttcgctggc aaagggcaag aaggaggggg 660

gagctccacc ctggggtgcc cctcccctcc catccc 696

<210> 7

<211> 66

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 7

gtgaaacaga ctttgaattt tgaccttctg aagttggcag gagacgttga gtccaaccct 60

gggccc 66

<210> 8

<211> 235

<212> PRT

<213>Homo sapiens (Homo sapiens)

<400> 8

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe

20 25 30

Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu

35 40 45

Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu

50 55 60

Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln

65 70 75 80

Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly

85 90 95

Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala

100 105 110

Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser

115 120 125

Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp

130 135 140

Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro

145 150 155 160

Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala

165 170 175

Thr Ala Pro Thr Ala Pro Gln Pro Pro Leu Leu Leu Leu Leu Leu Leu

180 185 190

Pro Val Gly Leu Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp Gln

195 200 205

Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro Val

210 215 220

Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His

225 230 235

Claims

1. including the composition of immune effector cell, the immune effector cell has targets identification tumour antigen or pathogen anti- Former receptor, the immune effector cell with the tested of the relevant disease with the expression of tumour antigen or pathogen antigen The agent for the effect of increasing the immune effector cell in the treatment of person is applied in combination, wherein：

(i) receptor has extracellular region, transmembrane region and intracellular region, wherein tumour antigen that extracellular region has identification described or The antigen-binding domains of pathogen antigen,

(ii) agent for increasing immunocyte effect is toll-like receptor agonist.

2. composition as described in claim 1, the toll-like receptor agonist is Toll-like receptor 3 (TLR3) agonist.

3. composition as claimed in claim 2, the TLR3 agonist is selected from natural or synthetic double stranded RNA, excellent Select poly I:poly C (poly (I:C)).

4. the receptor of composition a method according to any one of claims 1-3, the identification tumour antigen or pathogen antigen includes Chimeric antigen receptor (CAR), T cell (antigen) receptor (TCR), T cell fusion protein (TFP) or T cell antigen coupler (TAC)。

5. composition as claimed in claim 4, which is characterized in that the Chimeric antigen receptor includes：

(i) antibody, the transmembrane region of CD28, the costimulatory signal structural domain of CD28 and the CD3 ζ endochylema of the antigen are specifically bound Signal transduction structural domain；Or

(ii) antibody, the transmembrane region of CD28, the costimulatory signal structural domain of CD137 and the CD3 ζ born of the same parents of the antigen are specifically bound Starch signal transduction structural domain；Or

(iii) antibody of the antigen, the transmembrane region of CD28, the costimulatory signal structural domain of CD28, CD137 are specifically bound Costimulatory signal structural domain and CD3 ζ endochylema signal transduction structural domain.

6. composition as claimed in claim 4, the TFP include:

(a) TCR subunit, the TCR subunit include：

The part TCR extracellular domain, transmembrane domain and TCR intracellular domain, the intracellular domain include that stimulus signal passes Transduction domain；

(b) antigen-binding domains, the TCR subunit and the antigen-binding domains effectively connect,

Wherein, the extracellular of TCR subunit, cross-film and intracellular signal structural domain derive from CD3 ε or CD3 γ, the TFP integration The TCR expressed on into T cell.

7. composition as claimed in claim 4, the TAC include：

(a) extracellular domain：The extracellular domain includes having antigen-binding domains and the single-chain antibody in conjunction with CD3；

(b) transmembrane region；

(c) intracellular domain, the intracellular domain connect protein kinase LCK.

8. composition as claimed in claim 1, which is characterized in that the immunocyte is selected from T cell, kills naturally Hurt (NK) cell, natural killer T (NKT) cell, mast cell or bone marrow derived phagocyte.

The combination of 9.TOLL sample receptor stimulating agent and immune effector cell is preparing anti-tumor drug or antipathogen drug or is resisting Purposes in the drug of immunocompromised, the immune effector cell have targets identification tumour antigen or pathogen antigen by Body, this receptor have extracellular region, transmembrane region and intracellular region, and the extracellular region has in conjunction with the tumour antigen or pathogen The antigen-binding domains of antigen；

The preferred Chimeric antigen receptor of the receptor (CAR), T cell (antigen) receptor (TCR), T cell fusion protein (TFP), Or T cell antigen coupler (TAC).

10. purposes as claimed in claim 9, which is characterized in that the toll-like receptor agonist and the immunological effect are thin Born of the same parents are prepared into parenteral administration, are preferably injected intravenously the drug with intratumor injection administration.

11. purposes as claimed in claim 9, which is characterized in that the immunocyte is selected from T cell, natural kill (NK) Cell, natural killer T (NKT) cell, mast cell or bone marrow derived phagocyte.

12. the purposes as described in claim 9-11 is any, which is characterized in that the T of the immunocyte selection expression CAR is thin Born of the same parents, the NK cell for expressing CAR or the T cell for expressing TCR.

13. the purposes as described in claim 9-11 is any, the toll-like receptor agonist swashs for Toll-like receptor 3 (TLR3) Dynamic agent, it is more preferably poly I:poly C that the TLR3 agonist, which preferably is selected from natural or synthetic double stranded RNA, (poly(I:C))。

14. composition as described in claim 1, which is characterized in that it is exogenous that the immune effector cell also expresses other Albumen, it is preferred that the extrinsic protein is selected from the group：FLT3L, interferon, interleukin, interleukin-2-receptor, PD-1 inhibitor, PD-L1 inhibitor, or combinations thereof.

15. composition as described in claim 1, which is characterized in that the composition further includes MDSC inhibitor, and/or exempts from Epidemic disease checkpoint inhibitor.

16. composition as claimed in claim 15, which is characterized in that the MDSC inhibitor is selected from the group：Anti- Gr1 antibody, Cyclooxygenase-2 Inhibitor, prostanoid stem cell factor inhibitor, macrophage colony stimulating factor inhibitor, grain monokaryon Colony-stimulating factor inhibitor, vascular endothelial growth factor inhibitor, or combinations thereof.

17. composition as claimed in claim 15, which is characterized in that the immunologic test point inhibitor is selected from the group：PD-1 Inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, or combinations thereof.

18. a kind of composition product, which is characterized in that including：

(i) the first pharmaceutical composition, first pharmaceutical composition includes at least one immune effector cell synergist, described to exempt from Epidemic disease effector cell's synergist includes toll-like receptor agonist；And pharmaceutically acceptable carrier；

(ii) the second pharmaceutical composition, second pharmaceutical composition includes immune effector cell, the immune effector cell tool There is the receptor of targets identification tumour antigen or pathogen antigen；And pharmaceutically acceptable carrier；With

(iii) third pharmaceutical composition, the third pharmaceutical composition include MDSC inhibitor or immunologic test point inhibitor；With Pharmaceutically acceptable carrier.

19. a kind of purposes of composition product described in claim 18, which is characterized in that be used to prepare anti-tumor drug or anti- Pathogen drug or the low drug of anti-immunity.

20. a kind of method for improving immune effector cell vigor, which is characterized in that including step：

(i) immune effector cell is applied to the object of needs；(ii) immune effector cell synergist；And/or (iii) is optional MDSC inhibitor or immunologic test point inhibitor, wherein the immune effector cell has targets identification tumour antigen or pathogen The receptor of antigen, the immune effector cell synergist are toll-like receptor agonist.

21. method as claimed in claim 20, which is characterized in that compared with immune effector cell is administered alone, application (i) is exempted from Epidemic disease effector cell；(ii) immune effector cell synergist；(iii) optional MDSC inhibitor or immunologic test point inhibitor Tumour inhibiting rate improves >=20%, preferably, >=30%.

22. method as claimed in claim 20, which is characterized in that compared with immune effector cell is administered alone, application (i) is exempted from Epidemic disease effector cell；(ii) immune effector cell synergist；(iii) optional MDSC inhibitor or immunologic test point inhibitor Interferon emission levels improve >=20%, preferably, 30%, more preferably, >=50%.