CN106047934B

CN106047934B - Construction and application of multi-coding-frame non-integrative lentiviral vector

Info

Publication number: CN106047934B
Application number: CN201610651245.XA
Authority: CN
Inventors: 李因传
Original assignee: Individual
Current assignee: Guangzhou Xiling Biotechnology Co ltd
Priority date: 2016-08-09
Filing date: 2016-08-09
Publication date: 2020-04-07
Anticipated expiration: 2036-08-09
Also published as: CN106047934A

Abstract

The invention discloses construction and application of a multi-coding-frame non-integrative lentiviral vector, and relates to the technical field of gene/cell therapy, wherein the lentiviral vector sequentially comprises a 5 '-LTR sequence, a promoter sequence, a multiple cloning site 1, one or more IRES, multiple cloning site 2 and 3' -LTR sequence from upstream to downstream; also includes a viral packaging signal sequence for enhancing nuclear penetration; the multiple cloning site 1 and the multiple cloning site 2 are used for loading target exogenous expression genes. Experiments show that the cell immunotherapy vector for solid tumors, blood tumors, autoimmune diseases, stem cell therapy, viral diseases and genetic diseases can be effectively constructed on the basis of the lentiviral vector.

Description

Construction and application of multi-coding-frame non-integrative lentiviral vector

Technical Field

The invention relates to the field of gene therapy and cell therapy, in particular to a multi-target non-integrative lentiviral vector and application thereof in constructing an immunotherapy vector aiming at various solid tumors and blood tumors, autoimmune diseases, stem cell therapy, viral diseases and genetic diseases.

Background

The virus-mediated gene transfer technology is a key tool technology for cell therapy and gene targeting operation at present, and has the advantages of high efficiency and stable integration and expression of loaded genes.

At present, vector systems such as adenovirus, adeno-associated virus, retrovirus and lentivirus are mainly developed internationally, wherein the adenovirus system can not integrate genome effectively and is only used for transient expression, the retrovirus has high infection and integration efficiency but relatively high risk, the adeno-associated virus has low immunogenicity and has the safety advantage of site-specific integration and is regarded as an ideal tool for gene therapy, but the retrovirus has the defects of relatively low infection efficiency and limited vector capacity, and the lentivirus system has strong infection capacity and large vector capacity and is spotlighted. However, similar to retroviruses, there are disadvantages of high random integration of genome, which may interfere with the expression of nearby genes and cause mutation threat to the gene inserted into the site. In order to overcome the defects of lentivirus, a safer and more effective viral vector product is developed, which is a development direction of precise treatment.

In the field, DNA sequences called scaffold/matrix attachment regions (S/MARs) are widely distributed at certain positions of a genome, play a certain role in regulating and controlling the expression of genes, and a considerable part of S/MARs can enhance the expression of the genes. It is necessary to try to construct such vectors for cellular introduction of genes. And the possibility that the enzymes involved in the genome integration of the vector-introduced gene are involved in the genome integration can be further eliminated by carrying out site mutation on the enzymes of the lentivirus which are critical to the genome integration.

Since lentiviruses generally use 5 '-LTR as the promoter sequence signal and 3' -LTR as the terminator sequence signal for the gene, the loading of multi-target genes generally requires the integration of one or more enhancement and promoter elements internally, which requires modification of the 5 '-LTR and 3' -LTR to eliminate the inhibitory effect on internal promoter elements, and in addition, the use of ribosome internal binding site sequences can reduce the number of internal promoter elements and increase the effective capacity of vector loading. In addition, the transportation of the viral cell nucleus also needs to add some special elements to increase the nuclear entry efficiency.

Current CAR-T cell technology and targeted modification of genes both rely on viral-mediated cellular introduction of genetic elements.

To inhibit CD47, a secreted single chain antibody molecule or the CD47 binding domain of a soluble SIRPa extracellular region may be used. In order to make it secreted extracellularly, a secretory signal peptide needs to be added. In addition, the CAR mainly uses a murine scFV antibody chain of carcinoembryonic antigen CEA, and is subjected to humanized modification, and the antigen is glycoprotein and widely distributed in colon cancer, pancreatic cancer, rectal cancer, gastric cancer, lung cancer, biliary tract cancer, breast cancer, bladder cancer and the like; the scFv sequences of murine antibodies directed against mucin1 and mucin5a were also humanised and adapted to recognise tumours predominantly of pancreatic, mucinous cystadenomas, intrapancreatic mucinous tumours and intraductal papillary myxomas.

The application of chimeric antigen receptor T cells (CAR-T cells) in tumor therapy has recently received a great deal of attention, and the chimeric antigen receptor T cells are expected to be a class 4 effective tool for tumor therapy. However, due to the constantly variable nature of tumors, heterogeneous populations of tumor cells appear during growth, resulting in limitations of conventional single-target CAR-T therapy. Multiple target CAR-T fits exactly the therapeutic needs, and to achieve this, multiple CAR-T mixtures or multiple CAR elements can be transferred into T cells simultaneously, designing a multiple coding frame virus, which facilitates the realization of this design concept.

In view of the above, there is a need to design a non-integrated lentiviral vector, which has both the advantages of non-integrated genome and the characteristics of multi-coding-frame expression, and multiple targeting vectors derived from the lentiviral vector can simultaneously target the immunosuppressive function of macrophages and the CAR of anti-tumor targets, so as to achieve both the immunosuppressive function and the tumor cell killing function.

Disclosure of Invention

Based on the blank and the requirement of the field, the invention integrates a plurality of factors to construct a vector which not only can be efficiently packaged, transfected and introduced into cell nucleus, but also can prevent the DNA sequence of the virus from being integrated into genome and avoid or reduce the interference and/or mutation effect on normal genes. The technical scheme provided by the invention is as follows:

a multi-reading frame genome non-integrative expressed lentiviral vector, which is characterized in that: the slow virus vector comprises a 5 '-LTR sequence, a promoter sequence, a multiple cloning site 1, one or more IRES elements, a multiple cloning site 2 and a 3' -LTR sequence from upstream to downstream;

also includes a viral packaging signal sequence for enhancing nuclear penetration;

the multiple cloning site 1 and the multiple cloning site 2 are used for loading target exogenous expression genes.

The lentiviral vectors of the invention use one or more IRES sequences for the expression of multiple independent coding frames, particularly those containing an N-terminal signal peptide.

Further preferably, the viral packaging signal sequence comprises cPPT/CTS, RRE and/or WPRE sequences, and the packaging sequence may further comprise a nucleic acid sequence for a CAR coding sequence, receptor or ligand.

Further preferably, the promoter sequence comprises one or more of EF-1, alpha-actin, beta-actin, UBC, PGK, beta-tubulin, hU6, CMV, SV 40.

Further preferably, any of the lentiviral vectors described above, wherein: also included are S/MAR sequences for enhancing adhesion in the nuclear matrix and the ability to passage with mitosis. The S/MAR sequence, as long as it functions to enhance the adhesion in the nuclear matrix and the ability to passage with cells, may be suitable for the construction of the vector of the invention.

Further preferably, the S/MAR sequence is the human IFN-beta 1S/MAR sequence, the nucleotide sequence of which is shown in Seq ID No. 19.

Further preferably, said multiple cloning site 1 comprises the nucleotide sequence of Seq ID No.21 and/or said multiple cloning site 2 comprises the nucleotide sequence of Seq ID No. 22.

Preferably, the nucleotide sequence of the above-mentioned lentiviral vector is Seq ID No. 1.

The invention also provides a HIV-1 integrase sequence coding for mutation in Gag-pol coding frame, which is characterized in that the mutation site of the sequence coding for HIV-1 integrase comprises simultaneous point mutation of D64, D116 and E152, preferably the mutation sites of D64K, D116Q and E152Q, and the nucleotide sequence of the HIV-1 integrase is shown as Seq ID No. 10. The packaging vector constructed by the sequence is used together with the non-integrative vector of the invention, can be used for preventing the nuclear virus vector sequence from integrating into a cell genome, and codon optimization has the function of improving the yield of the packaging virus.

In another aspect of the present invention, a series of cellular immunotherapy vectors useful for the treatment of diseases are constructed on the basis of the above-described lentiviral vectors: namely, by means of the application of the newly constructed vector to CAR-T technology, aiming at the immune evasion effect of M2 macrophages in solid tumors, a composite CAR-T lentiviral vector aiming at M2 macrophages is designed, the vector is mainly designed aiming at CD47 (GENBANK: NM-001777) and SIRPa (GENBANK: NM-001040022) to play the role of 'eat me' signal in the immune effect of tumor evasion, CD47 expressed on the surface of tumor cells is combined with SIRPa expressed on the surface of macrophages to activate 'eat me' signal in the macrophages, so that the phagocytosis of the macrophages is inhibited, and the vector also aims at inhibiting CD47 while constructing the tumor target of CAR-T cells. Wherein the inhibition of CD47 or SIRPa is beneficial to inhibit the tumor cells from binding to SIRPa on the surface of macrophages through CD47 on the surface of the tumor cells, and the combination of the two is an important signal mechanism for triggering the effect of eating alone. The method comprises the following specific steps:

a cell immunotherapy vector, wherein a target gene selected from one or more of the following genes is loaded at the multiple cloning site of any one of the above lentiviral vectors:

a nucleotide sequence encoding an antibody molecule or an scFV thereof;

a nucleotide sequence encoding a secreted inhibitory effector molecule;

a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) targeting element.

Further preferably, it is characterized in that,

the antibody molecule or scFV thereof refers to secretory antibody or scFV molecule of anti-CD 47, and the antibody is murine antibody, human antibody or chimeric antibody;

the secretory inhibitory effector molecule refers to the human SIRPa extracellular domain, the nucleotide sequence of which is shown in SeqID No.18, or refers to an artificial SIRPa extracellular domain obtained by modifying or restructuring the human SIRPa extracellular domain without influencing the function;

the CAR targeting element refers to anti-carcinoembryonic antigen CEA, and has a nucleotide sequence shown in Seq ID No.23, wherein a framework region in a variable region of a murine antibody of the anti-carcinoembryonic antigen CEA is subjected to partial humanized transformation and is connected with a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region, so that the CAR targeting element is obtained by performing partial humanized transformation on the framework region in the variable region of the anti-carcinoembryonic antigen CEA antibody and is connected with a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region.

The CAR targeting element refers to a nucleic acid sequence of anti-aglycosylated Mucin antibody scFV, has a nucleotide sequence shown in Seq ID No.24, and is obtained by performing partial humanized transformation on a variable region framework region of the anti-aglycosylated Mucin murine monoclonal antibody, connecting a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region, and connecting a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region in the variable region of the anti-carcinoembryonic antigen CEA antibody.

I.e., inhibition of M2-type macrophages, a secreted anti-CD 47 antibody can be cloned. Enhancing the target killing effect of the CAR-T cells on tumor cells. Antibodies against CD47, with or without S/MAR sequences, are secreted, murine or human, and also include scFV molecular sequences of chimeric anti-CD 47 antibodies, for reducing immune evasion and tolerance effects in solid tumors, increasing tumor cytotoxicity of CAR-T cells.

Aiming at the inhibition effect of M2 type macrophages, the vector can also clone a SIRPa extracellular domain for blocking CD47, the domain is added with an N-terminal signal peptide, can be expressed in CAR-T cells and secreted to the outside of the cells, and aims to relieve the inhibition effect of tumor cells on the macrophages and enhance the target killing effect of the CAR-T cells on the tumor cells. But are not limited to, a secreted extracellular domain of SIRPa, but also include modified or engineered SIRPa extracellular domains for secretory expression in T cells, with or without S/MAR sequences. Used for reducing immune evasion and tolerance effects of macrophages helping solid tumors and increasing tumor cytotoxicity effect of CAR-T cells.

The vector can clone and load a CAR targeting element, such as scFV of an antibody against carcinoembryonic antigen CEA, and a framework region in a variable region of the antibody is subjected to partial humanized transformation to connect a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region. The vector tests a CAR aiming at carcinoembryonic antigen and verifies the in vitro expression and the tumor cell killing capacity of the CAR in the vector.

Preferably, the cellular immunotherapy vector comprises the construct of any one of Seq ID No.2 to 9, which is composed of the gene of interest loaded in the multiple cloning site and the IRES sequence and/or S/MAR sequence in the lentiviral vector.

Preferably, any of the above vectors is constructed from a plasmid based on a vector containing the pBR322 origin of replication and a penicillin resistance gene.

As some embodiments of the invention, the lentiviral expression vector and vectors derived therefrom have:

an empty vector map containing a multiple cloning site and IRES sequence of Seq ID No.1

(ii) deriving a vector map comprising the CAR coding sequence, IRES, anti-CD 47 single chain full length IgG1 antibody, the nucleotide sequence of the S/MAR being Seq ID No. 3.

(iii) deriving a vector map comprising the CAR coding sequence, an IRES, a SIRPa extracellular region CD47 binding domain nucleotide sequences Seq ID No.2 and Seq ID No. 4.

(iv) deriving a vector map containing multiple cloning sites, IRES and S/MAR sequences, the nucleotide sequence being

Seq ID No. 5; on this basis, the multiple cloning site was loaded with the CAR element expression sequence that recognizes specific mucins of cancer cells, the nucleotide sequence being shown in Seq ID No. 7.

(V) deriving a vector map containing the multiple cloning site, IRES, SIRPa extracellular region CD47 binding domain, and the nucleotide sequences of the S/MARs are Seq ID No.6, Seq ID No.8 and Seq ID No. 9.

(VI) the derivative map has IRES, S/MAR, SIRPa extracellular region modification sequence for blocking CD47 for inhibiting 'eat me' signal response, and CAR element expression sequence for recognizing cancer cell specific non-glycosylated extracellular region of Mucin, wherein the CAR element expression sequence comprises a scFv sequence for recognizing Mucin, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB auxiliary activation signal region and a CD3zeta signal structure domain, and the nucleotide sequences are Seq ID No.6, Seq ID No.8 and Seq ID No. 9.

In still another aspect of the present invention, there is provided a kit for disease treatment, comprising any of the above lentiviral vectors, or any of the above cellular immunotherapeutic vectors; the disease includes solid tumors, hematologic tumors, autoimmune diseases, stem cell therapy, viral diseases, and genetic diseases.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1: a partial vector map obtained by the present invention, wherein

1a, pLV-CMV-MCS1-IRES-MCS2(7473bp) map;

1b, pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD (9375bp) map;

1c, pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47_ IgG1(10704bp) map;

1d, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD (9390bp) map;

1e, pLV-CMV-MCS-IRES-S/MAR (9659bp) map;

1f, pLV-CMV-MCS-IRES-SIRPaECD-S/MAR (10124bp) map;

1g, pLV-CMV-CAR _ T _ Muc-IRES-S/MAR (11096bp) map;

1h, pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR (11561bp) map;

1i, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD-S/MAR (11576bp) map;

FIG. 2: the in vitro secretion of anti _ CD47 antibody and the in vitro secretion expression identification result of SIRPaECD, wherein

(A) 1: supernatant extracts of infected pLV-CMV-MCS1-IRES-MCS cells;

2: cell supernatant extracts infected with pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47_ IgG1, anti-CD 47IgG1 antibody about 65 kD.

(B) The concentration of supernatant antibody infecting pLV-CMV-MCS1-IRES-MCS cells and the concentration of cells infecting pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47_ IgG1 virus reached a secretion concentration of 220ng/ml, with an error line of + SD.

(C)1，pLV-CMV-MCS1-IRES-MCS；

2，pLV-CMV-CAR_T_Muc-IRES-SIRPaECD；

3，pLV-CMV-CAR_T_CEA-IRES-SIRPaECD；

4，pLV-CMV-MCS-IRES-SIRPaECD-S/MAR；

5，pLV-CMV-CAR_T_Muc-IRES-SIRPaECD-S/MAR；

6，pLV-CMV-CAR_T_CEA-IRES-SIRPaECD-S/MAR。

The concentrated supernatant protein is dyed by an anti-C-MYC label antibody WB, the molecular weight is about 20kD, 4 expression vectors are normally expressed, the adoption and construction of elements of the vectors are reasonable, and the addition of an S/MAR element does not influence the expression of genes.

(D) In the mixed sample of the culture supernatant after 293T cells are infected with virus for 2 days and 3 days, the secretion and concentration of SIRPaECD are measured, the normal expression of several SIRPaECD positioned behind IRES elements is not affected, and the error line is + SD.

FIG. 3: experimental results for cytotoxic effects of anti-CD 47 antibody, wherein

The antibody against CD47, which was added to HUVEC cells at 1. mu.g/ml, and 20-or 50-fold human peripheral blood mononuclear cells, was tested for ADCC effect by measuring LDH in the supernatant, and the anti-CD 47IgG1 antibody showed a stronger ADCC effect than the IgG1 antibody control.

FIG. 4: (ii) results of the S/MAR non-integrative passage ability assay experiment, wherein

(A)1, control of empty vector sample digested with XhoI;

2, XhoI digested genomic DNA of infectious virus pLV-CMV-MCS1-IRES-MCS 2-S/MAR;

3, XhoI digested genomic DNA infected with pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR virus; 2 and 3, the DNA was cleaved to show that the plasmid was still stable in the genome after 50 generations and no integration occurred.

(B)1, empty vector sample control digested with EcoRI;

2 EcoRI digested pLV-CMV-MCS1-IRES-MCS2-S/MAR virus infected genomic DNA;

3 EcoRI digested genomic DNA of pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR virus; southern blot showed 2 and 3, neither of which was integrated into the genome. According to XhoI and EcoRI cut genomic DNA, the inserted S/MAR sequence can effectively inhibit the corresponding plasmid element from being inserted into the genome and can be normally passed to daughter cells along with the mitosis of the maternal cells. FIG. 5: comparative experiment of cytokine release induced by CAR _ T _ Muc and CAR _ T _ CEA T cells constructed with different elements in the Presence of target cells

The figure shows that more IFN γ is produced except in the empty vector, so plasmids with and without the S/MAR element had similar effects on cytokine production.

FIG. 6: results of in vitro expression and toxicity validation of CAR _ T _ Muc and CAR _ T _ CEA

(A) The MTT-detected CAR _ T _ Muc has cytotoxic effect on PanC1 cells, all constructed plasmids containing the CAR _ T _ Muc can generate the cytotoxic effect, and the cytotoxic effect of the plasmids added with S/MAR sequences is similar to that of the plasmids without the S/MAR sequences.

(B) MTT-detected CAR _ T _ CEA toxic Effect on HUVEC cells overexpressing CEACAM1, 2 constructed CAR _ T _ Muc-containing plasmids were all able to produce cytotoxic effects.

Detailed Description

The invention discloses a multi-coding frame vector and construction of CAR-T element by using the same, and the technical scheme obtained by modifying or properly changing and combining the method and the vector without departing from the content and the scope of the invention is within the protection scope of the invention.

Example 1 construction of vectors and sequencing identification

A basic plasmid containing a pBR322 replication origin and a penicillin resistance gene is selected as a basis, a linear basic plasmid sequence is constructed by PCR, a 5 '-LTR sequence, a virus packaging signal sequence, a Rev response element RRE sequence, a provirus integration element nuclear-entering enhancement sequence cPPT/CTS sequence and a multiple cloning site of a lentivirus are integrated into a circular plasmid sequence, and then a regulatory sequence WPRE and a 3' -LTR sequence after transcription of the lentivirus of the woodchuck hepatitis virus are cloned into the downstream of the multiple cloning site of the plasmid by PCR.

A series of therapeutic vectors with specific functions are constructed by taking the plasmid as a basic plasmid sequence.

On the basis of the above basic plasmid, the S/MAR sequence Seq ID No.19 of human IFN-beta 1 (293T cell genome was amplified by PCR to obtain a 2.2kb sequence with XbaI at the upstream and PacI at the downstream) was subjected to double digestion with XbaI and PacI, and the resulting 2.2kb fragment was inserted into the multiple cloning site of the above basic plasmid.

The primer sequence is as follows:

S/MAR-S (XbaI) 5' -ATACTCGAGAGCAAGGTCGCCA and

S/MAR-R (PacI): 5' -CGCTTAATTAATATCAAGATATTTAAAGA, sequencing to identify positive clones.

The plasmid was then further digested with XbaI, the IRES element as shown in Seq ID No.20 was PCR amplified, the fragment was digested with SpeI and XbaI, inserted into the XbaI site, and sequenced to identify forward and reverse.

Primer sequences for cloning IRES:

IRES-S(SpeI):5’-ACAACTAGTGCCCCTCTCCCT-3’；

IRES-R(XbaI):5’-CTGTCTAGAATTATCATCGTGTTTTTC-3’，

sequencing primer: 5'-CTAG T CTC GAG GGG CCC GGG TGA TC-3' are provided.

Multiple cloning site of pLV-CMV-MCS1-IRES-MCS 2:

MCS1：

MCS2：

in order to improve the packaging efficiency of the virus, the length of the sequence between the 5 '-LTR and the 3' -LTR is as short as possible within 8.5 kb.

The structure of the resulting base plasmid sequence is shown in FIG. 1a, wherein the sequence of one of the base plasmids is shown in Seq ID No.1, wherein 5' -LTR: 1-636; viral packaging signal: 686 + 823; 1304-1536 for RRE; 2028-; the CMV promoter: 2185-2788; MCS 1: 2799 2831; IRES: 2838 3413; MCS 2: 3414-3439; WPRE 3442 and 4033; 4237-4873' -LTR; pUC origin of replication: 5343-6013; penicillin resistance gene Amp^r：6158-7154。

On the basis of the resulting base plasmid, the gene encoding the scFV molecular sequence of the secretory antibody against CD47, the gene encoding the SIRPa extracellular domain and/or the gene encoding the CAR targeting element were loaded at the multiple cloning site.

The scFV molecule of the secretory antibody of the anti-CD 47 is derived from a murine, human or chimeric anti-CD 47 monoclonal antibody;

the amino acid sequence of the SIRPa extracellular domain is shown as Seq ID No. 18. An original SIRPa extracellular domain or an artificial SIRPa extracellular domain obtained by modifying or restructuring the original SIRPa extracellular domain without affecting the function;

the CAR targeting element refers to an anti-carcinoembryonic antigen CEA antibody scFV, and is obtained by connecting a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region through partial humanized modification of a framework region in a variable region of the anti-carcinoembryonic antigen CEA antibody.

Specifically, according to the above design concept, several cell immunotherapy vectors for therapy are obtained, as shown in FIGS. 1b, 1c, 1d, 1e, 1f, 1g, 1h and 1i, and comprise a construction sequence having a nucleotide sequence shown in Seq ID No. 2-9.

FIG. 1b, pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD; the construction process comprises the following steps: artificially synthesized CAR _ T _ Muc (2805 and 4277), wherein the insertion sites are XhoI and EcoRI; the artificially synthesized SIRPaECD (4857) -5315) insertion site is XbaI, and sequencing is carried out to identify the positive and negative of the insertion direction; wherein the nucleotide sequence of the CAR _ T _ Muc-IRES-SIRPaECD structure is shown as Seq ID No. 2.

FIG. 1c, pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47_ IgG 1; the construction process comprises the following steps: the artificially synthesized CAR _ T _ Muc (2805 and 4277) has XhoI and EcoRI insertion sites; the insertion site of anti _ CD47_ IgG1(4857-6644) is XbaI, and sequencing is carried out to identify the positive and negative of the insertion direction, the SCFV sequence (GENBANK: NM-001777) of the antibody molecule is added with the constant region sequence of human IgG1, and the full-length single-chain anti-human CD47 antibody molecule is obtained by a DNA synthesis method; wherein the nucleotide sequence of CAR _ T _ Muc-IRES-anti _ CD47_ IgG1 is shown as Seq ID No. 3.

FIG. 1d, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD; the construction process comprises the following steps: the artificially synthesized CAR _ T _ CEA sequence (2805-4277) is inserted into the XhoI and EcoRI sites of the basic plasmid pLV-CMV-MCS1-IRES-MCS 2; then inserting the synthesized SIRPaECD sequence (4872-5330) into the XbaI site, and carrying out forward and reverse identification; wherein the CAR _ T _ CEA-IRES-SIRPaECD structure has a nucleotide sequence such as Seq ID No.4

FIG. 1e, pLV-CMV-MCS-IRES-S/MAR; the construction process comprises the following steps: the human genome DNA PCR amplifies S/MAR sequence, both ends of the upstream and downstream primers are respectively provided with XbaI and PacI restriction sites, after restriction enzyme, the XbaI and PacI sites of the basic plasmid pLV-CMV-MCS1-IRES-MCS2 are inserted, and the position of the S/MAR insertion sequence (3420-; wherein the nucleotide sequence of MCS1-IRES-S/MAR structure is shown as Seq ID No. 5.

FIG. 1f, pLV-CMV-MCS-IRES-SIRPaECD-S/MAR; constructing process, artificially synthesizing SIRPaECD (3420-3878), inserting XbaI site of pLV-CMV-MCS-IRES-S/MAR plasmid, and making forward and reverse identification; wherein the nucleotide sequence of the MCS-IRES-SIRPaECD-S/MAR structure is shown as Seq ID No. 6.

FIG. 1g, pLV-CMV-CAR _ T _ Muc-IRES-S/MAR; the construction process is that the artificially synthesized CAR _ T _ Muc (2805-; wherein the nucleotide sequence of the CAR _ T _ Muc-IRES-S/MAR structure is shown as Seq ID No. 7.

FIG. 1h, pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR;

the forward and reverse orientation was identified by inserting the SIRPaECD sequence (4857-5315) into the XbaI site on the basis of the pLV-CMV-CAR _ T _ Muc-IRES-S/MAR plasmid shown in FIG. 1 g;

wherein the nucleotide sequence of the CAR _ T _ Muc-IRES-SIRPaECD-S/MAR structure is shown as Seq ID No.8

FIG. 1i, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD-S/MAR; on the basis of pLV-CMV-MCS-IRES-S/MAR plasmid shown in FIG. 1e, CAR _ T _ CEA (2805-

Example 2: in vitro secretion identification of anti _ CD47 antibody and in vitro secretion expression identification of SIRPaECD

293T cells in DMEM medium (containing 5-10% FBS, penicillin chain and mycin) and 5% CO₂Under the conditions of 37 ℃ culture, with pLV-CMV-MCS-IRES, pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47 packaging respectively infected 293T cells, infection 48 hours after cell lysis.

The lysate is a medium-strength RIPA lysate (50mM Tris-Cl, 150mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1mM MgCl)₂5mM EDTA, 5% glycerol, with the addition of the Roche protease inhibitor cocktail).

After SDS-PAGE electrophoresis, a PVDF membrane is converted into a membrane, after the protein is sealed by skimmed milk, the membrane is incubated by goat anti-human IgG1 constant region antibody marked by horseradish peroxidase HRP, the PVDF membrane is treated by a luminescent substrate, an X-ray film is sensitized, and the X-ray film is treated by a developing solution and a fixing solution.

To test the effectiveness of IRES on its expression of the following coding frame, the following was used

pLV-CMV-MCS1-IRES-MCS2、

pLV-CMV-CAR_T_Muc-IRES-SIRPaECD、

pLV-CMV-CAR_T_CEA-IRES-SIRPaECD、

pLV-CMV-MCS-IRES-SIRPaECD-S/MAR、

pLV-CMV-CAR_T_Muc-IRES-SIRPaECD-S/MAR、

And pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD-S/MAR packaged lentivirus respectively infects 293T cells, 36 hours, 48 hours and 72 hours after infection, cell culture fluid is collected, mixed, added with protease inhibitor, and subjected to freeze drying concentration, protein is subjected to Western blot, and since SIRPaECD contains a C-terminal C-Myc label, primary antibody incubation is carried out by using a rat anti-C-Myc label antibody, then sheep anti-rat antibody labeled with HRP is used for incubation, and finally X-ray film exposure development and fixation are carried out.

The results show the supernatant extracts of pLV-CMV-MCS1-IRES-MCS cells infected as shown in FIG. 2(A) 1; 2, cell supernatant extract infected with pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47_ IgG1, anti-CD 47IgG1 antibody about 65 kD.

To measure the secretion concentration of anti-CD 47 antibody, virus-infected 293T cell supernatant mixtures (48 and 72 hours of infection) were coated in 96-well plates for ELISA detection, incubated with biotin-labeled anti-human IgG, then with HRP-labeled streptavidin, followed by TMB substrate addition, after termination of the reaction, absorbance was measured, and the anti-CD 47 antibody calibration curve was measured at known concentrations.

As shown in FIG. 2(B), the concentration of supernatant antibody infecting pLV-CMV-MCS1-IRES-MCS cells and the concentration of cells infecting pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD 47-IgG virus reached a secretion concentration of 220ng/ml, and the error line was + SD.

As shown in FIG. 2(C)

1，pLV-CMV-MCS1-IRES-MCS；

2，pLV-CMV-CAR_T_Muc-IRES-SIRPaECD；

3，pLV-CMV-CAR_T_CEA-IRES-SIRPaECD；

4，pLV-CMV-MCS-IRES-SIRPaECD-S/MAR；

5，pLV-CMV-CAR_T_Muc-IRES-SIRPaECD-S/MAR；

6, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD-S/MAR, concentrated supernatant protein is stained by anti-C-MYC tag antibody WB, the molecular weight is about 20kD, 4 expression vectors have normal expression, the element adoption and construction of the vector are reasonable, and the expression of genes is not influenced by the addition of S/MAR elements.

To determine the concentration of SIRPaECD in the cell supernatant, the virus-infected 293T cell supernatant mixture (48 and 72 hours infected) was subjected to ELISA assay, the cell culture supernatant was coated on the culture plate, incubated with the biomarker anti-C-MYC tag antibody, incubated with HRP-labeled streptavidin, TMB substrate was added, after the reaction was stopped, the absorbance was measured and purified SIRPaECD of known concentration was used to calibrate the concentration profile.

Results as shown in figure 2(D) secretion and concentration measurement of SIRPaECD in mixed samples of culture supernatants 2 days and 3 days after 293T cell infection with virus, normal expression of SIRPaECD after several IRES elements was unaffected, with an error line of + SD.

Example 3: cytotoxic Effect (ADCC) of anti-CD 47 antibody

The virus encoding the CD47 antibody (pLV-CMV-CAR-T-Muc-IRES-anti-CD 47-IgG 1) infected 293T cells, and the supernatant was collected after 48 hours and 72 hours, purified by a protein A column (GE Health Co.), eluted, dialyzed, and the resulting concentrated antibody against IgG1 of CD47 was quantified, a small amount for Western blot detection, and the remainder for ADCC detection described below.

Construction of HUVEC target cells: primers were designed based on the sequence of human CD47 (GENBANK: NM-001777), and the coding cassette sequence was PCR-amplified using human CD47 plasmid (Sinobiological) as a template, which was ligated into our constructed lentiviral vector pLV-CMV-MCS1-IRES-MCS, to obtain HUVEC cells stably transfected with CD 47.

Primers hCD47a-S (XhoI) 5'-GGCCTCGAGATGTGGCCCCTGGTA-3'; hCD47a-R (BamHI) 5'-CGTGGATCCAGTTATTCATCATTCATC-3';

ADCC was determined using a conventional Peripheral Blood Mononuclear Cell (PBMC) method, which was taken from healthy volunteers. HUVEC target cells were added to a 96-well plate, 1. mu.g/ml anti-CD 47 antibody was added, PBMC was added at a ratio of 10:1,20:1 and 50:1 to effector PBMC and HUVEC target cells, and the cells were cultured in an incubator at 37 ℃ for 6 hours as a control of human nonspecific IgG.

The lactate dehydrogenase LDH activity in the supernatant was then determined.

As shown in fig. 3. anti-CD 47 antibody 1. mu.g/ml was added to HUVEC cells, 10, 20 or 50 fold human peripheral blood mononuclear cells were added, and ADCC effect was measured by measuring LDH in the supernatant, and anti-CD 47IgG1 antibody showed stronger ADCC effect than IgG1 antibody control.

Example 4: detection of non-integration passage ability of S/MAR

To examine the effectiveness of S/MARs on non-integration and passaging, we performed Southern blot analysis, and multi-generation passaging analysis.

pLV-CMV-MCS1-IRES-MCS2-S/MAR and

pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR were transfected into 293T cells with the third generation packaging plasmid of type I mutant HIV integrase (shown in Seq ID No. 10), respectively, to obtain virus particles, and the culture medium was collected at 48 hours and 72 hours after transfection.

Infecting HepG2 cells by using collected culture solution, passing the infected cells once every 2-3 days for 50 generations, then harvesting partial cells, extracting genome DNA, carrying out enzyme digestion on the genome DNA by using restriction enzymes NotI and PacI, and electrically transferring the cell to a nylon membrane after electrophoresis.

The probe is connected into a pGEM-T vector by a 1kb fragment (without an S/MAR sequence) of a common sequence part of the vector, the probe integrated with digoxin nucleotide is obtained by in vitro transcription of T7 polymerase, and after the probe is hybridized, the probe is incubated with biotin-labeled anti-digoxin antibody and HRP-labeled streptavidin in sequence, exposed by an X-ray film, developed and fixed.

The results are shown in FIG. 4, (A)1, control of empty vector samples digested with XhoI; 2, XhoI digested genomic DNA of infectious virus pLV-CMV-MCS1-IRES-MCS 2-S/MAR; 3, XhoI digested genomic DNA infected with pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR virus; the digested DNA showed no integration of 2 and 3 in the genome after passage 50.

(B)1, empty vector sample control digested with EcoRI; 2 EcoRI digested pLV-CMV-MCS1-IRES-MCS2-S/MAR virus infected genomic DNA; 3 EcoRI digested genomic DNA of pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR virus; southern blot showed no genomic integration of both 2 and 3. According to XhoI and EcoRI cut genomic DNA, the inserted S/MAR sequence can effectively inhibit the corresponding plasmid element from being inserted into the genome and can be normally passed to daughter cells along with the mitosis of the maternal cells.

Example 5: cytotoxicity test of CAR _ T _ Muc and CAR _ T _ CEA T cells constructed with different elements

Experimental materials:

the vectors used contained the sequences shown in FIGS. 1a, 1b, 1c, 1d, 1h, and 1 i.

CAR _ T _ Muc T cells (i.e., T cells infected with pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD, pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47_ IgG1 and pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR virus, respectively)

Target cell: 2X10³PanC1 cell (ATCC)

HUVEC cells overexpressing human CEACAM1 plasmid (nano Biological);

CAR _ T _ CEA cells (i.e., T cells infected with pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD-S/MAR, respectively)

The method comprises the following steps:

t cells provided by healthy volunteers were infected.

The target cell is 2X10³The PanC1 cells were cultured in the presence of a recombinant plasmid,

controls are T cells infected with pLV-CMV-MCS-IRES-S/MAR virus and target cells PanC1 cells,

following the conventional procedures of CAR-T cell culture, viral infection and cytokine stimulation, T cells were isolated from blood of healthy volunteers, sorted with anti-human CD3/CD28Dynabead magnetic beads (Thermofeisher), activated CD3+ cells (beads/cells 1:1 or 2:1) in a culture system: AIM-V serum-free medium (GIBCO), 5% heat inactivated human AB serum (GIBCO), 1% Glutamax (GIBCO), 40IU/ml IL-2(EMD Millipore), cell initiation densityDegree of 1X 10⁶cells/ml, culture vessel as culture bag); day 2 or day 3: the retrovirus is infected once. Culturing for 7-10 days, supplementing fresh culture medium every other day, and maintaining cell density at 0.4 × 10⁶cells/ml. CAR-T cells (CAR _ T _ CEA T cells and CAR _ T _ Muc T cells) were obtained with cytotoxicity.

HUVEC cells overexpressing human CEACAM1 plasmid (nano Biological) were seeded in 96-well plates, and CAR _ T _ Muc and CAR _ T _ CEA T cells: the target cells were inoculated into the above 96-well plate at a ratio of 10:1,20:1 or 50:1, cultured for 12 hours, and the culture supernatant was taken and detected using an ELISA kit for IFN γ (Peprotech).

The ELISA detects the cytokine effects produced by CAR _ T _ Muc and CAR _ T _ CEA, respectively.

Results FIG. 5 shows that more IFN γ is produced except in the empty vector, and thus plasmids with and without the S/MAR element had a similar effect on cytokine production.

Example 6 in vitro expression and toxicity validation of CAR _ T _ Muc and CAR _ T _ CEA

Day 0: PBMCs were collected from healthy volunteers (a portion was cryopreserved),

CD3+ cells were sorted and activated with anti-human CD3/CD28Dynabead magnetic beads (Thermofeisher) (beads/cells2:1, 600X 10)⁶)，

The culture system is as follows: AIM-V serum-free medium (GIBCO), 5% heat-inactivated human AB serum (GIBCO), 1% Glutamax (GIBCO), 40IU/ml IL-2(EMD Millipore), cell initiation density of 1X 10⁸cells/ml, culture vessel is culture bottle or culture bag;

day 2 and 3: lentivirus supernatants were infected once each.

Continuing to culture for 6-8 days, supplementing fresh culture medium every other day, and maintaining cell density at 0.4 × 10⁶cells/ml。

The toxic effect of CAR _ T _ Muc on cells was detected by infection with pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD, pLV-CMV-CAR _ T _ Muc-IRES-anti _ CD47_ IgG1, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD, pLV-CMV-CAR _ T _ Muc-IRES-SIRPaECD-S/MAR, pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD-S/MAR,

the control was pLV-CMV-MCS-IRES-S/MAR virus infected T cells,

the target cell is a human pancreatic cancer cell PanC 1;

the toxic effect of CAR _ T _ CEA on target cells is through

pLV-CMV-CAR _ T _ CEA-IRES-SIRPaECD-S/MAR, control as above, target cells were Huvec cells overexpressing CEACAM1 (ATCC).

As shown in FIG. 6, (A) the cytotoxic effect of CAR _ T _ Muc detected by MTT (thiazole blue) on PanC1 cells, all plasmids containing CAR _ T _ Muc were constructed to produce the cytotoxic effect, and the cytotoxic effect of the plasmid with the added S/MAR sequence was similar to that of the plasmid without the added S/MAR sequence. (B) MTT-detected CAR _ T _ CEA toxic Effect on HUVEC cells overexpressing CEACAM1, 2 constructed CAR _ T _ Muc-containing plasmids were all able to produce cytotoxic effects.

Claims

1. A multi-coding-frame, non-integrating lentiviral vector, wherein: from upstream to downstream comprising a 5 '-LTR sequence, a promoter sequence, a multiple cloning site 1, one or more IRES elements, multiple cloning site 2 and 3' -LTR sequences, a cPPT/CTS, RRE and/or WPRE sequence;

the multiple cloning site 1 and the multiple cloning site 2 are used for loading target exogenous expression genes;

the multiple cloning site 1 contains the nucleotide sequence shown in Seq ID No.21, and/or the multiple cloning site 2 contains the nucleotide sequence shown in Seq ID No. 22.

2. The lentiviral vector of claim 1, wherein: also contains S/MAR sequences for enhanced adhesion in the nuclear matrix and passage with the cells.

3. The lentiviral vector of claim 1, wherein: the nucleotide sequence is shown as Seq ID No. 1.

4. A cellular immunotherapy vector further comprising a gene of interest loaded at the multiple cloning site of the lentiviral vector of any one of claims 1 to 3; the target gene is selected from one or more of the following genes:

a nucleotide sequence encoding an antibody molecule or an scFV thereof;

a nucleotide sequence encoding a secretory synergistic or inhibitory effector molecule;

a nucleic acid sequence encoding a CAR targeting element.

5. The cellular immunotherapy vector according to claim 4,

the antibody molecule or scFV thereof refers to secretory antibody or scFV molecule of anti-CD 47, and the antibody is murine, humanized or chimeric antibody;

the secretory inhibitory effector molecule refers to the human SIRPa extracellular domain, the nucleotide sequence of which is shown in Seq ID No.18, or refers to an artificial SIRPa extracellular domain obtained by modifying or restructuring the human SIRPa extracellular domain without affecting the function;

the CAR targeting element refers to anti-carcinoembryonic antigen CEA, and has a nucleotide sequence shown in Seq ID No.23, wherein a framework region in a variable region of a murine antibody of the anti-carcinoembryonic antigen CEA is subjected to partial humanization modification and is connected with a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region, so that the CAR targeting element is obtained by performing partial humanization modification on the framework region in the variable region of the anti-carcinoembryonic antigen CEA antibody and is connected with a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region.

The CAR targeting element refers to a nucleic acid sequence of anti-aglycosylated Mucin antibody scFV, has a nucleotide sequence shown in Seq ID No.24, and is obtained by performing partial humanized transformation on a variable region framework region of the anti-aglycosylated Mucin murine monoclonal antibody, connecting a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region, and connecting a CD8 hinge region, a transmembrane region, a 4-1BB coactivation signal region and a CD3zeta signal region in the variable region of the anti-carcinoembryonic antigen CEA antibody through partial humanized transformation.

6. The cellular immunotherapy vector according to claim 4, characterized in that: a construct comprising any of Seq ID No.2-9, said construct consisting of said gene of interest loaded into said multiple cloning site and an IRES sequence and/or S/MAR sequence in said lentiviral vector.

7. A kit for the treatment of a disease comprising the lentiviral vector of any one of claims 1 to 3, or the cellular immunotherapeutic vector of any one of claims 4 to 6, the disease comprising a solid tumor, a hematological tumor, an autoimmune disease, stem cell therapy, a viral disease, and a genetic disease.