CN111206043A

CN111206043A - Improved PB transposon system and application thereof

Info

Publication number: CN111206043A
Application number: CN201811392822.3A
Authority: CN
Inventors: 刘韬; 刘辉; 金华君; 王超; 何周; 何江川; 吴碧寒; 刘天怡; 钱其军
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2020-05-29

Abstract

The present invention relates to an improved PB transposon system and its use. Specifically, the invention provides a nucleic acid construct which sequentially comprises a promoter for controlling the expression of PiggyBac transposase, a PiggyBac transposase coding sequence, a PiggyBac transposon 5 'terminal repetitive sequence, an optional polyA tailing signal sequence 1, a polyclonal insertion site, a polyA tailing signal sequence 2 and a PiggyBac transposon 3' terminal repetitive sequence. The recombinant vector constructed by using the nucleic acid construct of the present invention has significantly improved integration efficiency.

Description

Improved PB transposon system and application thereof

Technical Field

The present invention relates to an improved PB transposon system and its use.

Background

The expression form of the foreign gene in the host cell can be divided into transient expression and stable expression, wherein the stable expression refers to: (1) the expression of the foreign gene after transfection into eukaryotic cells and integration into the genome. The stable expression level of the recombinant gene is generally 1-2 orders of magnitude lower than that of transient expression. (2) Although the host cell is subjected to multiple passages or condition changes, the expression level of the host cell is still stable.

In view of the fact that stable expression can maintain long-term sustained expression of foreign genes with cell division, it is of great significance in ex vivo cell modification (ex vivo), such as the study of transgenic Chimeric Antigen receptor T cells (CAR-T) therapy. The CAR-T cell can specifically recognize and kill tumor cells expressing specific cell surface antigens with high efficiency, and has remarkable clinical curative effect. For example, CAR-T against CD19 can kill B cell lymphoma expressing CD19 surface antigen with high efficiency, and the effective remission rate of patients with advanced refractory B cell lymphoma reaches 90% (Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, ZhengZ, Lacey SF, Mahnke YD, Melenhorst JJ, Rheinoloid SR, Shen A, Teache DT, LevineBL, June CH, Porter DL, Grupp SA., Chimeric anti receptor T cells for refractory remissions in Leukemia, N Engl J Med 2014. (16): 1507-17).

To achieve stable expression of foreign genes in host cells, commonly used vector systems include: 1. retroviral system: can effectively infect host cells and mediate the high-efficiency integration of the genome of an exogenous gene expression frame, but has limited loading capacity and complex preparation process of recombinant virus particles. 2. Eukaryotic expression plasmid system: the preparation process is relatively simple, but the integration efficiency is extremely low when the DNA is inserted into a host genome by random DNA recombination. 3. Transposon system: the plasmid system is adopted, the preparation process is relatively simple, and the integration efficiency is relatively low by integrating the exogenous gene into the genome through the transposase.

The mammalian transposon system used in the earliest was the fish-derived "sleeping beauty" transposon (sleeping beauty), but the "sleeping beauty" transposon has the defects of excessive inhibitory effect and small carrying fragment (about 5 kb), which limits its application in the transgenic field. The piggybac (pb) transposon derived from a lepidopteran insect is currently the most active transposon in mammals. The host range is extremely wide, and the single-cell organisms to mammals can play a role; can carry large exogenous DNA fragments, and when the transposition fragment is within 14kb, the transposition efficiency is not reduced obviously. The PB transposon mainly adopts a 'cut-past' mechanism to carry out transposition, a spot (focprint) cannot be left at an in-situ point after a transposition fragment is excised, a genome can be accurately repaired after excision, and the PB transposon has an important role in application of reversible genes. In addition, the PB transposase has high plasticity, and can change the activity and action mode of the transposase and improve the targeting property of foreign gene transposition by fusing with other functional proteins or changing the functional region of the transposase. In recent years, the integration efficiency of PB in mammalian cells is further improved through codon optimization, site-specific mutation of specific site amino acids, introduction of corresponding nuclear localization tags and the like, so that the system is widely applied to the fields of genome research, gene therapy, cell therapy, stem cell induction, post-induction differentiation and the like.

The conventional PB transposable system employs a binary transposition system composed of a donor plasmid (containing terminal repeat sequences recognized by PB integrase at both ends of an exogenous gene expression cassette) and a transposase helper plasmid (providing PB transposase). In the binary transposition system, in order to achieve effective integration of the foreign gene expression cassette, it is necessary to satisfy the condition that two plasmids are transfected into the same cell, and only a part of cells can achieve the integration in the transfection process (other cells either have one plasmid without successful transfection or only transfect one plasmid, and cannot achieve effective integration), thereby reducing the integration efficiency to some extent. Meanwhile, since the PB transposon system takes place in a completely reversible "cut-past" form, as long as the integrase maintains expression, it still has the possibility of recutting the foreign gene expression cassette that has been integrated into the genome, resulting in genomic instability and actually lowering the integration efficiency. In order to improve the integration efficiency of a PB transposable system, one strategy is to incorporate a donor plasmid and a transposase helper plasmid into the same plasmid, and set a Self-inactivation (Self-inactivation) mechanism of transposase at the same time, so as to ensure that the expression of transposase can be timely turned off after the integration of exogenous genes is realized. For example, the PB expression cassette is placed in the same orientation as the foreign gene expression cassette and shares the same polyA tailing signal sequence, once the foreign gene expression cassette is excised from the plasmid and integrated into the genome, the PB expression cassette will be defective in the polyA tailing signal sequence, causing the transcribed mRNA to be unstable and rapidly degraded, and PB expression to be turned off (Chakraborty S, Ji H, Chen J, Gersbach CA, Leong KW, Vector modifications topelimate delivery expression following piggy Bac-mediated transformation, SciRep.2014; 4: 7403).

Another drawback of PB transposable systems is that: the length of the wild-type 5 'and 3' Inverted Terminal Repeats (ITRs) is over 700bp, and the total length is about 1.5 kb. These sequences are necessary for the integration of a foreign gene into the genome by transposition. However, once transposition is complete, the 1.5kb sequence will no longer function and may potentially increase the risk of cell transformation due to its own promoter and enhancer activity. In addition, longer ITR sequences increase the load burden during transfection, affecting transfection efficiency. While shortening The ITR length results in a significant decrease in transposition efficiency (Zhuang L, Wei H, Lu C, Zhong B., The relationship between transposition internal domain sequences of piggyBacand transposition efficiency in BmNcells and Bombyx mori, Acta Biophys Sin (Shanghai) 2010; 42: 426. sup. 431; LiX, Harrell RA, Handler AM, Beam T, HennessK, Fraser Jr. MJ., piggyBac interaction area for efficiency transformation of target genes, InsectMol Biol 2005; 14: 17-30.).

Solodushko et al reported a minimized PB transposition system terminal repeat (minTR) with most of the wild type ITR removed, wherein the 5 'minTR is 35bp long and the 3' minTR is 63bp long, which is much shorter than the wild type 5 'and 3' ITRs, but the transposition process cannot be completed only by the presence of minTR itself. The sequence of a full-length ITR, even if present in a vector outside the transposable system, is still an element necessary for transposition to occur (Solodushko V, Bitko V, Fouty B, minimum piggyBacvectors for chromatography integration, Gene ther. 2014Jan; 21(1): 1-9). Although this minimized PB transposition system terminal repeat can significantly reduce the integration length of the ITR sequence into the target cell genome and thus the risk of transformation of the target cell, the nucleic acid load required to be transferred into the cell remains large and the transfection efficiency is still compromised because the full-length ITR sequence still needs to be present somewhere in the overall system and needs to be introduced into the cell together by methods such as viral or electrotransfer.

We have previously provided a highly efficient and safe transposon integration system which employs a monad transposition system, in which a vector contains a foreign gene and a PB transposase gene in opposite directions, and shares a bidirectional polyA sequence (CN 105154473A). In this system only minimal 5 'and 3' terminal repeats are used, and no full length sequences containing 5 'and 3' ITRs are used, but the PB transposition system still works normally effectively. Compared with the traditional binary PB transposition system, the transposition system has obviously higher transposition efficiency, but the transposition efficiency of the CAR-T cell preparation is still to be further improved.

Disclosure of Invention

The first aspect of the invention provides a nucleic acid construct, which comprises a promoter for controlling expression of PiggyBac transposase, a PiggyBac transposase coding sequence, a PiggyBac transposon 5 'terminal repeat sequence, an optional polyA tailing signal sequence 1, a polyclonal insertion site, a polyA tailing signal sequence 2 and a PiggyBac transposon 3' terminal repeat sequence which are connected in sequence.

In one or more embodiments, the PiggyBac transposon 5 'terminal repeat sequence is a truncated PiggyBac transposon 5' terminal repeat sequence.

In one or more embodiments, the PiggyBac transposon 3 'terminal repeat sequence is a truncated PiggyBac transposon 3' terminal repeat sequence.

In one or more embodiments, the nucleic acid construct does not contain the polyA tailed signal sequence 1.

In one or more embodiments, the nucleic acid construct has a deletion of TTAA sequences other than those contained in the 5 'terminal repeat and the 3' terminal repeat.

In one or more embodiments, the promoter controlling expression of PiggyBac transposase is selected from the group consisting of CMV promoter, SV40 promoter, PGK promoter, and chicken β -actin promoter, preferably CMV promoter, more preferably the sequence of the promoter is shown in SEQ ID No. 1.

In one or more embodiments, the PiggyBac transposase coding sequence comprises a nuclear localization signal selected from the group consisting of c-myc nuclear localization signal and SV40 nuclear localization signal; preferably, the piggyBac transposase coding sequence is shown as SEQ ID NO. 2; preferably, the nuclear localization signal is located at the N-terminus of the PiggyBac transposase coding sequence.

In one or more embodiments, the PiggyBac transposon 5' terminal repeat sequence is set forth in SEQ ID No. 3.

In one or more embodiments, the polyA tailed signal sequence 1 is as set forth in SEQ ID No. 4.

In one or more embodiments, the polyclonal insertion site is as set forth in SEQ ID NO 5.

In one or more embodiments, the polyA tailed signal sequence 2 is an SV40polyA tailed signal sequence; preferably, the polyA tailing signal sequence 2 is shown as SEQ ID NO 6.

In one or more embodiments, the 3' terminal repeat of the PiggyBac transposon is set forth in SEQ ID No. 7.

In one or more embodiments, the nucleic acid construct is selected from the group consisting of:

(a) a nucleic acid construct comprising sequentially linked SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, optionally SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7;

(b) a nucleic acid construct in which TTAA other than that contained in SEQ ID NO. 3 and SEQ ID NO. 7 is deleted from the nucleic acid construct shown in (a).

In one or more embodiments, the multiple cloning site of the nucleic acid construct is operably inserted with, or replaced with, one or more identical or different expression cassettes.

In a second aspect, the present invention provides a recombinant vector comprising a nucleic acid construct according to any of the embodiments herein.

In one or more embodiments, the multiple cloning site of the nucleic acid construct is operably inserted with, or replaced with, one or more of the same or different exogenous gene and optionally a promoter controlling expression of the exogenous gene.

In one or more embodiments, the recombinant vector is a recombinant cloning vector, a recombinant eukaryotic expression plasmid, or a recombinant viral vector.

In one or more embodiments, the recombinant cloning vector is a recombinant vector obtained by recombining a nucleic acid construct described in any of the embodiments herein with a vector of the pUC18, pUC19, pMD18-T, pMD19-T, pGM-T, pUC57, pMAX, or pDC315 series;

in one or more embodiments, the recombinant expression vector is a recombinant expression vector obtained by recombining the nucleic acid construct described in any one of the embodiments herein with a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, a pMAX vector, or a pDC315 series vector;

in one or more embodiments, the recombinant viral vector is a recombinant adenoviral vector, a recombinant adeno-associated viral vector, a recombinant retroviral vector, a recombinant herpes simplex viral vector, or a recombinant vaccinia viral vector.

In a third aspect, the invention provides a recombinant cell comprising a nucleic acid construct or a recombinant vector according to any one of the embodiments herein.

In one or more embodiments, the recombinant host cell is a recombinant mammalian cell, such as a recombinant primary culture T cell, Jurkat cell, K562 cell, embryonic stem cell, tumor cell, HEK293 cell, or CHO cell.

In a fourth aspect, the present invention provides a method for preparing a cell having a foreign gene expression cassette integrated into its genome, the method comprising the steps of transfecting a recombinant vector according to any one of the embodiments herein into a cell of interest, and culturing the transfected cell; preferably, the cells are cultured to more than three generations.

In a fifth aspect, the invention provides a cell having an exogenous gene expression cassette integrated into its genome, obtained by a method as described herein.

In a sixth aspect, the invention provides the use of a nucleic acid construct and a recombinant vector according to any one of the embodiments herein, selected from the group consisting of:

(1) use in the manufacture of a medicament or agent for integrating an exogenous gene expression cassette into the genome of a host cell;

(2) use as a means for integrating a foreign gene expression cassette into the genome of a host cell;

(3) the use in the preparation of a medicament or formulation for genomic research, gene therapy, cell therapy or stem cell induction and differentiation following induction;

(4) use as a tool for genomic research, gene therapy, cell therapy or stem cell induction and differentiation following induction.

The seventh aspect of the invention provides the use of one or more of the nucleic acid construct, the recombinant vector and the cell of any one of the embodiments herein in the manufacture of a medicament for the treatment of cancer.

In an eighth aspect, the present invention provides a method for treating cancer, comprising the steps of: administering to an individual having cancer a medicament containing one or more of the nucleic acid construct, the recombinant vector, and the cell of any embodiment herein.

In one or more embodiments, the cancer is one or more of CD19, CD20, CEA, GD2, GPC3, FR, PSMA, gp100, CA 3, CD 171/L3-CAM, IL-13R 3, MART-1, ERBB3, NY-ESO-1, MAGE family protein, BAGE family protein, GAGE family protein, AFP, MUC 3, CD44v 3/8, CD3, VEGFR 3, IL-11R 3, EGP-2, EGP-40, FBP, GD3, PSCA, FSA, FRA-72, PSA 5T 3, fetal acetylcholine receptor, LeY, EpLN, MSLN, IGFR 3, EGFR, RvEGFRIII, ER3672, POBB 3-125, MUTAG-72, GCT 3, VEGFR 3, GFR 3, GFR, EGF-72, EGF-III, EGF-72, EGF III, EGF, EG.

In one or more embodiments, the cancer is selected from one or more of acute B-lymphocytic leukemia, chronic B-lymphocytic leukemia, mantle cell lymphoma, non-hodgkin's lymphoma, multiple myeloma lung cancer, liver cancer, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, osteosarcoma, pancreatic cancer, and prostate cancer.

The invention has the beneficial effects that compared with the existing PB transposon vector system, the transposon integration system with higher transposition integration efficiency can further obviously improve the proportion of the exogenous gene integrated into a target cell (such as immune effector cell), and simultaneously improve the expression strength of the exogenous gene in the target cell, thereby providing a new tool and means based on non-viral vectors for cell therapy.

Drawings

FIG. 1: vector structure schematic of pNBC vector.

FIG. 2: vector structure schematic diagram of pNBC (-) vector.

FIG. 3: schematic vector structure of pNBC-AD vector.

FIG. 4: schematic vector structure of pNBC-D3 vector.

FIG. 5: schematic vector structure of pNBC-D5 vector.

FIG. 6: the vector structure of pNB338CFL-EGFP (-).

FIG. 7A: integration efficiency of pNB 338C-EGFP.

FIG. 7B: integration efficiency of pNB338C-EGFP (-).

FIG. 7C: integration efficiency of pNB 338C-EGFP-AD.

FIG. 7D: integration efficiency of pNB 338B-EGFP.

FIG. 7E: integration efficiency of pNB 338B-EGFP-D3.

FIG. 7F: integration efficiency of pNB 338B-EGFP-D5.

FIG. 7G: integration efficiency of pNB338CFL-EGFP (-).

FIG. 7H: the proportion of EGFP positive cells detected by flow cytometry at day 13 after transfection, after 3 passages, was changed.

FIG. 8: and (3) detecting the expression level of the PD-1 antibody after PBMCs from different individual sources are transfected with a vector containing the PD-1 single-chain antibody coding sequence.

FIG. 9: results of testing for CD19CAR integration efficiency following transfection of PBMCs from different individual sources with CD19 CAR-encoding sequences.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

Some terms related to the present invention are explained below.

In the present invention, the term "single chain antibody" (scFv) refers to an antibody fragment having the ability to bind to an antigen, which is formed by connecting an amino acid sequence of a light chain variable region (VL region) and an amino acid sequence of a heavy chain variable region (VH region) via a hinge. In certain embodiments, the single chain antibody (scFv) of interest is from an antibody of interest. The antibody of interest can be a human antibody, including human murine chimeric antibodies and humanized antibodies. The antibody may be secreted or membrane anchored.

The term "expression cassette" refers to the complete elements required for expression of a gene, including the promoter, gene coding sequence, PolyA tailing signal sequence.

The term "nucleic acid construct" is defined herein as a single-stranded or double-stranded nucleic acid molecule, preferably referring to an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises operably linked 1 or more control sequences capable of directing the expression of the coding sequence in a suitable host cell under conditions compatible therewith. Expression is understood to include any step involved in the production of a protein or polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "operably inserted/linked" is defined herein as a conformation in which a control sequence is located at an appropriate position relative to the coding sequence of a DNA sequence such that the control sequence directs the expression of a protein or polypeptide. In the nucleic acid construct of the present invention, for example, a promoter of a foreign gene and a coding sequence of the foreign gene are placed at the multiple cloning site by a DNA recombination technique. Said "operably linked" may be achieved by means of DNA recombination, in particular, the nucleic acid construct is a recombinant nucleic acid construct.

The term "coding sequence" is defined herein as that portion of a nucleic acid sequence whose amino acid sequence of the protein product is directly determined. The boundaries of the coding sequence are generally determined by a ribosome binding site immediately upstream of the 5 'open reading frame of the mRNA (for prokaryotic cells) and a transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term "control sequences" is defined herein to include all components necessary or advantageous for expression of a polypeptide of interest. Each control sequence may be native or foreign to the nucleic acid sequence encoding the protein or polypeptide. These regulatory sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. In order to introduce specific restriction sites for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein or polypeptide, regulatory sequences with linkers may be provided.

The promoters useful in the present invention include, but are not limited to, EF1 α promoter, PGK promoter, β -actin promoter, CMV promoter, EEF2 promoter, CAG promoter, U6 promoter, SV40 promoter and the like, preferred promoters are EF1 α promoter, CMV promoter and β -actin promoter, and different promoters may be selected for different host cells based on different coding sequences and different host cells as is well known in the art.

The control sequence may also be a suitable transcription termination sequence, i.e., a sequence recognized by a host cell to terminate transcription. The termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein or polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that functions in the host cell of choice may be used in the present invention.

The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of a protein or polypeptide and which directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding region of the nucleic acid sequence may naturally contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding region may contain a signal peptide coding region which is foreign to the coding sequence. Where the coding sequence does not normally contain a signal peptide coding region, it may be desirable to add a foreign signal peptide coding region. Alternatively, the native signal peptide coding region may simply be replaced by a foreign signal peptide coding region in order to enhance polypeptide secretion. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of interest may be used in the present invention.

The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide. A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

Where the polypeptide has both a signal peptide and a propeptide region at the amino terminus, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences which regulate the expression of the polypeptide depending on the growth of the host cell. Examples of regulatory systems are those that respond to a chemical or physical stimulus, including in the presence of a regulatory compound, to open or close gene expression. Other examples of regulatory sequences are those which enable gene amplification. In these instances, the nucleic acid sequence encoding the protein or polypeptide should be operably linked to the control sequence.

As used herein, the term "highly expressed" refers to a gene, such as a tumor-associated antigen gene, that is expressed at a level significantly higher than the level of expression of the corresponding gene in normal tissue.

Herein, the terms "individual", "subject" and "patient" have the same meaning and refer to an animal, in particular a mammal, more preferably a human.

The nucleic acid construct of the invention can contain a promoter for controlling expression of PiggyBac transposase, a PiggyBac transposase coding sequence, a PiggyBac transposon 5 'terminal repetitive sequence, an optional polyA tailing signal sequence 1, a polyclonal insertion site, a polyA tailing signal sequence 2 and a PiggyBac transposon 3' terminal repetitive sequence which are connected in sequence.

Promoters for PiggyBac transposase expression include a variety of promoters known in the art to be suitable for PiggyBac transposase expression, including, but not limited to, the EF1 α promoter, the PGK promoter, the β -actin promoter, the CMV promoter, the EEF2 promoter, the CAG promoter, the U6 promoter, and the SV40 promoter.

Herein, the PiggyBac transposase is a PiggyBac transposase well known in the art, including its various mutants having a desired transposase function well known in the art. Generally, in the nucleic acid construct of the present invention, a coding sequence for a Nuclear Localization Signal (NLS) may also be included between the promoter for expression of the PiggyBac transposase and the PiggyBac transposase coding sequence. The nuclear localization signal is typically a short amino acid sequence that interacts with nuclear entry vectors to allow the protein to be transported into the nucleus. The nuclear localization signal is usually composed of 4-8 amino acids, containing Pro, Lys and Arg. Nuclear localization signals well known in the art can be used in the present invention. Exemplary nuclear localization signals include, but are not limited to, the c-myc nuclear localization signal and the SV40 nuclear localization signal. The coding sequence for PiggyBac transposase is operably linked to one or more copies of the coding sequence for the nuclear localization signal sequence. The coding sequence of an exemplary PiggyBac transposase comprising a nuclear localization signal coding sequence can be shown in SEQ ID No. 2.

The nucleic acid construct of the invention also contains 3 'and 5' terminal repeat sequences of the PiggyBac transposon. Full length 3 'and 5' terminal repeats may be used. Exemplary full-length 5 'and 3' terminal repeats can be shown in SEQ ID NOs 10 and 11. Truncated, functional 3 'and 5' terminal repeats known in the art are preferably used herein. Exemplary truncated 5 'and 3' terminal repeats are shown in SEQ ID NOs 3 and 7, respectively, of the present application; alternatively, other known truncated 5 'and 3' terminal repeats known in the art may be used. For example, truncated 5 'and 3' terminal repeats disclosed in CN105154473A (incorporated herein by reference in its entirety) can be used (SEQ ID NOS: 1 and 4 in that application).

polyA tailing signal sequences known in the art may be used in the present invention, including but not limited to SV40polyA tailing signal sequences as well as various truncated forms of polyA tailing signals known in the art. Exemplary polyA tailing signal sequences can be set forth in SEQ ID NOS: 4 and 6. The nucleic acid construct of the invention may contain two polyA tail signals, referred to herein as polyA tail signal 1 and polyA tail signal 2, respectively, which may be the same or different. In a preferred embodiment, the nucleic acid construct of the invention comprises only polyA tailing signal 2.

The nucleic acid constructs of the invention also contain a polyclonal insertion site for insertion of an expression cassette of interest therein or replacement of the polyclonal insertion site with an expression cassette of interest.

In a preferred embodiment of the present invention, the nucleic acid construct of the present invention comprises a CMV promoter sequence (SEQ ID NO:1), a PiggyBac transposase coding sequence containing a c-myc nuclear localization signal (SEQ ID NO:2), a PiggyBac transposon 5 'terminal repeat sequence (SEQ ID NO:3), a polyA tailing signal sequence 1(SEQ ID NO:4), a multiple cloning insertion site (SEQ ID NO:5), a polyA tailing signal sequence 2(SEQ ID NO:6), and a PiggyBac transposon 3' terminal repeat sequence (SEQ ID NO:7) connected in this order. In certain embodiments, the nucleic acid constructs of the invention comprise a CMV promoter sequence (SEQ ID NO:1), a PiggyBac transposase coding sequence comprising a c-myc nuclear localization signal (SEQ ID NO:2), a PiggyBac transposon 5 'terminal repeat (SEQ ID NO:3), a polyclonal insertion site (SEQ ID NO:5), a polyA tailing signal sequence 2(SEQ ID NO:6), and a PiggyBac transposon 3' terminal repeat (SEQ ID NO:7) in sequential linkage.

In certain preferred embodiments, all TTAA except those contained within the 5 'and 3' terminal repeats are deleted from the nucleic acid construct of each embodiment of the present invention. Thus, in certain preferred embodiments, the nucleic acid constructs of the invention comprise, in sequence, a CMV promoter sequence (SEQ ID NO:1), a PiggyBac transposase coding sequence containing a c-myc nuclear localization signal (SEQ ID NO:2), a PiggyBac transposon 5 'terminal repeat (SEQ ID NO:3), a polyclonal insertion site (SEQ ID NO:5), a polyA tailing signal sequence 2(SEQ ID NO:6), and a PiggyBac transposon 3' terminal repeat (SEQ ID NO:7), with all TTAA except those contained within the 5 'and 3' terminal repeats being deleted.

In certain embodiments, the nucleic acid constructs of the invention have one or more expression cassettes of the same or different gene of interest inserted at, or replaced by, the polyclonal insertion site. In general, the number of expression boxes can be set as desired, e.g., 1, 2, 3, or more. The gene of interest typically expresses a polypeptide of interest, such as an active protein, including antibodies, such as an scFv. Exemplary polypeptides of interest include, but are not limited to: various reporters such as luciferin reporter proteins (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and the like), luciferases (e.g., firefly luciferase, renilla luciferase, and the like); functional proteins having a biological function of interest, such as TP53, GM-CSF, OCT4, SOX2, Nanog, KLF4, c-Myc, and the like; an antibody, such as an scFv, or a functional fragment thereof, such as an anti-PD 1 antibody or an scFv thereof; various types of Chimeric Antigen Receptors (CARs), such as CARs against CD19 or PD1, and the like. Sequences that can be inserted into or substituted for a polyclonal insertion site also include RNAi genes.

In certain embodiments, the nucleic acid construct of the invention is a recombinant vector. The recombinant vector may be a recombinant cloning vector, a recombinant eukaryotic expression plasmid, or a recombinant viral vector. The backbone vector of the recombinant cloning vector may be derived from pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vectors, pUC57, pMAX or pDC315 series vectors. The backbone vector of the recombinant expression vector can be derived from pCDNA3 series vectors, pCDNA4 series vectors, pCDNA5 series vectors, pCDNA6 series vectors, pRL series vectors, pUC57 vectors, pMAX vectors or pDC315 series vectors. The backbone vector of the recombinant viral vector can be derived from a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retrovirus vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector.

The recombinant vectors of the present invention can be constructed using methods well known in the art. For example, the nucleic acid construct of the present invention may be incorporated into a backbone vector after adding appropriate cleavage sites to each end of the backbone vector, depending on the cleavage sites contained in the backbone vector.

The nucleic acid constructs and recombinant vectors of the invention can be used as a tool for integrating foreign gene expression cassettes into the genome of host cells, or as a tool for genomic studies, gene therapy, cell therapy, or stem cell induction and differentiation after induction. In certain embodiments, the nucleic acid constructs and recombinant vectors of the invention may also be used in the manufacture of a medicament or agent for integration of a foreign gene expression cassette into the genome of a host cell, or in the manufacture of a medicament or formulation for genomic studies, gene therapy, cell therapy, or stem cell induction and differentiation following induction.

Thus, in certain embodiments, the invention also provides a recombinant cell comprising or transformed with a nucleic acid construct or recombinant vector according to any embodiment of the invention. Suitable recombinant cells can be any cell of interest, including but not limited to mammalian cells, such as primary culture T cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells, CHO cells, or the like. In certain embodiments, the recombinant cell has integrated into its genome an expression cassette of interest that is integrated into the genome of the recombinant cell via a nucleic acid construct or recombinant vector of the invention.

Thus, the invention also includes methods of making cells having a foreign gene expression cassette integrated into their genome and recombinant cells obtained thereby. The method of the present invention comprises transfecting a recombinant vector of the present invention containing an expression cassette of interest into a cell of interest, and culturing the transfected cell. Generally, transfected cells are cultured for more than three generations and cells with stably integrated expression cassettes into the genome are obtained. Methods for cell transfection and culture are conventional in the art, and suitable methods for transfection and culture can be selected according to different cell types.

In certain embodiments, when the vector of the invention comprises an expression frame for a therapeutic protein, the vector, or a cell comprising the vector, or a cell having integrated the expression frame in its genome via the vector, is useful for a medical use, for example, for treating a disease treatable by the therapeutic protein, or for preparing a medicament or formulation for treating a disease treatable by the therapeutic protein, suitable therapeutic proteins are well known in the art, including various antibodies (including single chain antibodies), chimeric antigen receptors, cytokines, and the like, in certain embodiments, a therapeutic protein suitable for the invention may be a protein known in the art for treating high expression CD19, CD20, CEA, GD2, GPC3, FR, PSMA, gp100, CA 3, CD 171/L3-CAM, IL-13R 3, MART-1, ERBB, NY-ESO-1, MAGE family protein, BAEGGFE family protein, GAEGGFE family protein, MUGFP, EGFP-72, EGCG-72, EGCG-14, EGCG-72, EGCG, VEGFR-72, EGCG, VEGFA, EGCG, EG.

Thus, the invention also includes the use of one or more of the various nucleic acid constructs, recombinant vectors, and cells of the invention in the preparation of a medicament for treating cancer, as well as methods of treating cancer in an individual using one or more of the various nucleic acid constructs, recombinant vectors, and cells of the invention or pharmaceutical compositions thereof. Preferably, the nucleic acid constructs, recombinant vectors, and cells contain an expression cassette for a therapeutic protein; more preferably, the therapeutic protein specifically binds or targets one or more of the proteins highly expressed in cancer described above (which binding or targeting would result in the inhibition of growth, metastasis, or killing of cancer cells that highly express the protein). Preferably, the cancer may be one or more of the cancers described hereinbefore which highly express one or more of the proteins, or may be selected from acute B-lymphocyte leukemia, chronic B-lymphocyte leukemia, mantle cell lymphoma, non-hodgkin's lymphoma, multiple myeloma lung cancer, liver cancer, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, osteosarcoma, pancreatic cancer and prostate cancer.

Administration may be by various modes of administration known in the art, including but not limited to topical administration and the like; the specific administration mode can be determined according to different drug types and disease types. The dose to be administered can be determined by those skilled in the art according to the age, sex, severity of disease, drug used, etc. of the patient.

The pharmaceutical compositions can contain a therapeutically effective amount of the nucleic acid constructs, vectors, and/or cells described herein. The therapeutically effective amount is an amount effective to treat or alleviate one or more symptoms of the disease and can be determined by one of skill in the art using routine methods. The pharmaceutical composition may also contain a suitable pharmaceutically acceptable carrier or excipient. For example, for pharmaceutical compositions containing cells, suitable carriers or excipients suitable for maintaining cell viability and compatibility with the animal body may be included.

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 construction of the vector

Construction of pNBC vector

The sequence of CMV promoter sequence (SEQ ID NO:1), the sequence of PiggyBac transposase coding sequence containing c-myc nuclear localization signal (SEQ ID NO:2), PiggyBac transposon 5 'terminal repeat sequence (SEQ ID NO:3), synthetic polyA tailing signal sequence (SEQ ID NO:4), multiple cloning insertion site (SEQ ID NO:5), SV40polyA tailing signal sequence (SEQ ID NO:6) and PiggyBac transposon 3' terminal repeat sequence (SEQ ID NO:7) were synthesized by Shanghai Jervey Biotech, and PacI and AscI cleavage sites were added to both ends, pUC57 (purchased from Shanghai Jervey), the obtained vector was named pNBC vector, and the vector structure was shown in FIG. 1.

Construction of pNBC (-) vector

The construction was carried out in a manner similar to that of pNBC except that the synthetic polyA tailing signal sequence (SEQ ID NO:4) was not included, and the fragment was inserted into pUC57 (purchased from George, Shanghai) and named pNBC (-) vector, the vector structure of which is shown in FIG. 2.

Construction of pNBC-AD vector

The first half was constructed as pNBC (-), except that after pNBC (-) was obtained, "TTAA" was deleted in pNBC (-) except for the 5 'terminal repeat and the 3' terminal repeat, including 2 in SV40poly A tailing sequence, 3 between the replication origin and the CMV promoter, and 1 between the replication origin and the kanamycin resistance gene.

The BGH poly (A) tailing signal sequence (SEQ ID NO:8) is entrusted to the Shanghai Jiehy Biotech limited to be synthesized to replace the SV40poly A tailing signal sequence; the name pNBC-i (-).

Synthesis of primers (jerry):

Nde I-CMV：TTGGCATATGATACACTTGATGTACTG(SEQ ID NO:15)

Xho I-CMV：GCCTCTCGAGGACATTGATTATTGAC(SEQ ID NO:16)

Xho I-ori：TGTCCTCGAGAGGCCTCACGTGACATGT(SEQ ID NO:17)

Inter-R：ATGACCAAAATCCCCGTGAGTTTTCGTTCC(SEQ ID NO:18)

Inter-F：AACGAAAACTCACGGGGATTTTGGTCATGC(SEQ ID NO:19)

Nco I-KanR：GTGACCCATGGCGATGCCT(SEQ ID NO:20)

PCR amplification was carried out using pNBC (-) as a template and primers Nde I-CMV/Xho I-CMV, Xho I-ori/Inter-R and Inter-F/Nco I-KanR, respectively, in accordance with

The HS DNA polymerase kit is used for explaining the operation, 264bp, 742bp and 274bp fragments are respectively amplified, and the amplification is carried out after the fragments are respectively recovered by electrophoresis in a ratio of 1:1, uniformly mixing the mixture as a template, performing pull-in PCR amplification by using a primer Nde I-CMV/Nco I-KanR to obtain 1238bp fragment double-stranded DNA, and recovering the fragment after double enzyme digestion by Nde I + NcoI (NEB);

carrying out ASC I + Nde I double enzyme digestion on the plasmid pNBC-I (-) and respectively recovering a 2568bp fragment and a 1860bp fragment; the 1860bp fragment is cut by Nco I enzyme, and a 623bp fragment is recovered; the 2568bp fragment, the 623bp fragment and the 1238bp fragment after enzyme digestion are subjected to three-fragment ligation (solution I) to obtain a vector which is named as pNBC-AD and is deleted with an unnecessary TTAA sequence, and the vector structure is shown in figure 3.

Construction of pNBC-D3 vector

The construction method is the same as that of pNBC (-), and is different from that of the pNBC (-), in that the 5 'terminal repetitive sequence (SEQ ID NO:3) of the PiggyBac transposon is also replaced by a 3' terminal repetitive sequence (SEQ ID NO:7), the fragment is loaded into pUC57 (purchased from Shanghai Jieri organisms) and is named as a pNBC-D3 vector, and the vector structure is shown in figure 4.

Construction of pNBC-D5 vector

The construction method is the same as that of pNBC (-), and is different from that of the pNBC (-), in that the 3 'terminal repetitive sequence (SEQ ID NO:7) of PiggyBac transposon is replaced by a 5' terminal repetitive sequence (SEQ ID NO:3), the fragment is loaded into pUC57 (purchased from Shanghai Jieri organisms) and is named as a pNBC-D5 vector, and the vector structure is shown in figure 5.

Example 2: construction of vector containing foreign Gene expression cassette

1. Construction of pNBC vector containing EGFP expression cassette

(1) The sequence of the DTS EF1 α promoter (shown in SEQ ID NO:8) was synthesized by Shanghai Jiehy Biotech Co., Ltd., Xba I and EcoR I restriction sites were added to both ends, and the vectors prepared in example 1 were inserted to obtain pNB338C, pNB338C (-), pNB338C-AD, pNB338C-D3 and pNB338C-D5, respectively.

(2) And (2) entrusting Shanghai Jiehy Biotechnology limited company to synthesize an EGFP coding sequence (SEQ ID NO:9), adding EcoR I and Sal I enzyme cutting sites at two ends respectively, filling the obtained vectors in the step (1) to obtain corresponding vectors which are named as pNB338C-EGFP, pNB338C-EGFP (-), pNB338C-EGFP-AD, pNB338C-EGFP-D3 and pNB338C-EGFP-D5 respectively.

2. Construction of pNB338B-EGFP vector

Based on a pNB328-EGFP vector disclosed by CN105154473A, an EF1a promoter sequence in the pNB328-EGFP is replaced by a DTS EF1a promoter with the sequence of SEQ ID NO. 8, so that the pNB338B-EGFP vector is obtained.

3. Construction of pNB338CFL-EGFP (-) vector

Shanghai Jiehy Biotech Co., Ltd was entrusted to synthesize the full-length 5 'ITR sequence (SEQ ID NO:10) and the full-length 3' ITR sequence (SEQ ID NO:11) of the PB transposon, and the 5 'ITR and the 3' ITR of the vector pNB338C-EGFP (-) were replaced with each other, to obtain the full-length ITR PB vector pNB338CFL-EGFP (-) containing the EGFP expression cassette. The structure of the carrier is shown in FIG. 6.

4. Construction of pNBC vector containing PD-1 single-chain antibody coding sequence

The method is the same as the construction method of the EGFP-containing expression cassette vector, and the coding sequence of EGFP in pNB338C-EGFP, pNB338C-EGFP (-), pNB338C-EGFP-AD, pNB338C-EGFP-D3 and pNB338C-EGFP-D5 are replaced by the coding sequence of PD-1 single-chain antibody (the sequence is shown as SEQ ID NO: 12), so that pNB338C-PD1scFv, pNB338C-PD1scFv (-), pNB338C-PD1scFv-AD, pNB338C-PD1scFv-D3 and pNB338C-PD1scFv-D5 are obtained respectively.

5. Construction of pNB338B-PD1scFv vector

Based on a pNB328-EGFP vector disclosed by CN105154473A, an EF1a promoter sequence in the pNB328-EGFP is replaced by a DTS EF1a promoter with a sequence of SEQ ID NO. 8, and an EGFP coding sequence is replaced by a PD-1 single-chain antibody coding sequence with a sequence of SEQ ID NO. 12, so that the pNB338B-PD1scFv vector is obtained.

6. Construction of pNBC vector containing CD19CAR coding sequence

The same method as the above-mentioned EGFP expression cassette-containing vector was followed, and the coding sequence of EGFP in pNB338B-EGFP, pNB338C-EGFP (-) and pNB338C-EGFP-AD was replaced by the coding sequence of CD19CAR (the sequence is shown in SEQ ID NO: 21), to obtain pNB338B-CD19CAR, pNB338C-CD19CAR (-), and pNB338C-CD19CAR-AD, respectively.

Example 3: detection of integration efficiency of EGFP-containing expression cassette vector in CHO cells

Preparation of 2X 10⁶The CHO cells with vigorous metabolism were transfected into the cell nucleus by a Lonza-Nucleofector 2b instrument with pNB338B-EGFP, pNB338C-EGFP, pNB338C-EGFP (-), pNB338C-EGFP-AD, pNB338C-EGFP-D3, pNB338C-EGFP-D5 and pNB338CFL-EGFP (-) all having a mass of 6. mu.g (Nucleofector)^TM2b procedure H-014), standing at 37 ℃ with 5% CO₂And (5) incubator culture. After the cells are full, subculturing according to the ratio of 1: 5. Changes in the proportion of EGFP positive cells were detected by flow cytometry at day 13 after transfection, after 3 passages, and CHO cells without transfected plasmid served as controls for flow cytometry detection.

When the cells were diluted for passage at a ratio of 1:5, the non-integrated plasmid was lost rapidly as the cells divided. After 3 passages, cells positive for green fluorescence can be considered to have stably integrated the green fluorescence expression cassette. The efficiency of integration can be determined by flow-detecting the proportion of green fluorescent positive cells.

As shown in FIG. 7, FIGS. 7A-7C show that the integration efficiencies of pNB338C-EGFP, pNB338C-EGFP (-), and pNB338C-EGFP-AD were 49.8%, 59.64%, and 54.43%, respectively (FIGS. 7A-7C), which were higher than 32.15% of pNB338B-EGFP (FIG. 7D). Whereas, when both ITRs were 3' terminal repeats, the integration efficiency was about 35% (FIG. 7E). When both ITRs were 5' terminal repeats, no integration was observed (FIG. 7F), which is substantially consistent with the control results of flow assay. When both ITRs were full-length sequences, the integration efficiency was 54.11% (fig. 7G), which was not significantly different from the minimal ITR integration efficiency. The quantitative histogram results of FIGS. 7A-7G are shown in FIG. 7H. The integration efficiency of pNB338C-EGFP (-) with pNB338C-EGFP-AD was relatively highest; the integration efficiency of pNB338CFL-EGFP (-) was substantially equivalent to that of pNB338C-EGFP (-) and pNB 338C-EGFP-AD.

pNB338C-EGFP (-), pNB338C-EGFP-AD were roughly equivalent in integration efficiency, but the fluorescence intensity of pNB338C-EGFP-AD was overall stronger than that of pNB338C-EGFP (-), indicating that the expression intensity of pNB338C-EGFP-AD was significantly higher than that of pNB338C-EGFP (-).

Example 4: detection of PD-1 antibody expression quantity after PBMC (peripheral blood mononuclear cell) from different individual sources is transfected with pNBC (pNBC) vector containing PD-1 single-chain antibody coding sequence

The pNB338C-PD1scFv, pNB338C-PD1scFv (-), pNB338C-PD1scFv-AD, pNB338C-PD1scFv-D3, pNB338C-PD1scFv-D5 and pNB338B-PD1scFv containing the PD-1 single-chain antibody coding sequence constructed in example 2 were electroporated by a Lonza2 b-Nuclear organism instrument with PBMCs (Nuclear organism) derived from 5 donor subjects (all 5 donor subjects were healthy adults)^TM2b procedure U-014), activated T cells obtained by electroporation at 2X 10⁶Cell number cells were harvested and counted at 2X 10⁶Cells/well, plated in 6-well plates with 3ml AIM-V culture medium, placed at 37 deg.C and 5% CO₂Culturing in an incubator for 24 hr, collecting cell supernatant, and storing at-20 deg.C.

The expression level of PD-1scFv was measured by ELISA. An ELISA plate is coated by using purified human PD-1 antigen recombinant protein, a mouse anti-human IgG4mAb with an HRP label is used as a secondary antibody, and a standard curve is prepared by using the purified PD-1 single-chain antibody as a standard substance. The purified human PD-1 antigen is prepared by the following method: the synthetic sequence is SEQ ID NO: 13 comprising at the 5' end a light chain signal peptide coding sequence, atThe 5 'end and the 3' end are respectively connected with an EcoRI enzyme cutting site linker and an XbaI enzyme cutting site linker, and then are connected to a pCDNA3.4 vector to construct an expression vector for over-expressing the human PD-1 antigen. According to ExpicHO^TMExpression System (available from Thermo Fisher company, Cat. No.: A29133) instructions, ExpicHO was used^TMAfter the expression system carries out overexpression on the fusion protein, the expression product is purified by using MabSelect affinity chromatography resin of GE Healthcare according to the operation steps of the instruction, and the human PD-1 antigen protein is obtained. The purified PD-1 single-chain antibody was prepared in a similar manner as described above, with SEQ ID NO: 13 substitution to SEQ ID NO: 14 comprising a light chain signal peptide coding sequence at the 5' end, and purification steps of other homologous PD-1 antigens. The expression amount of PD-1 single-chain antibodies derived from T cells of five patients, which are electrically transferred with pNB338C-PD1scFv, pNB338C-PD1scFv (-), pNB338C-PD1scFv-AD, pNB338C-PD1scFv-D3, pNB338C-PD1scFv-D5 and pNB338B-PD1scFv, was quantitatively detected after 50-fold dilution of the test sample.

As shown in FIG. 8, the median values of the expression amounts of PD-1 single-chain antibody in primary T cells of 5 donor subjects electroporated with pNB338C-PD1scFv (-) and pNB338C-PD1scFv-AD vectors were 96.06ng/mL and 97.66ng/mL, respectively, which were higher than the median value of the expression amounts of PD-1 single-chain antibody in primary T cells of five patients electroporated with pNB338B-PD1scFv vector, 77.94 ng/mL. The median value of the expression level of PD-1 antibody in primary cells of 5 donor subjects electroporated with the pNB338C-PD1scFv vector was 81.45ng/mL, slightly higher than that of the pNB338B-PD1scFv vector. The median value of the PD-1 antibody expression of primary cells of 5 donor subjects transfected with the pNB338C-PD1scFv-D3 vector was 38.50ng/mL, which was significantly lower than the median value of the PD-1 antibody expression of the pNB338C-PD1scFv (-) vector. And the cells electroporated with pNB338C-PD1scFv-D5 detected almost no expression of the expression level of the PD-1 single-chain antibody.

Example 5: pNBC vector CD19CAR-T cell ratio detection by transfection of CD19 CAR-encoding sequence-containing pNBC vector PBMC from different individual sources

(1) Wrapping a plate: a6-well plate was added with 1ml of PBS solution, and CD19 antigen (Acro, cat # CD9-H5259) and anti-CD 28 antibody (Merck Millipore, cat # CBL517) were added to the plate at final concentrations of 5. mu.g/ml each, at 4 ℃ and overnight.

(2) PBMC were isolated from blood of 3 donor subjects (3 donor subjects were healthy adults), 5ml of 2% FBS-containing AIM-V medium was added to a15 ml centrifuge tube, cells were transferred to the centrifuge tube, 1200rpm, and centrifuged for 5 min; the supernatant was discarded, transferred to a six-well plate containing 3ml of 2% FBS AIM-V medium, and cultured for 20-30 min.

(3) Collecting the cells into a15 ml centrifuge tube, centrifuging at 1200rpm for 5min, discarding the supernatant, and resuspending and counting 5ml of 0.9% physiological saline; the cells were added to 1.5ml centrifuge tubes at 2X 10 per tube⁶Centrifuging the cells at 2000rpm for 4 min; the supernatant was discarded.

(4) Preparing electrotransfer solution by using a Lonza electrotransfer kit, wherein each part of electrotransfer solution is 100 mu l (18 mu l Nucleofector)^TMSupplement+82μl Nucleofector^TMSolution), plasmids pNB338B-CD19CAR, pNB338C-CD19CAR (-) and pNB338C-CD19CAR-AD were added at 6. mu.g each, and the cells were resuspended in electrotransformation, and the cells were electrotransferred using the U-014 program. The cells after electroporation were added to 12-well plates, 1ml of AIM-V (containing 2% FBS) medium per well, and placed in an incubator for 4-6 hours.

(5) Discarding the coating solution in the coating pore plate, transferring the T cells which are electrically transferred and placed for 4-6 hours into the coated 6-pore plate; each well was supplemented with 2% FBS-containing AIM-V medium to 3ml, IL-2(500IU/ml) was added and mixed gently as day 0.

(6)37℃，5％CO₂Placing in a cell culture box for 5 days; on day 5, the cells were transferred out of the coated well plate and cultured; cell density was observed under a microscope, subcultured every 1-2 days, and culture was continued using AIM-V medium containing 2% FBS supplemented with IL-2(50 IU/ml).

CAR-T cells were taken on day 13 and their positive rate was flow-tested.

(1) Adding 1X 10 to a flow pipe⁶(ii) individual cells; adding 1ml PBS phosphate buffer solution for washing, centrifuging for 5min at 400g, and discarding the supernatant; adding 100 ul PBS phosphate buffer solution for resuspension;

(2) adding 2 μ l of biotinylated human CD19, Fc label, primary amine labeled (Acro, CD-H8259) flow antibody, mixing, placing in refrigerator at 2-8 deg.C, and incubating in dark for 30 min; setting a group of blank controls without adding reagents;

(3) adding 1ml PBS phosphate buffer solution, centrifuging for 5min at 400g, discarding the supernatant, adding 1ml PBS phosphate buffer solution again, centrifuging for 5min at 400g, discarding the supernatant;

(4) adding 100 μ l PBS phosphate buffer solution to resuspend cells, adding 1 μ l PE streptavidin flow antibody (BD, 554061), mixing, placing in refrigerator at 2-8 deg.C, and incubating for 30min in dark;

(5) adding 1ml PBS phosphate buffer solution, centrifuging for 5min at 400g, discarding the supernatant, adding 1ml PBS phosphate buffer solution again, centrifuging for 5min at 400g, discarding the supernatant; the cells were resuspended in 400. mu.l PBS phosphate buffer and examined by flow cytometry.

The results are shown in fig. 9, where the positive rates of CAR-T cells were 64.21% for the pNB338B-CD19CAR group, 73.34% and 77.90% for the pNB338C-CD19CAR (-) group and the pNB338C-CD19CAR-AD group, respectively, and there was a significant difference between the pNB338C-CD19CAR-AD group and the pNB338B-CD19CAR group (p ═ 0.0417). The results in fig. 9 show that the integration efficiency of pNB338C-CD19CAR (-) with pNB338C-CD19CAR-AD is significantly higher than that of pNB338B-CD19 CAR.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

Sequence listing

<110> Shanghai cell therapy group Co., Ltd

SHANGHAI CELL THERAPY Research Institute

<120> an improved PB transposon system and the use thereof

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gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 300

tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 360

cgcaacgggt ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca 420

cgcgcccgcc gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct 480

cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg taagtagctc aggtcgagac 540

cgggcctttg tccggcgctc ccttggagcc tacctagact cagccggctc tccacgcttt 600

gcctgaccct gcttgctcaa ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca 660

gatccaagct gtgaccggcg cctac 685

<210>9

<211>720

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210>10

<211>311

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt 60

tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc 120

ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt 180

gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa 240

ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt 300

atagatacct c 311

<210>11

<211>230

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata aataaacata 60

aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata aaacttaata 120

tctattcaaa ttaataaata aacctcgata tacagaccga taaaacacat gcgtcaattt 180

tacgcatgat tatctttaac gtacgtcaca atatgattat ctttctaggg 230

<210>12

<211>1488

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagcaa aggtgtcagt acatctggct atagttattt gcactggtat 180

caacagaaac ctggccaggc tcccaggctc ctcatctatc ttgcatccta cctagaatct 240

ggcgtcccag ccaggttcag tggtagtggg tctgggacag acttcactct caccatcagc 300

agcctagagc ctgaagattt tgcagtttat tactgtcagc acagcaggga ccttccgctc 360

acgttcggcg gagggaccaa agtggagatc aaaggtggag gcggttcagg cggaggtggc 420

agcggcggtg gcgggtcgca ggtgcagctg gtgcagtccg gcgtggaggt gaagaagcct 480

ggcgcctccg tcaaggtgtc ctgtaaggcc tccggctaca ccttcaccaa ctactacatg 540

tactgggtgc ggcaggcccc aggccaggga ctggagtgga tgggcggcat caacccttcc 600

aacggcggca ccaacttcaa cgagaagttc aagaaccggg tgaccctgac caccgactcc 660

tccaccacaa ccgcctacat ggaactgaag tccctgcagt tcgacgacac cgccgtgtac 720

tactgcgcca ggcgggacta ccggttcgac atgggcttcg actactgggg ccagggcacc 780

accgtgaccg tgtcctccga gtccaaatat ggtcccccat gcccaccatg cccagcacct 840

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 900

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 960

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1200

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1380

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1440

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatga 1488

<210>13

<211>924

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

cagatcccac aggcgccctg gccagtcgtc tgggcggtgc tacaactggg ctggcggcca 120

ggatggttct tagactcccc agacaggccc tggaaccccc ccaccttctc cccagccctg 180

ctcgtggtga ccgaagggga caacgccacc ttcacctgca gcttctccaa cacatcggag 240

agcttcgtgc taaactggta ccgcatgagc cccagcaacc agacggacaa gctggccgcc 300

ttccccgagg accgcagcca gcccggccag gactgccgct tccgtgtcac acaactgccc 360

aacgggcgtg acttccacat gagcgtggtc agggcccggc gcaatgacag cggcacctac 420

ctctgtgggg ccatctccct ggcccccaag gcgcagatca aagagagcct gcgggcagag 480

ctcagggtga cagagagaag ggcagaagtg cccacagccc accccagccc ctcacccagg 540

tcagccggcc agttccaaac cctggtggtt ggtgtcgtgg gcggcctgct gggcagcctg 600

gtgctgctag tctgggtcct ggccgtcatc tgctcccggg ccgcacgagg gacaatagga 660

gccaggcgca ccggccagcc cctgaaggag gacccctcag ccgtgcctgt gttctctgtg 720

gactatgggg agctggattt ccagtggcga gagaagaccc cggagccccc cgtgccctgt 780

gtccctgagc agacggagta tgccaccatt gtctttccta gcggaatggg cacctcatcc 840

cccgcccgca ggggctcagc tgacggccct cggagtgccc agccactgag gcctgaggat 900

ggacactgct cttggcccct ctga 924

<210>14

<211>1545

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaagccccag ctcagcttct cttcctcctg ctactctggc tcccagatac caccggagaa 120

attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 180

tcctgcaggg ccagcaaagg tgtcagtaca tctggctata gttatttgca ctggtatcaa 240

cagaaacctg gccaggctcc caggctcctc atctatcttg catcctacct agaatctggc 300

gtcccagcca ggttcagtgg tagtgggtct gggacagact tcactctcac catcagcagc 360

ctagagcctg aagattttgc agtttattac tgtcagcaca gcagggacct tccgctcacg 420

ttcggcggag ggaccaaagt ggagatcaaa ggtggaggcg gttcaggcgg aggtggcagc 480

ggcggtggcg ggtcgcaggt gcagctggtg cagtccggcg tggaggtgaa gaagcctggc 540

gcctccgtca aggtgtcctg taaggcctcc ggctacacct tcaccaacta ctacatgtac 600

tgggtgcggc aggccccagg ccagggactg gagtggatgg gcggcatcaa cccttccaac 660

ggcggcacca acttcaacga gaagttcaag aaccgggtga ccctgaccac cgactcctcc 720

accacaaccg cctacatgga actgaagtcc ctgcagttcg acgacaccgc cgtgtactac 780

tgcgccaggc gggactaccg gttcgacatg ggcttcgact actggggcca gggcaccacc 840

gtgaccgtgt cctccgagtc caaatatggt cccccatgcc caccatgccc agcacctgag 900

ttcctggggg gaccatcagt cttcctgttc cccccaaaac ccaaggacac tctcatgatc 960

tcccggaccc ctgaggtcac gtgcgtggtg gtggacgtga gccaggaaga ccccgaggtc 1020

cagttcaact ggtacgtgga tggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1080

gagcagttca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1140

ctgaacggca aggagtacaa gtgcaaggtc tccaacaaag gcctcccgtc ctccatcgag 1200

aaaaccatct ccaaagccaa agggcagccc cgagagccac aggtgtacac cctgccccca 1260

tcccaggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctac 1320

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1380

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaggct aaccgtggac 1440

aagagcaggt ggcaggaggg gaatgtcttc tcatgctccg tgatgcatga ggctctgcac 1500

aaccactaca cacagaagag cctctccctg tctctgggta aatga 1545

<210>15

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ttggcatatg atacacttga tgtactg 27

<210>16

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

gcctctcgag gacattgatt attgac 26

<210>17

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

tgtcctcgag aggcctcacg tgacatgt 28

<210>18

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

atgaccaaaa tccccgtgag ttttcgttcc 30

<210>19

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

aacgaaaact cacggggatt ttggtcatgc 30

<210>20

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

gtgacccatg gcgatgcct 19

<210>21

<211>1461

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacta agttggaaat aacaggtgga ggcggttcag gcggaggtgg cagcggcggt 420

ggcgggtcgg aggtgaaact gcaggagtca ggacctggcc tggtggcgccctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gtcaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccctggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaagctcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgctg a 1461

Claims

1. A nucleic acid construct comprises a promoter for controlling expression of PiggyBac transposase, a PiggyBac transposase coding sequence, a PiggyBac transposon 5 'terminal repeat sequence, an optional polyA tailing signal sequence 1, a polyclonal insertion site, a polyA tailing signal sequence 2 and a PiggyBac transposon 3' terminal repeat sequence which are connected in sequence.

2. The nucleic acid construct of claim 1,

the 5 'terminal repetitive sequence of the PiggyBac transposon is a truncated 5' terminal repetitive sequence of the PiggyBac transposon; and/or

The 3 'terminal repetitive sequence of the PiggyBac transposon is a truncated 3' terminal repetitive sequence of the PiggyBac transposon; and/or

The nucleic acid construct does not contain the polyA tailed signal sequence 1; and/or

The nucleic acid construct has the TTAA sequence deleted except the TTAA sequence contained in the 5 'terminal repeat and the 3' terminal repeat.

3. The nucleic acid construct of any one of claims 1-2, wherein the nucleic acid construct has one or more of the following characteristics:

(1) the promoter for controlling the expression of PiggyBac transposase is selected from a CMV promoter, an SV40 promoter, a PGK promoter and a chicken β -actin promoter, preferably the CMV promoter, and more preferably the sequence of the promoter is shown as SEQ ID NO. 1;

(2) the PiggyBac transposase coding sequence contains a nuclear localization signal, and the nuclear localization signal is selected from a c-myc nuclear localization signal and an SV40 nuclear localization signal; preferably, the piggyBac transposase coding sequence is shown as SEQ ID NO. 2; preferably, the nuclear localization signal is located at the N-terminus of the PiggyBac transposase coding sequence;

(3) the 5' terminal repetitive sequence of the PiggyBac transposon is shown as SEQ ID NO. 3;

(4) the polyA tailing signal sequence 1 is shown as SEQ ID NO. 4;

(5) the polyclonal insertion site is shown as SEQ ID NO. 5;

(6) the polyA tailing signal sequence 2 is an SV40polyA tailing signal sequence; preferably, the polyA tailing signal sequence 2 is shown as SEQ ID NO 6; and

(7) the 3' terminal repetitive sequence of the PiggyBac transposon is shown as SEQ ID NO. 7.

4. The nucleic acid construct of claim 1, wherein said nucleic acid construct is selected from the group consisting of:

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the multiple cloning site of the nucleic acid construct is operably inserted with one or more identical or different expression cassettes or the multiple cloning site is replaced with one or more identical or different expression cassettes.

6. A recombinant vector comprising the nucleic acid construct of any one of claims 1-5;

preferably, the recombinant vector is a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant viral vector; preferably, the recombinant cloning vector is a recombinant vector obtained by recombining the nucleic acid construct of any one of claims 1 to 5 with a vector of the pUC18, pUC19, pMD18-T, pMD19-T, pGM-T, pUC57, pMAX or pDC315 series; the recombinant expression vector is obtained by recombining the nucleic acid construct of any one of claims 1 to 5 with a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, a pMAX vector or a pDC315 series vector; the recombinant virus vector is a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retrovirus vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector.

7. A recombinant cell comprising the nucleic acid construct of any one of claims 1-5 or the recombinant vector of claim 6 or 7; preferably, the recombinant host cell is a recombinant mammalian cell, such as a recombinant primary culture T cell, Jurkat cell, K562 cell, embryonic stem cell, tumor cell, HEK293 cell, or CHO cell.

8. A method for preparing a cell having a foreign gene expression cassette integrated into its genome, the method comprising the steps of transfecting the recombinant vector of claim 6 or 7 into a cell of interest, and culturing the transfected cell; preferably, the cells are cultured to more than three generations.

9. A cell having an exogenous gene expression cassette integrated into its genome obtained by the method of claim 8.

10. Use of the nucleic acid construct of any one of claims 1 to 5 or the recombinant vector of claim 6 or 7, selected from the group consisting of:

11. Use of one or more of the nucleic acid construct of any one of claims 1-5, the recombinant vector of claim 6 or 7, and the cell of claim 9 in the preparation of a medicament for treating cancer.

12. The use according to claim 11, wherein the cancer is one or more of CD19, CD20, CEA, GD2, GPC3, FR, PSMA, gp100, CA 3, CD 171/L3-CAM, IL-13R 3, MART-1, ERBB3, NY-ESO-1, MAGE family protein, BAGE family protein, GAGE family protein, AFP, MUC 3, CD44v 3/8, CD3, VEGFR 3, IL-11R 3, EGP-2, EGP-40, FBP, 3, PSCA, FSA, PSA, TAG-72, h5T 3, fetal acetylcholine receptor, LeY, EpCAM, IGFR 3, EGFR, EGFRvIII, ERBB3, ERBB, TSGD 72, TSHA-3, GCR 3, GFR 3, EGFRV 3, EGFP, EGFRV 3, GFR, VEGFR, EGFRS, EGFRP 3, EGFRV 3, EGFCS-72, EGFCS, EGFRV 3, EGFCS-72, EGFCH 5, EGFCS-72, EG.

13. Use according to claim 11 or 12, wherein the cancer is selected from one or more of acute B-lymphocytic leukemia, chronic B-lymphocytic leukemia, mantle cell lymphoma, non-hodgkin's lymphoma, multiple myeloma lung cancer, liver cancer, lymphoma, colon cancer, large bowel cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, osteosarcoma, pancreatic cancer and prostate cancer.

14. A method of treating cancer, comprising the steps of: administering to an individual having cancer a medicament comprising one or more of the nucleic acid construct of any one of claims 1-5, the recombinant vector of claim 6 or 7, and the cell of claim 9.

15. The method of claim 14, wherein the cancer is one or more of CD19, CD20, CEA, GD2, GPC3, FR, PSMA, gp100, CA 3, CD 171/L3-CAM, IL-13R 3, MART-1, ERBB3, NY-ESO-1, MAGE family protein, BAGE family protein, GAGE family protein, AFP, MUC 3, CD44v 3/8, CD3, VEGFR 3, IL-11R 3, EGP-2, EGP-40, FBP, 3, PSCA, FSA, PSA, TAG-72, h5T 3, fetal acetylcholine receptor, LeY, EpCAM, IGFR 3, EGFR, EGFRvIII, ERBB3, ERBB, TSGD 72, TSCA-72, GCR 3, GFR, EGFRV 3, EGFP, EGFRV 3, GFR, VEGFR, GFR, EGFRV 3, VEGFR, GFR, EGFCS, EGFRV 3, EGFCS-72, EGFCH 5, EGFCS-72, EGFCH.

16. The method of treatment according to claim 11 or 12, wherein the cancer is selected from one or more of acute B-lymphocytic leukemia, chronic B-lymphocytic leukemia, mantle cell lymphoma, non-hodgkin's lymphoma, multiple myeloma lung cancer, liver cancer, lymphoma, colon cancer, large bowel cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, renal cancer, glioma, melanoma, osteosarcoma, pancreatic cancer, and prostate cancer.