CN114181311A

CN114181311A - Fully human anti-DLL 3scFv and application thereof in CART cell treatment

Info

Publication number: CN114181311A
Application number: CN202111560685.1A
Authority: CN
Inventors: 朱红佳; 徐南; 康立清; 余宙; 沈文燕; 闫志强; 谭靖雯; 叶晶; 贾裕杰; 方小燕
Original assignee: East China Normal University; Shanghai Unicar Therapy Bio Medicine Technology Co Ltd
Current assignee: East China Normal University; Shanghai Unicar Therapy Bio Medicine Technology Co Ltd
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-03-15
Anticipated expiration: 2041-12-20
Also published as: CN114181311B

Abstract

The invention discloses an anti-DLL 3scFv capable of specifically binding DLL 3; two sets of complementarity determining region sequences for the heavy and light chains are shown. The invention also discloses an anti-DLL 3CAR containing the anti-DLL 3scFv, a plasmid of related nucleotide sequence and a recombinant lentiviral vector. The invention verifies the specific killing of anti-DLL 3CAR-T on DLL3 positive tumor cells and the cytokine secretion after receiving the stimulation of target cells; lays a foundation for developing fully human DLL3 antibody and later DLL3 antibody to clinical development.

Description

Fully human anti-DLL 3scFv and application thereof in CART cell treatment

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an anti-DLL3 single-chain antibody (scFv) and a chimeric antigen antibody (CAR), a vector, a cell, a preparation method and application thereof.

Background

Lung cancer is the most common malignant tumor worldwide and has become the biggest killer of cancer worldwide. The World Health Organization (WHO) international agency for research on cancer (IARC) released the latest cancer data worldwide in 2020, and lung cancer mortality remained the first. Lung cancer is mainly divided into two major types: non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), with NSCLC accounting for approximately 85% of all lung cancer cases and SCLC accounting for 15%. Due to the extreme invasiveness, most lung cancer patients are diagnosed at an advanced stage, and if the lung cancer patients do not have corresponding precise treatment drugs, the lung cancer patients can die within 1 to 2 years, and the five-year survival rate is less than 15%.

In the field of lung cancer, the success of PD- (L)1 mab-based immunotherapy has dominated the field of lung cancer, particularly NSCLC, in recent years, and other classes of immunotherapy continue to dominate the lung cancer news, including combination therapies. There are currently 4 cancer immunotherapies based on the PD1/PDL1 immune checkpoint that have obtained FDA approval for the treatment of non-small cell lung cancer, among which the BMS product PD-1 inhibitor Opdivo, the roche PD-L1 inhibitor Tecentriq, the Merck & Co mordanto PD-1 inhibitor pembrolizumab Keytruda, for the treatment of small cell lung cancer by FDA approval in 2018, 3 months 2019 and 6 months 2019, respectively. Not only the immunotherapy makes great progress in the field of lung cancer, but also the research on the targeted therapy of lung cancer is always a research hotspot, and the targeted drugs are divided into two categories, namely antibody drugs and small molecule inhibitor drugs. The small molecule inhibitor drug targets the driver gene mutation on lung cancer cells, and from the current clinical practice application, the main gene variations in lung cancer include: EGFR, ALK, ROS1, BRAF, MET, ROS1, RET, KRAS, HER2 and the like, 5 targeted EGFR small molecule inhibitors (gefitinib and the like) are approved to be marketed for treating NSCLC, and EGFR-TKI becomes a first-line standard treatment scheme for patients with EGFRM + advanced NSCLC. For lung cancer patients with positive sensitive gene mutation, the targeted small molecule inhibitor greatly improves the overall remission rate of NSCLC. Small molecule inhibitors typically target downstream signaling pathways conducted by cells, while monoclonal antibodies target surface markers and receptors on the cell surface. For the patients with late-stage lung cancer with negative driver genes and drug resistance of small molecule inhibitors, cancer immunotherapy and targeted therapy become indispensable choices.

The main targets of the current monoclonal antibody medicines for targeted therapy of solid tumors are VEGF, EGFR, EpCAM, CEACAM5 and the like, wherein the total survival time (OS) of the monoclonal antibody Necitumumab which is approved to be marketed in 2015 is only increased by 1.6 months, and the EGFR is widely expressed in normal tissues and has the risk of fatal blood clotting. This suggests that we need to find specific tumor antigens that can be identified and identified.

According to the classification of tumor antigen specificity, tumor antigens can be classified into tumor-associated antigen TAA and tumor-specific antigen TSA. Tumor Specific Antigens (TSA) are "expressed" only in tumor cells, but not in any normal cells at different developmental stages, and are antigens produced by the accumulation of genetic mutations in cancer cells (e.g., Kras mutations), with high individual specificity; tumor-associated antigens (TAA) are present in normal cells in low amounts and are highly expressed in Tumor cells (CD19, CD20, CD38, Her 2). The selection of target points in immunotherapy is important, and the antigen which is highly expressed in tumor and not expressed in normal tissues is usually selected as the target point, so that the toxic and side effects of the drug on normal tissues and organs are avoided.

Delta-like canonical Notch ligand 3 (DLL 3) is a single transmembrane glycoprotein specifically overexpressed in tumor tissues but underexpressed or even not expressed in normal tissues, and belongs to a member of the DSL (Delta, serum, lag-2) protein family. The human DLL3 gene was located at 19q13 and its open reading frame length was approximately 1800 bp. Two transcriptional variants encoding different subtypes have been identified for this gene. As a ligand of the Notch signaling pathway, it plays a role in inhibiting or promoting cancer in various tumors and is involved in the development of tumors. DLL3 is a single-chain transmembrane protein with a gene encoding 618 amino acids in length. It is highly conserved during evolution, with 82% homologous sequences in the protein encoded by the human and murine DLL3 genes. The extracellular segment of DLL3 contains 6 Epidermal Growth Factor (EGF) -like repeats necessary for binding to Notch receptors, and 1 conserved DSL (Delta, Serrate, Lag2) domain consisting of 40 amino acids (UniProt/Swiss-Prot Q9NYJ 7). There are 5 DSL ligands in mammals, Delta-like Notch ligand 1(DLL1), DLL3, DLL4, Jagged1(JAG1) and Jagged 2(JAG 2). Of these, DLL3 is most structurally inconsistent. In recent years, numerous studies have shown that the Notch signaling pathway is closely related to the onset of small cell lung cancer. Among them, DLI3(DeltaLike3) as one of the Notch ligands may be associated with neuroendocrine phenotype, and promotes the development of small cell lung cancer by participating in the Notch signaling pathway. Research shows that DLL3 protein can be detected in small cell lung cancer and large cell neuroendocrine tumor tissues, but is not expressed in normal lung tissues, so DLL3 is expected to be a potential target for treating lung cancer.

More and more researchers are focusing on the preparation of antibodies, which can achieve the purpose of preventing and treating diseases by the competitive binding of antibodies with target proteins and further blocking and interfering signal pathways, or can trigger therapeutic effects by using ADCC and CDC effects specifically mediated by antibodies. At present, the hybridoma antibody technology is relatively perfect, a plurality of mouse-derived monoclonal antibody medicines are applied to clinic, but the mouse-derived monoclonal antibody medicines have immunogenicity, so that the development prospect is greatly limited.

With the development of DNA recombination technology and the elucidation of antibody gene structures, genetic engineering antibody technology has been widely used, and phage display technology is one of the important technologies. The phage display technology is a biological technology that can insert the DNA sequence of the fully human protein or polypeptide into the proper position of the structural gene of the coat protein of the phage, so that the fully human gene is expressed along with the expression of the coat protein, and simultaneously, the fully human protein is displayed on the surface of the phage along with the reassembly of the phage. The technology does not need to produce antibodies through animal immunization, really realizes the dream of full humanization of the antibodies, and simultaneously, the screened antibodies have low immunogenicity; easy passing through blood vessel wall, penetrating solid tumor, etc. In addition, the phage display technology links the genotype and the phenotype of the antibody, and the screening period is short, so that the method is an ideal mode for screening the antibody at present.

As mentioned above, ADC drugs targeting DLL3 have entered much of the clinical research phase, however research on Chimeric Antigen Receptor (CAR) modified immune cells targeting DLL3 is still in the research phase due to three challenges with CAR-T treatment of solid tumors: the first is to find tumor-specific antigens that are not or poorly expressed in normal tissues, the second is the high heterogeneity of solid tumors, and the high complexity of the solid tumor immune microenvironment limits many therapeutic approaches to solid tumors, and the third is the difficulty of therapeutic drugs entering tumor tissues.

Disclosure of Invention

To explore CAR-T treatment of solid tumors, the present invention provides an anti-DLL 3scFv capable of specifically binding to DLL 3;

the amino acid sequence of the complementarity determining region CDR of the heavy chain in the anti-DLL 3scFv comprises the CDR1 shown in SEQ ID NO.15, the CDR2 shown in SEQ ID NO.16, the CDR3 shown in SEQ ID NO. 17; the CDR amino acid sequence of the complementarity determining region of the light chain in the anti-DLL 3scFv comprises CDR1 shown in SEQ ID NO.18, CDR2 shown in SEQ ID NO.19, CDR3 shown in SEQ ID NO. 20;

or,

the CDR amino acid sequence of the heavy chain complementarity determining region in the anti-DLL 3scFv comprises CDR1 shown in SEQ ID NO.21, CDR2 shown in SEQ ID NO.22 and CDR3 shown in SEQ ID NO. 23; the CDR amino acid sequence of the complementarity determining region of the light chain in the anti-DLL 3scFv comprises CDR1 shown in SEQ ID NO.24, CDR2 shown in SEQ ID NO.25 and CDR3 shown in SEQ ID NO. 26.

In some embodiments, the anti-DLL 3scFv comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region and the light chain variable region are connected by a flexible linker;

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12;

or,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14.

In some embodiments, the heavy chain variable region has the nucleotide sequence set forth in SEQ ID No.7, and the light chain variable region has the nucleotide sequence set forth in SEQ ID No. 8;

or

The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.9, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 10.

In some embodiments, the amino acid sequence of the anti-DLL 3scFv is set forth in SEQ ID No. 4;

or,

shown as SEQ ID NO. 5.

The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the amino acid sequence of the anti-DLL 3scFv of claim 4; the nucleotide sequence is shown as SEQ ID NO.1 or SEQ ID NO. 2.

Nucleotide sequence

The invention also provides an anti-DLL 3CAR, wherein the anti-DLL 3CAR has the structure of CD8leader-DLL3 scFv-CD8 Hinge-CD8 TM-costimulatory domain-intracellular signal peptide, and comprises a CD8leader membrane receptor signal peptide, the single-chain variable fragment of claim 1, a CD8 Hinge region of a hind chimeric receptor, a CD8 TM chimeric receptor transmembrane region, a costimulatory domain, and an intracellular signal peptide which are connected in series in sequence; the co-stimulatory domain is 4-1 BB; the intracellular signal peptide is CD3 zeta,

the anti-DLL 3CAR target plasmid comprises a lentiviral backbone vector sequence, and a CAR portion, the amino acid sequence comprising:

1) the amino acid sequence of the CD8leader is SEQ ID NO. 50;

2) the amino acid sequence of CD8 Hinge is SEQ ID NO. 51;

3) the amino acid sequence of the CD8 TM transmembrane region is SEQ ID NO. 52;

4) the amino acid sequence of the 41BB co-stimulatory domain is SEQ ID NO. 53;

5) the amino acid sequence of CD3 zeta is SEQ ID NO. 54;

the anti-DLL3 CAR-related element nucleotide sequence corresponds to the amino acid sequence described above;

the method specifically comprises the following steps:

1) the nucleotide sequence of the CD8leader is SEQ ID NO. 35;

2) the nucleotide sequence of CD8 Hinge is SEQ ID NO. 36;

3) the nucleotide sequence of the CD8 TM transmembrane region is SEQ ID NO. 37;

4) the nucleotide sequence of the 41BB co-stimulatory domain is SEQ ID NO. 38;

5) the nucleotide sequence of CD3 zeta is SEQ ID NO. 39;

the present invention also provides a plasmid comprising the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region of DLL3scFv as described above.

The present invention also provides a recombinant lentiviral vector comprising the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region of DLL3scFv as described above.

The invention also provides a cell having modified thereon a chimeric antigen receptor which is an anti-DLL 3CAR as described above.

The invention also provides the use of an anti-DLL 3scFv as described above, or a nucleic acid molecule as described above, or an anti-DLL 3CAR as described above, or a plasmid as described above, or a recombinant lentiviral vector as described above, or a cell as described above, in the preparation of an anti-tumour drug, the tumour being small cell lung cancer.

As used herein, phage display is the process of cloning the gene segment encoding the polypeptide or protein into the proper position of the structural gene of the coat protein of phage to form the correct reading frame, so that the exogenous polypeptide or protein is expressed in fusion with the coat protein, and the expression systems of coat protein PII and PVI are available. The fusion protein is displayed on the surface of the bacteriophage, and the antibody molecule part of the fusion protein can form independent space structure and antigen-specific recognition and binding. After the target antigen and the phage antibody library interact for a period of time, unbound free phage are washed away, screened phage are used for the next round of screening after infecting host cells and carrying out propagation and amplification, and the phage specifically bound with the target molecule can be obtained by enrichment after multiple times of adsorption-elution-amplification. The biggest advantage of the technology is that the genotype and the phenotype are directly linked together, the genotype of the antibody can be known while the target antibody protein is obtained by screening, and the application of downstream genetic engineering technologies such as purification and the like is facilitated. Meanwhile, the monoclonal antibody obtained by the phage display technology, especially the fully human antibody, reduces the immunogenicity of the antibody and has better application value compared with the murine monoclonal antibody.

As used herein, the term "recombinant protein" refers to an artificially designed/constructed protein, rather than a naturally occurring protein. The "recombinant" in the "recombinant protein" of the present invention does not represent the manner in which it is produced, and is used merely to indicate that the "recombinant protein" does not naturally occur. The recombinant protein of the present invention may be an expressed protein, and may be an assembled protein.

As used herein, the term "Fc region" (Fc) consists of the IgG constant regions CH2, CH3 domain, and hinge region.

As used herein, the terms "treatment," "therapy," and "treating" are used interchangeably. The term "treating" includes controlling the progression of a disease, disorder, condition, and associated symptoms, preferably reducing the effect of one or more symptoms of the disease, disorder, condition. This term includes curing the disease or eliminating the symptoms altogether. The term includes relief of symptoms. This term also includes, but is not limited to, non-curative palliative treatment. The term "treating" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the invention to prevent or delay, alleviate or ameliorate the progression of a disease, disorder, condition, or the effects of one or more symptoms of a disease, disorder, condition.

The information represented by the base and amino acid sequences referred to in the invention is shown in the following table, and the detailed sequences are shown in the nucleotide and amino acid sequence table of the specification.

TABLE 1 nucleotide sequence of scFv antibody of 2 positive clones obtained by phage display screening

TABLE 2 scFv antibody amino acid sequences of 2 positive clones obtained by phage display screening

Sequence numbering	Amino acid sequence name
		SEQ ID NO.4	A7-scFv
SEQ ID NO.5	G8-scFv
		SEQ ID NO.6	Linker

TABLE 3 heavy and light chain nucleotide sequences of 2 positive clones obtained by phage display screening

Sequence numbering	Nucleotide sequence name
		SEQ ID NO.7	A7-VH
SEQ ID NO.8	A7-VL
		SEQ ID NO.9	G8-VH
SEQ ID NO.10	G8-VL

TABLE 4 heavy and light chain amino acid sequences of 2 positive clones obtained by phage display screening

TABLE 5 Complementarity Determining Region (CDR) sequences in anti-DLL 3scFv heavy chain light chain

TABLE 6 PUTAM003 expression vector sequences

Sequence numbering	Amino acid sequence name
		SEQ ID NO.27	PUT AM003 vector sequence

TABLE 7 anti-DLL3 CART nucleotide sequence (containing lentiviral backbone)

TABLE 8 anti-DLL3 CART amino acid sequence

The technical scheme of the invention has the following advantages: the invention screens two fully human scFv antibodies targeting DLL3 by using phage display technology. The invention takes DLL3 extracellular segment as antigen, enriches and screens 2 pieces of full-human anti-DLL 3scFv (single chain variable fragment) by phage display technology, has no artificial immunity, belongs to full-human natural phage antibody, and lays a foundation for developing full-human DLL3 antibody and later-stage DLL3 antibody to clinical development; 2 of the fully human anti-DLL 3scFv obtained by screening were identified by Elisa and flow cytometry and were identified to be capable of specifically binding to DLL3 antigen.

The invention verifies the specific killing of anti-DLL 3CAR-T on DLL3 positive tumor cells and the cytokine secretion after receiving the stimulation of target cells; lays a foundation for developing fully human DLL3 antibody and later DLL3 antibody to clinical development.

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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an enrichment screen of phage antibody libraries.

FIG. 2 phase Elisa identifies the specific binding activity of monoclonal phage to the extracellular domain of DLL 3.

FIG. 3 prokaryotic expression of anti-DLL3 soluble antibody A7G 8, identified its ability to bind to antigen.

FIG. 4 PCR amplified A7G 8scFv fragment electropherogram.

FIG. 5 vector fragments of PUTAM003 vector BspEI and EcoRV after cleavage.

FIG. 6 demonstrates the binding of the A7 scFv-hFc, G8scFv-hFc recombinant protein to antigen.

FIG. 7 is a flow cytometer of FIG. 7 detecting the specific binding of A7 scFv-hFc and G8scFv-hFc to the small cell lung cancer cell line SHP-77.

FIG. 8 DLL3 CART architecture

FIG. 9 transduction efficiency of D14, anti-DLL3 and anti-CD19 CART after lentivirus transduction.

FIG. 10 LDH killing levels of anti-DLL3 and anti-CD19 CART

FIG. 11 measures cytokine levels released after incubation of effector cells anti-DLL3-CART and anti-CD19 CART with target cell SHP-77.

Detailed Description

For the purposes of promoting an understanding of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It should be understood, however, that these specific embodiments are not intended to limit the scope of the invention. Any alterations and further modifications in the described embodiments, and any further applications of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates.

Example 1

Screening of anti-DLL3 phage antibody scFv

The phage antibody library is constructed by amplifying antibody heavy chain variable region (VH) and light chain variable region (VL) genes from healthy human B lymphocytes by using a PCR technology and expressing scFv segments on the surface of phage. The fully human scFv phage antibody library is from Shanghai Youcandi biomedical company, and is subjected to DLL3 antibody screening by using a phage display technology; His-DLL3 extracellular domain protein: human DLL3 Protein, His Tag (MALS verified), cat # DL3-H52H4-100ug, purchased from ACRO, was used for screening anti-DLL3 specific antibodies; coli Tg1(E.coli Tg1, available from the manufacturer Lucigen, cat # 60502-2); pancreatin (trypsin, cat # and specification T1426-250mg) purchased from Sigma-Aldrich;

coating His-DLL3 extracellular region protein in enzyme-linked immunosorbent assay (ELISA) plate, incubating the phage antibody library and target protein molecule (His-DLL3 extracellular region protein), washing away unbound free phage, and digesting the bound phage with pancreatin. The phages eluted by the pancreatin digestion were further infected with e.coli Tg1 for amplification, and then subjected to the next round of screening.

As a result: the His-DLL3 extracellular region protein is used as a solid phase antigen to carry out 4 rounds of 'adsorption-elution-amplification' enrichment screening on the fully human scFv phage antibody library, the input/output (output/input) ratio of the phage is continuously improved, and the yield of the phage antibody is increased from 0.14x10 of the 1 st round^-7128.75x10 to round 4^-7The increase was 1019.27-fold (see FIG. 1), and phages specifically binding to the target protein were highly enriched.

Example 2

Activity of anti-DLL3 Phage was identified by Phage enzyme-linked immunosorbent assay (Phage elisa)

The source of the His-DLL3 extracellular domain protein is the same as that in example 1; 96 well plates (cat # 3590) were purchased from Costar corporation; mouse Anti-M13 antibody (HRP) (cat # S004H-250uL) was purchased from Dow-Dow organisms; m13KO7 Helper Phage (N0315S) was purchased from NEB. The formula of the 2xYT culture medium comprises 16g/L tryptone, 10g/L yeast powder and 5g/L sodium chloride; putting part of double distilled water into a beaker, weighing the peptone, the yeast powder and the sodium chloride according to the proportion, pouring the mixture into the beaker, stirring the mixture until the peptone, the yeast powder and the sodium chloride are dissolved repeatedly, and clearing the mixture. Placing the weighed agar powder into a conical flask, pouring the culture solution, sealing by a sealing film, and sterilizing for 20 minutes by using an autoclave at 121 ℃. And adding corresponding antibiotics after cooling to below 55 ℃.

2xYTA agar culture plate, tryptone 10g/L, yeast powder 5g/L, sodium chloride 8g/L, agar powder 15 g/L. Pouring the solid culture medium into a flat plate, putting part of double distilled water into a beaker, weighing the peptone, the yeast powder and the sodium chloride in the proportion, pouring the mixture into the beaker, and stirring the mixture until the peptone, the yeast powder and the sodium chloride are dissolved repeatedly, so as to obtain the clear liquid. Placing the weighed agar powder into a conical flask, pouring the culture solution, sealing with a sealing film, and sterilizing with high pressure steam. Sterilizing with high pressure steam, cooling to about 55 deg.C (hand back tolerance), and adding antibiotic to prevent antibiotic inactivation due to high temperature. According to the following steps: ampicillin is added in 1000 portions, for example 400ml of medium plus 400. mu.l of ampicillin. Too high a temperature can result in reduced or inactivated antibiotic activity, and most antibiotics are not resistant to high temperatures. When the plate is poured, air bubbles are avoided, and a plurality of plates are overlapped together, so that excessive water drops are prevented from being condensed due to too cold plate covers. After the agar was completely solidified and inverted, the agar was placed at room temperature for one day to avoid premature storage at 4 ℃ (after incubation at 37 ℃ too early, there were often water droplets to fuse bacterial colonies.) and was placed on a clean bench overnight before being taken into the refrigerator, which was then taken care of sterility. Making a mark

From the bacterial colonies obtained after the last round (round 4) of screening, 96 clones were randomly selected, amplified and then the affinity of the Phage supernatant to the extracellular domain protein of His-DLL3 was identified by Phage ELISA. The Output phage antibody library obtained from the 4 th round of screening is diluted in gradient (10)²Dilution by fold, 10⁴Dilution by fold, 10⁶Dilution by fold, 10⁷Dilution by fold, 10⁸Double dilution), infected with 200ul log phase Ecoli. Tg1, plated with 2YTA plates (ampicillin 100mg/ml), and incubated overnight at 37 ℃.2 pieces of 96 deep-well plate, each hole adding 300ul 2YTGA (1% glucose, 100mg/ml ampicillin), preparation of 60ml 2YTAG, from the last round of screening plate randomly choose 92 clones to the deep-well plate, the remaining 2 TG1 control (i.e. blank Escherichia coli control) and 2YTA control (i.e. blank medium control), the row gun blow, 37 degrees C shaking 3.5-4 h. Preserving bacteria by using a 96-well cell culture plate, taking 100ul of bacteria from a 96-deep-well plate, adding 100ul of 50% glycerol for preserving bacteria, and storing at-20 ℃. Adding 50ul of Helper phase into 3.3ml of 2YT non-resistant culture medium, mixing, adding 30ul of the culture medium added with the Helper phase into each hole of a deep hole plate, extending to the liquid level, and mixing. The incubator was left at 37 ℃ for 30 minutes. 220rpm at 37 degrees celsius for 1 hour. The deep-well plate was supplemented with 400ul 2YT AMP + KANA + medium (ampicillin, kanamycin double antibody) per well, and cultured overnight at 30 ℃ and 220r for a final volume of 600ul per well. Antigen coating, antigen DLL3-His one 96-well Elisa plate, BSA coatingA96-well Elisa plate (for nonspecific binding) at 1ug/ml at 4 ℃ overnight. Phage Elisa identification step 1) the next day, a 96-deep-well plate for culturing Phage is taken out and placed still, thalli are precipitated, and a supernatant is taken. 2) Antigen-coated 4 ℃ overnight 96-well Elisa plates were blocked with 200ul 2% skim milk per well, 1h, and washed 3 times with PBST. 3) During the Elisa plate milk sealing period, 120ul of Phage supernatant is taken, 120ul of 2% skimmed milk is added, a dilution plate is used for sealing the Phage supernatant, after the sealing is finished, the supernatant is correspondingly added to serve as a primary antibody, a deep-well plate and the supernatant added to a 96-well Elisa plate correspond one by one, 100 ul/well are incubated for 1.5h, and PBST is washed for 6 times. 4) Anti M13-HRP (anti M13 phage) was added and incubated for 1h at 1:5000 dilution with 2% skim milk at 100 ul/well. 5) TMB 100 ul/well, color development 10 minutes. 6)2M H2SO 4100 ul/well, stop color development, plate reader reading OD450nm, negative standard OD450<0.2. 7) Positive clones shown by Phage Elisa results are selected and sent to the Producer organism Limited company for sequencing, and the diversity of the sequences of the positive clones is identified.

Results, 61 positive clones which are specifically combined with DLL3 are detected by phase Elisa (figure 2), and are identified as 2 sequences by biological sequencing, and the sequences are respectively: the nucleotide sequences of the scFv obtained by the identification of the anti-DLL3 positive clone A7 and the anti-DLL3 positive clone G8 through the biological sequencing are shown in Table 1, the amino acid sequences of the scFv are shown in Table 2, the nucleotide sequences of the heavy chain and the light chain are shown in Table 3, the amino acid sequences of the heavy chain and the light chain are shown in Table 4, and the amino acid sequences of the Complementarity Determining Regions (CDR) of the heavy chain and the light chain are shown in Table 5.

Example 3

Prokaryotic expression and primary activity identification of anti-DLL 3scFv

Plasmid DNA mini-drawer kit purchased from kangning; escherichia coli Rosetta competence was purchased from Tiangen Biochemical technology Ltd; HRP-labeled anti-c-myc antibody was purchased from Bethyyl; TMB was purchased from eBioscience.

1. Extraction and purification of A7G 8 Tg1 monoclonal plasmid: A7G 8 monoclonal, identified by Phage elisa as having specific binding, was used to preserve glycerol bacteria (ratio of 50% glycerol to bacterial solution 1: 1). 20ul of the stored positive clone glycerol was cultured overnight in 5ml of 2YTA medium (Ecoli. Tg1) at 220r and 37 ℃. Collecting 1-4ml overnight bacterial liquid in 2YTA culture medium, 12000g for 1min, discarding supernatant, and sucking filtrate with filter paper. 250 mul of Buffer S1 was added and the mixture was blown and stirred until suspension was uniform and no lumps were formed. Add 250. mu.l Buffer S2, gentle (prevent genomic DNA fragmentation) and fully flip up and down 4-6 times, mix well to fully lyse the thallus until a clear solution is formed. Mu.l of Buffer S3 was added and the precipitate appeared, and the mixture was stirred with addition. Preventing local over-concentration, mostly changing into linear and open-loop structure, mixing for 6-8 times, 12000g, 1min, keeping supernatant, and discarding protein flocculent precipitate. The supernatant was aspirated and transferred to a spin column (in a 2ml centrifuge tube) at 12000g for 1 min; 500 mul Buffer W1, 12000g, 1min, and discarding the filtrate; 700 mul Buffer W2, 12000g, 1min, discarding the filtrate, 700 mul Buffer W2, 12000g, 1min, discarding the filtrate; idling, 12000g, 1min, discarding the filtrate (5 min of air blowing on an ultra-clean bench to remove ethanol); the Spin column was transferred into a new 1.5ml EP tube, 60-80. mu.l of Eluent or deionized water (eluted without the cationic DNA repelling the Silica) was added to the center of the membrane, and allowed to stand for 2 minutes (DNA lysis) 12000g for 1 min. Eluent or ddH20(double distilled H2O) was heated to 65 ℃ in advance to improve the elution efficiency. The concentration of double-stranded DNA was measured using a Nano drop and the concentration and A260/280 were recorded. 10ul of plasmid was sent to BioProducer Biometrics, Inc. for sequencing.

2. Sequencing identified that the sequence of scFv was correct and then transformed into ecoli. 1) The plasmid transformation efficiency was influenced by the cold condition of Rosetta (DE3) taken out of-80 and rapidly placed on ice at 4 ℃. 2) Rosetta (DE3) was competent to add 200ng of the extracted correctly sequenced plasmid per 50ul, mix gently, and stand on ice at 4 ℃ for 30 minutes. 3) Heat activation, 42 ℃, 60 seconds, taking out, putting on ice for 2 minutes, adding 700 microliters of sterile 2YT medium without antibiotics, and mixing evenly. 4) The cells were cultured at 37 ℃ for 1 hour (220rpm) with shaking, and plated after 1 hour. 5) The plasmid-transferred bacterial solution was taken out, centrifuged at 3500rpm for 5 minutes, in order to increase the bacterial concentration. 6) After centrifugation, a portion of the supernatant, generally 750, was aspirated, 490 microliters of the supernatant was discarded, and the remaining liquid was mixed with a 200 microliter tip to increase the conversion concentration. 7) Sucking 200 microliter of bacterial liquid and uniformly coating the bacterial liquid on a 2YT agar medium plate containing the aminobenzene antibiotics to uniformly distribute the bacterial liquid. The culture medium is placed at 37 ℃ until the liquid is absorbed, and then placed in a 37 ℃ incubator for overnight culture. 8) The next day, 3 monoclonals were selected for sequencing by engineering biotechnology.

3. IPTG induced expression of soluble antibodies: 1) sequencing-correct A7G 8 monoclonal glycerol strain 30ul was transferred to a 3ml-2YTA culture tube, and cultured overnight at 37 ℃ with 50% glycerol. 2) According to the following steps: 100 were transferred to a shake overnight strain and grown for 3h to 3.5h in 100ul of ampicillin-resistant medium, followed by induction after logarithmic growth phase (OD450 nm: 0.4 to 0.5). 3) Centrifugation, discarding supernatant, changing to fresh medium, 10ml 2YT medium plus 100ul IPTG (0.1 m concentration as 1: 100) and 10ul of ampicillin (1: 1000) the cells were resuspended, and expression was induced overnight at 30 ℃ and 220 rpm. 4) Extraction of periplasmic proteins (on 4 degree ice): the expressed bacteria were induced overnight and centrifuged at 3500 ℃ for 10 minutes. 5) 1ml of lysozyme-added TES buffer (lysozyme is prepared fresh on ice, 1mg/ml lysozyme is added on ice), resuspended, and placed on ice for 0.5 h. The bacterial solution was added with lysozyme and then left on ice for 1 hour to stabilize the protein. 6)3500,4 ℃ centrifugation for 10 minutes, transfer the supernatant containing peripherin to a 2ml centrifuge tube, label, store in-20 ℃ refrigerator for peripherin ELASA.

4. Week to week protein Elisa identified the affinity of the antibody to DLL3 antigen: DLL3-His antigen was coated at 1ug/ml in 96-well Elisa plates, sealed membrane plates, and overnight at 4 ℃. After antigen coating overnight on Elisa plates, 0.05% PBST was washed once and 2% skim milk was blocked for 1h, during which time 2% skim milk was used to block proteins. After the end of the Elisa plate blocking, add primary antibody, 100 ul/well, and incubate primary antibody for 1 hour. 0.05% PBST 3 washes. Using 2% skim milk at 1: adding HRP labeled anti-c-myc secondary antibody at the ratio of 1000, 100 ul/hole, and incubating for 1 h. 0.05% PBST 3 washes. 100 ul/well TMB plus 100ul, development for 10min, and color too dark can be stopped. The chromogenic reaction was stopped at 100ul of sulfuric acid (2M) per well. The microplate reader performed photometric measurements (450 nm).

As a result: after extracting A7G 8 plasmid, transforming the plasmid into escherichia coli Rosetta competence, carrying out sequencing to identify the correct sequence, carrying out amplification culture, inducing the prokaryotic expression soluble antibody of the plasmid through IPTG, extracting escherichia coli Rosetta peri-protein, and then carrying out peri-protein Elisa to identify the binding activity of the escherichia coli Rosetta competence. The week to protein Elisa results show that both scFv, a7 and G8, can specifically bind to DLL3 antigen (fig. 3), and the purpose of the Elisa plate coating BSA (bovine serum albumin) and IgG (immunoglobulin) is to see if specific binding of anti-DLL3 antibody (a 7G 8) will occur. The results show (fig. 3) that anti-DLL3 scFvA 7G 8 can specifically bind to DLL3 antigen and does not produce non-specific binding (does not bind to unrelated antigens such as BSA).

Example 4

Construction of anti-DLL3 Single chain antibody (scFv-hFc)

The seamless cloning kit was purchased from nunoprazan; pr imeStar DNA polymerase was purchased from TAKARA; PUTAM003 expression vector (Youcandi biological Co., Ltd., sequence shown in Table 6); TOP10 was competently purchased from Tiangen biol.

In the present invention we also constructed recombinant single chain antibodies against DLL3, fusing the scFv fragment of a 7G 8 to the IgG Fc fragment using seamless cloning. The plasmids A7 and G8 extracted in example 3 were used to perform PCR amplification with the following primers:

A7-F(SEQ ID NO.58)：accggcgtgcactccgatatcCAGGTTCAGCTGGTGCAGTCT。

A7-R(SEQ ID NO.59)：tgtgtgagttttgtctccggaTTTGATCTCCACCTTGGTCCC。

G8-F(SEQ ID NO.60)：accggcgtgcactccgatatcCAGGTACAGCTGCAGCAGTCAG。

G8-R(SEQ ID NO.61)：tgtgtgagttttgtctccggaTAGGACGGTCAGCTTGGTCCC。

and (3) PCR amplification system: 100ng of the target plasmid, 2ul of the F sense primer, 2ul of the R antisense primer, 25ul of PrimeStar, and 50ul of ddH 2O. And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10 sec, annealing at 55 ℃ for 10 sec, extension at 72 ℃ for 15S, (denaturation-annealing-extension 35cycle), and extension at 72 ℃ for 5 min. The scFv fragments of A7 and G8 were obtained by amplification, and the nucleotide sequences are shown in Table 1, and the electrophoretogram is shown in FIG. 4. Recovering PCR product and scFv segment obtained by PCR amplification. Electrophoresis and glue recovery: a) preparing 1% Agarose gel, 25ml-TAE, 0.25g-Agarose, heating in a microwave oven for 90s to ensure that Agarose is completely dissolved; b) taking out PCR products from a PCR instrument, wherein 50ul of the whole system is obtained, 5.5ul of loading buffer (10X) is added to each antisense system, 10ul of D15000+2000 (Tiangen) marker is added to the first hole, and then the samples are sequentially loaded; c) and (4) replacing the electrophoresis solution until the gel is over, adjusting the voltage to 120v, and performing constant-voltage running electrophoresis. d) The indicator ran to position 2/3 to turn off the electrophoresis to prevent running through the lost DNA, and the digital gel image processing system tan 2500 photographed and compared to the marker to find the expected band. e) Cutting, recovering glue, loading into EP tube, peeling and weighing, adding 200ul NTI per 100mg, and melting at 50 deg.C for 10min to completely melt the glue. f) Purifying silica gel column, centrifuging, 11000g,30s, and discarding supernatant. g) 700ul of NT3 was added and washed twice, centrifuged for 11000, 30s, and the supernatant discarded. h) Idling is 11000g for 1min, and the NT3 is volatilized after the mixture is placed in a water bath kettle at 65 ℃ for 5 min. Sterilized water was preheated to 65 ℃ in advance. i) Transferring to 1.5ml EP tube, adding 50ul ddH2O in the center of the membrane, standing for 2min, centrifuging 11000g for 1min, transferring to new 1.5EP tube after centrifugation, and measuring the concentration of the recovered product by Nano drop. The mammalian cell expression vector PUTAM003 containing the Fc fragment of human IgG1 was derived from the Youcandi biopharmaceutical, with partial sequences as shown in Table 6. The PUTAM003 vector was linearized by enzymatic digestion with both BspEI and EcoRV. Vector enzyme digestion system: PUTAM 00310 ug, BspEI 10ul, EcoRV 10ul, Cutsmart Buffer (10X)20ul, enzyme free water to 200ul, water bath at 37 ℃ for 30 min. After the enzyme digestion, the product of the enzyme digestion is subjected to agarose gel electrophoresis, the carrier fragment after the enzyme digestion is recovered according to the steps, and the electrophoresis picture of the product after the enzyme digestion is shown in FIG. 5. And carrying out seamless cloning homologous recombination, and cloning the scFv segment obtained by PCR amplification into a PUTAM003 expression vector. Preparing a reaction system on ice: 140ng of vector, 14.4ng of insert, 4ul of 5 × CE II Buffer, 2ul of Exnase II without enzyme water to 20 ul. Reaction conditions are as follows: 30min at 37 ℃, standing on ice for 5min after connection, converting 5ul of connection product into 50ul of TOP10 competent state, standing on ice for 30min, thermally activating for 60s at 42 ℃, standing on ice for 2min, adding 700ul of non-resistant 2YT culture medium, culturing by shaking table at 37 ℃ for 60min and 3000g, centrifuging for 5min, discarding 400ul of supernatant, uniformly blowing the rest 200ul, coating a flat plate, uniformly distributing by using a coating rod, inverting the flat plate, and culturing overnight. Each plate was picked 3 single clones and sent for bioengineering sequencing.

As a result: primers are designed, PCR is carried out to amplify scFv sequences of A7 and G8, agarose gel electrophoresis is carried out, and the result is shown in FIG. 4, wherein the size of the scFv fragment is consistent with the expected size; the PUTAM003 expression vector was double digested with BspEI and EcoRV and agarose gel electrophoresis showed the size of the cleaved fragment to be consistent with that expected (FIG. 5). After homologous recombination of the scFv segment and the vector after enzyme digestion, the recombinant monoclonals of A7 scFv-hFc and G8scFv-hFc are correctly sequenced and identified by the company Biotech.

Example 5

Eukaryotic expression and further Activity identification of anti-DLL3 Single chain antibodies (Elisa, flow-through)

The extraction kit in the endo-free plasmid DNA purification is purchased from MACHEREY-NAGEL;

DMEM from Gibco; mouse anti-human IgG-Fc (HRP) from Bethyyl; PE anti-human Fc antibody was purchased from Biolegend; 293T cells were purchased from ATCC, and SHP-77 Small cell Lung cancer cell lines were purchased from Shanghai Med.

1. A large amount of plasmids are needed for mass transfection, so the invention uses an Endotoxin-free plasmid extraction kit, and the Endotoxin-free plasmid DNA purification to carry out mass plasmid extraction, and the steps are as follows 1) after the TOP10 monoclonal inoculation of A7 and G8 with correct sequencing is enlarged and cultured, the sequence is as follows 1: 100 inoculated in 200ml 2YTA medium, 220r, 37 ℃, overnight culture. 2) The bacterial liquid is collected from the conical flask and centrifuged, 8000g, 4 ℃, 15min, and the supernatant is discarded. 3) Adding solution I (suspension) and Buffer RES-EF 8mL, shaking and mixing uniformly to disperse and prevent bacterial liquid from aggregating. 4) Adding 8ml of solution II and Buffer LYS-EF (lysis solution with blue indicator), and gently turning and mixing up and down (preventing genome DNA from being broken); this was completed within 5 minutes at room temperature. 5) 2 centrifugation cartridges were mounted on a white disc and placed in a 200ml Erlenmeyer flask for leakage, rinsed with 15ml Buffer EQU-EF

An Xtra column, a equilibration column and a filter membrane. Note that the holes are not aligned along the sides of the posts. 6) Adding solution III and 8ml of Buffer NEU-EF per tube, turning upside down and mixing uniformly, and eliminating blue color to obtain white flocculent precipitate. Standing on ice for 5 min. Centrifuge 6000g,1 min. 7) Transferring the supernatant to

5ml of buffer FIL-EF was added to the Xtra column, and the column was filled up to the end of the run-outThe paper film was discarded. 8) 35ml Buffer ENDO-EF was added. 9) After the Buffer ENDO-EF leakage is finished, 15ml of Buffer WASH-EF is added. When the dripping is finished, the centrifugal tube needs to be replaced by a new centrifugal tube. 10)5ml of Elution buffer ELU-EF, elute (fresh centrifuge tube). 11) After the elution was complete, the column was removed, 3.5ml isopropanol was added and the plasmid precipitated, left at-20 ℃ for 20 minutes, centrifuged at 12000rpm at 4 ℃ for 10min, the supernatant discarded and placed on a three-fold paper and drained (at this time the plasmid was on the wall, being careful not to pour the plasmid). 12) Washing and drying with 2ml 70% EtOH ethanol, 12000g, centrifuging at room temperature for 5min, discarding supernatant, 12000g, 1min, sucking out residual ethanol, and air drying at room temperature for 10-15 min. 13) After the precipitate was air-dried and became transparent, 800ul of TE-EF was added to the precipitate to dissolve and mix the precipitate, and the DNA concentration was measured by NANO DROP. 5ul of plasmid sequence extracted in the sequencing identification of the biological engineering is taken for determination.

2. Eukaryotic expression of single chain antibodies, plasmid transfection of 293T by calcium chloride, the procedure was as follows: 1) correctly sequenced A7 scFv-hFc and G8scFv-hFc recombinant plasmids were expressed as 1: 1 proportion cotransfects 293T cell, 293 cell 1x107 cell/dish, 80% cell confluence, change culture medium before transfection (DMEM + 4% FBS), plasmid 22 ug/dish, the total volume of water, calcium chloride, plasmid is 500 ul/dish, HBS 500 ul/dish, transfection reagent is added 10ml cell culture solution after shaking fully mixing, 3 dishes of 293 cell of cotransfection altogether. 2) After the plasmid transfects the cells for 8h, the culture medium (DMEM + 4% FBS) is changed, 10ml of the culture medium is supplemented every 24h twice, finally 30ml of culture supernatant is added to each dish of cells, the culture supernatant is collected, and the cells are removed by filtering with a 0.45um filter membrane.

3. Elisa identified 293 the affinity of the antibodies in the culture supernatant for DLL3 antigen: DLL3-His antigen was coated at 1ug/ml in 96-well Elisa plates, sealed membrane plates, and overnight at 4 ℃. After antigen coating overnight on Elisa plates, 0.05% PBST was washed once and 2% skim milk was blocked for 1h, during which time 2% skim milk was used to block proteins. After the end of the Elisa plate blocking, add primary antibody, 100 ul/well, and incubate primary antibody for 1 hour. 0.05% PBST 3 washes. Using 2% skim milk at 1: mouse anti-human IgG-Fc (HRP) secondary antibody was added at a ratio of 1000, 100 ul/well and incubated for 1 h. 0.05% PBST 3 washes. 100 ul/well TMB plus 100ul, development for 10min, and color too dark can be stopped. The chromogenic reaction was stopped at 100ul of sulfuric acid (2M) per well. The microplate reader performed photometric measurements (450 nm).

4. Detecting the binding activity of the antibody in 293 culture supernatant and the SHP-77 cell surface antigen by a flow cytometry: collecting suspension of SHP-77 cells, discarding culture supernatant, infiltrating the bottom of the dish with 10mL of physiological saline, discarding physiological saline, adding 1mL of pancreatin digestive cells into each dish of cells, determining digestion time according to cell line characteristics, observing cell non-adherence under a microscope, suspending and rounding the shape, and adding 5mL of complete culture medium to terminate digestion. Centrifuging at 1500rpm for 5min, discarding the supernatant, adding 5mL of PBS for resuspension, centrifuging at 1500rpm for 5min, discarding the supernatant, suspending the cells with 1mL of complete culture medium, counting trypan blue, taking 2x106 cells for resuspension in 100ul of PBS, adding A7 scFv-hFc and G8scFv-hFc antibody, mixing uniformly, incubating at room temperature for 45min, washing with PBS once, discarding the supernatant, adding 2ul of secondary antibody PE anti-human Fc antibody, incubating at room temperature for 25 min, washing with PBS once, discarding the supernatant, and resuspending in 200ul of PBS for detection. The results are shown in FIG. 7.

As a result: 293T cells were transfected with plasmids A7 scFv-hFc and G8scFv-hFc using calcium chloride transfection, respectively, and scFv-Fc single-chain antibodies were expressed in the culture supernatant, which was subjected to Elisa. Fig. 6Elisa results show that both scFv-hFc, a7 and G8, can specifically bind to DLL3 antigen (fig. 6), and that the purpose of the Elisa plate coating BSA (bovine serum albumin) is to see if the anti-DLL3 antibody (a 7G 8 cFv-hFc) binds non-specifically to non-target antigens. The results show (fig. 6) that anti-DLL3 scFvA 7G 8 can specifically bind to DLL3 antigen and no non-specific binding (no binding to BSA) occurs. FIG. 7 flow cytometry detection of scFv binding specifically to cells shows that both the A7 scFv-hFc and the G8scFv-hFc proteins bind specifically to DLL3 antigen expressed on small cell lung cancer cell line SHP-77, with weaker binding of G8scFv-hFc (FIG. 7), and thus A7 scFv was selected for experiments following the present invention.

Example 6

Construction of anti-DLL 3CAR and packaging of lentiviruses

The lentiviral backbone plasmid vector was PSB1819, from eucardia; both pPac-R, pPac-GP and pEnv-G are from Youcadi; TOP10 was competently purchased from Tiangen biol.

The scFv fragment of the anti-DLL3 positive clone A7 obtained by screening in example 5 was seamlessly cloned into a lentiviral backbone plasmid vector PSB1819, which is derived from Youcadi, as shown in FIG. 8. referring to example 4, TOP10 E.coli was transformed after the plasmid recombination was successful, after the culture was expanded, plasmid extraction was performed, and after plasmid extraction was completed, 293T cells were transfected with lentiviral backbone plasmid, lentiviral packaging plasmid pPac-R, lentiviral packaging plasmid pPac-GP and lentiviral packaging plasmid pEnv-G. The packaging steps are as follows: plating 293T cells to be transfected by using a 10cm culture dish, replacing a culture medium with a serum-free culture medium when the cell density reaches about 70%, and packaging lentiviruses: in the first step, the culture medium is replaced by a serum-free medium, CaCl2 solution and a certain volume ratio of four plasmids are added into a centrifuge tube, water for bacterial endotoxin detection (BETW) is added, and HBS is added while vortex. Adding a proper amount of prepared transfection reagent into the transfected cells in a way of adding 2mL of CaCl ₂3 packaging plasmids (13. mu.g pPac-R, 20. mu.g pPac-GP, 5. mu.g pEnv-G) and 20. mu.g of the plasmid of interest (anti-DLL3 CAR), supplemented with bacterial endotoxin examination water to 4mL]Gently shaking and mixing, and adding CO₂And (5) changing the liquid after waiting for 6 hours in the incubator. And changed to DMEM complete medium after 6 hours. Collecting virus supernatant at 4 ℃ for storage after 24 hours, 48 hours and 72 hours of culture, filtering the virus supernatant by using a 0.45um filter after the collection of the virus supernatant is finished, filtering residues of cells, adding PEG8000 (polyethylene glycol 8000) to concentrate the virus, incubating at 4 ℃ for 12 hours, centrifuging at 3000rpm for 15 minutes, discarding the supernatant after centrifugation, adding a proper amount of sterile PBS to dissolve virus precipitates, subpackaging the dissolved virus and storing in a refrigerator at-80 ℃ to obtain the anti-i-DLL 3 recombinant lentiviral vector.

As a result: anti-DLL 3CAR-T cells and anti-CD19 CAR-T cells CAR was constructed as shown in FIG. 8, and the nucleotide sequences of the important elements (CD 8leader, CD8 transmembrane domain, 41BB co-stimulatory domain, etc.) in the CAR vector are shown in Table 7, and the amino acid sequences are shown in Table 8. Lentiviral packaging was performed according to the above procedure, with CD19 CAR set as a negative CAR control in the present invention.

Example 7

Preparation and characterization of anti-DLL 3CAR-T cells

FITC-Protein L Protein was purchased from Biolegend.

CD4+ CD8+ T cells are separated from human peripheral blood mononuclear cells by using CD4 and CD8 magnetic beads, the separated cells are placed into a T25 bottle coated with CD3 antibody CD28 antibody for activation for 24h, the recombinant lentiviral vector prepared in example 2 is added according to the MOI of 120 after the T cells are completely activated, PBS is washed for 2 times after 48h of infection, new complete culture medium is replaced for re-suspension, and then the transfection efficiency of the lentivirus transfected T cells is detected by FITC-Protein L Protein by using a flow cytometer. Protein L is an immunoglobulin-binding Protein that specifically binds to an immunoglobulin light chain.

CAR construction against DLL3CAR-T cells and against CD19 CAR-T cells as in figure 8. After T cells were transfected with lentiviruses, the infection efficiency was examined on day 14. Wherein the day of T cell activation is D0, transfection is performed after 24h of T cell activation, and the day of lentivirus transfection is D1.

As a result: as shown in FIG. 9, the transduction efficiency of anti-DLL3 CART cells was 46.94% at day 14, and that of anti-CD19 CART was 61.99%.

Example 8

Detection of LDH killing level of anti-DLL 3CAR-T cells on different target cells

Cytotox 96Non-Radioactive cytoxicity Assay, cat # REF: g1782, available from Promega. A549 is from cell bank of Shanghai Chinese academy of sciences

The Lactate Dehydrogenase (LDH) release method measures the killing efficiency of effector T cells against target cells. The suspensions of the target cells and effector cells were collected separately, centrifuged at 1500rpm for 5min, the supernatant was discarded, 3mL of PBS was added for resuspension, and centrifuged at 1200rpm for 5 min. Effector cells and target cells were resuspended separately in 1mL of AIM-V complete medium, mixed well and counted using a hemocytometer. Setting effector cells: when the ratio of target cells (E: T) is 2.5:1, 1X10 target cells are added to each well of a 96-well plate⁴Effector cells were added 2.5x10 per well⁴The volume is 50 ul. When the ratio of E to T is set to 5:1, 1x10 target cells are added to each well of a 96-well plate⁴Effector cells per wellAdd 5x10⁴The volume is 50 ul. When the ratio of E to T is set to 10:1, 1x10 target cells are added to each well of a 96-well plate⁴Effector cell add 1x10 per well⁵The volume is 50 ul. The LDH killing experiment needs to be provided with three multiple holes, effector cells and target cells are prepared and then are paved into a 96-hole plate, the 96-hole plate is sealed by a sealing film and then is placed into a centrifuge, 250g of the mixture is put into the centrifuge, the mixture is lifted by 3 and is descended by 1 for centrifugation for 5min, and the mixture is placed into a constant temperature incubator at 37 ℃ for incubation for 24 h. And (3) adding 10ul of lysate into each maximum lysis group well 45min before detection, putting the maximum lysis group wells into a constant-temperature incubator at 37 ℃ for further incubation for 45 minutes, adding an LDH substrate, then incubating for 10 minutes, and reading the absorbance reading at the wavelength of 490nm by using an enzyme-linked immunosorbent assay. Only the largest lysis group of target cells needs lysis buffer, and the other groups do not.

As a result: the LDH killing result shows that compared with a control group of anti-CD19 CAR-T cells without DLL3 target spots, the anti-DLL 3CAR-T cells have higher specific killing effect on small cell lung cancer cell line SHP-77 highly expressing DLL3 and have obvious dose dependence, the higher the amount of the CAR-T cells is, the higher the killing level is, and the DLL3 negative lung cancer cell strain A549 has no killing effect on any effector cell target cell ratio (10:1,5:1,2.5:1) (figure 10). The surface anti-DLL3 CART cells can kill DLL3 positive tumor cells specifically and have dose dependence.

Example 9

Cytokine secretion by anti-DLL 3CAR-T cells during killing

CBA cytokine detection kit was purchased from BD Bioscience.

Cell suspensions of the target cell SHP-77 and effector cells Anti-CD19 CART and Anti-DLL3 CART were collected, centrifuged at 1500rpm for 5min, the supernatant was discarded, 3mL of PBS was added for resuspension, and centrifuged at 1200rpm for 5 min. Effector cells and target cells were resuspended separately in 1mL of AIM-V complete medium, mixed well and counted using a hemocytometer. Setting effector cells: when the ratio of target cells (E: T) is 10:1, 1X10 target cells are added to each well of a 96-well plate⁴Effector cell add 1x10 per well⁵The volume is 50 ul. Spreading the prepared effector cells and target cells in 96-well plate, sealing the 96-well plate with sealing film, centrifuging at 250g for 5min, lifting 3, lowering 1, and placing at 37 deg.CAnd (5) incubating for 6h in a constant temperature incubator. Supernatants were collected for cytokine detection in 1.5mL EP tubes. a, sample treatment: after centrifugation of the cell suspension at 1500rpm for 3min, the supernatant was collected in a new 1.5mL EP tube. b, preparing a standard substance: and adding 2mL of standard product diluent to dilute the standard product freeze-dried powder, and standing at room temperature for 15 min. The standard was then diluted in 2-fold gradients (10 gradients from maximum to blank dilution). c Add 50. mu.L of standard or sample to the new EP tube, followed by 50. mu.L of magnetic beads and 50. mu.L of detection antibody, mix well in a vortex apparatus, and incubate at room temperature for 3 h. d adding Wash Buffer to Wash twice, discarding supernatant, adding 200 μ L of Wash Buffer to resuspend the machine to detect cytokines IL-2/IL-4/IL-6/IL-10/IFN-. gamma./TNF. The supernatant was tested for CBA content in 3 replicates.

As a result: the cytokine secretion levels after the effector cells Anti-CD19 CART and Anti-DLL3 CART were incubated with the target cell SHP-77 for 6h were measured by flow cytometry, and the results are shown in FIG. 11, in which IL-2, IFN-r and TNF-a were secreted in large amounts after the Anti-DLL3 CART was incubated with the DLL3 positive target cell SHP-77 for 6h, and in which the control group CD19 CART secreted a small amount of cytokines. As shown in the CBA detection result, after the anti-DLL3 CART is contacted with DLL3 positive cells, a large amount of anti-tumor factors IL-2, IFN-r and TNF-a are secreted by the antigen, so that target cells are killed.

The in vitro results prove that the anti-DLL 3CAR-T cells can be stimulated and activated by target cells which are positive to DLL3 in vitro, and can generate a series of cytokines relevant to immune activation, and simultaneously achieve good killing effect on the target cells.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, any theory, mechanism, proof, or finding stated herein is meant to further enhance understanding of the present invention, and is not intended to limit the present invention in any way to such theory, mechanism, proof, or finding. While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character.

Sequence listing

<110> university of east China

SHANGHAI UNICAR-THERAPY BIO-MEDICINE TECHNOLOGY Co.,Ltd.

<120> fully human anti-DLL 3scFv and application thereof in CART cell treatment

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gcaaagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240

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ggggtgactg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagggagt 360

gcatccgccc caacccttgg tggaggcggt tcaggcggag gtggcagcgg cggtggcggg 420

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Lys Lys Phe

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Ala Arg Gly Gly Gly Val Thr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Gly Gly

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Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Thr Val Arg Thr Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr Gly Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe

210 215 220

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

225 230 235 240

Gly Gly Thr Lys Val Glu Ile Lys

245

<210> 5

<211> 259

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asp

20 25 30

Ser Val Ala Trp Ser Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala

50 55 60

Ala Ser Val Lys Ser Arg Ile Ser Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Pro Leu Arg Tyr Gly Ser Ser Trp Trp Asp

100 105 110

Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Ile Thr Val Ser Ser Gly

115 120 125

Ser Ala Ser Ala Pro Thr Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Pro Ser Val

145 150 155 160

Ser Gly Ser Pro Gly Gln Ala Val Thr Ile Ser Cys Thr Gly Thr Ser

165 170 175

Ser Asp Val Gly Arg Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro

180 185 190

Gly Lys Val Pro Lys Leu Ile Leu Phe Asp Val Ser Ser Arg Pro Ser

195 200 205

Gly Val Ser His Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

210 215 220

Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

225 230 235 240

Thr Ser Tyr Arg Ser Gly Ser Glu Val Phe Gly Gly Gly Thr Lys Leu

245 250 255

Thr Val Leu

<210> 6

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gly Ser Ala Ser Ala Pro Thr Leu Gly Gly Gly Gly Ser Gly Gly Gly

1 5 10 15

Gly Ser Gly Gly Gly Gly Ser

20

<210> 7

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caggttcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180

gcaaagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240

atggaactga acagggtgac atctgacgac acggccgtgt attactgtgc gagaggcggt 300

ggggtgactg ggtttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354

<210> 8

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctttaggaga cagagtcacc 60

atcacttgcc gggcaagtca gaccgttaga acttatttaa attggtatca gcagaaacca 120

gggaaagccc ctaacctcct aatctatggt gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtgggtc tgggacagat ttcactctaa ccatcaacag tctgcaacct 240

gaggattttg gaacctacta ctgtcaacag agtgacactc ccccgctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 9

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcgacagtg ttgcttggag ctggatcaga 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

tatgattatg cagcatctgt gaaaagtcga ataagcatca acccagacac atccaagaac 240

cagttctccc tgcagttgaa ttctgtgact cccgaggaca cggctgtcta ttactgtgca 300

agagagcccc tccgttatgg gagcagctgg tgggatgctt ttgatatctg gggccaaggg 360

acaatgatca ccgtctcttc a 381

<210> 10

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cagtctgccc tgactcagcc tccctcagtg tccgggtctc ctggacaggc agtcaccatc 60

tcctgcactg gaaccagcag tgatgttggt cgttataatt atgtctcctg gtaccaacaa 120

cacccaggca aagtccccaa actcatcctt tttgatgtct ctagtcggcc ctcaggggtt 180

tctcatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc acctcatata gaagtggcag cgaggtcttc 300

ggcggaggga ccaagctgac cgtccta 327

<210> 11

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Lys Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Asn Arg Val Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Gly Val Thr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 12

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Val Arg Thr Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln Gln Ser Asp Thr Pro Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asp

20 25 30

Ser Val Ala Trp Ser Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala

50 55 60

Ala Ser Val Lys Ser Arg Ile Ser Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Pro Leu Arg Tyr Gly Ser Ser Trp Trp Asp

100 105 110

Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Ile Thr Val Ser Ser

115 120 125

<210> 14

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ala Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Arg Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu

35 40 45

Ile Leu Phe Asp Val Ser Ser Arg Pro Ser Gly Val Ser His Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Tyr Arg Ser Gly

85 90 95

Ser Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 15

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gly Tyr Thr Phe Thr Ser Tyr Tyr

1 5

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Ile Asn Pro Ser Gly Gly Ser Thr

1 5

<210> 17

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Ala Arg Gly Gly Gly Val Thr Gly Phe Asp Tyr

1 5 10

<210> 18

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gln Thr Val Arg Thr Tyr

1 5

<210> 19

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Ala Ser

1

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Gln Gln Ser Asp Thr Pro Pro Leu Thr

1 5

<210> 21

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Gly Asp Ser Val Ser Ser Asp Ser Val Ala

1 5 10

<210> 22

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr

1 5

<210> 23

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ala Arg Glu Pro Leu Arg Tyr Gly Ser Ser Trp Trp Asp Ala Phe Asp

1 5 10 15

Ile

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Ser Ser Asp Val Gly Arg Tyr Asn Tyr

1 5

<210> 25

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Asp Val Ser

1

<210> 26

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Thr Ser Tyr Arg Ser Gly Ser Glu Val

1 5

<210> 27

<211> 1621

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Arg Ala Ala Arg Ala His Gln Thr Leu Thr Asp Gln Thr Val Pro Phe

1 5 10 15

His Gly Ser Phe Leu Gln Ser Pro Ser Ser Thr Arg Ala Ala Thr Met

20 25 30

Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly Val

35 40 45

His Ser Asp Ile Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser

50 55 60

Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ile Thr

65 70 75 80

Ser Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro

85 90 95

Gly Arg Val Leu Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val

100 105 110

Thr Thr Val Ser Asp Thr Thr Lys Arg Asn Asn Met Asp Phe Ser Ile

115 120 125

Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile

130 135 140

Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala

145 150 155 160

Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser Ser Gly Asp Lys Thr

165 170 175

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

180 185 190

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

195 200 205

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

210 215 220

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

225 230 235 240

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

245 250 255

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

260 265 270

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

275 280 285

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

290 295 300

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

305 310 315 320

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

325 330 335

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

340 345 350

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

355 360 365

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

370 375 380

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

385 390 395 400

Ser Arg Pro Leu Thr Arg Pro Trp Ile Gln Ile Cys Cys Ala Phe Leu

405 410 415

Pro Ala Ile Cys Cys Leu Pro Leu Pro Arg Ala Phe Leu Asp Pro Gly

420 425 430

Arg Cys His Ser His Cys Pro Phe Leu Ile Lys Gly Asn Cys Ile Ala

435 440 445

Leu Ser Glu Val Ser Phe Tyr Ser Gly Gly Trp Gly Gly Ala Gly Gln

450 455 460

Gln Gly Gly Gly Leu Gly Arg Gln Gln Ala Cys Trp Gly Cys Gly Gly

465 470 475 480

Leu Tyr Gly Tyr Pro Gly Ala Glu Glu Leu Thr Arg Phe Leu Leu Gly

485 490 495

Gln Lys Glu Ala Gly Thr Ser Pro Ser Leu His Thr Leu Ser Thr Pro

500 505 510

Leu Val Leu Ser Ser Ser Pro Thr His Arg Thr Leu Ile Ala Gln Glu

515 520 525

Gly Ser Ala Phe Asn Pro Thr Arg Ser Thr Trp Ser Gly Leu Ser Leu

530 535 540

Pro His Gln Pro Thr Lys Pro Asn Leu Ala Ser Lys Ser Gly Lys Lys

545 550 555 560

Leu Lys Gln Asp Arg Leu Leu Ser Ala Glu Gly Glu Lys Met Pro Pro

565 570 575

Thr Cys Glu Glu Val Met Arg Glu Ile Ile Glu Phe Gly His Asp Leu

580 585 590

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

595 600 605

Ser Val Val Arg Leu Arg Arg Ala Val Ser Ala His Ser Lys Ala Val

610 615 620

Ile Arg Leu Ser Thr Glu Ser Gly Asp Asn Ala Gly Lys Asn Met Ala

625 630 635 640

Lys Gly Gln Gln Lys Ala Arg Asn Arg Lys Lys Ala Ala Leu Leu Ala

645 650 655

Phe Phe His Arg Leu Arg Pro Pro Asp Glu His His Lys Asn Arg Arg

660 665 670

Ser Ser Gln Arg Trp Arg Asn Pro Thr Gly Leu Arg Tyr Gln Ala Phe

675 680 685

Pro Pro Gly Ser Ser Leu Val Arg Ser Pro Val Pro Thr Leu Pro Leu

690 695 700

Thr Gly Tyr Leu Ser Ala Phe Leu Pro Ser Gly Ser Val Ala Leu Ser

705 710 715 720

His Ser Ser Arg Cys Arg Tyr Leu Ser Ser Val Val Val Arg Ser Lys

725 730 735

Leu Gly Cys Val His Glu Pro Pro Val Gln Pro Asp Arg Cys Ala Leu

740 745 750

Ser Gly Asn Tyr Arg Leu Glu Ser Asn Pro Val Arg His Asp Leu Ser

755 760 765

Pro Leu Ala Ala Ala Thr Gly Asn Arg Ile Ser Arg Ala Arg Tyr Val

770 775 780

Gly Gly Ala Thr Glu Phe Leu Lys Trp Trp Pro Asn Tyr Gly Tyr Thr

785 790 795 800

Arg Arg Thr Val Phe Gly Ile Cys Ala Leu Leu Lys Pro Val Thr Phe

805 810 815

Gly Lys Arg Val Gly Ser Ser Ser Gly Lys Gln Thr Thr Ala Gly Ser

820 825 830

Gly Gly Phe Phe Val Cys Lys Gln Gln Ile Thr Arg Arg Lys Lys Gly

835 840 845

Ser Gln Glu Asp Pro Leu Ile Phe Ser Thr Gly Ser Asp Ala Gln Trp

850 855 860

Asn Glu Asn Ser Arg Gly Ile Leu Val Met Arg Leu Ser Lys Arg Ile

865 870 875 880

Phe Thr Ile Leu Leu Asn Lys Ser Phe Lys Ser Ile Ser Ile Tyr Glu

885 890 895

Thr Trp Ser Asp Ser Tyr Gln Cys Leu Ile Ser Glu Ala Pro Ile Ser

900 905 910

Ala Ile Cys Leu Phe Arg Ser Ser Ile Val Ala Leu Gly Gly Gly Gly

915 920 925

Arg Gly Leu Pro Arg Glu Glu Gly Val Ala Asp Ser Tyr Gln Ala Ile

930 935 940

Ala Pro Ser Ser Ser Gln Lys Val Arg Glu Pro Arg Leu Met Arg Ala

945 950 955 960

Leu Leu Val Asp Gln Leu Val Ile Leu Asn Phe Cys Phe Ala Thr Glu

965 970 975

Arg Ser Ala Leu Ser Gly Arg Cys Val Ile Ser Phe Asn Ser Ala Lys

980 985 990

Val Arg Phe Ile Gln Gln Ser Arg Arg Pro Val Lys Ser Ala Cys Ser

995 1000 1005

Ala Ser Val Thr Thr Asn Pro Ile Leu Ile Arg Lys Thr His Arg Ala

1010 1015 1020

Ser Asn Glu Thr Ala Ile Tyr Ser Tyr Gln Asp Tyr Gln Tyr His Ile

1025 1030 1035 1040

Phe Glu Lys Ala Val Ser Val Met Lys Glu Lys Thr His Arg Gly Ser

1045 1050 1055

Ser Ile Gly Trp Gln Asp Pro Gly Ile Gly Leu Arg Phe Arg Leu Val

1060 1065 1070

Gln His Gln Tyr Asn Leu Leu Ile Ser Pro Arg Gln Lys Gly Tyr Gln

1075 1080 1085

Val Arg Asn His His Glu Arg Leu Asn Pro Val Arg Met Ala Lys Ala

1090 1095 1100

Tyr Ala Phe Leu Ser Arg Leu Val Gln Gln Ala Ser His Tyr Ala Arg

1105 1110 1115 1120

His Gln Asn His Ser His Gln Pro Asn Arg Tyr Ser Phe Val Ile Ala

1125 1130 1135

Pro Glu Arg Asp Glu Ile Arg Asp Arg Cys Lys Asp Asn Tyr Lys Gln

1140 1145 1150

Glu Ser Asn Ala Thr Gly Ala Gly Thr Leu Pro Ala His Gln Gln Tyr

1155 1160 1165

Phe His Leu Asn Gln Asp Ile Leu Leu Ile Pro Gly Met Leu Phe Ser

1170 1175 1180

Arg Gly Ser Gln Trp Val Thr Met His His Gln Glu Tyr Gly Asn Ala

1185 1190 1195 1200

Trp Ser Glu Glu Ala Ile Pro Ser Ala Ser Leu Val Pro Ser His Leu

1205 1210 1215

His His Trp Gln Arg Tyr Leu Cys His Val Ser Glu Thr Thr Leu Ala

1220 1225 1230

His Arg Ala Ser His Thr Ile Asp Arg Leu Ser His Leu Ile Ala Arg

1235 1240 1245

His Tyr Arg Glu Pro Ile Tyr Thr His Ile Asn Gln His Pro Cys Trp

1250 1255 1260

Asn Leu Ile Ala Ala Ser Ser Lys Thr Phe Pro Val Glu Tyr Gly Ser

1265 1270 1275 1280

His Pro Leu Tyr Tyr Cys Leu Cys Lys Gln Thr Val Leu Leu Phe Met

1285 1290 1295

Met Ile Tyr Phe Tyr Leu Val Gln Cys Asn Ile Arg Asp Phe Glu Thr

1300 1305 1310

Gln Arg Gly Phe Pro Pro Pro Pro Ile Ile Glu Ala Phe Ile Arg Val

1315 1320 1325

Ile Val Ser Ala Asp Thr Tyr Leu Asn Val Phe Arg Lys Ile Asn Lys

1330 1335 1340

Gly Phe Arg Ala His Phe Pro Glu Lys Cys His Leu Thr Ser Lys Lys

1345 1350 1355 1360

Pro Leu Leu Ser His Pro Ile Lys Ile Gly Val Ser Arg Gly Pro Phe

1365 1370 1375

Val Ser Arg Val Ser Val Met Thr Val Lys Thr Ser Asp Thr Cys Ser

1380 1385 1390

Ser Arg Arg Arg Ser Gln Leu Val Cys Lys Arg Met Pro Gly Ala Asp

1395 1400 1405

Lys Pro Val Arg Ala Arg Gln Arg Val Leu Ala Gly Val Gly Ala Gly

1410 1415 1420

Leu Thr Met Arg His Gln Ser Arg Leu Tyr Glu Cys Thr Ile Cys Gly

1425 1430 1435 1440

Val Lys Tyr Arg Thr Asp Ala Gly Glu Asn Thr Ala Ser Asp Trp Leu

1445 1450 1455

Leu Glu Arg Ser Arg Ser Arg Gly Ala Gly Ala Arg Ala Cys Pro Trp

1460 1465 1470

Ala Pro Arg Ala Arg Thr Pro Pro His Pro Ser Cys Gly Met Cys Val

1475 1480 1485

Ser Gly Val Glu Ser Pro Gln Ala Pro Gln Gln Ala Glu Val Cys Lys

1490 1495 1500

Ala Cys Ile Ser Ile Ser Gln Gln Pro Gly Val Glu Ser Pro Gln Ala

1505 1510 1515 1520

Pro Gln Gln Ala Glu Val Cys Lys Ala Cys Ile Ser Ile Ile Gly Gln

1525 1530 1535

Pro Ser Arg Pro Leu Arg Pro Ser Arg Pro Leu Arg Pro Val Pro Pro

1540 1545 1550

Ile Leu Arg Pro Met Ala Asp Phe Phe Leu Phe Met Gln Arg Pro Arg

1555 1560 1565

Pro Pro Arg Pro Leu Ser Tyr Ser Arg Ser Ser Glu Glu Ala Phe Leu

1570 1575 1580

Glu Ala Ala Phe Ala Lys Thr Ser Leu Gly Pro Pro Leu Lys Thr Pro

1585 1590 1595 1600

Trp Pro Pro Pro Gln Ser Ile Asn Leu Pro Gly Lys Trp Val Gln Lys

1605 1610 1615

Tyr Met Pro Gln Gly

1620

<210> 28

<211> 227

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 60

cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 120

tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 180

gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacg 227

<210> 29

<211> 180

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 60

tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 120

gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 180

<210> 30

<211> 353

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc 60

ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg 120

agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa 180

tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata 240

atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag 300

ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caa 353

<210> 31

<211> 233

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcctcaat 60

gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120

gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180

gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcc 233

<210> 32

<211> 495

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 60

gttggagtaa taaatctctg gaacagattg gaatcacacg acctggatgg agtgggacag 120

agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca 180

agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt 240

taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt 300

aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc 360

accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 420

agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg 480

acggtatcgg ttaac 495

<210> 33

<211> 118

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60

agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaattca aaatttta 118

<210> 34

<211> 1178

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60

gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120

atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180

tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240

tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300

cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360

agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420

ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480

tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540

aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600

cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660

cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720

gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780

ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840

cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900

cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960

agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020

agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080

tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140

tggttcaaag tttttttctt ccatttcagg tgtcgtga 1178

<210> 35

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 36

<211> 141

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta c 141

<210> 37

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60

tactgc 66

<210> 38

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 39

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 40

<211> 591

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc t 591

<210> 41

<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

tatcatgtct gg 132

<210> 42

<211> 164

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 60

ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 120

ctattccaga agtagtgagg aggctttttt ggatagactt ttgc 164

<210> 43

<211> 73

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Val Val Leu Cys Asn Thr Leu Val Val Leu Gln His Gly Asn Asp Glu

1 5 10 15

Leu Ala Thr Cys Leu Thr Arg Arg Glu Lys Ala Pro Cys Met Pro Ile

20 25 30

Gly Gly Ser Lys Val Val Arg Ser Cys Leu Ile Arg Lys Ala Thr Asp

35 40 45

Gly Ser Asp Met Asp Trp Thr Asn His Ile Ala Ala Leu Gln Arg Tyr

50 55 60

Cys Ile Val Pro Ser Ser Ile His Lys

65 70

<210> 44

<211> 57

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Gly Leu Ser Gly Thr Arg Ser Glu Pro Gly Ser Ser Leu Ala Asn Gly

1 5 10 15

Thr His Cys Leu Ser Leu Asn Lys Ala Cys Leu Glu Cys Phe Lys Cys

20 25 30

Val Pro Val Cys Cys Val Thr Leu Val Thr Arg Asp Pro Ser Asp Pro

35 40 45

Phe Ser Gln Cys Gly Lys Ser Leu Ala

50 55

<210> 45

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Asp

1 5 10 15

Gly Lys Lys Phe Gly Gly Gln Gly Glu Arg Lys Asn Ile Asn Asn Ile

20 25 30

Tyr Gly Gln Ala Gly Ser Asn Asp Ser Gln Leu Ile Leu Ala Cys Lys

35 40 45

His Gln Lys Ala Val Asp Lys Tyr Trp Asp Ser Tyr Asn His Pro Phe

50 55 60

Arg Gln Asp Gln Lys Asn Leu Asp His Tyr Ile Ile Gln Gln Pro Ser

65 70 75 80

Ile Val Cys Ile Lys Gly Arg Lys Thr Pro Arg Lys Leu Thr Arg Arg

85 90 95

Lys Ser Lys Thr Lys Val Arg Pro Pro His Ser

100 105

<210> 46

<211> 76

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

Arg Ser Phe Val Pro Trp Val Leu Gly Ser Ser Arg Lys His Tyr Gly

1 5 10 15

Arg Ser Leu Asn Asp Ala Asp Gly Thr Gly Gln Thr Ile Ile Val Trp

20 25 30

Tyr Ser Ala Ala Ala Glu Gln Phe Ala Glu Gly Tyr Gly Ala Thr Ala

35 40 45

Ser Val Ala Thr His Ser Leu Gly His Gln Ala Ala Pro Gly Lys Asn

50 55 60

Pro Gly Cys Gly Lys Ile Pro Lys Gly Ser Thr Ala

65 70 75

<210> 47

<211> 158

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

Trp Gly Phe Gly Val Ala Leu Glu Asn Ser Phe Ala Pro Leu Leu Cys

1 5 10 15

Leu Gly Met Leu Val Gly Val Ile Asn Leu Trp Asn Arg Leu Glu Ser

20 25 30

His Asp Leu Asp Gly Val Gly Gln Arg Asn Gln Leu His Lys Leu Asn

35 40 45

Thr Leu Leu Asn Arg Ile Ala Lys Pro Ala Arg Lys Glu Thr Arg Ile

50 55 60

Ile Gly Ile Arg Met Gly Lys Phe Val Glu Leu Val His Asn Lys Leu

65 70 75 80

Ala Val Val Tyr Lys Ile Ile His Asn Asp Ser Arg Arg Leu Gly Arg

85 90 95

Phe Lys Asn Ser Phe Cys Cys Thr Phe Tyr Ser Glu Ser Ala Gly Ile

100 105 110

Phe Thr Ile Ile Val Ser Asp Pro Pro Pro Asn Pro Glu Gly Thr Arg

115 120 125

Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gln Arg Gln

130 135 140

Ile His Ser Ile Ser Glu Arg Ile Ser Thr Val Ser Val Asn

145 150 155

<210> 48

<211> 37

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

Phe Lys Lys Arg Gly Asp Trp Gly Val Gln Cys Arg Gly Lys Asn Ser

1 5 10 15

Arg His Asn Ser Asn Arg His Thr Asn Arg Ile Thr Lys Thr Asn Tyr

20 25 30

Lys Ile Gln Asn Phe

35

<210> 49

<211> 382

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

Ala Pro Val Pro Val Ser Gly Gln Ser Ala His Arg Pro Gln Ser Pro

1 5 10 15

Arg Ser Trp Gly Glu Gly Ser Ala Ile Glu Pro Val Pro Arg Glu Gly

20 25 30

Gly Ala Gly Thr Gly Lys Val Met Ser Cys Thr Gly Ser Ala Phe Phe

35 40 45

Pro Arg Val Gly Glu Asn Arg Ile Val Gln Ser Pro Thr Phe Phe Phe

50 55 60

Ala Thr Gly Leu Pro Pro Glu His Arg Val Pro Cys Val Val Pro Ala

65 70 75 80

Gly Leu Ala Ser Leu Arg Val Met Ala Leu Ala Cys Leu Glu Leu Leu

85 90 95

Pro Pro Gly Cys Ser Thr Phe Leu Ile Pro Ser Phe Gly Leu Glu Val

100 105 110

Gly Gly Arg Val Arg Gly Leu Ala Leu Lys Glu Pro Leu Arg Leu Val

115 120 125

Leu Glu Leu Arg Pro Gly Leu Gly Ala Gly Ala Ala Ala Cys Glu Ser

130 135 140

Gly Gly Thr Phe Ala Pro Val Ser Leu Leu Ser Ile Ser Leu Pro Phe

145 150 155 160

Lys Ile Phe Asp Asp Leu Leu Arg Arg Phe Phe Ser Gly Lys Ile Val

165 170 175

Leu Met Arg Ala Lys Ile Cys Thr Leu Val Phe Arg Phe Leu Gly Pro

180 185 190

Arg Ala Ala Thr Gly Pro Val Arg Pro Ser Ala His Val Arg Arg Gly

195 200 205

Gly Ala Cys Glu Arg Gly His Arg Glu Ser Asp Gly Gly Ser Leu Lys

210 215 220

Leu Ala Gly Leu Leu Trp Cys Leu Ala Ser Arg Arg Arg Val Ser Pro

225 230 235 240

Arg Pro Gly Arg Gln Gly Trp Pro Gly Arg His Gln Leu Arg Glu Arg

245 250 255

Lys Asp Gly Arg Phe Pro Ala Leu Leu Gln Gly Ala Gln Asn Gly Gly

260 265 270

Arg Gly Ala Arg Glu Ser Gly Arg Val Ser His Pro His Lys Gly Lys

275 280 285

Gly Pro Phe Arg Pro Gln Pro Ser Leu His Val Thr Pro Leu Ser Thr

290 295 300

Gly Arg Arg Pro Gly Thr Ser Ile Ser Ser Arg Ala Phe Gly Val Arg

305 310 315 320

Arg Leu Val Gly Gly Arg Gly Phe Met Arg Trp Ser Phe Pro Thr Leu

325 330 335

Ser Gly Trp Arg Leu Lys Leu Gly Gln Leu Gly Thr Cys Asn Ser Pro

340 345 350

Trp Asn Leu Pro Phe Leu Ser Leu Asp Leu Gly Ser Phe Ser Ser Leu

355 360 365

Arg Gln Trp Phe Lys Val Phe Phe Phe His Phe Arg Cys Arg

370 375 380

<210> 50

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 51

<211> 47

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

35 40 45

<210> 52

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

1 5 10 15

Val Ile Thr Leu Tyr Cys

20

<210> 53

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 54

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 55

<211> 197

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

Asn Gln Pro Leu Asp Tyr Lys Ile Cys Glu Arg Leu Thr Gly Ile Leu

1 5 10 15

Asn Tyr Val Ala Pro Phe Thr Leu Cys Gly Tyr Ala Ala Leu Met Pro

20 25 30

Leu Tyr His Ala Ile Ala Ser Arg Met Ala Phe Ile Phe Ser Ser Leu

35 40 45

Tyr Lys Ser Trp Leu Leu Ser Leu Tyr Glu Glu Leu Trp Pro Val Val

50 55 60

Arg Gln Arg Gly Val Val Cys Thr Val Phe Ala Asp Ala Thr Pro Thr

65 70 75 80

Gly Trp Gly Ile Ala Thr Thr Cys Gln Leu Leu Ser Gly Thr Phe Ala

85 90 95

Phe Pro Leu Pro Ile Ala Thr Ala Glu Leu Ile Ala Ala Cys Leu Ala

100 105 110

Arg Cys Trp Thr Gly Ala Arg Leu Leu Gly Thr Asp Asn Ser Val Val

115 120 125

Leu Ser Gly Lys Ser Ser Ser Phe Pro Trp Leu Leu Ala Cys Val Ala

130 135 140

Thr Trp Ile Leu Arg Gly Thr Ser Phe Cys Tyr Val Pro Ser Ala Leu

145 150 155 160

Asn Pro Ala Asp Leu Pro Ser Arg Gly Leu Leu Pro Ala Leu Arg Pro

165 170 175

Leu Pro Arg Leu Arg Leu Arg Pro Gln Thr Ser Arg Ile Ser Leu Trp

180 185 190

Ala Ala Ser Pro Pro

195

<210> 56

<211> 43

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Asn Leu Phe Ile Ala Ala Tyr Asn Gly Tyr Lys Ser Asn Ser Ile Thr

1 5 10 15

Asn Phe Thr Asn Lys Ala Phe Phe Ser Leu His Ser Ser Cys Gly Leu

20 25 30

Ser Lys Leu Ile Asn Val Ser Tyr His Val Trp

35 40

<210> 57

<211> 50

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

Ile Pro Pro Leu Thr Pro Pro Ile Pro Pro Leu Thr Pro Pro Ser Ser

1 5 10 15

Ala His Ser Pro Pro His Gly Leu Ile Phe Phe Ile Tyr Ala Glu Ala

20 25 30

Glu Ala Ala Ser Ala Ser Glu Leu Phe Gln Lys Gly Gly Phe Phe Gly

35 40 45

Thr Phe

50

<210> 58

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

accggcgtgc actccgatat ccaggttcag ctggtgcagt ct 42

<210> 59

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

tgtgtgagtt ttgtctccgg atttgatctc caccttggtc cc 42

<210> 60

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

accggcgtgc actccgatat ccaggtacag ctgcagcagt cag 43

<210> 61

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

tgtgtgagtt ttgtctccgg ataggacggt cagcttggtc cc 42

Claims

1. An anti-DLL 3scFv, wherein the anti-DLL 3scFv is capable of specifically binding to DLL 3;

or,

2. The anti-DLL 3scFv of claim 1, wherein the anti-DLL 3scFv comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region and the light chain variable region are connected by a flexible linker;

or,

3. The anti-DLL 3scFv of claim 2, wherein the heavy chain variable region has the nucleotide sequence set forth in SEQ ID No.7 and the light chain variable region has the nucleotide sequence set forth in SEQ ID No. 8;

or

4. The anti-DLL 3scFv of claim 1, wherein the amino acid sequence of the anti-DLL 3scFv is set forth in SEQ ID No. 4;

or,

shown as SEQ ID NO. 5.

5. A nucleic acid molecule characterized in that its nucleotide sequence encodes the amino acid sequence of the anti-DLL 3scFv of claim 4; the nucleotide sequence is shown as SEQ ID NO.1 or SEQ ID NO. 2.

6. An anti-DLL 3CAR, wherein the anti-DLL 3CAR has the structure CD8leader-DLL3 scFv-CD8 Hinge-CD8 TM-costimulatory domain-intracellular signal peptide, comprising, in series, in order, a CD8leader membrane receptor signal peptide, the single-chain variable fragment of claim 1, a CD8 Hinge chimeric receptor Hinge region, a CD8 TM chimeric receptor transmembrane region, a costimulatory domain, and an intracellular signal peptide; the co-stimulatory domain is 4-1 BB; the intracellular signal peptide is CD3 zeta,

the anti-DLL 3CAR target plasmid comprises a lentivirus backbone vector sequence and a CAR part, and an amino acid sequence comprises:

1) the amino acid sequence of the CD8leader is SEQ ID NO. 50;

2) the amino acid sequence of CD8 Hinge is SEQ ID NO. 51;

3) the amino acid sequence of the CD8 TM transmembrane region is SEQ ID NO. 52;

4) the amino acid sequence of the 41BB co-stimulatory domain is SEQ ID NO. 53;

5) the amino acid sequence of CD3 zeta is SEQ ID NO. 54;

the method specifically comprises the following steps:

1) the nucleotide sequence of the CD8leader is SEQ ID NO. 35;

2) the nucleotide sequence of CD8 Hinge is SEQ ID NO. 36;

3) the nucleotide sequence of the CD8 TM transmembrane region is SEQ ID NO. 37;

4) the nucleotide sequence of the 41BB co-stimulatory domain is SEQ ID NO. 38;

5) the nucleotide sequence of CD3 zeta is SEQ ID NO. 39.

7. A plasmid comprising the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region of DLL3scFv of claim 3.

8. A recombinant lentiviral vector comprising the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region of DLL3scFv of claim 3.

9. Use of the anti-DLL 3scFv of claim 1 or the nucleic acid molecule of claim 5 or the anti-DLL 3CAR of claim 6 or the plasmid of claim 7 or the recombinant lentiviral vector of claim 8 in the preparation of an anti-tumor medicament, said tumor being small cell lung cancer.