CN113710253A

CN113710253A - Nucleic acid sequences encoding chimeric antigen receptor and short hairpin RNA sequences for IL-6 or checkpoint inhibitors

Info

Publication number: CN113710253A
Application number: CN202080027442.9A
Authority: CN
Inventors: 吴力军; 维塔·格鲁博斯卡娅
Original assignee: Primabo Biotechnology; Promab Biotechnologies Inc
Current assignee: Primabo Biotechnology; Promab Biotechnologies Inc
Priority date: 2019-02-04
Filing date: 2020-02-03
Publication date: 2021-11-26
Also published as: WO2020163222A1

Abstract

The present invention relates to a nucleic acid sequence comprising: (a) a first polynucleotide encoding a Chimeric Antigen Receptor (CAR) fusion protein comprising, from N-terminus to C-terminus: (i)) Comprising V_HAnd V_LThe single-chain variable fragment (scFv) of (a), wherein the scFv specifically binds to a tumor antigen, (ii) a transmembrane domain, (iii) at least one costimulatory domain, and (iv) an activation domain; and (b) a second polynucleotide encoding an IL-6 short hairpin RNA (shRNA) sequence or a checkpoint inhibitor shRNA, wherein the checkpoint inhibitor is PD-1, CTLA-4, TIM-3, TIGIT, or LAG-3.

Description

Nucleic acid sequences encoding chimeric antigen receptor and short hairpin RNA sequences for IL-6 or checkpoint inhibitors

Sequence listing, table, or computer program reference

The text file in the Sequence table in ASCII format is submitted through EFS-Web with the specification at the same time, with the file name Sequence listing. txt, creation date of 2020, 2, 3 and size of 39.8 kbytes. The sequence listing submitted by EFS-Web is part of the specification and is hereby incorporated by reference in its entirety.

Technical Field

The present invention relates to nucleic acid sequences comprising a first polynucleotide encoding a chimeric antigen receptor, and a second polynucleotide encoding a short hairpin rna (shrna) that down-regulates IL-6 or a checkpoint protein.

Background

Immunotherapy is becoming a very promising approach to cancer treatment. T cells or T lymphocytes are the armed force of our immune system, constantly looking for foreign antigens and differentiating abnormal cells (cancer or infected cells) from normal cells [1 ]. Genetic modification of T cells with CARs is the most common approach to designing tumor-specific T cells. CAR-T cells targeting Tumor Associated Antigens (TAAs) can be infused into patients (called adoptive cell transfer or ACT), which represents an effective immunotherapeutic approach [1, 2 ]. The advantage of CAR-T technology over chemotherapy or antibodies is that reprogrammed T cells can proliferate and persist in the patient ("an active drug") [3], [4 ].

CARs (chimeric antigen receptors) generally consist of: monoclonal antibody-derived single chain variable fragments (scFv) are linked by hinges to several intracellular signaling domains via transmembrane domains: a single, cell-activating, CD 3-zeta domain; and a CD28, CD137(4-1BB) or other costimulatory domain (CD27 signaling domain has also been used in place of the CD28 or CD137 domains) in tandem with the CD 3-zeta domain (fig. 1) [3], [5 ]. The evolution of CARs goes from first generation (without costimulatory domains) to second generation (with one costimulatory domain) to third generation CARs (with several costimulatory domains). The generation of CARs with multiple co-stimulatory domains (so-called third generation CARs) leads to increased CAR-T cell killing activity and significantly improves CAR-T cell persistence, thereby enhancing its anti-tumor activity.

The major problem with CAR-T cell therapy is that patients treated with CAR-T cells, due to high secretion of IL-6, produce Cytokine Release Syndrome (CRS); CRS is associated with high fever and poor neurological symptoms. IL-6 is secreted by T cells, stimulating immune response of one of the main cytokines. IL-6 is an important regulator of fever and plays an important role in the pathogenesis of various diseases such as multiple myeloma, autoimmune diseases and prostate cancer. IL-6 secretion and CRS all symptoms can be reduced with Tolizumab (a humanized antibody to the IL-6 receptor) which blocks inflammatory IL-6 cytokine production.

Checkpoint inhibitor therapy is one form of cancer immunotherapy. The therapy targets immune checkpoints, which are key regulators of the immune system, that when activated can suppress the immune response to immune stimuli. Some cancers may protect themselves from attack by stimulating immune inspection targets. Checkpoint therapy can block inhibitory checkpoints and restore immune system function. Currently approved checkpoint inhibitor targeting molecules are CTLA4, PD-1 and PD-L1.

CTLA4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that serves as an immune checkpoint and down regulates immune responses. CTLA4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells upon activation — a phenomenon particularly pronounced in cancer. When bound to CD80 or CD86 on the surface of an antigen presenting cell, it acts as an "off" switch.

PD-1 is a transmembrane programmed cell death 1 protein that interacts with PD-L1(PD-1 ligand 1). PD-L1 on the cell surface binds to PD1 on the surface of immune cells, inhibiting immune cell activity. Plays a key role in the regulation of T cell activity in PD-L1 function. Upregulation of PD-L1 on the cell surface may inhibit the original attack of T cells. Antibodies that bind to PD-1 or PD-L1 and thus block the interaction may enable T cells to attack tumors.

TIGIT (also known as T cell immunoreceptor, with Ig and ITIM domains) is an immunoreceptor present on some T cells and natural killer cells. TIGIT binds with high affinity to CD155(PVR) on Dendritic Cells (DC), macrophages, etc., and also binds with lower affinity to CD112(PVRL 2).

T cell immunoglobulins and mucin-domain-3 (Tim-3) -containing proteins are type I transmembrane proteins. Tim-3 plays a key role in inhibiting Th1 responses and the expression of cytokines such as TNF and INF-gamma. Dysregulation of Tim-3 expression is associated with autoimmune diseases.

Studies have shown that TIGIT-Fc fusion proteins can interact with PVR on dendritic cells and increase/decrease their IL-10/IL-12 secretion levels upon LPS stimulation and also inhibit T cell activation in vivo [1 ]. Inhibition of NK cytotoxicity by TIGIT can be blocked by antibodies directed against its interaction with PVR, and this activity is directed by its ITIM domain. [4]

RNA silencing or RNA interference refers to gene silencing family effects by which gene expression is negatively regulated by non-coding RNAs such as micrornas. RNA silencing works by repressing translation or cleaving messenger RNA (mrna). A common example of RNA silencing is RNA interference (RNAi), in which endogenously expressed micrornas (mirnas) or exogenous small interfering RNAs (sirnas) induce degradation of complementary messenger RNAs. RNA silencing pathways are associated with regulatory activity of small non-coding RNAs (approximately 20-30 nucleotides in length) as factors involved in inactivation of homologous sequences, promotion of endonuclease activity, translational arrest, and/or chromatin or DNA modification.

There is a need for a method of generating safer and more effective CAR-T cells in CAR-T therapy to overcome CRS in a clinical setting.

Drawings

FIG. 1 shows the structure of the CAR. The left panel shows the structure of the first generation CAR (no co-stimulatory domain). The middle panel shows the structure of a second generation CAR (one co-stimulatory domain). The right panel shows a third generation CAR (two or several co-stimulatory domains) [3 ].

FIG. 2 shows the construct of CD-19-CAR with IL-6shRNA or checkpoint inhibitor protein (PD-1, TIGIT). The CAR sequence with CD19-CAR contained a Flag tag after the scFv. Each shRNA contains sense, loop, antisense and termination signals. The CAR sequence may contain a Flag tag or TF tag before or after ScFv to facilitate detection of CAR expression.

FIG. 3 shows a real-time cytotoxicity assay (RTCA) with CD19 positive Hela cervical cancer cells. The effector/target ratio was 10: 1. T cells, mock-CAR-T cells, were used as negative control cells against Hela-CD19 positive cells. CD19-IL-6shRNA-CAR-T cells and CD19-CAR-T cells efficiently killed HeLa-CD19 positive cells. CD19F refers to the Flag tag after CD19 scFv.

FIG. 4 shows that CD19-IL-6shRNA-CAR-T cells secreted significantly less IL-6 than CD19-CAR-T cells against Raji cells. P <0.025, CD19-IL-6shRNA-CAR-T cells vs CD19-CAR-T cells.

FIG. 5 shows that CD19-IL-6shRNA-CAR-T cells did not reduce IFN- γ levels in Raji cells. The E: T ratio was 1: 1.

FIG. 6 shows an RTCA assay against Hela-CD19 target cells with CD19TF-CAR-T cells and CD19TF-IL-6shRNA-CAR-T cells.

FIG. 7 shows that CD19TF-IL6shNA-CAR-T cells secreted significantly less IL-6 than CD19TF-CAR-T cells against CD19 positive Raji target cells.

Figure 8 shows RTCA assays against Hela-CD19 target cells with CD19-CAR-T cells (PMC193), CD19TF-PD1 shRNA-CAR-T cells (PMC317), and CD19-TF-TIGIT shRNA-CAR-T cells (PMC 316).

Figure 9 shows RTCA assays against Raji target cells with CD19-CAR-T cells (PMC193), CD19TF-PD1 shRNA-CAR-T cells (PMC317), and CD19-TF-TIGIT shRNA-CAR-T cells (PMC 316).

FIG. 10 shows that CD19-TF-PD-1 shRNA-CAR-T cells and CD19-TF-TIGIT shRNA-CAR-T cells secrete high levels of IFN- γ against target HeLa-CD19 cells.

FIG. 11 shows the high in vivo efficacy of CD19-TF-PD-1 shRNA CAR-T cells and CD19-TF-TIGIT shRNA CAR-T cells. Imaging showed a significant reduction in bioluminescence in the Raji-luciferase positive xenograft mouse model treated with CD19-TF-PD-1 shRNA-CAR T cells and CD19-TF-TIGIT shRNA CAR-T cells. P-0.006, CAR-T vs T cells, student T-test.

Figure 12 shows that CD19-TF-PD-1 shRNA-CAR-T cells and CD19-TF-TIGIT shRNA CAR-T cells significantly prolonged mouse survival in Raji xenograft NSG models. P <0.05 relative to T cells and CD19TF-CAR-T cells.

Detailed Description

Definition of

As used herein, a "Chimeric Antigen Receptor (CAR)" is a receptor protein that has been engineered to give T cells a new ability to target specific proteins. The receptors are chimeric in that they combine antigen binding and T cell activation functions into a single receptor. A CAR is a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are sometimes referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". "extracellular domain capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to an antigen. By "intracellular domain" is meant any oligopeptide or polypeptide known to function as a domain that transmits a signal to activate or inhibit a biological process in a cell.

As used herein, "domain" means a region in a polypeptide that folds into a particular structure independently of other regions.

As used herein, "shRNA" or "short hairpin RNA" or "small hairpin RNA" refers to an RNA molecule having a hairpin-like structure; this molecule is slightly larger than the siRNA molecule and, unlike siRNA, is produced in the nucleus inside the cell. shRNA transcripts were constructed by ligating the sense and antisense strands of the siRNA duplexes to loop sequences so that a single transcript can fold back into the duplex structure upon transcription. After transcription, the shRNA is processed into siRNA by Dicer enzyme. shRNA is a means of preparing siRNA sequences for delivery to cells that can be expressed in situ from plasmid DNA or virus-derived constructs. shRNA is a means of inducing RNA interference-mediated post-transcriptional gene silencing against target genes.

As used herein, "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody that retains the ability to bind antigen. Examples of scfvs include antibody polypeptides formed by recombinant DNA techniques, and in which the Fv variable regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via spacer sequences. Various methods for making scFv are known to those skilled in the art.

As used herein, "tumor antigen" means an antigenic biomolecule, the expression of which results in cancer.

The invention relates to a nucleic acid sequence comprising (a) a first polynucleotide encoding a Chimeric Antigen Receptor (CAR) fusion protein and (b) a second polynucleotide encoding a short hairpin IL-6shRNA sequence, wherein the fusion protein comprises from N-terminus to C-terminus: (i) comprising V_HAnd V_LThe single-chain variable fragment (scFv) of (i), the scFv specifically binds to a tumor antigen, (ii) the transmembrane domain, (iii) the at least one costimulatory domain, and (iv) the activation structureA domain. The first polynucleotide and the second polynucleotide are transcribed from the same construct. The short hairpin IL-6shRNA sequence is capable of silencing expression of IL-6 and alleviating cytokine release syndrome in a patient treated with CAR-T cells.

The invention also relates to a nucleic acid sequence comprising (a) a first polynucleotide encoding a Chimeric Antigen Receptor (CAR) fusion protein and (b) a second polynucleotide encoding a short hairpin shRNA sequence of a checkpoint inhibitor, wherein the fusion protein comprises from N-terminus to C-terminus: (i) comprising V_HAnd V_LThe single-chain variable fragment (scFv) of (i), the scFv specifically binding to a tumor antigen, (ii) the transmembrane domain, (iii) the at least one costimulatory domain, and (iv) the activation domain, and the checkpoint inhibitor is PD-1, CTLA-4, TIM-3, TIGIT, or LAG-3. The first polynucleotide and the second polynucleotide are transcribed from the same construct. Checkpoint inhibitor shRNA sequences are capable of silencing the expression of checkpoint inhibitor proteins and prevent T cell failure in patients treated with CAR-T cells.

In one embodiment, the second polynucleotide is downstream of the first polynucleotide.

In one embodiment, the first polynucleotide and the second polynucleotide each have their own promoter to initiate transcription.

Insertion of short hairpin shRNA sequences in CAR constructs provides stable knockdown cell lines and eliminates the need for multiple rounds of siRNA oligonucleotide addition by transfection, which dilutes the siRNA in each round of replication. Inclusion of shRNA in lentiviral CAR constructs increases reproducibility of results. The use of lentiviral, adenoviral or retroviral vectors to deliver shRNA generates cells with stable shRNA expression. Lentiviral delivery using shRNA is preferred because of its low cytotoxicity. The potential beneficial effects of shRNA expression are: the RNAi effect is more durable than the delivery of synthetic nucleotide-based sirnas.

The invention provides a nucleic acid sequence that encodes a CAR with a short hairpin shRNA sequence that silences the expression of II-6 or a checkpoint inhibitor. The structure of the short nucleic acid construct is shown in FIG. 2. FIG. 2 shows CD19-CAR-shRNA (IL-6, PD-1, TIGIT), but the same design can be used for tumor antigens other than CD19 and checkpoint inhibitors other than PD-1 and TIGIT. The CAR sequence with CD19-CAR contained a Flag tag after the scFv. Short hairpin IL-6, PD-1 or TIGIT shRNA sequences were inserted under the H1 promoter, and each shRNA contained sense, loop, antisense and termination signals. The CAR sequence may contain a Flag tag or TF (transferrin) tag before or after the ScFv to facilitate detection of CAR expression.

In one embodiment, the tumor antigen is selected from the group consisting of: BCMA (CD269, TNFRSF17), CD19, CD22, VEGFR-2, CD4, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD47, CD52, CD56, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, CD133, EGFR, GPC 3; PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2/HER-2, ErbB3/HER3, ErbB 3/HER-4, EphA 3, IGF 13, GD3, O-acetyl GD3, GHRHR, GHR, FLT 3, KDR, FLT 3, CD44v 3, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR 3, IL 63, gpl3, Lewis A, Lewis Y, TNFR 3, PD-L3, HVEM, MAGE-A, mesothelin, NY-ESO-1, PSMA, RORl, TNFRSF 3, CD3, MULRP 3, MUZT-L3, TW 3679, TRPC 3, TRPC 3679, TRPR 3, TRPC 3679, TRPC 3, TRPR 3679, TROCH-3, TRPC 3679, TROCH-3, TRPC 3, TRPC 3679, TRPC 3, TRPC, TROCH-3, TRPC, TROCH-3, TRPC 3, TRPC 3, TRPC. In a preferred embodiment, the tumor antigen is CD19 or CD 22.

In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP 10. A preferred co-stimulatory domain is CD 28.

A preferred activation domain is CD3 ζ (CD 3Z or CD3 ζ).

The transmembrane domain may be derived from a native polypeptide, or may be artificially designed. The transmembrane domain derived from a native polypeptide may be from any membrane-bound or transmembrane protein. For example, transmembrane domains of the T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR may be used. Artificially designed transmembrane domains are polypeptides that predominantly include hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. In particular, linker sequences with glycine-serine stretches may be used.

Sequences such as Flag tags or transferrin tags can be inserted to detect ScFv expression and to detect CAR expression.

Nucleic acids encoding shRNA containing CARs can be prepared by conventional methods. The base sequence encoding the amino acid sequence may be derived from the NCBI RefSeq ID or GenBank accession number described above for the amino acid sequence of each domain, and the nucleic acids of the invention may be prepared using standard molecular biology and/or chemical procedures. For example, a nucleic acid can be synthesized based on a base sequence, and the nucleic acid of the present invention can be prepared by binding DNA fragments obtained from a cDNA library using Polymerase Chain Reaction (PCR). shRNA sequences can be designed using different software, using, for example, IL-6mRNA or other sequences as target sequences for silencing.

Compositions comprising the nucleic acids of the invention as active ingredients can be administered for the treatment of, for example, cancers such as leukemia, solid tumors, and the like. The administration mode of the composition comprising the nucleic acid of the present invention as an active ingredient may be intradermal, intramuscular, subcutaneous, intraperitoneal, intravenous, intratumoral, or into afferent lymphatic vessels, by parenteral administration, for example, by injection or infusion, and the administration route is not particularly limited.

Nucleic acids encoding the CARs and shrnas of the invention can be inserted into a vector, and the vector can be introduced into a cell. For example, viral vectors such as retroviral vectors (including tumor retroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian viral vectors, vaccinia viral vectors, or sendai viral vectors, epstein-barr virus (EBV) vectors, and HSV vectors can be used. As the viral vector, it is preferable to use a viral vector which lacks replication ability and thus cannot replicate in an infected cell by itself.

The mechanism by which shRNA causes gene silencing through transcriptional repression is as follows: long dsRNA from the following sources: hairpin, complementary RNA, RNA-dependent RNA polymerase. Long dsRNA is cleaved by endoribonucleases known as Dicer. Dicer cleaves long dsRNA to form short interfering RNA or siRNA; this enables the molecule to form an RNA-induced silencing complex (RISC).

1. Once the shRNA is transported from the nucleus to the cytoplasm, it is incorporated into other proteins to form RISC.

2. Once the shRNA becomes part of the RISC complex, the shRNA unfolds to form a single-stranded siRNA.

3. The thermodynamically less stable strand due to its base pairing at the 5' end is selected to retain part of the RISC complex.

4. Single stranded siRNA, which are part of the RISC complex, can now scan and find complementary mRNA.

5. Once the single stranded siRNA (part of the RISC complex) binds to its target mRNA, it induces mRNA cleavage.

6. The mRNA is now cleaved and recognized as abnormal by the cell. This causes degradation of the mRNA and does not in turn translate the mRNA into amino acids and hence proteins. Thus, the target gene encoding the mRNA is silenced.

Short hairpin RNAs (shrnas) are a class of RNA molecules that have a length of 19-23 base pairs and function within the RNA interference (RNAi) pathway. shrnas can be up to 30 base pairs, but shorter 19-23 base pairs are preferred because they generally have no immune response. shRNA is able to interfere with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription and preventing translation.

In the present invention, IL-6shRNA targets IL-6 mRNA. As an example, the sequence encoding IL-6mRNA is shown below, with one targeting sequence shown in bold and underlined.

In one embodiment, the shRNA structure inserted into the lentiviral construct includes a sense strand (as bolded and underlined above), an antisense strand (complementary to the sense strand), and a loop (sequence between the sense and antisense sequences). For example, the loop is a 5-10 nucleotide spacer, such as TTGATATCCG, CCACC, CTCGAG, AAGCTT, CCACACC, TTCAAGAGA (SEQ ID NO. 2-7). The loop connects the sense and antisense strands. The termination signal tttttttt follows the antisense sequence. Short hairpin shRNA constructs can be initiated from either sense or antisense sequences without affecting gene silencing.

RNA polymerase III preferably initiates transcription of short hairpin siRNA constructs with purines (G or A). If the short hairpin insert does not start with a "G" or "A", an additional "G" is added to the 5' end of the hairpin insert sequence. Once the shRNA is transcribed under the H1 promoter (or other RNA polymerase III promoter), it forms a double-stranded hairpin, and the Dicer endonuclease binds to and cleaves a shorter shRNA that forms a complex with RISC (RNA-induced silencing complex). The shRNA is converted to a single stranded siRNA, which remains part of the RISC complex. Once the siRNA-RISC complex binds to the target mRNA in the cell for silencing, it degrades the mRNA and causes gene silencing due to lack of translation.

For example, when a retroviral vector is used, the method of the present invention can be carried out by selecting an appropriate packaging cell based on the LTR sequence and the packaging signal sequence possessed by the vector, and producing a retroviral particle using the packaging cell. Examples of packaging cells include PG13(ATCC CRL-10686), PA317(ATCC CRL-9078), GP + E-86 and GP + envAm-12 and Psi-Crip. Also 293 cells or 293T cells with high transfection efficiency can be used for the preparation of retroviral particles. A variety of retroviral vectors, produced on the basis of retroviruses and packaging cells (vectors that can be used to package retroviruses), are widely commercially available from a number of companies.

When the CAR binds to a specific antigen on the cell surface, a signal is transmitted into the cell and the cell is thereby activated. Activation of the CAR-expressing cell varies depending on the kind of host cell and the intracellular domain of the CAR, and can be confirmed based on, for example, release of cytokines, improvement in cell proliferation rate, change in cell surface molecules, and the like as indicators. For example, release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, IL-6, etc.) from activated CAR-T cells destroys antigen-expressing target cells. In addition, the release of cytokines or changes in cell surface molecules stimulate other immune cells such as B cells, dendritic cells, NK cells and macrophages.

Cells (e.g., T cells or NK cells) modified to express the CAR can be used as therapeutic agents for diseases. The therapeutic agent includes CAR-expressing cells as an active ingredient, and may further comprise a pharmaceutically acceptable excipient (such as a medium or buffer optionally with added components (cytokines, growth factors)) to dilute the cells.

The invention provides T cells or natural killer cells (NK cells) modified to express a CAR as described above. The CAR-T cells or CAR-NK cells of the invention bind to a specific tumor antigen via the scFv of the CAR, thereby transmitting a signal into the cell, thereby activating the cell. Activation of the CAR-expressing cell varies depending on the kind of host cell and the intracellular domain of the CAR, and can be confirmed based on, for example, release of cytokines, improvement of cell proliferation rate, change in cell surface molecules, killing of target cells, and the like as indicators.

The invention further provides adoptive cell therapy methods for treating cancer. The method comprises the following steps: obtaining a CAR-T cell or NK-cell modified to express a nucleic acid sequence of the invention, administering the CAR-T cell or CAR-NK cell to a subject having cancer, wherein the cancer cells of the subject overexpress a tumor antigen, and the CAR-T cell or CAR-NK cell binds to the cancer cells to kill the cancer cells.

The inventors have found that the addition of IL-6shRNA in CAR lentiviral constructs blocks the secretion of IL-6 while maintaining the activity of CAR-T cells. The inventors have generated CD19-IL-6shRNA-CAR T cells against hematological malignancies (leukemias, lymphomas, and myelomas) that have high killing activity against cancer cells that overexpress CD 19. The inventors confirmed the high cytotoxicity of CD19-Flag-CAR-T cells or CD19-TF-CAR-T cells with IL-6shRNA-CAR-T cells by real-time cytotoxicity assays against cervical cancer cell line Hela stably overexpressing CD19 antigen, and hematologic cancer Raji cells endogenously overexpressing CD19 antigen.

Insertion of short hairpin IL-6shRNA sequences in CAR constructs reduces IL-6 secretion, thereby increasing the safety of CAR-T cells to tumor cells. Clinically, the main problem with CAR-T cell delivery to patients is that CAR-T cells cause cytokine release syndrome or "cytokine storm". The primary inducer of cytokine release symptoms is IL-6 cytokine.

CAR-T cells are produced by T cells isolated from the blood of a patient. CAR-T cells are transduced with lentiviruses, retroviruses, or other viral or plasmid-based vectors, and these engineered CAR-T cells are typically injected into patients by intravenous injection. Such CAR-T cell therapy can cause adverse cytokine release syndrome or adverse neurological symptoms. The present invention generates CAR-T cells that are more safe in the clinic, reducing the production of IL-6 cytokines in patients to reduce cytokine release syndrome.

CD19 IL-6shRNA-CAR-T cells secreted significantly less IL-6 than CD19-CAR-T cells.

Insertion of the human IL-6shRNA sequence in the nucleic acid sequence encoding the CAR does not generate an adverse immune response in humans. The same strategy can be applied to CAR constructs using natural killer cells (primary human natural killer cells and NK-92 cells) or macrophages.

During the process of manufacturing CD19-IL-6shRNA-CAR T cells, enriched cell and memory T cell subsets can be selected to increase the efficacy and cytotoxicity of T cell production.

Combination therapy of CAR-T cells with IL-6shRNA, either bispecific CD19-CD22-CAR-T or bispecific BCMA-plus other ScFv against any MM marker CD38, CD319, CD138, CD33, can be used to increase the activity of single CAR-T cell therapy while reducing IL-6 cytokine secretion.

Combination therapy of CD19-IL-6shRNA CAR cells with chemotherapy or immune checkpoint inhibitors (PD-1, CTLA-4, TIM-3, TIGIT, LAG-3, and others) can be used to increase the activity of a single CAR. Tumors use checkpoint proteins to protect themselves from immune cell attack. Checkpoint immunotherapy blocks inhibitory checkpoints, restoring immune cell activation. The most well-known ligand-receptor interactions are the transmembrane programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) expressed in T cells with its frequently overexpressed ligand in tumors: interaction between PD-1 ligand 1(PD-L1, CD 274). Cell surface protein PD-L1 binds to PD1 on the surface of immune cells, inhibiting immune cell activity. Cancer-dependent upregulation of PD-L1 on the cell surface inhibited T cell activity. Different PD-1 or PD-L1 antibodies that bind to PD-1 or PD-L1 block this interaction, enabling T cells to attack the tumor. Similar mechanisms act on other interactions between CTLA-4, TIM-3 and other T cell receptors described above and tumor cell surface ligands. Downregulation of these receptors with shRNA will block the interaction with ligands and T cell activity will be higher than for T cells with checkpoint receptors.

The inventors demonstrated the cytotoxicity of CD19-TF-PD-1 shRNA CAR-T cells and CD19-TF-TIGIT shRNA CAR-T cells against target cancer cells in vitro and in vivo.

The inventors demonstrated that CD19TF-PD-1 shRNA-CAR T cells and CD19-TF-TIGIT shRNA CAR-T cells had higher in vivo efficacy against target cells than CD19TF-CDR T cells.

CAR-T cells targeting different tumor antigens can be used with IL-6 shRNA.

In addition to IL6, shRNAs for other cytokines (IFN-. gamma., IL-2, IL-10, MCP-1, others) may also be used in the CAR.

Other techniques for silencing IL-6, PD-1, or TIGIT, such as CRISPR/Cas-9, Talen, and SFN, can be used in CAR.

The following examples further illustrate the invention. These examples are intended to be merely illustrative of the invention and should not be construed as limiting.

Examples

Example 1 CAR constructs

The mouse FMC63 anti-CD 19 scFv (Kochenderfer et al (2009), i.immunother, 32:689-702) was inserted into a second generation CAR cassette containing a signaling peptide from GM-CSF, a hinge region, a transmembrane domain and a costimulatory domain from CD28, and a CD3 ζ activation domain; this CAR is referred to herein as a CD19 CAR. After insertion of the IL-6shRNA sequence under an independent promoter into the CAR. In the same way, a- "mock" CAR with scFv-specific for intracellular proteins and therefore not reacting with intact cells was constructed and used as a negative control CAR.

Example 2 sequence of CAR construct.

The amino acid sequence for each segment of the CD19-CAR construct in our experiments is shown below. Each segment may be replaced with an amino acid sequence having at least 95% identity.

(a)CD19-Flag CAR

Human GM-CSF Signal peptide-anti-CD 19 scFv (VL-linker-VH) -Flag-CD28 hinge, transmembrane CD 28-costimulatory CD28, CD 3-zeta

< human GM-CSF signal peptide > SEQ ID NO: 8

MLLLVTSLLLCELPHPAFLLIP

< FMC063 anti-CD 19 scFv (VL-linker-VH) >

<VL>SEQ ID NO：9

< linker > SEQ ID NO: 10

<VH>SEQ ID NO：11

In our construct, VH is followed by 3 amino acids AAA.

< Flag tag > SEQ ID NO: 12

D Y K D D D D K

< CD28 hinge > SEQ ID NO: 13

< transmembrane domain CD28> SEQ ID NO: 14

< co-stimulatory domain CD28> SEQ ID NO: 15

< activation domain CD3- ζ > SEQ ID NO: 16

CD19-CAR as shown below, the Flag tag is underlined, SEQ ID NO: 17

(b)CD19-Flag-IL-6shRNA CAR

The CD19-Flag CAR nucleotide sequence with IL-6shRNA sequence inside a lentiviral vector under H1 promoter is shown below as SEQ ID NO: 20, the IL-6shRNA, shown in bold, has the following structure: sense (by bold capital) -loop- (antisense capital, bold italics), termination sequence (underlined), H1 promoter shown in lower case letters 5' upstream of shRNA:

< Kpn I site >5' -GGTACC

< H1 promoter > SEQ ID NO: 18

< Xho I site > CTCGAG

< DNA encapsulation (encapsulation) IL-6 short hairpin shRNA > sense (bold) -loop-antisense (bold italics) and stop signal (underlined), SEQ ID NO: 19

< Nhe I site > GCTAGC-3'

Constructs of the DNA sequences inserted into lentiviral vectors encoding CAR and IL-6shRNA are shown below. The 5'-Xba I and 3' EcoR I sites (underlined) flank the Flag-tag containing CD19-CAR sequence (italic, underlined). The ATG initiation codon of the CAR sequence is shown in bold (underlined). The H1 promoter and short hairpin shRNA sequences are then shown in bold, larger font, starting from the Kpn I site (underlined) and ending with the Nhe I site (underlined) GCTAGC. The Kpn I site (lower case, underlined) is followed by the H1 promoter (in lower case) and then the Xho site (italics, upper case); sense (capital letters, underlined), loop (capital) and antisense (capital, italic, underlined) and termination (capital) sequences, ending with the Nhe I site.

(c)CD19-TF-IL-6shRNA-CAR

We prepared a CD19-TF (transferrin) tag-CAR with IL-6. The CAR sequence starts with the ATG start codon (underlined) followed by a CAR with TF underlined in italics. The H1 promoter and short hairpin shRNA sequences are shown in bold, starting from the Kpn I site (underlined) and ending with the Nhe I site (underlined) GCTAGC. The Kpn I site (lower case, underlined) is followed by the H1 promoter (lower case) and then the Xho site (italics, upper case); sense (capital letters, underlined), loop (capital) and antisense (capital, italic, underlined) and termination (capital) sequences, ending with the Nhe I site.

(d)IL-6shRNA

The IL-6shRNA targets the coding sequence of the mRNA for the IL-6 sequence, starting from ATG and ending with the GTA stop codon (GenBank: M54894.1; Wong, G.G., Witek-Giannotti, J., Hewick, R.M., Clark, S.C. and Ogawa, M. Interleukin 6: identified as hematopoietic colony stimulating factor Behring Inst.Mitt.83, 40-47(1988) PUBMED:3266463)。

the coding sequence of the IL-6mRNA is as set forth in SEQ ID NO: 1 is shown. Within the coding sequence, 4 targeting regions corresponding to the sense regions of the 4 IL-6shRNA are shown in bold and underlined, with overlapping sequences in the two targeting regions. The shRNA targets the IL-6mRNA and results in reduced transcription and expression of IL-6. Underlined larger font targeting sequences were used to prepare CARs with IL-6shRNA in the examples.

SEQ ID NO.22-25 are examples of four DNA sequences encoding IL-6shRNA (sense-loop-antisense-stop signal) flanked by BamH1 sites at the 5 'and 3' positions.

(SEQ ID NO: 22, used in the examples to prepare CAR)

Addition of CG to RNA polymerase III to increase transcription efficiency

(e)PD-1 shRNA

The coding sequence of PD-1(GeneBank ID: L27440.1) is shown below as SEQ ID NO: shown at 26. Within the coding sequence, 3 targeting regions corresponding to the sense regions of the three PD-1 shrnas are shown in bold and underlined, with overlapping sequences in the two targeting regions.

The coding sequence of PD-1shRNA has the structure of sense (bold underlined), loop, antisense (italic, bold), termination sequence (SEQ ID NO: 27-29)

(SEQ ID NO: 27, used in the examples)

(f)CTLA-4shRNA

CTLA-4cDNA, Gene ID1493The nucleotide sequence targeted by the shRNA is underlined and in bold.

The DNA sequence encoding the CTLA-4shRNA is shown below.

(g)TIM-3shRNA_

TIM-3DNA, ID: AK314406, the nucleotide sequence targeted by shRNA is underlined and bolded.

The DNA sequence encoding the TIM-3shRNA is shown below.

(h)LAG-3shRNA

LAG-3DNA, Gene ID: 3902, the nucleotide sequence targeted by the shRNA is underlined and in bold.

The DNA sequence encoding LAG-3shRNA is shown below.

(h)TIGIT shRNA

TIGIT DNA, gene ID: 3902, the nucleotide sequence targeted by the shRNA is underlined and in bold. In the TIGIT DNA sequence, 3 targeting regions corresponding to the sense regions of the three TIGIT shrnas are shown in bold and underlined, with overlapping sequences in the two targeting regions.

The DNA sequence encoding TIGIT shRNA is shown below.

(SEQ ID NO: 40, used in the examples for CAR constructs)

The shRNAs for BTLA (Gene ID: 151888) and other targets can be prepared in a similar manner.

We generated a CD19-TF tag-CAR with PD-1 shRNA. The XbaI and EcoRI sites flanking the CAR are underlined and the ATG start codon of the CAR is shown in bold. The CD19-CAR portion was identical to CD19-CAR with IL-6shRNA, except that the TF (transferrin) tag replaced the Flag tag. The TF tag is underlined in italics.

The H1 promoter and short hairpin shRNA sequences are then shown in bold in larger font, starting from the Kpn I site GGTACC (underlined) and ending with the Nhe I site (lower case, underlined) GCTAGC. Following the Kpn I site is the H1 promoter (in lower case, underlined), followed by sense (upper case, underlined), loop (upper case) and antisense (upper case, italic, underlined) and termination sequences (upper case), ending with the Nhe I site.

(i)CD19-TF-PD-1 shRNA CAR

The nucleic acid sequence of the CAR with PD-1shRNA (large font, bold), starting with an XbaI site (underlined) and ending with an Eco RI site (underlined) is shown below. The TF tag is in large font and underlined.

The transferrin tag can be used to detect CAR and it can reduce cytokines, which is clinically beneficial for reducing cytokine release syndrome.

CD19-TF CAR the TF tag(s) ((R))KNPDPWAKNLNEKDYSEQ ID NO: 44) is underlined.

We prepared CD19-TF tag-CAR with TIGIT shRNA in the same manner as described above. The sequence is shown below, starting with the underlined ATG start codon, the TF tag is underlined in italics, and the TIGIT siRNA is flanked by bold Kpn I and Nhe I sites. Following the Kpn I site is the H1 promoter (lower case, underlined), followed by sense (upper case, underlined), loop (upper case) and antisense (upper case, italic, underlined) and termination (upper case) sequences, ending with the Nhe I site (lower case, underlined).

Example 3 preparation of CAR-encoding lentiviruses

DNA encoding CAR was synthesized by Syno Biological (beijing, china) and subcloned into a third generation lentiviral vector with EF1a promoter. All CAR lentiviral constructs were sequenced in both directions to confirm the CAR sequence and used for lentiviral production. Will be 1 × 10⁷Stagnant HEK293FT cells (Thermo Fisher) were seeded into T75 flasks and cultured overnight, then transfected with CalPhos transfection kit (Takara, Mountain View, Calif.) by adding pPACKH1 Lentivector packaging mix (System Biosciences, Palo Alto, Calif.) and 10 μ g of each lentiviral vector. The next day, the medium was replaced with fresh medium for 48 hoursThe lentivirus-containing medium is then collected. The medium was cleared of cell debris by centrifugation at 2100g for 30 minutes. Viral particles were collected by centrifugation at 112,000g for 100 min, suspended in AIM V medium, aliquoted and frozen at-80 ℃. The titer of the virus preparation was determined by quantitative RT-PCR according to the manufacturer's protocol and 7900HT thermal cycler (Thermo Fisher) using the Lenti-XqRT-PCR kit (Takara). The lentivirus titer is>1×10⁸pfu/ml。

Example 4 preparation and expansion of CAR-T cells

PBMC at 1X 10⁶Cells/ml were suspended in AIM V-Albumax medium (Thermo Fisher) containing 10% FBS and 300U/ml IL-2(Thermo Fisher), mixed with an equal number (1:1 ratio) of CD3/CD28 Dynabeads (Thermo Fisher), and cultured in untreated 24-well plates (0.5 ml/well). Lentiviruses were added to the culture at multiplicity of infection (MOI)5 at 24 and 48 hours, along with 1. mu.l of TransPlus transduction enhancer (AlStem). As T cells proliferate in the next two weeks, cells were counted every 2-3 days, and fresh medium with 300U/ml IL-2 was added to the culture to maintain cell density at 1-3X 10⁶Individual cells/ml.

Example 5 flow cytometry

To measure CAR expression, 0.5X 10⁶Individual cells were suspended in 100. mu.l buffer (PBS containing 0.5% BSA) and incubated with 1. mu.l human serum (Jackson Immunoresearch, West Grove, Pa.) on ice for 10 min. Then 1 μ l Allophycocyanin (APC) -labeled anti-CD 3(eBioscience, san diego, CA) and 2 μ l Phycoerythrin (PE) -labeled anti-Flag or anti-FAB or its isotype control antibody were added and the cells were incubated on ice for 30 minutes. Cells were washed with 3ml buffer, then suspended in buffer and assayed using FACSCalibur (BD biosciences). CD3 stained cells were analyzed against Flag or FAB staining or isotype control staining.

Example 6 preparation of Stable HeLa-CD19 cell line

To prepare HeLa cells stably expressing human CD19, DNA encoding the open reading frame of human CD19 was synthesized by Syno Biological and subcloned into the pCD510 lentiviral vector (systems Biosciences). Lentiviruses containing the vector were prepared as described above. HeLa cells were infected with a lentivirus with an MOI of 5 and cultured in the presence of 1. mu.g/ml puromycin to produce resistant cells, herein referred to as HeLa-CD 19. Expression of CD19 was confirmed by flow cytometry (BioLegend) using CD19 antibody.

Example 7 real-time cytotoxicity assay (RTCA)

Adherence target cells (HeLa or HeLa-CD19) at 1X 10⁴Individual cells/well were seeded into 96-well E-plates (ace Biosciences, san diego, CA) and monitored overnight in culture with an impedance-based real-time cell assay (RTCA) icelligene system (ace Biosciences). The following day, the medium was removed and replaced with 10% FBS. + -. 1X 10⁵AIM V-AlbuMAX medium of individual effector cells (CAR-T cells or non-transduced T cells) in triplicate. Cells in the E-plate were monitored for an additional 2-3 days with the RTCA system and impedance plotted against time. Cell lysis was calculated as (impedance of target cells without effector cells-impedance of target cells with effector cells) x 100/impedance of target cells without effector cells. For nonadherent target cells (Raji), E plates were first coated with anti-CD 40 antibody (Acea Biosciences) to match CD40⁺Raji cell binding. Then spread the plate 1X 10⁴Individual Raji cells/well and RTCA assay was performed as described above.

Example 8 cytokine Induction assay (ELISA)

Target cells (Raji or HeLa-CD19) and effector cells (CAR-T cells or non-transduced T cells) in a ratio of 1:1 (1X 10 each)⁴Individual cells) were cultured in triplicate in U-bottom 96-well plates in 200 μ l of AIM V-AlbuMAX medium containing 10% FBS. After 16 hours, the top 150 μ l of medium was transferred to V-bottom 96-well plates and centrifuged at 300g for 5 minutes to pellet any remaining cells. The top 120. mu.l of supernatant was transferred to a new 96-well plate and human IFN-. gamma.and IL-6 levels were analyzed by ELISA using a kit from Thermo Fisher according to the manufacturer's protocol.

Example 9 statistical analysis

Data were analyzed and plotted using Prism software (GraphPad, San Diego, CA). Comparisons were made between the two groups by unpaired T-test (Student's T test). p <0.05 was considered significant.

Example 10, CD19-IL-6shRNA-CAR-T cells demonstrated high cytotoxicity against CD19 positive Hela-CD19 cells.

The high cytotoxicity of CD19-IL 6shRNA-CAR-T cells against CD19 positive Hela cells was confirmed by a real-time high-sensitivity cytotoxicity assay (fig. 3). Similar to CD19-CAR-T cells, CD19-IL-6shRNA specifically killed Hela-CD19 positive cells.

Example 11, CD19-IL-6shRNA-CAR-T cells secreted significantly less IL-6 than CD19-CAR-T cells in Raji leukemia cells.

CD19-IL-6shRNA CAR-T cells were cytotoxic against Raji cells, with endogenous expression of CD19 similar to CD19-CAR-T cells (data not shown). We performed ELISA assays for CD19-CAR-T cells and CD19-IL 6shRNA-CAR-T cells targeting IL-6 secretion by Raji cells (fig. 4). The secretion of IL-6 by CD19-IL-6shRNA-CAR-T cells against target Raji cells was significantly less (>1.6 fold, p <0.025) than by CD19-CAR-T cells (FIG. 4).

The reduction of IL-6 obtained with CD19-IL-6shRNA-CAR-T cells was specific for silencing of IL-6, but had no effect on other cytokines. CD19 IL-6shRNA-CAR-T and CD19-CAR-T cells secreted the same level of IFN- γ (fig. 5). Thus, the IL-6shRNA effect is highly specific and only reduces IL-6.

Example 12, CD19TF-IL 6shRNA-CAR-T cells and CD19TF-CAR-T cells have the same cytotoxicity.

We used Hela-CD19 as target cells at different E: T (effector/target) ratios: 20:1, 10:1 and 5:1, RTCA cytotoxicity assays as described above were performed on CD19-TF-IL-6shRNA-CAR-T cells and CD19TF-CAR-T cells, followed by quantitative analysis of cytotoxicity. There was no significant difference in cytotoxicity between these CAR-T cells at each E: T ratio (figure 6).

Example 13 CD19TF-IL-6shRNA CAR-T cells secreted significantly less IL-6 than CD19TF CAR-T cells

We assayed IL-6 by ELISA and demonstrated that CD19TF-IL-6shRNA CAR-T cells secreted significantly less IL-6 than CD19-CAR-T cells against Hela-CD19 cells. (FIG. 7).

Example 14, CD19-TF-PD-1 shRNA and CD19TF-TIGIT shRNA-CAR-T cells were as highly cytotoxic as CD19-CAR T cells without shRNA.

We performed RTCA assays of CD19-CAR T cells, CD19-TF-PD-1 shRNA-CAR T cells, and CD19TF-TIGIT shRNA-CAR-T cells using Hela-CD19 cells as target cells. All CAR-T cells were equally highly cytotoxic against Hela-CD19 cells (fig. 8) and Raji lymphoma cells (fig. 9). PD-1 and TIGIT shRNA (not shown) reduced the levels of PD-1 and TIGIT after coculture with Hela-CD19 cells.

CAR-T cells had high levels of IFN- γ secretion by Hela-CD19 target cells, but minimal secretion by Hela cells (figure 10).

Example 15, CD19-TF-PD-1 shRNA-CAR-T cells and CD19-TF-TIGIT shRNA-CAR-T cells had higher efficacy in vivo than conventional CD19-CAR-T cells.

We will 5X 10⁵Each Raji-luciferase cell was injected intravenously into the tail vein of NSG mice, and the next day, 1X 10 cells were injected in the same manner⁷CD19-TF-PD-1 shRNA-CAR-T cells or CD19-TF-TIGIT shRNA-CAR-T cells. We performed IVIS imaging and detected a significant reduction in bioluminescence in Raji NSG mouse models using CD19-TF-PD-1 shRNA-CAR-T cells or CD19-TF-TIGIT shRNA-CAR-T cells (figure 11). In addition, both CAR-T cells significantly prolonged mouse survival compared to CD19-TF-CAR-T cells (figure 12). These data show that CD19-TF-PD-1 shRNA-CAR-T cells and CD19-TF-TIGIT shRNA-CAR-T cells are more advantageous than conventional CD19-CAR-T cells, likely due to decreased T cell depletion and increased in vivo activity.

Reference to the literature

Gross, G. and Eshhar, Z. (2016). Therapeutic patent of T Cell Clinical Antibiotic Receptors (CARs) in Cancer Treatment. coupling of Off-Tumor toxins for Safe CAR T Cell therapy. Annu Rev Pharmacol toxin 56,59-83.

Maus, M.V., Grupp, S.A., Porter, D.L., and June, C.H, (2014.) anti-modified T cells, CARs take the front seat for pharmaceutical industries blood 123, 2625. supplement 2635.

Maus, M.V., Haas, A.R., Beatty, G.L., Albelda, S.M., Levine, B.L., Liu, X, ZHao, Y., Kalos, M.and June, C.H, (2013). T cells expressing polymeric adhesives receptors can be used as fuels in humans, cancer Immunol Res 1,26-31.

Kochenderfer, J.N., Dudley, M.E., Kassim, S.H., Somerville, R.P., Carpenter, R.O., Stetler-Stevenson, M.A., Yang, J.C., Phan, G.Q., Hughes, M.S., Sherry, R.M. et al (2015), Chemoterpathy-regenerative direct large B-cell lymphoma and index B-cell maligngances can be used for expressing an anti-CD 19 chimeric antigen Clin on col 33, 540. f.

Golubvskaya, V. and Wu, L. (2016.) Differencen Subsets of T Cells, Memory, Effect Functions, and CAR-T immunology. cameras (Basel)8.

6.Maus, M.V. and June, C.H. (2013). Zoom Zoom: racing CARs for multiple Myeloma. Clin Cancer Res 19, 1917-.

Maus, M.V. and June, C.H. (2014). CARTs on the road for myeloma. Clin Cancer Res 20, 3899-.

Kochenderfer, J.N. and Rosenberg, S.A. (2013). Treting B-cell candidate with Tcells expressing anti-CD 19 polymeric anti-cancer receptors Nat Rev Clin Oncol 10,267-276.

Sequence listing

<110> Promega Biotech Co

PROMAB BIOTECHNOLOGIES, Inc.

<120> nucleic acid sequence encoding a short hairpin RNA sequence of a chimeric antigen receptor and IL-6 or a checkpoint inhibitor

<130> 119995-8014.WO01

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accattatcc atgtgaaagg gaaacacctt tgtccaagtc ccctatttcc cggaccttct 960

aagccctttt gggtgctggt ggtggttggg ggagtcctgg cttgctatag cttgctagta 1020

acagtggcct ttattatttt ctgggtgagg agtaagagga gcaggctcct gcacagtgac 1080

tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1140

ccaccacgcg acttcgcagc ctatcgctcc agagtgaagt tcagcaggag cgcagacgcc 1200

cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1260

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1320

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1380

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1440

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1500

cctcgctaag aattcggatc cgcggccgcg aaggatctgc gatcgctccg gtgcccgtca 1560

gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg 1620

aacgggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct 1680

ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt 1740

tctttttcgc aacgggtttg ccgccagaac acagctgaag cttcgagggg ctcgcatctc 1800

tccttcacgc gcccgccgcc ctacctgagg ccgccatcca cgccggttga gtcgcgttct 1860

gccgcctccc gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct 1920

caggtcgaga ccgggccttt gtccggcgct cccttggagc ctacctagac tcagccggct 1980

ctccacgctt tgcctgaccc tgcttgctca actctacgtc tttgtttcgt tttctgttct 2040

gcgccgttac agatccaagc tgtgaccggc gcctacgcta gatgaccgag tacaagccca 2100

cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 2160

tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 2220

ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 2280

cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 2340

tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 2400

aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 2460

ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 2520

gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 2580

acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 2640

gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg agtcgacaat caacctctgg 2700

attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 2760

gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 2820

tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 2880

ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 2940

ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 3000

aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 3060

attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca 3120

cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 3180

ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 3240

agacgagtcg gatctccctt tgggccgcct ccccgcctgg taccgaacgc tgacgtcatc 3300

aacccgctcc aaggaatcgc gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc 3360

gccctggcag gaagatggct gtgagggaca ggggagtggc gccctgcaat atttgcatgt 3420

cgctatgtgt tctgggaaat caccataaac gtgaaatgtc tttggatttg ggaatcttat 3480

aagttctgta tgagaccacc tcgaggagta acatgtgtga aagcttgata tccggctttc 3540

acacatgtta ctcttttttg ctagc 3565

<210> 21

<211> 2405

<212> DNA

<213> Intelligent people

<400> 21

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120

gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180

aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240

ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300

gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360

ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420

ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480

ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540

gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600

agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660

tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720

tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780

acctcagtca ccgtctcctc agcggccgca aaaaacccgg atccgtgggc gaaaaacctg 840

aacgaaaaag attatattga agttatgtat cctcctcctt acctagacaa tgagaagagc 900

aatggaacca ttatccatgt gaaagggaaa cacctttgtc caagtcccct atttcccgga 960

ccttctaagc ccttttgggt gctggtggtg gttgggggag tcctggcttg ctatagcttg 1020

ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1080

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1140

tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1200

gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1260

agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1320

ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1380

gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1440

ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1500

ctgccccctc gctaagaatt cgtcgacaat caacctctgg attacaaaat ttgtgaaaga 1560

ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 1620

cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 1680

tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 1740

actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 1800

tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 1860

gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 1920

aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 1980

tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 2040

ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 2100

tgggccgcct ccccgcctgg taccgaacgc tgacgtcatc aacccgctcc aaggaatcgc 2160

gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag gaagatggct 2220

gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt tctgggaaat 2280

caccataaac gtgaaatgtc tttggatttg ggaatcttat aagttctgta tgagaccacc 2340

tcgaggagta acatgtgtga aagcttgata tccggctttc acacatgtta ctcttttttg 2400

ctagc 2405

<210> 22

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 22

gagtaacatg tgtgaaagct tgatatccgg ctttcacaca tgttactctt tttt 54

<210> 23

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 23

ggagacatgt aacaagagtt tgatatccga ctcttgttac atgtctcctt tttt 54

<210> 24

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 24

gatggatgct tccaatctgt tgatatccgc agattggaag catccatctt tttt 54

<210> 25

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 25

cgttcggtac atcctcgacg gttgatatcc gccgtcgagg atgtaccgaa tttttt 56

<210> 26

<211> 867

<212> DNA

<213> Intelligent people

<400> 26

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggtcagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atctgctccc gggccgcacg agggacaata 600

ggagccaggc gcaccggcca gcccctgaag gaggacccct cagccgtgcc tgtgttctct 660

gtggactatg gggagctgga tttccagtgg cgagagaaga ccccggagcc ccccgtgccc 720

tgtgtccctg agcagacgga gtatgccacc attgtctttc ctagcggaat gggcacctca 780

tcccccgccc gcaggggctc agctgacggc cctcggagtg cccagccact gaggcctgag 840

gatggacact gctcttggcc cctctga 867

<210> 27

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 27

ggcgcagatc aaagagagct tgatatccgg ctctctttga tctgcgcctt tttt 54

<210> 28

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 28

aaggcgcaga tcaaagagag cttgatatcc ggctctcttt gatctgcgcc tttttttt 58

<210> 29

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 29

aacacatcgg agagcttcgt gttgatatcc gcacgaagct ctccgatgtg tttttttt 58

<210> 30

<211> 672

<212> DNA

<213> Intelligent people

<400> 30

atggcttgcc ttggatttca gcggcacaag gctcagctga acctggctac caggacctgg 60

ccctgcactc tcctgttttt tcttctcttc atccctgtct tctgcaaagc aatgcacgtg 120

gcccagcctg ctgtggtact ggccagcagc cgaggcatcg ccagctttgt gtgtgagtat 180

gcatctccag gcaaagccac tgaggtccgg gtgacagtgc ttcggcaggc tgacagccag 240

gtgactgaag tctgtgcggc aacctacatg atggggaatg agttgacctt cctagatgat 300

tccatctgca cgggcacctc cagtggaaat caagtgaacc tcactatcca aggactgagg 360

gccatggaca cgggactcta catctgcaag gtggagctca tgtacccacc gccatactac 420

ctgggcatag gcaacggaac ccagatttat gtaattgatc cagaaccgtg cccagattct 480

gacttcctcc tctggatcct tgcagcagtt agttcggggt tgttttttta tagctttctc 540

ctcacagctg tttctttgag caaaatgcta aagaaaagaa gccctcttac aacaggggtc 600

tatgtgaaaa tgcccccaac agagccagaa tgtgaaaagc aatttcagcc ttattttatt 660

cccatcaatt ga 672

<210> 31

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 31

gatccttgca gcagttagtt tgatatccga ctaactgctg caaggatctt tttt 54

<210> 32

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 32

aaatcaagtg aacctcacta tttgatatcc gatagtgagg ttcacttgat tttttttt 58

<210> 33

<211> 906

<212> DNA

<213> Intelligent people

<400> 33

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cggatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactctg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgat ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag attggccaat gacttacggg actctggagc aaccatcaga 600

ataggcatct acatcggagc agggatctgt gctgggctgg ctctggctct tatcttcggc 660

gctttaattt tcaaatggta ttctcatagc aaagagaaga tacagaattt aagcctcatc 720

tctttggcca acctccctcc ctcaggattg gcaaatgcag tagcagaggg aattcgctca 780

gaagaaaaca tctataccat tgaagagaac gtatatgaag tggaggagcc caatgagtat 840

tattgctatg tcagcagcag gcagcaaccc tcacaacctt tgggttgtcg ctttgcaatg 900

ccatag 906

<210> 34

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 34

ggatttccgc aaaggagatt tgatatccga tctcctttgc ggaaatcctt tttt 54

<210> 35

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 35

gtccctgacc atagagaatt tgatatccga ttctctatgg tcagggactt tttt 54

<210> 36

<211> 1578

<212> DNA

<213> Intelligent people

<400> 36

atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg 60

aagcctctcc agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc 120

cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg 180

gtcacttggc agcatcagcc agacagtggc ccgcccgctg ccgcccccgg ccatcccctg 240

gcccccggcc ctcacccggc ggcgccctcc tcctgggggc ccaggccccg ccgctacacg 300

gtgctgagcg tgggtcccgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc 360

cagctggatg agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg 420

cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc cctctcctgc 480

cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca gccccccagg atctctcaga 540

gcctccgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg 600

cattggttcc ggaaccgggg ccagggccga gtccctgtcc gggagtcccc ccatcaccac 660

ttagcggaaa gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc 720

tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct cactgttctg 780

ggtctggagc ccccaactcc cttgacagtg tacgctggag caggttccag ggtggggctg 840

ccctgccgcc tgcctgctgg tgtgggtacc cggtctttcc tcactgccaa gtggactcct 900

cctgggggag gccctgacct cctggtgact ggagacaatg gcgactttac ccttcgacta 960

gaggatgtga gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag 1020

cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca 1080

cctggatccc tggggaagct gctttgtgag gtgactccag tatctggaca agaacgcttt 1140

gtgtggagct ctctggacac cccatcccag aggagtttct caggaccttg gctggaggca 1200

caggaggccc agctcctttc ccagccttgg caatgccagc tgtaccaggg ggagaggctt 1260

cttggagcag cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga 1320

gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcacccttgg tgtcctttct 1380

ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca 1440

agacgatttt ctgccttaga gcaagggatt caccctccgc aggctcagag caagatagag 1500

gagctggagc aagaaccgga gccggagccg gagccggaac cggagcccga gcccgagccc 1560

gagccggagc agctctga 1578

<210> 37

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 37

gcatcctcac ctacagagat caagagtctc tgtaggtgag gatgcttttt t 51

<210> 38

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 38

ggagacaatg gcgactttat caagagtaaa gtcgccattg tctccttttt t 51

<210> 39

<211> 734

<212> DNA

<213> Intelligent people

<400> 39

atgcgctggt gtctcctcct gatctgggcc caggggctga ggcaggctcc cctcgcctca 60

ggaatgatga caggcacaat agaaacaacg gggaacattt ctgcagagaa aggtggctct 120

atcatcttac aatgtcacct ctcctccacc acggcacaag tgacccaggt caactgggag 180

cagcaggacc agcttctggc catttgtaat gctgacttgg ggtggcacat ctccccatcc 240

ttcaaggatc gagtggcccc aggtcccggc ctgggcctca ccctccagtc gctgaccgtg 300

aacgatgcag gggagtactt ctgcatctat cacacctacc ctgatgggac gtacactggg 360

agaatcttcc tggaggtcct agaaagctca gtggctgagc acggtgccag gttccagatt 420

ccattgcttg gagccatggc cgcgacgctg gtggtcatct gcacagcagt catcgtggtg 480

gtcgcgttga ctagaaagaa gaaagccctc agaatccatt ctgtggaagg tgacctcagg 540

agaaaatcag ctggacagga ggaatggagc cccagtgctc cctcaccccc aggaagctgt 600

gtccaggcag aagctgcacc tgctgggctc tgtggagagc agcggggaga ggactgtgcc 660

gagctgcatg actacttcaa tgtcctgagt tacagaagcc tgggtaactg cagcttcttc 720

acagagactg gtta 734

<210> 40

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 40

gagaaaggtg gctctatcat tgatatccgt gatagagcca cctttctctt tttt 54

<210> 41

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 41

gtggctctat catcttacat tgatatccgt gtaagatgat agagccactt tttt 54

<210> 42

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> composition

<400> 42

gctgcatgac tacttcaatt tgatatccgg ctctctttga tctgcgcctt tttttt 56

<210> 43

<211> 2414

<212> DNA

<213> Intelligent people

<400> 43

tctagagccg ccaccatgct tctcctggtg acaagccttc tgctctgtga gttaccacac 60

ccagcattcc tcctgatccc agacatccag atgacacaga ctacatcctc cctgtctgcc 120

tctctgggag acagagtcac catcagttgc agggcaagtc aggacattag taaatattta 180

aattggtatc agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga 240

ttacactcag gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttattctctc 300

accattagca acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg 360

cttccgtaca cgttcggagg ggggactaag ttggaaataa caggctccac ctctggatcc 420

ggcaagcccg gatctggcga gggatccacc aagggcgagg tgaaactgca ggagtcagga 480

cctggcctgg tggcgccctc acagagcctg tccgtcacat gcactgtctc aggggtctca 540

ttacccgact atggtgtaag ctggattcgc cagcctccac gaaagggtct ggagtggctg 600

ggagtaatat ggggtagtga aaccacatac tataattcag ctctcaaatc cagactgacc 660

atcatcaagg acaactccaa gagccaagtt ttcttaaaaa tgaacagtct gcaaactgat 720

gacacagcca tttactactg tgccaaacat tattactacg gtggtagcta tgctatggac 780

tactggggtc aaggaacctc agtcaccgtc tcctcagcgg ccgcaaaaaa cccggatccg 840

tgggcgaaaa acctgaacga aaaagattat attgaagtta tgtatcctcc tccttaccta 900

gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 960

cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 1020

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1080

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1140

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1200

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1260

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1320

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1380

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1440

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1500

cttcacatgc aggccctgcc ccctcgctaa gaattcgtcg acaatcaacc tctggattac 1560

aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 1620

tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 1680

tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 1740

cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 1800

acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 1860

atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 1920

gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg 1980

attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 2040

tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 2100

agtcggatct ccctttgggc cgcctccccg cctggtaccg aacgctgacg tcatcaaccc 2160

gctccaagga atcgcgggcc cagtgtcact aggcgggaac acccagcgcg cgtgcgccct 2220

ggcaggaaga tggctgtgag ggacagggga gtggcgccct gcaatatttg catgtcgcta 2280

tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg atttgggaat cttataagtt 2340

ctgtatgaga ccacggcgca gatcaaagag agcttgatat ccggctctct ttgatctgcg 2400

ccttttttgc tagc 2414

<210> 44

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> composition

<400> 44

Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr

1 5 10 15

<210> 45

<211> 504

<212> PRT

<213> Intelligent people

<400> 45

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

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

20 25 30

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

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

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

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Lys Asn

260 265 270

Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr Ile Glu Val

275 280 285

Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

355 360 365

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

370 375 380

Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala

385 390 395 400

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

405 410 415

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

420 425 430

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

435 440 445

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

450 455 460

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

465 470 475 480

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

485 490 495

His Met Gln Ala Leu Pro Pro Arg

500

<210> 46

<211> 2399

<212> DNA

<213> Intelligent people

<400> 46

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120

gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180

aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240

ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300

gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360

ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420

ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480

ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540

gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600

agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660

tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720

tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780

acctcagtca ccgtctcctc agcggccgca aaaaacccgg atccgtgggc gaaaaacctg 840

aacgaaaaag attatattga agttatgtat cctcctcctt acctagacaa tgagaagagc 900

aatggaacca ttatccatgt gaaagggaaa cacctttgtc caagtcccct atttcccgga 960

ccttctaagc ccttttgggt gctggtggtg gttgggggag tcctggcttg ctatagcttg 1020

ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1080

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1140

tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1200

gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1260

agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1320

ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1380

gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1440

ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1500

ctgccccctc gctaagaatt cgtcgacaat caacctctgg attacaaaat ttgtgaaaga 1560

ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 1620

cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 1680

tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 1740

actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 1800

tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 1860

gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 1920

aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 1980

tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 2040

ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 2100

tgggccgcct ccccgcctgg taccgaacgc tgacgtcatc aacccgctcc aaggaatcgc 2160

gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag gaagatggct 2220

gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt tctgggaaat 2280

caccataaac gtgaaatgtc tttggatttg ggaatcttat aagttctgta tgagaccacg 2340

agaaaggtgg ctctatcatt gatatccgtg atagagccac ctttctcttt tttgctagc 2399

Claims

1. A nucleic acid sequence, comprising:

(a) a first polynucleotide encoding a Chimeric Antigen Receptor (CAR) fusion protein comprising, from N-terminus to C-terminus:

(i) comprising V_HAnd V_LThe single-chain variable fragment (scFv) of (1), wherein the scFv specifically binds to a tumor antigen,

(ii) (ii) a transmembrane domain which is capable of,

(iii) at least one co-stimulatory domain, and

(iv) an activation domain; and

(b) a second polynucleotide encoding an IL-6 short hairpin RNA (shRNA) sequence.

2. The nucleic acid sequence of claim 1, wherein the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 22. 23, 24 or 25.

3. A nucleic acid sequence comprising:

(ii) (ii) a transmembrane domain which is capable of,

(iii) at least one co-stimulatory domain, and

(iv) an activation domain; and

(b) a second polynucleotide encoding an shRNA sequence of a checkpoint inhibitor, wherein the checkpoint inhibitor is PD-1, CTLA-4, TIM-3, TIGIT, or LAG-3.

4. The nucleic acid sequence of claim 3, wherein the second polynucleotide encodes PD-1shRNA and comprises the nucleotide sequence of SEQ ID NO: 27. 28 or 29.

5. The nucleic acid sequence of claim 3, wherein the second polynucleotide encodes a TIGIT shRNA and comprises the nucleic acid sequence of SEQ ID NO: 40. 41 or 42.

6. The nucleic acid sequence of claim 3, wherein the second polynucleotide encodes a CTLA-4shRNA and comprises the nucleic acid sequence of SEQ ID NO: 31 or 32.

7. The nucleic acid sequence of claim 3, wherein the second polynucleotide encodes TIM-3shRNA and comprises SEQ ID NO: 34 or 35.

8. The nucleic acid sequence of claim 3, wherein the second polynucleotide encodes a LAG-3shRNA and comprises the nucleotide sequence of SEQ ID NO: 37 or 38.

9. The nucleic acid sequence of claim 1 or 3, wherein the second polynucleotide is downstream of the first polynucleotide and each polynucleotide has its own promoter.

10. The nucleic acid sequence of claim 1 or 3, wherein the tumor antigen is selected from the group consisting of: CD19, CD22, BCMA, VEGFR-2, CD20, CD30, CD25, CD28, CD30, CD33, CD47, CD52, CD56, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, CD133, EGFR, GPC 3; PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2/HER-2, ErbB3/HER3, ErbB 3/HER-4, EphA 3, IGF 13, GD3, O-acetyl GD3, GHRHR, GHR, FLT 3, KDR, FLT 3, CD44v 3, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR 3, IL 63, gpl3, Lewis A, Lewis Y, TNFR 3, PD-L3, HVEM, MAGE-A, mesothelin, NY-ESO-1, PSMA, RORl, TNFRSF 3, CD3, MULRP 3, MUZT-L3, TW 3679, TRPC 3, TRPC 3679, TRPR 3, TRPC 3679, TRPC 3, TRPR 3679, TROCH-3, TRPC 3679, TROCH-3, TRPC 3, TRPC 3679, TRPC 3, TRPC, TROCH-3, TRPC, TROCH-3, TRPC 3, TRPC 3, TRPC.

11. The nucleic acid sequence of claim 10, wherein the tumor antigen is CD 19.

12. The nucleic acid sequence of claim 10, wherein the co-stimulatory domain is CD28, 4-1BB, ICOS-1, CD27, OX-40, GITR, or DAP 10.

13. The nucleic acid sequence of claim 10, wherein the activation domain is CD3 ζ.

14. The nucleic acid sequence of claim 11, wherein the scFv that binds CD19 further comprises the amino acid sequence of SEQ ID NO: 12 or the Flag tag of SEQ ID NO: 44, transferrin tag.

15. The nucleic acid sequence of claim 14, wherein the CAR comprises SEQ ID NO: 17 or 45.

16. A T cell or NK cell modified to express the nucleic acid sequence of any one of claims 1-15.