NUCLEIC ACID SEQUENCE ENCODING CHIMERIC ANTIGEN RECEPTOR AND A SHORT HAIRPIN RNA SEQUENCE FOR IL-6 OR A

CHECKPOINT INHIBITOR

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM The Sequence Listing is concurrently submitted herewith with the specification as an ASCII formatted text file via EFS-Web with a file name of Sequence Listing.txt with a creation date of February 3, 2020 and a size of 39.8 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby i ncorporated in its entirety by reference herein.

FIELD OF THE INVENTION

The present invention relates to a nucleic acid sequence comprising a first

polynucleotide encoding a chimeric antigen receptor and a second polynucleotide encoding short hairpin ENA (shRNA) down-regulating IL-6 or a checkpoint protein.

BACKGROUND OF THE INVENTION

Immunotherapy is emerging as a highly promi sing approach for the treatment of cancer. T cells or T lymphocytes, the armed forces of our immune system that constantly looks for foreign antigens and discriminates abnormal (cancer or infected cells) from normal cells [1] Genetically modifying T cells with CARs is the most common approach to design tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAA) can be infused into patients (called adoptive cell transfer or ACT) representing an efficient immunotherapy approach [1 , 2]. The advantage of CAR-T technology compared with chemotherapy or antibody is that reprogrammed engineered T cells can proliferate and persist in the patient (“a living drug”)[3], [4]

CARs (Chimeric antigen receptors) usually consist of a monoclonal antibody-derived single-chain variable fragment (scFv) linked by a hinge and then transmembrane domain to a variable number of intracellular signaling domains: a single, cellular activating, CD3-zeta domain; and CD28, CD137 (4-lBB) or other co-stimulatory domains, in tandem with a CD3-zeta domain (the CD27 signaling domain has also been used in the place of either the i CD28 or CD137 domain) (FIG. 1) [3], [5] The evolution of CARs went from first generation

(with no costimulation domains) to second generation (with one co-stimulation domain) to third generation CAR (with several co-stimulation domains). Generating CARs with multiple costimulatory domains (the so-called 3 generation CAR) have led to increased cytolytic activity, and significantly improved persistence of CAR-T cells that demonstrate augmented antitumor activity.

The major problem with CAR-T cell therapy is that patients treated with CAR-T cells develop cytokine release syndrome (CRS) due to high secretion of IL-6; CRS is accompanied with high fever and adverse neurological symptoms. IL-6 is one of the major cytokines secreted by T cells to stimulate immune response. IL-6 is an important modulator of fever and plays major role in pathogenesis of many diseases such as multiple myeloma, autoimmune diseases and prostate cancer. IL-6 secretion and all symptoms of CRS can be decreased with

Tocilizumab (a humanized antibody against IL-6 receptor) that blocks inflammatory IL-6 cytokine production.

Checkpoint inhibitor therapy is a form of cancer immunotherapy. The therapy targets immune checkpoints, key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function. Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1 .

CTLA4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD 152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA4 is const! tutively expressed in regulator} T cells but only upregulated in conventional T cells after activation - a phenomenon which is particularly notable in cancers. It acts as an "off switch when bound to CD80 or CD86 on the surface of antigen-presenting cells.

PD-1 is the transmembrane programmed cell death 1 protein, which interacts with PD- L1 (PD-1 ligand 1). PD-Ll on the cell surface binds to PD1 on an immune cell surface, which inhibits immune ceil activity. Among PD-Ll functions is a key regulatory role on T cell activities. Deregulation of PD-Ll on the cell surface may inhibit T cells that might otherwise attack. Antibodies that bind to either PD-1 or PD-Ll and therefore block the interaction may allow ^? the T-cells to attack the tumor. TIGIT (also called T cell immunoreceptor with Ig and GPM domains) is an immune receptor present on some T cells and Natural Killer Cells. TIGIT binds to CD155 (PVR) on dendritic cells (DCs), macrophages, etc. with high affinity, and also to CD112 (PVRL2) with lower affinity.

T cell immunoglobulin and mucin-domain containing-3 (Tim-3) is a type I trans membrane protein. Tim-3 plays a key role in inhibiting Thl responses and the expression of cytokines such as TNF and INF-g. Dysregulation of Tim-3 expression has been associated with autoimmune diseases.

Research has shown that TIGIT-Fc fusion protein could interact with PVR on dendritic cells and increase its IL-IQ secretion level/decrease its IL-12 secretion level under EPS stimulation, and also inhibit T cell activation in vivo.[l] TIGIT' s inhibition of NK cytotoxicity' can be blocked by antibodies against its interaction with PVR and the activity is directed through its GPM domain. [4]

RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing functions by repressing translation or by cleaving messenger RNA (niRNA). A common example of RNA silencing is RNA interference (RNAi), in which endogenously expressed microRNA (mlRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. RNA silencing pathways are associated with the regulatory activity of small non-coding RNAs (approximately 20-30 nucleotides in length) that function as factors involved in inactivating homologous sequences, promoting endonuclease activity, translational arrest, and/or chromatic or DNA modification

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

BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 illustrates the structures of CAR. The left panel shows the structure of the first generation of CAR (no costimulatory domains). The middle panel shows the structure of the second generation of CAR (one co-stimulation domain). The right panel show's the third generation of CAR (two or several co-stimulation domains) [3]

FIG. 2 illustrates the construct of CD- 19-CAR with IL-6 shRNA or a checkpoint inhibitor protein (PD-1 , TIGIT). The CAR sequence with CD 19-CAR contained Flag tag after scFv. Each shRNA contained sense, loop, antisense and termination signal. The CAR sequence may contain either a Flag tag or a TF tag before or after ScFv for an easy detection of CAR expression.

FIG. 3 shows the real-time cytotoxicity assay (RTCA) with CD 19-positive Hela cervical cancer cells 10: 1 ratio of Effector to Target cells was used. T cells, Mock-CAR-T cells used as negative control cells against Hela-CD19-posirive ceils. CD19-IL-6 shRNA- CAR-T cells and CD19-CAR-T cells effectively killed Flela-CD 19-positive cells. CD19F refers to Flag tag after CD 19 scFv.

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

FIG. 5 shows that CD19-IL-6 shRNA-CAR-T cells did not decrease IFN-ga ma level in Raji cells. E:T ratio was 1 : 1

FIG. 6 shows an RTCA assay with CD 19TF -CAR-T cells and CD19TF-IL-6 shRNA- CAR-T cells against Hela-CD19 target cells.

FIG. 7 shows that CD 19TF -IL6shNA-C AR-T cells secreted significantly less IL-6 than CD 19TF -CAR-T cells against CD19-positive Raji target cells.

FIG 8 shows RTCA assays with CD19-CAR-T cells (PMC 193), CD19TF-PD1 shRNA-CAR-T cells (PMC317), and CD19-TF-TIGIT shRNA-CAR-T cells (PMC316) against Hela-CD19 target cells.

FIG 9 shows RTCA assays with CD 19-C AR-T cel 1 s (PMC 193), CD 19TF-PD 1 shRNA-CAR-T cells (PMC317), and CD 19-TF-TIGIT shRNA-CAR-T cells (PMC316) against Raji target cells.

FIG 10 shows that CD19-TF-PD-1 shRNA-CAR-T cells and CD 19-TF-TIGIT shRNA-CAR-T cells secreted high level of IFN-gamma with target He! a-CD 19 cells

FIG. 11 show ^? s high in vivo efficacy of CD19-TF-PD-1 shRNA CAR-T cells and

CD19-TF-TIGIT shRNA CAR-T cells. The imaging show's a significant decrease of biolurninescence by CD19-TF-PD-1 shRNA-CAR T cells and CD 19-TF-TIGIT shRNA CAR-T cells in Raji-luciferase+ xenograft mouse model * P=0.006, CAR-T versus T cells, Student’ s t-test.

FIG. 12 shows that CD19-TF-PD-1 shRNA-CAR-T cells and CD! 9-TF-TIGIT shRNA CAR-T cells significantly prolonged mouse survival in Raji xenograft NSG model. p<Q.()5 versus T cells and CD 19TF -CAR-T cells. DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein that has been engineered to give T cells the new ability to target a specific protein. The receptor is chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. CAR is a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. The "chimeric antigen receptor (CAR)" is sometimes called a "chimeric receptor", a "T-body", or a "chimeric immune receptor (OR) " The "extracellular domain capable of binding to an antigen” means any oligopeptide or polypeptide that can bind to a certain antigen. The "intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

As used herein, a "domain" means one region in a polypeptide which is folded into a particular structure independently of other regions.

As used herein,“shKNA”, or“short hairpin RNA”, or“small hairpin RNA”, refers to an RNA molecule with a hairpin-like structure; the molecule is slightly larger than siRNA molecules and, unlike siRNA, is produced inside the cell in the nucleus. shRNA transcripts are constructed by connecting the sense and antisense strands of an siRNA duplex with a loop sequence, allowing a single transcript to fold back into a duplex structure on being transcribed. After transcription, shRNAs are processed into siRNAs by the Dicer enzyme. shRNA is a way to prepare siRNA sequences for delivery to cells that can be expressed in situ from plasmid DNA or from virus-derived constructs. shRNA is a way of inducing RNA interference-mediated posttranscriptional gene silencing for target genes.

As used herein, a "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv variable regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for preparing an scFv are known to a person skilled in the art.

As used herein, a "tumor antigen" means a biological molecule having antigenicity, expression of which causes cancer. The present invention relates to a nucleic acid sequence comprising a first

polynucleotide encoding a chimeric antigen receptor and a second polynucleotide encoding a short hairpin RNA (shRNA) down-regulating IL-6 or a checkpoint protein.

The present invention is directed to a nucleic acid sequence comprising: (a) a first polynucleotide encoding a chimeric antigen receptor (CAR) fusion protein comprising from the N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) comprising VH and V _L , wherein scFv specifically binds to a tumor antigen, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain, and (b) a second

polynucleotide encoding a short hairpin IL-6 shRNA sequence. The first and the second polynucleotides are transcribed from the same construct. The short hairpin IL-6 shRNA sequence is capable to silence the expression of 11-6 and to reduce cytokine release syndrome in patients treated with CAR-T cells.

The present invention is also directed to a nucleic acid sequence comprising: (a) a first polynucleotide encoding a chimeric antigen receptor (CAR) fusion protein comprising from the N-terminus to C-terminus: (i) a single-chain variable fragment (scFv) comprising VH and VL, wherein scFv specifically binds to a tumor antigen, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain, and (b) a second

polynucleotide encoding a short hairpin shRNA sequence of a check point inhibitor, wherein the check point inhibitor is PD-1, CTLA-4, TIM-3, TIGIT, or LAG-3. The first and the second polynucleotides are transcribed from the same construct. The checkpoint inhibitor shRNA sequence is capable to silence the expression of the checkpoint inhibitor protein, and to overcome T cell exhaustion in patients treated with CAR-T cells.

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

polynucleotide.

In one embodiment, each of the first and the second polynucleotides has its own promoter to initiate the transcription.

The insertion of a short hairpin shRNA sequence in the CAR construct provides stable knockdown cell lines and eliminates the need for multiple rounds of adding siRNA oligonucleotides by transfection, which dilutes siRNA with each round of replication.

Including shRNA in a lentivira! CAR construct increases reproducibility of results. The use of lenti viral, adenoviral or retroviral vector to deliver shRNA generates cells with stable shRNA expression. The lentivira! delivery of shRNA used is preferred because of its low toxicity to cells. A potentially beneficial effect of shRNA expression is that RNAi effects are more sustained than delivery of synthetic nucleotide-based siRNAs.

The present invention provides a nucleic acid sequence encoding a CAR with a short hairpin shRNA sequence that silences the expression of 11-6 or a checkpoint inhibitor. The structure of short the nucleic acid construct is shown on FIG. 2. FIG. 2 illustrates CD 19- CAR-shRNA (EL-6, PD-1, TIGIT), but the same design can be used for tumor antigens other than CD 19 and checkpoint inhibitors other than PD-1 and TIGIT. The CAR sequence with GDI 9-CAR contained Flag tag after scFv. The short hairpin EL-6, PD-1 or TIGIT shRNA sequences are inserted under HI promoter and each shRNA contains sense, loop, antisense, and termination signal. The CAR sequences may contain a Flag tag or TF (transferrin) tag before or after ScFv for easier detection of CAR expression.

In one embodiment, the tumor antigen is selected from the group consisting of:

BCMA (CD269, TNFRSFI7), CD 19, CD22, VEGFR-2, CD4, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD47, CD52, CD56, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, CP I 33. EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvffl, ErbB2/HER-2, ErbB3/HER3, ErbB4/HER-4, EphA2, IGF 1 R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA,

TGFBR2, TGFBRl, IL6R, gp!30, Lewis A, Lewis Y, TNFRl, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40,

CD 137, TWEAK-R, LTPR, LIFRP, LRP5, MUCl , TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robol, a, Frizzled, 0X40, CD79b, and Notch-1-4. In a preferred embodiment, the tumor antigen is CD 19 or CD22.

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

A preferred activating domain is CD3 zeta (CD3 Z or K03z).

The trans-membrane domain may be derived from a natural polypeptide, or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a

transmembrane domain of a T cell receptor alpha or beta chain, a CD3.zeta. chain, CD28, CD3. epsilon., CD45, CD4, CDS, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD 154, or a GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is

Ί found at each end of the synthetic transmembrane domain. Particularly, a linker sequence having a glycine-serine continuous sequence can be used.

A sequence such as Flag tag or a transferrin tag can be inserted to detect ScFv expression and detect CAR expression.

The nucleic acid encoding the CAR containing shKNA can be prepared by a conventional method. A base sequence encoding an amino acid sequence can be obtained from the aforementioned NCBI RefSeq IDs or accession numbers of GenBank for an amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using a standard molecular biological and/or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR). ShRNA sequence can be designed using different software using, for example, IL-6 mRNA, or other sequence as a target sequence to silence.

A composition comprising the nucleic acid of the present invention as an active ingredient can be administered for treatment of, for example, a cancer such as a blood cancer (leukemia), a solid tumor etc. A composition comprising the nucleic acid of the present invention as an active ingredient can be administered intradermally, intramuscularly, subcutaneously, intraperitoneally, intravenously, intratumorally, or into an afferent lymph vessel, by parenteral administration, for example, by injection or infusion, although the administration route is not particularly limited.

The nucleic acid encoding the CAR and shRNA of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, and an HSV vector can be used. As the virus vector, a virus vector lacking the replicating ability so as not to self-replicate in an infected cell is preferably used.

The mechanism by which shRNA causes gene silencing through repression of transcription occurs as follows: Long dsRNA which can come from the following sources: hairpin, complementary RNAs, RNA dependent RNA polymerases. The long dsRNA is cleaved by an endo-ribonuclease called Dicer Dicer cuts the long dsRNA to form short interfering RNA or siRNA; which enables the molecules to form the RNA-Induced Silencing Complex (RISC). 1. Once shRNA is transported from the nucleus to the cell cytoplasm, it gets

incorporated into other proteins to form the RISC.

2. Once the shRNA is part of the RISC complex, the shRNA is unwound to form single stranded siRNA.

3. The strand that is thermodynamically less stable due to its base pairing at the 5 ^' end is chosen to remain part of the RISC-complex.

4. The single stranded siRNA which is part of the RISC complex now can scan and find a 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 cut and recognized as abnormal by the cell. This causes

degradation of the mRNA and in turn no translation of the mRNA into amino acids and then proteins. Thus, the target gene that encodes that mRNA is silenced.

Short hairpin RNA (shRNA) is a class of RNA molecules, which have 19-23 base pairs in length and operate within the RNA interference (RNAi) pathway. The shRNA can be up to 30 base pairs, but shorter 19-23 base pairs are preferred because they typically have no immune response. The shRNA interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription and

preventing translation.

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

ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCT

CCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTACCCCCAGGAGAAGATTCCAAA

GATGTAGCCGCCCCACACAGACAGCCACTCACCTCTTCAGAACGAATTGACAAA

CAAATTCGGTACATCCTCGACGGCATCTCAGCCCTGAGAAAGGAGACATGTAAC

AAGAGTAACATGTGTGAAAGCAGCAAAGAGGCACTGGCAGAAAACAACCTGA

AC C TT C C A A AG AT GGC T G A A A A AG AT GG AT GC TT C C A AT C T GG AT T C A AT G AGG

AGACTTGCCTGGTGAAAATCATCACTGGTCTTTTGGAGTTTGAGGTATACCTAGA

GT AC C TC C AG A AC AGATTT GAGAGT AGT G AGGA AC A AGC C AGAGC T GT GC AGAT

GAGTACAAAAGTCCTGATCCAGTTCCTGCAGAAAAAGGCAAAGAATCTAGATGC

AATAACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGACGAAGCTGCAGGC

ACAGAACCAGTGGCTGCAGGACATGACAACTCATCTCATTCTGCGCAGCTTTAAG

GAGTTCCTGCAGTCCAGCCTGAGGGCTCTTCGGCAAATGTA (SEQ ID NO: 1)

In one embodiment, the structure of shRNA insert in a lenti viral construct includes sense strand (as above in bold and underlined), anti-sense strand (complementary to the sense strand) and the loop (sequence between sense and antisense sequences). For example, the loop is a 5-10 nucleotide spacer such as TTGATATCCG, CCACC, CTCGAG, AAGCTT, CCACACC, TTCAAGAGA (SEQ ID NQs. 2-7). The loop connects the sense and anti-sense strands. The termination signal TTTTTT is located after anti-sense sequence. The short hairpin shRNA construct can start either with a sense sequence or an antisense sequence with no impact of silencing of the gene

RNA polymerase III prefers to initiate transcription of the short hairpin siRNA construct with a purine (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 a shRNA is transcribed under HI promoter (or other RNA polymerase ID promoters), it forms double- stranded hairpin that Dicer endonuclease binds and cleaves to shorter shRNA that forms complex with RISC (the RNA-Induced Silencing Complex). The shRNA is transformed to single stranded siRNA that remains part of the RISC complex. Once siRNA-RISC complex binds to the intracellular target mRNA to silence, it degrades this mRNA and cause gene silencing due to absence of translation.

For example, when a retrovirus vector is used, the process of the present invention can be carried out by selecting a suitable packaging cell based on the LTR sequence and a packaging signal sequence possessed by the vector and preparing a retrovirus particle using the packaging cell. Examples of the packaging cell include PG13 (ATCC CRL-10686),

PAS 17 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. A retrovirus particle can also be prepared using a 293 cell or a 293T cell having high transfection efficiency. Many kinds of retrovirus vectors produced based on retroviruses and packaging cells that can be used for packaging of the retrovirus vectors are widely commercially available from many companies.

When CAR binds to a specific antigen on a cell surface, a signal is transmitted into the cell, and as a result, the cell is activated. The activation of the cell expressing the CAR is varied depending on the kind of a host ceil and an intracellular domain of the CAR, and can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, or the like as an index. For example, release of a cytotoxic cytokine (a tumor necrosis factor, lymphotoxin, 11.-6 etc.) from the activated CAR- T cell causes destruction of a target cancer cell expressing an antigen. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune ceils, for example, a B cell, a dendritic cell, a NK cell, and a macrophage.

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

The present invention provides T cells or natural killer cells (NK cells) modified to express the CAR as described above. CAR-T cells or CAR-NK cells of the present invention bind to a specific tumor antigen via the scFv of CAR, thereby a signal is transmitted into the cell, and as a result, the cell is activated. The activation of the cell expressing the CAR is varied depending on the kind of a host cell and an intracellular domain of the CAR, and can be confirmed based on, for example, release of a cytokine, improvement of a cell proliferation rate, change in a ceil surface molecule, killing target cells, or the like as an index.

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

The inventors have discovered that adding an 1L-6 shRNA in a CAR lentiviral construct blocks secretion of IL-6 while maintaining the activity of CAR-T cells. The inventors have generated CD19-IL-6 shRNA-CAR T cells against hematologic malignancies (leukemia, lymphoma, and myeloma), which have high killing activity against cancer cells overexpressing CD 19. The inventors have demonstrated high cytotoxic activity of CD 19- Flag-CAR-T cells or CD19-TF-CAR-T cells with IL-6 shRNA-CAR-T ceils by real-time cytotoxicity assay against cervical cancer cell line Hela stably overexpressing CD19 antigen and hematological cancer Raji cells endogenously overexpressing CD19 antigen.

The insertion of a short hairpin IL-6 shRNA sequence in the CAR construct decreases IL-6 secretion, and thus increases safety of CAR-T cells against tumor cells. In clinic, a major problem with CAR-T cell deliver} _' to patients is that CAR-T cells cause a cytokine release syndrome or "cytokine storm". The major inducer of cytokine release symptom is IL- 6 cytokine.

CAR-T cells are generated from the isolation of T cells from the blood of a patient. The CAR-T cells are transduced with !entiviral, retroviral or other virus-or plasmid-based vector, and these engineered CAR-T cells are injected to the patient usually by intravenous injection. This CAR-T cells therapy can cause adverse cytokine release syndrome or adverse neurological symptoms. The present invention generates safer CAR-T cells in clinic with decreased production of IL-6 cytokine in a patient to reduce cytokine release syndrome.

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

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

CD19-IL-6 shRNA-CAR T cells can be used in manufacturing process with selection of enriched cells together with memory T cell subsets for increasing efficacy of T cell production and cytotoxicity.

Combination therapy with bi-specific CDI9-CD22- CAR-T or bi-specific BCMA-plus other ScFv against any of MM markers CD38, CD319, CD138, CD33 CAR-T cells with IL-6 shRNA can be used to increase activity of single CAR-T cell-therapy with less secretion of IL-6 cytokine.

Combination therapy with CD19-1L-6 shRNA CAR cells and chemotherapy or inhibitors of immune checkpoints (PD-1, CTLA-4, TIM-3, TIGIT, LAG-3 and other) can be used to increase activity of single CAR. Tumors use checkpoint proteins to protect themselves from immune cell attack. Checkpoint immunotherapy blocks inhibitory _' checkpoints, restoring immune cell activation. The most known ligand-receptor interaction is the interaction between the transmembrane programmed cell death 1 protein (PDCDl, PD-1; also known as CD279) that is expressed in T cells and its ligand that is often overexpressed in tumors: PD-1 ligand 1 (PD-L1, CD274). Cell surface protein PD-L1 binds to PD1 on an immune cell surface, which inhibits immune cell activity. Cancer-dependent upregulation of PD-L1 on the cell surface inhibits activity of T cells. Different PD-1 or PD-L1 antibodies that bind to either PD-1 or PD-L1 block this interaction allowing the T-cells to attack the tumor. Similar mechanisms are for other interactions described above between CTLA-4, TIM-3 and other T cells receptors with tumor cell surface ligands. The down-regulation of these receptors with shRNA will block interaction with the ligands and T ceil activity will be higher than T cells with checkpoint receptors. The inventors demonstrate 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 demonstrate higher in vivo efficacies of CD19TF-PD-1 shRNA-CAR T cells and CD 19-TF-TIGIT shRNA CAR-T cells than CD19TF-CDR T ceils against target cells.

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

In addition to IL6, shRNAs of other cytokines can he used in CAR (IFN-gamma, IL-2, IL-10, MCP-I, other).

Other technologies to silence 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 present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being 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, transmembrane domain and costimulatory domain from CD28, and the CD3 zeta activation domain; this CAR is herein called the CD! 9 CAR. The IL-6 shRNA sequence was inserted after CAR under an independent promoter. A‘"mock” CAR with an scFv specific for an intracellular protein - and thus not reactive with intact cells --- was constructed in the same manner and used as a negative control CAR.

Example 2. Sequences of CAR Constructs.

The amino acid sequences of each segment of CD 19-CAR constructs were used in our experiments and shown below. Each segment can be replaced with amino acid sequence with at least 95% identity.

(a) CD 19-Flag CAR

Human GM-CSF Signal peptide- anti-CD 19 scFv (VT. -Linker- VH)-Flag-CD28 hinge, Transmembrane CD28-Co-stimuiating CD28, CD3-Zeta <Human GM-CSF Signal peptide> SEQ E) NO: 8

MLLLVTSLLLCELPHPAFLLIP

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

<VL> SEQ ID NO: 9

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDG

T V K L L I Y H T S R L H S G V P S R F S G S G S G T D Y S L T ISNL E Q E D!AT Y F C Q Q G N T L P Y T F G G G T K L E I T

- linker SEQ ID NO: 10

G S T S G S G K P G S G E G S T K G

<VH> EQ ID NO: 11

E V K L Q E S G P G L V A P S Q S L S V TCI V S G V S L P D Y G V S W I R Q P P R K G L E W L G V I G S E T T Y Y N S A L K S R L T I I K D N S K S Q V F L K Met N S L Q T D D T A I Y Y C A K H Y Y Y G G S Y A M D Y W G Q G T S V T V S S

In our construct, we have 3 amino acids AAA after VH.

<Flag Tag> SEQ ID NO: 12

DYKDDDDK

<CD28 hinge> SEQ ID NO: 13

I E V M Y P P P Y LDNEKSNGTimVKGKHL C P S P L F P G P S K P

<Transmembrane Domain CD28> SEQ ID NO: 14

F W V L V V V G G V L A C Y S L L V T V A F I I F W V

<Co-stimuiating domain CD28 > SEQ ID NO: 15

R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S

< Activation domain CD3-zeta> SEQ ID NO: 16

RVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER

RRGK

The GDI 9-CAR is shown below, Flag tag is underlined, SEQ ID NO: 17

MLLL V TSLLLCELPH P A F L L I P D I Q M T Q T T SSLS A S L G D R V T I SCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQED!ATYFCQQGNT L P Y TFGGGTKL E I T G S T S G S G KPGSG E G S T K G E V K L Q E S G P G LVAPSQSLS V T C T

V S G V S L P D Y G V S W I R Q P P R K G L E W L G V I W G S E T T Y Y NSALK SRLTIIKDNSKSQ VFLKMNSLQTDDTAIYYC AKHYYYGGS Y A M DYWGQGTS V T V S S A A A DYKDDDDK I E V M Y P P P Y L D N E K S N G T 11 H V K G K 11 L C P S P L F P G P S K P F W V L V V V G G V L A C Y S L L

V T V A F 11 F W V RSKRSRLLHSDY M N M T P R R P G P T R K H Y Q P Y A PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREE

V D V L D K R R G R D P E M G G K P R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R

(b) CD 19-FIag-IL-6 shRNA CAR

The CD 19-Flag CAR nucleotide sequence with IL~6 shRNA sequence under the HI promoter inside lentiviral vector is shown below as SEQ ID NO: 20. , IL-6 shRNA is shown in bold, with structure: sense (by bold uppercase font)-foop-(antisense uppercase font, bold Italic), termination sequence (underlined), Hi promoter is shown in lowercase letters 5 ^! upstream of shRNA:

<Kpn I site> 5'- GGTACC

<H1 promoter> SEQ ID NO: 18

Gaacgctgacgtcatcaacccgctccaaggaatcgcgggcccagtgtcactaggcgggaa cacccagcgcgcgtgcgccctggca ggaagatggctgtgagggacaggggagtggcgccctgcaatatttgcatgtcgctatgtg ttctgggaaatcaccataaacgtgaaatgt ctttggattgggaatctataagttctgtatgagaccac

<Xho I site> CTCGAG

<DNA encloding 1L-6 short hairpin shRNA> sense (bold)-loop-anti-sense (bold Italic) and termination signal (underlined), SEQ ID NO: 19

GAGTAACATGTGTGAAAGCTTGATATCCGGCTTTCACACATGTTACTCTTrni

Nhel site> GCTAGC-3' The construct of DNA sequence inserted into a lentivirai vector encoding CAR and IL-6 shRNA is shown below. The 5'-Xba I and 3' EcoR I sites (underlined) flank CD 19-CAR sequence containing Flag tag (in Italic, underlined). ATG start codon of CAR sequence is shown in bold (underlined). Then HI promoter and short hairpin shRNA sequence is shown in bold, in larger font starting from Kpn I site (underlined) and ending with Nhe I site (underlined) GCTAGC After Kpn I site (lowercase font, underlined) is HI promoter (in lowercase font), then Xho site (Italics, uppercase); sense (uppercase, underlined), loop sequence (uppercase font) and antisense sequences (uppercase font, Italics, underlined), and termination sequence (uppercase font), ends with Nhe I site. t_ct_agaGCCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCAC ACCCAGCATT CCTCCTGATCCCAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGG AGACAGAG TCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGA AACCAGAT GGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGG TTCAGTGG CAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGC CACTTACT TTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAA CAGGCTCC ACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTG CAGGAGTC AGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGT CTCATTAC CCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAG TAATATGG GGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGAC AACTCCAA GAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTG TGCCAAAC ATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCG TCTCCTCA GCGGCCGCAGACTACAAAGACGATGACGACAAGATTGAAGTTATGTATCCTCCTCCTTAC CTAGACAA TGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCT ATTTCCCG GACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCT TGCTAGTA ACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGAC TACATGAA CATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCG CAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG GCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA CGTGGCCG GGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA ACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG CAGGCCCT GCCCCCTCGCTAAGAATTCggatccgcggccgcgaaggatctgcgatcgctccggtgccc gtcagtgg gcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacg ggtgccta gagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcc cgagggtg ggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttg ccgccaga acacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctga ggccgcca tccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtc cgccgtct aggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctac ctagactc agccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttt tctgttct gcgccg11.acagatccaagctgtgaccggcgcctacg ctagatgaccgagt acaagcccacggtgcgc ctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgac taccccgc cacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaact c11cctca cgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcgg tctggacc acgccggagagcgtcgaagcgggggcggtg 1.1cgccgagatcggcccgcgcatggccgag11gagcgg ttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaagga gcccgcgt gg11 cctggccaccg tcggcgtctcgcccgaccaccagggcaagggtctgggcagegccgt cg tgctc cccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccc cgcaacct ccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggacc gcgcacct ggtgcatgacccgcaagcccggtgcctgagtcgacaatcaacctctggattacaaaattt gtgaaaga ttgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatg cctttgta tcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgct gtctcttt atgaggag11gtggcccg11g tcaggcaacgtggcgtgg t.gtgcact.gtg111gct.gacgcaaccccc actggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctc cctattgc cacggcggaact cat.cgccgcctgccttgcccgctgctggacaggggctcggctg 1.1gggcactgaca attccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgcca cctggatt ctgcgcgggacgt cc1.1ctgctacgtccc11cggccctcaatccagcggacc11cc11cccgcggcct gctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctc cctttggg ccgcctccccgcctggtaccgaacgctgacgtcatcaacccgctccaaggaatcgcgggc ccagtgtc actaggcgggaacaaccagcgcgagtgcgcactggcaggaagatggatgtgagggacagg ggagtggc gccctgcaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatgtc tttggatt tgggaatcttataagttGtgtatgagaccacCTCGAQGAGTAACATGTGTGASAGCTTGA TATCCGGC

TTTCACACATGTTACTCTTTTTTGCTAGC

(SEQ ID NO: 20)

(c) CD19-TF-I L-6 shRNA-CAR

We also generated CD19-TF (transferrin) tag-CAR with IL-6. The CAR Sequence starts with ATG start codon (underlined), then CAR with TF in italics underlined. The HI promoter and short hairpin shRNA sequence are shown in bold starting from Kpn I site (underlined) and ending with Nhe I site (underlined) GCTAGC. After Kpn I site (lowercase font, underlined) is HI promoter (in lowercase font), then Xho site (Italics, uppercase); sense (uppercase, underlined), loop sequence (uppercase font) and antisense sequences (uppercase font, Italics, underlined), and termination sequence (uppercase font), ends with Nhe I site.

ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTC CTGATCCCAGA

CATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCAC CATCAGTTGCA

GGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAA CTGTTAAACTC

CTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGT GGGTCTGGAAC

AGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTG CCAACAGGGTA

ATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCT CTGGATCCGGC

AAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGA CCTGGCCTGGT

GGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGA CTATGGTGTAA

GCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTA GTGAAACCACA

TACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGC CAAGTTTTCTT

AAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTA TTACTACGGTG

GTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGG CCGCA

aaaaacccgga tccgtgggcgaaaaacctgaacgaaaaagatta t

ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATC CATGTGAA AGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCT GGTGGTGG TTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGG TGAGGAGT AAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCC ACCCGCAA GCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTT CAGCAGGA GCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG GACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG AGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTG GGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA CAGCCACC AAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAGAATTCg tcgacaatcaacc tctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttac gctatgtg gatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttct cctccttg tataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggc gtggtgtg cactg tg 111gctgacgcaacccccactgg 1.1ggggca11gccaccacctgtcagctcc111ccggga ctttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgct gctggaca ggggctcggct g11gggcactgacaa11ccgtggtg 11gtcggggaaatcat cg t.cctttcctt.ggct gctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggc cctcaatc cagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgcc ttcgccct cagacgagtcggatctccctttgggccgcctccccgcct ggtace

gaacgclgacgteatcaacccgctccaaggaategcgggcccagtglcaclaggcgg gaacacccagcgcgegigcgccctggcagga agatggctgtgagggacaggggagtggcgccctgcaatatttgcatgtcgctatgtgttc tgggaaatcaccataaacgtgaaatgtcttt ggatttgggaatcttataagttctgtatgagaccacCTCG^GGAGTAACATGTGTGAAAG CTTGATATCCGG T TCACACATGTTACTCTTTTTT GCTAGC

(SEQ ID NO: 21)

(d) IL-6 sfaRNA

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

The coding sequence of IL-6 mRNA is shown below in SEQ ID NO: 1. Within the coding sequence, the 4 targeted areas that correspond to the sense regions of four IL-6 shRNAs are shown in bold and underlined, with overlapping sequences in two target areas. shRNA targets IL-6 mRNA and causes decreased transcription and expression of II, -6. The underlined larger font targeted sequence w'as used to prepare for CAR with IL-6 shRNA in the examples.

ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCT

CCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTACCCCCAGGAGAAGATTCCAAA

GATGTAGCCGCCCCACACAGACAGCCACTCACCTCTTCAGAACGAATTGACAAA

CAAATTCGGTACATCCTCGACGGCATCTCAGCCCTGAGAAAGGAGACATGTAA

CAAGAGTAACATGTGTGAAAGCAGCAAAGAGGCACTGGCAGAAAACAAC

CTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAAT

GAGGAGACTTGCCT GGTGAAAAT C ATC ACTGGT CTTTTGGAGTTT GAGGTATACC

TAGAGTACCTCCAGAACAGATTTGAGAGTAGTGAGGAACAAGCCAGAGCTGTGC

AGATGAGTACAAAAGTCCTGATCCAGTTCCTGCAGAAAAAGGCAAAGAATCTAG

ATGCAATAACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGACGAAGCTGC

AGGCACAGAACCAGTGGCTGCAGGACATGACAACTCATCTCATTCTGCGCAGCTT

TAAGGAGTTCCTGCAGTCCAGCCTGAGGGCTCTTCGGCAAATGTA (SEP ID NO:

1)

SEQ ID NOs. 22-25 are examples of four DNA sequences encoding IL-6 shRNAs (sense-loop-antisense-termination signal) that are flanked by BamHl site at 5’ and at 3’ site.

GAGTAACATGTGTGAAAGCTTGATATCCGGC77TC4C4C4r(yjX4CrCrTTTTT (SEQ ID NO: 22, used in preparing CAR in the examples)

GGAGACATGT AAC A AGAGTTT GAT AT CCG4 CTCTTGTTA CA TGTCTCCIUTTT

(SEQ ID NO: 23) GATGGATGCTFCCAATCTGTTGATATCCGCAGATTGGAAGCATCCATCTTmT (SEQ ID NO: 24)

* CGTTCGGTAC ATCCT CGACGGTT G AT ATC CGCCGTCGA GGA TGTA CCG2L4 TTTT TT (SEQ ID NO: 25)

*CG is added for R A polymerase III to have more efficient transcription

(e) PD~1 sfaRNA

The coding sequence of PD-1 (GeneBank ID: L27440.1) is shown below in SEQ ID

NO: 26. Within the coding sequence, the 3 targeted areas that correspond to the sense regions of three PD-1 shRNAs are shown in bold and underlined, with overlapping sequences in two target areas.

atgcag atcccacagg cgccctggcc agtcgtctgg

gcggtgctac aactgggctg gcggccagga tggttcttag actccccaga caggccctgg aaccccccca ccttctcccc agccctgctc gtggtgaccg aaggggacaa cgccaccttc acctgcagct t ct.c.c.aacac atcggagagc ttcgtgcta?! actggtaccg catgagcccc agcaaccaga cggacaagct ggccgccttc cccgaggacc gcagccagcc cggccaggac tgccgc11;cc gtgtcacaca act;gcccaac gggcgtga ct tccacatgag cgtggtcagg gcccggcgca atgacagcgg cacctacctc tgtggggcca tctccctggc ^c cccaaggeg

cagatcaaag agagcctgcg ggcagagctc agggtgacag agagaagggc agaagtgccc

acagcccacc ccagcccctc acccaggtca gccggccagt tccaaaccct ggtggttggt gtcgtgggcg gcctgctggg cagcctggtg ctgctagtct gggtcctggc cgtcatctgc tcccgggccg cacgagggac aa taggagcc aggcgcaccg gccagcccct gaaggaggac ccctcagccg tgcctgtgt:t ctctgt:ggac tatggggagc tggatttcca gtggcgagag aagaccccgg agccccccgt gccctgtgtc cctgagcaga cggagtatgc caccattgtc tttcctagcg ga tgggcac ctcatccccc gcccgcaggg gctcagctga cggccctcgg agtgcccagc cactgagg cc tgagga tgga cactgctctt ggcccctctg a

(SEQ ID NO: 26)

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

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

AAGGCGCAGATCAAAGAGAGCTFGATATCCGGCTCTCTTTGATCTGCGCCTT TTTTTT (SEQ ID NO: 28) AACACATCGGAGAGCTTCGTGTTGATATCCG CACGAAGCTCTCCGATGTGTT

TTTTTT (SEQ ID NO: 29)

(f) CTLA-4 shRNA

CTLA-4 cDNA, gene ED 1493 the nucleotide sequences targeted by shRNAs is underlined and bolded

atggc ttgccttgga tttcagcggcacaaggctca gctgaacctg gctaccagga cctggccctg cactctcctg ttttttcttc tcttcatccc tgtcttctgc aaagcaatgc acgtggccca gcctgctgtg gtactggcca gcagccgagg catcgccagc tttgtgtgtg agtatgcatc tccaggcaaa gccactgagg tccgggtgac agtgcttcgg caggctgaca gccaggtgac tgaagtctgt gcggcaacct acatgatggg gaatgagttg accttcctag atgattccat ctgcacgggc acctccagtg gaaatcaagt gaacctcact atccaaggac tgagggccat ggacacggga ctctacatct

gcaaggtgga gctcatgtac ccaccgccat actacctggg cataggcaac ggaacccaga tttatgtaat tgatccagaa ccgtgcccag attctgactt cctcctctgg atorttgcag^agtagttc ggggttgttt ttttatagct ttctcctcac agctgtttct ttgagcaaaa tgctaaagaa aagaagccct cttacaacag gggtctatgt gaaaatgccc ccaacagagc

cagaatgtga aaagcaattt cagccttatt ttattcccat caattga (SEQ ID NO: 30)

DNA sequences encoding CTLA-4 shRNA are shown below.

GATCC T TGCAGCAGT TAGT T T GAT AT C C GACTAACTGCTGCAAGGA T T T T T

(SEQ ID NO: 31)

A A AT C A AGTGA AC CTC ACT AT T T GAT AT C C GATAGTGAGGT TCAC TTGAT TTTTTTTT

( SEQ ED NO: 32)

(g) TIM-3 shRNA

TIM-3 DNA, ED: AK314406, the nucleotide sequences targeted by shRNA are underlined and bolded.

atgttttca catcttccct ttgactcrtgt cctactgctg ctgctgctac tacttacaag

qtcctcagaa gtggaataca gagcggaggt cggtcagaat gcctatctgc cctgcttcta

caccccagcc gccccaggga acctcgtgcc cgtctgctgg ggcaaaggag cctgtcctgt

gtttgaatgt ggcaacgtgg tgctcaggac tgatgaaagg gatgtgaatt attggacatc

cagatactgg ctaaatqqgg atttccgcaa aggagatqtcf tcectgacea tagagaatqt

gactctagca gacagtggga tctactgctg ccggatccaa atcccaggca taatgaatga

tgaaaaattt; aacctgaagt: tggtcatcaa accagccaag gtcacccctg caccgactct

gcagagagac ttcactgcag cctttccaag gatgcttacc accaggggac atggcccagc

agagacacag acactgggga gcctccctga tataaatcta acacaaatat ccacattggc

caatgagtta cgggactcta gattggccaa tgacttacgg gactctggag caaccatcag

aataggcatc tacatcggag cagggatctg tgctgggctg gctctggctc ttatcttcgg

cgctttaatt ttcaaatggt attctcatag caaagagaag atacagaatt taagcctcat

ctctttggcc aacctccctc cctcagga.tt ggcaa.atgca gtagcagagg gaattcgctc

agaagaaaac atctatacca ttgaagagaa cgtatatgaa gtggaggagc ccaatgagta

ttattgctat gtcagcagca ggcagcaacc ctcacaacct ttgggttgtc gctttgcaat

gccatag

(SEQ ID NO: 33)

DNA sequences encoding TIM-3 shRNA are shown below. GGATTTCCGCAAAGGAGATTTGATATCCGATCTCCTTTGCGGAAATCCTTim

(SEQ ID NO: 34)

GTCCCTGACCATAGAGAATTTGATATCC GA TTCTCTA TGGTCAGGGACTTTTTT

(SEQ ID NO: 35)

(h) LAG-3 shRNA

LAG-3 DNA, Gene ID: 3902, the nucleotide sequences targeted by shRNAs are underlined and bolded.

atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg aagcctctcc agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg gtcacttggc agcatcagcc agacagtggc ccgcccgctg ccgcccccgg ccatcccctg gcccccggcc ctcacccggc ggcgccctcc tcctgggggc ccaggccccg ccgctacacg gtgctgagcg tgggtcccgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc cagctggatg agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg cgcgcggacg ccggcgagta cegegcegeg gtgcacctca gggaccgcgc cctctcctgc cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca gccccccagg atctctcaga gcctccgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg cattggttcc ggaaccgggg ccagggccga gtccctgtcc gggagtcccc ccatcaccac tagcggaaa gcttcc ctt cctgceccaa gtcagcccca tggactctgg gccctggggc tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct cactgttctg ggtctggagc ccccaactcc cttgacagtg tacgctggag caggttccag ggtggggctg ccctgccgcc tgcctgctgg tgtgggtacc cggtctttcc tcactgccaa gtggactcct cctgggggag gccctgacct cctggtgact ggagacaatg gcgaetttac ccttcgacta gaggatgtga gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca cctggatccc tggggaagct gctttgtgag gtgactccag tatctggaca agaaegettt gtgtggagct ctctggacac cccatcccag aggagtttct caggaccttg gctggaggca caggaggccc agctcctttc ccagccttgg caatgccagc tgtaccaggg ggagaggett cttggagcag cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga gccccaggtg ccctcccaqc aggccacctc ctgctgtttc tcacccttgg tgtcctttct ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca agacgatttt ctgccttaga gcaagggatt eaccctccgc aggctcagag caagatagag gagc ggagc aagaaccgga gccggagccg gagccggaac cggagcccga gcccgagccc gageeggage agctctga (SEQ ID NO: 36) DNA sequences encoding LAG-3 shRNA are shown below.

GCATCCTCACCTACAGAG ATC AAGAGrC C G J GGTGA GGA TTgCTTTTTT

(SEQ ID NO: 37)

(fa) TIGIT shRNA

TIGIT DNA, Gene ID: 3902, the nucleotide sequences targeted by shRNA are underlined and bolded. Within the TIGIT DNA sequence, the 3 targeted areas that correspond to the sense regions of three TIGIT shRNAs are shown in bold and underlined, with overlapping sequences in two target areas.

Atgcgctggtgtctcctcctgatctgggcccaggggctgaggcaggctcccctcgcc tcaggaatgatgacaggcacaatagaaaca acuuuaaacatttctucagilgfOiaggTggLlCltlfcfl/c/tocoatutcacctctc ctccaccacaacacaaatuacccav gtcaactgggagcagcaggaccagcttctggccatttgtaatgctgacttggggtggcac atctccccatccttcaaggatcgagtggcc ccaggtcccggcctgggcctcaccctccagtcgctgaccgtgaacgatgcaggggagtac ttctgcatctatcacacctaccctgatgg gacgtacactgggagaatcttcctggaggtcctagaaagctcagtggctgagcacggtgc caggttccagattccattgcttggagccat ggccgcgacgctggtggtcatctgcacagcagtcatcgtggtggtcgcgttgactagaaa gaagaaagccctcagaatccattctgtgg aaggtgaccteaggagaaaateagctggacaggaggaatggagccccagtgctccctcac ccccaggaagctgtgtccaggcagaa gctgcacctgctgggctctgtggagagcageggggagaggactgtgccgagctgcatgac tacttcaatgtcctgagttacagaagc ctgggtaactgeagcttetteacagagactggtta

(SEQ ID NO: 39)

DNA sequences encoding TIGIT shRNAs are shown below.

GAGAAAGGTGGCTCTATC ATTGAT AT CCGTGATA GA GCCA CCTTTCTCITTTTT

(SEQ ID NO: 40, used for CAR construct in the example)

GTGGCTCTATCATCTTACAT T GAT AT C C G TGTAAGA TGA TAG A GCCA CT T T T T T

(SEQ ID NO: 41)

GC TGCATGAC TAG TTCAAT T T GAT AT C C G GCTCTCTTTGATCTGCGCCTTT: T T T T T (SEQ ID NO: 42) shRNA of BTLA (Gene ID: 151888) and other targets can he generated with similar approach.

We generated CD19-TF tag-CAR with PD-1 shRNA. The Xbal and EcoRI sites flanking CAR are underlined, ATG start codon of CAR is shown in bold. The CD 19-CAR part was the same as the that with IL-6 shRNA, except TF (Transferrin) tag replaced Flag tag TF tag is underlined in italics.

Then HI promoter and short hairpin shRNA sequence is shown in bold, in larger font starting from Kpn I site GGTACC (underlined) and ending with Nhe I site (lowercase font, underlined) GCT ^' AGC. After Kpn I site is HI promoter (in lowercase font, underlined), then sense (uppercase, underlined), loop sequence (uppercase font) and antisense sequences (uppercase font, Italics, underlined), and termination sequence (uppercase font), ends with Nhe I site.

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

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

tcta qaGCCGCCACCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAG CATTCCTCCTGATCCCAGA CATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCAT CAGTTGCAGGGCAAGTCAGGACA TTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCT ACCATACATCAAGATTACACTCA GGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGC AACCTGGAGCAAGAAGATATTGC CACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTT GGAAATAACAGGCTCCACCTCTG GATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGT CAGGACCTGGCCTGGTGGCGCCC TCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTA AGCTGGATTCGCCAGCCTCCACG AAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGC TCTCAAATCCAGACTGACCATCA TCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACA CAGCCATTTACTACTGTGCCAAA CATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACC GTCTCCTCAGCGGCCGCA

aaaaacccggatccgtgggcgaaaaacctgaacgaaaaaga ttat

ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATC CATGTGAAAGGGAAACACCTTTG TCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGG AGTCCTGGCTTGCTATAGCTTGC TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACA GTGACTACATGAACATGACTCCC CGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCA GCCTATCGCTCCAGAGTGAAGTT CAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGT ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA AGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGA TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCA GGCCCTGCCCCCTCGCTAAGAAT TCgtcga caatca cctctggattacaa aa11tgtgaaaga11gactggta11c11aactatg11gctcc1111acgctatgt ggat acg c.tgc111aatg c.c111gtatcatgcta 1.1 gc11.cccgtatggc1.11ca1.111ctcctcc1.1 gtat.aaatcctgg11. gctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgt gtttgctgacgcaacccccactg g11ggggca11gccacca cctgtcagctcc111ccggga c111cgc111ccccctcccta 11gcca cggcggaactcatcgcc gcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtg ttgtcggggaaatcatcgtcctt tccttggctgctcgcctgtgttgccacctggattctgcgcggg cgtccttctgctacgtcccttcggccctcaatccagcgg acc11cc11cc.cgcggcctg ctgccggctctgcggcctc11cc.gcgtc.11 cgcc11cg c.cctcagacgagt cggat ctcc.c11 tgggccgcctccccgcct ggtacc- gaacgctgacgtcatcaacccgctccaaggaatcgcgggcccagtgtcactaggcgggaa cacccagcgcgcgtgcgccctggcaggaagatggctg tgagggacaggggagtggcgccctgcaatatttgcatgtcgctatgtgttctgggaaatc accataaacgtgaaatgtctttggatttgggaatcttataa gttctgtatgagaccacGGCGCAGATCAAAGAGAGCTTGATATCCGGCrcrCTT TGATCTGCGCCrrrm' zct c

(SFiQ ID NO: 43)

Transferrin tag can be used for detecting CAR, and it can decrease cytokines that can be advantageous in clinic to reduce cytokine release syndrome.

The CD19-TF CAR is shown below, TF tag (KNPDP W AKNLNEKD Y. SEQ ID NO:

44) is underlined.

MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYYYGGSYAMDYWGOGTSVTVSSAAAKNPDPW AKNLNEKD YIEV MYPPPYLDNEKSNGTHHVKGKHLCPSPLFPGPSKPFWVLVVVGGYLACYSLLVTVAF IIFW VRSKRSRLLHSD YMNMTPRRPGPTRKHY QP YAPPRDF AAYRSRVKF SRS AD AP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (SEQ ID NO:

45)

We generated CD19-TF tag-CAR with TIGIT shRNA same way as described above.

The sequence is shown below starting with ATG start codon underlined, TF tag in italics underlined, TIGIT siRNA flanked with Kpn I and Nhe I site in bold. After Kpn I site is HI promoter (in lowercase font, underlined), then sense (uppercase, underlined), loop sequence

(uppercase font) and antisense sequences (uppercase font, Italics, underlined), and termination sequence (uppercase font), ends with Nhe I site (lowercase font, underlined).

ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCT CCTGATCCCAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTG GGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTA AATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACAT CAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAG ATT ATTCTCTC AC C ATT AGC AACC TGGAGC A AGAAGAT ATTGC C AC TT ACTTTTG CCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAAT AACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAA GGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAG CCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGC TGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGT AGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGG ACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACA

CAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGA

CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCA

aaaaacccggatccgtgggcgaaaaacctgaacgaaaaagattat

ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCA TTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCT AAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTAT AGCTTGC TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCT GCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCA TTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTAT AACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGT GGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG C CTGT AC AAT GAACT GC AGAAAGAT AAGAT GGCGGAGGC C T AC AGTGAGATT GG GATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCT CAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGCTAAGAATTCgtcgacaatcaacctctggattacaaaatttgtgaaagattgactggt attcttaactatgttgctccttttacg ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttc attttctcctccttgtataaatcctggttgctgtctctt tatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgac gcaacccccactggttggggcattgccacc acctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactc atcgccgcctgccttgcccgctgctggacag gggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttc cttggctgctcgcctgtgttgccacctggatt ctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcc cgcggcctgctgccggctctgcggcctctt ccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcct ggtacc

gaacgctgacgtcatcaacccgctccaaggaatcgcgggcccagtgtcactaggcgg gaacacccagcgcgcgtgcgccctg gcaggaagatggctgtgagggacaggggagtggcgccctgcaatatttgcatgtcgctat gtgttctgggaaatcaccataaa cgtgaaatgtctttggatttgggaatcttataagttctgtatgagaccacGAGAAAGGTG GCTCTATCATTG AT AT CCG TGA TA GA GCCA CCTTT CT CTTTTTT gctagc

(SEQ ID NO: 46) Example 3, Generation of CAR-encoding lentivirus

DNAs encoding the CARs were synthesized and subcloned into a third-generation lentiviral vector with EFla promoter by Syno Biological (Beijing, China). All CAR lenti viral constructs were sequenced in both directions to confirm CAR sequence and used for lentivirus production. Ten million growth-arrested HEK293FT cells ( Thermo Fisher) were seeded into T75 flasks and cultured overnight, then transfected with the pPACKHl

Lentivector Packaging mix {System Biosciences , Palo Alto, CA) and 10 pg of each lentiviral vector using the CalPhos Transfection Kit ( Takara , Mountain View, CA). The next day the medium was replaced with fresh medium, and 48 h later the lentivirus-containing medium was collected. The medium was cleared of cell debris by centrifugation at 2100 g for 30 min. The vims particles were collected by centrifugation at 112,000 g for 100 min, suspended in AIM V medium, aliquoted and frozen at -80 °C. The titers of the virus preparations were determined by quantitative RT-PCR using the Lenti -X qRT-PCR kit ( Takara ) according to the manufacturer’s protocol and the 7900HT thermal cycler {Thermo Fisher) The lentiviral titers were >lxl0 ⁸ pfu/ml. Example 4. Generation and expansion of CAR-T cells

PBMC were suspended at i x 10 ⁶ cells/ml in AIM V-A!buMAX medium (Thermo Fisher) containing 10% FBS and 300 U/rnl IL-2 (Thermo Fisher), mixed with an equal number (1 : 1 ratio) of CD3/CD28 Dynabeads ( Thermo Fisher ), and cultured in non-treated 24- well plates (0.5 ml per well). At 24 and 48 hours, !entivirus was added to the cultures at a multiplicity of infecti on (MOI) of 5, along with J m! of TransPlus transducti on enhancer (AlStem). As the T cells proliferated over the next two weeks, the cells were counted every 2- 3 days and fresh medium with 300 U/ml IL-2 was added to the cultures to maintain the cell density at 1-3 x 10 ⁶ cells/ml.

Example 5, Flow cytometry

To measure CAR expression, 0.5 million cells w^ere suspended in 100 mΐ of buffer (PBS containing 0.5% BSA) and incubated on ice with 1 mΐ of human serum (Jackson Immunoresearch, West Grove, PA) for 10 min. Then 1 m! of a!lophycoeyanin (APC)-!abeled anti-CD3 ( eBioscience , San Diego, CA), and 2 mΐ of either phycoerythrin (PE)-labeled anti- Fiag or anti -FAB or its isotype control antibody was added, and the cells were incubated on ice for 30 min. The cells were rinsed with 3 ml of buffer, then suspended in buffer and acquired on a FACSCa!ibur (BD Biosciences). Cells were analyzed for CD3 staining versus Flag or Fab staining or isotype control staining

Example 6, Generation of the stable HeLa-CD19 cell line

To generate HeLa cells stably expressing human CD 19, a DNA encoding the human GDI 9 open reading frame w'as synthesized and subcloned into the pCD51() lentiviral vector (System Biosciences) by Syno Biological. Lentivirus containing the vector was made as described above. HeLa cells were infected with the lentivirus at an MOI of 5 and cultured in the presence of 1 pg/ml puromycin to generate resistant cells, herein called HeLa-CD19. The expression of CD 19 was confirmed by flow cytometry with a CD 19 antibody (BioLegend).

Example 7, Real-time cytotoxicity assay (RTCA)

Adherent target cells (HeLa or HeLa-CD19) were seeded into 96-well E-plates (Ace a

Biosciences, San Diego, CA) at 1 x 10 ⁴ cells per well and monitored in culture overnight with the impedance-based real-time cell analysis (RTCA) iCELLigence system (Acea

Biosciences). The next day, the medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± l x 10 ³ effector cells (CAR-T cells or non-transduced T cells), in triplicate. The cells in the E-plates were monitored for another 2-3 days with the RTCA system, and impedance was plotted over time Cytolysis was calculated as (impedance of target cells without effector cells - impedance of target cells with effector ceils) xlOO / impedance of target cells without effector cells. For non-adherent target cells (Raji), the E- plates were first coated with an anti-CD40 antibody (Acea Biosciences) to bind to the CD40 ⁺ Raji cells. Then 1 x ! 0 ⁴ Raji cells w'ere plated per well and the RTCA assay was performed as described above.

Example 8. Cytokine induction assay (ELISA)

The target cells (Raji or HeLa-CDl 9) w'ere cultured with the effector cells (CAR-T cells or non-transduced T cells) at a 1 : 1 ratio (1 x 10 ⁴ cells each) in U-bottom 96-well plates with 200 mΐ of AIM V-AibuMAX medium containing 10% FBS, in triplicate. After 16 h the top 150 m! of medium was transferred to V-bottom 96-well plates and centrifuged at 300 g for 5 min to pellet any residual cells. The top 120 mΐ of supernatant was transferred to a new 96- weil plate and analyzed by ELISA for human IF ^' N-g and IL-6 levels using kits from Thermo Fisher according to the manufacturer’s protocol.

Example 9, Statistical analysis

Data were analyzed and plotted with Prism software ( GraphPad , San Diego, CA). Comparisons between two groups were performed by unpaired Student’s t test. p<0.05 was considered significant.

Example 10. CD19-IL-6 sh RNA-CAR-T ^' cells demonstrate high cytotoxicity against CD19-po$itive Heia-CD19 cells.

The real-time highly sensitive cytotoxicity assay demonstrated high activity of CD 19- IL6 shRNA-CAR-T ceils against CD 19-positive Hela cells (FIG. 3) CD19-IL-6 shRNA specifically killed Hela-CD19-positivel cells similarly to CD19-CAR-T ceils.

Example 11. CD19-IL-6 sh RNA-CAR-T cells secreted significantly less IL-6 than

CD19-CAR-T cells in Raji leukemia cells.

CD19-IL-6 shRNA CAR-T cells were cytotoxic against Raji cells with endogenous expression of CD 19 similarly to CD19-CAR-T ceils (data not shown). We performed ELISA assay for IL-6 secretion by CD19-CAR-T cells and CD! 9--IL6 shRNA-CAR-T cells against Raji cells (FIG. 4). CD19-IL-6 shRNA-CAR-T cells secreted significantly less (> 1.6-fold, p<0.025) IL-6 than CD19-CAR-T cells against target Raji cells (FIG. 4).

The decrease of IL-6 by CD19-IL-6 shRNA-CAR-T cells was specific to silencing of IL-6, and not other cytokine. The same level of IFN-gamma was secreted by CD 19 IL-6 shRNA-CAR-T and CD 19-CAR- T cells (FIG. 5). Thus, IL-6 shRNA effect was highly specific and decreased only IL-6.

Example 12, CD19TF-IL6 shRNA-CAR-T cells and as CD 19TF-C AR-T cells had the same cytotoxicity,

We performed RTCA cytotoxicity assay as above with CD19-TF-IL-6shRNA-CAR-T cells and CD19TF-CAR-T cells using Hela-CDl 9 target cells at different E:T (effector to target ceils) ratio: 20: 1; 10: 1 and 5: 1 and then performed quantification of cytotoxicity'. There were no significant difference between these CAR-T cells in cytotoxicity at each E:T ratio (FIG. 6).

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

CD19TF CAR-T cells

We performed ELISA assay with IL-6 and demonstrated that CD19TF~IL-6shRNA CAR-T cells secreted significantly less IL-6 than CDI 9-CAR-T cells against Hela-CDl 9 cells. (FIG. 7).

Example 14. CD19-TF-PD-1 shRNA and CD19TF-TIGIT shRNA-CAR-T cells had high cytotoxicity as CD19-CAR T cells with no shRNA,

We tested CD 19-CAR T cells, CD19-TF-PD-1 shRNA-CAR T cells, and CD19 TF- TIGIT shRNA-CAR-T cells RTCA with target Hela-CDl 9 cells. All CAR-T cells had the same high cytotoxic activity against Hela-CDl 9 cells (FIG. 8), and against Raji lymphoma cells (FIG. 9). The level of PD-1 and TIGIT level was decreased by PD-1 and TIGIT shRNA (not shown) after co-culturing with Hela-CDl 9 cells.

The CAR-T cells had high level of IFN-gamma secretion with Hela-CDl 9 target cells, but minimal secretion with Hela cells (FIG. 10)

Example 15, CD19-TF-PD-1 shRNA-CAR-T cells and CD 19-TF- TIGIT shRNA-CAR-T cells had higher efficacy in vivo than regnlar CD19-CAR-T cells.

We injected 5xl0 ^A 5 Raji-luciferase cells into vein tail of NSG mice intravenously, and next day injected same way Ixl0 ^/V 7 of CDI9-TF-PD-1 shRNA-CAR-T ceils or CDI9- TF-TIGIT shRNA-CAR-T cells. We performed IVIS imaging and detected significant decrease of bioluniinescence in Raji NSG model by both CD19-TF-PD-1 shRNA-CAR-T cells or CD19-TF-TIGIT shRNA-CAR-T cells (FIG. 11). In addition, both CAR-T cells significantly prolonged mouse survival compared with CD19-TF-CAR-T ceils (FIG. 12). These data show' that CD19-TF-PD-1 shRNA-CAR-T cells and CD19-TF-TIGIT shRNA- CAR-T cells had advantage versus regular CD19-CAR-T cells probably due to decreased exhaustion of T ceils and increased their activity in vivo.

References

1. Gross, G., and Eshhar, Z. (2016). Therapeutic Potential of T Cell Chimeric Antigen Receptors (CARs) in Cancer Treatment: Counteracting Off-Tumor Toxicities for Safe CAR T Cell Therapy. Annu Rev Pharmacol Toxicol 56, 59-83.

2. Mans, M.V., Grupp, S.A., Porter, D.L., and June, C.H. (2014). Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood 123, 2625-2635

3. 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 chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 1, 26-31.

4. Kochenderfer, J.N., Dudley, M.E., Kassirn, S.H., Somerville, R.P., Carpenter, R.O., Stetler-Stevenson, M., Yang, J.C., Phan, G.Q., Hughes, M.S., Sherry', R.M., et al. (2015). Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti- CD 19 chimeric antigen receptor. J Clin Oncol 33, 540-549.

5. Golubovskaya, V., and Wu, L. (2016). Different Subsets of T Ceils, Memory', Effector Functions, and CAR-T Immunotherapy. Cancers (Basel) 8.

6. Maus, M. V., and June, C.H. (2013). Zoom Zoom: racing CARs for multiple myeloma.

Clin Cancer Res 19, 1917-1919

7. Maus, M. V., and June, C.H. (2014). CARTs on the road for myeloma. Clin Cancer Res 20, 3899-3901.

8. Kochenderfer, J.N., and Rosenberg, S.A. (2013). Treating B-cell cancer with T cells expressing an ti -CD 19 chimeric antigen receptors Nat Rev Clin Oncol 10, 267-276.