WO2020163222A1 - Séquence d'acide nucléique codant pour un récepteur d'antigène chimérique et séquence d'arn en épingle à cheveux courte d'il-6 ou inhibiteur de point de contrôle - Google Patents

Séquence d'acide nucléique codant pour un récepteur d'antigène chimérique et séquence d'arn en épingle à cheveux courte d'il-6 ou inhibiteur de point de contrôle Download PDF

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WO2020163222A1
WO2020163222A1 PCT/US2020/016375 US2020016375W WO2020163222A1 WO 2020163222 A1 WO2020163222 A1 WO 2020163222A1 US 2020016375 W US2020016375 W US 2020016375W WO 2020163222 A1 WO2020163222 A1 WO 2020163222A1
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car
cells
shrna
nucleic acid
acid sequence
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PCT/US2020/016375
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Lijun Wu
Vita Golubovskaya
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Promab Biotechnologies, Inc.
Forevertek Biotechnology Co., Ltd
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Priority to CN202080027442.9A priority Critical patent/CN113710253A/zh
Publication of WO2020163222A1 publication Critical patent/WO2020163222A1/fr

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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A—HUMAN NECESSITIES
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
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    • C12N2510/00—Genetically modified cells

Definitions

  • 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.
  • shRNA short hairpin ENA
  • 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].
  • adoptive cell transfer or ACT adoptive cell transfer
  • 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
  • CRS cytokine release syndrome
  • 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
  • CD 152 cluster of differentiation 152
  • CTLA4 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.
  • 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
  • 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.
  • 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).
  • niRNA messenger RNA
  • RNA silencing RNA interference
  • mlRNA endogenously expressed microRNA
  • siRNA exogenously derived small interfering 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
  • 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.
  • 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
  • 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
  • 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.
  • a “domain” means one region in a polypeptide which is folded into a particular structure independently of other regions.
  • 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.
  • scFv single chain variable fragment
  • 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.
  • H chain immunoglobulin heavy chain
  • L chain light chain
  • 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.
  • shRNA short hairpin RNA
  • 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 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 transme
  • 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 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 transmembr
  • 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.
  • the second polynucleotide is downstream of the first
  • 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.
  • 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 EGFR
  • the tumor antigen is CD 19 or CD22.
  • 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
  • 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.
  • 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.
  • 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.
  • a virus vector such as a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector
  • AAV adeno-associated virus
  • simian virus vector a vaccinia virus vector or a sendai virus vector
  • ESV Epstein-Barr virus
  • HSV vector an HSV vector
  • 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
  • shRNA is part of the RISC complex, the shRNA is unwound to form single stranded siRNA.
  • 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.
  • the single stranded siRNA which is part of the RISC complex now can scan and find a complementary mRNA.
  • siRNA part of the RISC complex
  • the target gene that encodes that mRNA is silenced.
  • Short hairpin RNA 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
  • IL-6 shRNA targets IL-6 mRNA.
  • sequence coding IL-6 mRNA is shown below with one targeted sequence shown in bold and underlined.
  • 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).
  • 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.
  • 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.
  • 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.
  • suitable packaging cell include PG13 (ATCC CRL-10686),
  • 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.
  • CAR 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.
  • release of a cytotoxic cytokine a tumor necrosis factor, lymphotoxin, 11.-6 etc.
  • 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
  • the cells 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.
  • IL-6 shRNA sequence decreases IL-6 secretion, and thus increases safety of CAR-T cells against tumor cells.
  • CAR-T cell deliver ⁇ ' to patients 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 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.
  • shRNAs of other cytokines can he used in CAR (IFN-gamma, IL-2, IL-10, MCP-I, other).
  • 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.
  • RVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER
  • the GDI 9-CAR is shown below, Flag tag is underlined, SEQ ID NO: 17
  • 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:
  • Nhel site> GCTAGC-3' The construct of DNA sequence inserted into a lentivirai vector encoding CAR and IL-6 shRNA is shown below.
  • ATG start codon of CAR sequence is shown in bold (underlined).
  • 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
  • Kpn I site lowercase font, underlined
  • Xho site Italics, uppercase
  • sense uppercase, underlined
  • loop sequence uppercase font
  • antisense sequences uppercase font, Italics, underlined
  • termination sequence uppercase font
  • CD19-TF (transferrin) tag-CAR with IL-6 was also generated.
  • 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.
  • Kpn I site lowercase font, underlined
  • Xho site Italics, uppercase
  • sense uppercase, underlined
  • loop sequence uppercase font
  • antisense sequences uppercase font, Italics, underlined
  • termination sequence ends with Nhe I site.
  • 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.
  • 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.
  • GGAGACATGT AAC A AGAGTTT GAT AT CCG4 CTCTTGTTA CA TGTCTCCIUTTT
  • 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)
  • CTLA-4 cDNA, gene ED 1493 the nucleotide sequences targeted by shRNAs is underlined and bolded
  • CTLA-4 shRNA DNA sequences encoding CTLA-4 shRNA are shown below.
  • TIM-3 shRNA DNA sequences encoding TIM-3 shRNA are shown below. GGATTTCCGCAAAGGAGATTTGATATCCGATCTCCTTTGCGGAAATCCTTim
  • 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.
  • TIGIT shRNAs DNA sequences encoding TIGIT shRNAs are shown below.
  • 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.
  • 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.
  • Kpn I site is HI promoter (in lowercase font, underlined)
  • sense uppercase, underlined
  • loop sequence uppercase font
  • antisense sequences uppercase font, Italics, underlined
  • termination sequence uppercase font
  • 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:
  • 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.
  • PBMC peripheral blood mononuclear cells
  • 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).
  • !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).
  • MOI multiplicity of infecti on
  • AlStem TransPlus transducti on enhancer
  • 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).
  • Adherent target cells (HeLa or HeLa-CD19) were seeded into 96-well E-plates (Ace a).
  • 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 m ⁇ 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 m ⁇ of supernatant was transferred to a new 96- well 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 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.
  • CD19-CAR-T cells in Raji leukemia cells 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).
  • 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).
  • IL-6 shRNA effect was highly specific and decreased only IL-6.
  • CD19-TF-PD-1 shRNA and CD19TF-TIGIT shRNA-CAR-T cells had high cytotoxicity as CD19-CAR T cells with no shRNA
  • 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)
  • 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.
  • 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.
  • T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 1, 26-31.

Abstract

La présente invention concerne une séquence d'acide nucléique comprenant : (a) un premier polynucléotide codant pour une protéine de fusion de récepteur d'antigène chimérique (CAR) comprenant de l'extrémité N-terminale à l'extrémité C-terminale : (i) un fragment variable à chaîne unique (scFv) comprenant VH et VL, le scFv se liant spécifiquement à un antigène tumoral, (ii) un domaine transmembranaire, (iii) au moins un domaine de co-stimulation, et (iv) un domaine d'activation ; et (b) un second polynucléotide codant pour une séquence d'ARN en épingle à cheveux courte (ARNsh) d'IL-6, ou un ARNsh d'inhibiteur de point de contrôle, l'inhibiteur de point de contrôle étant PD-1, CTLA-4, TIM-3, TIGIT, ou LAG-3.
PCT/US2020/016375 2019-02-04 2020-02-03 Séquence d'acide nucléique codant pour un récepteur d'antigène chimérique et séquence d'arn en épingle à cheveux courte d'il-6 ou inhibiteur de point de contrôle WO2020163222A1 (fr)

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