# US 11,066,680 B2 · IL6R block CAR-T transgenic vector for alleviating CRS, preparation method thereof

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- Number: US 11,066,680 B2
- Document ID: US11066680B2
- Status: Granted
- Kind: B2
- Application number: 16/464,680
- Filed: 2017-11-13
- Published: 2019-09-26
- Granted: 2021-07-20
- Earliest claimed: 2016-12-05
- Assignees: Shanghai Unicar-Therapy Bio-Medicine Technology Co., Ltd
- Applicants: Shanghai Unicar-Therapy Bio-Medicine Technology Co., Ltd
- Inventors: Lei Yu; Liqing Kang; Zhou Yu; Nan Xu
- Web page: https://patentgrep.com/patent/US11066680B2

## CPC

- C12N 15/86: Viral vectors
- A61K 38/1774: {Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4211: {CD19 or B4}
- A61K 40/4217: {Receptors for interleukins [IL]}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- C07K 14/5412: {IL-6}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C12N 15/113: 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 (when used in plants C12N15/8218)}
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12N 15/867: Retroviral vectors
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/21: from primates, e.g. man
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2760/00043: viral genome or elements thereof as genetic vector
- C12N 2760/00051: Methods of production or purification of viral material
- C12N 2800/107: for mammalian
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

An IL6R block CAR-T transgenic vector for alleviating CRS includes: AmpR sequence containing ampicillin resistance gene (SEQ ID NO: 1); prokaryotic replicon pUC Ori sequence (SEQ ID NO: 2); virus replicon SV40 Ori sequence (SEQ ID NO: 3); eWPRE enhanced posttranscriptional regulatory element of hepatitis B virus (SEQ ID NO: 11); human EF1a promoter (SEQ ID NO: 12); lentiviral packaging cis-elements for lentiviral packaging; humanized single-chain antibody fragment IL6RscFv1 (SEQ ID NO: 21), IL6RscFv2 (SEQ ID NO: 22), or IL6RscFv3 (SEQ ID NO: 23) of human IL6R; IRES ribosome binding sequence (SEQ ID NO: 25); IL6 signal peptide (SEQ ID NO: 26); human antibody Fc segment (SEQ ID NO: 27); and chimeric antigen receptors of the second or third generation CAR for integrating recognition, transmission and initiation. A preparation method of the IL6R block CAR-T transgenic vector and an application thereof in a preparation of drugs for alleviating CRS.

## Claims

### Claim 1

A CAR-T transgenic vector for alleviating cytokine release syndrome (CRS) by blocking IL6R, said vector comprising:
an AmpR sequence comprising ampicillin resistance gene having the sequence of SEQ ID NO. 1 for amplifying target bacterial strains;
a prokaryotic replicon pUC Ori sequence having the sequence of SEQ ID NO: 2 for plasmid replication;
a SV 40 Ori sequence of viral replicator having the sequence of SEQ ID NO: 3 for enhancing replication in eukaryotic cells;
an eWPRE enhanced posttranscriptional regulatory element of groundhog hepatitis B virus having the sequence of SEQ ID NO: 11 for enhancing the expression efficiency of transgene;
a human EF1α promoter having the sequence of SEQ ID NO. 12 for eukaryotic transcription of chimeric antigen receptor genes;
a lentivirus packaging cis-element for lentivirus packaging;
a nucleotide sequence encoding a humanized single chain antibody fragment of human IL6R, wherein the humanized single chain antibody fragment of the human IL6R is selected from the group consisting of (i) IL6RscFv1 encoded by the sequence of SEQ ID NO: 21, (ii) IL6RscFv2 encoded by the sequence of SEQ ID NO: 22 and (iii) IL6RscFv3 encoded by the sequence of SEQ ID NO: 23;
an IRES ribosome binding sequence having the sequence of SEQ ID NO. 25 for co-transcription and expression of proteins;
a nucleotide sequence having the sequence of SEQ ID NO 26 encoding IL-6 signal peptide;
a nucleotide sequence having the sequence of SEQ ID NO: 27 encoding human antibody FC segment; and
a nucleotide sequence encoding a second-generation chimeric antigen receptor (CAR) or a third generation CAR.

### Claim 2 (depends on 1)

The CAR-T transgenic vector of claim 1, wherein the lentivirus packaging cis-element is a second-generation lentivirus vector, and the second-generation lentivirus vector comprises: a lentivirus 5 terminal LTR having the sequence of SEQ ID NO: 5, a lentivirus 3 terminal self-Inactivating LTR having the sequence of SEQ ID NO: 6, a Gag cis-element having the sequence of SEQ ID NO: 7, a RRE cis-element having the sequence of SEQ ID NO: 8, an env cis-element having the sequence of SEQ ID NO: 9, and a cPPT cis-elements having the sequence of SEQ ID NO: 10.

### Claim 3 (depends on 1)

The CAR-T transgenic vector of claim 1, wherein the lentivirus packaging cis-element-is a third-generation lentivirus vector, and the third generation lentivirus vector comprises: a lentivirus 5 terminal LTR having the sequence of SEQ ID NO: 5, a lentivirus 3 terminal self-Inactivating LTR having the sequence of SEQ ID NO: 6, a Gag cis-element having the sequence of SEQ ID NO: 7, a RRE cis-element having the sequence of SEQ ID NO: 8, an env cis-element having the sequence of SEQ ID NO: 9, a cPPT cis-elements having the sequence of SEQ ID NO: 10, and a RSV promoter having the sequence of SEQ ID NO: 4.

### Claim 4

The CAR-T transgenic vector of 1, wherein the chimeric antigen receptors of the second-generation CAR comprises: a CD8 leader chimeric receptor signal peptide encoded by the sequence of SEQ ID NO: 13, a CD19 single chain antibody fragment light chain VL encoded by the sequence of SEQ ID NO: 14, an Optimal Linker C encoded by the sequence of SEQ ID NO: 15, a CD19 single chain antibody fragment heavy chain VH encoded by the sequence of SEQ ID NO: 16, a CD8 Hinge chimeric receptor hinges encoded by the sequence of SEQ ID NO: 17, a CD8 transmembrane chimeric receptor transmembrane regions encoded by the sequence of SEQ ID NO: 18, a CD137 chimeric receptor costimulatory factors encoded by the sequence of SEQ ID NO: 19, and a TCR chimeric receptor T cell activation domains encoded by the sequence of SEQ ID NO: 20;
the chimeric antigen receptors of the third-generation CAR comprises: a CD8 leader chimeric receptor signal peptide encoded by the sequence of SEQ ID NO: 13, a CD19 single chain antibody fragment light chain VL encoded by the sequence of SEQ ID NO: 14, an Optimal Linker C encoded by the sequence of SEQ ID NO: 15, a CD19 single chain antibody fragment heavy chain VH encoded by the sequence of SEQ ID NO: 16, a CD8 Hinge chimeric receptor hinges encoded by the sequence of SEQ ID NO: 17, a CD8 transmembrane chimeric receptor transmembrane regions encoded by the sequence of SEQ ID NO: 18, a CD137 chimeric receptor costimulatory factors encoded by the sequence of SEQ ID NO: 19, a TCR chimeric receptor T cell activation domains encoded by the sequence of SEQ ID NO: 20, and a CD28 chimeric receptor costimulatory factors encoded by the sequence of SEQ ID NO: 28.

### Claim 5 (depends on 1)

A preparation method of the CAR-T transgenic vector of claim 1, comprising the following steps:
(i) modifying a lentiviral skeleton plasmid to carry the ampR sequence containing ampicillin resistance gene having the sequence of SEQ ID NO: 1, the prokaryotic replicon pUC Ori sequence having the sequence of SEQ ID NO:2, the vims replicon SV40 Ori sequence having the sequence of SEQ ID NO:3, the lentiviral packaging cis-element for lentiviral packaging, and the eWPRE enhanced posttranscriptional regulatory element of the groundhog hepatitis B virus having the sequence of SEQ ID NO: 11;
(ii) combining the human EF1α promoter having the sequence of SEQ ID NO: 12 and the chimeric antigen receptors of the second-generation CAR or third-generation CAR for integrating recognition, transmission and initiation to form a second-generation CAR design scheme or a third-generation CAR design scheme; and cloning the second generation CAR design scheme or the third-generation CAR design scheme into lentivirus skeleton plasmids by enzymatic digestion, ligation and recombination reaction to obtain recombinant lentivirus plasmids designed by the second-generation CAR or the third generation CAR;
(iii) cloning the nucleotide sequence encoding humanized single-chain antibodies fragment IL6RscFv1, IL6RscFv2, or IL6RscFv3 of human IL-6R, the IRES ribosome binding sequence, the nucleotide sequence encoding IL6 signal peptide and the nucleotide sequence encoding human antibody Fc fragment into recombinant lentiviral plasmids respectively to obtain IL6R recombinant lentiviral plasmids pCAR19-IL6RscFv1, pCAR19-IL6RscFv2, or pCAR19-IL6RscFv3;
(iv) transfecting the IL6R recombinant lentiviral plasmid pCAR19-IL6RscFv1, pCAR19-IL6RscFv2, or pCAR19-IL6RscFv3 into HEK293T/17 cells with lentiviral packaging plasmids pPac-GP and pPac-R and membrane protein pEnv-G respectively; wherein after the recombinant lentiviral plasmids are transcribed and expressed in the HEK.293T/1 7 cells, and recombinant lentiviral vectors are packaged successfully are released into a supernatant of a HEK293T/17 cells culture medium; collecting the supernatant containing the recombinant lentiviral vectors; and
(v) purifying the supernatants by column purification of filtration, adsorption and elution to obtain the recombinant lentiviral vector respectively.

### Claim 6 (depends on 5)

The preparation method of claim 5, wherein in step (5), the step of the filtration comprises: controlling a volume of the supernatant from 200 ml to 2000 ml, a vacuum degree from −0.5 MPA to 0.9 MPA to prevent a loss of vectors caused by blockage; the step of the adsorption comprises: controlling a pH value of solution from 6 to 8, and preventing the recombinant lentiviral vectors from inactivating due to a change of pH; and the step of the elution comprises: controlling an ionic strength of eluent at 0.5 M-1.0 M, and preventing a change of the ionic strength from leading to incomplete elution or inactivation of the recombinant lentiviral vectors.

### Claim 7 (depends on 5)

The preparation method of claim 5, wherein the lentivirus packaging cis-element is a second-generation lentivirus vector, and the second-generation lentivirus vector comprises: a lentivirus 5 terminal LTR having the sequence of SEQ ID NO: 5, a lentivirus 3 terminal self-Inactivating LTR having the sequence of SEQ ID NO: 6, a Gag cis-element having the sequence of SEQ ID NO: 7, a RRE cis-element having the sequence of SEQ ID NO: 8, an env cis-element having the sequence of SEQ ID NO: 9, and a cPPT cis-elements having the sequence of SEQ ID NO: 10.

### Claim 8 (depends on 5)

The preparation method of claim 5, wherein the lentivirus packaging cis-element-is a third-generation lentivirus vector, and the third generation lentivirus vector comprises: a lentivirus 5 terminal LTR having the sequence of SEQ ID NO: 5, a lentivirus 3 terminal self-Inactivating LTR having the sequence of SEQ ID NO: 6, a Gag cis-element having the sequence of SEQ ID NO: 7, a RRE cis-element having the sequence of SEQ ID NO: 8, an env cis-element having the sequence of SEQ ID NO: 9, a cPPT cis-elements having the sequence of SEQ ID NO: I 0, and a RSV promoter having the sequence of SEQ ID NO: 4.

### Claim 9 (depends on 5)

The preparation method of claim 5, wherein the chimeric antigen receptors of the second-generation CAR comprises: a CD8 leader chimeric receptor signal peptide encoded by the sequence of SEQ ID NO: 13, a CD19 single chain antibody fragment light chain VL encoded by the sequence of SEQ ID NO: 14, an Optimal Linker C encoded by the sequence of SEQ ID NO: 15, a CD19 single chain antibody fragment heavy chain VH encoded by the sequence of SEQ ID NO: 16, a CD8 Hinge chimeric receptor hinges encoded by the sequence of SEQ ID NO: 17, a CD8 transmembrane chimeric receptor transmembrane regions encoded by the sequence of SEQ ID NO: 18, a CD137 chimeric receptor costimulatory factors encoded by the sequence of SEQ ID NO: 19, and a TCR chimeric receptor T cell activation domains encoded by the sequence of SEQ ID NO: 20;
the chimeric antigen receptors of the third-generation CAR comprises: a CD8 leader chimeric receptor signal peptide encoded by the sequence of SEQ ID NO: 13, a CD19 single chain antibody fragment light chain VL encoded by the sequence of SEQ ID NO: 14, an Optimal linker C encoded by the sequence of SEQ ID NO: 15, a CD19 single chain antibody fragment heavy chain VH encoded by the sequence of SEQ ID NO: 16, a CD8 Hinge chimeric receptor hinges encoded by the sequence of SEQ ID NO: 17, a CD8 transmembrane chimeric receptor transmembrane regions encoded by the sequence of SEQ ID NO: 18, a CD137 chimeric receptor costimulatory factors encoded by the sequence of SEQ ID NO: 19, a TCR chimeric receptor T cell activation domains encoded by the sequence of SEQ ID NO: 20, and a CD28 chimeric receptor costimulatory factors encoded by the sequence of SEQ ID NO: 28.

### Claim 10 (depends on 7)

The preparation method of claim 7, wherein in step (5), the step of the filtration comprises: controlling a volume of the supernatant from 200 ml to 2000 ml, a vacuum degree from −0.5 MPA to 0.9 MPA to prevent a loss of vectors caused by blockage; the step of the adsorption comprises: controlling a pH value of solution from 6 to 8, and preventing the recombinant lentiviral vectors from inactivating due to a change of pH; and the step of the elution comprises: controlling an ionic strength of eluent at 0.5 M-1.0 M, and preventing a change of the ionic strength leading to incomplete elution or inactivation of the recombinant lentiviral vectors.