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WO2020061796A1 - Récepteur d'antigène chimérique à double ciblage bcma et cd19 et utilisations associées - Google Patents

Récepteur d'antigène chimérique à double ciblage bcma et cd19 et utilisations associées Download PDF

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WO2020061796A1
WO2020061796A1 PCT/CN2018/107520 CN2018107520W WO2020061796A1 WO 2020061796 A1 WO2020061796 A1 WO 2020061796A1 CN 2018107520 W CN2018107520 W CN 2018107520W WO 2020061796 A1 WO2020061796 A1 WO 2020061796A1
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set forth
bcma
human
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Yarong Liu
Feng He
Haiying Wang
Zixiao SHI
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Hrain Biotechnology Co Ltd
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Hrain Biotechnology Co Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
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    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
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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
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7153—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for colony-stimulating factors [CSF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • 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
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to the field of cell therapy. Specifically, it relates to a BCMA-and-CD19 dual-target chimeric antigen receptor and uses thereof.
  • Chimeric Antigen Receptor-T-cells are the T-cells that are genetically modified to be capable of non-MHC-restricted antigen recognition and sustained activation to proliferation.
  • CAR-T-cell autologous infusion is presently the anti-tumor immune therapy with the most definite efficacy.
  • Extensive studies have shown that CAR-T-cells can effectively recognize tumor antigens, elicit specific anti-tumor immune response and significantly improve patients'well-being.
  • Chimeric antigen receptor is core to CAR-T, which render T-cells the HLA-independent recognition of tumor antigens. This expands the spectrum of targets for CAR-modified T-cells, compared to native T-cell surface receptors (TCR) .
  • a design of CAR may basically include a tumor-associated antigen (TAA) binding domain, which is usually derived from a scFV fragment of the antigen-binding domains of a monoclonal antibody, an extracellular hinge domain, a transmembrane region and an intracellular signaling domain. Selection of the target antigen is crux for specificity and efficacy of the CAR, as well as safety of the genetically modified T-cells per se.
  • TAA tumor-associated antigen
  • CD19 is a 95kDa glycoprotein on the surface of B-cells, which is expressed by B-cells from the early stage of development until differentiation into plasma cells.
  • CD19 is a member of the superfamily of immunoglobulins (Igs) and a component of the B-cell surface signaling complex, which participates in regulation of B-cell receptor signaling.
  • Igs immunoglobulins
  • a component of the B-cell surface signaling complex which participates in regulation of B-cell receptor signaling.
  • Igs immunoglobulins
  • CD19 expression is limited to B-cell lineage, which is not seen on surface of multipotent hematopoietic stem cells.
  • CD19 expression has also been observed on cells in most B cell lymphoma, mantle cell lymphoma, ALLs, CLLs, hairy cell leukemia and part of acute myeloid leukemia. Accordingly, for leukemia/lymphoma, CD19 is a valuable target of immunotherapy. Importantly, CD19 is not expressed on most normal cells (including multipotent hematopoietic stem cells) other than B-cells, and this makes it a safer therapy target, which minimizes the risk of autoimmune diseases and irreversible toxic injury to bone marrow in patients. Up till now, anti-CD19 antibodies and scFv fragments have been developed with promising future in applications as tested in mouse and human beings/primates.
  • CD19 CAR T-cells have been quite competitive, while many large pharmaceutical companies have partnership ties with research institutes. In recurrent and refractory acute B cell lymphoma in children and adults, CD19 CAR T-cells expressing CD28 or 4-1BB provide a complete response rate of around 90%. Recently, CD19 CAR T-cell therapy is reported with an overall response rate of 50%-100%in diffuse large B cell lymphoma, follicular lymphoma or chronic lymphoma. CD19 CAR T-cell therapy has an clinical edge in multiple myeloma because terminally differentiated plasma cells do not express CD19, and malignant B cell precursors continuously produce malignant plasma cells.
  • B-cell maturation antigen also known as CD269, consists of 184 amino acid residues, which comprises an intracellular domain comprising 80 amino acid residues and a very short extracellular domain comprising a single carbohydrate-recognition domain as a B-cell surface molecule.
  • BCMA is a type I transmembrane signaling protein lacking signal peptide and is a member of the family of tumor necrosis factor receptor s (TNFRs) .
  • BCMA B cell activation factor
  • APRIL proliferation-induced ligand
  • BCMA is ubiquitously expressed in multiple myeloma cell lines, which has been confirmed by the detection in multiple myeloma patients.
  • Kochenderfer et al. on basis of the previously reported results, further explored the expression profile of BCMA using multiple techniques including Q-PCR, Flow Cytometry and immunohistochemistry, and concluded that BCMA is not expressed on normal human tissues except for mature B cells and plasmacytes, and is not expressed on CD34+ hematopoietic cells.
  • BCMA-deficiency has no impact on the number of B-cells in mouse and is not fatal.
  • BCMA is a potential target of CAR-T-cells for use in cellular immunotherapy of multiple myeloma.
  • the present invention utilizes a CAR component dually targeting CD19 and BCMA.
  • the anti-CD19-BCMA bi-specific CAR T-cells exhibit a strong killing of target cells, which sets a sound basis for future clinical trials and therapy.
  • the present disclosure provides a polynucleotide sequence selected from the group consisting of:
  • polynucleotide sequence comprising the followings linked in sequence: sequences encoding an anti-BCMA and an anti-CD19 single chain antibodies, a sequence encoding the hinge region of human IgG4, a sequence encoding the transmembrane region of human CD28, a sequence encoding the intracellular domain of human 41BB, a sequence encoding the intracellular domain of human CD3 ⁇ and optionally a sequence encoding a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV; and
  • the sequence encoding the light chain variable region of said anti-BCMA single chain antibody has the sequence of nucleotides 64-396 as set forth in SEQ ID NO: 1.
  • the sequence encoding the heavy chain variable region of said anti-BCMA single chain antibody has the sequence of nucleotides 442-792 as set forth in SEQ ID NO: 1.
  • the sequence encoding the heavy chain variable region of said anti-CD19 single chain antibody has the sequence of nucleotides 853-1212 as set forth in SEQ ID NO: 1.
  • the sequence encoding the light chain variable region of said anti-CD19 single chain antibody has the sequence of nucleotides 1267-1587 as set forth in SEQ ID NO: 1.
  • the sequence encoding said hinge region of human IgG4 has the sequence of nucleotides 1588-1623 as set forth in SEQ ID NO: 1.
  • the sequence encoding the transmembrane region of human CD28 has the sequence of nucleotides 1627-1707 as set forth in SEQ ID NO: 1.
  • the sequence encoding the intracellular domain of human 41BB has the sequence of nucleotides 1708-1833 as set forth in SEQ ID NO: 1. In one or more embodiments, the sequence encoding the intracellular domain of human CD3 ⁇ has the sequence of nucleotides 1834-2169 as set forth in SEQ ID NO: 1.
  • the present disclosure provides a fusion protein selected from the group consisting of:
  • a fusion protein comprising the followings linked in sequence: an anti-BCMA single chain antibody and an anti-CD19 single chain antibody, the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB and the intracellular domain of human CD3 ⁇ ; and
  • (2) a fusion protein derived from (1) , comprising one or more substitution (s) , deletion (s) or addition (s) in the amino acid sequence of (1) while retaining the activity of T-cell activation;
  • the anti-CD19 single chain antibody is of the anti-CD19 monoclonal antibody FMC63;
  • the anti-BCMA single chain antibody is of the anti-BCMA monoclonal antibody C11D5.3.
  • the present disclosure provides a nucleic acid construct comprising the polynucleotide sequence according to the present disclosure.
  • the nucleic acid construct is a vector. In one or more embodiments, the nucleic acid construct is a retrovirus vector comprising an origin of replication, a 3’LTR, a 5’LTR and the polynucleotide sequence of the present disclosure, as well as an optional selection marker.
  • the present disclosure provides a retrovirus which comprises the nucleic acid construct, preferably the vector, more preferably the retrovirus vector according to the present disclosure.
  • the present disclosure provides a pharmaceutical composition comprising the genetically modified T-cell according to the present disclosure.
  • the present disclosure provides use of the polynucleotide sequence, the fusion protein, the nucleic acid construct or the retrovirus according to the present disclosure for producing activated T-cells.
  • the present disclosure provides use of the polynucleotide sequence, the fusion protein, the nucleic acid construct, the retrovirus, the genetically modified T-cell or the pharmaceutical composition according to the present disclosure for manufacturing medicaments for treating diseases mediated by BCMA.
  • the disease mediated by BCMA is multiple myeloma.
  • FIG. 1 schematically depicts the retrovirus expression vector RV-BCMA-CD19 -BBz.
  • FIG. 2 shows the flow cytometry of BCMA-CD19 -BBz CART expression by T-cells infected with the retrovirus for 72 hours.
  • FIG. 3 shows the flow cytometry of CD19 expression on surface of the target cell MM. 1S-CD19.
  • FIG. 4 shows the CD107a expression by a 5-day preparation of BCMA-CD19 CART-cells incubated with different target cells for 5 hours.
  • FIG. 5 shows the INF- ⁇ secretion by a 5-day preparation of BCMA-CD19 CART-cells incubated with different target cells for 5 hours.
  • FIG. 6 shows the killing of tumor cells by a 5-day preparation of BCMA-CD19 CART-cells after incubation with different target cells for 5 hours.
  • the present disclosure provides a chimeric antigen receptor (CAR) dually targeting BCMA and CD19.
  • the CAR comprises the followings linked in sequence: an anti-BCMA single chain antibody and an anti-CD19 single chain antibody, the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB, the intracellular domain of human CD3 ⁇ and optionally a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV of the receptor.
  • a suitable anti-BCMA single chain antibody may be one derived from any of the anti-BCMA monoclonal antibodies known to a person in the art.
  • a suitable anti-CD19 single chain antibody may be one derived from any of the anti-CD19 monoclonal antibodies known to a person in the art.
  • the light chain variable region and the heavy chain variable region may be joined by a linker.
  • the anti-BCMA single chain antibody has a light chain variable region having the sequence of amino acids 22-132 as set forth in SEQ ID NO: 2.
  • the anti-BCMA single chain antibody has a heavy chain variable region having the sequence of amino acids 148-264 as set forth in SEQ ID NO: 2.
  • the anti-CD19 single chain antibody has a heavy chain variable region having the sequence of amino acids 285-404 as set forth in SEQ ID NO: 2.
  • the anti-CD19 single chain antibody has a light chain variable region having the sequence of amino acids 423-529 as set forth in SEQ ID NO: 2.
  • the hinge region of human IgG4 useful in the present disclosure may have the sequence of amino acids 530-541 as set forth in SEQ ID NO: 2.
  • the transmembrane region of human CD28 useful in the present disclosure may be any one of the human CD28 transmembrane sequences useful in CARs.
  • the transmembrane region of human CD28 has the sequence of amino acids 532-569 as set forth in SEQ ID NO: 2.
  • the 41BB useful in the present disclosure may be any one of the 41BB molecules useful in CARs.
  • the 41BB used in the present disclosure has the sequence of amino acids 570-611 as set forth in SEQ ID NO: 2.
  • the intracellular domain of human CD3 ⁇ useful in the present disclosure may any one of the intracellular domains of human CD3 ⁇ useful in CARs.
  • the intracellular domain of human CD3 ⁇ has the sequence of amino acids 612-723 as set forth in SEQ ID NO: 2.
  • linkers may be any of those used in antibodies, like those comprising G and S.
  • linkers comprise repeats of one or more motifs. Examples of the motif include GGGS, GGGGS, SSSSG, GSGSA and GGSGG.
  • the motifs are adjacent one another in a linker, without in-between amino acid residues.
  • the linker may comprise 1, 2, 3, 4 or 5 repeats of a motif.
  • the linker may be 3-25 amino acid residues, for example, 3-15, 5-15, 10-20 amino acid residues in length.
  • the linker is a poly (glycine) linker.
  • There is no limit on the number of glycine residues in the linker while the number is usually in the range of 2-20, for example, 2-15, 2-10 and 2-8.
  • the linker may further comprises some additional amino acid residues, like alanine (A) , leucine (L) , threonine (T) , glutamic acid (E) , phenylalanine (F) , arginine (R) , glutamine (Q) , etc.
  • the light chain variable region and the heavy chain variable region are linked via (GGGGS) n , wherein n is an integer from 1 to 5.
  • the CAR of the present disclosure may further comprises in its amino acid sequence a fragment of EGFR comprising the extracellular domain III and the extracellular domain IV of the receptor, the signal peptide thereof and a linker.
  • the expressed amino acid sequence will include one or more irrelevant residues at the end (s) , which will not interfere the activity of the sequence of interests.
  • the protein may need to include some additional amino acids at the N-terminal, the C-terminal of some other region of the fusion protein as appropriate.
  • the additional amino acids include but are not limited to a linker peptide, a signal peptide, a leader, a terminal extension.
  • the fusion protein (i.e., the CAR) of the present disclosure may further include at the N-or the C-terminal one or more polypeptide fragment (s) as protein tag (s) .
  • Any suitable tags are useful in the present disclosure.
  • the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags are useful in protein purification.
  • the present disclosure further includes variants of the CAR having the sequence of amino acids 22-723 as set forth in SEQ ID NO: 2, the CAR having the sequence of amino acids 1-723 as set forth in SEQ ID NO: 2 or the CAR having the amino acid sequence of SEQ ID NO: 2.
  • the variant includes a amino acid sequence that has a sequence identity of at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%to the specified CAR and the same biological activity (e.g., of T-cell activation) as the specified CAR.
  • Sequence identity can be calculated, for example, using BLASTp from NCBI.
  • Variants also include those having one or a plurality of mutation (s) (insertion, deletion or substitution) in the sequence of amino acids 22-723 as set forth in SEQ ID NO: 2, the sequence of amino acids 1-723 as set forth in SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 and remaining the biological activity of the CAR.
  • the "plurality” normally refers to a number raging from 1 to 10, such as from 1 to 8, from 1 to 5 or from 1 to 3.
  • the substitution is preferably a conservative one. For instance, conservative substitution between amino acids close or similar in property is known as will not change the effect of the protein or polypeptide.
  • amino acids close or similar in property include, for example, a family of amino acid residues having similar side chains.
  • amino acids with a basic side chain e.g., lysine, arginine, histidine
  • amino acids with an acidic side chain e.g., aspartic acid, glutamic acid
  • amino acids with an uncharged polar side chain e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • amino acids with a nonpolar side chain e.g., alanine, valine, leucine, isoleucine valine, phenylalanine, methionine, tryptophan
  • amino acids with a ⁇ -branched side chain e.g., threonine, proline, isoleucine
  • amino acids with an aromatic side chain e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the present disclosure includes polynucleotide sequences encoding the fusion proteins according to the present disclosure.
  • the polynucleotide sequences of the present disclosure may be in the form of DNA or RNA.
  • the term "DNA" includes cDNA, genomic DNA or artificial synthetic DNA.
  • the DNA may be a single-strand or a doubled-strand DNA.
  • the DNA may be the coding strand or the non-coding strand.
  • the present disclosure further includes degenerate variants of the polynucleotide sequence encoding the fusion protein, i.e., different nucleotide sequences that encode the same amino acid sequence.
  • the polynucleotide sequences according to the present disclosure can be prepared by PCR amplification. Specifically, the sequence can be amplified using primers designed according to the given nucleotide sequence (particularly the Open Reading Frames) and commercially available cDNA libraries or self-made cDNA libraries as templates. For longer sequences, two or more runs will be needed, and fragments from each run are then assembled into the correct sequence.
  • the polynucleotide sequence encoding the fusion protein of the present disclosure has the sequence of nucleotides 64-2169 as set forth in SEQ ID NO: 1 or the sequence of nucleotides 1-2169 as set forth in SEQ ID NO: 1.
  • the present disclosure further includes a nucleic acid construct, which comprises the polynucleotide sequence according to the present disclosure operably linked to one or more regulatory sequence (s) .
  • the polynucleotide sequence according to the present disclosure can be manipulated in various ways to ensure a successful expression of the fusion protein (CAR) .
  • the nucleic acid construct Before being inserted into a vector, the nucleic acid construct may be adaptively processed according to the selected expression vector. The recombinant DNA techniques useful to modify polynucleotide sequences are already known.
  • the regulatory sequence may be a proper promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter may be any of the nucleotide sequences that exhibit transcription activity in the host, which includes mutated, truncated or hybrid promoters, and which may be obtained from the gene of an extracellular or intracellular polypeptide that is homologous or heterologous to the host.
  • a regulatory sequence may also be a transcription terminator sequence as appropriate, which terminates transcription upon recognition by the host cell.
  • the terminator sequence is operably linked to the 3’-end of the nucleotide sequence encoding the polypeptide. Any terminator functional in a selected host is useful in the present disclosure.
  • the regulatory sequence may also be a leader sequence as appropriate, which is a untranslated region of mRNA important for translation in the host cell.
  • the leader sequence is operably linked to the 5′-end of the nucleotide sequence encoding the polypeptide. Any leader sequence functional in a selected host is useful in the present disclosure.
  • the nucleic acid construct is a vector.
  • the polynucleotide sequence according to the present disclosure is operably link to the promoter, and the construct is incorporated into an expression vector to obtain an effective expression of the polynucleotide sequence according to the present disclosure.
  • the vector may be one suitable for replication in and integration into an eukaryotic cell.
  • a cloning vector comprises a transcription terminator, a translation terminator, an initiation region and a promoter to modulate the desired expression of a nucleic acid sequence.
  • the polynucleotide sequence according to the present disclosure may be cloned into various types of vectors. For example, it can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus or a cosmid.
  • the vector may be an expression vector.
  • the expression vector may be delivered into the cell in form of a virus vector.
  • Viral vector technology is already known and has been described in, for example, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Sambrook et al., 2001, New York) and many other virology and molecular biology manuals.
  • Viruses that are useful as vectors include but are not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and lentivirus.
  • a suitable vector comprises a replication origin, promoter sequence, convenient restriction site and one or more selectable markers that are functional in at least one organism (e.g., WO 01/96584; WO01/29058; and US patent No. 6,326,193) .
  • a retrovirus vector which comprises an origin of replication, a 3’LTR, a 5’LTR, the polynucleotide sequence according to the present disclosure, and optionally a selectable marker.
  • cytomegalovirus CMV
  • EF-1 ⁇ extension growth factor-1 ⁇
  • constitutive promoter sequences may also be used, including but not limited to: simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, EB virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, such as actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, also contemplated are inducible promoters.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukosis virus promoter
  • EB virus immediate early promoter EB virus immediate early promoter
  • Rous sarcoma virus promoter Rous sarcoma virus promoter
  • human gene promoter such
  • Inducible promoters provide a molecular switch, which can turn on the expression of the polynucleotide sequence operably linked to the inducible promoter as desired, and turn off when the expression is not desired.
  • inducible promoter include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter.
  • the expression vector to be introduced into cells may further comprise a selectable marker gene or a reporter gene or both, such that expression cells can be identified or selected from the cell population transfected or infected with the virus vector.
  • the selectable marker may be carried on a separate DNA sequence for use in co-transfection. Both the selectable marker and the reporter genes may be flanked by one or more regulatory sequences for expression in host cells.
  • Useful selectable markers include for example antibiotics resistance genes, like neo, etc.
  • Reporter genes are used to identify potentially transfected cells and to assess functionality of the regulatory sequences. After DNA being transferred into the recipient cells, the reporter gene may be detected at an appropriate time point. Suitable reporter genes may include those encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein. Suitable expression systems are already known and can be prepared using existing techniques or commercially obtained.
  • Vectors can be conveniently delivered into host cells, like mammalian, bacterial, yeast or insect cells, using various methods as known to a person in the art.
  • an expression vector may be transferred into the host cell using a physical, chemical or biological means.
  • Physical methods of transferring polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc.
  • Biological methods of transferring polynucleotides into host cells involve DNA and RNA vectors.
  • Chemical methods of transferring polynucleotides into host cells involve colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres and beads; and lipid-based systems, including oil in water emulsion, micelle, mixed micelles and liposomes.
  • virus vectors especially retrovirus vector, which has been widely used in gene integration into mammalian cells, like human cells.
  • Additional virus vectors may be those derived from lentivirus, poxvirus, simple herpes virus I, adenovirus, adeno-associated virus, etc.
  • virus-based systems have been developed for use in gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene transfer systems. A selected gene may be inserted into a vector and then packaged into a retrovirus particle using techniques as known to a person in the art. The recombinant virus may then be isolated and transferred into cells from a subject in vivo or ex vivo.
  • adenovirus vectors may be used.
  • adenovirus vectors as known to a person in the art.
  • a lentivirus vector is used.
  • the present disclosure further provides a retrovirus useful in T-cell activation, wherein the virus comprises the retrovirus vector according to the present disclosure and corresponding package genes, like gag, pol and vsvg.
  • T-cells useful in the present disclosure can be those of any origins and of any types.
  • T-cells may be those from PBMC from a patient having B-cell malignant tumor.
  • the obtained T-cells may first be stimulated using an appropriate amount (such as 30-80ng/ml, e.g., 50ng/ml) of an anti-CD3 antibody, and then cultured in a medium supplemented with an appropriate amount (such as 30-80IU/ml, e.g., 50IU/ml) of IL2.
  • an appropriate amount such as 30-80ng/ml, e.g., 50ng/ml
  • the present disclosure provides a genetically modified T-cell, which comprises the polynucleotide sequence according to the present disclosure or the retrovirus vector according to the present disclosure, or is infected with the retrovirus vector according to the present disclosure, or is prepared by the method according to the present disclosure, or stably expresses the fusion protein according to the present disclosure.
  • the CAR-T-cells according to the present disclosure may undergo robust in vivo T-cell expansion, sustain in blood and bone marrow for a prolonged time period, and form specific memory T-cells. Without being bound to any particular theory, after encountering and depleting the target cells expressing the antigen substitute, the CAR-T-cells according to the present disclosure can differentiate into the central memory status in vivo.
  • the present disclosure further includes a kind of cell therapy, wherein T-cells are genetically modified to express the CAR and optionally the tEGFR according to the present disclosure, and the CAR-T-cells are infused into a recipient in need of such a therapy.
  • the infused cells kill tumor cells in the recipient.
  • CAR-T-cells are capable of in vivo replication and production, which leads to a long-term sustained control of tumor.
  • the CAR-T-cells-mediated anti-tumor immune response may be an active or a passive one. Additionally, the CAR-mediated immune response may be part of an adoptive immunotherapy, wherein the CAR-T-cells induce an immune response with a specificity defined by the antigen-binding part of the CAR.
  • the diseases that can be treated using the CAR, the sequence encoding same, the nucleic acid construct, the expression vector, the virus and the CAR-T-cells according to the present disclosure are preferably diseases mediated by BCMA.
  • the CAR-modified T-cells according to the present disclosure may be used alone or in form of a pharmaceutical composition and in combination with a diluent and/or other components like relevant cytokine (s) or cell population (s) .
  • the pharmaceutical composition according to the present disclosure may comprise the CAR-T-cells according to the present disclosure in combination with one or more pharmaceutically or physiologically acceptable carrier (s) , diluent (s) or excipient (s) .
  • the composition may comprise a buffer solution, such as a neutral buffered saline, sulfate buffered saline, etc; a carbohydrate, such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid, such as glycine; an antioxidant; a chelator, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide) ; and a preservative.
  • a buffer solution such as a neutral buffered saline, sulfate buffered saline, etc
  • a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol
  • a protein such as glycine
  • a polypeptide or an amino acid such as glycine
  • an antioxidant such as glycine
  • a chelator such as EDTA or glutathione
  • an adjuvant e.g.,
  • the pharmaceutical composition according to the present disclosure may be administered in a manner as appropriate for the disease that is to be treated or prevented.
  • the amount and frequency of administration will be determined by known factors, like the medical condition of the patient and the classification and severity of the disease.
  • an immunologically effective amount an anti-tumor effective amount
  • a tumor-inhibition effective amount a therapeutically effective amount
  • the exact amount at which the composition according to the present disclosure is to be administered will be determined by a physician on an individual basis with considerations including patient's (subject's) age, body weight, tumor size, degree of invasion or metastasis.
  • the pharmaceutical composition comprising the T-cell may be administered at an dosage ranging from 10 4 to 10 9 cells/kg bodyweight, preferably 10 5 to 10 6 cells/kg bodyweight.
  • the T-cell composition may also be administered multiple times by repeating the specified dosage.
  • the cells may be administered using conventional infusion techniques as known in immunotherapy (see for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988) .
  • the optimal dosage and regimen for a particular patient may be conveniently determined by monitoring the patient's signs of disease and making adjustment accordingly.
  • the composition may be administered in any way as convenient, like aerosol, injection, swallowing, infusion, implantation or transplantation.
  • the composition may be administered to a patient subcutaneously, intradermally, intratumorally, intraductally, intraspinally, intramuscularly, intravenously or intraperitoneally.
  • the T-cell composition according to the present disclosure is administered via intradermal or subcutaneous injection.
  • the T-cell composition is preferably administered via intravenous injection.
  • the T-cell composition may be directly injected into the tumor, lymph nodes or sites of infection.
  • the CAR-T-cells or the composition may be supplied in combination with an additional therapy.
  • the additional therapy may include but is not limited to chemotherapy, radiation and immunosuppressants.
  • the additional therapy may be any of the radio-or chemo-therapies known as useful in diseases mediated by BCMA.
  • anti-tumor effect refers to a biological effect that is characterized in decreased tumor size, reduced number of tumor cells, reduced metastasis, increased life expectancy or improvement in any physiological symptoms associated with cancer.
  • patient refers to a living organism, like an mammalian, in which the immune response can be induced. Examples include but are not limited to human beings, dogs, cats, mice, rats and corresponding transgenic species.
  • the present invention may provide inter alia the following advantages.
  • the present invention advantageously synthesizes the whole gene sequence of the chimeric antigen receptor, which may be expressed by BCMA scFV-CD19scFV-41BB-CD3 ⁇ , using the gene sequences of BCMA scFV+CD19 scFV, and sequences of the hinge region of human IgG4, the transmembrane region of human CD28, the intracellular domain of human 41BB and the intracellular domain of human CD3 ⁇ from NCBI GenBank.
  • the whole gene sequence is then incorporated into a retrovirus vector (RV) , which enables delivery of the nucleic acid sequence of interests, i.e., the sequence encoding CAR.
  • RV retrovirus vector
  • the recombinant plasmids are packaged into viruses in 293T-cells, to infect T-cells to express the chimeric antigen receptor.
  • the chimeric antigen receptor-genetically modified T lymphocytes are transformed using a retrovirus-based process, which advantageously provides inter alia a high efficiency of transformation, a stable expression of the exogenous gene and a shortened period of in vitro culturing before reaching a clinical-grade number of T lymphocytes.
  • the transferred nucleic acid is transcribed and expressed on surface of the transgenic T lymphocytes.
  • the CAR expression retroviruses according to the invention were used to prepare CAR-T-cells according to the Retronectin method.
  • CAR-T-cells of 3 days post-infection are assayed for infection efficiency for CAR by flowmetry.
  • CAR-T-cells of 5 days post-infection were incubated in vitro with CD19-positive or BCMA-positive tumor cells (K562-CD19, MM. 1S, MM. 1S-CD19) for 5 hours before detection of CD107a expression and IFN ⁇ secretion.
  • CAR-T-cells of 5 days post-infection were incubated in vitro with CD19-positive or BCMA-positive tumor cells (K562-CD19, MM. 1S, MM. 1S-CD19) for 5 hours before detection of specific killing of tumor cells by the CAR-T-cells (cytotoxicity) .
  • the CD19-BCMA-BBz CART according to the present disclosure is potentially useful in treatment of multiple myeloma.
  • the specified sequences were linked in sequence, with distinct restriction sites introduced at each of the adjunctions between sequences, whereby to generate the complete sequence of BCMA -CD19-BBz.
  • the obtained recombinant plasmid was sequenced by Sangon Biotech (Shanghai) Co., Ltd. .
  • the result was aligned to the to-be-synthesized BCMA -CD19-BBz sequence to confirm the correct sequence.
  • the sequencing primers are:
  • Fig. 1 The plasmid constructed according to this example is schematically depicted in Fig. 1.
  • Example 2 Construction of viral vector comprising the CAR molecule
  • the nucleotide sequence of the CAR molecule prepared according to Example 1 was double digested with NotI (NEB) and EcoRI (NEB) , and inserted into the retrovirus RV vector at the NotI-EcoRI site using T4 ligase (NEB) .
  • the vector was then transferred into the competent Escherichia coli. strain (DH5 ⁇ ) .
  • the plasmids were purified using the plasmid purification kit from Qiagen.
  • the purified plasmids were transferred into 293T-cells using the calcium phosphate-method for retrovirus packaging.
  • Day 1 The 293T-cells should be within the 20 th passage and not over-confluent.
  • the cells were plated at 0.6 ⁇ 10 6 cells/ml on a 10cm dish containing 10ml DMEM medium. The cells were mixed well until uniform, and cultured at 37°C over night;
  • CD3+T-cells were purified using Ficcol solution (Tian Jin Hao Yang Biological Manufacture Co., Ltd) and conditioned in X-VIVO (LONZA) medium supplemented with 5%AB serum till cell density of 1 ⁇ 10 6 /mL.
  • the cells were inoculated at 1ml/well onto a plate pre-treated with 50ng/ml anti-human CD3 antibody (Beijing T&L Biotechnology Co. Ltd) and 50ng/ml CD28 antibody (Beijing T&L Biotechnology Co. Ltd) , followed by addition of 100IU/ml IL-2 (Beijing SL Pharmaceutical Co. Ltd) . After cultivation under stimulation for 48 hours, the cells were infected with the viruses.
  • a 24-well non-tissue treated plates (corning) were prepared by coating with 250 ⁇ l/well of Retronectin (Takara) diluted in PBS to the final concentration of 15 ⁇ g/ml. The plates were kept in dark at 4°Covernight for use.
  • T-cell culturing medium supplemented with 100IU/ml of IL-2 was added as appropriate to maintain T-cell density at around 5 ⁇ 10 5 /ml and to effect cell expansion.
  • Example 5 Flow cytometric assay of CAR expression on surface of T-lymphocytes
  • the BCMA-CD19-BBz cells were collected by centrifugation 72 hours after the infection The cells were washed once with PBS and the supernatant was discarded. The cells were then exposed to corresponding antibodies in dark for 30min, washed again with PBS, re-suspended and assayed via flow cytometry. CAR + was detected using anti-mouse IgG F (ab') antibody (Jackson Immunoresearch) .
  • the percentage of BCMA-CD19-tEGFR CART positive is at least 50%.
  • the target cell MM. 1S-CD19 expresses both CD19 and BCMA.
  • the cell line was constructed in-lab.
  • MM. 1S-CD19 expresses CD19 at the efficiency of 99.6%.
  • CART/NT-cells (2 ⁇ 10 5 cells) and target cells (K562-CD19, MM. 1S, MM. 1S-CD19, 2 ⁇ 10 5 cells) /control cells (K562, 2 ⁇ 10 5 cells) re-suspended to 200 ⁇ l in IL-2-free X-VIVO complete medium supplemented with BD GolgiStop (containing monesin, 1 ⁇ l BD GolgiStop/1ml medium) .
  • 2ul/well anti-CD107a antibody (1: 50) was added. After incubation at 37°C for 4 hours, cells were collected.
  • CD19-BCMA CART-cells express CD107a at a percentage around 80%when co-incubated with single-target target cells (K562-CD19, MM. 1S) , around 80%with the dual-target target cell (MM. 1S-CD19) , and almost null with the control (K562) .
  • the BCMA-CD19 dual-target CART's activity in vitro on the single-target target cells MM. 1S and K562-CD19 is comparable to the single-target BCMA CART or the CD19 CART in terms of CD107a expression.
  • Example 8 INF- ⁇ secretion by CAR-T-cell co-incubated with target cells
  • test groups comprised in each well 2 ⁇ 10 5 target cells (K562-CD19, MM. 1S, MM. 1S-CD19) or negative control cells (K562) , 2 ⁇ 10 5 CAR-T-cells and 200 ⁇ l Lonza medium free of IL-2.
  • the mixture was added onto a 96-well plate.
  • BD GolgiPlug (with monesin, 1 ⁇ l BD GolgiPlug/1ml cell culture) was added and mixed well. The mixture was then incubated at 37°C for 5 hours. The cells were collected as the test group.
  • Fixation/Permeabilization solution was added at 250 ⁇ l/EP tube followed by incubation at 4°C for 20 min to fix the cells and to disrupt cell membrane.
  • the cells were washed twice with 1 ⁇ BD Perm/Wash TM buffer, 1mL each time.
  • the cells were stained for intracellular cytokines: certain amount of IFN- ⁇ cytokine fluorescent antibody or negative control was diluted in BD Perm/Wash TM buffer to 50 ⁇ l. Cells after fixation and membrane disruption were re-suspended in this diluted antibody solution, and incubated at 4°C in dark for 30min, washed twice with 1 ⁇ BD Perm/Wash TM buffer 1mL/time, and re-suspended in PBS.
  • CD19-BCMA CART-cells express IFN- ⁇ at a percentage around 70%when co-incubated with single-target target cells (K562-CD19, MM. 1S) , around 70%with the dual-target target cell (MM. 1S-CD19) , and almost null with the control (K562) .
  • the BCMA-CD19 dual-target CART's activity in vitro on the single-target target cells MM. 1S and K562-CD19 is comparable to the single-target BCMA CART or the CD19 CART in terms of IFN- ⁇ expression
  • Example 9 Tumor-specific killing by CAR-T-cells incubated with target cells
  • K562 cells (CD19 or BCMA free, serving as the negative control) were re-suspended in serum-free medium (1640) , with cell concentration adjusted to 1 ⁇ 10 6 /ml, followed by addition of Fluorescent dye BMQC (2, 3, 6, 7-tetrahydro-9-bromomethyl-1H, 5H-quinolizino (9, 1-gh) coumarin) to the final concentration of 5 ⁇ M.
  • Fluorescent dye BMQC 2, 3, 6, 7-tetrahydro-9-bromomethyl-1H, 5H-quinolizino (9, 1-gh) coumarin
  • the cells were re-suspended in the cytotoxicity medium (phenol red-free 1640+5%AB serum) and incubated at 37°C for 60min.
  • cytotoxicity medium phenol red-free 1640+5%AB serum
  • the cells were washed twice with fresh cytotoxicity medium and re-suspended in fresh cytotoxicity medium to 1 ⁇ 10 6 cells/ml.
  • MM. 1S-CD19 cells (expressing CD19 and BCMA, serving as target cells) were re-suspended in PBS supplemented with 0.1%BSA, and cell concentration was adjusted to 1 ⁇ 10 6 cells/ml.
  • Fluorescent dye CFSE (carboxyfluorescein diacetate succinimidyl ester) was added to the final concentration of 1 ⁇ M.
  • the cells were washed and re-suspended in the fresh cytotoxicity medium to 1 ⁇ 10 6 cells/ml.
  • the effector T-cells were washed and re-suspended in the cytotoxicity medium and the concentration was adjusted to 5 ⁇ 10 6 cells/ml.
  • CAR-T cell cytotoxicity of the CD19-BCMA-BBz CAR-infected T-cells (CAR-T cell) was compared with cytotoxicity of the uninfected negative control effector T-cells (NT-cells) , both coming from the same patient.
  • the target cells were 100,000 (50 ⁇ l) in amount
  • the negative control cells were K562 cells (100,000 cells, 50 ⁇ l) .
  • another group is designed to merely comprise target cells and K562 negative control cells.
  • the cells were co-incubated at 37°C for 5hrs.
  • 7-AAD (7-aminoactinomycin D) was added according to instruction immediately after the cells were washed with PBS, and then incubated on ice for 30min.
  • the cells were directly loaded onto the flow cytometer without washing, and the data were analyzed using Flow Jo.
  • the analysis was gated by 7AAD-negative living cells to detect the percentage of living target cells and the percentage of living negative control cells after co-incubation of the T-cells and the target cells.
  • cytotoxicity killing % (1- (living target cells in presence of effector /living K562 cells in presence of effector) / (living target cells in absence of effector /living K562 cells in absence of effector) ) ⁇ 100%.

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Abstract

L'invention concerne un récepteur d'antigène chimérique à double ciblage BCMA et CD19 et ses utilisations. La présente invention concerne une séquence polynucléotidique sélectionnée dans le groupe constitué de : (1) une séquence polynucléotidique comprenant, dans l'ordre séquentiel, des séquences codant pour des anticorps de chaîne unique anti-BCMA et anti-CD19, une séquence codant pour la région charnière d'IgG4 humaine, une séquence codant pour la région transmembranaire de CD28 humaine, une séquence codant pour le domaine intracellulaire de 41BB humaine, une séquence codant pour le domaine intracellulaire de CD3ζ humaine et éventuellement une séquence codant pour un fragment de l'EGFR comprenant le domaine extracellulaire III et le domaine extracellulaire IV; et (2) une séquence complémentaire de la séquence polynucléotidique de (1). L'invention concerne en outre des protéines de fusion et des vecteurs correspondants comprenant la séquence codante, et les utilisations des protéines de fusion et des vecteurs associées.
PCT/CN2018/107520 2018-09-26 2018-09-26 Récepteur d'antigène chimérique à double ciblage bcma et cd19 et utilisations associées Ceased WO2020061796A1 (fr)

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WO2024170001A1 (fr) * 2023-04-07 2024-08-22 科弈(浙江)药业科技有限公司 Lymphocytes car-t bispécifiques ciblant le bcma et le cd19
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US12311022B2 (en) 2023-03-31 2025-05-27 AbelZeta Inc. Bispecific chimeric antigen receptors targeting CD20 and BCMA
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WO2022007650A1 (fr) * 2020-07-06 2022-01-13 四川科伦博泰生物医药股份有限公司 Récepteur antigénique chimérique car ou construction de car ciblant bcma et cd19 et utilisation associée
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US12311022B2 (en) 2023-03-31 2025-05-27 AbelZeta Inc. Bispecific chimeric antigen receptors targeting CD20 and BCMA
US12458667B2 (en) 2023-03-31 2025-11-04 AbelZeta Inc. Bispecific chimeric antigen receptors targeting CD20 and BCMA
WO2024170001A1 (fr) * 2023-04-07 2024-08-22 科弈(浙江)药业科技有限公司 Lymphocytes car-t bispécifiques ciblant le bcma et le cd19
WO2025031469A1 (fr) * 2023-08-10 2025-02-13 Gracell Biotechnologies (Shanghai) Co., Ltd. Utilisation de cellules car-t à double cible dans le traitement de maladies auto-immunes à lymphocytes b

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