WO2019129174A1 - Cellule car-t ciblant le cd19 et exprimant un anticorps cd40 à un niveau élevé de stabilité, et son utilisation - Google Patents

Cellule car-t ciblant le cd19 et exprimant un anticorps cd40 à un niveau élevé de stabilité, et son utilisation Download PDF

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WO2019129174A1
WO2019129174A1 PCT/CN2018/124680 CN2018124680W WO2019129174A1 WO 2019129174 A1 WO2019129174 A1 WO 2019129174A1 CN 2018124680 W CN2018124680 W CN 2018124680W WO 2019129174 A1 WO2019129174 A1 WO 2019129174A1
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amino acid
antibody
sequence
cell
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钱其军
金华君
唐熙
何周
刘祥箴
李林芳
崔连振
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上海细胞治疗研究院
上海细胞治疗集团有限公司
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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
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • 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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    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention pertains to genetic engineering and immunology, and relates to CAR-T cells that target CD19 and stably express CD40 antibodies at high levels and uses thereof.
  • Tumor immunotherapy is one of the most promising research directions in the field of cancer treatment. "Science” magazine ranked tumor immunotherapy as the top ten scientific breakthroughs in 2013. There are many types of tumor immunotherapy. To date, the most popular immunotherapy with the best efficacy and the most potential to overcome tumor TB is chimeric antigen receptor T cells (CAR-T). Novartis's Kymriah and Kite's Yescarta two CAR-T products have been approved for marketing.
  • CAR-T chimeric antigen receptor T cells
  • the chimeric antigen receptor is a synthetic receptor that typically comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signal transduction region.
  • CAR antibody single-chain variable region
  • ITAM immunoimmunoreceptor tyrosine-based activation motifs
  • TAA tumor associated antigen
  • the gene is recombined in vitro to generate a recombinant plasmid.
  • This plasmid is then transferred into T cells by gene transduction, such that the genetically engineered T cells are referred to as CAR-T cells. After extensive expansion in vitro, CAR-T cells are returned to the patient and can exhibit potent anticancer effects in a non-MHC-restricted mode.
  • HL Hodgkin's lymphoma
  • NHL non-Hodgkin's lymphoma
  • B-cell lymphoma can be seen in both the Hodgkin's lymphoma and the non-Hodgkin's lymphoma.
  • lymphoma treatments for lymphoma include cytotoxic drugs such as glucocorticoids and alkylating agents, and targeted drugs based on specific molecular targets (such as rituximab, etc.), wherein combination chemotherapy based on targeted drugs significantly improves partial lymphoma.
  • cytotoxic drugs such as glucocorticoids and alkylating agents
  • targeted drugs based on specific molecular targets (such as rituximab, etc.)
  • combination chemotherapy based on targeted drugs significantly improves partial lymphoma.
  • Some new treatments (such as cellular immunotherapy) have relieved and prolonged survival in patients with partially relapsed or refractory lymphoma.
  • CAR-Ts There are many types of CAR-Ts currently being developed for hematological malignancies, including the use of anti-CD19, anti-CD20, anti-Kappa light chain, anti-CD22, anti-CD23, anti-CD30, anti-CD70 and other antibodies to construct CAR-modified T cells. Anti-tumor studies were conducted in which anti-CD19 and anti-CD20 monoclonal antibodies were the most popular.
  • CD19CAR-T is widely used for malignancy such as acute B lymphocytic leukemia (B-ALL), chronic B lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL), NHL, and multiple myeloma (MM). Clinical trial of B cell lymphoma.
  • B-ALL acute B lymphocytic leukemia
  • B-CLL chronic B lymphocytic leukemia
  • MCL mantle cell lymphoma
  • NHL multiple myeloma
  • CD40 antigen is a cell surface molecule belonging to the TNFR superfamily. It is a type I transmembrane glycoprotein with a molecular weight of 48KD and is widely expressed in T cells, antigen presenting cells, hematopoietic cells, granulocytes and the like.
  • CD40L is a type II transmembrane glycoprotein belonging to the TNF superfamily and mainly expressed in CD4+ helper T cells (Th cells).
  • Th cells CD40-CD40L is a pair of extremely important costimulatory molecules in the immune response with a wide range of biological effects.
  • the CD40-CD40L interaction transmits signals, which up-regulates IL-12 levels, activates DC cells, enhances the ability of APC to present antigens, and CD40-CD40L also promotes the secretion of a large number of cytokines by T cells, such as GM-CSF, IL. -4, TNF-a, IFN- ⁇ , thereby enhancing the CTL effect of CD8+ T cells and enhancing the killing effect on tumors.
  • T cells such as GM-CSF, IL. -4, TNF-a, IFN- ⁇
  • APX005M NCT024821678 developed by Apexigen, a US biopharmaceutical research and development company, has good inhibitory effects on non-small cell carcinoma, melanoma, urothelial carcinoma, high frequency microsatellite instability (MSI-H), head and neck cancer.
  • the present invention provides a T cell that self-expresses a CD40 activating antibody and targets CD19.
  • the expression cassette of the CD40 activating antibody and the expression cassette of the chimeric antigen receptor recognizing CD19 are integrated into the genome of the T cell.
  • the amino acid sequence of the CD40 activating antibody is set forth in amino acid residues 21 to 497 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the coding sequence of the CD40 activating antibody is set forth in the base sequence of positions 61-1491 of SEQ ID NO: 4, or as set forth in SEQ ID NO: 4.
  • the chimeric antigen receptor recognizing CD19 in sequence, comprises an optional signal peptide, an anti-CD19 single chain antibody, a hinge region, a transmembrane region, intracellular costimulation from N-terminus to C-terminus. Signal domain and intracellular signal domain.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide; more preferably a CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide Shown as amino acid residues 1 to 21 of SEQ ID NO: 1.
  • the amino acid sequence of the scFv is as shown in amino acid residues 22-263 of SEQ ID NO: 1.
  • the hinge region is selected from the group consisting of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge of CD4.
  • the transmembrane region is a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region.
  • a CD8 transmembrane region preferably having an amino acid sequence as shown in amino acid residues 309-332 of SEQ ID NO: 1.
  • the intracellular costimulatory signal domain comprises an intracellular domain of a costimulatory signaling molecule, including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase
  • a costimulatory signaling molecule including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase
  • the intracellular signal domain is a CD3 sputum intracellular signal domain or an Fc ⁇ RI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ. ID NO: 1 shows the amino acid residues at positions 375-486.
  • the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 22 to 486 of SEQ ID NO: 1, or as shown in SEQ ID NO: 1; preferably, The coding sequence of the chimeric antigen receptor is shown as bases 64-1 to 458 of SEQ ID NO: 3, or as shown in SEQ ID NO: 3.
  • the invention also provides a composition
  • a composition comprising: a vector comprising an expression cassette of a chimeric antigen receptor of the invention, the vector for integrating the expression cassette into the genome of a host cell; A vector comprising an expression cassette for a CD40 activating antibody, the vector being used to integrate the expression cassette into the genome of a host cell.
  • the amino acid sequence of the CD40 activating antibody is set forth in amino acid residues 21 to 497 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the coding sequence of the CD40 activating antibody is set forth in the base sequence of positions 61-1491 of SEQ ID NO: 4, or as set forth in SEQ ID NO: 4.
  • the invention also provides a kit, the kit comprising:
  • a vector comprising an expression cassette of a chimeric antigen receptor of the present invention, which vector is used to integrate the expression cassette into the genome of a host cell;
  • a vector comprising an expression cassette of a CD40 activating antibody, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the amino acid sequence of the CD40 activating antibody is set forth in amino acid residues 21 to 497 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the coding sequence of the CD40 activating antibody is set forth in the base sequence of positions 61-1491 of SEQ ID NO: 4, or as set forth in SEQ ID NO: 4.
  • the present invention also provides a pharmaceutical composition comprising the T cell of the present invention.
  • the invention also provides the use of a T cell as described herein in the manufacture of a medicament for the treatment or prevention of a malignancy.
  • the malignant tumor is a malignant B cell lymphoma, including acute B lymphocytic leukemia (B-ALL), chronic B lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL), NHL and multiple bone marrow Tumor (MM).
  • B-ALL acute B lymphocytic leukemia
  • B-CLL chronic B lymphocytic leukemia
  • MCL mantle cell lymphoma
  • NHL multiple bone marrow Tumor
  • Figure 1 Schematic diagram of the gene structure of pNB328-CD19CAR, pS328- ⁇ CD40-wt, pS328- ⁇ CD40, pNB328-CD19CAR-2A- ⁇ CD40, pNB328- ⁇ CD40-IRES-CD19CAR.
  • Figure 2A Positive rates of three CAR-T cells, CD19CAR-2A- ⁇ CD40, ⁇ CD40-IRES-CD19CAR, and CD19CAR- ⁇ CD40, constructed by different methods.
  • Figure 2B CD19CAR-2A- ⁇ CD40, ⁇ CD40-IRES-CD19CAR, CD19CAR- ⁇ CD40 three CAR-T cell positive rate antibody secretion levels constructed by different methods.
  • 3A-3B Comparison of CD19CAR- ⁇ CD40T cell positive rate and antibody secretion amount constructed under different conditions of CAR and CD40 antibody plasmid.
  • Figure 4 Comparison of CD19CAR-secretion levels IL-2, IL-4, IL-6, IL-10, TNF-, and IFN-, cytokine secretion changes under CD19 antigen stimulation.
  • Figure 5 Proliferation assay of CD19CAR T cells and CD19 CAR- ⁇ CD40 T cells.
  • Figure 6 Therapeutic effect of CD19CAR T cells, CD19CAR- ⁇ CD40-wt T, CD19CAR- ⁇ CD40T cells on a mouse Raji-luc xenograft model.
  • expression cassette refers to an entire element required for expression of a gene, including a promoter, a gene coding sequence, and a transcription terminator sequence such as (PolyA tailing signal sequence).
  • coding sequence is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, CAR, single chain antibody, hinge region, and transmembrane region).
  • the boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • Fc fragment crystallizable (Fc) of an antibody
  • Fc fragment crystallizable
  • costimulatory molecule refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal.
  • the proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules.
  • the costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell.
  • B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.
  • linker or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide.
  • exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (KD) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • KD Affinity
  • pharmaceutically acceptable excipient refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include, but are not limited to, sodium chloride.
  • the term "effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • subject or “patient” may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog. , monkeys, cattle, horses, etc.
  • CAR chimeric antigen receptor
  • T cells immune cells
  • CAR typically comprises, in turn, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signaling region.
  • a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a membrane antigen that is widely expressed by tumor cells with moderate affinity.
  • the polypeptide that binds to the tumor cell membrane antigen may be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.
  • single-chain antibody refers to an antibody fragment which is obtained by hinge-ligating an amino acid sequence of an antibody light chain variable region (VL region) and a heavy chain variable region (VH region), and having antigen-binding ability.
  • the single chain antibody of interest is from an antibody of interest.
  • Antibodies of interest may be human antibodies, including human murine chimeric antibodies and humanized antibodies.
  • the antibody may be secreted or membrane anchored; preferably a membrane anchored.
  • the inventors unexpectedly discovered during the research of CAR-T that the addition of CD40 antibody significantly improved the proliferation ability and killing ability of CAR-T.
  • the invention is expensive, and the present inventors express CD40 antibody on the existing CD19 CAR-T cells, and when combined with the CD40 antigen, can initiate the costimulatory signal, promote the activation and proliferation of CAR-T cells in vivo, and increase the anti-tumor of cytotoxic T cells. The killing effect, thereby improving the efficacy of specific killing of tumors.
  • the IgG4 Fc fragment of CD40-activating antibody is easily phagocytosed by monocyte/macrophage recognition, and the CD40-activating antibody of the CD40-activated antibody IgG4Fc fragment of the present invention can be modified to meet the T-cell self-expression. It works well and does not cause ADCC reactions.
  • the present invention provides a CD40 activating antibody which up-regulates the levels of TNF- ⁇ , TRAIL and FasL, inhibits the growth of tumor cells, and promotes the proliferation efficiency of CAR-T cells by regulating cell cycle/proliferation. , prolonging the action time in the body, thereby enhancing the CAR-T effect in multiple aspects.
  • the CD40 activating antibody of the present invention contains an anti-CD40 single chain antibody and IgG4Fc.
  • the amino acid sequence of the IgG4 Fc is set forth in amino acid residues 269-497 of SEQ ID NO: 2; preferably, the coding sequence thereof is set at bases 805-1491 of SEQ ID NO: Shown.
  • the antibody light chain variable region (VL region) amino acid sequence of the anti-CD40 single-chain antibody is represented by amino acid residues 21 to 146 of SEQ ID NO: 2; preferably, Its coding sequence is shown in nucleotide sequence 64-438 of SEQ ID NO:4.
  • the heavy chain variable region (VH region) amino acid sequence of the anti-CD40 single-chain antibody is set forth in amino acids at positions 161-168 of SEQ ID NO: 2; preferably, the coding sequence thereof is The nucleotide sequence at positions 481 to 804 of SEQ ID NO: 4 is shown.
  • the amino acid sequence of the anti-CD40 single chain antibody is set forth as amino acid residues 21 to 268 of SEQ ID NO: 2; preferably, the coding sequence thereof is SEQ ID NO: 4, 61-804 The base sequence is shown.
  • the CD40 antibody further comprises a light chain signal peptide.
  • the CD40 antibody comprises, from the N-terminus to the C-terminus, a light chain signal peptide, an anti-CD40 single chain antibody, and an IgG4 Fc in sequence.
  • the amino acid sequence of the light chain signal peptide is represented by amino acid residues 1-20 of SEQ ID NO: 2; preferably, the coding sequence of the indicated light chain signal peptide is SEQ ID NO: 4 The base sequence of the 1-60th base is shown.
  • amino acid sequence of the CD40 activating antibody is set forth in amino acid sequence 21-497 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the invention also encompasses a coding sequence for the CD40 antibody or a complement thereof, the coding sequence comprising at least the coding sequence for an IgG4 Fc described herein or a complement thereof.
  • the coding sequence of the CD40 antibody comprises the sequence set forth in bases 61-1491 of SEQ ID NO:4, preferably comprising the sequence set forth in SEQ ID NO:4.
  • the present invention also encompasses a nucleic acid construct comprising the coding sequence of a CD40 antibody of the present invention or a complement thereof.
  • the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or its complement into a host cell.
  • the invention also provides a host cell comprising a nucleic acid construct as described herein.
  • the invention also provides the use of the CD40 antibody, its coding sequence or complementary sequence, a nucleic acid construct, and a host cell for the preparation or treatment of a malignant tumor, particularly a CD40-associated tumor, including but not limited to Various malignant tumors.
  • the present invention also provides a pluripotent T cell which is modified by the CD19CAR gene and can express a CD40 activating antibody, and the T cell can stably express the CD19CAR gene and the CD40 activating antibody at a high level, and the exogenously expressed CD19CAR gene can accurately target To CD19 antigen, enhance the proliferation of T cells and the secretion of cytokines, the expression of CD40-activating antibodies can help CAR-T cells to break through the inhibition of tumor microenvironment, thereby enhancing the killing of tumor cells by CAR-T cells, and by enhancing The immune response exerts an anti-tumor effect.
  • the exogenous CAR gene and the CD40 activating antibody gene can be integrated into the genome of the T cell via the PB transposase system, thereby stably and continuously expressing in the T cell.
  • the high level of T cells stably expressing the CAR gene and the CD40 activating antibody gene obtained by the present invention can be used for the treatment of a variety of CD19-expressing malignant lymphomas.
  • the CAR of the invention typically contains an optional signal peptide sequence, an scFv that recognizes the CD19 antigen, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.
  • a signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of newly synthesized proteins to the secretory pathway, often referred to as the N-terminal amino acid sequence in the newly synthesized polypeptide chain that directs transmembrane transfer (localization) of the protein. (Sometimes not necessarily at the N-terminus), it is responsible for directing proteins into subcellular organelles with different membrane structures.
  • the signal peptide can be a secreted signal peptide or a membrane-bound signal peptide.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide, or a CD4 signal peptide; more preferably a CD8 signal peptide.
  • the amino acid sequence of the CD8 signal peptide can be as shown in amino acid residues 1-21 of SEQ ID NO: 1; in certain embodiments, the coding sequence is set forth in bases 1-63 of SEQ ID NO: 3.
  • the scFv recognizing the CD19 antigen may be a scFv that recognizes the CD19 antigen commonly used in the art.
  • the amino acid sequence of the scFv is as shown in amino acid residues 22-263 of SEQ ID NO: 1; in certain embodiments, the coding sequence is SEQ ID NO: 3, pp. 64-789 The base is shown.
  • the hinge region refers to the region between the functional regions of the immunoglobulin heavy chain CH1 and CH2, which is rich in proline, does not form an alpha helix, is prone to stretching and is somewhat distorted, and is beneficial to the antigen binding site of the antibody. Complementary binding between epitopes.
  • the hinge region suitable for use herein may be selected from any one or more of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4. .
  • the hinge region is preferably a hinge region that is longer than 50 amino acid residues, more preferably 80 amino acids or longer.
  • a CD8 alpha hinge region or an IgG4 Fc CH2CH3 hinge region is used herein.
  • the amino acid sequence of the exemplary CD8 alpha hinge region is shown in amino acid residues 264 to 308 of SEQ ID NO: 1, and the coding sequence thereof is shown in positions 790-924 of SEQ ID NO: 3.
  • the transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD8 transmembrane region
  • the amino acid sequence thereof is represented by amino acid residues 309-332 of SEQ ID NO: 1; in certain embodiments, the coding sequence is set forth in bases 925-996 of SEQ ID NO: 10.
  • the intracellular co-stimulatory signal domain including the intracellular domain of the costimulatory signaling molecule may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T cell costimulation. Intracellular domain of factor (ICOS) and DNAX activator protein 10 (DAP10).
  • the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD137/4-1BB; preferably, the amino acid sequence of the CD137/4-1BB is SEQ ID NO: 1 Shown at amino acid residues 333-374; in certain embodiments, the coding sequence is set forth in bases 997-1122 of SEQ ID NO: 3.
  • the intracellular signal domain is preferably an immunoreceptor tyrosine activation motif, which may be a CD3 sputum intracellular signal domain or an Fc ⁇ RI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain As described in amino acid residues 375-486 of SEQ ID NO: 1; in certain embodiments, the coding sequence is set forth in bases 1123-1458 of SEQ ID NO: 3.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, an scFv, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB and a CD3 intracellular signal domain; preferably, the inlay
  • the amino acid sequence of the antigen receptor is shown as amino acid residues 22 to 486 of SEQ ID NO: 1.
  • the chimeric antigen receptor further comprises a signal peptide, preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 1-21 of SEQ ID NO: 1.
  • the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.
  • linker sequence can be a linker sequence suitable for use in antibodies well known in the art, such as linker sequences comprising G and S.
  • the linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, and 10 to 20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8.
  • the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.
  • a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest.
  • promote expression of a recombinant protein obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein.
  • Other suitable regions include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like.
  • the amino or carboxy terminus of a CAR herein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in this article.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • polynucleotide sequences encoding the chimeric antigen receptor.
  • the polynucleotide sequence herein may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the polynucleotide sequences described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequences disclosed herein, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.
  • the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the polynucleotide sequence encoding a fusion protein described herein is set forth in SEQ ID NO:3.
  • nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor described herein or a polynucleotide sequence encoding the CD40 activating antibody, and one or operably linked to the sequences Multiple regulatory sequences.
  • the nucleic acid constructs of the invention are expression cassettes.
  • the control sequence can be a suitable promoter sequence.
  • the promoter sequence is typically operably linked to the coding sequence of the protein to be expressed.
  • the promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and may be derived from an extracellular or heterologous source encoding the host cell. Or the gene of the intracellular polypeptide is obtained.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used herein.
  • the nucleic acid construct is a vector.
  • the coding sequences of the CARs herein or the coding sequences for CD40 activating antibodies can be cloned into many types of vectors, for example, but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • the vector can be an expression vector.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism.
  • the invention employs a retroviral vector containing a replication initiation site, a 3'LTR, a 5' LTR, a coding sequence for a CAR described herein, or a CD40 activating antibody A coding sequence, and optionally a selectable marker.
  • Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme Promoter and creatine kinase promoter. Further, an inducible promoter can also be considered.
  • SV40 prion 40
  • MMTV mouse breast cancer virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • EB virus immediate early promoter EB virus immediate early promoter
  • Russ sarcoma virus promoter avian leukemia virus promoter
  • an inducible promoter can also be considered.
  • an inducible promoter provides a molecular switch that is capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter upon expression of the term, and shutting down expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • various promoter sequences disclosed in CN201510021408.1 can be used, including but not limited to the CCEF promoter containing the mCMV enhancer, the hCMV enhancer, and the EF1 ⁇ promoter shown in SEQ ID NO: 5 of the application.
  • the selectable marker includes either or both of the selectable marker genes or reporter genes to facilitate identification and selection of the expressed cells from the population of cells infected by the viral vector.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neo and the like.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.
  • the coding sequence of the chimeric antigen receptor described herein and the coding sequence of a CD40 activating antibody can be separately cloned into a vector for integration of the nucleic acid sequence of interest into the genome of the host cell (also referred to as In order to integrate vectors, in particular transposon vectors.
  • the transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty.
  • Such transposon vectors contain the 5' inverted terminal repeat (5' LTR) of the corresponding transposon and the 3' inverted terminal repeat (3' LTR) of the corresponding transposon.
  • the transposase can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system.
  • the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to the transposition system, which can be readily determined by those skilled in the art.
  • LTR is the expression cassette for the CAR or antibody of the invention, including the corresponding promoter sequence, the coding sequence for the CAR or antibody, and the transcription terminator sequence (e.g., the polyA tailing signal sequence).
  • the transposase is a transposase from a piggybac transposition system.
  • the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively.
  • the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference.
  • the transposon 3' inverted terminal repeat is as shown in CN 201510638974.7 SEQ ID NO: 4.
  • the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence.
  • the coding sequence for the piggybac transposase is as shown in CN 201510638974.7 SEQ ID NO: 5.
  • the promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling expression of the transposase coding sequence.
  • the expression of the transposase coding sequence is controlled using a CMV promoter.
  • the sequence of the CMV promoter can be as shown in CN 201510638974.7 SEQ ID NO: 6.
  • the vector of the present invention comprising a coding sequence for a chimeric antigen receptor is the pNB328 vector disclosed in CN 201510638974.7.
  • the coding sequence of the chimeric antigen receptor of the present invention can be prepared by a method conventional in the art and cloned into a suitable vector.
  • the vector for integrating a gene of interest into the genome of a host cell does not contain a transposase coding sequence.
  • such vectors can be obtained by removing the transposase coding sequence based on the pNB328 vector.
  • such vectors are used to integrate the coding sequence for a CD40 activating antibody and a signal peptide coding sequence (such as the coding sequence for a light chain signal peptide) into the genome of a host cell.
  • a T cell modified by a CD19 CAR gene and capable of expressing a CD40 activating antibody as described herein can be transduced into a transposase comprising a chimeric antigen receptor coding sequence for integration into the T cell genome.
  • a vector encoding a sequence, and a vector comprising no transposase coding sequence for integration into the coding sequence of a CD40 activating antibody described herein in the T cell genome can be transduced into a transposase comprising a chimeric antigen receptor coding sequence for integration into the T cell genome.
  • the T cell is transformed into a vector containing a chimeric antigen receptor coding sequence constructed using the pNB328 vector as a backbone vector and constructed using a pS328 vector (with no transposase coding sequence compared to pNB328) as a backbone vector.
  • a vector containing a CD40 activating antibody coding sequence is set forth in the base sequence of SEQ ID NO: 3, positions 64-1458, or as set forth in SEQ ID NO: 3; the CD40 activating antibody The coding sequence is shown as the base sequence of positions 61-1491 of SEQ ID NO:4.
  • the signal peptide of the CD40 activating antibody is a light chain signal peptide.
  • the amino acid sequence of an exemplary light chain signal peptide can be as shown in amino acid residues 1-20 of SEQ ID NO:2.
  • the vector comprising a transposase coding sequence that incorporates a chimeric antigen receptor coding sequence in a T cell genome comprises a 5' LTR, a promoter, a CD8 signal peptide coding sequence , coding sequence of scFv recognizing CD19 antigen, coding sequence of CD8 hinge region, coding sequence of CD8 transmembrane region, coding sequence of 4-1BB, coding sequence of CD3 intracellular signal domain, polyA tailing signal sequence, 3'LTR And a transposase coding sequence and a promoter sequence thereof; said vector comprising a transposase coding sequence encoding a coding sequence for a CD40 activating antibody as described herein in a T cell genome at 5'LTR and 3'LTR
  • the coding sequence of the promoter, the light chain signal peptide, the coding sequence of the CD40 activating antibody, and the polyA tailing signal sequence are sequentially contained between them.
  • the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the CD40 activating antibody coding sequence is from 1 to 7:1 to 7, preferably from 1 to 3:1 to 3, preferably 1 : 1 to 3, more preferably 1:1 to 2, still more preferably 1:1.
  • Methods of transfection are routine methods in the art including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation.
  • the vector is transfected into a cell of interest using electroporation.
  • the cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, ⁇ T cells, and cytokines.
  • T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL).
  • the invention also provides a composition comprising a vector comprising an expression cassette for a chimeric antigen receptor described herein and a vector comprising an expression cassette for a CD40 activating antibody described herein.
  • Suitable agents may also be included in the compositions including, but not limited to, transfection reagents.
  • the invention also provides a kit comprising a vector comprising an expression cassette for a chimeric antigen receptor described herein and a vector comprising an expression cassette for a CD40 activating antibody described herein, or a combination as described herein Things.
  • a reagent or instrument for transferring the vector into a cell can also be provided in the kit.
  • the expression cassette contains at least a suitable promoter and transcription terminator sequence (e.g., a polyA tail signal sequence) in addition to the coding sequence comprising a chimeric antigen receptor or a CD40 activating antibody.
  • a suitable promoter and transcription terminator sequence e.g., a polyA tail signal sequence
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a T cell as described herein. Suitable pharmaceutically acceptable carriers or excipients may be included in the pharmaceutical compositions.
  • the pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells. The therapeutically or prophylactically effective amount of the T cell can be determined according to factors such as the condition of the patient.
  • the invention also provides the use of a T cell or a pharmaceutical composition thereof as described herein for the manufacture of a medicament for the treatment or prevention of a malignancy.
  • the present invention also provides a method of treating or preventing a malignant tumor, which comprises administering to a subject in need thereof a therapeutically or prophylactically effective amount of a T cell of the present invention.
  • Malignant tumors are malignant B-cell lymphomas, including acute B-lymphocytic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL), NHL, and multiple myeloma (MM).
  • Example 1 Construction of recombinant plasmids pNB328-CD19CAR, pS328- ⁇ CD40, pS328- ⁇ CD40-wt, pNB328- ⁇ CD40-IRES-CD19CAR, pNB328-CD19CAR-2A- ⁇ CD40 and acquisition of chimeric antigen receptor-modified T cells
  • CD19CAR (nucleotide sequence as shown in SEQ ID NO: 3, amino acid sequence as shown in SEQ ID NO: 1), anti-CD40 ( ⁇ CD40) (sequence as shown in SEQ ID NO: 4) , amino acid sequence as shown in SEQ ID NO: 2), anti-CD40-wt ( ⁇ CD40-wt) (nucleotide sequence shown in SEQ ID NO: 9, amino acid sequence shown in SEQ ID NO: 8), CD19CAR -2A- ⁇ CD40 (the nucleotide sequence of 2A is shown in SEQ ID NO: 5, the amino acid sequence is shown in SEQ ID NO: 6) and the ⁇ CD40-IRES-CD19CAR gene (the nucleotide sequence of IRES is SEQ ID NO: 7)), its structural pattern is shown in Figure 1, which was inserted between pNB328, pS328 vector EcoRI and SalI restriction sites, and the recombinant
  • pNB328 The structure and sequence of pNB328 are described in CN 201510638974.7, the entire disclosure of which is hereby incorporated by reference in its entirety in its entirety in the in the in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in in The promoter sequence and the polyA tailing signal sequence are not shown in the respective structural pattern diagrams, which are located between the 5'LTR and the signal peptide sequence and before the 3' LTR, respectively.
  • PBMCs Peripheral blood mononuclear cells
  • the PBMCs were cultured for 2-4 h, and the unattached suspension cells were the initial T cells.
  • the suspension cells were collected into 15 ml centrifuge tubes, centrifuged at 1200 rmp for 3 min, the supernatant was discarded, physiological saline was added, and centrifuged at 1200 rmp for 3 min to abandon the physiology.
  • CD19CAR T cells were constructed using pNB328-CD19CAR (6 ug) constructed in Example 1; Mock T cells were constructed using pNB328 blank plasmid (6 ug); pNB328-CD19CAR (4 ug) constructed using Example 1 was used.
  • CD19CAR- ⁇ CD40-wt T cells were constructed with pS328- ⁇ CD40-wt (4 ug).
  • Example 3 CAR T cell positive rate and antibody secretion targeting CD19 from CD40 antibody-expressing
  • the CD19CAR- ⁇ CD40T cells, CD19CAR-2A- ⁇ CD40T cells and ⁇ CD40-IRES-CD19CAR T cells prepared in Example 2 were collected and divided into two portions, each of which was 1 ⁇ 10 6 cells, washed twice with physiological saline, and 100 ul physiologically.
  • the cells were resuspended in saline, one portion was added with 1 ug of CD19-biotin, and the other was added without incubation at 30 °C for 30 minutes.
  • the saline was washed twice, and the cells were resuspended again with 100 ul of physiological saline, and 1 ul of streptomycin-PE antibody was added thereto, and incubated at 4 ° C for 30 minutes.
  • the saline was washed twice, and the upper machine was tested, and only the secondary antibody was added as a control, and the results are shown in Fig. 2A.
  • the OD value was measured at 450 nm on a microplate reader, a standard curve was drawn, and the CD40 antibody concentration was calculated.
  • Example 4 Different plasmid ratio test of pNB328-CD19CAR and pS328- ⁇ CD40
  • the amount of pNB328-CD19CAR and pS328- ⁇ CD40 plasmids constructed in Example 1 were set to 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, respectively.
  • the ratio of CAR T cells was constructed in the same manner as in Example 2.
  • the positive rate of CAR T cells and the amount of antibody secretion were measured under the seven ratios (the same method as in Example 3), and the results are shown in Figs. 3A and 3B.
  • Example 5 Comparison of cytokine release by CD19CAR and CD19CAR- ⁇ CD40T cells under specific stimulation of CD19 antigen
  • the 96-well plate was coated with 2 ug/ml of CD19 antigen, coated overnight at 4° C., washed 3 times with PBS, and 1 ⁇ 10 5 of CD19CAR and CD19CAR- ⁇ CD40T cells constructed in Example 2 and control Mock T cells were added and cultured. Cell supernatants were collected after 24 h. The cytokine secretion of these three T cells stimulated by CD19 antigen was detected by BD CBA Human Th1/Th2Cytokine Kit II. The specific steps are as follows:
  • Th1/Th2 cytokine standard double dilution 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml) , 20pg/ml, 0pg/ml) and 50ul of the sample to be tested (diluted by dilution 2 times);
  • CD19CAR T cells, CD19 CAR- ⁇ CD40 T cells and Mock T cells which were cultured on the 8th day were cultured in the same manner, and 3 ⁇ 10 5 cells were taken and placed in a 12-well plate, and the culture volume was 1 ml.
  • Example 7 In vivo functional assay of CD19CAR T cells and CD19 CAR- ⁇ CD40 T cells.
  • mice of 4-6 weeks old Twelve NSG completely immunodeficient mice of 4-6 weeks old were used, with an average weight of 22-27 g, provided by Beijing Biotech Biotechnology Co., Ltd., and raised in SPF animal laboratory.

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Abstract

L'invention concerne une cellule CAR-T qui cible le CD19 et qui exprime un anticorps CD40 à un niveau élevé de stabilité, et son utilisation. La cellule CAR-T contient une séquence de codage pour un récepteur d'antigène chimère qui reconnaît le CD19 et une séquence de codage pour un anticorps d'activation de CD40 ; et/ou un récepteur d'antigène chimère qui exprime et reconnaît le CD19 et un anticorps d'activation du CD40. Après que l'anticorps et l'antigène CD40 sont combinés, un signal co-stimulateur peut être lancé, favorisant l'activation et la prolifération de cellules CAR-T in vivo, et augmentant l'effet anti-tumoral des cellules T cytotoxiques, ce qui permet d'améliorer l'efficacité de la destruction spécifique des tumeurs.
PCT/CN2018/124680 2017-12-28 2018-12-28 Cellule car-t ciblant le cd19 et exprimant un anticorps cd40 à un niveau élevé de stabilité, et son utilisation WO2019129174A1 (fr)

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KEVIN J CURRANT: "Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD 40L Expression", MOLECULAR THERAPY, 10 February 2015 (2015-02-10), XP002789068, ISSN: 1525-0016 *

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CN113307871A (zh) * 2020-02-27 2021-08-27 福州拓新天成生物科技有限公司 新型抗cd19抗体和cd19-car-t细胞的制备及其应用
CN113307871B (zh) * 2020-02-27 2023-05-02 福州拓新天成生物科技有限公司 新型抗cd19抗体和cd19-car-t细胞的制备及其应用
CN115667317A (zh) * 2020-06-30 2023-01-31 南京驯鹿医疗技术有限公司 全人源的靶向cd19和cd22双特异抗原嵌合受体及其应用
US11667723B2 (en) 2020-08-17 2023-06-06 Utc Therapeutics (Shanghai) Co., Ltd. Lymphocytes-antigen presenting cells co-stimulators and uses thereof

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