WO2021031113A1 - Anticorps anti-bcma et utilisation associé dans le domaine des car-t - Google Patents

Anticorps anti-bcma et utilisation associé dans le domaine des car-t Download PDF

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WO2021031113A1
WO2021031113A1 PCT/CN2019/101542 CN2019101542W WO2021031113A1 WO 2021031113 A1 WO2021031113 A1 WO 2021031113A1 CN 2019101542 W CN2019101542 W CN 2019101542W WO 2021031113 A1 WO2021031113 A1 WO 2021031113A1
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region
antigen
antibody
bcma
seq
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PCT/CN2019/101542
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Chinese (zh)
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侯勇
赵正琦
李波
王飞
徐曲苗
葛玉萍
朱琳楠
李毅坚
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武汉华大吉诺因生物科技有限公司
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Priority to CN201980099439.5A priority Critical patent/CN114222758A/zh
Priority to PCT/CN2019/101542 priority patent/WO2021031113A1/fr
Priority to TW109105464A priority patent/TW202115113A/zh
Publication of WO2021031113A1 publication Critical patent/WO2021031113A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors

Definitions

  • the present invention relates to the field of biomedicine, in particular to an anti-BCMA antibody and its application in the CAR-T field.
  • Chimeric antigen receptor T cell refers to a T cell into which chimeric antigen receptor (CAR, Chimeric antigen receptor) genes are transferred through gene modification technology.
  • CAR-T immunotherapy belongs to adoptive immune cell therapy. The peripheral blood lymphocytes of the patient are extracted and modified in vitro to express the CAR gene, and then amplified in vitro and then transferred back to the patient.
  • the modified CAR-T cells can be MHC Recognize and activate specific antigens on the surface of tumor cells in a non-restricted way, and a large number of them are amplified in the body.
  • the tumor cells are directly killed by releasing perforin and granzyme B, and at the same time by releasing cytokines IFN- ⁇ , IL-2, etc. recruit human endogenous immune cells to kill tumor cells, so as to achieve the purpose of treating tumors.
  • CAR chimeric antigen receptor
  • CAR is a fusion protein, including extracellular target binding domain, extracellular hinge domain, transmembrane domain and intracellular domain.
  • the extracellular target binding region is usually derived from the single-chain variable fragment (scFv, Single-chain variable fragment) of an antibody that recognizes tumor-associated antigen (TAA, Tumor-associated antigen), and its binding affinity to tumor antigen is much higher than that of MHC polypeptide
  • TAA tumor-associated antigen
  • TCR T cell receptor
  • the sequence of the extracellular hinge region is derived from the constant region of IgG or CD8, etc.
  • the transmembrane region generally comes from the transmembrane region of receptors such as CD8, CD28, or CD3, which has a certain impact on the stability of the CAR structure.
  • Car-T cells for tumor immunotherapy needs further improvement.
  • an object of the present invention is to provide an anti-BCMA antibody and its application in the CAR-T field.
  • the present invention provides an anti-BCMA antibody or antigen-binding fragment, isolated polynucleotides, expression vectors, recombinant cells, kits containing anti-BCMA antibodies or antigen-binding fragments, pharmaceutical compositions, and an anti-BCMA Chimeric antigen receptors and Car-T cells, etc., also provide a method for treating tumors in the subject.
  • BCMA B-cell maturation antigen
  • TNF tumor necrosis factor
  • BAFF B-cell activating factor
  • APRIL proliferation inducing ligand
  • Nanjing Legend reported the clinical data of a CAR-T therapy targeted to BCMA. According to data, in a clinical trial involving 35 patients with relapsed or drug-resistant multiple myeloma, the objective remission rate of this therapy reached 100%. Among the 19 patients who received treatment at the earliest, Nanjing Legend has been observed and followed up for more than 4-14 months. Among them, 14 patients continued to meet the strict diagnostic criteria for complete response, and 5 patients experienced partial remission.
  • an anti-BCMA antibody or antigen-binding fragment thereof as well as isolated nucleotides, expression vectors, recombinant cells, etc. are provided.
  • the anti-BCMA antibody or its antigen-binding fragment provided by the present invention can specifically bind to the BCMA antigen. It can be applied to the targeted therapy of tumors, or made into a kit for specific detection of BCMA antigen, etc., which has important value.
  • the present invention provides an anti-BCMA antibody or antigen-binding fragment, comprising at least one of the following: (1) a heavy chain variable region having the amino acid sequence shown in GYTFTSYV, IIPYNDDT and ARWNYDGYFDV, and The light chain variable region of the amino acid sequence shown in QSLVHSNGNTY, YKVS and SQITHVPYT; compared with (1), an amino acid sequence with at least one conservative amino acid substitution.
  • the aforementioned anti-BCMA antibody or antigen-binding fragment may further include the following technical features:
  • the anti-BCMA antibody or antigen-binding fragment includes at least one of the following: (a) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2.
  • the variable region of the light chain showing the amino acid sequence; compared with (a), the amino acid sequence has at least one conservative amino acid substitution.
  • the present invention provides an isolated polynucleotide encoding the antibody or antigen-binding fragment of the first aspect of the present invention.
  • the isolated polynucleotide described above may further include the following technical features:
  • the polynucleotide is a nucleotide sequence having at least one of the following: the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4
  • the original sequence is preferably a sequence with more than 98% homology, and more preferably a sequence with more than 99% homology; compared with the nucleotide sequence of the light chain variable region shown in SEQ ID NO: 4 , A sequence with 90% or more homology, optionally a sequence with 95% or more homology, preferably a sequence with 98% or more homology, and more preferably a sequence with 99% or more homology.
  • the present invention provides an expression vector comprising the polynucleotide according to the second aspect of the present invention.
  • the above-mentioned expression vector may further include the following technical features:
  • the above-mentioned expression vector may further include a control element operably linked to the polynucleotide for controlling the expression of the polynucleotide in the host cell.
  • control element includes at least one of the following: a promoter, an enhancer, and a terminator.
  • the host cell is a mammalian cell.
  • the present invention provides a recombinant cell comprising the expression vector according to the third aspect of the present invention.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment according to the first aspect of the present invention.
  • the kit is used for the diagnostic detection of BCMA antigen.
  • the antibodies or antigen-binding fragments in the kit can be used for the diagnosis and detection of BCMA antigen, for example, it can be applied to immunoblotting, immunoprecipitation or ELISA detection and so on.
  • the present invention provides a pharmaceutical composition, comprising: the antibody or antigen-binding fragment of the first aspect of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides the use of an antibody or antigen-binding fragment in the preparation of a medicament for the treatment of tumors, and the antibody or antigen-binding fragment is the one described in the first aspect of the present invention Antibody or antigen-binding fragment.
  • the tumor is multiple myeloma.
  • the present invention provides an anti-BCMA chimeric antigen receptor, comprising: an extracellular target binding region, an extracellular hinge region, a transmembrane region and an intracellular region, the extracellular target binding Region, the extracellular hinge region, the transmembrane region, and the intracellular region are sequentially connected; the extracellular target binding region includes an antigen or antigen-binding fragment, and the antibody or antigen-binding fragment is a single chain, The antibody or antigen-binding fragment is the antibody or antigen-binding fragment described in the first aspect of the present invention.
  • the above-mentioned anti-BCMA chimeric antigen receptor may further include the following technical features:
  • the antibody or antigen-binding fragment is a single-chain fragment shown in SEQ ID NO: 5.
  • the extracellular hinge region further includes a human CD8 extracellular hinge region, and the amino acid sequence of the human CD8 extracellular hinge region is preferably as shown in SEQ ID NO: 6.
  • the transmembrane region is a human CD28 transmembrane region, and the amino acid sequence of the human CD28 transmembrane region is preferably as shown in SEQ ID NO:7.
  • the intracellular region includes a human CD28 intracellular region and a human CD3 ⁇ intracellular region.
  • the amino acid sequence of the human CD28 intracellular region is preferably as shown in SEQ ID NO: 8, the human CD3 ⁇
  • the amino acid sequence of the intracellular region is preferably as shown in SEQ ID NO: 9.
  • the nucleic acid sequence encoding the antibody or antigen-binding fragment is shown in SEQ ID NO: 10; the nucleic acid sequence encoding the human extracellular hinge region is shown in SEQ ID NO: 11; encoding The nucleic acid sequence of the human CD28 transmembrane region and the human CD28 intracellular region is shown in SEQ ID NO: 12; the nucleotide sequence encoding the human CD3 ⁇ intracellular region is shown in SEQ ID NO: 13.
  • the present invention provides a Car-T cell that expresses the anti-BCMA chimeric antigen receptor of the eighth aspect of the present invention.
  • the antibody or antigen-binding fragment provided by the present invention is used as a single-chain antibody to perform gene recombination in vitro with the extracellular hinge region, transmembrane region and intracellular region, such as immunoreceptor tyrosine activation motif protein (which can be CD3 ⁇ ), Recombinant plasmids are generated, and then transfected into the patient’s T cells by transfection technology in vitro to allow the patient’s T cells to express tumor antigen receptors. After transfection, the purified and large-scale amplified T cells are called chimeric antigen receptors. Somatic T cells (Car-T cells). Infusing these Car-T cells back into the patient's body can achieve the therapeutic effect of identifying and killing cancer cells, just like installing a GPS navigation system on the patient's T cells to accurately identify and kill cancer cells.
  • Car-T cells Somatic T
  • the present invention provides a method for treating tumors in a subject, comprising administering to the subject an effective amount of Car-T cells, the Car-T cells being the ninth aspect of the present invention Car-T cells.
  • the tumor is multiple myeloma.
  • Multiple myeloma is a malignant B-cell lymphoma caused by malignant clonal proliferation of bone marrow plasma cells, accompanied by monoclonal immunoglobulin or light chain (M protein) overproduction.
  • M protein monoclonal immunoglobulin or light chain
  • the common clinical manifestations are Anemia, bone pain, renal insufficiency, infection, bleeding, neurological symptoms, hypercalcemia, amyloidosis, etc.
  • Traditional treatment methods such as chemotherapy, radiotherapy, and corticosteroids can alleviate the condition, but it will almost eventually relapse, and the survival rate within five years is only 20-30%.
  • Fig. 1 is a graph showing the binding and dissociation curves of BCMA antigen and the biosensor loaded with mouse anti-human BCMA antibody at 25, 50, and 100 nM concentration after homogenization with a negative control sample provided by an embodiment of the present invention.
  • Fig. 2 is a flow cytometry result diagram of an anti-BCMA antibody provided according to an embodiment of the present invention after incubation with the MM.1S cell line.
  • Fig. 3 is a flow cytometry result diagram after incubating the anti-BCMA antibody and the RPMI8226 cell line according to an embodiment of the present invention.
  • Fig. 4 is a flow cytometry result diagram of anti-BCMA antibodies after incubation with negative control K562 cells according to an embodiment of the present invention.
  • Figure 5 is a flow cytometer according to an embodiment of the present invention to detect the expression of CAR protein on the surface of T cells after infection.
  • Fig. 6 is a graph showing the secretion results of IFN-gamma after different cells are co-cultured with three MM target cells and negative control cells according to an embodiment of the present invention.
  • Fig. 7 is a graph showing the secretion of IL-2 after different cells are co-cultured with three MM target cells and negative control cells according to an embodiment of the present invention.
  • Fig. 8 is a graph showing the killing effect of different cells on CHO-BCMA cells according to an embodiment of the present invention.
  • Fig. 9 is a graph showing the killing effect of different cells on negative CHO cells according to an embodiment of the present invention.
  • Fig. 10 is a graph showing changes in tumor size in mice after injection of different cells according to an embodiment of the present invention.
  • the term "antibody” is an immunoglobulin molecule capable of binding to a specific antigen. It includes two light chains with a lighter molecular weight and two heavy chains with a heavier molecular weight.
  • the heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule.
  • the amino terminal (N-terminal) amino acid sequence of the peptide chain has great changes, called the variable region (V region), and the carboxyl terminal (C-terminal) is relatively stable with little change, and is called the constant region (C region).
  • the V regions of the L chain and H chain are called VL and VH, respectively.
  • variable region the amino acid composition and arrangement sequence of certain regions have a higher degree of variation, which is called hypervariable region (Hypervariable region, HVR).
  • the hypervariable region is the position where antigen and antibody bind, so it is also called determinant complementation Complementarity-determining region (CDR).
  • CDR complementation Complementarity-determining region
  • the anti-BCMA antibody provided by the present invention can specifically bind to BCMA antigen, and can be used to prepare BCMA CAR-T cells to realize targeted therapy of tumors.
  • the present invention provides an anti-BCMA antibody or antigen-binding fragment.
  • the CDR regions on the heavy chain variable region of the antibody or antigen-binding fragment are respectively CDR1 being GYTFTSYV, CDR2 being IIPYNDDT and CDR3 being ARWNYDGYFDV;
  • the CDR regions on the light chain variable region are respectively CDR1 for QSLVHSNGNTY, CDR2 for YKVS and CDR3 for SQITHVPYT.
  • Antigen-binding fragment herein refers to an amino acid fragment that has the ability to specifically bind to BCMA antigen.
  • anti-BCMA antibodies can also be referred to as BCMA antibodies, both of which refer to immunoglobulin molecules that can bind to BCMA antigen.
  • the antibody or antigen-binding fragment has a heavy chain variable region shown in SEQ ID NO: 1 and a light chain variable region shown in SEQ ID NO: 2.
  • the heavy chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution, such as one conservative amino acid substitution, compared with the amino acid sequence shown in SEQ ID NO:1.
  • the light chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO: 2, for example, has one conservative amino acid substitution, and has two A conservative amino acid substitution, or even three conservative amino acid substitutions. Of course, these conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment. In some embodiments, these conservative amino acid substitutions can occur on amino acids other than the CDR regions in the heavy chain variable region and the light chain variable region.
  • “conservative amino acid substitution” refers to the substitution of an amino acid with a residue that is biologically, chemically or structurally similar to another amino acid.
  • Biologically similar means that the substitution does not destroy the biological activity of the BCMA antibody or the BCMA antigen.
  • Structurally similar means that amino acids have side chains of similar length, such as alanine, glycine or serine, or side chains of similar size.
  • Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other.
  • use polar amino acids such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine and so on.
  • amino acid sequence of the variable region of the heavy chain (SEQ ID NO:1)
  • polynucleotides expressing these antibodies can be connected to different vectors to obtain corresponding antibodies.
  • the present invention also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment described above.
  • nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 of the variable region of the heavy chain is (SEQ ID NO: 3):
  • the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 of the light chain variable region is (SEQ ID NO: 4):
  • the isolated polynucleotide has at least 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 3, preferably more than 95% homology, more preferably 98%, 99% or more homology. In at least some embodiments, the isolated polynucleotide has at least more than 90% homology, preferably more than 95%, compared with the nucleotide sequence shown in SEQ ID NO: 4 above , More preferably 98%, 99% or more homology.
  • sequences that have homology with the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 can express amino acids similar to SEQ ID NO: 1 and SEQ ID NO: 2, so that they can be specific to the BCMA antigen Sexual binding to achieve the targeted function of antibodies.
  • the present invention also provides an expression vector comprising the above-mentioned isolated polynucleotide.
  • the isolated polynucleotide When the isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide.
  • these control elements can come directly from the carrier itself, or they can be exogenous, that is, not from the carrier itself.
  • the polynucleotide can be operably linked to the control element.
  • operably linked refers to the connection of a foreign gene to a vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the foreign gene. Function.
  • control elements in the vector such as transcription control sequences and translation control sequences, etc.
  • the polynucleotides used to encode the heavy chain and light chain of an antibody can be inserted into different vectors independently, and it is common to insert into the same vector.
  • Commonly used vectors can be, for example, plasmids, phages, lentiviruses, and so on.
  • the present invention also provides a recombinant cell, which contains the expression vector.
  • the expression vector can be introduced into mammalian cells to construct recombinant cells, and then use these recombinant cells to express the antibodies or antigen-binding fragments provided by the present invention. By culturing the recombinant cells, corresponding antibodies can be obtained.
  • These usable mammalian cells can be, for example, 293F cells, CHO cells and the like.
  • the present invention also provides a pharmaceutical composition, which comprises the aforementioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
  • the anti-BCMA antibodies provided herein can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • these pharmaceutical compositions include the anti-BCMA antibodies provided herein and a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable carrier” may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof.
  • isotonic agents are included in the pharmaceutical composition, such as sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride.
  • the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to extend the shelf life or efficacy of the antibody.
  • the antibodies of the invention may be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, intramuscular
  • These pharmaceutical compositions can be prepared in various forms.
  • liquid, semi-solid and solid dosage forms including but not limited to liquid solutions (for example, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.
  • Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions.
  • the antibody can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • kits including the above-mentioned BCMA antibody.
  • kit including the above-mentioned BCMA antibody.
  • kit provided by the present invention, for example, it can be used for immunoblotting, immunoprecipitation, etc., which involve the use of the specific binding properties of BCMA antigen and antibody for detection.
  • kits may contain any one or more of the following: antagonists, anti-BCMA antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc.
  • Anti-BCMA antibodies can be used in different types of diagnostic tests, for example, it can detect the presence of various diseases or drugs, toxins or other proteins in vitro or in vivo. For example, it can be used to test related diseases by testing the subject's serum or blood. Such related diseases may include BCMA related diseases, such as various cancers or tumors. Of course, the antibodies provided herein can also be used for radioimmunoassay and radioimmunotherapy of cancer.
  • cancers or tumors can be any unregulated cell growth. Specifically, it may be lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, and so on.
  • the anti-BCMA antibody provided by the present invention can be provided to the subject.
  • the present invention provides a method for treating cancer, which comprises administering the antibody or antigen-binding fragment thereof provided by the present invention to a subject in need.
  • this application has developed antibody-based immunotherapy methods based on BCMA antigens against multiple tumor cells and even tumor stem cells, including monoclonal antibodies against BCMA and Car-T cell therapy technology based on the antibodies.
  • monoclonal hybridomas capable of producing anti-BCMA antibodies are obtained.
  • the anti-BCMA antibody produced by one of the monoclonal hybridomas was sequenced, and the nucleotide sequence of the heavy chain variable region was shown in SEQ ID NO: 3, and the nucleotide sequence of the light chain variable region was shown in SEQ ID NO: 4 shown.
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1
  • the amino acid sequence of the light chain variable region is SEQ ID NO: 2.
  • Example 2 the affinity of the anti-BCMA antibody obtained in Example 1 to the antigen and the affinity to the MM cell line were measured.
  • Biofilm layer interference technology detects the affinity of anti-BCMA antibody and antigen
  • the biofilm layer interference technology combines biomolecules on the surface of the sensor to form a biofilm.
  • the biofilm will cause interference with the light waves passing through the sensor. This interference will be detected in a phase shifting manner, so that the binding can be detected.
  • the changes in the number of molecules on the sensor surface can be used to study the effects of antibodies and antigens.
  • Example 2 Dilute the mouse anti-human BCMA antibody obtained in Example 1 to 0.1 mg/mL in a buffer solution, incubate with the sensor for more than 30 minutes, and then put the sensor in the buffer solution and wash three times for 10 minutes each.
  • Control (negative control) is a buffer without antigen.
  • Figure 1 shows the binding and dissociation curves of BCMA antigen and the biosensor loaded with mouse anti-human BCMA antibody at 25, 50, and 100 nM concentration after normalization with a negative control (Control) sample.
  • the X axis represents the entire time course of the sensor from the baseline to the combination and then to dissociation.
  • the Y axis represents the strength of the combined signal.
  • the corresponding concentration refers to the sample loading concentration
  • KD represents the affinity constant
  • Ka represents the binding rate constant
  • Kd represents the dissociation rate constant
  • Rmax represents the maximum response obtained by data fitting
  • R equilibrium represents equilibrium.
  • the maximum response at time, R 2 represents the correlation coefficient.
  • the detection result of biofilm layer interference technology shows that the affinity constant value KD of anti-BCMA antibody and antigen is in the order of 10 -9 M.
  • MM Multiple myeloma
  • M protein monoclonal immunoglobulin or light chain
  • Figure 2 shows that the anti-BCMA antibody specifically recognizes the cell line MM.1S with high expression of BCMA;
  • Figure 3 shows that the anti-BCMA antibody specifically recognizes the cell line RPMI8226 with low expression of BCMA;
  • Figure 4 shows that the anti-BCMA antibody does not recognize cells that do not express BCMA. Department of K562.
  • the curve marked with an arrow in the figure is the fluorescence intensity curve detected after the anti-BCMA antibody is incubated with the corresponding cell line
  • the curve not marked with an arrow is the staining situation of the isotype control and the corresponding cell line, that is, using the anti-BCMA antibody extract
  • Immunoglobulins of the same species source, the same subtype, the same dose, and the same immunoglobulin and subtype are used to eliminate background staining due to the non-specific binding of antibodies to the cell surface.
  • the anti-BCMA antibody of the present invention specifically binds to the MM cell line (MM.1S, RPMI8226) but does not recognize the control cell line K562.
  • the targeted BCMA CAR designed in the present invention contains an anti-BCMA single-chain antibody, a human CD8 extracellular hinge region, a human CD28 transmembrane region, a human CD28 intracellular region and a human CD3 ⁇ intracellular region connected in sequence.
  • amino acid sequence of the anti-BCMA single-chain antibody is as follows (SEQ ID NO: 5):
  • amino acid sequence of the extracellular hinge region of human CD8 is as follows (SEQ ID NO: 6):
  • the amino acid sequence of human CD28 transmembrane region is as follows (SEQ ID NO: 7):
  • the amino acid sequence of human CD28 intracellular region is as follows (SEQ ID NO: 8):
  • amino acid sequence of the intracellular region of human CD3 ⁇ is as follows (SEQ ID NO: 9):
  • nucleotide sequences encoding these amino acid sequences are:
  • the nucleotide sequence encoding the amino acid sequence of the anti-BCMA single-chain antibody is (SEQ ID NO: 10):
  • nucleotide sequence encoding the amino acid sequence of the extracellular hinge region of human CD8 is (SEQ ID NO: 11):
  • the nucleotide sequence encoding the amino acid sequence of the transmembrane and intracellular regions of human CD28 is (SEQ ID NO: 12):
  • the nucleotide sequence encoding the amino acid sequence of the intracellular region of human CD3 ⁇ is (SEQ ID NO: 13):
  • BCMA CAR nucleic acid sequence (sequence includes BCMA scFv, human CD8 extracellular hinge region, human CD28 penetrating region and intracellular region, and human CD3 ⁇ intracellular region, each sequence is connected in sequence) about 1.5kb, with NheI at both ends And EcoRI restriction endonuclease digestion sites, the BCMA CAR nucleic acid fragment with sticky ends was obtained by double digestion, and the pCDH-EF1 ⁇ -MCS vector was double digested with NheI and EcoRI restriction enzymes (vector from System The vector fragments obtained by Biosciences) were ligated, transformed into DH5 ⁇ competent cells, selected single clones for sequencing identification, and selected the vector with the correct sequence.
  • Day 1 The 293T cells should be in good condition, less than 20 generations, and not overgrown. Plate the plate at 0.3-0.4 ⁇ 10 6 cells/mL, add 10 mL of DMEM+10% FBS complete medium to a 10 cm culture dish, mix the cells thoroughly, and place them in a 37°C, 5% CO 2 incubator overnight.
  • 293T cells reach a confluence of about 60-70% for transfection; take Lipofectamine3000 transfection reagent as an example, prepare a tube of plasmid and P3000 complex, the amount of each plasmid is 12ug BCMA CAR shuttle plasmid, PsPAX2 7.8ug, pMD2.G 4.2ug, P3000 48uL, add DMEM medium to make up to 300uL; another tube is diluted 36uL Lipofectamine3000 to 300uL with DMEM (serum-free) medium. Mix the two tubes separately, combine them, mix well, incubate at room temperature for 15-20 minutes, gently add the mixture to the 293T Petri dish. After incubating at 37°C for 6 hours, remove the medium and re-add the pre-warmed fresh medium.
  • Day 4-5 Collect the supernatant for 48-72 hours after transfection, remove floating cells by centrifugation at 3000 rpm for 10 minutes, filter with 0.45um filter, ultracentrifugation at 35000 rpm for 90 minutes, discard the supernatant, and use 100uL DMEM medium Resuspend the virus pellet, and freeze it at -80°C after aliquoting.
  • T cells After T cells are activated, count the cells again. According to the count results, inoculate 2x106 cells/well in a six-well plate. Add 2ml AIM-V medium + 10% FBS (containing 50ng/mL anti-human CD3 antibody, 50ng/mL anti-human CD28 antibody, 200IU/mL interleukin 2) medium, the virus solution prepared in item 8 and polybrene (Sigma), pipet and mix well. Move the above-mentioned six-well plate into a plate centrifuge, centrifuge at 800g for 30 minutes, and transfer it to an incubator to continue culturing.
  • FBS containing 50ng/mL anti-human CD3 antibody, 50ng/mL anti-human CD28 antibody, 200IU/mL interleukin 2
  • NT cells and mock CAR-T cells are defined in Example 3.
  • the test results are shown in Figure 6 and Figure 7.
  • Figure 6 represents the results of interferon- ⁇ (IFN-gamma) secretion after co-cultivation of different cells with three MM target cells and negative control cells
  • Figure 7 represents the co-cultivation of different cells with three MM target cells and negative control cells
  • IL-2 interleukin-2
  • Target cell alone in Figure 6 and Figure 7 refers to only culturing target cells or negative control cells, without using NT cells, mock CAR-T cells or BCMA CAR-T cells to co-culture.
  • test results showed that compared with the control group, the BCMA CAR-T cells in the experimental group were incubated with MM target cells and the secretion of IFN-gamma and interleukin-2 increased significantly, while the secretion of cytokines did not increase after incubation with the negative control cell line , Indicating that BCMA CAR-T cells can specifically recognize target cells and are activated to release corresponding cytokines.
  • FIG. 8 the killing effect of different cells on CHO-BCMA cells is shown.
  • the results in Figure 8 show that the target cells continued to increase in cell index before CAR-T cells were added. After adding BCMA CAR-T cells (pointed by the arrow in the figure), most of the CHO-BCMA target cells were killed within 24 hours. The index continued to decrease; while the control mock CAR-T cells or NT cells had no killing effect.
  • Each graph line shows the average value of three-well experiment data.
  • the RPMI8226 cell line was used to evaluate the resistance of CAR-T in a preclinical animal model of multiple myeloma. Tumor activity.
  • the RPMI8226 cell line is a human multiple myeloma cell line, purchased from ATCC.
  • mice Fifteen NSG mice (NODscid gamma mice, purchased from Jackson Laboratory) were divided into three groups, 5 mice in each group, and 1 ⁇ 10 7 RPMI8226 tumor cells were inoculated subcutaneously; on the 18th and 22nd day after tumor inoculation Day, the three groups of mice were injected intravenously with PBS (PBS group), 2 ⁇ 10 7 mock CAR-T cells (mock CAR-T group), 2 ⁇ 10 7 BCMA CAR-T cells (BCMA-CAT-T group, according to Prepared by infection with the method described in item 8, CAR-positive cells are about 25-30%).
  • the tumor volume (unit: mm 3 ) was measured every four days. Compare the tumor growth status of the control group, mock CAR-T group and BCMA-CAR-T group. As shown in Figure 10.
  • the abscissa represents the days of injection of RPMI8226 tumor cells into mice, and the abscissa represents the tumor volume.
  • the results in Figure 10 show that compared with the PBS group and the mock CAR-T group, the tumor growth of the mice infused with BCMA CAR-T was significantly inhibited. On the 36th day, the tumor size of the mice in each group was detected. The tumor size of mice in the mock CAR-T group reached about 2500-3000 mm 3 , while the tumors in the BCMA-CAR-T group did not grow and were significantly reduced.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, "a plurality of” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the terms “installed”, “connected”, “connected”, “fixed” and other terms should be understood in a broad sense. For example, they may be directly connected or indirectly through an intermediary. The connection may be the internal communication between two elements or the interaction relationship between the two elements, unless specifically defined otherwise. When two nucleic acid sequences or nucleotide sequences are connected, they can be connected by a 3'-5' phosphodiester bond. For those of ordinary skill in the art, the specific meaning of the above-mentioned terms in the present invention can be understood according to specific circumstances.

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Abstract

La présente invention a trait au domaine de la biomédecine et concerne en particulier un anticorps anti-BCMA et son utilisation dans le domaine des CAR-T. L'anticorps anti-BCMA selon l'invention comprend au moins l'un des éléments suivants : (1) une région variable de chaîne lourde avec des séquences d'acides aminés telles que représentées dans GYTFTSYV, IIPYNDDT et ARWNYDGYFDV, et une région variable de chaîne légère avec des séquences d'acides aminés telles que représentées dans QSLVHSNGNTY, YKVS et SQITHVPYT ; et une séquence d'acides aminés avec au moins une substitution conservatrice d'acides aminés par rapport au point (1). L'anticorps anti-BCMA selon l'invention peut se lier spécifiquement à un antigène BCMA et peut par conséquent être utilisé dans le traitement ciblé de tumeurs.
PCT/CN2019/101542 2019-08-20 2019-08-20 Anticorps anti-bcma et utilisation associé dans le domaine des car-t WO2021031113A1 (fr)

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WO2023138666A1 (fr) * 2022-01-19 2023-07-27 Utc Therapeutics (Shanghai) Co., Ltd. Arn circulaire et son utilisation

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