WO2020248156A1 - Agent de liaison ciblant pd-l1 et son utilisation - Google Patents

Agent de liaison ciblant pd-l1 et son utilisation Download PDF

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WO2020248156A1
WO2020248156A1 PCT/CN2019/090878 CN2019090878W WO2020248156A1 WO 2020248156 A1 WO2020248156 A1 WO 2020248156A1 CN 2019090878 W CN2019090878 W CN 2019090878W WO 2020248156 A1 WO2020248156 A1 WO 2020248156A1
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amino acid
seq
acid sequence
cancer
residue substitutions
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PCT/CN2019/090878
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English (en)
Chinese (zh)
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徐建青
黄杨
郜明泉
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苏州工业园区唯可达生物科技有限公司
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Priority to PCT/CN2019/090878 priority Critical patent/WO2020248156A1/fr
Publication of WO2020248156A1 publication Critical patent/WO2020248156A1/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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of biomedicine, and specifically relates to targeted binding agents for PD-L1, nucleic acids encoding them and uses thereof.
  • the present invention relates to monoclonal antibodies against PD-L1, nucleic acids encoding them, and uses of these antibodies.
  • the invention also relates to cell lines expressing the targeted binding agent or antibody.
  • the present invention also relates to the use of the targeted binding agent for diagnosis and treatment of diseases related to the activity and/or expression of PD-L1.
  • the adaptive immune response involves the activation, selection and clonal proliferation of two main types of lymphocytes, T cells and B cells. After encountering an antigen, T cells proliferate and differentiate into antigen-specific effector cells, while B cells proliferate and differentiate into antibody secreting cells.
  • T cell activation is a multi-step process that requires some signal transduction events between T cells and antigen presenting cells (APC). For T cell activation to occur, two types of signals must be delivered to resting T cells. The first type is mediated by antigen-specific T cell receptors (TCR) and confers specificity for immune responses. The second type of co-stimulation regulates the size of the response and is delivered via a co-receptor on T cells.
  • TCR antigen-specific T cell receptors
  • the main costimulatory signal is delivered by binding to the ligand B7-1 or B7-2 on APC to activate the CD28 receptor on T cells.
  • the binding of the same B7-1 or B7-2 ligand to the inhibitory CTLA-4 receptor results in a weakening of the T cell response. Therefore, the CTLA-4 signal opposes CD28-mediated costimulation.
  • CD28 costimulation surpasses CTLA-4 inhibition.
  • the temporary regulation of the expression of CD28 and CTLA-4 maintains the balance between the activation signal and the inhibitory signal and ensures the formation of an effective immune response, while preventing the formation of autoimmunity.
  • PD-L1 also known as B7-H1
  • B7-H1 is a type I transmembrane protein with a size of about 53kD.
  • PD-L1 is expressed on a variety of immune cell types, including activated T cells, anergic/depleted T cells, natural B cells and activated B cells, as well as on myeloid dendritic cells (DC), monocytes and mast cells. It is also expressed on non-immune cells, including pancreatic islets, liver Kupffer Cells, vascular endothelial cells, and epithelial cells, such as tracheal epithelial cells and renal tubular epithelial cells, where its expression during inflammatory episodes is Enhanced.
  • DC myeloid dendritic cells
  • PD-L1 expression levels are up-regulated in a variety of tumors, including but not limited to breast cancer, colorectal cancer, lung cancer, kidney cancer, gastric cancer, bladder cancer, liver cancer, and pancreatic cancer, as well as black Prime tumor.
  • PD-L1 is a member of the B7 family of proteins, which contains two extracellular immunoglobulin (Ig) domains, an N-terminal V-type domain, followed by a C-type domain.
  • the 30 amino acid long intracellular domain does not contain obvious signal transduction motifs, but contains possible sites for protein kinase C phosphorylation.
  • Mouse PD-L1 has 69% amino acid homology with human PD-L1 and shares a conserved structure.
  • PD-L1 binds to two alternative ligands, one of which is PD-1, a 50-55kD type I transmembrane receptor, which was originally identified in T cells undergoing activation-induced apoptosis.
  • PD-1 is expressed in activated T cells, B cells, monocytes and other cells of the immune system, and binds to PD-L1 and PD-L2.
  • the second is the B7 family member B7-1, which is expressed on activated T cells, B cells, monocytes and antigen presenting cells.
  • PD-1 is a member of the immunoglobulin superfamily, which contains a single Ig V-like domain in its extracellular domain.
  • the cytoplasmic domain of PD-1 contains two tyrosines, and the tyrosine closest to the membrane is located in the immunoreceptor tyrosine-based inhibitory motif (Immunoreceptor Tyrosine-Based Inhibitory Motif, ITIM).
  • ITIM immunoreceptoreceptor Tyrosine-Based Inhibitory Motif
  • Human and murine PD-1 proteins share about 60% amino acid homology, of which 4 N-glycosylation sites and amino acid residues that define the Ig-V domain are conserved.
  • the ITIM in the cytoplasmic region and the ITIM-like motif around the tyrosine at the carboxyl terminal of PD-L1 are also conserved between humans and mice.
  • PD-L1 expressed in tumor cells helps the detection and elimination of tumor escape from the immune system.
  • PD-L1 plays a role in this aspect through some selectable mechanisms, including driving the failure and anergy of tumors infiltrating T lymphocytes, stimulating the secretion of immunosuppressive cytokines into the tumor microenvironment, and stimulating inhibitory regulation T cells act and prevent tumor cells expressing PD-L1 from being lysed by tumor cell-specific cytotoxic T cells.
  • the present invention relates to a targeted binding agent that specifically binds to PD-L1 and inhibits the biological activity of PD-L1.
  • the present invention relates to a targeted binding agent that specifically binds to PD-L1 and thereby inhibits the activity of PD-L1.
  • the present invention relates to a targeted binding agent that specifically binds to PD-L1 and thereby inhibits the binding of PD-L1 to PD-1.
  • the present invention relates to a targeted binding agent that blocks PD-L1 induced T cell suppression and thereby enhances anti-tumor immunity.
  • the present invention also relates to a targeted binding agent that can further stimulate one or more of the following activities, including T cell proliferation in mixed lymphocyte reaction, IFN- ⁇ Or IL-2 secretion.
  • Embodiments of the present invention relate to targeted binding agents that specifically bind to PD-L1 and inhibit the biological activity of PD-L1.
  • the targeted binding agent inhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25% of the biological activity of PD-L1 compared with the absence of the targeted binding agent. , At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.
  • Embodiments of the present invention relate to targeted binding agents that specifically bind to PD-L1 and thereby inhibit the activity of PD-L1.
  • the targeted binding agent inhibits PD-L1 activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 % Or at least 95%.
  • Embodiments of the present invention relate to targeted binding agents that specifically bind to PD-L1 and thereby inhibit the binding to PD-1.
  • the targeted binding agent inhibits the binding of PD-L1/PD-1 receptor ligand by at least 5%, at least 10%, at least 15%, at least 20% compared to when the targeted binding agent is not present. %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, or at least 95%.
  • Another embodiment of the present invention relates to a targeted binding agent that specifically binds to B7-H1 and thereby enhances anti-tumor immunity.
  • the targeted binding agent enhances anti-tumor immunity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25% compared to what would happen in the absence of the targeted binding agent , At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.
  • Another embodiment of the present invention relates to a targeted binding agent that specifically binds to B7-H1 and thereby inhibits cell proliferation.
  • the targeted binding agent inhibits cell proliferation by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% Or at least 95%.
  • an antibody of the present invention is less than or equal to 100nM, EC 50nM 1nM or 50. Further, in another embodiment, the antibodies of the invention having from about 1nM to about 100nM of up to 50 EC; EC or about 1nM to about 50nM up to 50; or from about 1nM to about 20nM up to 50 EC; or of about 50nM up to about 100nM EC 50; or from about 70nM to about 100nM until the EC 50.
  • Further embodiments of the present invention relates specifically binds to PD-L1 and EC 50 of less than or equal to 100nM inhibiting targeted binding agent inhibits proliferation of PD-L1-mediated T cell.
  • the antibody of the invention has an EC50 of less than or equal to 100 nM, for example, 90, 80, 70, 60, 50 , 40, 30, 20, or 10 nM.
  • the antibodies of the invention having about 100nM 10nM up to about 50 EC; EC or from about 50nM to about 10nM up to 50; or from about 20nM to about 10nM up to 50 EC; or of about 50nM up to about 100nM EC 50; or from about 70nM to about 100nM until the EC 50; or from about 80nM to about 100nM until the EC 50.
  • Targeted binding agents also inhibit tumor cell adhesion, motility, invasion and cell metastasis, and targeted binding agents are useful for reducing tumor growth. Mechanisms that can achieve this may include, but are not limited to, inhibition of PD-L1 activity.
  • the targeted binding agent is an antibody. In one embodiment of the invention, the targeted binding agent is a monoclonal antibody. In one embodiment of the invention, the targeted binding agent is a humanized antibody or fragment thereof.
  • the monoclonal antibody herein may refer to the anti-PD-L1 antibody or the antibody of the present invention.
  • Antibodies, monoclonal antibodies and human monoclonal antibodies include IgG1, IgG2, IgG3, and IgG4 isotype antibodies.
  • the targeted binding agent is a humanized monoclonal antibody of the IgG1 isotype.
  • the IgG1 isotype improves the antibody-dependent cellular cytotoxicity (ADCC) of the antibody.
  • the IgG1 isotype has high stability.
  • the targeted binding agent has a desired therapeutic property selected from one or more of the following: high affinity for PD-L1, inhibiting PD-L1 activity in vitro or in vivo Ability, the ability to inhibit PD-L1-mediated tumor cell survival and the ability to inhibit PD-L1-mediated tumor-reactive T cell suppression, which can further reduce tumor cell proliferation, motility, invasion, metastasis and tumor growth.
  • the present invention includes antibodies that specifically bind to PD-L1 with very high affinity.
  • the targeted binding agent binds PD-L1 with a binding affinity (Kd) of less than 5 nM. In other embodiments, the targeted binding agent binds with a Kd of less than 4 nM, 3 nM, 2 nM, or 1 nM.
  • the antibody of the present invention is used at about 5 nM to about 1 nM; or about 5 nM to about 2 nM; or about 5 nM to about 3 nM; or about 5 nM to about 4 nM; or about 3 nM to about 1 nM; or about A Kd of 2nM to about 1nM binds PD-L1.
  • the targeted binding agent binds PD-L1 with a Kd of less than 950pM, 900pM, 800pM, 700pM, 600pM, 500pM, 400pM, 300pM, 200pM, or 100pM.
  • the antibody of the present invention is at about 900pM to about 100pM; or about 900pM to about 200pM; or about 900pM to about 300pM; or about 900pM to about 400pM; or about 900pM to about 500pM; or about 900pM to about 600pM; or about 900pM to about 700pM; or about 200pM to about 100pM; or about 300pM to about 200pM; or about 400pM to about 300pM Kd binds PD-L1.
  • the targeted binding agent binds PD-L1 with a Kd of less than 90pM, 80pM, 70pM, 60pM, 55pM, or 50pM. In some other embodiments, the targeted binding agent binds PD-L1 with a Kd of less than 60 pM. In some other embodiments, the targeted binding agent binds PD-L1 with a Kd of less than 55 pM.
  • the antibody of the present invention is used at about 100 pM to about 50 pM; or about 100 pM to about 70 pM; or about 100 pM to about 80 pM; or about 100 pM to about 90 pM; or about 70 pM to about 50 pM; or about 60pM to about 50pM; or about 55pM to about 50pM Kd binds PD-L1.
  • Kd can be evaluated using the methods described herein or well known to those of ordinary skill in the art (for example, the BIAcore method) (Biacore International AB, Uppsala, Sweden).
  • the targeted binding agent or antibody comprises a sequence comprising any one of the heavy chain sequences of antibodies 1C2, 1D4, 1G11, 2F7, 3D7, or 6A3.
  • Light chain promiscuity is very well established in the art. Therefore, a targeted binding agent containing any one of the sequences of the heavy chain sequence of the antibody 1C2, 1D4, 1G11, 2F7, 3D7 or 6A3, another antibody disclosed herein, or The antibody may also comprise any one of 1C2, 1D4, 1G11, 2F7, 3D7, or 6A3 shown in Table 2, the light chain sequence (VL) of other antibodies disclosed herein.
  • the targeted binding agent or antibody comprises a sequence comprising any of the heavy chain sequences of antibodies 1C2, 1D4, 1G11, 2F7, 3D7 or 6A3 and further comprising antibodies 1C2, 1D4, 1G11, The corresponding light chain sequence of 2F7, 3D7 or 6A3.
  • the antibody is a fully human monoclonal antibody.
  • the targeted binding agent or antibody comprises a sequence comprising any one of the light chain sequences shown in Table 2. In another embodiment, the targeted binding agent or antibody comprises a sequence comprising any one of the light chain sequences of antibodies 1C2, 1D4, 1G11, 2F7, 3D7, or 6A3.
  • Another embodiment is a targeted binding agent or antibody that specifically binds to PD-L1, and includes a sequence that includes one of the CDR2 sequences and one of the CDR3 sequences shown in Table 2.
  • the targeted binding agent or antibody further comprises a sequence comprising the CDR3 sequence shown in Table 1.
  • the targeted binding agent or antibody further comprises a sequence comprising the CDR2 sequence and CDR3 sequence as shown in Table 1 and/or Table 2.
  • the targeted binding agent or antibody further comprises a sequence comprising: CDR1, CDR2 and CDR3 sequences as shown in Table 1 and/or Table 2.
  • the targeted binding agent or antibody may comprise a sequence comprising the CDR1 of any one of monoclonal antibodies 1C2, 1D4, 1G11, 2F7, 3D7, or 6A3 as shown in Table 1. Either CDR2 or CDR3.
  • the targeted binding agent or antibody may comprise a sequence comprising CDR1, CDR2 of any one of monoclonal antibodies 1C2, 1D4, 1G11, 2F7, 3D7 or 6A3 as shown in Table 2.
  • the targeted binding agent or antibody may comprise a sequence comprising CDR1, CDR2 of any one of monoclonal antibodies 1C2, 1D4, 1G11, 2F7, 3D7 or 6A3 as shown in Table 1.
  • the targeted binding agent or antibody may comprise a sequence comprising CDR1, CDR2 of any one of monoclonal antibodies 1C2, 1D4, 1G11, 2F7, 3D7 or 6A3 as shown in Table 2.
  • the targeted binding agent or antibody may comprise a sequence comprising CDR1, CDR2 of any one
  • the targeted binding agent or antibody comprises a sequence comprising the CDR1, CDR2, and CDR3 sequences of the heavy chain of monoclonal antibody 1C2 as shown in Table 1 and the single sequence as shown in Table 2.
  • the CDR1, CDR2 and CDR3 sequences of the light chain of antibody 1C2 were cloned.
  • the targeted binding agent or antibody comprises a sequence comprising the CDR1, CDR2 and CDR3 sequences of the heavy chain of monoclonal antibody 1D4 as shown in Table 1 and the single sequence as shown in Table 2.
  • the CDR1, CDR2 and CDR3 sequences of the light chain of antibody 1D4 were cloned.
  • the targeted binding agent or antibody comprises a sequence comprising the CDR1, CDR2 and CDR3 sequences of the heavy chain of monoclonal antibody 1G11 as shown in Table 1 and the single sequence as shown in Table 2.
  • the CDR1, CDR2 and CDR3 sequences of the light chain of antibody 1G11 were cloned.
  • the targeted binding agent or antibody comprises a sequence comprising the CDR1, CDR2 and CDR3 sequences of the heavy chain of monoclonal antibody 2F7 as shown in Table 1 and the single sequence as shown in Table 2.
  • the CDR1, CDR2 and CDR3 sequences of the light chain of antibody 2F7 were cloned.
  • the targeted binding agent or antibody comprises a sequence comprising the CDR1, CDR2 and CDR3 sequences of the heavy chain of the monoclonal antibody 3D7 as shown in Table 1 and the single sequence as shown in Table 2.
  • the CDR1, CDR2 and CDR3 sequences of the light chain of the antibody 3D7 were cloned.
  • the targeted binding agent or antibody comprises a sequence comprising the CDR1, CDR2 and CDR3 sequences of the heavy chain of monoclonal antibody 6A3 as shown in Table 1 and the single sequence as shown in Table 2.
  • the CDR1, CDR2 and CDR3 sequences of the light chain of antibody 6A3 were cloned.
  • the antibody is a humanized antibody.
  • the targeted binding agent or antibody of the present invention comprises the CDR3 sequence as shown in Table 1 or Table 2; or any of the CDR1, CDR2 or CDR3 sequences as shown in Table 1 or Table 2.
  • the antibody is a humanized antibody.
  • the targeted binding agent or antibody, or antigen-binding portion thereof comprises a heavy chain variable region having at least 90% homology with the amino acid of SEQ ID NO: 1, and comprises a heavy chain variable region with SEQ ID NO:
  • the amino acid sequence of 2 has a light chain variable region with at least 90% homology.
  • the targeted binding agent or antibody, or antigen binding portion thereof comprises a heavy chain variable region having at least 90% homology with the amino acid of SEQ ID NO: 3, and comprises a heavy chain variable region with SEQ ID NO:
  • the amino acid sequence of 4 has a light chain variable region with at least 90% homology.
  • the targeted binding agent or antibody, or antigen-binding portion thereof comprises a heavy chain variable region having at least 90% homology with the amino acid of SEQ ID NO: 5, and comprises a heavy chain variable region with SEQ ID NO: 5
  • the amino acid sequence of 6 has a light chain variable region with at least 90% homology.
  • the targeted binding agent or antibody, or antigen-binding portion thereof comprises a heavy chain variable region having at least 90% homology with the amino acid of SEQ ID NO: 7, and comprises the same as SEQ ID NO:
  • the amino acid sequence of 8 has a light chain variable region with at least 90% homology.
  • the targeted binding agent or antibody, or antigen-binding portion thereof comprises a heavy chain variable region having at least 90% homology with the amino acid of SEQ ID NO: 9 and comprises the same as SEQ ID NO:
  • the amino acid sequence of 10 has a light chain variable region with at least 90% homology.
  • the targeted binding agent or antibody, or antigen-binding portion thereof comprises a heavy chain variable region having at least 90% homology with the amino acid of SEQ ID NO: 11, and comprises a heavy chain variable region with SEQ ID NO:
  • the amino acid sequence of 12 has a light chain variable region with at least 90% homology.
  • the targeted binding agent or antibody comprises a variant or derivative of the CDR disclosed herein, spanning the framework region of the light chain sequence or heavy chain sequence disclosed herein and the CDR or the framework of the antibody disclosed herein Regions and CDRs (especially from FR1 to FR4 or CDR1 to CDR3).
  • Variants include a targeted binding agent or antibody, the targeted binding agent or antibody comprising a sequence having the continuous sequence of CDR1, CDR2 or CDR3 as shown in Table 1 or Table 2, the light disclosed herein Chain sequence or heavy chain sequence, or any of the monoclonal antibodies disclosed herein, up to 20, 16, 10, 9 or less, for example, 1, 2, 3, 4, 5 or 6 amino acid additions, substitutions, deletions and/or insertions.
  • Variants include a targeted binding agent or antibody, the targeted binding agent or antibody comprising a sequence having the continuous sequence of CDR1, CDR2 or CDR3 as shown in Table 1 or Table 2, the light disclosed herein Chain sequence or heavy chain sequence, or 1, 2, 3 amino acid additions, substitutions, deletions and/or insertions in any of the monoclonal antibodies disclosed herein.
  • Variants include a targeted binding agent or antibody, the targeted binding agent or antibody comprising a sequence that is consistent with the continuous sequence of CDR1, CDR2 or CDR3 as shown in Table 1 or Table 2, and the light disclosed herein.
  • Chain sequence or heavy chain sequence or any of the monoclonal antibodies disclosed herein, have at least about 60%, 70%, 80%, 85%, 90%, 95%, 98%, or about 99% amino acid homology Sex.
  • the percent homology of two amino acid sequences can be determined by any method known to those skilled in the art, including but not limited to, paired protein alignment.
  • the variant includes an alteration in the CDR sequence or light chain sequence or heavy chain sequence disclosed herein, the alteration is naturally occurring or through the use of recombinant DNA technology or mutation technology to engineer the natural sequence in vitro To introduce.
  • Naturally occurring variants include those produced in vivo in the corresponding germline nucleotide sequence during the production of antibodies against foreign antigens.
  • the targeted binding agent comprises:
  • CDR1 having the amino acid sequence shown in SEQ ID NO: 13, 16, 19, 22, 25, or 28,
  • CDR2 having the amino acid sequence shown in SEQ ID NO: 14, 17, 20, 23, 26, or 29, or having SEQ ID NO: CDR3 of the amino acid sequence shown in 15, 18, 21, 24, 27 or 30, or one or more of their variants;
  • CDR1 having the amino acid sequence shown in SEQ ID NO: 31, 34, 37, 40, 43, or 46
  • CDR2 having the amino acid sequence shown in SEQ ID NO: 32, 35, 38, 41, 44, or 47, or having SEQ ID NO: CDR3 of the amino acid sequence shown in NO: 33, 36, 39, 42, 45 or 48, or one or more of their variants;
  • the sequence of the variant includes up to 20, 16, 10, 9 or less, for example, an increase of 1, 2, 3, 4, 5 or 6 amino acids , Substitution, deletion and/or insertion, or the sequence of the variant and any of the sequences have at least 60%, 70%, 80%, 85%, 90%, 95%, 98% Or 99% homology.
  • the targeted binding agent is a PD-L1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein:
  • CDR1 has the same amino acid sequence as SEQ ID NO: 13, 16, 19, 22, 25, or 28 or contains 1, 2, or relative to SEQ ID NO: 13, 16, 19, 22, 25 or 28 Amino acid sequence with 3 amino acid residue substitutions;
  • CDR2 has the same amino acid sequence as SEQ ID NO: 14, 17, 20, 23, 26 or 29 or contains 1, 2, or relative to SEQ ID NO: 14, 17, 20, 23, 26 or 29 Amino acid sequence with 3 amino acid residue substitutions;
  • CDR3 has the same amino acid sequence as SEQ ID NO: 15, 18, 21, 24, 27 or 30 or contains 1, 2 or 1 relative to SEQ ID NO: 15, 18, 21, 24, 27 or 30 Amino acid sequence with 3 amino acid residue substitutions;
  • the light chain variable region comprises CDR1, CDR2 and CDR3, wherein:
  • CDR1 has the same amino acid sequence as SEQ ID NO: 31, 34, 37, 40, 43 or 46 or contains 1, 2 or 1 relative to SEQ ID NO: 31, 34, 37, 40, 43 or 46 Amino acid sequence with 3 amino acid residue substitutions;
  • CDR2 has the same amino acid sequence as SEQ ID NO: 32, 35, 38, 41, 44 or 47 or contains 1, 2 or 1 relative to SEQ ID NO: 32, 35, 38, 41, 44 or 47 Amino acid sequence with 3 amino acid residue substitutions;
  • CDR3 has the same amino acid sequence as SEQ ID NO: 33, 36, 39, 42, 45 or 48 or contains 1, 2 or 1 relative to SEQ ID NO: 33, 36, 39, 42, 45 or 48 Amino acid sequence with 3 amino acid residue substitutions.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO:1 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:1;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 2 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 2.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 3 or an amino acid sequence containing 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 3;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 4 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 4.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 5 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 5;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 6 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 6.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 7 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 7;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 8 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 8.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 9 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 9;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 10 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 10.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 11 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 11;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 12 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 12.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 13 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 13;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 14 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 14;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 15 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 15;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 31 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 31;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 32 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 32;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 33 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 33.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 16 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 16;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 17 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 17;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 18 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 18;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 34 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 34;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 35 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 35;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 36 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 36.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 19 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 19;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 20 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 20;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 21 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 21;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 37 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 37;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 38 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 38;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 39 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 39.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 22 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 22;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 23 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 23;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 24 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 24;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 40 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 40;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 41 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 41;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 42 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 42.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 25 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 25;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 26 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 26;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 27 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 27;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 43 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 43;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 44 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 44;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 45 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 45.
  • the variant includes a targeted binding agent or antibody, which comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 28 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 28;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 29 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 29;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 30 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 30;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 46 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 46;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 47 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 47;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 48 or an amino acid sequence containing 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 48.
  • the antibody or antigen-binding fragment thereof comprises the heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO: 13, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 15
  • the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO: 31 the light chain CDR1 of the amino acid sequence shown in SEQ ID NO: 32, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO: 33.
  • the antibody or antigen-binding fragment thereof comprises: the heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO: 16, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO: 17, the amino acid sequence of SEQ ID NO: 18
  • the antibody or antigen-binding fragment thereof comprises: heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO: 19, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO: 20, and amino acid sequence of SEQ ID NO: 21
  • the antibody or antigen-binding fragment thereof comprises: heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO: 22, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO: 23, and amino acid sequence of SEQ ID NO: 24
  • the antibody or antigen-binding fragment thereof comprises: the heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO: 25, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence shown in SEQ ID NO: 27
  • the antibody or antigen-binding fragment thereof comprises: heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO: 28, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO: 29, and amino acid sequence of SEQ ID NO: 30
  • the derivative may be a heterogeneous antibody, that is, an antibody in which two or more antibodies are linked together.
  • Derivatives include antibodies that have been chemically modified. Examples include covalent bonding of one or more polymers, such as water-soluble polymers, N-linked or O-linked carbohydrates, sugars, phosphates, and/or other such molecules. Derivatives are modified in such a way that the type or location of the bound molecule is different from the naturally occurring antibody or the starting antibody. Derivatives also include the deletion of one or more chemical groups naturally occurring in the antibody.
  • an isolated nucleic acid encoding the targeted binding agent, vectors and host cells containing such nucleic acid, and recombinant technology for producing the targeted binding agent are provided.
  • the coding nucleic acid sequence of the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 71; preferably, the coding nucleic acid sequence of the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 72;
  • the coding nucleic acid sequence of the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 73; preferably, the coding nucleic acid sequence of the amino acid sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 74;
  • the coding nucleic acid sequence of the amino acid sequence shown in SEQ ID NO: 5 is shown in SEQ ID NO: 75; preferably, the coding nucleic acid sequence of the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 76;
  • the coding nucleic acid sequence of the amino acid sequence shown in SEQ ID NO: 7 is shown in SEQ ID NO: 77; preferably, the coding nucleic acid sequence of the amino acid sequence shown
  • a vector is provided, which is suitable for expressing a nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies.
  • the vector further comprises a host cell suitable for expressing the nucleic acid.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary (CHO).
  • a method for preparing an anti-PD-L1 antibody or antigen-binding fragment thereof includes culturing a nucleic acid containing a form suitable for expression under conditions suitable for the production of such antibody or fragment.
  • the nucleic acid encodes any of the aforementioned anti-PD-L1 antibodies or antigen-binding fragments, and the antibodies or fragments are recovered.
  • the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression.
  • the DNA encoding the monoclonal antibody can be easily separated and sequenced using conventional procedures (for example, using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).
  • Many carriers are available.
  • the choice of vector depends in part on the host cell to be used. Generally, the preferred host cell is of prokaryotic or eukaryotic (usually mammalian) origin.
  • a chimeric antigen receptor of a PD-L1 antibody which comprises a PD-L1 antibody variable region sequence.
  • the chimeric antigen receptor of the PD-L1 antibody further comprises one or more elements selected from the group consisting of: signal peptide, linker sequence, transmembrane domain, internal domain and costimulatory Structure domain.
  • the chimeric antigen receptor of the PD-L1 antibody comprises the CD8 antigen signal peptide, the light chain variable region of the PD-L1 antibody, the heavy chain variable region of the PD-L1 antibody, the CD28 element, the 4- 1BBL element and CD3 ⁇ element.
  • the coding nucleic acid sequence of the CD8 antigen signal peptide is shown in SEQ ID NO: 51, and the amino acid sequence is shown in SEQ ID NO: 52.
  • the coding nucleic acid sequence of the CD28 element is shown in SEQ ID NO:53, and the amino acid sequence is shown in SEQ ID NO:54.
  • the coding nucleic acid sequence of the 4-1BBL element is shown in SEQ ID NO: 55, and the amino acid sequence is shown in SEQ ID NO: 56.
  • the coding nucleic acid sequence of the CD3 ⁇ element is shown in SEQ ID NO: 57, and the amino acid sequence is shown in SEQ ID NO: 58.
  • the nucleic acid sequence encoding the chimeric antigen receptor is selected from the sequence shown in 59, 61, 63, 65, 67 or 69, and the corresponding amino acid sequence is selected from 60, 62, 64, 66, 68 or 70 shown in the sequence.
  • a nucleic acid encoding a chimeric antigen receptor of a PD-L1 antibody is provided.
  • a recombinant vector, expression cassette, recombinant virus or recombinant cell containing the nucleic acid is provided.
  • a CAR construct comprising a nucleic acid encoding a chimeric antigen receptor.
  • a T cell is provided, which is transduced with the CAR construct.
  • a method for preparing PD-L1 CAR-T cells includes the following steps:
  • the vector can be a eukaryotic expression vector or a lentiviral vector, preferably a lentiviral vector, Preferably, the vector is a lentiviral vector pHAGE-EF1 ⁇ -MCS-ZsGreen;
  • step (b) Transfect or infect host T cells with the vector constructed in step (a) to produce CAR-T cells with CAR elements that can recognize human PD-L1.
  • a method for preparing PD-L1 CAR-T cells includes the following steps:
  • the vector can be a eukaryotic expression vector or a lentiviral vector, preferably a lentiviral vector;
  • the vector is a lentiviral vector pHAGE-EF1 ⁇ -MCS-ZsGreen;
  • step (b) Transfect the vector constructed in step (a) into a packaging cell line to produce lentiviral particles with CAR elements that can recognize human PD-L1;
  • the CAR element comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO:1 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:1;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 2 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 2;
  • the CAR element comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 3 or an amino acid sequence containing 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 3;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 4 or an amino acid sequence containing 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 4;
  • the CAR element comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 5 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 5;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 6 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 6;
  • the CAR element comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 7 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 7;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 8 or an amino acid sequence containing 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 8;
  • the CAR element comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 9 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 9;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 10 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 10;
  • the CAR element comprises the following sequence:
  • VH sequence having the same amino acid sequence as SEQ ID NO: 11 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 11;
  • VL sequence having the same amino acid sequence as SEQ ID NO: 12 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 12;
  • the CAR element comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 13 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 13;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 14 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 14;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 15 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 15;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 31 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 31;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 32 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 32;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 33 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 33.
  • the CAR element comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 16 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 16;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 17 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 17;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 18 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 18;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 34 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 34;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 35 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 35;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 36 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 36.
  • the CAR element comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 19 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 19;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 20 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 20;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 21 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 21;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 37 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 37;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 38 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 38;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 39 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 39.
  • the CAR element comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 22 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 22;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 23 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 23;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 24 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 24;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 40 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 40;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 41 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 41;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 42 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 42.
  • the CAR element comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 25 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 25;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 26 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 26;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 27 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 27;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 43 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 43;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 44 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 44;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 45 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 45.
  • the CAR element comprises the following sequence:
  • VH CDR1 having the same amino acid sequence as SEQ ID NO: 28 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 28;
  • VH CDR2 having the same amino acid sequence as SEQ ID NO: 29 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 29;
  • VH CDR3 having the same amino acid sequence as SEQ ID NO: 30 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 30;
  • VL CDR1 having the same amino acid sequence as SEQ ID NO: 46 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 46;
  • VL CDR2 having the same amino acid sequence as SEQ ID NO: 47 or an amino acid sequence with 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 47;
  • VL CDR3 having the same amino acid sequence as SEQ ID NO: 48 or an amino acid sequence containing 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 48.
  • CDR region or “CDR” is intended to mean the hypervariable regions of the heavy and light chains of an antibody that confer antigen binding specificity to the antibody.
  • CDR can be defined according to the Kabat system (Kabat, E.A., etc. (1991) Sequences of Proteins of Immunological Interest, 5th edition. US Department of Health and Human Services, Public Service, NIH, Washington) and more recent versions.
  • Antibodies usually contain 3 heavy chain CDRs and 3 light chain CDRs.
  • the term CDR is used herein to indicate that, depending on the situation, one of these regions or some or even all of these regions contains most of the amino acid residues responsible for binding by the affinity of the antibody to the antigen or epitope recognized by the antibody.
  • the third CDR (HCDR3) of the heavy chain has a larger size of variability (larger diversity is basically due to the gene arrangement mechanism that makes it produced). Although the longest known size is 26, it can be as short as 2 amino acids.
  • the length of the CDR can also vary according to the adaptable length of the specific basic framework. Functionally, HCDR3 plays an important role in determining the specificity of antibodies (Segal et al., PNAS, 71: 4298-4302, 1974, Amit, Science, 233: 747-753, 1986, Chothia, J. Mol.
  • CDR group includes CDR1, CDR2 and CDR3. Therefore, the HCDR group refers to HCDR1, HCDR2, and HCDR3, and the LCDR group refers to LCDR1, LCDR2, and LCDR3.
  • VH and VL regions and CDR variants of the present invention including those whose amino acid sequences are listed herein and which can be applied to PD-L1 targeted binding agents and antibodies, can be required by sequence change or mutation methods and screening. The characteristics of antigen targeting are obtained.
  • required properties include, but are not limited to: increased binding affinity to antigen relative to known antibodies specific to antigen; neutralization of increased antigen activity relative to known antibodies specific to antigen (if the activity is already Known); specific ability to compete with known antibodies or ligands for antigen at a specific molar ratio; the ability to immunoprecipitate ligand-receptor complexes; the ability to bind to specific epitopes; linear epitopes, For example, peptide sequences identified by peptide binding scanning, for example, peptides screened in linear and/or defined conformations; conformational epitopes, formed by non-contiguous residues; ability to modulate new biological activities of B7-H1 or downstream molecules ; The ability to bind and/or neutralize B7-H1 and/or any other required properties.
  • the properties of antibodies can be derived from empirical and theoretical models of antibody sequence, functional structure, and three-dimensional structure (for example, analysis of possible contact residues or calculated physicochemical properties, and these properties can be considered individually and in combination.
  • the antibody antigen binding site composed of VH domain and VL domain is usually formed by 6 polypeptide loops: 3 from the light chain variable domain (VL) and three from the heavy chain variable domain (VH).
  • VL light chain variable domain
  • VH heavy chain variable domain
  • the binding site loop has a small amount of main One of the chain conformations: canonical structure. It has been shown that the canonical structure formed in a specific loop is determined by its size and the presence of certain residues at key positions in both the loop and the framework region.
  • sequence-structure relationship can be used to predict those residues in antibodies of known sequences (except for the unknown three-dimensional structure), which are important in maintaining the three-dimensional structure of its CDR loops and thereby maintaining binding specificity. These predictions can be supported by comparing the predictions with the output from the leading optimal experiment.
  • any freely available or commercial software package such as WAM
  • WAM any freely available or commercial software package
  • a protein visualization and analysis software package such as Insight II (Accelrys, Inc.) or Deep View, can then be used to evaluate the possible substitutions at each position in the CDR. This information can then be used to perform substitutions that may have minimal or beneficial effects on activity or impart other desired properties.
  • polypeptide fragment refers to a polypeptide having amino-terminal and/or carboxy-terminal deletions, but where the remaining amino acid sequence is the same as the corresponding position in the deduced naturally occurring sequence, for example, from a full-length cDNA sequence. Fragments are generally at least 5, 6, 8, or 10 amino acids in length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length, and even more preferably at least in length 70 amino acids.
  • analog refers to a polypeptide consisting of a fragment of at least 25 amino acids that has sufficient identity with a portion of the deduced amino acid sequence, and which has at least one of the following properties: (1) In appropriate Under binding conditions, specific binding to PD-L1, (2) the ability to block appropriate PD-L1 protein binding, or (3) the ability to inhibit PD-L1 activity.
  • polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) with respect to naturally occurring sequences.
  • the analog is generally at least 20 amino acids in length, preferably at least 50 amino acids or longer in length, and can generally be as long as a full-length naturally occurring polypeptide.
  • Peptide analogs are often used in the pharmaceutical industry as non-peptide drugs with properties similar to template peptides. These types of non-peptide compounds are referred to as "peptidomimetics" or "peptoids". Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS page 392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference. Computer-based molecular modeling is often used to develop such compounds. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce equivalent therapeutic or preventive effects.
  • the systematic substitution of one or more amino acids of the consensus sequence with the same type of D-amino acid for example, D-lysine instead of L-lysine
  • D-lysine instead of L-lysine
  • antibody used herein can be a single chain antibody, oligoclonal antibody, polyclonal antibody, monoclonal antibody (including full-length monoclonal antibody), camelized, alone or in combination with other amino acid sequences provided by known techniques.
  • Antibodies can be derived from any species.
  • the antibody comprises a polypeptide or a group of polypeptides, which consists of at least one binding domain formed from the folding of a polypeptide chain with a three-dimensional binding space, wherein the inner surface shape and charge distribution are complementary to the characteristics of the antigen's epitope .
  • Antibodies generally have a tetrameric form, consisting of two identical pairs of polypeptide chains, each pair having a "light” chain and a "heavy” chain. The variable region of each light/heavy chain forms an antibody binding site. Natural antibodies are usually heterotetrameric glycoproteins with a molecular weight of about 150Kd, composed of two identical light chains and two identical heavy chains.
  • Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bonds.
  • Each heavy chain has a variable region (VH) at one end, followed by a constant region.
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is the same as the variable region of the heavy chain. District relative.
  • the light chain is classified as lambda chain or kappa chain based on the amino acid sequence of the light chain constant region.
  • the variable region of the kappa light chain may also be referred to herein as VK.
  • variable region can also be used to describe the variable region of a heavy or light chain. Specific amino acid residues form the boundary between the variable region of the light chain and the variable region of the heavy chain.
  • the variable region of each light/heavy chain pair forms an antibody binding site.
  • the antibody can be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice and the like.
  • antibody includes binding fragments of the antibodies of the present invention, and exemplary fragments include single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fv fragments, Fab fragments, F(ab)' fragments , F(ab′)2 fragments, antibody fragments showing the required biological activity, disulfide bond stabilized variable regions (dsFv), dimeric variable regions (diabodies), anti-idiotypic antibodies, intracellular antibodies , Linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments and any of the above epitope binding fragments.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, that is, molecules containing antigen binding sites.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), of any type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.
  • Enzyme (papain) digestion of the antibody produces two identical antigen-binding fragments, also called “Fab” fragments, and "Fc” fragments, which have no antigen-binding activity but have the ability to crystallize.
  • Enzyme (pepsin) digestion of the antibody produces F(ab')2 fragments, where the two arms of the antibody molecule remain connected and contain two antigen binding sites. F(ab')2 fragments have the ability to cross-link antigens.
  • Fv refers to the smallest fragment of an antibody that retains both the antigen recognition site and the antigen binding site. This region is composed of a dimer of a heavy chain variable region and a light chain variable region that are tightly non-covalently bonded or covalently bonded.
  • the 3 CDRs of each variable region interact to define the antigen binding site on the surface of the VH-VL dimer.
  • the 6 CDRs confer antigen binding specificity to the antibody.
  • a single variable region or only half of the 3 CDRs specific to the antigen still has the ability to recognize and bind to the antigen.
  • Fab refers to a fragment of an antibody, which, in addition to the heavy chain variable region and the light chain variable region, also includes the light chain constant domain and the CH1 region of the heavy chain.
  • dAb refers to a fragment of an antibody that is the smallest functional binding unit of a human antibody.
  • DAb is a single domain antibody and contains the VH of the antibody heavy chain or the VL of the antibody light chain.
  • Each dAb contains 3 of the 6 naturally occurring CDRs (Ward et al., Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544-546 (1989); Holt et al., Domain antibodies: protein for therapy , Trends Biotechnol. 21, 484-49 (2003)).
  • Their molecular weight ranges from 11kDa to 15kDa, they are 4 times smaller than the antigen-binding fragment (Fab) 2 and half the molecular size of the single-chain Fv (scFv).
  • camel origin refers to antibody molecules composed of heavy chain dimers, which lack light chains but have a large number of antigen-binding components (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature363:446-448).
  • diabodies refers to small antibody fragments with two antigen binding sites, the fragments of which include the heavy chain variable region connected to the light chain variable region in the same polypeptide chain. By using a sufficiently short linker to pair two domains on the same chain, the domains are paired with the complementary domains of another chain and two antigen binding sites are constructed. Diabodies are more fully described in, for example, EP404,097; WO93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
  • binding fragments are (Ward, ES, et al., (1989) Nature 341, 544-546) Fab fragments composed of VL, VH, CL and CH1 domains; (McCafferty et al., (1990) Nature, 348, 552-554) by VH (Holt et al., (2003) Trends in Biotechnology 21,484-490) Fv fragment composed of the VL and VH domains of a single antibody; dAb fragment (Ward, ES etc., Nature 341,544-546 ( 1989), McCafferty et al., (1990) Nature, 348,552-554, Holt et al., (2003) Trends in Biotechnology 21, 484-490), which consist of VH or VL domains; isolated CDR regions; F(ab')2 fragments, A bivalent fragment containing two linked Fab fragments; a single-chain Fv molecule (sc
  • Fv, scFv or diabody molecules can be stabilized by the incorporation of a disulfide bridge connecting the VH and VL domains (Reiter, Y. et al., Nature Biotech, 14, 1239-1245, 1996).
  • Minibodies containing scFvs that bind to the CH3 domain can also be produced (Hu, S. et al., (1996) Cancer Res., 56, 3055-3061).
  • binding fragments are Fab', which differ from Fab fragments due to the addition of several residues to the carboxy terminus of the CH1 domain of the heavy chain, the residues including one or more cysteines from the hinge region of an antibody , And Fab'-SH, which are Fab' fragments in which the cysteine residues of the constant domain carry free thiol groups.
  • variable refers to the fact that certain parts of antibody variable regions differ greatly in sequence between antibodies and are responsible for the binding specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed in the variable domains of antibodies. It is concentrated in fragments called complementarity determining regions (CDR) in both the light chain variable domain and the heavy chain variable domain. The highly conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the heavy and light chains of natural antibodies each contain 4 FR regions, which adopt a ⁇ -sheet configuration to a large extent and are connected by 3 CDRs, which form a connected ⁇ -sheet structure and in some In this case, a loop that forms part of the ⁇ -sheet structure.
  • the CDRs in each chain are closely combined with the CDRs from other chains through the FR region, which helps to form the antigen binding site of the antibody (see, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service , National Institutes of Health, Bethesda, MD (1991)).
  • the constant region generally does not directly participate in antigen binding, but can affect antigen binding affinity and can exhibit various effector functions, such as ADCC, CDC, and/or antibody involvement in apoptosis.
  • hypervariable region refers to the amino acid residues of an antibody related to its binding to an antigen.
  • the hypervariable region includes amino acid residues of the "complementarity determining region” or "CDR" (e.g., residues 24-34 (L1), residues 50-56 (L2), and 89 of the light chain variable region). -97 residues (L3) and 31-35 residues (H1), 50-65 residues (H2) and 95-102 residues (H3) of the heavy chain variable region (Kabat et al. , Sequences of Proteins of Immunological Interest, 5th edition.
  • CDR complementarity determining region
  • Targeted binding protein refers to agents that preferentially bind to a target site, such as antibodies or binding fragments thereof.
  • the targeted binding agent is specific for only one target site. In other embodiments, the targeted binding agent is specific for more than one target site.
  • the targeted binding agent may be a monoclonal antibody and the target site may be an epitope.
  • the targeted binding agent may comprise at least one antigen binding domain (e.g., CDR) of an antibody, wherein the domain is fused to or contained in a heterologous protein scaffold, for example, a non-antibody protein scaffold.
  • CDR antigen binding domain
  • binding fragment of the antibody is produced by recombinant DNA technology, or by enzymatic or chemical cleavage of the intact antibody. Binding fragments include Fab, Fab', F(ab')2, Fv, dAb, and single chain antibodies. Antibodies other than "bispecific” or “bifunctional” antibodies are understood to have the same each binding site. When excess antibody reduces the amount of receptor bound to the counter receptor by at least about 20%, 40%, 60%, or 80%, and more usually more than about 85% (as measured in an in vitro competitive binding assay ), the antibody basically inhibits the binding of the receptor to the counter receptor.
  • epitope includes any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor.
  • Epitope determinants usually consist of surface groups (such as amino acids or sugar side chains) of chemically active molecules, and may (but not always) have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • the dissociation constant is ⁇ 1 ⁇ M, preferably ⁇ 100 ⁇ M and most preferably ⁇ 10 ⁇ M, the antibody is said to specifically bind to the antigen.
  • the term "medicament” represents a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • the "active" or “active” of the PD-L1 polypeptide refers to the part of the PD-L1 polypeptide that has the biological activity or immunological activity of the natural PD-L1 polypeptide.
  • "Biological” as used herein refers to the biological function caused by the activity of the native PD-L1 polypeptide. Preferred biological activities of PD-L1 include, for example, cell proliferation, cell adhesion and invasion induced by PD-L1.
  • mammal refers to any animal that is considered a mammal. Preferably, the mammal is a human.
  • animal includes animals that are considered mammals.
  • the animal is a human.
  • patient includes humans and test animals.
  • mAb refers to a monoclonal antibody.
  • liposome refers to a small vesicle that can be used to deliver a drug that can contain the PD-L1 polypeptide of the present invention or an antibody to such a PD-L1 polypeptide to a mammal.
  • Label refers to the addition of a detectable moiety to a polypeptide, such as a radioactive label, a fluorescent label, an enzyme label, a chemiluminescent label, or a biotin-based.
  • the radioisotope or radionuclide may include 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, the fluorescent label may include rhodamine, rare earth phosphor or FITC, and the enzyme Labels may include horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase.
  • Additional markers include, by way of example and not limitation: enzymes such as glucose-6-phosphate dehydrogenase, ⁇ -D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, Lysozyme, malate dehydrogenase and peroxidase; dyes; additional fluorescent labels or fluorescent agents include, for example, fluorescein and its derivatives, fluorochromes, GFP (green fluorescent protein), dansyl, umbelliferone , Phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescein; fluorophores such as cryptate rare earth compounds and chelates, such as europium, etc.
  • enzymes such as glucose-6-phosphate dehydrogenase, ⁇ -D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase
  • chemiluminescence labeling or chemical Luminescent substances such as isoluminol, luminol and dioxane; sensitizers; coenzymes; enzyme substrates; particles, such as latex particles or carbon particles; metal sols; microcrystals; liposomes; cells, etc.
  • toxin moieties such as those selected from Pseudomonas exotoxin (PE or its cytotoxicity) Fragment or mutant), diphtheria toxin or its cytotoxic fragment or mutant, botulinum toxin A, B, C, D, E or F, ricin or its cytotoxic fragment such as ricin A, acacia toxin Or its cytotoxic fragment, saporin or its cytotoxic fragment, pokeweed antiviral toxin or its cytotoxic fragment, and bryodin1 or its cytotoxic fragment.
  • Pseudomonas exotoxin PE or its cytotoxicity
  • diphtheria toxin or its cytotoxic fragment or mutant diphtheria toxin or its cytotoxic fragment or mutant
  • botulinum toxin A, B, C, D, E or F botulinum toxin A, B, C, D, E or F
  • ricin or its cytotoxic fragment such as ricin A, acacia
  • pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • Other chemical terms used herein are used according to conventional usage in the field, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., McGraw-Hill, San Francisco (1985)).
  • substantially pure means that the subject substance is the main substance present (that is, its moles in the composition are more abundant than any other individual substance), and preferably the substantially pure composition
  • a fraction is a composition in which the subject substance accounts for at least 50% by mole of all macromolecular substances present.
  • a substantially pure composition will account for greater than about 80% of all macromolecular species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%.
  • the subject substance is purified to be substantially homogeneous (contaminant substances cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular substance.
  • ADCC antibody-dependent cytotoxicity
  • non-specific cytotoxic cells such as natural killer (NK) cells, monocytes, neutrophils
  • FcR Fc receptors
  • the main cells that mediate ADCC are NK cells, which only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 9 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • an in vitro ADCC assay can be performed, such as described in US Patent No. 5,500,362 or No. 5,821,337. Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and NK cells.
  • PBMC peripheral blood mononuclear cells
  • NK cells Alternatively, or in addition, the ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1988).
  • C1q the first component of complement
  • C1q is a large, ⁇ 410kD complex glycoprotein (Cooper, N.R.1985.Adv.Immunol.37:151) that exists in human serum at a concentration of 70 ⁇ g/mL.
  • C1r, C1s, and C1q together with two serine proteases form the first component of complement—complex C1.
  • At least two of the N-terminal spherical heads of C1q must bind to the Fc of Igs for C1 activation to initiate the complement cascade (Cooper, N.R. 1985. Adv. Immunol. 37: 151).
  • antibody half-life means the pharmacokinetic properties of the antibody, which is a measure of the average survival time of the antibody molecule after administration.
  • Antibody half-life can be expressed as the time required to eliminate 50% of the known amount of immunoglobulin from the patient's body or its specific compartments or other tissues, for example, as measured in serum or plasma, ie circulating half life.
  • the half-life may vary according to the immunoglobulin or the type of immunoglobulin. Generally, an increase in the half-life of an antibody results in an increase in the circulating mean residence time (MRT) of the administered antibody.
  • MRT mean residence time
  • isotype refers to the classification of antibody heavy chain constant regions or light chain constant regions.
  • the constant region of an antibody does not participate in binding antigen, but exhibits a variety of effector functions.
  • a given human antibody or immunoglobulin can be assigned to one of the five main immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM.
  • Some of these classes can be further divided into subclasses (isotypes), for example, IgG1 ( ⁇ 1), IgG2 ( ⁇ 2), IgG3 ( ⁇ 3) and IgG4 ( ⁇ 4), and IgA1 and IgA2.
  • the heavy chain constant regions corresponding to different types of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the structures and three-dimensional configurations of different kinds of immunoglobulins are well known.
  • human immunoglobulin classes only human IgG1, IgG2, IgG3, IgG4 and IgM are known to activate complement. It is known that human IgG1, IgG2, IgG3 and IgG4 bind to Fc ⁇ receptors, which mediate multiple effector functions including ADCC.
  • the light chain constant region can be classified into two main categories, kappa and lambda.
  • therapeutic antibodies require complement-dependent cytotoxicity.
  • complement-dependent cytotoxicity There are many antibody isotypes with complement-dependent cytotoxicity, including but not limited to the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgA, human IgG1, and human IgG3.
  • therapeutic antibodies need to bind to Fc receptors on effector cells and participate in antibody-dependent cytotoxicity.
  • antibody isotypes with antibody-dependent cytotoxic antibodies including but not limited to the following: murine IgG2a, murine IgG2b, murine IgG3, human IgG1, and human IgG3.
  • the antibody produced initially does not have to have the said isotype, and the antibody can be isotype switched using conventional techniques well known in the art.
  • the technologies include direct recombination technology (see U.S. Patent No. 4,816,397) and the use of cell-cell fusion technology (see U.S. Patent Nos. 5,916,771 and 6,207,418).
  • the “whole blood assay” uses unfractionated blood as a source of natural effectors. Blood contains complement in plasma, as well as FcR-expressing cellular effectors, such as polymorphonuclear leukocytes (PMN) and monocytes (MNC). In this way, the whole blood assay allows the simultaneous evaluation of the synergy of the effector mechanisms of ADCC and CDC in vitro.
  • PMN polymorphonuclear leukocytes
  • MNC monocytes
  • a “therapeutically effective” amount is an amount that provides some improvement or benefit to the subject.
  • a “therapeutically effective” amount is an amount that provides a certain alleviation, relief, and/or reduction of at least one clinical symptom.
  • the clinical symptoms associated with the disease that can be treated by the method of the present invention are well known to those skilled in the art. Moreover, those skilled in the art will understand that the therapeutic effect need not be thorough or curative, as long as it provides certain benefits to the subject.
  • Exemplary cancers include bladder tumors, breast tumors, prostate tumors, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain and CNS cancer (e.g. glioma), cervical cancer, choriocarcinoma, colon and rectal cancer , Connective tissue cancer, cancer of the digestive system; endometrial cancer, esophageal cancer; eye cancer, head and neck cancer, gastric cancer; intraepithelial tumor; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (such as small cell and non-small cell lung cancer) ); Lymphoma, including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma; melanoma; myeloma, neuroblastoma, oral cancer (for example, lips, tongue, mouth and pharynx); ovarian cancer; Pancreatic cancer, retinoblastoma; rhabdomyosarcoma; rectal cancer, kidney cancer,
  • Exemplary chronic infections include HIV infection, hepatitis B virus (HBV) infection, and hepatitis C virus (HCV) infection.
  • Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25kD) and a "heavy” chain (about 50-70kD).
  • the amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition.
  • the carboxy terminal part of each chain defines the constant region that is mainly responsible for effector functions.
  • Light chains are classified into kappa light chains and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha or epsilon, and define the isotype of the antibody as IgM, IgD, IgA, and IgE, respectively.
  • variable and constant regions are connected by a "J" region of about 12 or more amino acids, where the heavy chain also includes a "D” region of about 10 or more amino acids.
  • J Joint Immunology Ch. 7
  • D double-chain region
  • a complete antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.
  • the chains all show the same general structure: relatively conservative framework regions (FR) connected by 3 hypervariable regions (also called CDRs).
  • the CDRs from the two chains of each pair are aligned by the framework regions to enable binding to a specific epitope.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains From N-terminus to C-terminus, both the light chain and the heavy chain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains.
  • amino acids are assigned to each domain Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987) ; Chothia et al., Nature 342:878-883 (1989).
  • Bispecific or bifunctional antibodies are artificial hybrid antibodies with two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by various methods including fusion of hybridomas or linking of Fab' fragments. See Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148: 1547-1553 (1992).
  • Bispecific antibodies do not exist in the form of fragments with a single binding site (for example, Fab, Fab' and Fv).
  • VH and VL are paired to provide an antibody antigen binding site, although a separate VH or VL can be used to bind the antigen.
  • Human antibodies avoid certain problems associated with antibodies with mouse or rat variable and/or constant regions.
  • Mouse or rat-derived antibodies can lead to rapid elimination of antibodies or can lead to an immune response by the patient against the antibodies.
  • fully human antibodies can produce fully human antibodies by introducing functional human antibody loci into rodents, other mammals, or animals. To generate.
  • the recovered lymphocytes are fused with a bone marrow type cell line to prepare a permanent hybridoma cell line.
  • These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the target antigen.
  • Provided herein are methods for the generation of multiple hybridoma cell lines specific for PD-L1.
  • the characterization of the antibodies produced by the cell line including the nucleotide and amino acid sequence analysis of the heavy and light chains of the antibody.
  • B cells can be assayed directly.
  • CD19+ B cells can be isolated from hyperimmunized mice and allowed to proliferate and differentiate into antibody-secreting plasma cells. The antibody from the cell supernatant was then screened for reactivity against PD-L1 immunogen by ELISA. The supernatant can also be screened for immunoreactivity against PD-L1 fragments to further mark different antibodies that bind to the target functional domain on B7-H1.
  • Antibodies can also be screened for other related human proteins and directed against rats, mice, and non-human primates, such as cynomolgus monkeys, orthologs of B7-H1, and ultimately determine species cross-reactivity.
  • B cells from wells containing the antibody of interest can be immortalized by a variety of methods, including fusion to generate hybridomas from separate wells or from combined wells, or by infection with EBV or by known immortalization
  • the gene is transfected and then inoculated in a suitable medium.
  • the B7-H1 specific hemolytic plaque assay is then used to isolate individual plasma cells that secrete antibodies with desired properties (see, for example, Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)).
  • the cells targeted for lysis are preferably sheep red blood cells (SRBC) covered with B7-H1 antigen.
  • SRBC sheep red blood cells
  • antibodies as described herein can be expressed in cell lines other than hybridoma cell lines. Sequences encoding specific antibodies can be used to transform suitable mammalian host cells. Transformation can be performed by any known method for introducing polynucleotides into host cells, including, for example, packaging the polynucleotides in a virus (or in a viral vector) and transducing with the virus (or vector) Host cells, or by transfection procedures known in the art, as shown in U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are incorporated herein by reference). The transformation method used depends on the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, and protoplast fusion , Electroporation, encapsulation of polynucleotides in liposomes, and direct DNA microinjection into the nucleus.
  • myeloma, CHO cells or other cell lines with heavy chains of any desired isotype are prepared, and myeloma, CHO cells or other cell lines with light chains are prepared.
  • the cells can then be fused and cell lines expressing intact antibodies can be isolated.
  • antibody candidates are generated that meet the required "structural" attributes as described above, and through isotype conversion they can generally have at least some of the required "functional” attributes.
  • a myeloma, CHO cell or other cell line with a heavy chain of the desired isotype is prepared and a myeloma, CHO cell or other cell line with a light chain is prepared.
  • the cells can then be fused and cell lines expressing intact antibodies can be isolated.
  • antibody candidates are generated that meet the required "structural" attributes as described above, and through isotype conversion they can generally have at least some of the required "functional” attributes.
  • Embodiments of the invention include the antibodies listed below.
  • the underline indicates the CDR region.
  • the CDR sequence and its numbering of each antibody heavy chain are shown in Table 1, and the CDR sequence and its numbering of the antibody light chain are shown in Table 2.
  • the embodiment of the present invention includes a pharmaceutical preparation of a PD-L1 targeted binding agent for treating diseases; preferably, the PD-L1 targeted binding agent is an anti-PD-L1 antibody.
  • the formulation will inhibit the binding of PD-L1 to one or more of its cognate ligands, thereby treating pathological conditions.
  • the antibody of the present invention preferably has sufficient affinity to effectively inhibit the activity of PD-L1, or inhibit the binding of PD-L1 to one or more of its cognate ligands, and preferably has a sufficient duration of action to allow in humans Infrequent administration. The extended duration of action will allow for a less frequent and more convenient dosing regimen via alternate parenteral routes such as subcutaneous or intramuscular injections.
  • Sterile preparations can be produced, for example, by filtration through a sterile filter membrane before or after freeze-drying and reconstitution of the antibody.
  • Antibodies are generally stored in lyophilized form or in solution.
  • the therapeutic antibody composition is generally placed in a container with a sterile access port, for example, an intravenous solution pack or a vial with a connector that allows the preparation to be retrieved, such as a stopper pierceable by a hypodermic injection needle.
  • the PD-L1 targeted binding agent can be used to treat cancer. It includes administering an effective amount of PD-L1 targeted binding agent or a composition containing the same to the patient.
  • the cancer is selected from the group consisting of: bladder tumor, breast tumor, prostate tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain and CNS cancer (e.g.
  • glioma glioma), cervical cancer, choriocarcinoma Cancer, colon and rectal cancer, connective tissue cancer, cancer of the digestive system; endometrial cancer, esophageal cancer; eye cancer, head and neck cancer, gastric cancer; intraepithelial tumor; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (such as small Cell and non-small cell lung cancer); lymphoma, including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma; melanoma; myeloma, neuroblastoma, oral cancer (for example, lip, tongue, mouth and Pharynx); ovarian cancer; pancreatic cancer, retinoblastoma; rhabdomyosarcoma; rectal cancer, kidney cancer, cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer, cancer of the urinary system, and Other cancers and s
  • the PD-L1 targeted binding agent can be used in combination with a chemotherapeutic agent.
  • the PD-L1 targeted binding agent can be used to treat infections and can be used in combination with antibiotics.
  • the antibiotic is an antiviral agent; also preferably, the antiviral agent is a reverse transcriptase inhibitor; more preferably, the reverse transcriptase inhibitor is a polymerase inhibitor.
  • the infection is a chronic infection, such as HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) infection.
  • the infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi and protozoa.
  • the PD-L1 targeted binding agent can also be used in combination with at least one vaccine.
  • Figure 1 shows the results of purification of PD-L1.
  • Figure 2 is a graph showing the results of affinity determination of anti-PD-L1 antibodies.
  • Figure 3 is an immunofluorescence staining image of anti-PD-L1 antibody.
  • Figure 4 is a graph showing the results of the EC50 determination of immunofluorescence titer of anti-PD-L1 antibody.
  • Figure 5 is a graph showing the results of affinity determination of the humanized anti-PD-L1 antibody.
  • Figure 6 is a graph showing the EC50 determination result of immunofluorescence titer of humanized anti-PD-L1 antibody.
  • Figure 7 shows the results of the K562-PD-L1 film surface binding test.
  • Figure 8 is a graph showing the results of the PD-L1 binding test on the surface of Raji-PD-L1 membrane.
  • Figure 9 shows the results of the PD-L1 binding test on the membrane surface.
  • Figure 10 is a schematic diagram of the PD-L1 CAR element structure.
  • Figure 11 is a graph of PD-L1 chimeric antigen receptor (PD-L1 CAR) activity.
  • Example 1 Expression and purification of recombinant human PD-L1-his protein
  • the pVKD1.0-hPD-L1-his vector was transfected into 293T cells mediated by PEI, the supernatant was collected, and purified by nickel column chromatography, and then purified by ultrafiltration to obtain the purified human PD-L1-his recombinant protein, the specific method Please refer to the kit instructions (GE Healthcare, article number: 17-5247-01). The purification results are shown in Figure 1.
  • Example 2 Mouse immunity, hybridoma fusion and clone screening
  • mice Five female BALB/c mice aged 6-8 weeks were purchased from the Animal Experiment Center of Soochow University and raised in the SPF animal room of the Animal Experiment Center of Soochow University. After fully emulsifying the human PD-L1-his recombinant protein in Example 1 with complete Freund's adjuvant, complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), the back was injected subcutaneously at 10 ⁇ g/mouse. Immunize BALB/c mice 3 to 4 times at intervals of one month.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice were immunized with 50 ⁇ g human PD-L1-His recombinant protein by intraperitoneal injection. The immunized mice were sacrificed by cervical dislocation, and spleen cells of the mice were obtained. The mouse spleen cells and SP 2/0 myeloma cells were washed three times with serum-free DMEM medium, then centrifuged at 500 g for 5 minutes, resuspended in 10 mL serum-free DMEM medium, and the cells were counted.
  • SP 2/0 myeloma cells 5:1 and mix, centrifuge at 500 g for 5 minutes, remove the supernatant and use 1 mL of 50% PEG for cell fusion. After the fusion is completed, centrifuge at 500g for 5 minutes, remove the supernatant, resuspend in DMEM medium containing 20% FBS, add 2 ⁇ 10 5 cells/well to a 96-well plate, and incubate overnight at 37°C. On the next day, HAT-containing hybridoma selection medium (Sigma, catalog number A9666) was added, 100 ⁇ L/well. Afterwards, every 3 days, half of the medium was removed and supplemented with hybridoma selection medium. On the 14th day, 100 ⁇ L of supernatant was taken from each well, and the antibody activity in the hybridoma supernatant was screened by ELISA.
  • HAT-containing hybridoma selection medium Sigma, catalog number A9666
  • the ELISA method is well known in the art and is briefly described as follows.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument Factory. Recombinant human PD-L1-his protein was provided by Suzhou Industrial Park Weike Biotechnology Co., Ltd.
  • the protein was coated with NaHCO 3 buffer (pH 9.6) at 4°C overnight, and the coating concentration was 1 ⁇ g/mL.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • PBST phosphate buffered saline
  • Tween 20 phosphate buffered saline
  • the wells with an OD value> 1.0 were defined as positive clones.
  • the positive cell clones were picked from the 96-well cell culture plate and transferred to 24-well culture. Two days later, the above-mentioned ELSIA method was used to retest the supernatant antibody activity. Retest positive clones were transferred to a 6-well plate for culture, and then the cells were collected for subsequent experiments. The results of the titration after the retest are shown in Table 3. Select clones with titer> 1000 after retest for further study.
  • the hybridoma cells were lysed and treated with Trizol reagent (Thermo, catalog number 15596026), and then the hybridoma cell RNA was extracted.
  • Trizol reagent Thermo, catalog number 15596026
  • the specific method is well known in the art, please refer to the Trizol reagent manual.
  • the amplified product is ligated to the pMD19T vector (Bao Biological, Item No. 6013) and sent for sequencing. At least 10 clones of each VH and VL are sent for sequencing to obtain antibody VH and VL nucleic acid sequences. And analyze the CDR region sequence of each antibody, see Table 1 and Table 2 for the CDR region sequence of each antibody.
  • the hybridoma cells obtained in Example 2 were amplified with DMEM medium containing 10% FBS, and the hybridoma supernatant was collected and purified with a protein A column (GE Healthcare, catalog number: 28985254).
  • a protein A column GE Healthcare, catalog number: 28985254
  • the kit instructions to obtain The EC50 titer of each purified mouse monoclonal antibody was determined by ELISA method.
  • the method of ELISA is well known to those skilled in the art and is briefly described as follows.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument Factory.
  • the recombinant human PD-L1-his protein prepared in Example 1.
  • the protein was coated with NaHCO 3 buffer (pH 9.6) at 4°C overnight, and the coating concentration was 1 ⁇ g/mL.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • PBST phosphate buffered saline
  • TMB 3,3,5,5-tetramethylbenzidine
  • the EC50 titers of anti-PD-L1 antibodies are shown in Table 4.
  • Friguet method J Immunol Methods. 1985; 77: 305-19 was used to determine the equilibrium dissociation constant Kd of each antibody, that is, the affinity of the antibody.
  • the main steps are briefly described as follows.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument Factory.
  • the recombinant human PD-L1-his protein was prepared in Example 1.
  • the protein was coated with NaHCO 3 buffer (pH 9.6) at 4°C overnight, and the coating concentration was 5 ⁇ g/mL.
  • PBS phosphate buffered saline
  • FBS fetal bovine serum
  • PBS phosphate buffered saline
  • FBS fetal bovine serum
  • the VH regions of each antibody gene obtained in Example 3 were cloned into the vector Abvec-hIgG1 (GenBank: FJ475055.1) with the human IgG1 CH1 structural region and its Fc structural region by molecular biology techniques well known in the art.
  • the VL regions of each antibody gene obtained in Example 3 were cloned into the vector AbVec-hIgKappa (GenBank: FJ475056.1) with human kappa light chain constant region by molecular biology techniques well known in the art. Then, the plasmids expressing the humanized heavy chain and the humanized light chain were co-transfected into 293T cells, and the supernatant was collected to obtain the anti-PD-L1 humanized antibody.
  • the cell transfection supernatant containing the humanized antibody obtained in Example 5 was purified by a protein A column (GE Healthcare, catalog number: 17-0402-01), and the specific method refers to the kit instructions. Each purified humanized antibody was obtained, and the results are shown in Table 7.
  • Example 7 Determination of EC50 titer of humanized antibody
  • the EC50 activity of anti-PD-L1 antibody was determined by ELISA.
  • the method of ELISA is well known to those skilled in the art and is briefly described as follows.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument Factory.
  • the recombinant human PD-L1-his protein prepared in Example 1 was coated with NaHCO 3 buffer (pH 9.6) at 4°C overnight, and the coating concentration was 1 ⁇ g/mL.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • PBST phosphate buffered saline
  • TMB 3,3,5,5-tetramethylbenzidine
  • Example 8 Affinity determination of humanized anti-PD-L1 antibody
  • the Kd value of the humanized antibody was measured by the Friguet method in Example 4. The results are shown in Figure 5, and the results are summarized in Table 9.
  • Example 9 Affinity of humanized anti-PD-L1 antibody (immunofluorescence method)
  • Example 4 The immunofluorescence method in Example 4 was used to determine the affinity of the humanized antibody for binding to natural PD-L1.
  • Example 10 Ability of anti-PD-L1 antibody to bind to PD-L1 antigen on the surface of tumor cell membrane
  • Flow cytometry was used to detect the ability of anti-PD-L1 antibody to bind the PD-L1 antigen on the surface of tumor cell membrane.
  • the total volume of the staining system is 50 ⁇ L of PBS buffer (staining buffer) containing 2% FBS.
  • the primary antibody is the anti-PD-L1 antibody obtained in Example 2.
  • the final concentration of the antibody is adjusted to 2 ⁇ g/mL and incubated at 4°C 30min. After finishing, add 500 ⁇ L of staining buffer to each tube for elution twice, and centrifuge at 800g for 3 minutes each time. After the primary antibody staining, prepare the secondary antibody staining mixture, add the secondary antibody donkey anti-mouse-IgG (H+L) (AF647 Conjugate, Abcam) at 1:1000, and the total volume of the staining system is also 50 ⁇ L of staining buffer. Stain for 30 minutes at 4°C, add 500 ⁇ L of staining buffer to each tube for elution twice, and centrifuge at 800g for 3 minutes each time.
  • H+L secondary antibody donkey anti-mouse-IgG
  • Example 11 Chimeric antigen receptor (CAR) activity of anti-PD-L1 antibody
  • VL and VH regions of the PD-L1 antibody were spliced for gene synthesis, and the synthesized PD-L1 chimeric antigen receptor genes were all transferred to the lentiviral vector pHAGE-EF1 ⁇ -MCS-ZsGreen (purchased from Harvard Medical School, catalog number :EvNO00061636), so as to obtain the CAR that can recognize human PD-L1 based on 6 PD-L1 monoclonal antibodies: 1C2 CAR, 1D4 CAR, 1G11 CAR, 2F7 CAR, 3D7 CAR and 6A3 CAR (PD-L1 CAR element structure diagram see Figure 10).
  • CD8 SP represents the sequence derived from the human CD8 antigen signal peptide (CD8 SP)
  • PD-L1 VL represents the VL sequence of the anti-PD-L1 antibody
  • PD-L1 VH represents the VH sequence of the anti-PD-L1 antibody
  • CD28, 4 -1BBL and CD3 ⁇ represent their respective components, which are commonly used components for CAR construction.
  • the coding nucleic acid sequence of CD8 SP is shown in SEQ ID NO: 51, and the amino acid sequence is shown in SEQ ID NO: 52;
  • the coding nucleic acid sequence of CD28 element is shown in SEQ ID NO: 53, and the amino acid sequence is shown in SEQ ID NO.
  • the coding nucleic acid sequence of the 4-1BBL element is shown in SEQ ID NO: 55, and the amino acid sequence is shown in SEQ ID NO: 56; the coding nucleic acid sequence of the CD3 ⁇ element is shown in SEQ ID NO: 57, and the amino acid sequence is shown in SEQ ID NO: 57. As shown in SEQ ID NO: 58.
  • the coding nucleic acid sequence of 1C2 CAR is shown in SEQ ID NO: 59, and the amino acid sequence is shown in SEQ ID NO: 60; the coding nucleic acid sequence of 1D4 CAR is shown in SEQ ID NO: 61, and the amino acid sequence is shown in SEQ ID NO: 62 Show; 1G11 CAR coding nucleic acid sequence is shown in SEQ ID NO: 63, amino acid sequence is shown in SEQ ID NO: 64; 2F7 CAR coding nucleic acid sequence is shown in SEQ ID NO: 64, and amino acid sequence is shown in SEQ ID NO: 66; 3D7 CAR coding nucleic acid sequence is shown in SEQ ID NO: 67, amino acid sequence is shown in SEQ ID NO: 68; 6A3 CAR coding nucleic acid sequence is shown in SEQ ID NO: 69, and amino acid sequence is shown in SEQ ID NO: 70 shown.
  • Transfect the lentivirus packaging plasmid into 293T cells collect the virus supernatant after 48 hours of transfection, centrifuge at 3000 rpm, 4°C for 10 minutes to remove cell debris, filter through a 0.45 ⁇ m filter, and add lentivirus concentrate at a volume ratio of 1:5 PEG8000 (5 ⁇ ), concentrated overnight, 4000g, 4°C for 20 minutes to obtain lentivirus precipitation, resuspend the lentivirus in RPMI1640 medium, and store at -80°C.
  • JE6-1 a concentrated virus to infect Jurkat E6-1 (ATCC catalog number: TIB-152 TM , referred to as JE6-1), control the number of cells during infection to 2 ⁇ 10 5 , and add the final concentration of 10 ⁇ g/mL sulfuric acid, a pro-infection reagent Fish protein.
  • Use centrifugal infection method 1000g, centrifugal infection at 32°C for 1 hour. 6-8 hours after the end of the centrifugal infection, change to a fresh medium RPMI1640, and continue to grow and expand the infected JE6-1. After the cells are expanded to a certain number, the PD-L1 CAR positive cells are sorted by flow cytometry.

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Abstract

La présente invention concerne un agent de liaison ciblant PD-L1 et son utilisation. L'agent de liaison ciblant PD-L1 se lie spécifiquement à PD-L1 et inhibe sa bioactivité, et peut être utilisé pour inhiber la survie des cellules tumorales induites par PD-L1 et la suppression médiée par PD-L1 des lymphocytes T réactifs aux tumeurs, de manière à réduire la prolifération, la motilité, l'invasion et la migration de cellules tumorales et la croissance tumorale.
PCT/CN2019/090878 2019-06-12 2019-06-12 Agent de liaison ciblant pd-l1 et son utilisation WO2020248156A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101248089A (zh) * 2005-07-01 2008-08-20 米德列斯公司 抗程序性死亡配体1（pd-l1）的人单克隆抗体
CN102245640A (zh) * 2008-12-09 2011-11-16 霍夫曼-拉罗奇有限公司 抗-pd-l1抗体及它们用于增强t细胞功能的用途
CN102264762A (zh) * 2008-09-26 2011-11-30 达纳-法伯癌症研究公司 人抗pd-1、pd-l1和pd-l2的抗体及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101248089A (zh) * 2005-07-01 2008-08-20 米德列斯公司 抗程序性死亡配体1（pd-l1）的人单克隆抗体
CN102264762A (zh) * 2008-09-26 2011-11-30 达纳-法伯癌症研究公司 人抗pd-1、pd-l1和pd-l2的抗体及其应用
CN102245640A (zh) * 2008-12-09 2011-11-16 霍夫曼-拉罗奇有限公司 抗-pd-l1抗体及它们用于增强t细胞功能的用途

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