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WO2022121846A1 - Anticorps se liant au pd-l1 et son application - Google Patents

Anticorps se liant au pd-l1 et son application Download PDF

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WO2022121846A1
WO2022121846A1 PCT/CN2021/135769 CN2021135769W WO2022121846A1 WO 2022121846 A1 WO2022121846 A1 WO 2022121846A1 CN 2021135769 W CN2021135769 W CN 2021135769W WO 2022121846 A1 WO2022121846 A1 WO 2022121846A1
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seq
amino acid
acid sequence
cancer
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Chinese (zh)
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王�忠
朱梓焱
李玉娟
张盟
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Bj Bioscience Inc
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Bj Bioscience Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present invention relates to monoclonal antibodies that bind to human programmed death ligand 1, antigen-binding fragments thereof, pharmaceutical compositions thereof, and their use in the treatment of cancer.
  • PD-L1 Programmed death-ligand 1
  • CD274 cluster of differentiation 274
  • B7 homolog 1 B7 homolog1, B7-H1
  • the necrosis factor superfamily is a type I transmembrane glycoprotein composed of 290 amino acid residues, including an lgV-like region, an lgC-like region, a transmembrane hydrophobic region and an intracellular tail of 30 amino acids.
  • the complete molecular weight is 40kDa.
  • PD-L1 is also able to provide costimulatory signals to T cells.
  • PD-L1 mRNA is expressed in almost all tissues, but PD-L1 protein is only continuously expressed in a small number of tissues, including liver, lung, tonsil, and immune amnesty tissues such as eye and placenta. PD-L1 is also expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, and the like. Under normal physiological conditions, PD-L1 mRNA is under strict post-transcriptional regulation, but PD-L1 protein is abundantly expressed on the cell surface of various human cancers.
  • PD-1 The receptor for PD-L1 is PD-1.
  • Programmed death-1 (PD-1) is a member of the CD28 superfamily, which also includes CD28, CTLA-4, ICOS and BTLA.
  • PD-1 is a type I transmembrane protein in the immunoglobulin superfamily. It has 288 amino acids and is one of the known major immune checkpoints. It is mainly expressed in CD4+T cells, CD8+T cells, NKT cells, B Surface of immune cells such as cells and activated monocytes.
  • the binding of PD-L1 to PD-1 can limit the interaction between antigen-presenting cells or dendritic cells and T cells, further inhibit the metabolism of T cells, inhibit the secretion of anti-apoptotic cells Bcl-X2, and reduce the effector cytokine IL -2.
  • the secretion of IFN- ⁇ induces T cell exhaustion and apoptosis, thereby reducing the immune response involved in immune T cells and exerting a negative regulatory function.
  • PD-L1 The expression of PD-L1 at tumor sites can protect tumor cells from damage through multiple pathways. Studies have shown that tumor cell-associated PD-L1 can increase apoptosis of tumor-specific T cells, while PD-L1 monoclonal antibodies can attenuate this effect. In cancer, the PD-L1 and PD-1/PD-L1 pathways protect tumors from cytotoxic T cells, ultimately suppressing antitumor immune responses by inactivating cytotoxic T cells in the tumor microenvironment, and Prevents the priming and activation of new T cells in the lymph nodes and their subsequent recruitment to the tumor. PD-L1 is not only a ligand of PD-1, it can also act as a receptor to transmit reverse signals to protect tumor cells from apoptosis induced by other anti-tumor pathways such as FAS-FASL.
  • PD-L1 plays an important role in tumor immunity by increasing the apoptosis of antigen-specific T cell clones.
  • PD-L1 has been found to be expressed in tissues of various tumor patients, including non-small cell lung cancer, lung cancer, gastric cancer, colon cancer, liver cancer, intrahepatic cholangiocarcinoma, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, head and neck squamous cell carcinoma Squamous cell carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, bladder cancer, skin cancer, renal cell carcinoma, oral squamous cell carcinoma, urothelial cell carcinoma, etc.
  • new protein molecules will be produced due to gene mutation and other reasons, and some peptide fragments of these proteins can be expressed on the cell surface after being degraded in the cell to become tumor antigens.
  • the immune system can recognize tumor antigens and eliminate tumor cells through immune surveillance, and tumor cells can use PD-L1 to evade immune attack.
  • PD-L1 is called a hot target for developing tumor immunotherapy drugs. Inhibition of PD-L1 signaling has been proposed as a way to enhance T-cell immunity to treat cancer.
  • PD-L1 products there are only three approved PD-L1 products in the world, namely Durvalumab from AstraZeneca, Atezolizumab from Roche, and the combination of Merck and Pfizer. Avelumab was developed. Therefore, it is necessary to provide more PD-L1 antibodies to meet the market demand.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: (1) heavy chain complementarity determining regions CDR1, CDR2, and CDR3, wherein the Described CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, described CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, described CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; And (2) light chain Complementarity determining regions CDR1', CDR2', CDR3', the CDR1' comprises the amino acid sequence shown in SEQ ID NO:4, the CDR2' comprises the amino acid sequence shown in SEQ ID NO:5, and the CDR3' comprises the SEQ ID NO:5 The amino acid sequence shown in ID NO:6.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the amino acid sequence shown in SEQ ID NO: 22, and the light chain constant region is The amino acid sequence shown in SEQ ID NO:23.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region.
  • VH heavy chain variable region
  • variable region wherein the heavy chain variable region comprises a variable region selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14
  • the amino acid sequence shown, the light chain variable region comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID The amino acid sequence shown in NO:21.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the amino acid sequence shown in SEQ ID NO: 22, and the light chain constant region is The amino acid sequence shown in SEQ ID NO:23.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain is SEQ ID
  • the heavy chain is SEQ ID
  • the amino acid sequence shown in NO:24, the light chain is the amino acid sequence shown in SEQ ID NO:25.
  • the present invention provides a monoclonal antibody that binds to human PD-L1 or an antigen-binding fragment thereof, wherein the antigen-binding fragment is selected from scFv, (scFv) 2 , Fab, and Fab′ of the PD-L1 antibody or F(ab') 2 .
  • the present invention also provides a pharmaceutical composition for treating cancer in a subject, comprising the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 and a pharmaceutically acceptable carrier.
  • the present invention also provides a pharmaceutical composition for treating cancer in a subject, comprising the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 according to the present invention and a second therapeutic agent.
  • the present invention also provides the use of the monoclonal antibody or its antigen-binding fragment that binds to human PD-L1 in the preparation of a medicament for treating cancer.
  • the present invention also provides a nucleotide sequence encoding the aforementioned monoclonal antibody or an antigen-binding fragment thereof.
  • the present invention also provides a vector comprising the aforementioned nucleotide sequence.
  • the present invention also provides a non-human host cell comprising the aforementioned vector.
  • the present invention provides cell lines that produce the antibodies of the present invention or antigen-binding fragments thereof, recombinant expression vectors comprising the nucleotides of the present invention, and methods for producing antibodies by culturing antibody-producing cell lines.
  • the present invention also provides a method of treating cancer in a subject, comprising administering a therapeutically effective amount of any one of the monoclonal antibodies of the present invention or antigen-binding fragments thereof or any one of the pharmaceutical compositions of the present invention to the object.
  • the monoclonal antibody provided by the present invention binds human PD-L1 with high affinity, exhibits a good dose-dependence with the secretion level of IL-2, and enhances the immune response of T cells.
  • These antibodies can be used as anti-tumor immune enhancers, and can also be used as diagnostic reagents to detect human PD-L1 in the blood or tissues of patients with cancer or other diseases.
  • Figure 1 Biomacromolecule interaction analysis results of 025 monoclonal antibody.
  • FIG. 1 Biomacromolecular Interaction Analysis Results of Atezolizumab.
  • FIG. 3 Biomacromolecule interaction analysis results of hIgG1.
  • Figure 4 Equilibrium dissociation constants of the tested monoclonal antibody (025), positive control (Atezolizumab) and negative control (hIgGl). *: Not available because not bound to PD-L1.
  • Figure 5 The binding activity of the humanized PD-L1 monoclonal antibody was detected by ELISA.
  • Figure 6 Cell-based blocking assay to test humanized PD-L1 monoclonal antibodies.
  • Figure 7 Repetition of a cell-based blocking assay to test humanized PD-L1 monoclonal antibodies.
  • Figure 8 Jurkat luciferase assay for detection of humanized PD-L1 monoclonal antibody.
  • Figure 9 The effect of humanized PD-L1 monoclonal antibody on the secretion level of IL-2.
  • Figure 10 The effect of humanized PD-L1 monoclonal antibody on the secretion level of IFN-g.
  • Figure 11 Detection of the binding activity of humanized PD-L1 monoclonal antibodies by FACS using the CHOK1-PD-L1 cell line.
  • Figure 12 Efficacy of anti-PD-L1 antibodies on tumor growth and effect on mouse body weight.
  • antibody refers to any form of antibody that exhibits a desired biological activity (eg, inhibition of binding of a ligand to its receptor or by inhibition of ligand-induced receptor signaling).
  • Antibody fragment and “antigen-binding fragment” refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of the parent antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, antibody fragments retain at least 10% of the parent binding activity when the activity is expressed on a molar basis.
  • the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody for the target.
  • examples of antibody fragments include, but are not limited to: Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies; domain antibodies; and multispecific antibodies formed from antibody fragments.
  • a "Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • a "Fab'fragment” contains a light chain and part of a heavy chain comprising the VH and CH1 domains and the region between the CH1 and CH2 domains, thus allowing between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F(ab') 2 molecules.
  • an “F(ab') 2 fragment” contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains.
  • an F(ab') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • “Fv regions” comprise variable regions from both heavy and light chains, but lack constant regions.
  • Single-chain Fv antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides contain an additional polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • humanized antibody refers to an antibody comprising the CDRs of an antibody derived from a mammal other than a human, as well as the framework regions (FR) and constant regions of a human antibody.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against only a single determinant on an antigen, as opposed to conventional (polyclonal) antibody preparations, which typically include multiple different antibodies directed against multiple different determinants (epitopes).
  • the modifier “monoclonal” refers to the properties of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method to prepare said antibody. For example, monoclonal antibodies for use in the present invention can be prepared by hybridoma or recombinant DNA methods. Monoclonal antibodies can include "chimeric" antibodies.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 of the present invention comprises: (1) heavy chain complementarity determining regions CDR1, CDR2, CDR3, said CDR1 comprising SEQ ID NO: 1
  • the amino acid sequence shown the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2
  • the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3
  • the CDR1' comprises the amino acid sequence shown in SEQ ID NO:4
  • the CDR2' comprises the amino acid sequence shown in SEQ ID NO:5
  • the CDR3' comprises the amino acid sequence shown in SEQ ID NO:6 amino acid sequence.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the amino acid sequence shown in SEQ ID NO: 22, and the light chain constant region is The amino acid sequence shown in SEQ ID NO:23.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 of the present invention comprises: (1) a heavy chain variable region comprising a variable region selected from the group consisting of SEQ ID NO: 9, SEQ ID NO : 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or the amino acid sequence shown in SEQ ID NO: 14; and (2) a light chain variable region comprising a variable region selected from the group consisting of SEQ ID NO: 15.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the amino acid sequence shown in SEQ ID NO: 22, and the light chain constant region is The amino acid sequence shown in SEQ ID NO:23.
  • variable region of the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 is selected from the following combinations: (a) the heavy chain variable region is the amino acid shown in SEQ ID NO:9 sequence, the light chain variable region is the amino acid sequence shown in SEQ ID NO: 15; or (b) the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region is SEQ ID NO: The amino acid sequence shown in 16; (c) the variable region of the heavy chain is the amino acid sequence shown in SEQ ID NO: 11, and the variable region of the light chain is the amino acid sequence shown in SEQ ID NO: 17; or (d) the heavy chain The variable region is the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 18.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the amino acid sequence shown in SEQ ID NO: 22, and the light chain constant region is The amino acid sequence shown in SEQ ID NO:23.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 of the present invention comprises: (1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 24; and (2) ) light chain comprising the amino acid sequence shown in SEQ ID NO:25.
  • the binding domain of the monoclonal antibody or antigen-binding fragment thereof of the present invention may carry a signal peptide, which is usually located at the N-terminus of the secreted protein and generally consists of 15-30 amino acids.
  • SRP signal recognition granule
  • protein synthesis is suspended or slowed down, the signal recognition granule carries the ribosome to the endoplasmic reticulum, and the protein synthesis restarts.
  • the newly synthesized protein enters the endoplasmic reticulum cavity, and the signal peptide sequence is cleaved under the action of the signal peptidase.
  • termination transit sequence exists at the C-terminus of the nascent peptide chain, it may not be cleaved by the signal peptidase, for example, ovalbumin contains an internal signal peptide. Neither its precursor nor the mature form is cleaved by signal peptidase.
  • Specifically binds means that a monoclonal antibody or antigen-binding fragment thereof of the invention is capable of specifically binding to at least two, three, four, five, six, seven, eight or more of each human target molecule. More amino acid interactions.
  • the "specific binding" of an antibody is primarily characterized by two parameters: a qualitative parameter (binding epitope or antibody binding site) and a quantitative parameter (binding affinity or binding strength).
  • Antibody binding epitopes can be determined by FACS, peptide dot epitope mapping, mass spectrometry, or peptide ELISA.
  • the Biacore method and/or ELISA method can measure the binding strength of an antibody to a specific epitope.
  • Signal-to-noise ratios are often calculated as a representative measure of binding specificity.
  • the signal represents the strength of antibody binding to the target epitope
  • the noise represents the strength of antibody binding to other non-target epitopes.
  • a signal-to-noise ratio for the epitope of interest of about 50 can be considered to bind the antibody under evaluation to the epitope of interest in a specific manner, ie "specifically binds".
  • an antigen-binding protein (including an antibody) is "specific for an antigen” if it binds to the antigen with high binding affinity as determined by the value of the dissociation constant (KD, or corresponding Kb, as defined below) combined".
  • the dissociation constant is ⁇ 5.662 ⁇ 10 ⁇ 11 M.
  • KD as used herein refers to the equilibrium dissociation constant for a particular antibody-antigen interaction.
  • sequence “variant” refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.
  • Constantly modified variants or “conservative amino acid substitutions” refer to amino acid substitutions known to those of skill in the art, making such substitutions that generally do not alter the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity.
  • the % identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. MoI. Biol. 48:444-453 (1970)), which has been introduced into the GAP program of the GCG software package (available available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.
  • binding refers to a binding reaction that determines the presence or absence of a protein and/or other biological agent in a heterogeneous population of said protein.
  • a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in significant amounts.
  • an “equivalent variant” is one that is identical or substantially similar in biological activity and function to the indicated sequence (eg, amino acid sequence) but is about 80%, about 85%, about 90%, about 95% similar in sequence to the indicated sequence. %, about 96%, about 97%, about 98%, or about 99% identical sequences.
  • antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, particulate debris (host cells or lysed fragments) is removed as a first step, eg, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from the expression system is typically first concentrated using a commercially available protein concentration filter (eg, Amicon or Millipore Pellicon ultrafiltration units). Protease inhibitors (eg PMSF) can be used in any of the preceding steps to inhibit proteolysis, and antibiotics can be used to prevent the growth of foreign contaminants.
  • a commercially available protein concentration filter eg, Amicon or Millipore Pellicon ultrafiltration units.
  • Protease inhibitors eg PMSF
  • PMSF can be used in any of the preceding steps to inhibit proteolysis, and antibiotics can be used to prevent the growth of foreign contaminants.
  • the glycoprotein can be purified by adsorbing the glycoprotein to a lectin substrate (eg, a lectin affinity column) to remove the fucose-containing glycoprotein from the preparation and thereby enrich the Collection of fucose-free glycoproteins.
  • a lectin substrate eg, a lectin affinity column
  • “Pharmaceutical composition” refers to a pharmaceutical formulation for use in humans.
  • the pharmaceutical composition comprises a monoclonal antibody or antigen-binding fragment thereof of the invention that binds to human PD-L1 and a suitable formulation of a carrier, stabilizer and/or excipient.
  • the present invention provides pharmaceutical formulations comprising the monoclonal antibodies of the present invention or antigen-binding fragments thereof.
  • To prepare a pharmaceutical or sterile composition the antibody or antigen-binding fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient.
  • Formulations of therapeutic and diagnostic agents in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers.
  • Toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with immunosuppressants can be determined in cell cultures or experimental animals by standard pharmaceutical methods, such as for determining LD50 (lethal to 50% of a population). dose) and ED50 (the dose effective to treat 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio of LD50 to ED50.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed.
  • Suitable routes of administration include parenteral (eg, intramuscular, intravenous or subcutaneous) and oral administration.
  • Antibodies for use in pharmaceutical compositions or for practicing the methods of the invention can be administered in a variety of conventional ways, such as by oral ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, parenteral, intraarterial or Intravenous injection.
  • the binding compounds of the present invention are administered intravenously.
  • the binding compounds of the present invention are administered subcutaneously.
  • one can administer the antibody in a local rather than systemic manner usually in a depot or sustained release formulation), eg, via direct injection of the antibody into the site of action.
  • Appropriate doses are determined by the clinician, eg, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment. Typically, the starting dose is slightly lower than the optimal dose and is increased by small amounts thereafter until the desired or optimal effect is achieved with respect to any adverse side effects.
  • Important diagnostic measurements include measuring, for example, inflammatory symptoms or levels of inflammatory cytokines produced.
  • Antibodies, antibody fragments, and cytokines can be provided by continuous infusion or by administration at intervals (eg, one day, one week, or 1-7 times per week). Dosages can be provided intravenously, subcutaneously, intraperitoneally, transdermally, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinal or by inhalation.
  • a preferred dosage regimen is one that includes the maximum dose or frequency of administration that avoids significant undesirable side effects.
  • the total weekly dose is usually at least 0.05 ⁇ g/kg body weight, more usually at least 0.2 ⁇ g/kg, most usually at least 0.5 ⁇ g/kg, typically at least 1 ⁇ g/kg, more typically at least 10 ⁇ g/kg, most typically at least 1 ⁇ g/kg is at least 109 ⁇ g/kg, preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg, ideally at least 10 mg/kg, more ideally at least 25 mg/kg, and Most desirably at least 50 mg/kg.
  • a small molecule therapeutic agent such as a peptidomimetic, natural product or organic chemical is approximately the same as that of an antibody or polypeptide.
  • the pharmaceutical composition of the present invention may also contain other agents, including but not limited to cytotoxic agents, cytostatic agents, anti-angiogenic or anti-metabolite drugs, targeted tumor drugs, immunostimulatory or immunomodulatory agents or in combination with cytotoxic agents, cellular Growth inhibitory or other toxic drug conjugated antibodies.
  • the pharmaceutical compositions can also be administered with other treatment modalities such as surgery, chemotherapy, and radiation.
  • Typical veterinary, experimental or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses and humans.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 of the present invention can be used in combination with a second therapeutic agent.
  • the second therapeutic agent and the monoclonal antibody or antigen-binding fragment thereof of the invention are administered at substantially the same time.
  • An individual will sometimes use a second therapeutic agent concurrently with the monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the second therapeutic agent or other agent typically administered to a cancer patient and the monoclonal antibody or antigen-binding fragment thereof of the invention may be combined into a pharmaceutical composition; in other specific embodiments, both applied separately.
  • second therapeutic agent is any agent that is advantageously combined with an anti-PD-L1 antibody.
  • agents that may be advantageously combined with anti-PD-L1 antibodies include, but are not limited to, other agents that inhibit PD-L1 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or interferences Reagents for upstream or downstream signaling of PD-L1.
  • One aspect of the present invention provides methods of treating cancer with the monoclonal antibodies or antigen-binding fragments thereof that bind to human PD-L1 of the present invention. More specifically, the present invention provides methods of enhancing T cell function and anti-tumor immunity, comprising administering a therapeutically effective amount of any of the above-described monoclonal antibodies that bind to human PD-L1, or antigen-binding fragments thereof, or pharmaceutical compositions.
  • Enhancing T-cell function and anti-tumor immunity provides a broad-spectrum approach to the treatment of cancer. Therefore, various types of cancers can be treated by administering the monoclonal antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present invention that bind to human PD-L1.
  • administer and “treat” in reference to an animal, human, subject, cell, tissue, organ or biological fluid, it refers to combining an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, or biological fluid. Person, cell, tissue, organ or biological fluid contact.
  • administering and “treatment” can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, eg, by agents, diagnostic agents, binding compositions, or by other cells.
  • the terms “inhibit” or “treating” include delaying the development of symptoms associated with a disease and/or reducing the severity of those symptoms that will or are expected to develop in the disease.
  • the term also includes alleviation of existing symptoms, prevention of additional symptoms, and alleviation or prevention of underlying causes of such symptoms.
  • the term indicates that a beneficial result has been conferred on a vertebrate subject suffering from a disease.
  • the term "therapeutically effective amount” or “effective amount” refers to when a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 of the invention is administered alone or in combination with an additional therapeutic agent In the case of a cell, tissue or subject, it is an amount effective to prevent or slow down the disease or disorder to be treated.
  • a therapeutically effective dose further refers to an amount of the compound sufficient to cause alleviation of symptoms, such as to treat, cure, prevent or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention or alleviation of the condition .
  • the therapeutically effective amount refers to that ingredient alone.
  • a therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether it is administered in combination, consecutively, or simultaneously.
  • a therapeutically effective amount will generally reduce symptoms by at least 10%; usually by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.
  • “about” means that the index value is within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, “about” or “substantially comprising” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean at most one order of magnitude or at most five times the value. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about” or “substantially comprising” should be assumed to be within an acceptable error range for the specific value.
  • the monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 of the present invention can be used to treat cancer.
  • the cancers include but are not limited to: gastric cancer, lung cancer, liver cancer, intrahepatic cholangiocarcinoma, colon cancer, pancreatic cancer, ovarian cancer, glioma, kidney cancer, urothelial cell carcinoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma squamous cell carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, bladder carcinoma, renal cell carcinoma, skin cancer and oral squamous cell carcinoma.
  • the cancer is a highly immunogenic cancer.
  • the cancer is a PD-L1 expressing cancer.
  • Test antibodies were purchased from Boji Biomedical Technology (Hangzhou) Co., Ltd.: 025 (Lot#P20170915007, IgG1, ⁇ ), and the heavy chain complementarity determining region sequence of the monoclonal antibody is shown in SEQ ID NOs: 1-3 , the light chain complementarity determining region sequence is shown in SEQ ID NO:4-6, the sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the sequence of the light chain variable region is shown in SEQ ID NO:8.
  • Atezolizumab (Lot#20140734A3, IgG1,k), hIgG1 (Lot#20170406004, hIgG1,k), hIgG4 (Lot#151031001, hIgG4,k) were purchased from ChemPartner (Shanghai).
  • Biacore The running buffer is HBS-EP+. The flow rate was 30 ⁇ L/min. The analyte (antigen) is titrated on the sensor chip surface with a ligand (antibody) captured by anti-human Fc IgG. Serial dilution samples were injected with a correlation phase of 180s. 10 ⁇ L/min for 30 s in glycine pH 1.5.
  • the machine model used for analysis is: T200, the analysis temperature is 25°C, and the analysis method is multi-cycle kinetics.
  • the equilibrium dissociation constant of 025 monoclonal antibody was 5.662 ⁇ 10 -11 M (Fig. 1); the equilibrium dissociation constant of Atezolizumab monoclonal antibody was 2.747 ⁇ 10 -10 M (Fig. 2).
  • the equilibrium dissociation constants of monoclonal antibody 025 were all lower than those of Atezolizumab (Fig. 4), indicating that 025 had higher binding affinity than Atezolizumab.
  • the isotype control hIgG1 did not bind to the PD-L1 antigen ( Figure 3), indicating that the monoclonal antibodies tested were specific for PD-L1.
  • the 025 monoclonal antibody was humanized.
  • the variable region sequence of the humanized antibody is shown in Table 2 below, and the constant region sequence of the antibody is shown in Table 3.
  • the antibody affinity analysis after humanization is shown in Table 4 below.
  • ELISA test ELISA method is performed to determine the interaction between monoclonal antibody and PD-L1 protein. Briefly, antibodies were coated in plates containing 50 ul lug/ml hPD-L1-ECD-hFc protein and incubated overnight at 4°C, and the plates were washed 3 times with PBST. Plates were then blocked with PBST containing 1% BSA for 2 hours at room temperature. Wash the plate 3 times with PBST, add 50ul/well of monoclonal antibody solution, and incubate at 37°C for 1 hour. The plate was washed 3 times with PBST, 50ul/well of secondary antibody was added, and incubated at 37°C for 0.5 hours.
  • the plate was washed 3 times with PBST, and then 100ul/well of TMB was added for 15min. Quench with 50ul/well 1N HCl. Plate readings at 450 nm were measured using a Molecular device spectra max plus384.
  • the monoclonal antibody antibodies L1-H1, L2-H2, L3-H3, L5-H5 and Atezolizumab all bound PD-L1 protein with similar strength.
  • L1-H1 has the relatively highest binding force.
  • hIgG1 and hIgG4 did not bind to PD-L1 protein, indicating that the monoclonal antibody has specific binding.
  • CHOK1-Mock-A1 CHOK1-PD-L1 cell lines were purchased from ChemPartner.
  • Cell blocking assay Cells were harvested by centrifugation at 1000 rpm for 5 minutes. Cells were then resuspended in 2E6 cells/ml FACS buffer (2% FBS in IX PBS) and plated in 96-well plates (100 ul/well) and incubated at room temperature for 20 minutes. Antibody solutions were prepared by diluting the antibody with FACS buffer. A 60 ⁇ g/ml (2 ⁇ ) solution of Bio-PD1-Fc was prepared using FACS buffer. Centrifuge at 300g for 5 minutes and discard the supernatant. Cells were then resuspended with 50 ⁇ l of antibody solution. Add 50 ⁇ l of Bio-PD1 solution and shake the plate gently to mix the solution well.
  • Hep3B-OS8-PDL1 cells were plated in 96-well plates and incubated overnight at 37°C.
  • prepare the antibody solution in the assay medium Media was removed from pre-plated Hep3B-OS8-PDL1 (purchased from ChemPartner) cells. 50 ⁇ l of antibody solution was then added to the plate. Incubate for 20-30 minutes.
  • Jurkat-NFAT-PD1 cells were harvested using assay medium instead of growth medium. 50 ⁇ l of Jurkat-NFAT-PD1 (purchased from ChemPartner) cells were then added to the plate. Incubate the assay plate in a humidified 37 °C, 5% CO2 incubator for 6 h. Luciferase activity was measured by the ONE-Glo TM Luciferase Assay System.
  • results The luciferase assay results of PD-L1 monoclonal antibody are shown in FIG. 8 . It can be seen from Figure 8 that the L1-H1 monoclonal antibody can better activate Jurkat cells. L2-H2, L3-H3 and L5-H5 monoclonal antibodies all better activate Jurkat cells.
  • MLR Mixed Lymphocyte Reaction
  • PBMC isolation First, dilute a blood sample from a single donor with the same volume of sterile PBS, eg, add 15 ml of sterile PBS to 15 ml of fresh whole blood and shake gently. Transfer 15ml of Ficoll-Pague medium to a new 50ml centrifuge tube, making sure that the volume ratio of Ficoll and blood is 3:4, then carefully add the diluted blood sample to the surface of the Ficoll medium by as soft as possible Mix to avoid mixing. This way the layers of the two liquids can be clearly seen. Gently move the test tube to the centrifuge, during the centrifugation process, centrifuge at 400 ⁇ g and 20°C for 30 min with the maximum acceleration and minimum deceleration settings.
  • a total of four interfaces can be observed after centrifugation, from top to bottom they are plasma, mononuclear cell layer, Ficoll medium and RBC. During this process, move the tube as gently as possible to avoid mixing the separated layers. Carefully aspirate the mononuclear cell layer and transfer it to another new sterile centrifuge tube. Another thing to note is that it is much better to aspirate a volume of plasma than Ficoll medium. Sterile PBS buffer was then added to the collected PBMCs and washed at a volume ratio of 3:1. Cells were washed twice with 50 ml of PBS and counted by flow cytometry. Centrifuge at 200xg, 20°C for 10 minutes at maximum acceleration and maximum deceleration settings.
  • DC culture Isolated human PBMCs were diluted to approximately 1E6 cells/ml in RPMI-1640 medium (serum free). It was then dispensed into 150 mm cell culture dishes (AXYGEN, Cat# 430599) at a concentration of 25 ml/dish and incubated in a 37 degree incubator for 3 hours. Aspirate and discard the supernatant from each petri dish, add fresh medium containing 250U/ml IL-4 and 500U/ml GM-CSF to RPMI-1640 medium containing 10% FBS, and incubate at 37°C Incubate for 1 day in the box.
  • DC (stimulator) was adjusted to a final concentration of 1 ⁇ 10 6 cells/ml, and the proliferation of DC was inhibited with 10ug/ml mitomycin C (1 ⁇ ) at 37°C for 45min. 500g x3, 5 minutes. DCs were then washed twice with 10 ml of PBS.
  • CD4+ T cell isolation Purify CD4+ T cells from freshly isolated human PBMC (EasySep TM Human CD4+ T Cell Isolation Kit) according to the manufacturer's guidelines.
  • Monoclonal Antibody Solution Preparation: Prepare monoclonal antibody dilutions in RPMI 1640 medium containing 10% FBS as arranged.
  • MLR Densities of CD4+ T cells (responders) and DCs (stimulators) were adjusted using X-VIVO15 medium (LONZA, Cat#04-418Q), and CD4+ T cells (responders) were adjusted to a final concentration of 1 ⁇ 10 6 cells/ml, DC (stimulator) was adjusted to a final concentration of 2 x 10 5 cells/ml. Mix stimulated and responder cells in a 1:1 volume ratio. Each mAb was diluted to a final concentration of 500 nM in X-VIVO15 medium. Add 50 ⁇ l of the diluted Ab solution to each well of a 96-well plate.
  • Quantitative ELISA operate according to the assay procedure of Human IL-2 DuoSet ELISA Kit (R&D, Cat#DY202) or Human IFN- ⁇ DuoSet ELISA Kit (R&D, Cat#DY285B).
  • FACS Cells were harvested by centrifugation at 1000 rpm for 5 minutes. Cells were then resuspended in FACS buffer (2% FBS in IX PBS) and placed in 96-well plates (100 ul/well) and incubated at room temperature for 20 minutes. Centrifuge at 300g for 5 minutes, then discard the supernatant. Cells were resuspended in FACS buffer containing primary antibody (100ul/well) and incubated at 4 degrees for 60 minutes. Cells were washed 2 times with PBS, centrifuged at 300 g for 5 min, and the supernatant was discarded. Cells were resuspended in FACS buffer containing secondary antibody (100ul/well) and incubated at 4 degrees for 60 minutes. Cells were washed 2 times with 1 ⁇ PBS and centrifuged at 300 g for 5 min, then the supernatant was discarded. Resuspend cells in 1x PBS. Perform FACS analysis on a computer.
  • Figure 11 shows that L1-H1, L2-H2 and L5-H5 specifically bind PD-L1 expressed on CHOK1 cells.
  • L1-H1, L2-H2, and L5-H5 all had EC50s comparable to the positive control, Atezolizumab, suggesting that all three humanized mAbs could bind efficiently/sensitively to PD-L1.
  • L1-H1, L2-H2 and L5-H5 had higher MFIs than the positive control Atezolizumab ( Figure 11), indicating that L1-H1, L2-H2 and L5-H5 were associated with PD-L1 compared with the positive control Atezolizumab higher binding affinity.
  • PD-L1 antibody significantly inhibits tumor growth in vivo

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Abstract

L'invention concerne un anticorps monoclonal qui se lie au PD-L1 humain, un fragment de liaison à l'antigène de celui-ci, une composition pharmaceutique de celui-ci, et une application de celui-ci dans le traitement du cancer. L'anticorps monoclonal présente une affinité élevée pour PD-L1, peut présenter une excellente dose-dépendance dans une réaction lymphocytaire mixte avec le niveau de sécrétion d'IL-2 et peut être utilisé pour traiter le cancer.
PCT/CN2021/135769 2020-12-08 2021-12-06 Anticorps se liant au pd-l1 et son application Ceased WO2022121846A1 (fr)

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CN106478819A (zh) * 2016-10-27 2017-03-08 常州费洛斯药业科技有限公司 一种针对pd‑l1的单克隆抗体或抗体片段
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WO2020057225A1 (fr) * 2018-09-17 2020-03-26 苏州泓迅生物科技股份有限公司 Anticorps anti-pd-l1, son procédé de préparation et son utilisation
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US20090055944A1 (en) * 2005-07-01 2009-02-26 Medarex, Inc. Human monoclonal antibodies to be programmed death ligand 1 (pd-l1)
CN107922503A (zh) * 2016-03-04 2018-04-17 四川科伦博泰生物医药股份有限公司 一种pdl‑1抗体、其药物组合物及其用途
CN106478819A (zh) * 2016-10-27 2017-03-08 常州费洛斯药业科技有限公司 一种针对pd‑l1的单克隆抗体或抗体片段
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