WO2021063350A1 - 一种融合蛋白及其应用 - Google Patents

一种融合蛋白及其应用 Download PDF

Info

Publication number
WO2021063350A1
WO2021063350A1 PCT/CN2020/118799 CN2020118799W WO2021063350A1 WO 2021063350 A1 WO2021063350 A1 WO 2021063350A1 CN 2020118799 W CN2020118799 W CN 2020118799W WO 2021063350 A1 WO2021063350 A1 WO 2021063350A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
antibody
amino acid
seq
acid sequence
Prior art date
Application number
PCT/CN2020/118799
Other languages
English (en)
French (fr)
Inventor
任舒文
何云
甘馨
李东海
陈飞
王玲
李瑾
戎一平
Original Assignee
和铂医药(苏州)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 和铂医药(苏州)有限公司 filed Critical 和铂医药(苏州)有限公司
Publication of WO2021063350A1 publication Critical patent/WO2021063350A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This application relates to the field of biomedicine, in particular to an anti-PD-L1 and anti-TGFB fusion protein and its application.
  • P-L1 Programmed death receptor ligand 1
  • CD274 cluster of differentiation 274
  • B7 homologous protein 1 B7-H1
  • PD-L1 is a 40KDa type I transmembrane protein, usually in activated T It is expressed on cells, B cells, monocytes, dendritic cells, macrophages and many non-hematopoietic cells.
  • PD-L1 can bind to programmed death receptor 1 (PD-1).
  • PD-L1/PD-1 signal pathway is a very important co-suppressive signal pathway in the immune response, which negatively regulates the T cell immune response, inhibits T cell activity, and weakens the secretion of cytokines.
  • Transforming growth factor-B TGFB is a powerful cytokine that has a significant impact on the immune system.
  • tumor cells use a variety of immunosuppressive mechanisms in the tumor microenvironment to evade recognition and attack by the body's immune system. These immunosuppressive mechanisms include immunosuppressive cytokines (such as TGFB), regulatory T cells (Tregs), and co-suppressive signaling pathways Molecules, myeloid inhibitory cells, etc.
  • immunosuppressive cytokines such as TGFB
  • Tregs regulatory T cells
  • the application provides a fusion protein, which includes human TGFBRII or a fragment thereof and an antibody or an antigen-binding fragment thereof that specifically binds to PD-L1.
  • the application also provides the application of the fusion protein in the prevention and treatment of tumors or cancers.
  • the application provides a fusion protein comprising: a) human TGFBRII or a fragment thereof; and b) an antibody or antigen-binding fragment thereof that specifically binds PD-L1; wherein the specific binding to PD-L1
  • the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27,
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 8
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 26
  • the LCDR3 includes The amino acid sequence shown in SEQ ID NO: 31;
  • the CDR sequence is defined using Chothia definition rules (see Table 1).
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes SEQ ID NO: 13
  • the amino acid sequence of, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 4.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 comprises an antibody heavy chain, and the antibody heavy chain is fused in frame with the human TGFBRII or a fragment thereof to form a fusion polypeptide.
  • the C-terminus of the antibody heavy chain is connected to the N-terminus of the human TGFBRII or a fragment thereof.
  • the human TGFBRII or fragments thereof comprise the extracellular domain of human TGFBRII.
  • the human TGFBRII or a fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 56-57.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 further comprises a heavy chain constant region, and the heavy chain constant region is a human IgG1 constant region.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 further comprises a light chain constant region.
  • the light chain constant region comprises a human Ig ⁇ constant region.
  • the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14; and the second polypeptide comprises The amino acid sequence shown in SEQ ID NO: 5.
  • the fusion protein comprises two of the first polypeptides and two of the second polypeptides.
  • the present application also provides an isolated nucleic acid molecule, which comprises a polynucleotide encoding the fusion protein or a functional fragment thereof.
  • the application also provides a pharmaceutical composition, which comprises the fusion protein, or the nucleic acid molecule, and optionally a pharmaceutically acceptable carrier.
  • the use of the fusion protein or the nucleic acid molecule for preparing a medicine which is used for treating cancer, inhibiting tumor growth and/or inhibiting tumor cell proliferation.
  • the tumor or cancer comprises colon cancer.
  • the tumor or cancer is a tumor or cancer in which PD-L1 expression is abnormal.
  • this application also provides the fusion protein, or the nucleic acid molecule, which is used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the tumor or cancer comprises colon cancer.
  • the tumor or cancer is a tumor or cancer in which PD-L1 expression is abnormal.
  • the present application also provides a method for treating cancer in a subject, inhibiting tumor growth in the subject, and/or inhibiting tumor cell proliferation, the method comprising administering to the subject or the tumor cells The fusion protein, or the nucleic acid molecule.
  • the tumor or cancer comprises colon cancer.
  • the tumor or cancer is a tumor or cancer in which PD-L1 expression is abnormal.
  • Figure 1 shows that the fusion protein described in this application binds to human PD-L1 in vitro
  • Figure 2 shows that the fusion protein described in this application binds to human TGFB1 in vitro
  • Figure 3 shows that the fusion protein described in this application blocks the binding of PD-L1 and PD-1 in vitro
  • Figure 4 shows that the fusion protein of the present application inhibits the activation of the TGFB/Smad signaling pathway
  • Figure 5 shows that the fusion protein of the present application enhances the secretion of cytokine IL-2 from activated T lymphocytes
  • Figure 6 shows that the fusion protein of the present application enhances the secretion of cytokine IFN- ⁇ from activated T lymphocytes
  • Figure 7 shows the pharmacokinetic results of the fusion protein described in this application.
  • Figure 8 shows the tumor inhibitory effect of the fusion protein described in this application on CT26-human PD-1/PD-L1 double knockout mice, A: tumor volume change, B: mouse body weight change.
  • antibody or antigen-binding fragment thereof generally refers to a protein containing a portion that binds to an antigen, and optionally allows the portion that binds to the antigen to adopt a scaffold or a conformation that promotes the binding of the antibody or its antigen-binding fragment to the antigen.
  • the skeleton part. may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4 .
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antibodies or antigen-binding fragments thereof include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), Immunoconjugates, multispecific antibodies (for example, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen-binding fragments
  • Fv fragments fragments
  • F(ab') 2 fragments
  • scFv di-scFv and/or dAb
  • Immunoconjugates multispecific antibodies (for example, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • fusion protein generally refers to a protein composed of two or more polypeptides, which are usually not combined in the natural state, but the respective amino and carboxyl termini can be directly or indirectly combined to form one Continuous peptides.
  • fusion protein can be used interchangeably with “protein” when referring to the fusion protein described in this application.
  • fusion protein can be used interchangeably with “antibody” when referring to the fusion protein described in this application.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' usually refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteine from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain
  • F(ab ') 2 generally refers to Fab' dimer antibody fragments comprising two Fab fragments by a disulfide bridge at the hinge region.
  • Fv generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site.
  • the fragment may be composed of a dimer in which a heavy chain variable region and a light chain variable region are tightly non-covalently bound;
  • dsFv generally refers to a disulfide bond-stabilized Fv fragment, The bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • dAb fragment generally refers to an antibody fragment composed of VH domains.
  • scFv generally refers to a monovalent molecule formed by covalently connecting and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • the more highly conserved parts of variable domains are called the framework (FR).
  • the variable domains of the natural heavy chain and light chain each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) Most of them adopt ⁇ -sheet configuration and are connected by three CDR structure loop regions.
  • the CDRs in each chain are close together through the FR region, and together with the CDR from the other chain form the antigen binding site of the antibody.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions. , Such as involved in antibody-dependent cytotoxicity.
  • the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., J Mol Biol 273:927-48, 1997).
  • the Combined definition rule including Kabat definition and Chothia definition is also used to determine the amino acid residues in the variable domain sequence and the full-length antibody sequence, see Table 1.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rules) to position bb (Chothia coding rules);
  • Haa-Hbb can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain , The amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule).
  • L24-L34 can refer to the amino acid sequence from the 24th to the 34th starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and following the Chothia coding rules The amino acid sequence from position 26 to position 32.
  • isolated protein generally refers to a protein that has been identified, separated, and/or recovered from a component of its production environment (eg, natural or recombinant).
  • the pollutant components of the environment are usually substances that interfere with its research, diagnostic or therapeutic use, and can include enzymes, hormones and other protein or non-protein solutes.
  • the isolated protein or antibody will usually be prepared through at least one purification step.
  • the term “specific binding” generally means that the antibody binds to the epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, when an antibody binds to an epitope through its antigen binding domain more easily than it will bind to a random, unrelated epitope, an antibody is said to “specifically bind” the antigen.
  • Epitope refers to a specific atomic group (for example, sugar side chain, phosphoryl group, sulfonyl group) or amino acid on an antigen that binds to an antibody.
  • KD and “K D” are used interchangeably, usually referring to the equilibrium dissociation constant
  • KD is the dissociation rate constant (kdis, also known as the “off-rate ) (koff)” or “kd") and the ratio of the association rate constant (kon, also known as “combination rate (kon)” or “ka”).
  • the binding rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD) can be used to express the binding affinity of the antibody to the antigen.
  • Methods for determining the rate constants of association and dissociation are well known in the art, including but not limited to Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , Co-immunoprecipitation (Co-IP) and protein chip technology. If measured under different conditions (such as salt concentration, pH), the measured affinity of a specific protein-protein interaction may be different.
  • PD-L1 generally refers to the programmed death ligand 1 protein, its functional variants and/or its functional fragments.
  • PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by the CD274 gene (in humans).
  • PD-L1 binds to its receptors, such as programmed death 1 (PD-1), which is expressed in activated T cells, B cells, and macrophages (Ishida et al., 1992 EMBO J, 11: 3887-3395; Okazaki et al., Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001; 291:319-22).
  • PD-1 programmed death 1
  • the complexation of PD-L1 and PD-1 exerts an immunosuppressive effect by inhibiting the proliferation of T cells and the production of cytokines IL-2 and IFN- ⁇ (Freeman et al., Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J.Exp.Med.2000,192:1027-1034; Carter et al., PD-1:PD-L inhibitory pathway affects both CD4(+)and CD8(+)T cells and is overcome by IL-2.Eur.J.Immunol.2002,32:634–643).
  • PD-L1 encompasses any natural PD-L1 of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • the term encompasses "full length", unprocessed PD-L1 and any form of PD-L1 produced by processing in the cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
  • the basic structure of PD-L1 includes 4 domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • the PD-L1 sequence is known in the art. For example, you can find information about the human PD-L1 gene (including genomic DNA sequence) under NCBI Gene ID No. 29126. For another example, you can find information about mouse PD-L1 gene (including genomic DNA sequence) under NCBI Gene ID No. 60533. For another example, you can find information about the cynomolgus monkey PD-L1 gene (including genomic DNA sequence) under NCBI Gene ID No. 102145573.
  • the amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI accession number NP_054862 or UniProt accession number Q9NZQ7.
  • Exemplary full-length mouse PD-L1 protein sequences can be found under NCBI accession number NP_068693 or Uniprot accession number Q9EP73.
  • An exemplary full-length cynomolgus monkey PD-L1 protein sequence can be found under NCBI accession number XP_005581836 or Uniprot accession number G7PSE7.
  • PD-1 generally refers to the programmed death 1 receptor (also known as CD279), its functional variants and/or its functional fragments.
  • PD-1 is usually expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DC).
  • DC dendritic cells
  • PD-1 can bind to its ligands PD-L1 and PD-L2.
  • variants that differ in amino acid sequence from naturally-occurring PD-1 but retain the ability to specifically bind to PD-L1.
  • a variant that enhances the biological activity of PD-L1 is further included.
  • the PD-1 sequence is known in the art.
  • an exemplary full-length human PD-1 protein sequence can be found under NCBI accession number NP_005009
  • an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found under NCBI accession number NP_001271065 or Uniprot accession number BOLAJ3.
  • TGFBRII generally refers to a transforming growth factor beta (TGFB) receptor II (also known as TGFBR2).
  • TGFBR transforming growth factor
  • the TGFBR on the cell surface can be combined and activated by transforming growth factor (TGFB), signal transmission through the SMAD pathway, and has the activity of regulating growth, anti-inflammatory and immune regulation.
  • TGFBRII usually consists of a C-terminal protein kinase domain and an N-terminal extracellular domain.
  • the complete human TGFBRII sequence can be found in Uniprot accession number P37173. Alternatively, the complete human TGFBRII sequence may be as shown in SEQ ID NO:53.
  • epitope generally refers to a certain region or region of an antigen to which an antibody specifically binds.
  • Epitopes usually consist of chemically active surface groups such as amino acids or carbohydrates or sugar side chain molecules, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • the epitope can be a "linear epitope” or a "configuration epitope”. In a linear epitope, all interaction points between a protein and an interacting molecule (such as an antibody) occur linearly along the protein's primary amino acid sequence. In a conformational epitope, the points of interaction crossover occur on amino acids on separate proteins.
  • epitope mapping Methods of which epitope a given antibody binds (e.g., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, e.g., testing overlapping or adjacent peptides (e.g., from PD-L1) Reactivity with a given antibody.
  • Methods for determining the spatial conformation of an epitope include techniques in the art and those described in this application, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS.
  • the term "incompletely overlapping" epitope generally means that when two or more antibodies are mentioned, each antibody binds to a different group of amino acid residues, as determined by a given method.
  • Techniques for determining whether the antibody described in this application overlaps with the reference antibody binding epitope include, for example, epitope mapping methods, such as x-ray analysis of crystals of antigen: antibody complexes, which provide atomic resolution of epitopes, And hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to antigen fragments or mutant variants of antigens, where the loss of binding due to modification of amino acid residues in the antigen sequence is often regarded as indicative of epitope components (e.g., alanine scanning mutagenesis -Cunningham and Wells (1985) Science 244:1081). In addition, the calculation combination method of epitope positioning can also be used.
  • epitope mapping methods such as x-ray analysis of crystals of antigen: antibody complexes, which provide atomic resolution of epitopes, And hydrogen/deuterium exchange mass spectrometry (HDX-MS).
  • Other methods monitor the binding of antibodies to antigen fragments or mutant variants of
  • Methods for determining epitopes in the art include, but are not limited to, synthetic peptide methods, immunoinformatics prediction methods, polypeptide activity determination, epitope peptide scanning technology, protein cleavage methods, phage display technology, X-ray diffraction and nuclear magnetic resonance analysis, and the use of computers
  • the software performs epitope prediction.
  • a biological membrane interferometry (BLI) Octet molecular interaction analysis platform can be used to perform competitive binding of two or more antibodies to an epitope. For example, the first antibody is mixed with the antigen, and then the second antibody is added. ForteBio Octet is used to determine the competitive inhibition rate of the second antibody against the first antibody. When the competitive inhibition rate is less than 85%, it can be said The first antibody and the second antibody bind different epitopes of the antigen.
  • BLI biological membrane interferometry
  • the term "subject” generally refers to a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as human and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). ).
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the term "cell” generally refers to an individual cell, cell line, or cell line that can or already contains a plasmid or vector containing the nucleic acid molecule described in this application, or capable of expressing the antibody or antigen-binding fragment thereof described in this application.
  • the cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells and the original parent cells may not necessarily be identical in morphology or genome, but they can express the antibodies or antigen-binding fragments described in this application.
  • the cells can be obtained by transfecting cells in vitro using the vectors described in this application.
  • the cell may be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or Myeloma cells).
  • the cell may be a mammalian cell.
  • the mammalian cell may be a CHO-K1 cell.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector is introduced.
  • the recombinant host cell includes not only certain specific cells, but also the progeny of these cells.
  • the term "pharmaceutical composition” generally refers to a preparation that exists in a form that allows the biological activity of the active ingredient to be effective, and does not contain unacceptable toxicity to the subject to which the composition will be administered. Additional ingredients.
  • the composition is sterile.
  • “Sterile” compositions are sterile or free of all living microorganisms and their spores.
  • treatment generally refers to the desire to change the natural course of the individual to be treated, and can be a clinical intervention to achieve prevention and treatment or in the course of clinical pathology.
  • Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.
  • antibodies e.g., anti-PD-L1 antibodies
  • administering generally refers to the subject (eg, patient) administering a certain dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition containing an anticancer therapeutic agent)
  • a compound eg, an anticancer therapeutic agent
  • a pharmaceutical composition eg, a pharmaceutical composition containing an anticancer therapeutic agent
  • Methods can be carried out by any suitable means, including parenteral, intrapulmonary and intranasal, and (if local treatment is required) intralesional administration.
  • Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the administration can be carried out by any suitable route, for example by injection (such as intravenous or subcutaneous injection).
  • Various administration procedures are covered herein, including but not limited to single administration or multiple administrations at various time points, bolus administration, and pulse infusion.
  • tumor generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues.
  • the tumor may include colon cancer.
  • the application may also include functional variants, derivatives, analogs, homologs and fragments thereof.
  • the term "functional variant” refers to a polypeptide that has substantially the same amino acid sequence as a naturally-occurring sequence or is encoded by substantially the same nucleotide sequence and is capable of having one or more activities of the naturally-occurring sequence.
  • a variant of any given sequence means that the specific sequence of residues (whether amino acid or nucleotide residues) has been modified so that the polypeptide or polynucleotide essentially retains at least one A sequence of endogenous functions.
  • the variant sequence can be obtained by the addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in the naturally-occurring protein and/or polynucleotide, as long as the The original functional activity is sufficient.
  • the term "derivative” generally refers to any substitution, variation, modification, substitution, deletion, and deletion of one (or more) amino acid residues of the self/pair sequence in the polypeptide or polynucleotide of the present application. /Or addition, as long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analog generally refers to a polypeptide or polynucleotide, including any mimic of a polypeptide or polynucleotide, that is, possessing at least one endogenous function of the polypeptide or polynucleotide mimicked by the mimic. Chemical compounds.
  • amino acid substitutions can be made, for example, at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions, as long as the modified sequence basically maintains the requirements The activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogs.
  • the protein or polypeptide used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Intentional amino acid substitutions can be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or similarity of the amphoteric properties of the residues, as long as the endogenous function is retained.
  • the negatively charged amino acids include aspartic acid and glutamic acid; the positively charged amino acids include lysine and arginine; and the amino acids with similar hydrophilicity values without an electrical head group include glutamic acid. Paraffin, glutamine, serine, threonine and tyrosine.
  • the term “homolog” generally refers to an amino acid sequence or a nucleotide sequence that has certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence.
  • the term “homology” can be equated with sequence "identity”.
  • the homologous sequence can include an amino acid sequence that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • the homologue will contain the same active site as the subject amino acid sequence, etc.
  • homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), and homology can also be expressed in terms of sequence identity.
  • any one of the SEQ ID NO of the mentioned amino acid sequence or nucleotide sequence has a percent identity sequence refers to a sequence having the percentage identity over the entire length of the mentioned SEQ ID NO the sequence of.
  • sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a fusion protein, which may comprise: a) human TGFBRII or a fragment thereof; and b) an antibody or an antigen-binding fragment thereof that specifically binds to PD-L1.
  • the human TGFBRII or fragments thereof of the fusion protein of the present application may comprise the extracellular domain of human TGFBRII.
  • the human TGFBRII or a fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NO: 56-57.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 15: GFTFSSY (SEQ ID NO: 15).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 27: KQEGSE (SEQ ID NO: 27).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 8: DRAVAGAFDI (SEQ ID NO: 8).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 41: RASQSIYIWLA (SEQ ID NO: 41).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 26: KASSLET (SEQ ID NO: 26).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 31: QQYYGSSRT (SEQ ID NO: 31).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds to PD-L1 of the fusion protein may include a heavy chain variable region VH, and the heavy chain variable region VH may include SEQ ID NO: 13
  • the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQEGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRAVAGAFDIWGQGTMVTVSS (SEQ ID NO: 13).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein may include a light chain variable region VL, and the light chain variable region VL may include SEQ ID NO: 4
  • the amino acid sequence of: DIQMTQSPSTLSASVGDRVTVTCRASQSIYIWLAWYQQKPGKAPNLLIYKASSLETGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFGQGTKVEIK (SEQ ID NO: 4).
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antibody or antigen-binding fragment thereof that specifically binds to PD-L1 of the fusion protein may include an antibody heavy chain constant region, and the heavy chain constant region may include a human IgG constant region or a mutant thereof.
  • the human IgG constant region may comprise a human IgG1 constant region or a mutant thereof.
  • the human IgG1 constant region of the fusion protein or a mutant thereof may include the amino acid sequence shown in SEQ ID NO: 54.
  • the antibody or antigen-binding fragment thereof that specifically binds to PD-L1 of the fusion protein may include an antibody light chain constant region, and the light chain constant region may include a human Ig ⁇ constant region.
  • the human Ig ⁇ constant region includes natural and artificial Ig ⁇ constant regions or mutants thereof.
  • the human Ig ⁇ constant region of the fusion protein or a mutant thereof may comprise the amino acid sequence shown in SEQ ID NO: 55.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-L1 of the fusion protein described in the present application may comprise an antibody heavy chain or a fragment thereof.
  • the antibody heavy chain or a fragment thereof may comprise the amino acid sequence shown below or a part thereof: SEQ ID NO:1.
  • the antibody or antigen-binding fragment thereof that specifically binds to PD-L1 of the fusion protein described in the present application may comprise an antibody light chain or a fragment thereof.
  • the antibody light chain or a fragment thereof may comprise the amino acid sequence shown below or a part thereof: SEQ ID NO: 5.
  • the heavy chain or fragment thereof of the fusion protein and the light chain or fragment thereof can form an antigen binding portion that specifically binds to PD-L1 after being combined.
  • the antibody heavy chain or fragment thereof of the antibody or antigen-binding fragment thereof of the fusion protein described herein that specifically binds to PD-L1 is fused in frame with the human TGFBRII or fragment thereof to form a fusion polypeptide.
  • the N-terminus of the antibody heavy chain or fragments thereof may be directly or indirectly connected to the C-terminus of the human TGFBRII or fragments thereof.
  • the C-terminus of the antibody heavy chain or fragments thereof may be directly or indirectly connected to the N-terminus of the human TGFBRII or fragments thereof.
  • the C-terminus of the antibody heavy chain or its fragment can be directly connected to the N-terminus of the human TGFBRII or its fragment.
  • the fusion protein may comprise a first polypeptide and a second polypeptide.
  • the first polypeptide may include the heavy chain or fragment thereof of the antibody or antigen-binding fragment thereof that specifically binds PD-L1 and the human TGFBRII or fragment thereof.
  • the first polypeptide from the N-terminus to the C-terminus may include the heavy chain or fragment thereof of the antibody or antigen-binding fragment thereof that specifically binds to PD-L1, and the human TGFBRII or fragment thereof .
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 14.
  • the second polypeptide may include the light chain of the antibody or antigen-binding fragment thereof that specifically binds to PD-L1, or a fragment thereof, for example, the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 5 .
  • the first polypeptide of the fusion protein may include the amino acid sequence set forth in SEQ ID NO: 14, and the second polypeptide may include the amino acid sequence set forth in SEQ ID NO: 5.
  • the fusion protein may comprise the same first polypeptide and second polypeptide as PR001902.
  • the fusion protein of the present application may include two of the first polypeptides and two of the second polypeptides.
  • a part of each heavy chain or light chain amino acid sequence of the fusion protein or antigen-binding fragment is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category.
  • the variable regions and constant parts of the light and heavy chains are all derived from the variable and constant regions of an antibody of an animal species (such as human).
  • the physical/chemical properties and/or biological activity of the fusion protein described in the present application can be identified, screened or characterized by various assays known in the art.
  • the application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (for example, Western blot), flow cytometry (for example, FACS), immunohistochemistry, immunofluorescence, etc.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting for example, Western blot
  • flow cytometry for example, FACS
  • immunohistochemistry for example, immunofluorescence, etc.
  • immunofluorescence etc.
  • the fusion protein can bind to human-derived PD-L1 and/or TGFB1 with a KD value of 1 ⁇ 10 -8 M or lower.
  • the binding affinity of the fusion protein to PD-L1 and/or TGFB1 can be determined by any method known in the art. In some cases, binding affinity can be measured by surface plasmon resonance (SPR), enzyme-linked immunoassay (ELISA), bound antigen precipitation, equilibrium dialysis, and biofilm interference (BLI). In some cases, the binding affinity and KD value of the fusion protein to PD-L1 and/or TGFB1 can be determined by biofilm interference (BLI). For example, the FortieBio Octet molecular interaction analyzer can be used to analyze the binding kinetics between antigen and antibody.
  • the fusion protein can bind to human-derived PD-L1 with a KD value of 1 ⁇ 10 -8 M or lower.
  • the value of KD may be about 1 ⁇ 10 -8 M or less, about 9 ⁇ 10 -9 M or less, about 8 ⁇ 10 -9 M or less, about 7 ⁇ 10 -9 M or less, about 6 ⁇ 10 -9 M or less, about 5 ⁇ 10 -9 M or less, about 4 ⁇ 10 -9 M or less, about 3 ⁇ 10 -9 M or less, about 2 ⁇ 10 -9 M or less, about Values of 1 ⁇ 10 -9 M, about 5 ⁇ 10 -10 M, or about 1 ⁇ 10 -10 M or less bind to human-derived PD-L1, for example, using a FortieBio Octet molecular interaction analyzer.
  • the fusion protein can bind to human-derived TGFB1 with a KD value of 1 ⁇ 10 -8 M or lower.
  • the value of KD may be about 1 ⁇ 10 -8 M or less, about 9 ⁇ 10 -9 M or less, about 8 ⁇ 10 -9 M or less, about 7 ⁇ 10 -9 M or less, about 6 ⁇ 10 -9 M or less, about 5 ⁇ 10 -9 M or less, about 4 ⁇ 10 -9 M or less, about 3 ⁇ 10 -9 M or less, about 2 ⁇ 10 -9 M or less, about A value of 1 ⁇ 10 -9 M, about 5 ⁇ 10 -10 M, or about 1 ⁇ 10 -10 M or less binds to human-derived TGFB1, for example, using a FortieBio Octet molecular interaction analyzer.
  • the binding activity of the fusion protein described in this application to PD-L1 can be detected using flow cytometry or enzyme-linked immunoassay.
  • the EC50 value of the fusion protein and PD-L1 can be between about 0.0001 nM to about 100 nM, for example, about 0.001 nM Between about 10nM, about 0.01nM to about 10nM, about 0.05nM to about 5nM, about 0.05nM to about 1nM, about 0.05nM to about 0.8nM, about 0.1nM to about 0.8nM between.
  • the binding activity of the fusion protein described in this application to TGFB1 can be detected using flow cytometry or enzyme-linked immunoassay.
  • the EC50 value of the fusion protein and TGFB1 can be between about 0.0001 nM to about 100 nM, for example, between about 0.001 nM to about 10 nM, and about 0.001 nM to about 5 nM. , Between about 0.01 nM and about 5 nM, between about 0.1 nM and about 3 nM.
  • the fusion protein described in the present application can block the binding of PD-1 and PD-L1.
  • the blocking of the binding of PD-1 and PD-L1 by the fusion protein can be determined by flow cytometry FACS and enzyme-linked immunoassay ELISA.
  • a host cell stably expressing PD-L1 such as CHOK1 cell
  • PD-L1 enzyme-linked immunoassay ELISA
  • a host cell stably expressing PD-L1 such as CHOK1 cell
  • a biotin-labeled PD-1 protein is incubated with a biotin-labeled PD-1 protein.
  • the cells were analyzed using FACS to confirm that the fusion protein blocked the binding of PD-1 to PD-L1.
  • the PD-L1 antigen protein is first coated on the plate, and a decreasing amount of the unlabeled fusion protein and the biotin-labeled PD-1 protein are mixed and incubated together. Then, the cells were analyzed using ELISA to confirm that the fusion protein can block the binding of PD-1 and PD-L1.
  • the fusion protein described in this application can activate T cells.
  • the fusion protein can stimulate the secretion of IFN- ⁇ and/or IL2 in immune cells.
  • the immune cells may include lymphocytes, such as B cells, T cells, natural killer cells, myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. Any method known to those skilled in the art can be used to measure the secretion of cytokines in immune cells, for example, by enzyme-linked immunoassay (ELISA) to quantitatively measure the proliferation of immune cells (such as T cells) or the cytokines produced by immune cells (such as IFN- ⁇ or IL-2 produced by T cells).
  • ELISA enzyme-linked immunoassay
  • the application also provides isolated one or more nucleic acid molecules.
  • the one or more nucleic acid molecules may encode the fusion protein or antigen-binding fragment described in this application.
  • each nucleic acid molecule in the one or more nucleic acid molecules may encode the complete fusion protein or antigen-binding fragment, or may encode a part thereof (for example, HCDR1-3, LCDR1-3, VL, VH , One or more of light chain or heavy chain).
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesis, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the antibody and its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides. Overlapping extension PCR. For specific operations, please refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.
  • this application provides one or more vectors, which comprise one or more isolated nucleic acid molecules described in this application.
  • Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcriptional expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters can include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part can be fused with a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), and it may or may not contain additional introns.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • the vector may include, for example, plasmids, cosmids, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application.
  • each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application.
  • each or each host cell may contain multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in this application can be introduced into the host cell, such as a eukaryotic cell, such as a plant-derived cell, fungus, or yeast cell.
  • the vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the application provides a method for preparing the fusion protein and/or antigen-binding fragment.
  • the method may include culturing the cell described in the present application under conditions that allow the expression of the fusion protein and/or antigen-binding fragment, and optionally harvesting the expressed fusion protein.
  • conditions that allow the expression of the fusion protein and/or antigen-binding fragment, and optionally harvesting the expressed fusion protein.
  • the method may further include the step of isolating and/or purifying the antibody or antigen-binding fragment thereof.
  • protein G-Sepharose or Protein A-Sepharose can be used for affinity chromatography, and gel electrophoresis and/or high performance liquid chromatography can also be used to purify and separate the antibodies or antigen-binding fragments described in this application. .
  • the present application provides a pharmaceutical composition, which may comprise the fusion protein and/or antigen-binding fragment described in the present application, the nucleic acid molecule, the vector, the host cell, and Optionally a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used, and may include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine ; Preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride) ), phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol) ; Low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gel or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamyl acid, day Winteryl acid, histidine
  • the pharmaceutical composition can be used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the tumor size (or even substantially eliminate the tumor), and/or can alleviate and/or stabilize the disease state.
  • the pharmaceutical composition described in the present application may include a preventive and/or therapeutically effective amount of the fusion protein or antigen-binding fragment.
  • the prophylactic and/or therapeutically effective amount is a dose required to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications thereof in a subject suffering from or at risk of development.
  • the application provides the use of the fusion protein in the preparation of medicines.
  • the drug can be used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the tumor or cancer comprises colorectal tumor or cancer.
  • the tumor or cancer is a tumor or cancer in which PD-L1 expression is abnormal.
  • This application also provides the method for detecting PD-L1 expression in biological samples as described below. In some cases, the method includes contacting a biological sample with the fusion protein described in this application under conditions that allow the fusion protein to bind PD-L1, and detecting whether the fusion protein is between the fusion protein and PD-L1 Form a complex.
  • the tumor or cancer is a tumor or cancer in which PD-L1 expression is increased compared to a non-tumor or cancer sample.
  • Such methods can be in vitro or in vivo methods.
  • the fusion protein described in the present application can be used in, for example, immunoassays, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (for example, western blotting), flow cytometry (for example, FAGS) And enzyme-linked immunosorbent assay (ELISA).
  • IHC immunohistochemistry
  • IF immunofluorescence
  • IF immunoblotting
  • flow cytometry for example, FAGS
  • ELISA enzyme-linked immunosorbent assay
  • the fusion protein is used to select subjects suitable for therapy with the fusion protein described in this application.
  • the application also provides the use of the fusion protein in a method for diagnosing a subject suffering from a disorder (for example, cancer or immune dysfunction), the method comprising: contacting a sample with the fusion protein of the present invention And detecting the presence of the bound fusion protein to determine the presence or expression level of PD-L1 in the sample obtained from the subject.
  • a disorder for example, cancer or immune dysfunction
  • the sample refers to a biological sample, and may include a tissue or cell sample.
  • a biological sample may include cells or tissues from normal or cancer patients.
  • the source of the tissue or cell sample can be, for example, solid tissue from fresh, frozen and/or preserved organ or tissue samples or biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, Amniotic fluid, peritoneal fluid, or interstitial fluid; cells from the subject at any time during pregnancy or development.
  • the biological sample is obtained from an in vitro tissue or cell culture.
  • biological samples in this application include, but are not limited to, tumor biopsy, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and Preserved tumor samples, such as formalin-fixed paraffin-embedded tumor samples or frozen tumor samples.
  • the application also provides the use of the fusion protein in a method for diagnosing a subject suffering from a tumor or cancer, the method comprising: contacting a sample with the fusion protein of the application and detecting the presence of the bound antibody The presence or expression level of PD-L1 in a sample obtained from a subject.
  • the sample can be selected from the group consisting of a tissue sample, a whole blood sample, a serum sample, and a plasma sample.
  • the tissue sample may be a tumor sample.
  • the tumor sample may include tumor infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.
  • the present application provides a method for treating cancer in a subject, inhibiting tumor growth in a subject, and/or inhibiting tumor cell proliferation, including administering the method of the present application to a subject in need or the tumor cell
  • the fusion protein, the molecular nucleic acid, the vector, the host cell and/or the pharmaceutical composition can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, transcutaneously, intraarterially, and intraperitoneally.
  • Intra-injury, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, and rectal Locally, intratumorally, peritoneally, subconjunctivally, intracapsular, mucosal, intrapericardial, intraumbilical, intraocular, intraorbital, orally Way, by topical way, by transdermal way, by intravitreal way (for example, by intravitreal injection), by eye drops, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by direct Bathe the local perfusion of target cells, through a catheter, through lavage, in the form of a cream or in the form of a lipid composition.
  • composition used in the methods described herein can also be administered systemically or locally.
  • the method of administration may vary depending on various factors (for example, the compound or composition being administered and the severity of the condition, disease, or disorder being treated).
  • intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, implantation, inhalation Intrathecal, intraventricular, or intranasal administration of anti-cancer therapy (e.g., anti-PD-L1 antibody).
  • anti-cancer therapy e.g., anti-PD-L1 antibody
  • the administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection.
  • Various dosing schedules are covered herein, including but not limited to a single administration or multiple administrations at various time points, bolus administrations, and pulse infusions.
  • the fusion protein or pharmaceutical composition described herein can be formulated, administered, and administered in a manner consistent with good medical practice.
  • the considerations in this situation include the specific condition being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of the condition, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner .
  • the therapeutic agent e.g., anti-PD-L1 antibody
  • the effective amount of such other agents depends on the amount of therapeutic agent (e.g., anti-PD-L1 antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above.
  • These agents can generally be used in any dosage that is empirically/clinically determined to be appropriate and through any route that is empirically/clinically determined to be appropriate.
  • the dose can be administered as a single dose or as multiple doses (e.g. 2 or 3 doses), such as an infusion. Compared with a single treatment, the dose of the antibody administered in the combination treatment can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.
  • the present application provides a kit, which may include the fusion protein described in this application, and/or the pharmaceutical composition described in this application. It can include the fusion protein described in this application and/or the pharmaceutical composition in a single common container, and can also be optionally combined with one or more therapeutic agents, and optionally formulated together in the pharmaceutical composition.
  • the kit may also include a device for administering the fusion protein or pharmaceutical composition described herein; for example, the device depends on the method of administration of the contents.
  • the kit may include a package insert that includes information about the fusion protein, pharmaceutical composition, and dosage form in the kit. Generally, this type of information helps patients and physicians use encapsulated antigen binding proteins, pharmaceutical compositions, and dosage forms effectively and safely.
  • the container used in such a kit can generally comprise at least one vial, test tube, flask, bottle, syringe, or other suitable container, in which one or more of the detection and/or therapeutic compositions can be placed.
  • the kit may also contain a second different container in which the second detection and/or therapeutic composition can be placed.
  • a second container in which the second detection and/or therapeutic composition can be placed.
  • multiple compounds can be prepared as a single pharmaceutical composition, and can be packaged in a single container device such as a vial, flask, syringe, bottle, or other suitable single container.
  • the present application provides a drug delivery device (such as a plastic or vial, such as a hollow pin or a syringe cylinder), which can be used to administer the fusion protein described in the present application or a pharmaceutical composition thereof.
  • the device can introduce a substance into the patient through parenteral routes (for example, intramuscular, subcutaneous, or intravenous).
  • the injection device may be a syringe (for example, a pre-filled syringe with the fusion protein described in this application or a pharmaceutical composition thereof, such as an auto-injector), which may include a fluid for containing the fluid to be injected (for example, the antigen binding device described in this application). Protein or its pharmaceutical composition) syringes and needles (which can be used to pierce the skin and/or blood vessels).
  • the mode of administration can be changed.
  • the route of administration may include oral, intramuscular injection, subcutaneous injection, and rectal administration.
  • the TGFBRII extracellular region truncated sequence (SEQ ID NO: 56) was constructed to the C-terminus of the heavy chain of the anti-PD-L1 antibody PR000265 to construct a bifunctional fusion protein molecule, and the plasmid encoding the polypeptide sequence was transfected into mammals Host cells (such as human embryonic kidney cells HEK293). HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, A1383504).
  • the fusion protein PR001902 of the present application was obtained and used in the following experiments.
  • the protein molecule M7824 also called PR001599, which is a bintrafusp alfa analog
  • the protein molecule Hengrui fusion protein 9 also called PR002466, whose sequence is derived from patent WO2018205985A1
  • TGFBRII Trap also called PR002040, which The sequence of the anti-chicken lysozyme antibody clone TEL16 was fused with the truncated sequence of the extracellular region of TGFBRII) as the control in the example.
  • the sequence of the above-mentioned fusion protein is shown in Table 2, and the CDR division is based on the Chothia definition rule as an example.
  • Example 2 Fusion protein binds to CHO-K1 cells overexpressing human PD-L1
  • CHO-K1 cells overexpressing human PD-L1 were used to carry out the antibody at the cellular level.
  • PR001902 can effectively bind to CHO-K1/hPD-L1 cells, and has a dose-dependent effect on drug concentration.
  • Protein sample EC50(nM) PR000265 0.258 Positive control M7824 0.718 PR001902 0.515 Human IgG1 /
  • TGFB1 Novoprotein, CA59
  • a 96-well plate was coated at 100 ⁇ l/well at 4°C overnight. Wash 3 times with 200 ⁇ l/well 1 ⁇ PBST, add 200 ⁇ l/well 2% BSA, and block at 37°C for 2 hours. Add 200 ⁇ l/well of 1xPBST to wash 3 times, add 5-fold concentration gradient antibody, wherein the highest final concentration of antibody is 100nM, and incubate at 37°C for 1 hour.
  • 200 ⁇ l/well 1 ⁇ PBST was washed 3 times, 100 ⁇ l/well anti-human FC-HRP secondary antibody (Sigma-Aldrich, A0170, diluted 1:4000) was added to each well, and incubated at 37°C for 1 hour. Wash 3 times with 200 ⁇ l/well 1 ⁇ PBST, add 100 ⁇ l/well TMB to each well, incubate at room temperature for 10 minutes, add 50 ⁇ l/well 1M ELISA stop solution to stop the reaction. Detect the absorbance at 450nm and 570nm on the microplate reader.
  • the software GraphPad Prism 8 is used for data processing and graphing analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve and EC50 value are obtained.
  • Example 4 Fusion protein blocks the binding of human PD-1 to CHO-K1 cells overexpressing human PD-L1
  • CHO-K1 cells (CHO-K1/hPD-L1, Nanjing GenScript, M00543) that highly express human PD-L1 were used to carry out antibody at the cellular level. Block the experiment. Digest the CHO-K1/hPD-L1 cells and resuspend them in F12K complete medium to adjust the cell density to 2x10 6 /mL. Inoculate a 96-well U bottom plate with 50 ⁇ l cells/well, and then add 100 ⁇ L/well, 5-fold concentration gradient dilution of the antibody to be tested, where the highest final concentration of the antibody is 100 nM.
  • Example 5 Using BLI to determine the affinity of the fusion protein to human PD-L1 or human TGFB1
  • the Octet molecular interaction analyzer (ForteBio, model Octet Red96e) was used to analyze the binding kinetics between antigen and antibody.
  • the affinity of the fusion protein to human TGFB1 protein was determined by Octet. Two rows of SA sensors are placed on the sensor rack, one row is used as the test SA sensor and the other row is used as the reference SA sensor. Use 1 ⁇ PBS with 0.02% Tween as the buffer, and first equilibrate the SA sensor in the buffer for 10 minutes; test the SA sensor to capture biotinylated human TGFB1 protein (NovoProtein, CA59) using biotinylation reagents Box (ThermoFisher, A39257, EZ-Link Sulfo-NHS-LC-Biotin) for biotinylation), set the capture height to 0.3nm; the reference SA sensor is immersed in the buffer for 30 seconds.
  • biotinylated human TGFB1 protein NovoProtein, CA59
  • biotinylation reagents Box ThermoFisher, A39257, EZ-Link Sulfo-NHS-LC-Biotin
  • the two rows of sensors then interact with the fusion protein to be tested in gradient dilutions (for example, the concentration of the fusion protein to be tested can be two-fold dilutions of 6.25 ⁇ 0.39nM and 0nM). Each sensor corresponds to a concentration.
  • the dissociation time is 180 seconds and 600 seconds, respectively.
  • the affinity of the fusion protein to human PD-L1 protein was determined by Octet.
  • Two rows of AHC sensors are placed on the sensor rack, one row is used as the test AHC sensor, and the other row is used as the reference AHC sensor.
  • the AHC sensor is first equilibrated in the buffer for 10 minutes; the test AHC sensor captures the fusion protein (concentration 40 nM) for 30 seconds, and the capture height is about 0.7 nm; the reference AHC sensor is immersed in the buffer for 30 seconds.
  • the two rows of sensors then interact with the human PD-L1 protein in a gradient dilution (for example, the PD-L1 protein concentration can be a two-fold dilution of 25 ⁇ 0.39nM and 0nM). Each sensor corresponds to a concentration.
  • the dissociation time is 300 seconds and 900 seconds respectively.
  • the ForteBio Octet platform was used to perform epitope competition experiments on the obtained fusion protein and the positive control M7824 (ie PR001599).
  • the first step is to obtain the 100% signal of the antibody: use the SA sensor to capture the biotinylated human PD-L1 protein (NovoProtein (NovoProtein, C315)) and use the biotinylation kit (ThermoFisher, A39257, EZ-Link Sulfo) -NHS-LC-Biotin) for biotinylation), the capture height is 0.25nm.
  • the sensor was immersed in the antibody (50 nM) for 500 seconds, and the final signal of the antibody binding to PD-L1 was recorded as the 100% signal of the antibody.
  • the second step, epitope competition experiment use SA sensor to capture biotinylated PD-L1 protein, the capture height is 0.25nm. Immerse the sensor in the first antibody (50nM) for 500 seconds, and then immerse the SA sensor in the mixture of the first antibody and the second antibody (the final concentration of both antibodies is 50nM) for 500 seconds, and record the final signal This is the signal of the second antibody.
  • the inhibition rate is calculated by the following formula,
  • A 100% signal of a certain antibody (obtained from the first step)
  • B signal of the antibody as a second antibody (obtained from the second step).
  • the obtained inhibition rate is greater than 85 (%), it means that the epitopes of the two antibodies completely overlap; if the inhibition rate is less than 85 (%), it means that the epitopes of the two antibodies are not completely overlapped.
  • HEK-Blue TM TGF- ⁇ cells that highly express human TGFBRI and Smad3/4 gene fused with the SEAP reporter gene were used to study the Smad signaling pathway induced by TGFB1 by the fusion protein at the cellular level. Inhibition of activation.
  • 10 ⁇ l/well of human TGFB1 protein R&D Systems, 240-B/F
  • 10 ⁇ l/well of human TGFB1 protein R&D Systems, 240-B/F
  • the anti-TGFB1 antibody (Biointron, B5484) was used as the control molecule. Inoculate 180 ⁇ l, 2.5 ⁇ 10 4 cells/well in a 96-well plate, and continue to incubate overnight in a carbon dioxide incubator. Then transfer 20 ⁇ l of cell supernatant from each well to another 96-well plate, and then add 180 ⁇ l of Quanti-Blue working solution (Invivogen, rep-qb1) to each well, and incubate for 1 hour at 37°C. Detect the absorbance value at 655nm on the microplate reader.
  • the software GraphPad Prism 8 is used for data processing and graphing analysis, through four-parameter nonlinear fitting, the binding curve and IC50 value and other parameters are obtained.
  • PR001902 inhibited the production of SEAP reporter induced by TGFB1 in a dose-dependent manner, and had comparable efficacy and IC50 to the positive control M7824.
  • the inhibitory activity and IC50 of PR001902 are 8 times stronger than the positive control Hengrui fusion protein 9 and TGFB1 antibody.
  • PBMC peripheral blood mononuclear cells
  • the cultured cells were collected by centrifugation, washed 4 times with PBS containing 2% FBS, resuspended in a fresh medium, adjusted to a density of 2 ⁇ 10 5 cells/mL, and inoculated to a 96-well cell culture plate with 100 ⁇ l/well.
  • Pan T cells (Meltenyi, 130-096-535) were isolated from fresh allogeneic PBMC and resuspended in fresh medium, the cell density was adjusted to 1 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was inoculated into a 96-well cell culture plate.
  • IFN- ⁇ ELISA kit Thermo, 88-7316-88 was used to detect the level of IFN- ⁇ in the supernatant after 120 hours; refer to the reagent instructions for specific operations.
  • PR001902 can dose-dependently enhance the secretion of cytokines IL-2 ( Figure 5) and IFN- ⁇ ( Figure 6) by activated T lymphocytes, and has a ratio
  • the positive control has a stronger activation effect.
  • This example tests the pharmacokinetic properties of the fusion protein.
  • the method is as follows, select 3 female C57BL/6 mice weighing 18-22 grams, and give PR001902 intravenously at a dose of 9.53 mg/kg. Collect whole blood before administration and 0.5 hours, 24 hours (day 1), day 2, day 4, day 7, day 10, and day 14 after administration, and let the whole blood stand for 30 minutes. It was coagulated, then centrifuged at 2,000 rpm at 4°C for 5 minutes and the separated serum samples were frozen at -80°C until analysis. In this example, two ELISA methods were used to quantitatively determine the drug concentration in mouse serum.
  • ELISA method one the Fc end detection method, captures the human Fc-containing fusion protein in mouse serum by goat anti-human Fc polyclonal antibody coated on a 96-well plate, and then adds HRP-labeled goat anti-human Fc second Antibody to detect;
  • ELISA method two TGFBRII end detection method, capture the fusion protein containing human TGFBRII domain in mouse serum by human TGFB1 protein coated on 96-well plate, and then add HRP-labeled goat anti-human Fc secondary antibody to detect.
  • NCA non-compartmental model
  • the results are shown in Figure 7 and Table 9.
  • the PK analysis results show that under the Fc-terminal detection method, the half-life of PR001902 in mice is about 11 days; the TGFBRII-end detection method shows that the half-life of PR001902 in mice is about 8.5 days .
  • Example 10 Anti-tumor activity of the fusion protein on the in vivo animal model of colon cancer CT26-human PD-1/PD-L1 double knockout mice
  • mice Inoculate CT26-human PDL1(tg)-mPDL1(KO) cells in logarithmic growth phase into humanized mouse BALB/c-human PD-1/PD-L1 right axillary subcutaneously, when the average tumor volume reaches 80- At 120 mm 3 , after removing the mice with too large tumor volume, the mice were randomly divided into 3 groups according to the tumor volume, with 6 mice in each group. The intraperitoneal administration was started on the day of grouping, and the drug was given once every three days for a total of 6 administrations (q3d ⁇ 6). After starting the administration, weigh the body weight and tumor volume twice a week.
  • tumor volume (mm 3 ) 0.5 ⁇ tumor long diameter ⁇ tumor short diameter 2 .
  • the tumor-bearing mice were euthanized and the tumor was stripped and weighed.
  • the experimental results such as the tumor volume and mouse body weight of each group of animals were calculated as mean ⁇ standard error (Mean ⁇ SEM).
  • Multi-group comparisons were performed using one-way ANOVA (one way ANOVA) test method to compare whether there were significant differences between the different treatment groups and the control group.
  • the data was analyzed using SPSS 18.0. P ⁇ 0.05 indicates a significant difference.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种融合蛋白，其包括人TGFBRII或其片段以及特异性结合PD-L1的抗体或其抗原结合片段。还提供了所述融合蛋白在制备药物中的应用，所述药物用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。

Description

一种融合蛋白及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种抗PD-L1和抗TGFB的融合蛋白及其应用。
背景技术
程序性死亡受体配体1(PD-L1)，也称为分化簇274(CD274)或B7同源蛋白1(B7-H1)，是一个40KDa的I型跨膜蛋白，一般在活化的T细胞、B细胞、单核细胞、树突状细胞、巨噬细胞及许多非造血细胞上表达。
PD-L1能与程序性死亡受体1(PD-1)结合。其中PD-L1/PD-1信号通路是免疫反应中非常重要的共抑制信号途径，负调节T细胞免疫应答，抑制T细胞活性，减弱细胞因子的分泌。研究发现PD-L1能在许多肿瘤组织中表达，包括胃癌、肺癌、乳腺癌、胰腺癌、卵巢癌、结肠癌、肥大细胞性肿瘤以及恶性黑色素瘤等，以及在浸润肿瘤微环境的骨髓细胞中表达，保护肿瘤细胞逃避免疫攻击。转化生长因子-B(TGFB)是对免疫系统有显著影响的强效细胞因子，参与许多增殖性和非增殖性细胞过程，如细胞增殖和分化、胚胎发育、细胞外基质形成、骨骼发育、伤口愈合、造血作用以及免疫和炎症反应。现有治疗技术手段无论是化学疗法或是肿瘤靶向疗法，影响其疗效的一个重要瓶颈是肿瘤细胞产生免疫耐受。肿瘤细胞在肿瘤微环境中利用多种免疫抑制机制来逃避机体免疫系统识别和攻击，这些免疫抑制机制包括免疫抑制型的细胞因子(如TGFB)、调节性T细胞(Tregs)、共抑制信号通路分子、髓系抑制性细胞等等。
免疫抑制的多种机制可能会阻碍免疫治疗具有有效性。在一些情况下，肿瘤对于单药免疫治疗来说是难治的并且只有一小部分的癌症有完全反应。因此，研发具备阻断PD-L1/PD-1信号通路能力的药物以及在抑制PD-1/PD-L1通路的基础上靶向中和肿瘤微环境的免疫抑制型细胞因子TGFB，能为肿瘤及多种免疫系统相关疾病的治疗带来全新的解决方法。
发明内容
本申请提供了一种融合蛋白，其包括人TGFBRII或其片段以及特异性结合PD-L1的抗体或其抗原结合片段。本申请还提供了所述融合蛋白在预防和治疗肿瘤或癌症中的应用。
一方面，本申请提供了一种融合蛋白，其包含：a)人TGFBRII或其片段；以及b)特异性结合PD-L1的抗体或其抗原结合片段；其中所述特异性结合PD-L1的抗体或其抗原结合片 段包含HCDR1，HCDR2和HCDR3，以及LCDR1，LCDR2和LCDR3；所述HCDR1包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2包含SEQ ID NO:27所示的氨基酸序列，所述HCDR3包含SEQ ID NO:8所示的氨基酸序列，所述LCDR1包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:31所示的氨基酸序列；所述CDR序列使用Chothia定义规则来定义(见表1)。
在某些实施方式中，所述特异性结合PD-L1的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:13所示的氨基酸序列，且所述轻链可变区包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述特异性结合PD-L1的抗体或其抗原结合片段包含抗体重链，且所述抗体重链与所述人TGFBRII或其片段框内融合而形成融合多肽。
在某些实施方式中，所述抗体重链的C端与所述人TGFBRII或其片段的N端连接。
在某些实施方式中，所述人TGFBRII或其片段包含人TGFBRII的胞外结构域。
在某些实施方式中，所述人TGFBRII或其片段包含SEQ ID NO:56-57中任一项所示的氨基酸序列。
在某些实施方式中，所述特异性结合PD-L1的抗体或其抗原结合片段还包含重链恒定区，且所述重链恒定区为人IgG1恒定区。
在某些实施方式中，所述特异性结合PD-L1的抗体或其抗原结合片段还包含轻链恒定区。
在某些实施方式中，所述轻链恒定区包含人Igκ恒定区。
在某些实施方式中，所述的融合蛋白包含第一多肽和第二多肽，其中所述第一多肽包含SEQ ID NO:14所示的氨基酸序列；且所述第二多肽包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述的融合蛋白包含两条所述第一多肽以及两条所述第二多肽。
另一方面，本申请还提供了分离的核酸分子，其包含编码所述的融合蛋白或其功能性片段的多核苷酸。
另一方面，本申请还提供了药物组合物，其包含所述的融合蛋白，或所述的核酸分子，以及任选地药学上可接受的载体。
所述的融合蛋白，或所述的核酸分子用于制备药物的用途，所述药物用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。
在某些实施方式中，所述肿瘤或癌症包括结肠癌。
在某些实施方式中，所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。
另一方面，本申请还提供了所述的融合蛋白，或所述的核酸分子，其用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。
在某些实施方式中，所述肿瘤或癌症包括结肠癌。
在某些实施方式中，所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。
另一方面，本申请还提供了治疗受试者中的癌症、抑制受试者中肿瘤生长和/或抑制肿瘤细胞增殖的方法，所述方法包括向所述受试者或所述肿瘤细胞施用所述的融合蛋白，或所述的核酸分子。
在某些实施方式中，所述肿瘤或癌症包括结肠癌。
在某些实施方式中，所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述融合蛋白体外结合人源PD-L1；
图2显示的是本申请所述融合蛋白体外结合人源TGFB1；
图3显示的是本申请所述融合蛋白体外阻断PD-L1和PD-1的结合；
图4显示的是本申请所述融合蛋白抑制TGFB/Smad信号通路的激活；
图5显示的是本申请所述融合蛋白增强激活的T淋巴细胞分泌细胞因子IL-2；
图6显示的是本申请所述融合蛋白增强激活的T淋巴细胞分泌细胞因子IFN-γ；
图7显示的是本申请所述融合蛋白药代动力学结果；
图8显示的是本申请所述融合蛋白对CT26-人PD-1/PD-L1双敲小鼠的肿瘤抑制效果，A：肿瘤体积变化，B：小鼠体重变化。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“抗体或其抗原结合片段”通常是指包含结合抗原的部分的蛋白质， 以及任选地允许结合抗原的部分采用促进抗体或其抗原结合片段与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR-1、CDR1、FR-2、CDR2、FR-3、CDR3和FR-4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体或其抗原结合片段的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“融合蛋白”通常是指由两种或多种多肽构成的蛋白质，在天然状态下通常不结合，但是各自的氨基和羧基末端可通过直接或间接结合在一起以形成一个连续多肽。在某些情形中，提及本申请所述的融合蛋白时，术语“融合蛋白”可与“蛋白质”互换的使用。在某些情形中，提及本申请所述的融合蛋白时，术语“融合蛋白”可与“抗体”互换的使用。
在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDR结构环区连接。每条链中的CDRs通 过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点，恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞毒性。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，J Mol Biol 273:927-48,1997)。在本申请中，还使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基，参见表1。
表1抗体CDR定义方法
  Kabat Chothia Combined
LCDR1 L24--L34 L24--L34 L24-L34
LCDR2 L50--L56 L50--L56 L50-L56
LCDR3 L89--L97 L89--L97 L89-L97
HCDR1 H31--H35 H26--H32 H26-H35
HCDR2 H50--H65 H52--H56 H50-H65
HCDR3 H95--H102 H95--H102 H95-H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。
在本申请中，术语“分离的”蛋白质通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的蛋白质。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的蛋白质或抗体通常将通过至少一个纯化步骤来制备。
在本申请中，术语“特异性结合”通常是指抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的、不相关的表位而言，更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗体结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“KD”、“K D”可互换地使用，通常是指平衡解离常数，“KD”是解 离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。可使用结合速率常数(kon)、解离速率常数(kdis)和平衡解离常数(KD)表示抗体对抗原的结合亲和力。确定结合和解离速率常数的方法为本领域熟知，包括但不限于生物膜干涉技术(BLI)、放射免疫法(RIA)、平衡透析法、表面等离子共振(SPR)、荧光共振能量迁移(FRET)、免疫共沉淀(Co-IP)以及蛋白质芯片技术。如果在不同的条件(例如盐浓度、pH)下测量，则所测得的某种特定蛋白-蛋白相互作用的亲和力可不同。
在本申请中，术语“PD-L1”通常是指程序性死亡配体1蛋白、其功能变体和/或其功能片段。PD-L1也称为分化簇274(CD274)或B7同源物1(B7-H1)，并且是由(人类中)CD274基因编码的蛋白。PD-L1结合其受体，例如程序性死亡1(PD-1)，所述PD-1在活化的T细胞、B细胞和巨噬细胞中表达(Ishida et al.,1992 EMBO J,11:3887-3395；Okazaki et al.,Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.Science,2001；291:319-22)。PD-L1和PD-1的络合通过抑制T细胞增殖和产生细胞因子IL-2和IFN-γ发挥免疫抑制作用(Freeman et al.,Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation,J.Exp.Med.2000,192:1027-1034；Carter et al.,PD-1:PD-L inhibitory pathway affects both CD4(+)and CD8(+)T cells and is overcome by IL-2.Eur.J.Immunol.2002,32:634–643)。术语“PD-L1”涵盖任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。PD-L1序列是本领域已知的。例如可在NCBI Gene ID No.29126下找到关于人PD-L1基因(包括基因组DNA序列)的信息。又例如可在NCBI Gene ID No.60533下找到关于小鼠PD-L1基因(包括基因组DNA序列)的信息。又例如可在NCBI Gene ID No.102145573下找到关于食蟹猴PD-L1基因(包括基因组DNA序列)的信息。示例性的全长人PD-L1蛋白的氨基酸序列可在NCBI登录号NP_054862或UniProt登录号Q9NZQ7下找到。示例性的全长小鼠PD-L1蛋白序列可在NCBI登录号NP_068693或Uniprot登录号Q9EP73下找到。示例性的全长食蟹猴PD-L1蛋白序列可在NCBI登录号XP_005581836或Uniprot登录号G7PSE7下找到。
在本申请中，术语“PD-1”通常是指程序性死亡1受体(也称为CD279)、其功能变体和/或其功能片段。PD-1通常在T细胞、B细胞、自然杀伤T细胞、活化的单核细胞和树突 细胞(DC)上表达。PD-1可以结合其配体PD-L1和PD-L2。在PD-1的定义之内还包括与天然存在的PD-1的氨基酸序列不同但保持特异性结合PD-L1的能力的变体。在PD-1的定义之内进一步包括增强PD-L1的生物活性的变体。PD-1序列是本领域已知的。例如，示例性的全长人PD-1蛋白序列可在NCBI登录号NP_005009下找到，示例性的全长食蟹猴的PD-1蛋白序列可在NCBI登录号NP_001271065或Uniprot登录号B0LAJ3下找到。
在本申请中，术语“TGFBRII”通常是指一种转化生长因子β(TGFB)的受体II(也称为TGFBR2)。细胞表面的TGFBR可以被转化生长因子(TGFB)结合并激活，通过SMAD通路进行信号传递，具有调节生长，抗炎和免疫调节的活性。TGFBRII通常由C端蛋白激酶域和N端胞外域组成。完整的人TGFBRII序列可以在Uniprot登录号P37173中找到。或者，完整的人TGFBRII序列可以如SEQ ID NO:53所示。
在本申请中，术语“表位”通常是指抗体特异性结合的抗原的某个区或区域。表位通常由诸如氨基酸或碳水化合物或糖侧链分子的化学活性表面群组组成，且通常具有特异性三维结构特征以及特异性电荷特征。表位可为“线性表位”或“构型表位”。在线性表位中，蛋白质与相互作用的分子(诸如抗体)之间的所有相互作用点沿蛋白质的一级氨基酸序列线性发生。在构型表位中，相互作用点于彼此分离的蛋白质上的氨基酸上交叉发生。给定抗体结合何种表位的方法(如，表位作图(Epitope Mapping))是本领域所公知的，包括例如免疫印迹和免疫沉淀测定，例如，测试重叠或邻接的肽(例如，来自PD-L1)与给定抗体的反应性。确定表位空间构象的方法包括本领域技术和本申请所述的那些技术，例如，X射线晶体学、二维核磁共振和HDX-MS。术语“不完全重叠”的表位通常是指在提及两种或多种抗体时，各抗体结合不同的氨基酸残基群，如通过给定方法所测定的。用于确定本申请所述抗体是否与参比抗体结合表位重叠的技术包括例如表位作图方法，诸如，抗原：抗体复合物的晶体的x射线分析，其提供表位的原子分辨率，和氢/氘交换质谱法(HDX-MS)。其他方法监测抗体与抗原片段或抗原的突变变化形式的结合，其中由于抗原序列内氨基酸残基的修饰所致的结合损失经常被视为可指示表位组分(例如，丙氨酸扫描诱变-Cunningham及Wells(1985)Science 244:1081)。另外，亦可使用表位定位的计算组合方法。本领域测定表位的方法包括但不限于合成肽法，免疫信息学预测法，多肽活性测定，表位肽扫描技术，蛋白质切割法，噬菌体展示技术，X射线衍射与核磁共振分析，以及利用计算机软件进行表位预测。在某些实施方式中，可以使用基于生物膜干涉法(BLI)Octet分子相互作用分析平台进行两种或多种抗体对抗原表位的竞争性结合。例如，将第一种抗体与抗原混合，再加入第二种抗体，利用ForteBio Octet测定第二种抗体对第一种抗体的竞争性抑制率，当竞争性抑制率低于85％时，可以说第一抗体与第二种抗体结合抗原的表位不同。
在本申请中，术语“受试者”通常是指哺乳动物。哺乳动物包括但不限于驯化动物(例如奶牛、绵羊、猫、狗和马)、灵长类(例如人和非人灵长类，诸如猴)、兔以及啮齿类(例如，小鼠和大鼠)。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。在本申请中，术语“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
在本申请中，术语“药物组合物”通常是指这样的制剂，其以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。所述组合物是无菌的。“无菌”组合物是灭菌的，或不含所有活的微生物及它们的孢子。
在本申请中，术语“治疗”通常是指期望改变所治疗个体的天然病程，且可为实现防治或在临床病变过程中进行的临床介入。合乎需要的治疗效果包括但不限于防止疾病发生或复发性、减轻症状、减弱疾病的任何直接或间接病理学后果、防止转移、降低疾病进展速率、改善或缓解疾病状态以及缓和或改善预后。在一些情形中，抗体(例如，抗PD-L1抗体)可用来延迟疾病发展或减缓疾病进展。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的化合物(例如，抗癌治疗剂)或药物组合物(例如，包含抗癌治疗剂的药物组合物)的方法。施用可通 过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。胃肠外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。部分地根据施用是否为短暂的或长期的，给药可通过任何适合的途径进行，例如通过注射(诸如静脉内或皮下注射)进行。本文涵盖各种给药过程，包括但不限于单次施用或各种时间点内的多次施用、推注施用和脉冲输注。
在本申请中，术语“肿瘤”通常是指所有赘生性细胞生长和增殖(无论恶性还是良性)以及所有癌前和癌性细胞和组织。在本申请中，肿瘤可以包括结肠癌。
除了本文提到的特定序列的蛋白质和核苷酸之外，本申请还可包括其功能性变体、衍生物、类似物、同源物及其片段。
术语“功能性变体”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的变体是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得变体序列，只要保持原来的功能活性即可。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。
在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序 列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
融合蛋白
另一方面，本申请提供了一种融合蛋白，其可包含：a)人TGFBRII或其片段；以及b)特异性结合PD-L1的抗体或其抗原结合片段。
本申请所述融合蛋白的所述人TGFBRII或其片段可包含人TGFBRII的胞外结构域。在某些情形中，所述人TGFBRII或其片段可包含SEQ ID NO:56-57中任一项所示的氨基酸序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含HCDR1，且所述HCDR1可包含SEQ ID NO:15所示的氨基酸序列：GFTFSSY(SEQ ID NO:15)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含HCDR2，且所述HCDR2可包含SEQ ID NO:27所示的氨基酸序列：KQEGSE(SEQ ID NO:27)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含HCDR3，且所述HCDR3可包含SEQ ID NO:8所示的氨基酸序列：DRAVAGAFDI(SEQ ID NO:8)。 例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含LCDR1，且所述LCDR1可包含SEQ ID NO:41所示的氨基酸序列：RASQSIYIWLA(SEQ ID NO:41)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含LCDR2，且所述LCDR2可包含SEQ ID NO:26所示的氨基酸序列：KASSLET(SEQ ID NO:26)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含LCDR3，且所述LCDR3可包含SEQ ID NO:31所示的氨基酸序列：QQYYGSSRT(SEQ ID NO:31)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含重链可变区VH，且所述重链可变区VH可包含SEQ ID NO:13所示的氨基酸序列：EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQEGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRAVAGAFDIWGQGTMVTVSS(SEQ ID NO:13)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含轻链可变区VL，且所述轻链可变区VL可包含SEQ ID NO:4所示的氨基酸序列：DIQMTQSPSTLSASVGDRVTVTCRASQSIYIWLAWYQQKPGKAPNLLIYKASSLETGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFGQGTKVEIK(SEQ ID NO:4)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含抗体重链恒定区，且所述重链恒定区可以包含人IgG恒定区或其突变体。在某些情形中，所述人IgG恒定区可包含人IgG1恒定区或其突变体。例如，所述融合蛋白的人IgG1恒定区或其突变体可包含SEQ ID NO:54所示的氨基酸序列。
所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含抗体轻链恒定区，且所述轻链恒定区可以包含人Igκ恒定区。所述人Igκ恒定区包括天然和人工合成的Igκ恒定区或其突变体。例如，所述融合蛋白的人Igκ恒定区或其突变体可包含SEQ ID NO:55所示的氨基酸序列。
本申请所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含抗体重链或其片段。例如，所述抗体重链或其片段可包含下述所示的氨基酸序列或其一部分：SEQ ID NO:1。本申请所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段可包含抗体轻链或其 片段。例如，所述抗体轻链或其片段可包含下述所示的氨基酸序列或其一部分：SEQ ID NO:5。所述融合蛋白的所述重链或其片段与所述轻链或其片段组合后可形成特异性结合PD-L1的抗原结合部分。本申请所述融合蛋白的特异性结合PD-L1的抗体或其抗原结合片段的所述抗体重链或其片段与所述人TGFBRII或其片段框内融合而形成融合多肽。在某些情形中，所述抗体重链或其片段的N端可以直接或间接与所述人TGFBRII或其片段的C端连接。在某些情形中，所述抗体重链或其片段的C端可以直接或间接与所述人TGFBRII或其片段的N端连接。例如，所述抗体重链或其片段的C端可以直接与所述人TGFBRII或其片段的N端连接。
在本申请中，所述融合蛋白可包含第一多肽和第二多肽。所述第一多肽可包含所述特异性结合PD-L1的抗体或其抗原结合片段的重链或其片段以及所述人TGFBRII或其片段。在某些情形中，所述第一多肽从N端至C端可以包括所述特异性结合PD-L1的抗体或其抗原结合片段的重链或其片段，以及所述人TGFBRII或其片段。例如，所述第一多肽可包含SEQ ID NO:14所示的氨基酸序列。所述第二多肽可包含所述特异性结合PD-L1的抗体或其抗原结合片段的轻链或其片段，例如，所述第二多肽可包含SEQ ID NO:5所示的氨基酸序列。
在某些情形中，所述融合蛋白的所述第一多肽可包含SEQ ID NO:14所述的氨基酸序列，且所述第二多肽可包含SEQ ID NO:5所述的氨基酸序列。例如，所述融合蛋白可包含与PR001902相同的第一多肽和第二多肽。
本申请的所述的融合蛋白可包含两条所述第一多肽以及两条所述第二多肽。
在本申请中，所述融合蛋白或抗原结合片段的每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别。例如，轻链和重链的可变区及恒定部分均来自一个动物物种(如人)的抗体的可变区及恒定区。
可通过本领域已知的各种测定鉴别、筛选或表征本申请所述融合蛋白的物理/化学特性和/或生物活性。
在一个方面，例如可通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请融合蛋白的抗原结合活性。
本申请中，所述融合蛋白能够以1×10 -8M或更低的KD值结合源自人的PD-L1和/或TGFB1。所述融合蛋白对PD-L1和/或TGFB1的结合亲和力可通过本领域已知的任何方法测定。在某些情形中，结合亲和力可通过表面等离子共振法(SPR)、酶联免疫法(ELISA)、结合抗原沉淀法、平衡透析法、生物膜干涉(BLI)测定。在某些情形中，所述融合蛋白对PD-L1和/或TGFB1的结合亲和力和KD值可通过生物膜干涉(BLI)测定。例如，可使用FortieBio  Octet分子相互作用分析仪，来进行抗原抗体之间的结合动力学分析。
本申请中，所述融合蛋白能够以1×10 -8M或更低的KD值结合源自人的PD-L1。例如，所述KD的值可以以约1×10 -8M或以下、约9×10 -9M或以下、约8×10 -9M或以下、约7×10 -9M或以下、约6×10 -9M或以下、约5×10 -9M或以下、约4×10 -9M或以下、约3×10 -9M或以下、约2×10 -9M或以下、约1×10 -9M、约5×10 -10M、约1×10 -10M或以下的值结合源自人的PD-L1，例如，使用使用FortieBio Octet分子相互作用分析仪所检测的。
本申请中，所述融合蛋白能够以1×10 -8M或更低的KD值结合源自人的TGFB1。例如，所述KD的值可以以约1×10 -8M或以下、约9×10 -9M或以下、约8×10 -9M或以下、约7×10 -9M或以下、约6×10 -9M或以下、约5×10 -9M或以下、约4×10 -9M或以下、约3×10 -9M或以下、约2×10 -9M或以下、约1×10 -9M、约5×10 -10M、约1×10 -10M或以下的值结合源自人的TGFB1，例如，使用使用FortieBio Octet分子相互作用分析仪所检测的。
在另一情形中，本申请所述融合蛋白与PD-L1的结合活性可使用流式细胞术或酶联免疫法测定进行检测。例如，在FACS检测中，使用稳定表达人PD-L1的宿主细胞(如CHOK1细胞)，所述融合蛋白与PD-L1的EC50值可以在约0.0001nM至约100nM之间，例如，约0.001nM至约10nM之间，约0.01nM至约10nM之间，约0.05nM至约5nM之间，约0.05nM至约1nM之间，约0.05nM至约0.8nM之间，约0.1nM至约0.8nM之间。
在另一情形中，本申请所述融合蛋白与TGFB1的结合活性可使用流式细胞术或酶联免疫法测定进行检测。例如，在ELISA中，使用人TGFB1蛋白，所述融合蛋白与TGFB1的EC50值可以在约0.0001nM至约100nM之间，例如，约0.001nM至约10nM之间，约0.001nM至约5nM之间，约0.01nM至约5nM之间，约0.1nM至约3nM之间。
在另一个方面，本申请所述的融合蛋白能够阻断PD-1与PD-L1的结合。在某些情形中，所述的融合蛋白阻断PD-1与PD-L1的结合可通过流式细胞技术FACS、酶联免疫法ELISA测定。例如，首先将稳定表达PD-L1的宿主细胞(如CHOK1细胞)与递减量的未标记的所述融合蛋白孵育，随后用生物素标记的PD-1蛋白孵育。然后，使用FACS分析细胞，以证实所述融合蛋白阻断PD-1与PD-L1结合。又例如，首先将PD-L1抗原蛋白包被在板上，将递减量的未标记的所述融合蛋白和生物素标记的PD-1蛋白混合后，共同孵育。然后，使用ELISA分析细胞，以证实所述融合蛋白可以阻断PD-1和PD-L1的结合。
本申请所述融合蛋白能够激活T细胞。在某些情形中，所述融合蛋白能够刺激免疫细胞中IFN-γ和/或IL2的分泌。所述免疫细胞可包括淋巴细胞，例如B细胞、T细胞，天然杀伤细胞，髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。可用本领域任何技术人员已知的方法测定免疫细胞中细胞因子的分泌，例如，通过酶联免疫法 (ELISA)定量测定免疫细胞(例如T细胞)增殖情况或由免疫细胞产生的细胞因子(例如由T细胞产生的IFN-γ或IL-2)。
核酸、载体、宿主细胞和制备方法
在另一个方面，本申请还提供了分离的一种或多种核酸分子。所述一种或多种核酸分子可编码本申请所述的融合蛋白或抗原结合片段。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述融合蛋白或抗原结合片段，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体、其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning，A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology，Greene Publishing and Wiley-Interscience，New York N.Y.，1993。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种分离的核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的 其他载体。例如，所述载体为表达载体。
在另一个方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
在另一个方面，本申请提供了制备所述的融合蛋白和/或抗原结合片段的方法。所述方法可包括，在使得所述的融合蛋白和/或抗原结合片段表达的条件下，培养所述本申请所述的细胞，以及任选地收获所表达的所述融合蛋白。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
在某些情形中，所述方法还可包括分离和/或纯化所述抗体或其抗原结合片段的步骤。例如，可以采用蛋白G-琼脂糖或蛋白A-琼脂糖进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述的抗体或其抗原结合片段。
药物组合物、方法、用途
在另一个方面，本申请提供了一种药物组合物，其可包含本申请所述的融合蛋白和/或抗原结合片段，所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的载体。
所述药学上可接受的载体在所采用的剂量和浓度下对接受者无毒性，并且可包括缓冲剂，诸如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(诸如十八烷基二甲基苄基氯化铵、氯化六烃季铵(hexamethonium chloride)、氯化苯二甲羟铵(benzalkonium chloride)、氯化苄甲乙氧铵(benzethonium chloride)、苯酚、丁醇或苄醇；对羟苯甲酸烷酯，诸如对羟苯甲酸甲酯或丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇和间-甲酚)；低分子量(小于约10个残基)多肽；蛋白质，诸如血清白蛋白、凝胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯基吡咯烷酮；氨基酸，诸如甘氨酸、谷酰氨酸、天冬酰氨酸、组氨酸、精氨酸或赖氨酸；单糖、双糖以及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，诸如EDTA；糖，诸如蔗糖、甘露醇、海藻糖或山梨醇；成盐反离子，诸如钠离子；金属络合物(例如，Zn-蛋白质络合物)；和/或非离子表面活性剂，诸如TWEEN TM、PLURONICS TM或聚乙二醇(PEG)。本申请中的药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量取决于例如制剂中存在 的拮抗剂的量和类型，以及受试者的临床参数。
所述药物组合物可以用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述融合蛋白或抗原结合片段。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的疾病或病症和/或其任何并发症而所需的剂量。
另一方面，本申请提供了所述融合蛋白在制备药物中的用途。所述药物可用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。在某些实施方式中，所述肿瘤或癌症包含结直肠肿瘤或癌症。在某些实施方式中，所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。本申请还提供了以下描述的检测生物样品中PD-L1表达的方法。在某些情形中，所述方法包括使生物样品与本申请所述的融合蛋白在容许所述融合蛋白结合PD-L1的条件下接触，和检测在所述融合蛋白与PD-L1之间是否形成复合物。例如，所述肿瘤或癌症为与非肿瘤或癌症样品相比，PD-L1表达升高的肿瘤或癌症。此类方法可以是体外或体内方法。本申请所述融合蛋白可用于例如免疫测定中，所述免疫测定包括例如免疫组织化学(IHC)、免疫荧光(IF)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FAGS)和酶联免疫吸附测定(ELISA)。在某些情形中，例如当PD-L1为用于选择患者的生物标记时，所述融合蛋白用来选择适于用本申请所述融合蛋白进行的疗法的受试者。本申请还提供了所述融合蛋白在诊断患有病症(例如，癌症或免疫功能失调)的受试者的方法中的用途，所述方法包括：通过使样品与本发明的所述融合蛋白接触并检测结合的所述融合蛋白的存在来确定获自受试者的样品中PD-L1的存在或表达水平。
在一些情况下，样品是指生物样品，可包括组织或细胞样品。例如，生物样品可包括来自正常或癌症患者的细胞或组织。在某些情况下，组织或细胞样品的来源可为如来自新鲜、冷冻和/或防腐器官或组织样品或活检物或吸出物的固体组织；血液或任何血液成分；体液，诸如脑脊髓液、羊水、腹膜液或间质液；来自受试者妊娠或发育的任何时间的细胞。在另一些情形中，所述生物样品获自体外组织或细胞培养物。本申请中的生物样品的实例包括但不限于肿瘤活检、循环肿瘤细胞、血清或血浆、循环血浆蛋白、腹水、来源于肿瘤的或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定石蜡包埋的肿瘤样品或冷冻的肿瘤样品。
本申请还提供了所述融合蛋白在诊断患有肿瘤或癌症的受试者的方法中的用途，所述方法包括：通过使样品与本申请的融合蛋白接触并检测结合的抗体的存在来确定获自受试者的 样品中PD-L1的存在或表达水平。在一些情况下，所述样品可选自由以下组成的组：组织样品、全血样品、血清样品和血浆样品。在一些情况下，所述组织样品可以为肿瘤样品。在一些情况下，所述肿瘤样品可包含肿瘤浸润性免疫细胞、肿瘤细胞、基质细胞及其任何组合。
另一方面，本申请提供了治疗受试者中的癌症、抑制受试者中肿瘤生长和/或抑制肿瘤细胞增殖的方法，包括向有需要的受试者或所述肿瘤细胞施用本申请所述的融合蛋白、所述的分子核酸、所述的载体、所述的宿主细胞和/或所述的药物组合物。可通过任何合适的方法来施用，所述合适的方法包括例如：以静脉内方式、以肌内方式、以皮下方式、以皮内方式、以经皮方式、以动脉内方式、以腹膜内方式、以损伤内方式、以颅内方式、以关节内方式、以前列腺内方式、以胸膜内方式、以气管内方式、以鞘内方式、以鼻内方式、以阴道内方式、以直肠内方式、以局部方式、以肿瘤内方式、以腹膜方式、以结膜下方式、以囊内方式、以粘膜方式、以心包内方式、以脐内方式、以眼内方式、以眶内方式、以口服方式、以局部方式、以透皮方式、以玻璃体内方式(例如，通过玻璃体内注射)、通过滴眼剂、通过吸入、通过注射、通过植入、通过输注、通过连续输注、通过直接沐浴靶细胞的局部灌注、通过导管、通过灌洗、以乳膏形式或以脂质组合物形式。用于本文描述的方法中的组合物还可以全身方式或以局部方式施用。施用方法可以根据各种因素(例如，所施用的化合物或组合物以及所治疗的病状、疾病或病症的严重性)而变化。在某些实施方案中，以静脉内方式、以肌内方式、以皮下方式、以局部方式、以口服方式、以透皮方式、以腹膜内方式、以眶内方式、通过植入、通过吸入、以鞘内方式、以心室内方式或以鼻内方式施用抗癌疗法(例如，抗PD-L1抗体)。部分地根据施用是否为短暂的或长期的，给药可通过任何适合的途径进行，例如通过注射，诸如静脉内或皮下注射。本文涵盖各种给药排程，包括但不限于单次施用或各种时间点内的多次施用、推注施用和脉冲输注。
本文所述的融合蛋白或药物组合物可以符合良好医疗实践的方式配制、给药和施用。在此情形下的考虑因素包括所治疗的特定病症、所治疗的特定哺乳动物、单个患者的临床病状、病症的病因、药剂递送部位、施用方法、施用排程和医学从业者已知的其他因素。治疗剂(例如，抗PD-L1抗体)无需但任选地与一种或多种当前用来预防或治疗所考虑的病症的药剂一起配制和/或同时施用。此类其他药剂的有效量取决于制剂中存在的治疗剂(例如，抗PD-L1抗体)的量、病症或治疗的类型以及以上论述的其他因素。这些药剂通常可以凭经验/临床上确定为适当的任何剂量且通过凭经验/临床上确定为适当的任何途径加以使用。剂量可作为单个剂量或作为多个剂量(例如2或3个剂量)施用，诸如输注。与单个治疗相比，可减少组合治疗中施用的抗体的剂量。通过常规技术易于监测此疗法的进展。
试剂盒
另一方面，本申请提供了一种试剂盒，其可以包含本申请所述的融合蛋白，和/或本申请所述的药物组合物。其可在单一常用容器中包括本申请所述的融合蛋白，和/或药物组合物，也可任选地与一种或多种治疗剂组合，任选地一起配制于药物组合物中。
在某些情形中，所述试剂盒还可以包括施用本申请所述融合蛋白，或药物组合物的装置；例如，所述装置取决于内容物的施用方式。在某些情形中，所述试剂盒可包括包装插页，其包括关于试剂盒中的融合蛋白、药物组合物和剂型的信息。通常，此类信息帮助患者和医师有效且安全地使用封装的抗原结合蛋白、药物组合物和剂型。用于此类试剂盒中的容器通常可包含至少一个小瓶、试管、烧瓶、瓶、注射器或其它适合容器，检测和/或治疗组合物中的一种或多种可置于其中。在还提供第二治疗剂的情况下，试剂盒也可含有第二不同容器，其中可放置该第二检测和/或治疗组合物。或者，多种化合物可制备成单一药物组合物，且可包装在单一容器装置诸如小瓶、烧瓶、注射器、瓶或其它适合单一容器中。
给药装置
另一方面，本申请提供了一种给药装置(例如塑料或小瓶，例如空心销或注射器圆筒)，它可以用来施用本申请所述的融合蛋白或其药物组合物。该设备可以通过肠胃外途径(例如肌肉内部，皮下或静脉内)将一种物质引入患者体内。例如，注射设备可以是注射器(例如预先填充的具有本申请所述融合蛋白或其药物组合物的注射器，例如自动注射器)，其可以包括用于容纳要注射的流体(例如本申请所述抗原结合蛋白或其药物组合物)的针筒和针(可以用于刺穿皮肤和/或血管)。给药方式可改变。给药途径可以包括口服，肌肉注射，皮下注射，经直肠给药等。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
下面显示的实施例意在说明本发明的具体实施方案，并且不意在以任何方式限制本说明书或权利要求书的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook，J.，Fritsch，E.F.and Maniais，T.(1989)MolecuLar Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例1融合蛋白质分子的制备
利用分子克隆技术，将TGFBRII胞外区截短序列(SEQ ID NO:56)构建到抗PD-L1抗体PR000265的重链C末端构建双功能融合蛋白分子，将编码多肽序列的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)。将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo，A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6～8x10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2x10 6细胞/ml。准备30ml培养的细胞。将上述编码抗体重链的质粒和编码抗体轻链的质粒以2:3的比例混合共计30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo，31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences，23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)缓冲液平衡含有MabSelect TM(GE Healthcare Life Science，71-5020-91AE)的重力柱(Bio-Rad，7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore，UFC901024)浓缩换液至PBS缓冲液，得到纯化的蛋白溶液。最后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。得到本申请的融合蛋白PR001902，用于下述各实施例实验中。另外，制备蛋白分子M7824(也称为PR001599，其为bintrafusp alfa类似物)，蛋白分子Hengrui融合蛋白9(也称为PR002466，其序列来源于专利WO2018205985A1)，以及TGFBRII Trap(也称为PR002040，其利用抗鸡溶菌酶的抗体克隆TEL16的序列与TGFBRII胞外区截短序列融合得到)作为实施例中的对照。上述融合蛋白的序列见表2，CDR划分以Chothia定义规则为例。
表2本申请所述融合蛋白PR001902和对照蛋白的序列
Figure PCTCN2020118799-appb-000001
Figure PCTCN2020118799-appb-000002
实施例2融合蛋白结合过表达人PD-L1的CHO-K1细胞
为了研究融合蛋白融合蛋白PR001902体外结合PD-L1的活性，采用过表达人PD-L1的CHO-K1细胞(CHO-K1/hPD-L1，南京金斯瑞，M00543)，进行细胞水平上的抗体结合实验。消化CHO-K1/hPD-L1细胞，并用F12K完全培养基重悬，将细胞密度调整为2x10 6/mL。以50μl细胞/孔接种于96孔U底板，随后加入100μL/孔，5倍浓度梯度稀释的待测抗体，其中抗体的最高终浓度为100nM。将细胞放置于4℃，避光孵育30分钟。之后，加入100μl/孔预冷PBS漂洗细胞两次，于500g，4℃下离心5分钟，弃上清。再加入100μL/孔二抗AF488山羊抗人lgG(H+L)(Invitrogen，A11013，1：1000)，4℃，避光孵育30分钟。用100μl/孔预冷PBS漂洗细胞两次，于500g，4℃下离心5分钟，弃上清。最后，200μl/孔预冷PBS重悬细胞。使用流式细胞仪BD FACS CANTOII读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
结果如图1和表3显示，PR001902与阳性对照相比，能够有效地与CHO-K1/hPD-L1细胞结合，并且有药物浓度剂量依赖效应。
表3融合蛋白体外结合PD-L1
蛋白样品 EC50(nM)
PR000265 0.258
阳性对照M7824 0.718
PR001902 0.515
人IgG1 /
实施例3融合蛋白体外结合TGFB1
以1μg/ml浓度的人源TGFB1(Novoprotein，CA59)为抗原，按100μl/孔包被96孔板，4℃过夜。200μl/孔1×PBST洗涤3次，加入200μl/孔2％BSA，37℃封闭2小时。加入200μl/孔1xPBST洗涤3次，加入5倍浓度梯度稀释的抗体，其中抗体的最高终浓度为100nM， 37℃孵育1小时。200μl/孔1×PBST洗涤3次，每孔加入100μl/孔抗人FC-HRP二抗(Sigma-Aldrich，A0170，以1:4000稀释)，37℃孵育1小时。200μl/孔1×PBST洗涤3次，每孔再加入100μl/孔TMB，室温孵育10分钟后加入50μl/孔1M ELISA终止液停止反应。酶标仪上检测450nm及570nm测吸收值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
结果如图2和表4所示，PR001902与阳性对照相比，能够有效地与TGFB1结合，并且有药物浓度剂量依赖效应。
表4融合蛋白体外结合TGFB1
蛋白样品 EC50(nM)
TGFBRII Trap对照 0.783
阳性对照M7824 1.546
PR001902 1.492
人IgG1 /
实施例4融合蛋白阻断人PD-1与过表达人PD-L1的CHO-K1细胞的结合
为了进一步研究融合蛋白体外阻断PD1/PDL1通路的活性，采用高表达人PD-L1的CHO-K1细胞(CHO-K1/hPD-L1，南京金斯瑞，M00543)，进行细胞水平上的抗体阻断实验。消化CHO-K1/hPD-L1细胞，并用F12K完全培养基重悬，将细胞密度调整为2x10 6/mL。以50μl细胞/孔接种于96孔U底板，随后加入100μL/孔，5倍浓度梯度稀释的待测抗体，其中抗体的最高终浓度为100nM。将细胞放置于4℃，避光孵育30分钟。之后，加入100μl/孔预冷PBS漂洗细胞，于500g，4℃下离心5分钟，弃上清。加入50μl/孔1μg/mL Biotin-hPD-1(Acrobiosystems，PD1-H82F2)，4℃，避光孵育30分钟。用100μl/孔预冷PBS漂洗细胞，于500g，4℃下离心5分钟，弃上清。再加入100μL/孔二抗PE Streptavidin(BD Pharmingen，554061，1:200稀释)，4℃，避光孵育30分钟。用100μl/孔预冷PBS漂洗细胞，于500g，4℃下离心5分钟，弃上清。最后，200μl/孔预冷PBS重悬细胞，使用BD FACS CANTOII读取荧光发光信号值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及IC50值等参数。
结果如图3和表5所示，PR001902与阳性对照均能够有效地阻断PD-1同CHO-K1/hPD-L1细胞结合，并且有药物浓度剂量依赖效应。
表5融合蛋白体外阻断PD1/PDL1
蛋白样品 IC50(nM)
PR000265 0.127
阳性对照M7824 0.323
PR001902 0.311
人IgG1 /
实施例5利用BLI测定融合蛋白与人PD-L1或人TGFB1的亲和力的结果
通过生物膜干涉(BLI)技术，使用Octet分子相互作用分析仪(ForteBio，型号Octet Red96e)，来进行抗原抗体之间的结合动力学分析。
通过Octet测定融合蛋白与人TGFB1蛋白的亲和力。传感器架上放置两列SA传感器，一列作为测试SA传感器，一列作为参照SA传感器。以0.02％吐温的1×PBS为缓冲液，先将SA传感器在缓冲液中平衡10分钟；测试SA传感器捕获生物素化的人TGFB1蛋白(人TGFB1蛋白(NovoProtein,CA59)利用生物素化试剂盒(ThermoFisher，A39257，EZ-Link Sulfo-NHS-LC-Biotin)进行生物素化)，设置捕获高度为0.3nm；参照SA传感器浸入缓冲液中30秒。两列传感器再与梯度稀释的待测融合蛋白(例如，待测融合蛋白浓度可以为6.25～0.39nM的两倍梯度稀释以及0nM)相互作用，每根传感器对应一个浓度，在程序中设置结合、解离时间分别为180秒和600秒。
通过Octet测定融合蛋白与人PD-L1蛋白(NovoProtein，C315)的亲和力。传感器架上放置两列AHC传感器，一列作为测试AHC传感器，一列作为参照AHC传感器。先将AHC传感器在缓冲液中平衡10分钟；测试AHC传感器捕获待测融合蛋白(浓度40nM)30秒，捕获高度约为0.7nm；参照AHC传感器浸入缓冲液中30秒。两列传感器再与梯度稀释的人PD-L1蛋白(例如，PD-L1蛋白浓度可以为25～0.39nM的两倍梯度稀释以及0nM)相互作用，每根传感器对应一个浓度，在程序中设置结合、解离时间分别为300秒和900秒。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，选择双扣除模式(double reference)扣除背景信号，选择“1:1Global fitting”方法进行数据拟合，计算出抗原(亲和对象)与待测融合蛋白结合的动力学参数，得到k on(1/Ms)值、k dis(1/s)值和K D(M)值。
结果如表6所示，PR001902与阳性对照相比，对人TGFB1以及人PD-L1都具极高的亲和力。
表6融合蛋白与人源TGFB1或人源PD-L1的体外亲和力
Figure PCTCN2020118799-appb-000003
实施例6利用BLI方法测定融合蛋白结合PD-L1的表位竞争
使用ForteBio Octet平台对得到的融合蛋白和阳性对照M7824(即PR001599)进行表位竞争实验。第一步，获取抗体的100％信号：用SA传感器捕获生物素化的人PD-L1蛋白(人PD-L1蛋白(NovoProtein,C315)利用生物素化试剂盒(ThermoFisher，A39257，EZ-Link Sulfo-NHS-LC-Biotin)进行生物素化)，捕获高度为0.25nm。将传感器浸入抗体中(50nM)，时间500秒，将该抗体与PD-L1结合的最终信号记录为该抗体的100％信号。第二步，表位竞争实验：用SA传感器捕获生物素化的PD-L1蛋白，捕获高度为0.25nm。将传感器浸入第一抗体中(50nM)，时间500秒，再将SA传感器浸入第一抗体和第二抗体的混合物中(两种抗体的终浓度均为50nM)，时间500秒，将最终信号记录为该第二抗体的信号。抑制率通过下式计算，
抑制率(％)＝(A–B)/A*100
A：某抗体的100％信号(从第一步中获得)，B：该抗体作为第二抗体的信号(从第二步获得)。
若得到的抑制率大于85(％)，则意味着两种抗体表位完全重叠；若抑制率小于85(％)，则意味着两种抗体的表位不完全重叠。
结果如表7所示，PR001902与对照分子PR001599的表位不完全重叠。
表7融合蛋白的表位竞争
Figure PCTCN2020118799-appb-000004
实施例7融合蛋白对TGFB/Smad信号通路激活的抑制作用
该实验使用高表达人源TGFBRI及融合SEAP报告基因的Smad3/4基因的HEK-Blue TM TGF-β细胞(Invivogen，hkb-tgfb)，在细胞水平上研究了融合蛋白对TGFB1诱导的Smad信号通路的激活的抑制作用。加入10μl/孔，5倍浓度梯度稀释的待测融合蛋白于96孔板中，最高终浓度为50nM。随后，加入10μl/孔，终浓度为1ng/ml的人TGFB1蛋白(R&D Systems，240-B/F)于96孔板中。以抗TGFB1抗体(Biointron，B5484)为对照分子。将180μl，2.5×10 4细胞/孔接种于96孔板中，在二氧化碳培养箱中继续孵育过夜。然后每孔移取20μl细胞上清入另一块96孔板中，然后每孔加入180μl Quanti-Blue工作液(Invivogen，rep-qb1)，37℃下孵育1小时。酶标仪上检测655nm测吸收值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及IC50值等参数。
结果如图4和表8所示，PR001902以剂量依赖性形式抑制TGFB1诱导的SEAP报告物的产生，且与阳性对照M7824具有相当的功效和IC50。另外，PR001902的抑制活性和IC50较阳性对照Hengrui融合蛋白9及TGFB1抗体强8倍。
表8融合蛋白抑制TGFB1诱导的Smad信号通路激活
样品 IC50(nM)
TGFBRII Trap对照 0.0093
阳性对照M7824 0.0090
阳性对照Hengrui融合蛋白9 0.0769
PR001902 0.0103
TGFB1抗体 0.0876
实施例8 MLR法体外检测融合蛋白对T细胞的激活作用
为了研究融合蛋白对T细胞的协同激活作用，收集和纯化人外周血单核细胞(PBMC)，分离单核细胞(Meltenyi，130-050-201)，采用完全培养基(RPI1640含50ng/mL IL-4,100ng/mL GM-CSF)培养6天，再加入1μg/ml LPS诱导24h,生成成熟的DC细胞。收集上述培养的细胞离心，用含2％FBS的PBS洗4次，重悬至新鲜的培养基中，调整密度为2×10 5细胞/mL，100μl/孔接种至96孔细胞培养板。同时，自异体新鲜PBMC中分离Pan T细胞((Meltenyi，130-096-535)新鲜培养基重悬，调整细胞密度为1×10 6细胞/ml，100μl/孔接种至96孔细胞培养板。将不同浓度的抗体50μl/孔，及终浓度为0.3ng/ml的人TGFB1蛋白 (R&D Systems，240-B/F)以50μl/孔分别加入上述96孔细胞培养板的对应孔中，并将细胞培养板置于37℃，5％CO 2培养箱孵育5天。分别收集72小时后和120小时后的上清液。采用IL2ELISA试剂盒(Thermo,88-7025-88)检测72小时后上清液中的IL-2的水平；采用IFN-γELISA试剂盒(Thermo,88-7316-88)检测120小时后上清液中的IFN-γ的水平；具体操作参考试剂说明书。
结果如图5和图6所示，在外加TGFB1的条件下，PR001902能够剂量依赖地增强激活的T淋巴细胞分泌细胞因子IL-2(图5)和IFN-γ(图6)，并且具有比阳性对照更强的激活作用。
实施例9融合蛋白的药代动力学研究
本实施例测试融合蛋白的药代动力学性能。其方法如下，选取体重18～22克的雌性C57BL/6小鼠3只，静脉注射给与PR001902，剂量9.53mg/kg。于给药前以及给药后0.5小时、24小时(第1天)、第2天，第4天、第7天、第10天和第14天采集全血，将全血静置30分钟使其凝固，随后在4℃下以2,000rpm离心5分钟并将分离的血清样品在-80℃下冻存直至分析。本实施例采用两种ELISA方法来定量测定小鼠血清中的药物浓度。ELISA方法一，即Fc端检测方法，通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的融合蛋白，然后加入HRP标记的山羊抗人Fc第二抗体来检测；ELISA方法二，即TGFBRII端检测方法，通过包被于96孔板的人TGFB1蛋白来捕获小鼠血清中的含有人TGFBRII结构域的融合蛋白，然后加入HRP标记的的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件6.4版，选用非房室模型(NCA)对血药浓度数据进行分析以评价其药代动力学。
结果如图7和表9所示，PK分析结果表明，Fc端检测方法下，PR001902在小鼠体内的半衰期约为11天；TGFBRII端检测方法显示，PR001902在小鼠体内的半衰期约为8.5天。
表9 PR001902的药代动力学
Figure PCTCN2020118799-appb-000005
Figure PCTCN2020118799-appb-000006
实施例10融合蛋白对结肠癌CT26-人PD-1/PD-L1双敲小鼠的动物体内模型的抗肿瘤活性
评价测试药物在小鼠结肠癌细胞CT26-人PDL1(tg)-小鼠PDL1(KO)(将小鼠结肠癌细胞CT26的内源性鼠PD-L1基因敲除并引入外源人PD-L1转基因，集萃药康生物科技)移植免疫检查点人源化小鼠BALB/c-人PD-1/PD-L1(将BALB/c小鼠同时引入外源人PD-1转基因和人PD-L1转基因，集萃药康生物科技)中的抗肿瘤作用。接种对数生长期的CT26-人PDL1(tg)-mPDL1(KO)细胞于人源化小鼠BALB/c-人PD-1/PD-L1腋右侧背部皮下，当平均肿瘤体积达到80-120mm 3时，去除肿瘤体积过大的小鼠个体后，小鼠根据肿瘤体积随机分成3组，每组6只。分组当天开始腹腔给药，每三天给一次药，共给药6次(q3d×6)。开始给药后，每周称量体重及瘤体积两次，瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×肿瘤长径×肿瘤短径 2。实验结束后将荷瘤小鼠安乐死并剥瘤称重。计算各组动物的肿瘤体积、小鼠体重等实验结果以平均值±标准误差(Mean±SEM)表示。多组比较采用单因素方差分析(one way ANOVA)检验方法比较不同治疗组与对照组相比有无显著性差异。数据使用SPSS 18.0进行分析。P<0.05为具有显著性差异。
结果如图8显示，PR001902与阳性对照相比，均能显著抑制CT26人PD-1/PD-L1小鼠皮下移植瘤的生长；且在实验过程中，各组动物体重均出现增长，表明动物对测试药物耐受良好，未对动物产生明显毒性作用，安全性好。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附权利要求和其等同方案的范围内。

Claims (17)

  1. 一种融合蛋白，其包含：a)人TGFBRII或其片段；以及b)特异性结合PD-L1的抗体或其抗原结合片段；其中所述特异性结合PD-L1的抗体或其抗原结合片段包含HCDR1,HCDR2和HCDR3，以及LCDR1，LCDR2和LCDR3；所述HCDR1包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2包含SEQ ID NO:27所示的氨基酸序列，所述HCDR3包含SEQ ID NO:8所示的氨基酸序列，所述LCDR1包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:31所示的氨基酸序列。
  2. 根据权利要求1所述的融合蛋白，其中所述特异性结合PD-L1的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:13所示的氨基酸序列，且所述轻链可变区包含SEQ ID NO:4所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的融合蛋白，其中所述特异性结合PD-L1的抗体或其抗原结合片段包含抗体重链，且所述抗体重链与所述人TGFBRII或其片段框内融合而形成融合多肽。
  4. 根据权利要求3所述的融合蛋白，其中所述抗体重链的C端与所述人TGFBRII或其片段的N端连接。
  5. 根据权利要求1-4中任一项所述的融合蛋白，其中所述人TGFBRII或其片段包含人TGFBRII的胞外结构域。
  6. 根据权利要求1-5中任一项所述的融合蛋白，其中所述人TGFBRII或其片段包含SEQ ID NO:56-57中任一项所示的氨基酸序列，优选SEQ ID NO:56所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的融合蛋白，其中所述特异性结合PD-L1的抗体或其抗原结合片段还包含重链恒定区，且所述重链恒定区为人IgG1恒定区。
  8. 根据权利要求1-7中任一项所述的融合蛋白，其中所述特异性结合PD-L1的抗体或其抗原结合片段还包含轻链恒定区，所述轻链恒定区包含人Igκ恒定区。
  9. 根据权利要求1-8中任一项所述的融合蛋白，其包含第一多肽和第二多肽，
    其中所述第一多肽包含SEQ ID NO:14所示的氨基酸序列；且
    所述第二多肽包含SEQ ID NO:5所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的融合蛋白，其包含两条所述第一多肽以及两条所述第二多肽。
  11. 分离的核酸分子，其包含编码权利要求1-10中任一项所述的融合蛋白或其功能性片段的多核苷酸。
  12. 药物组合物，其包含权利要求1-10中任一项所述的融合蛋白，或权利要求11所述的 核酸分子，以及任选地药学上可接受的载体。
  13. 权利要求1-10中任一项所述的融合蛋白，或权利要求11所述的核酸分子用于制备药物的用途，所述药物用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。
  14. 权利要求13所述的用途，其中所述肿瘤或癌症包括结肠癌。
  15. 权利要求13-14中任一项所述的用途，其中所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。
  16. 权利要求1-10中任一项所述的融合蛋白，或权利要求11所述的核酸分子，其用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。
  17. 治疗受试者中的癌症、抑制受试者中肿瘤生长和/或抑制肿瘤细胞增殖的方法，所述方法包括向所述受试者或所述肿瘤细胞施用权利要求1-10中任一项所述的融合蛋白，或权利要求11所述的核酸分子。
PCT/CN2020/118799 2019-09-30 2020-09-29 一种融合蛋白及其应用 WO2021063350A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910942734.4 2019-09-30
CN201910942734.4A CN112574314A (zh) 2019-09-30 2019-09-30 一种融合蛋白及其应用

Publications (1)

Publication Number Publication Date
WO2021063350A1 true WO2021063350A1 (zh) 2021-04-08

Family

ID=75116580

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/118799 WO2021063350A1 (zh) 2019-09-30 2020-09-29 一种融合蛋白及其应用

Country Status (3)

Country Link
CN (1) CN112574314A (zh)
TW (1) TW202126700A (zh)
WO (1) WO2021063350A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220411513A1 (en) * 2019-09-30 2022-12-29 Harbour Biomed (Shanghai) Co., Ltd Anti-pd-l1 antigen binding protein and application thereof
CN115322259A (zh) * 2021-05-11 2022-11-11 正大天晴药业集团南京顺欣制药有限公司 针对PD-1和TGF-β的双功能蛋白
CN115322257B (zh) * 2021-08-16 2023-05-30 上海优替济生生物医药有限公司 Bcma靶向抗体、嵌合抗原受体及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106939047A (zh) * 2016-01-04 2017-07-11 钜川生物医药 一种pd-l1抗体及其制备方法
WO2018064190A1 (en) * 2016-09-27 2018-04-05 Epicentrx, Inc. Immunomodulatory fusion proteins
WO2018218215A1 (en) * 2017-05-26 2018-11-29 The Johns Hopkins University Multifunctional antibody-ligand traps to modulate immune tolerance
CN109641963A (zh) * 2016-08-12 2019-04-16 默克专利有限公司 癌症的联合治疗
CN110198738A (zh) * 2017-01-07 2019-09-03 默克专利有限公司 用于靶向的TGF-β抑制的给药方案和剂型

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106939047A (zh) * 2016-01-04 2017-07-11 钜川生物医药 一种pd-l1抗体及其制备方法
CN109641963A (zh) * 2016-08-12 2019-04-16 默克专利有限公司 癌症的联合治疗
WO2018064190A1 (en) * 2016-09-27 2018-04-05 Epicentrx, Inc. Immunomodulatory fusion proteins
CN110198738A (zh) * 2017-01-07 2019-09-03 默克专利有限公司 用于靶向的TGF-β抑制的给药方案和剂型
WO2018218215A1 (en) * 2017-05-26 2018-11-29 The Johns Hopkins University Multifunctional antibody-ligand traps to modulate immune tolerance

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOCHEMS, C. ET AL.: "Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)", ONCOTARGET, vol. 8, no. 43, 8 September 2017 (2017-09-08), pages 75217 - 75231, XP055798126 *
PRINCIPE DANIEL R., PARK ALEX, DORMAN MATTHEW J., KUMAR SANDEEP, VISWAKARMA NAVIN, RUBIN JONATHAN, TORRES CAROLINA, MCKINNEY RONAL: "TGFβ Blockade Augments PD-1 Inhibition to Promote T-Cell–Mediated Regression of Pancreatic Cancer", MOLECULAR CANCER THERAPEUTICS, AMERICAN ASSOCIATION FOR CANCER RESEARCH [, US, vol. 18, no. 3, 1 March 2019 (2019-03-01), US, pages 613 - 620, XP055798139, ISSN: 1535-7163, DOI: 10.1158/1535-7163.MCT-18-0850 *

Also Published As

Publication number Publication date
TW202126700A (zh) 2021-07-16
CN112574314A (zh) 2021-03-30

Similar Documents

Publication Publication Date Title
US20240132603A1 (en) Ccr8 antibody and application thereof
WO2021063350A1 (zh) 一种融合蛋白及其应用
EP1871807A2 (en) Antibodies against cxcr4 and methods of use thereof
TWI793395B (zh) 結合ｐｄ-ｌ１和ｏｘ４０的雙特異性抗體
TWI832013B (zh) 一種抗pd-l1抗原結合蛋白及其應用
US20210032350A1 (en) Antibodies to galectin-3 and methods of use thereof
CN113735974B (zh) 针对Claudin18.2的抗体及其用途
JP2022523710A (ja) Ｃｄ４４に特異的な抗体
JP2022514693A (ja) Ｍｕｃ１８に特異的な抗体
KR20190127791A (ko) 항-pd-l1-항-tim-3 이중특이적 항체
JP2022514786A (ja) Ｍｕｃ１８に特異的な抗体
WO2020156507A1 (zh) 抗pd-l1的新型抗体及其用途
CN114729013A (zh) 抗cd22抗体及其用途
US20200024347A1 (en) Or10h1 antigen binding proteins and uses thereof
EP4293047A1 (en) Anti-pd-l1 antibody and use thereof
TWI837517B (zh) 抗claudin 18.2和cd3的雙特異性抗體以及其用途
WO2023109847A1 (zh) Pd-l1抗原结合蛋白及其用途
WO2022171104A1 (zh) 抗pd-1抗体及其用途
EP3904383A1 (en) Anti-ox40 monoclonal antibody and application thereof
BR112016029734B1 (pt) Anticorpo isolado ou fragmento de ligação ao antígeno deste e uso do mesmo, composição farmacêutica e método para detecção de níveis de expressão de il-21 em uma amostra

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20872337

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20872337

Country of ref document: EP

Kind code of ref document: A1