WO2020143720A1 - 阻断血管内皮细胞生长且活化t细胞的多靶向融合蛋白和包含其的药物组合物 - Google Patents

阻断血管内皮细胞生长且活化t细胞的多靶向融合蛋白和包含其的药物组合物 Download PDF

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WO2020143720A1
WO2020143720A1 PCT/CN2020/071213 CN2020071213W WO2020143720A1 WO 2020143720 A1 WO2020143720 A1 WO 2020143720A1 CN 2020071213 W CN2020071213 W CN 2020071213W WO 2020143720 A1 WO2020143720 A1 WO 2020143720A1
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antibody
fusion protein
domain
vegfr
seq
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French (fr)
Chinese (zh)
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胡品良
邹敬
洪伟东
何芸
白洁
宋凌云
杨文第
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Beijing Beyond Biotechnology Co Ltd
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Beijing Beyond Biotechnology Co Ltd
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Priority to EP20738951.1A priority Critical patent/EP3909986A4/en
Priority to US17/421,874 priority patent/US20220106389A1/en
Priority to JP2021538762A priority patent/JP2022517920A/ja
Publication of WO2020143720A1 publication Critical patent/WO2020143720A1/zh
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/2803—Immunoglobulins [IG], 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
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention generally relates to the field of medical biotechnology.
  • the present invention relates to a multi-target fusion protein comprising (i) vascular endothelial cell growth inhibitory domain; (ii) immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD)
  • the multi-target fusion protein and pharmaceutical composition of the present invention can treat or prevent cancerous diseases in individuals.
  • the tumor microenvironment is a complex environment on which tumor cells depend for survival and development. It consists of cellular components and non-cellular components. Among them, cellular components include tumor cells themselves, immune cells, endothelial cells, etc.; non-cellular components include cytokines, chemokines, etc. . With the study of tumors, people realize that the generation, growth and metastasis of tumors are regulated by the tumor microenvironment. The tumor microenvironment determines whether tumor cells can grow preferentially.
  • tumor cells express PD-L1, thereby utilizing the inhibitory signaling pathway of immune checkpoints (ie, PD-1/PD-L1 inhibitory Signaling pathway) to inhibit T lymphocyte activity, where PD-L1 is not expressed in normal human tissues.
  • T cell receptor T cell receptor
  • MHC major histocompatibility complex
  • PD-1 and CD28 co-localize on the T lymphocyte membrane, and the target of PD-1 mediated immunosuppression is mainly CD28, not TCR.
  • PD-1 rapidly recruits Shp2 phospholipase after binding to PD-L1, Shp2 phospholipase preferentially dephosphorylates CD28, which is stronger than dephosphorylation of TCR, thereby inhibiting T cell function by inactivating CD28 signaling (Hui E. et al., T cell costimulatory receptor, CD28 is a primary target for PD-1-mediated inhibition, Science, 2017, 355(6332): 1428-1433).
  • CTLA-4 expressed on the surface of T cells has a high degree of homology with the costimulatory molecule CD28 on the surface of T cells, and they have the same ligands CD86 (B7-2) or CD80 (B7-1).
  • the combination of CTLA-4 and B7 molecules usually inhibits the activation of T cells, so blocking the immune checkpoint B7/CTLA-4 pathway can enhance tumor-specific T cell activation.
  • tumor cells also release angiogenic factors, such as vascular endothelial growth factor (Vascular Endothelial Cell Growth Factor (VEGF)), resulting in a surge in the number of VEGF.
  • VEGF vascular endothelial growth factor
  • VEGFR cell surface receptor
  • Anti-PD-1 antibody drugs such as Bristol-Myers Squibb (BMS)'s Nivolumab and Merck's Pembrolizumab; Bristol-Myers Squibb (BMS)'s anti-PD-1 antibody CTLA-4 antibody Ipilimumab (trade name Yervoy); Genentech's human-mouse chimeric anti-VEGF antibody Bevacizumab (trade name Avastin); Sanofi-aventis and Regeneron Aflibercept and the like developed as VEGF-Trap.
  • BMS Bristol-Myers Squibb
  • BMS Bristol-Myers Squibb
  • CTLA-4 antibody Ipilimumab trade name Yervoy
  • Genentech's human-mouse chimeric anti-VEGF antibody Bevacizumab trade name Avastin
  • Sanofi-aventis and Regeneron Aflibercept and the like developed as VEGF-Trap.
  • multi-targeted fusion proteins can simultaneously specifically target multiple signaling pathways involved in tumorigenesis and development, there is a multi-targeted fusion that can improve the tumor microenvironment by blocking the growth of vascular endothelial cells and activating T cells Protein, and the need to combine the multi-target fusion protein with other anti-cancer drugs.
  • the inventors have developed a group of multi-targeted fusion proteins that block the growth of vascular endothelial cells and activate T cells through vigorous research, which include (i) vascular endothelial growth inhibitory domain; (ii) immunoglobulin Fc domain ; And (iii) CD80 extracellular domain (ECD).
  • the multi-target fusion protein can improve the tumor microenvironment and improve the effect of tumor immunotherapy from two aspects, one of which is to improve the tumor microenvironment through the CD80 extracellular domain (ECD) of the multi-target fusion protein )
  • ECD extracellular domain
  • the CD80 extracellular domain binds to PD-L1 as an immune checkpoint to alleviate the PD-1/PD-L1 inhibitory signaling pathway , "Brake" the immune system; through the CD80 extracellular domain binds CTLA-4 and exerts the same function as the anti-CTLA-4 antibody ipilimumab to inhibit regulatory T cells (Treg); through the CD80 extracellular domain
  • the inhibitor domain blocks the VECD80 extracellular domain (EC
  • the multi-target fusion protein of the present invention comprises (i) an antigen-binding fragment derived from an anti-VEGF antibody and/or an anti-VEGFR antibody and/or a VEGFR extracellular receptor functional region; (ii) an immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD).
  • the antigen-binding fragment derived from the anti-VEGF antibody contained in the multi-target fusion protein may be derived from any antigen-binding fragment of the anti-VEGF antibody, as long as it can bind to VEGF and thereby block or inhibit the binding of VEGF to its receptor VEGFR Antibody.
  • the anti-VEGF antibodies include anti-VEGF antibodies known in the art and anti-VEGF antibodies developed in the future.
  • the antigen-binding fragment of the anti-VEGF antibody is Fab, Fab′, F(ab′) 2 , Fv, single-chain Fv of the anti-VEGF antibody; preferably, the antigen-binding fragment of the anti-VEGF antibody Contains all 6 heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence selected from SEQ ID NO: 1/2 and 3/4, or with all 6 heavy chain CDRs and one or more of the light chain CDRs have a sequence of one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions); more preferably, the The antigen-binding fragment of the anti-VEGF antibody comprises the paired heavy chain variable region sequence/light chain variable region sequence selected from SEQ ID NOs: 1/2 and 3/4, or the paired heavy chain variable region sequence / Light chain variable region sequence having a sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the antigen-binding fragment derived from the anti-VEGFR antibody contained in the multi-target fusion protein may be derived from any antigen-binding fragment of the anti-VEGFR antibody, as long as it can bind to VEGFR and thereby block or inhibit the binding of VEGF to its receptor VEGFR Antibody.
  • the anti-VEGFR antibodies include anti-VEGFR antibodies known in the art and anti-VEGFR antibodies developed in the future.
  • the antigen-binding fragment of the anti-VEGFR antibody is Fab, Fab', F(ab') 2 , Fv, single-chain Fv of the anti-VEGFR antibody; preferably, the antigen-binding fragment of the anti-VEGFR antibody All 6 heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 5/6, or with all 6 heavy chain CDRs One or more CDRs in the light chain CDR have a sequence of one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions); more preferably, the anti-VEGFR antibody is antigen-binding
  • the fragment comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 5/6, or has at least 90% of the paired heavy chain variable region sequence/light chain variable region sequence, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequences with
  • the VEGFR extracellular receptor functional region contained in the multi-target fusion protein may be any VEGFR extracellular receptor functional region, as long as it is a VEGFR cell capable of binding VEGF and thereby blocking or inhibiting the binding of VEGF to its receptor VEGFR
  • the external receptor functional area is sufficient.
  • the VEGFR extracellular receptor functional area comprises VEGFR1 immunoglobulin-like domain 2 and VEGFR2 immunoglobulin-like domain 3; or the VEGFR extracellular receptor functional area comprises VEGFR1 immunoglobulin-like domain Domain 2 and immunoglobulin-like domain 3 of VEGFR2 and immunoglobulin-like domain 4 of VEGFR2; or the VEGFR extracellular receptor functional region contains immunoglobulin-like domain 2 of VEGFR1; more preferably, The VEGFR extracellular receptor functional region has any one selected from the amino acid sequence shown in SEQ ID NO: 7-9 or at least 90%, 91%, 92%, and the amino acid sequence shown in SEQ ID NO: 7-9. Amino acid sequences with 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the (ii) immunoglobulin Fc domain contained in the multi-target fusion protein may be any immunoglobulin Fc domain, in particular, the (ii) is a human immunoglobulin Fc domain.
  • the immunoglobulin Fc domain is the Fc domain of an IgG class antibody, in particular the Fc domain of an IgG 1 subclass, IgG 2 subclass, IgG 4 subclass antibody.
  • the immunoglobulin Fc domain contained in the multi-target fusion protein of the present invention is the Fc domain of an IgG 1 subclass antibody, particularly the Fc structure of a human IgG 1 subclass antibody area.
  • the immunoglobulin Fc domain contained in the multi-target fusion protein of the present invention is the Fc domain of an IgG 4 subclass antibody, in particular the Fc structure of a human IgG 4 subclass antibody area.
  • the (ii) immunoglobulin Fc domain in the multi-target fusion protein of the present invention comprises the Fc domain of the amino acid sequence shown in SEQ ID NO: 10, 11, or 12, or comprises the amino acid sequence of SEQ ID NO: Fc domain having an amino acid sequence shown in 10, 11 or 12 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity .
  • CD80 (ECD) contained in the multi-target fusion protein is part of the extracellular domain of CD80.
  • the CD80 ECD comprises the CD80 immunoglobulin V (IgV) region (CD80-IgV).
  • the CD80 ECD comprises CD80 immunoglobulin V and C regions (CD80-IgVIgC).
  • the CD80 ECD is a human CD80 ECD, preferably the CD80 ECD comprises human CD80 IgV.
  • the CD80-IgV has the amino acid sequence shown in SEQ ID NO: 13, or at least 90%, 91%, 92%, 93%, 94% with the amino acid sequence of SEQ ID NO: 13 , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences.
  • the CD80-IgVIgC has the amino acid sequence shown in SEQ ID NO: 14, or at least 90%, 91%, 92%, 93%, 94%, the amino acid sequence of SEQ ID NO: 14; Amino acid sequences of 95%, 96%, 97%, 98%, 99% or more identity.
  • the multi-target fusion protein further comprises a peptide linker between (i), (ii) and/or (iii); preferably, the peptide linker comprises one or more amino acids, More preferably it contains at least 5 amino acids and most preferably it contains a peptide linker selected from SEQ ID NO: 20-46.
  • the multi-target fusion protein is in the order of (i), (ii), and (iii) from N-terminus to C-terminus; (iii), (i), and (ii); or ( The order of iii), (ii) and (i) is effectively connected.
  • the multi-target fusion protein of the invention comprises
  • CD80 ECD an immunoglobulin Fc domain in the form of a dimer operatively linked at the C-terminus of CD80 ECD; and a derivative derived from an operative link at the C-terminus of the immunoglobulin Fc domain in the form of the dimer Anti-VEGF antibodies and/or antigen-binding fragments of anti-VEGFR antibodies;
  • CD80 ECD an immunoglobulin Fc domain in the form of a dimer operatively linked at the C-terminus of CD80 ECD; and VEGFR cells operatively linked at the C-terminus of the immunoglobulin Fc domain in the form of the dimer External receptor functional area;
  • the antibody is an IgG class antibody, in particular an IgG 1 subclass, IgG 2 subclass, IgG 4 subclass antibody, more particularly an IgG 4 subclass antibody; also preferably, the IgG 4 subclass antibody
  • the antibody comprises an amino acid substitution at position S228 in the Fc domain, more preferably the amino acid substitution S228P; further preferably, the light chain type of the antibody is kappa or lambda, preferably kappa;
  • the full-length anti-VEGF antibody is Bevacizumab
  • the full-length anti-VEGFR antibody is Ramucirumab
  • the multi-target fusion protein of the invention is selected from
  • a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 80 and the second subunit of the fusion protein of SEQ ID NO: 82;
  • a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 84 and the second subunit of the fusion protein of SEQ ID NO: 86;
  • a fusion protein comprising the fusion protein subunit of SEQ ID NO: 88.
  • the present invention also provides a polynucleotide encoding the multi-target fusion protein of the present invention, a vector comprising the polynucleotide encoding the multi-target fusion protein of the present invention, preferably an expression vector, and most preferably glutamine with a double expression cassette Amide synthase expression vector.
  • the present invention provides a host cell comprising the polynucleotide or vector of the present invention.
  • the host cell is a CHO, HEK293, or NSO cell.
  • the present invention also provides a method for producing the multi-target fusion protein of the present invention, which includes step (i) culturing the host cell of the present invention under conditions suitable for expressing the multi-target fusion protein of the present invention, and (ii ) Recovery of the multi-target fusion protein of the present invention.
  • the present invention also provides a pharmaceutical composition in which the multi-target fusion protein of the present invention is combined with an anti-PD-1 antibody.
  • the multi-target fusion protein of the present invention shows good synergy when used in combination with an anti-PD-1 antibody, thereby being able to better achieve the purpose of inhibiting tumor growth.
  • the anti-PD-1 antibody may be any anti-PD-1 antibody, as long as it can inhibit or reduce the binding of PD-1 to its ligand, including the prior art Known anti-PD-1 antibodies and anti-PD-1 antibodies developed in the future.
  • the anti-PD-1 antibody comprises SEQ ID NO: 47/48, 49/50, 51/52, 53/54, 55/56, 57/58, 59/60, 61/ All six heavy chain CDRs and light chains contained in the paired heavy chain variable region sequence/light chain variable region sequence of 62, 63/64, 65/66, 67/68, 69/70 and 71/72 CDR, or a sequence with one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions) from one or more of the six heavy chain CDRs and light chain CDRs ;
  • the anti-PD-1 antibody comprises selected from SEQ ID NO: 47/48, 49/50, 51/52, 53/54, 55/56, 57/58, 59/60, 61/62 , 63/64, 65/66, 67/68, 69/70 and 71/72 paired heavy chain variable region sequence/light chain variable region sequence, or the paired heavy chain variable region
  • the present invention provides the use of the multi-target fusion protein and pharmaceutical composition of the present invention for preparing a medicament for treating or preventing cancerous diseases (eg, solid tumors and soft tissue tumors) in an individual, preferably , Cancerous diseases are melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), kidney cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer , Pancreatic cancer, prostate cancer, head and neck cancer, stomach cancer, hematological malignancies (eg, lymphoma); in particular, the disease is colon cancer or triple negative breast cancer; preferably, wherein the individual is a mammal , More preferably human.
  • cancerous diseases are melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), kidney cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer , Pancre
  • FIGS. 1A and B provide schematic diagrams of the multi-target fusion protein of the present invention, wherein FIG. 1A illustrates a multi-target fusion protein containing an antigen-binding fragment of an antibody, an immunoglobulin Fc domain, and CD80 ECD from the N-terminus to the C-terminus.
  • FIG. 1B illustrates a schematic diagram of a fusion protein comprising the CD80, ECD, immunoglobulin Fc domain, and VEGFR extracellular receptor functional region from the N-terminus to the C-terminus.
  • Figure 2 Shows the results of each protein of the invention prepared and purified in Example 2 by SDS-PAGE electrophoresis and staining with Coomassie blue in the presence of a reducing agent (5mM 1,4-dithiothreitol) .
  • Lane 1 Protein molecular weight standard marker
  • Lane 2 Fusion protein BY24.4
  • Lane 3 Fusion protein BY24.5
  • Lane 4 Fusion protein BY24.12
  • Lane 5 Fusion protein BY31.19
  • Lane 6 Antibody BY18 .1
  • Figure 3 shows the effect of the multi-target fusion protein of the present invention and its combination with anti-PD-1 antibody on IFN- ⁇ secretion in the mixed lymphocyte reaction (MLR) of Example 5.
  • MLR mixed lymphocyte reaction
  • Figure 4 shows the inhibitory effect of the multi-target fusion protein of the present invention and its combination with anti-PD-1 antibody on tumor growth in the animal model of Example 6.
  • the present invention provides a multi-target fusion protein that blocks the growth of vascular endothelial cells and activates T cells and a pharmaceutical composition containing the multi-target fusion protein.
  • the invention also provides a method for producing the multi-target fusion protein, and the use of the multi-target fusion protein or pharmaceutical composition in the treatment or prevention of cancerous diseases in an individual.
  • the term “comprising” or “including” means including the recited element, integer, or step, but does not exclude any other element, integer, or step.
  • PD-1/PD-L1 inhibitory signaling pathway refers to any intracellular signaling pathway triggered by the binding of PD-1 to PD-L1.
  • misalignment As used herein, "mitigation,” “interference,” “inhibition,” or “blocking” of the PD-1/PD-L1 inhibitory signaling pathway can be used interchangeably and refers to (i) interfering with PD-1 and PD-L1. Interactions; and/or (ii) lead to the inhibition of at least one biological function of the PD-1/PD-L1 signaling pathway.
  • the "relief”, “interference”, “inhibition” or “blocking" of the PD-1/PD-L1 signaling pathway caused by the specific binding of the multi-target fusion protein of the present invention to PD-L1 need not be a complete remission , Interfere, inhibit or block.
  • CD28/B7 signaling pathway can be used interchangeably in this article, which means (i) the signaling pathway that stimulates cell activation through the binding of CD28 and CD80 ; And/or (ii) a signaling pathway that stimulates cell activation by binding CD28 to CD86.
  • CD80 and CD86 are both transmembrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF) with highly similar structures, and are also collectively called B7 molecules.
  • the extracellular domain of CD80 and CD86 is composed of an immunoglobulin V (IgV) domain and an immunoglobulin C (IgC) domain.
  • the mature CD80 molecule consists of 254 amino acids, of which the extracellular region consists of 208 amino acids, the transmembrane region has 25 amino acids and the intracellular region has 21 amino acids.
  • the mature CD86 molecule is composed of 303 amino acids, of which the extracellular region is composed of 222 amino acids, the transmembrane region is 20 amino acids and the intracellular region is 61 amino acids.
  • CD80 also known as B7.1
  • B7.1 is expressed on the surface of T cells, B cells, dendritic cells and monocytes, and binds CD28, PD-L1 and CTLA-4 with lower affinity through its immunoglobulin V (IgV) region ,
  • IgV immunoglobulin V
  • CD80 and CD28 is 4 ⁇ M
  • the binding affinity of CD80 and PD-L1 is ⁇ 1.7 ⁇ M
  • the binding affinity of CD80 and CTLA-4 is 0.2 ⁇ M
  • CD86 binds CD28 and CTLA-4, but not PD-L1.
  • Soluble CD80 (eg, CD80-Fc) can produce continuous activation of T lymphocytes through the CD28/B7 costimulatory pathway and stimulate the production of interferon.
  • CD80-Fc maintains T lymphocytes to produce interferon in vitro, which is even more effective than anti-PD-1 antibody or anti-PD-L1 antibody.
  • CD80-Fc soluble CD80
  • anti-PD-L1 antibodies Ostrand-Rosenberg S et al., Novel Strategies for inhibiting PD-1 pathway-mediated immune suppression
  • CD80-Fc can inhibit PD-1/PD-L1 pathway-mediated immunosuppression by binding to PD-L1, and deliver costimulatory signals to T cells activated by the CD28/B7 costimulatory pathway, thereby enhancing T lymphocyte activation.
  • CD80-Fc can alleviate the immunosuppressive effect of PD-1/PD-L1 pathway and activate tumor immunoreactive T cells.
  • soluble CD86 for example, CD86-Fc
  • CD86-Fc can also activate CD28, and even produce a 3-5 fold activation effect of CD80-Fc, but because CD86 does not bind PD-L1, the final CD80-Fc activation of T lymphocytes is strong
  • CD86-Fc Haile, ST, et al., Soluble, CD80, restores, T, cell, activation, and overcomes, tumor, programmed, death,ligand, 1-mediated immunity, depression, J. Immunol., September 1, 2013; 191(5): 2829-36
  • CD80-Fc has the following effects: (i) When CD80-Fc is used alone, its tumor suppressing effect is better than that of PD-L1 antibody (AACR ANNUAL MEETING, April 14-18, 2018, Illinois, USA (Chicago, California); (ii) CD80-Fc promotes lymphocytes to infiltrate tumor tissues, and the effect is better than PD-L1 antibody (Horn LA et al., Soluble CD80ProteinDelaysTumor Growth and PromotesTumor-InfiltratingLymphocytes, CancerImmunolRes.
  • CD80-Fc When CD80-Fc is used alone, it has a better tumor suppressing effect than PD-1/PD-L1 pathway inhibitors, and is compatible with PD-1 antibodies There is synergy when combined. Five Prime even believes that CD80-Fc is superior to T cell agonists such as GITRL, OX40L and 4-1BBL. Seeing the good immunotherapy effect of CD80-Fc, Five's Prime Therapeutics, Inc.'s CD80-Fc project FPT155 plans to conduct clinical trials in the near future.
  • B7/CTLA-4 pathway and “B7/CTLA-4 signaling pathway” are used interchangeably and refer to (i) signaling pathway caused by the binding of CD80 to CTLA-4; and/or ( ii) Signaling pathway caused by CD86 binding to CTLA-4.
  • VEGF/VEGFR pathway and "VEGF/VEGFR signaling pathway” are used interchangeably herein, and refer to binding to one or more of the cell surface receptor VEGFR family through one or more of the VEGF family Mediated signaling pathway.
  • the VEGF family contains six closely related polypeptides, which are highly conserved homodimeric glycoproteins, with six subtypes: VEGF-A, -B, -C, -D, -E, and placental growth factor ( placental growth factor (PLGF)), with molecular weights ranging from 35 to 44 kDa.
  • VEGF-A (including its splices such as VEGF 165 ) is related to the microvessel density of some solid tumors, and the concentration of VEGF-A in the tissue is related to the prognosis of solid tumors such as breast cancer, lung cancer, prostate cancer and colon cancer. .
  • each VEGF family member is mediated by one or more of the cell surface VEGF receptor (VEGFR) family, which includes VEGFR1 (also known as Flt-1), VEGFR2 (also known as KDR , Flk-1), VEGFR3 (also known as Flt-4), etc., where VEGFR1, VEGFR2 are closely related to angiogenesis, and VEGF-C/D/VEGFR3 is closely related to lymphangiogenesis.
  • VEGFR1 also known as Flt-1
  • VEGFR2 also known as KDR , Flk-1
  • VEGFR3 also known as Flt-4
  • the main biological functions of the VEGF family include: (1) Selectively promote vascular endothelial cell mitosis, stimulate endothelial cell proliferation and promote blood vessel formation; (2) Improve the permeability of blood vessels, especially microvessels, and deposit extravasated plasma macromolecules In the extravascular matrix, it provides nutrition for the growth of tumor cells and the establishment of a new capillary network; (3) promotes the proliferation and metastasis of tumors, which depend on the VEGF family for vascular endothelial cells to secrete collagenase and Plasminogen is used to degrade the vascular basement membrane. At the same time, the newly formed microvascular basement membrane inside the tumor tissue is imperfect.
  • VEGF can be used as an immunosuppressive molecule to suppress the body's immunity Response to promote infiltration and metastasis of malignant tumors (Lapeyre-Prost A et al., Immunomodulatory Activity of VEGF in Cancer, Int Rev Cell Mol Biol., 2017; 330:295-342); (5) Other effects: VEGF family can induce Epithelial cells have gaps and open windows, which can activate cytoplasmic vesicles and organelles of the epithelial cells; the VEGF family directly stimulates endothelial cells to release proteolytic enzymes, degrade the matrix, release more VEGF family molecules, accelerate tumor development, extracellular Proteases can also activate the binding of extracellular matrix and the release of VEGF family; VEGF family releases plasma proteins (including fibrinogen) by increasing vascular permeability to form a cellulose network, which provides good for tumor growth, development and metastasis Matrix; VEGF family promote
  • Bevacizumab brand name Avastin developed by Genentech is a recombinant human-mouse chimeric anti-VEGF antibody, which can prevent the activation of VEGFR by blocking the binding of VEGF-A to VEGFR. This plays an anti-angiogenic role. Bevacizumab is currently used for the treatment of metastatic colorectal cancer, lung cancer, breast cancer, pancreatic cancer, kidney cancer, etc.
  • Aflibercept developed by Sanofi-aventis and Regeneron is a kind of VEGF-Trap, which is obtained by fusing the second extracellular domain of VEGFR1 and the third extracellular domain of VEGFR2 with human IgG1 constant region
  • a kind of fusion protein can play an anti-tumor effect on some tumor patients by inhibiting angiogenesis.
  • the term "specific binding” means selective to the binding of an antigen or a molecule of interest and can be distinguished from unwanted or non-specific interactions.
  • the specific binding may be by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technique (analysis on BIAcore instrument) (Liljeblad et al., Analysis of ofagalacto- IgG in rhumatoid arthritis using surface plasmon resonance, Glyco J., 2000, 17, 323-329).
  • vascular endothelial growth inhibitory domain refers to the part of the vascular endothelial growth inhibitor that blocks VEGF/VEGFR signaling, and the part is a region that functions to inhibit vascular endothelial growth.
  • the vascular endothelial growth inhibitory domain may be, for example, one or more variable domains of anti-VEGF antibodies (also called antibody variable regions), one or more variable domains of anti-VEGFR antibodies, or VEGFR extracellularly. Provided by the body ribbon.
  • Treg regulatory T cells
  • the term "regulatory T cells (Treg)” represents a specific subset of T lymphocytes that are essential for maintaining self-tolerance.
  • the Treg cells with repressor function can be distinguished from other T lymphocytes by expressing the transcription factor FOXP3 and other cell markers such as CD127 low , CTLA-4 + , LAP, CD39 + , PD-1 + , GARP, etc. .
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k off and k on, respectively ).
  • K D dissociation constant
  • association rate constant k off and k on, respectively .
  • Affinity can be measured by common methods known in the art. A specific method for measuring affinity is surface plasmon resonance (SPR).
  • antibody is used in the broadest sense herein and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), as long as they exhibit the desired antigen binding activity .
  • the antibody may be a complete antibody of any type and subtype (eg, IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2) (eg, having two full-length light chains and two full-length heavy chains) chain).
  • whole antibody full length antibody
  • complete antibody complete antibody
  • whole antibody is used interchangeably herein to refer to an antibody that has a structure that is substantially similar to the structure of a natural antibody.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation, which normally determines the class of antibody to which it belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation.
  • ⁇ light chain and ⁇ light chain refer to the two main antibody light chain types.
  • Bispecific antibodies are artificial hybrid antibodies that have two different heavy/light chain pairs and have two different binding sites. Bispecific antibodies can be prepared by a variety of methods including fusion of hybridomas or ligation of Fab' fragments.
  • antigen-binding fragment of an antibody is a portion or segment of an antibody or antibody chain that has fewer amino acid residues than an intact or complete antibody or antibody chain, and is capable of binding antigen or binding to an intact antibody (ie, derived from an antigen-binding fragment Intact antibodies) compete for antigen binding.
  • Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to Fab, Fab', F(ab') 2 , Fv, and single-chain Fv.
  • the Fab fragment is a monovalent fragment composed of VL , VH , CL and CH1 domains.
  • Fab fragments can be obtained by papain digestion of complete antibodies.
  • the complete antibody is digested under the disulfide bond of the hinge region by pepsin to produce F(ab′) 2 , which is a dimer of Fab′ and is a bivalent fragment.
  • F(ab') 2 can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, thus converting the F(ab') 2 dimer to Fab' monomer.
  • Fab' monomers are basically Fab fragments with hinge regions (for more detailed descriptions of other antibody fragments, see: Fundamental Immunology, edited by WE Paul, Raven Press, NY (1993)).
  • the Fv fragment consisting of V L and V H domains of a single arm of an antibody composition.
  • the two domains V L and V H of the Fv fragment are encoded by independent genes, using recombinant methods, they can be connected by a synthetic linker that enables the two domains to be produced as a single protein chain.
  • the V L and V H regions of the single protein chain pair to form a single chain Fv.
  • the antibody fragment can be obtained by a chemical method, a recombinant DNA method, or a protease digestion method.
  • immunoglobulin refers to a protein having the structure of a naturally occurring antibody.
  • an IgG-like immunoglobulin is a heterotetrameric glycoprotein composed of two light chains and two heavy chains bonded by disulfide bonds of about 150,000 daltons. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 And CH3), also known as the heavy chain constant region.
  • VH variable region
  • CH1 And CH3 constant domains
  • each immunoglobulin light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light chain (CL)
  • VL variable region
  • CL constant light chain
  • the heavy chains of immunoglobulins can be assigned to one of five categories, called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG) or ⁇ (IgM), some of which can be further divided into sub Classes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG 3 ), ⁇ 4 (IgG 4 ), ⁇ 1 (IgA 1 ), and ⁇ 2 (IgA 2 ).
  • the light chain of an immunoglobulin can be divided into one of two types based on the amino acid sequence of its constant domain, called ⁇ and ⁇ .
  • An immunoglobulin is basically composed of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.
  • Fc domain or "Fc region” is used herein to define the C-terminal region of the immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the natural immunoglobulin "Fc domain” contains two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain.
  • the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of IgG, IgA, and IgD class antibodies; or The second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) from the two heavy chains of IgM and IgE class antibodies.
  • the numbering of amino acid residues in the Fc region or the heavy chain constant region is based on, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health, Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also called the EU index) is used for numbering.
  • Human immunoglobulin is an immunoglobulin that has an amino acid sequence corresponding to an immunoglobulin produced by a human or human cell or from a non-human using a human immunoglobulin library or other sequence encoding human immunoglobulin Source derivation.
  • the “percent identity (%)" of the amino acid sequence means that the candidate sequence is aligned with the specific amino acid sequence shown in this specification and, if necessary, after introducing gaps to achieve the maximum percentage of sequence identity, regardless of any When conservative substitutions are part of sequence identity, the percentage of amino acid residues in the candidate sequence that are the same as the amino acid residues of the specific amino acid sequence shown in this specification.
  • operably linked means that the specified components are in a relationship that allows them to function in the intended manner.
  • N-terminal refers to the last amino acid at the N-terminal
  • C-terminal refers to the last amino acid at the C-terminal
  • fusion refers to the direct connection of two or more components by peptide bonds or effective connection via one or more peptide linkers.
  • host cell refers to a cell into which a foreign polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom.
  • the host cell is any type of cell system that can be used to produce the multi-target fusion protein of the present invention.
  • the host cell includes a cultured cell, and also includes a transgenic animal, a transgenic plant, or a cell within a cultured plant tissue or animal tissue.
  • mammals include, but are not limited to domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, human and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large animals) mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, human and non-human primates such as monkeys
  • rabbits eg, mice and large animals
  • rodents eg, mice and large animals
  • treatment refers to a clinical intervention intended to alter the natural course of the disease in the individual being treated. 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 progression of the disease, improving or alleviating the disease state, and relieving or improving the prognosis.
  • the multi-target fusion protein or pharmaceutical composition of the invention is used to delay disease progression or to slow the progression of the disease.
  • anti-tumor effect refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduced tumor volume, reduced tumor cell number, reduced tumor cell proliferation, or reduced tumor cell survival.
  • tumor tumor cell proliferation
  • tumor cell survival a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduced tumor volume, reduced tumor cell number, reduced tumor cell proliferation, or reduced tumor cell survival.
  • tumor cancer
  • the present invention provides a novel multi-target fusion protein comprising (i) vascular endothelial growth inhibitory domain; (ii) immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD).
  • the (i), (ii) and/or (iii) are optionally operably linked by a peptide linker.
  • the multi-target fusion protein of the present invention is a heterotetrameric glycoprotein composed of two first subunits of fusion proteins and two second subunits of fusion proteins that are disulfide bonded.
  • the multi-target fusion protein of the invention is a homodimeric or heterodimeric glycoprotein bonded by disulfide bonds.
  • the multi-target fusion protein of the present invention blocks the growth of vascular endothelial cells and activates T lymphocytes.
  • the multi-target fusion protein can block the growth of vascular endothelial cells by blocking the VEGF/VEGFR pathway, on the other hand, it can inhibit the PD-1/PD-L1 inhibitory signaling pathway by activating the CD28/B7 costimulatory pathway, B7/CTLA-4 signaling pathway inhibits the function of regulatory T cells (Treg) to activate T lymphocytes, thereby improving the tumor microenvironment and improving the effect of tumor immunotherapy.
  • the multi-target fusion protein of the present invention binds to VEGF or VEGFR with a dissociation constant (K D ) of 10 -8 M or less, for example, 10 -9 M to 10 -12 M; and CD28, PD-L1 and CTLA-4 specifically bind.
  • K D dissociation constant
  • the "vascular endothelial growth inhibitory domain" in the multi-target fusion protein of the present invention can specifically bind to VEGF and/or VEGFR, including but not limited to antigen-binding fragments derived from anti-VEGF antibodies and/or anti-VEGFR antibodies and/or VEGFR extracellular receptor functional area.
  • the antigen-binding fragments derived from the anti-VEGF antibody and/or anti-VEGFR antibody contained in the multi-target fusion protein of the present invention enable the multi-target fusion protein of the present invention to have high affinity, for example, 10-8 M D or less, preferably with a K D of 10 -9 M to 10 -12 M, specifically binds to VEGF and/or VEGFR, and thereby blocks the signaling pathway mediated by the binding of VEGF to its receptor VEGFR.
  • the antigen-binding fragment of the multi-target fusion protein of the present invention comprises the heavy chain variable region (VH) and/or light chain variable region of the anti-VEGF antibody and/or anti-VEGFR antibody in Table 1.
  • VL amino acid sequence is substantially the same sequence, for example, with the paired heavy chain variable region sequence / light chain variable region sequence shown in Table 1 has at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or more sequences with sequence identity.
  • the antigen-binding fragment of the multi-target fusion protein of the present invention comprises a pair of heavy chain variable region sequence/light chain variable selected from SEQ ID NO: 1/2, 3/4, and 5/6 All six heavy chain complementarity determining regions (CDRs) and light chain CDRs contained in the region sequence.
  • CDRs heavy chain complementarity determining regions
  • Methods and techniques for identifying CDRs in the amino acid sequences of heavy chain variable regions and light chain variable regions are known in the art and can be used to identify specific heavy chain variable regions and/or light chains disclosed herein CDR in the amino acid sequence of the variable region.
  • Exemplary well-known techniques that can be used to identify CDR boundaries include, for example, Kabat definition method, Chothia definition method, and AbM definition method.
  • the anti-VEGF antibody or anti-VEGFR antibody as the source of the antigen-binding fragment in the multi-target fusion protein of the present invention can be classified into a ⁇ type or a ⁇ type based on the amino acid sequence of the light chain constant region, preferably a ⁇ type.
  • amino acid sequence of the light chain constant region of the anti-VEGF antibody or anti-VEGFR antibody are provided in Table 2 below.
  • the amino acid sequence of the anti-VEGF antibody or anti-VEGFR antibody derived from the antigen-binding fragment in the multi-target fusion protein of the present invention based on the constant region of its heavy chain is preferably an IgG class antibody, particularly an IgG 1 subclass and an IgG 2 subclass , IgG 4 subclass antibodies, more particularly IgG 4 subclass antibodies.
  • the IgG 4 subclass antibody contains an amino acid substitution that prevents arm-exchange at position S228 in the Fc region, in particular the amino acid substitution S228P.
  • amino acid sequence of the antibody heavy chain constant region are provided in Table 3 below.
  • the VEGFR extracellular receptor functional region contained in the multi-target fusion protein of the present invention is a part of the extracellular domain of VEGFR or a combination thereof.
  • the VEGFR receptor is a tyrosine kinase receptor located on the cell surface, and its extracellular region is composed of seven immunoglobulin (Ig)-like domains.
  • human VEGFR1 contains seven Ig-like domains numbered 1, 2, 3, 4, 5, 6, and 7, Ig-like domain 1 is at the N-terminus of the extracellular domain, and Ig-like domain 7 is extracellular C-terminal of the domain. Unless otherwise indicated herein, Ig-like domains are numbered sequentially from the N-terminus to the C-terminus of the VEGFR protein.
  • the VEGFR extracellular receptor functional region comprises at least one Ig-like domain of one or more VEGFRs selected from VEGFR1, VEGFR2, and VEGFR3. In some aspects, the VEGFR extracellular receptor functional region comprises at least 1, 2, 3, 4, 5, 6 but not more than 7 Ig-like domains of VEGFR. In another aspect, the VEGFR extracellular receptor functional region comprises 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 Ig-like domains of VEGFR.
  • VEGFR extracellular receptor functional region comprising at least one Ig-like domain of two or more VEGFRs.
  • the VEGFR extracellular receptor functional region comprises at least one Ig-like domain from two or more VEGFRs selected from VEGFR1, VEGFR2, and VEGFR3.
  • VEGFR extracellular receptor functional domains containing any combination of the seven Ig-like domains of each VEGFR.
  • the VEGFR extracellular receptor functional region may comprise Ig-like domain 2 of VEGFR1 (eg, human VEGFR1) and Ig-like domain 3 of VEGFR2 (eg, human VEGFR2).
  • the VEGFR extracellular receptor functional region may comprise Ig-like domains 1-3 of VEGFR1 (eg, human VEGFR1), Ig-like domains 2-3, VEGFR2 (eg, human) of VEGFR1 (eg, human VEGFR1) VEGFR2) Ig-like domain 1-3, VEGFR1 (eg human VEGFR1) Ig-like domain 2 and VEGFR2 (eg human VEGFR2) Ig-like domain 3-4, or VEGFR1 (eg human VEGFR1) Ig-like structure Domain 2 and Ig-like domain 3 of VEGFR3 (eg, human VEGFR3).
  • VEGFR1 eg, human VEGFR1
  • Ig-like domains 2-3 eg-like domains 2-3
  • Ig-like domains and other Ig-like domains that can be used as part of the VEGFR extracellular receptor functional area are described in more detail in U.S. Patent No. 7531173; Yu, DC, etc., Soluble, Vascular, Endothelial, Growth Factor, Decoy Receptor, FP3, Exerts, Potent Antiangiogenic Effects , Mol. Ther., 2012, 20(3): 938-947 and Holash, J. et al., VEGF-Trap: a VEGF blocker with potent antitumor effects, PNAS, 2002, 99(17): 11393-11398, all literature The overall introduction is used here as a reference.
  • the VEGFR extracellular receptor functional region has any one selected from the amino acid sequence shown in SEQ ID NO: 7-9 in Table 4 or at least 90 from the amino acid sequence shown in SEQ ID NO: 7-9 Amino acid sequences of %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the VEGFR extracellular receptor functional region in the multi-target fusion protein of the present invention can interact with VEGF with high affinity, for example, with a K D of 10 -8 M or less, preferably 10 -9 M to 10 -12 M.
  • the family binds specifically, and thereby inhibits the binding of the VEGF family to VEGFR on the cell surface and subsequent signaling.
  • the "immunoglobulin Fc domain" in the multi-target fusion protein of the present invention contains all the amino acid residues of the naturally occurring immunoglobulin Fc domain or a part of the amino acid residues of the naturally occurring immunoglobulin Fc domain.
  • the immunoglobulin Fc domain provides advantageous pharmacokinetic properties for the multi-target fusion protein of the invention, including but not limited to long serum half-life.
  • the immunoglobulin Fc domain also makes it possible to purify the multi-target fusion protein of the present invention by, for example, protein A affinity chromatography.
  • Immunoglobulin Fc domains are usually dimeric molecules.
  • the immunoglobulin Fc domain may be produced by papain digestion or trypsin digestion of intact (full-length) immunoglobulin or may be recombinantly produced, which comprises a CH2 domain, a CH3 domain, and an optional CH4 domain.
  • the IgG Fc region comprises IgG CH2 domain and IgG CH3 domain.
  • the immunoglobulin Fc domain has the amino acid sequence shown in SEQ ID NO: 10-12 in Table 5 or has at least 90%, 91%, 92% of the amino acid sequence shown in SEQ ID NO: 10-12 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences.
  • the IgG Fc region may also contain a peptide sequence obtained by performing additional sequence modification on SEQ ID NO: 10-12, for example, SEQ ID NO: 10-12
  • an amino acid substitution that prevents arm-exchange from occurring particularly amino acid substitution S228P, is included in the IgG Fc region at position S228.
  • the IgG4 Fc region comprises the amino acid substitution S228P.
  • the "extracellular domain (CDD) of CD80" in the multi-target fusion protein of the present invention contains all the amino acid residues of naturally occurring CD80 ECD or a part of the amino acid residues of naturally occurring CD80 ECD.
  • the CD80ECD comprises CD80 IgV.
  • the CD80ECD comprises human CD80 IgV.
  • the CD80ECD has the amino acid sequence shown in SEQ ID NO: 13 or 14 in Table 6.
  • CD80ECD can also contain peptide sequences obtained by additional sequence modification of SEQ ID NO: 13 and 14, for example, in SEQ ID NO: 13 and 14.
  • the modified peptide will retain the activity or function associated with the unmodified peptide.
  • the modified peptide usually has an amino acid sequence that is substantially homologous to the amino acid sequence of the unmodified sequence.
  • the amino acid sequence shown in SEQ ID NO: 13 or 14 has at least 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • vascular endothelial growth inhibitory domain vascular endothelial growth inhibitory domain
  • immunoglobulin Fc domain an immunoglobulin Fc domain
  • a "peptide linker" to which CD80 and ECD are optionally operatively linked are one or Peptides with multiple amino acids, generally about 2-20 amino acids. Peptide linkers are known in the art or described herein.
  • the peptide linker comprises at least 5 amino acids, preferably selected from AKTTPKLEEGEFSEAR (SEQ ID NO: 20); AKTTPKLEEGEFSEARV (SEQ ID NO: 21); AKTTPKLGG (SEQ ID NO: 22); SAKTTPKLGG ( SEQ ID NO: 23); SAKTTP (SEQ ID NO: 24); RADAAP (SEQ ID NO: 25); RADAAPTVS (SEQ ID NO: 26); RADAAAAGGPGS (SEQ ID NO: 27); RADAAAA (SEQ ID NO: 28) ); SAKTTPKLEEGEFSEARV (SEQ ID NO: 29); ADAAP (SEQ ID NO: 30); DAAPTVSIFPP (SEQ ID NO: 31); TVAAP (SEQ ID NO: 32); TVAAPSVFIFPP (SEQ ID NO: 33); QPKAAP (SEQ ID NO: 34); QPKAAPSVTLFPP (SEQ ID NO: 35); AKTTPP (SEQ ID NO: 36); A
  • multi-target fusion proteins comprising (i) vascular endothelial growth inhibitory domain; (ii) immunoglobulin Fc domain; and (iii) CD80 ECD in any order, including but not limited to multi-target fusion proteins From the N terminal to the C terminal in the order of (i), (ii) and (iii); the order of (iii), (i) and (ii); or the order of (iii), (ii) and (i) is valid connection.
  • the multi-target fusion protein comprises a full-length anti-VEGF antibody, a full-length anti-VEGFR antibody, or a full-length anti-VEGF and VEGFR bispecific antibody from N-terminus to C-terminus; and A CD80 ECD is effectively connected to the C-terminal of each heavy chain in the heavy chain.
  • the multi-target fusion protein comprises a full-length anti-VEGF antibody, a full-length anti-VEGFR antibody, or a full-length anti-VEGF and VEGFR bispecific antibody; each of the two heavy chains of the antibody One CD80 ECD operatively linked to the N-terminus of a heavy chain; and one CD80 ECD operatively linked to the N-terminus of each of the two light chains of the antibody.
  • the multi-target fusion protein comprises CD80 ECD from the N-terminus to the C-terminus; an immunoglobulin Fc domain in the form of a dimer operably linked at the C-terminus of CD80 ECD; and The C-terminus of the immunoglobulin Fc domain in the form of a polymer is operatively linked to an antigen-binding fragment derived from an anti-VEGF antibody and/or anti-VEGFR antibody.
  • the multi-target fusion protein comprises CD80 ECD from the N-terminus to the C-terminus; an immunoglobulin Fc domain in the form of a dimer operably linked at the C-terminus of CD80 ECD; and The C-terminus of the immunoglobulin Fc domain in the form of a polymer is effectively linked to the VEGFR extracellular receptor functional domain.
  • the multi-target fusion protein of the present invention can be obtained, for example, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production.
  • the polynucleotide encoding each subunit of the multi-target fusion protein is isolated and inserted into one or more vectors for further cloning and/or expression in the host cell.
  • the polynucleotide can be easily isolated and sequenced.
  • a vector comprising one or more polynucleotides of the present invention, preferably an expression vector.
  • the expression vector can be constructed using methods well known to those skilled in the art.
  • Expression vectors include but are not limited to viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • a glutamine synthetase high-efficiency expression vector with dual expression cassettes is used.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a suitable host cell for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.
  • a host cell comprising one or more polynucleotides of the invention.
  • host cells comprising the expression vector of the present invention are provided.
  • the term "host cell” refers to any kind of cellular system that can be engineered to produce the multi-target fusion protein of the present invention.
  • Host cells suitable for replicating and supporting the expression of the multi-target fusion protein of the present invention are well known in the art. As required, such cells can be transfected or transduced with a specific expression vector, and a large number of vector-containing cells can be cultivated for inoculation of large-scale fermenters to obtain a sufficient amount of the multi-target fusion protein of the present invention for clinical applications.
  • Suitable host cells include prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like.
  • prokaryotic microorganisms such as E. coli
  • eukaryotic microorganisms such as filamentous fungi or yeast
  • various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like.
  • a mammalian cell line suitable for suspension culture can be used.
  • Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2 ), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, Sp2/0 and so on.
  • the host cell is a CHO, HEK293 or NSO cell.
  • a method of producing a multi-target fusion protein of the invention comprising culturing a host cell as provided herein under conditions suitable for expression of the multi-target fusion protein,
  • the host cell contains a polynucleotide encoding the multi-target fusion protein, and the multi-target fusion protein is recovered from the host cell (or host cell culture medium).
  • the multi-target fusion protein prepared as described herein can be purified by known existing techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art.
  • the purity of the multi-target fusion protein of the present invention can be determined by any of a variety of well-known analysis methods including gel electrophoresis, high-performance liquid chromatography, and the like.
  • the physical/chemical properties and/or biological activities of the multi-target fusion proteins provided herein can be identified, screened, or characterized by various assays known in the art.
  • PD-1 is an immunosuppressive protein with two ligands, PD-L1 and PD-L2. It is known that the interaction between PD-1 and PD-L1 results in, for example, a reduction of tumor-infiltrating lymphocytes and/or immune escape of cancer cells. Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; when the interaction of PD-1 with PD-L2 is also blocked, the effect is additive (Iwai Y. etc. People, Involvement of PD-L1 tumors cells in the theescape from host immune system and tumor immunotherapy by PD-L1 blockade, Proc. Nat'l. Acad. Sci.
  • the present invention has developed a pharmaceutical composition for combination therapy, which comprises the multi-target fusion protein of the present invention and an anti-PD-1 antibody.
  • the pharmaceutical composition for combination therapy described herein can provide superior beneficial effects, such as enhanced anti-cancer effects, Reduced toxicity and/or reduced side effects.
  • the multi-target fusion protein and/or anti-PD-1 antibody of the present invention in a pharmaceutical composition can be administered at a lower dose or shorter administration time than is necessary to achieve the same therapeutic effect compared to monotherapy administration. Therefore, the present invention also discloses the use of pharmaceutical compositions for combination therapy to treat cancer. The effectiveness of the aforementioned pharmaceutical composition can be tested in cell models and animal models known in the art.
  • the anti-PD-1 antibody included in the combination therapy may be any anti-PD-1 antibody, as long as it can inhibit or reduce the binding of PD-1 to its ligand, including anti-PD-1 known in the art 1 Antibodies and anti-PD-1 antibodies developed in the future. Anti-PD-1 antibodies can specifically bind to PD-1 with a high affinity, for example at a K D of 10 -8 M or less, preferably from 10 -9 M to 10 -12 M, and thereby block The signal transduction pathway mediated by the binding of PD-1 to ligands PD-L1 and/or PD-L2.
  • the anti-PD-1 antibody in the combination therapy of the present invention comprises a sequence substantially the same as the amino acid sequence shown in Table 7, for example, the paired heavy chain variable region sequence shown in Table 7/
  • the light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the anti-PD-1 antibody in the combination therapy of the present invention comprises SEQ ID NO: 47/48, 49/50, 51/52, 53/54, 55/56, 57/58, 59/ All heavy chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of 60, 61/62, 63/64, 65/66, 67/68, 69/70 and 71/72 Light chain CDR.
  • Methods and techniques for identifying CDRs in the amino acid sequences of heavy chain variable regions and light chain variable regions are known in the art and can be used to identify specific heavy chain variable regions and/or light chains disclosed herein CDR in the amino acid sequence of the variable region.
  • Exemplary well-known techniques that can be used to identify CDR boundaries include, for example, Kabat definition, Chothia definition, and AbM definition. See, for example, Kabat, Sequences of Proteins, Immunological Interests, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., Standard conformations, for the immunological globulins, J. Mol. Biol. 273 -948 (1997); and Martin AC and others, Modeling antibody hypervariable loops: a combined algorithm, Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989).
  • the anti-PD-1 antibody in the combination therapy of the present invention is selected from nivolumab (Nivolumab), pidilizumab, and pembrolizumab.
  • the pharmaceutical composition of the present invention may contain a "therapeutically effective amount” or “prophylactically effective amount” of the multi-target fusion protein of the present invention and an anti-PD-1 antibody.
  • “Therapeutically effective amount” refers to an amount that is effective to achieve the desired therapeutic result at the required dose for the required period of time.
  • the therapeutically effective amount can be varied according to various factors such as disease state, individual's age, sex, and weight.
  • a therapeutically effective amount is any amount whose toxic or harmful effects are less than the beneficial effects of treatment.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more Preferably at least about 80%.
  • a measurable parameter eg, tumor growth rate
  • the ability of the pharmaceutical composition of the present invention to inhibit measurable parameters can be evaluated in an animal model system that predicts efficacy in human tumors.
  • prophylactically effective amount refers to an amount that is effective to achieve the desired preventive result at the required dose for the required period of time. Generally, since the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • the multi-target fusion protein and anti-PD-1 antibody treatment regimen of the combination therapy of the present invention can cooperate with each other, the pharmaceutical composition of the present invention is Tumor immune escape is beneficial.
  • Co-administration of the pharmaceutical composition comprising the multi-target fusion protein and the anti-PD-1 antibody of the present invention can be carried out by separately administering the multi-target fusion protein or the anti-PD-1 antibody, or the single-target multi-target fusion protein Combined preparation with anti-PD-1 antibody.
  • the administration of the multi-target fusion protein and anti-PD-1 antibody in the combination therapy of the present invention allows flexibility in the amount and schedule of administration.
  • the multi-target fusion proteins and pharmaceutical compositions disclosed herein have therapeutic and prophylactic uses for cancer.
  • the multi-target fusion protein and its composition with an anti-PD-1 antibody can be administered to cultured cells in vitro or ex vivo or to a subject, for example, a human subject, to treat and/or prevent multiple A cancerous disease.
  • the present invention relates to the use of a multi-target fusion protein or a pharmaceutical combination of the present invention to treat or prevent a disease that requires blocking the growth of vascular endothelial cells and activating T cells in a subject, thereby inhibiting or reducing related Diseases such as the growth or appearance, metastasis, or recurrence of cancerous tumors.
  • Multi-target fusion proteins can be used alone to inhibit the growth of cancerous tumors or prevent their appearance.
  • the multi-target fusion protein can be administered in combination with other cancer therapeutic/prophylactic agents (eg, anti-PD-1 antibodies). When the multi-target fusion protein of the present invention and anti-PD-1 antibody are administered, this combination may be administered in any order or simultaneously.
  • the present invention provides a method of inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of the multi-target fusion protein or pharmaceutical composition described herein .
  • the present invention provides a method of preventing the appearance or metastasis or recurrence of tumor cells in a subject, the method comprising administering to the subject a prophylactically effective amount of the multi-target fusion protein described herein or Pharmaceutical composition.
  • cancers treated and/or prevented with multi-target fusion proteins or pharmaceutical compositions include but are not limited to solid tumors, hematological cancers (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma) ) And metastatic lesions.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, such as sarcomas and cancers of multiple organ systems, such as invasion of the lung, breast, ovary, lymphoid, gastrointestinal (eg, colon), anus, genital, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (e.g., brain, nerve, or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated Metastatic or locally recurrent nasopharyngeal carcinoma) and those of the pancreas, as well as adenocarcinomas, including malignant tumors, such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer.
  • the cancer can be early, middle, or advanced or metastatic cancer.
  • the cancer is selected from melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC ), ovarian cancer, pancreatic cancer, prostate cancer, head and neck tumors, gastric cancer, hematological malignancies (eg, lymphoma).
  • Example 1 Construction of a glutamine synthetase high-efficiency expression vector containing the gene of interest
  • nucleotide sequence of the light chain (BY18.1L) of anti-PD1 antibody BY18.1 SEQ ID NO: 73:
  • Anti-PD1 antibody BY18.1 light chain (BY18.1L) amino acid sequence (SEQ ID NO: 74):
  • the nucleotide sequence of the heavy chain (BY18.1H) of anti-PD1 antibody BY18.1 (SEQ ID NO: 75):
  • Amino acid sequence of heavy chain (BY18.1H) of anti-PD1 antibody BY18.1 (SEQ ID NO: 76):
  • METDTLLLWVLLLWVPGSTG is the signal peptide sequence.
  • the BY18.1L encoding nucleotides were double digested with XhoI-EcoRI, and the glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.) was used with XhoI -EcoRI double digestion, and then ligase the XhoI-EcoRI double digested BY18.1L coding nucleotide into the XhoI-EcoRI double digested glutamine synthetase efficient expression vector with double expression cassette, Obtained a high-efficiency expression vector for glutamine synthetase with dual expression cassettes into which BY18.1L coding nucleotides have been introduced; then, the BY18.1H
  • the BY18.1L-encoding nucleotide can also be linked into a glutamine synthetase high-efficiency expression vector with dual expression cassettes into which the BY18.1H-encoding nucleotide has been introduced to express and obtain the antibody BY18.1.
  • CD80 extracellular domain in Table 1 and the IgG4Fc sequence in Table 6 it was optimized to be a nucleotide sequence suitable for expression in Chinese hamster ovary cancer cells (CHO), and entrusted to Shanghai Jierui Bioengineering Co., Ltd. for synthesis as follows SEQ ID NO: 77 polynucleotide sequence.
  • the CD80-Fc fusion protein produced after the expression of the nucleotide sequence is also referred to herein as fusion protein BY31.19.
  • Nucleotide sequence of fusion protein BY31.19 (CD80-Fc, IgG4) (SEQ ID NO: 77)
  • Amino acid sequence of fusion protein BY31.19 (CD80-Fc, IgG4) (SEQ ID NO: 78)
  • the amino acid sequence " METDTLLLWVLLLWVPGSTG” is a signal peptide.
  • the BY31.19 encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) From Beijing Biyang Biotechnology Co., Ltd.).
  • the recombinant vector was sequenced and verified to be correct for CD80-Fc fusion protein expression.
  • the expressed CD80-Fc fusion protein was named fusion protein BY31.19.
  • the sequence of the heavy chain variable region and the light chain variable region of the anti-VEGF antibody in Table 2 Based on the sequence of the CD80 extracellular domain in Table 1, the sequence of the heavy chain variable region and the light chain variable region of the anti-VEGF antibody in Table 2, the sequence of the heavy chain constant region of the antibody in Table 3, and the light chain of the antibody in Table 4
  • the sequence of the constant region and the peptide linker sequence of SEQ ID NO: 20-46 are optimized to the nucleotide sequence suitable for expression in Chinese hamster ovary cancer cells (CHO), and Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize the following SEQ ID NO: polynucleotide sequences shown in 79 and 81.
  • the anti-VEGF antibody-CD80 fusion protein produced after the expression of the nucleotide sequence is expressed herein as fusion protein BY24.4.
  • the amino acid sequence " METDTLLLWVLLLWVPGSTG” is a signal peptide.
  • the BY24.4L encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) (Beijing Biyang Biotechnology Co., Ltd.); by XbaI-SalI double digestion to clone BY24.4H encoding nucleotides to BY24.4L encoding nucleotides with a double expression cassette glutamine synthetase with high efficiency Expression vector; or vice versa.
  • the recombinant vector was sequenced and verified to be correct for expression.
  • the expressed anti-VEGF antibody-CD80 fusion protein was named fusion protein BY24.4.
  • sequence of the CD80 extracellular domain in Table 1 the sequence of the heavy chain variable region and the light chain variable region of the anti-VEGFR antibody in Table 2, the sequence of the heavy chain constant region of the antibody in Table 3, and the light chain of the antibody in Table 4
  • the sequence of the constant region and the peptide linker sequence of SEQ ID NO: 20-46 are optimized to be suitable for nucleotide sequence expression in Chinese hamster ovary cancer cells (CHO), and Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize the following SEQ ID NO: Polynucleotide sequences shown in 83 and 85.
  • the anti-VEGFR antibody-CD80 fusion protein produced after the expression of the nucleotide sequence is expressed herein as fusion protein BY24.5.
  • the amino acid sequence " METDTLLLWVLLLWVPGSTG” is a signal peptide.
  • the BY24.5L encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) (Beijing Biyang Biotechnology Co., Ltd.); then cloned the BY24.5H encoding nucleotide by XbaI-SalI double digestion into the glutamine synthetase with the double expression cassette linked to the BY24.5L encoding nucleotide. Expression vector; or vice versa. The recombinant vector was sequenced and verified to be correct for expression.
  • the expressed anti-VEGFR antibody-CD80 fusion protein was named fusion protein BY24.5.
  • CD80 extracellular domain in Table 1 the sequence of IgG4Fc in Table 6, and the sequence of VEGFR functional region in Table 5 (VEGFR1-D2/VEGFR2-D3), it is optimized to be suitable for nuclear expression in Chinese hamster ovary cancer cells (CHO) Nucleotide sequence, and entrusted Shanghai Jierui Biological Engineering Co., Ltd. to synthesize the polynucleotide sequence shown in SEQ ID NO: 87
  • the CD80-Fc-VEGFR fusion protein produced after the expression of the nucleotide sequence is also referred to herein as fusion protein BY24.12.
  • Nucleotide sequence of fusion protein BY24.12 (CD80-Fc-VEGFR) (SEQ ID NO: 87)
  • the BY24.12 encoding nucleotide was ligated to a glutamine synthetase high-efficiency expression vector with dual expression cassettes (patent authorization number: CN104195173B, obtained by XhoI-EcoRI double digestion) From Beijing Biyang Biotechnology Co., Ltd.).
  • the recombinant vector was sequenced and verified to be correct for protein expression.
  • the expressed fusion protein was named fusion protein BY24.12.
  • each of the recombinant expression vector plasmid DNA containing the target gene prepared in Example 1 was 250ug and polyethylenimine (PEI) (Sigma, catalog number: 408727) 500ug was added 1ml of serum-free CD 293 culture solution was mixed well. After standing at room temperature for 8 minutes, the PEI/DNA suspension was added dropwise to a shake flask containing 100ml of cell suspension. Mix gently and place in 5% CO 2 at 37°C for shaking (120 rpm). After 5 days, the culture supernatant was collected.
  • PEI polyethylenimine
  • the target protein present in the culture supernatant collected in Example 2(1) above was purified using a HiTrap MabSelect SuRe 1ml column (GE Healthcare Products Life Catalog Number: 11-0034-93) equilibrated with pH 7.4 PBS solution.
  • the HiTrap MabSelect SuRe 1ml column was equilibrated with 10 column bed volumes of PBS solution at pH 7.4 at a flow rate of 0.5ml/min; the culture supernatant collected in Example 2(1) above was filtered with a 0.45 ⁇ m filter Then, load the sample onto a HiTrap MabSelect SuRe 1ml column equilibrated with pH 7.4 PBS solution; after loading the supernatant, first wash the column with PBS solution pH 7.4 at a flow rate of 0.5ml/min to wash 5-10 column bed volumes, and It was then eluted with 100 mM citrate buffer (pH 4.0) at a flow rate of 0.5 ml/min. The elution peaks were collected, and the target proteins BY18.1, BY31.19, BY24.4, BY24.5, and BY24.12 existed in the elution peaks, respectively.
  • Recombinant human CD28 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 50103-M08H), recombined human PD-L1 (Beijing Bepsey Biotechnology Co., Ltd., catalog number: PD1-H5229) and recombined human CTLA-4 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11159-H08H) diluted to 0.5 ⁇ g/ml, 0.25 ⁇ g/ml, and 1.0 ⁇ g/ml and coated with 96-well ELISA plates (Corning Corporation, article number: 42592).
  • the fusion proteins BY24.4, BY24.5, BY24.12, and BY31.19 purified in the above Example 2(2) were diluted to 2000 ⁇ g/ml, and then serially diluted 3 times, a total of 16 gradients were diluted for each The concentration gradient was used for double well detection. 50 ⁇ l of the diluted sample was added to the above-mentioned 96-well plate coated with recombinant human CD28, recombinant human CTLA-4 or recombinant human PD-L1, and incubated at 37°C for 2 hours.
  • ELISA results show that the multi-target fusion proteins of the present invention BY24.4, BY24.5, BY24.12 and BY31.19 as positive controls can bind recombinant human PD-L1, recombinant human CD28 and CTLA-4.
  • Each fusion protein has the strongest binding ability to CTLA-4, followed by PD-L1, and the weakest to CD28.
  • Example 4 Using Biacore T100 to determine the affinity of the target protein of the present invention for the target
  • the anti-IgG antibody (GE Healthcare, Life Sciences, catalog number: BR-1008-39) was covalently fixed on the CM5 chip by amide coupling.
  • EDC N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • each target protein of the present invention prepared in Example 2 was diluted to 5 ⁇ g/ml, and the dilution was injected at a flow rate of 10 ⁇ L/min for 2 minutes.
  • Each target protein of the present invention prepared in Example 2 passed through its own Fc region. Covalently captured on the CM5 chip surface. The resulting complex is stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration.
  • antigen PD-1 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 10377-H08H
  • VEGF 165 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 11066-HNAH
  • mouse VEGF164 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 50159-MNAB
  • VEGFR2 Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. product, catalog number: 10012-H02H1
  • concentration gradients 7nM, 22nm, 66nM, 200nM , 600nM.
  • Binding was measured by injecting each concentration at a flow rate of 30 ⁇ l/min for 180 seconds and a dissociation time of 600 seconds. The surface was regenerated by washing with 3M MgCl 2 solution at a flow rate of 10 ⁇ L/min for 30 seconds. Data analysis was performed using BIA evaluation software (BIAevaluation 4.1 software from GE Healthcare Biosciences AB, Sweden) to obtain the affinity data shown in Table 10 below.
  • the KD(M) of the fusion proteins BY24.4, BY24.5, BY24.12 and their respective targets are lower than 10 -9 M, indicating that they have high affinity with their respective targets, especially the fusion protein BY24.12 can bind Both mouse and human VEGF-A bind with high affinity and the difference is not very large.
  • Example 5 Effect of the target protein of the present invention and its composition on IFN- ⁇ secretion in mixed lymphocyte reaction (MLR)
  • CD4 + T lymphocytes and dendritic cells were purchased from Beijing Shihe Biotechnology Co., Ltd.
  • the CD4 + T lymphocytes and dendritic cells (DC) were derived from different healthy persons.
  • CD4 + T lymphocytes and dendritic cells (DC) were plated in 96-well cell culture plates at 1 ⁇ 10 5 cells/well and 1 ⁇ 10 4 cells/well, respectively.
  • the experiment was divided into 7 groups, namely BY18.1 group (1.0 ⁇ g/ml), BY24.4 group (1.14 ⁇ g/ml), BY24.4 (1.14 ⁇ g/ml) + BY18.1 (1.0 ⁇ g/ml) group , BY24.5 (1.14 ⁇ g/ml) group, BY24.5 (1.14 ⁇ g/ml) + BY18.1 (1.0 ⁇ g/ml) group, BY24.12 (0.84 ⁇ g/ml) group and BY24.12 (0.84 ⁇ g) /ml)+BY18.1 (1.0 ⁇ g/ml) group.
  • test results are as follows: compared with the antibody BY18.1 group (2221.8 ⁇ 364.5pg/ml), the fusion protein BY24.4 group alone (924.1 ⁇ 221.9pg/ml), BY24.5 group (760.1 ⁇ 286.8pg/ml) And BY24.12 group (793.4 ⁇ 139.2pg/ml) IFN- ⁇ secretion were significantly lower than antibody BY18.1 group (P ⁇ 0.01); but fusion proteins BY24.4, BY24.5 and BY24.12 were added respectively After BY18.1, IFN- ⁇ secretion was significantly increased.
  • the IFN- ⁇ secretion of each group was: BY24.4+BY18.1 group (3494.2 ⁇ 364.5pg/ml), BY24.5+BY18.1 group (3523.8 ⁇ 465.1pg/ml) and BY24.12+BY18.1 group (3801.8 ⁇ 702.2pg/ml).
  • the fusion proteins BY24.4, BY24.5, and BY24.12 combined with BY18.1 can cause a significant increase in IFN- ⁇ secretion (P ⁇ 0.01), and indicate that the antibody BY18.1 (PD-1 antibody )
  • the combination with the multi-target fusion proteins BY24.4, BY24.5 and BY24.12 of the present invention has a clear synergistic effect on the secretion of IFN- ⁇ .
  • Example 6 Synergy of the multi-target fusion protein of the present invention and PD-1 antibody
  • the main purpose of this example is to explore the synergistic effect of the multi-target fusion protein of the present invention and the PD-1 antibody in anti-tumor in vivo, so the dosage of the PD-1 antibody and each fusion protein is a low dose because of the high The dose may have a good tumor suppression effect, and no synergy between the PD-1 antibody and each fusion protein was observed.
  • mouse colon cancer cells CT26 (ATCC) in RPMI-1640 medium were inoculated subcutaneously on the right anterior rib of 6-week-old female BALB/c mice, 100 ⁇ l/mouse, inoculation volume was 1 ⁇ 10 6 Cells/mouse.
  • the time of inoculation of mouse-derived colon cancer cell CT26 was set to day 0.
  • the tumor-bearing mice were randomly divided into 6 mice in each group, for a total of 5 groups. Calculate the molar amount based on the molecular weight, and administer the fusion proteins BY31.19, BY24.12 and anti-mPD-1 antibody in equivalent molar amounts.
  • the grouping and administration doses are: vehicle (PBS) control group; fusion protein BY31.19 (1.6mg/kg) group; fusion protein BY24.12 (2.5mg/kg,) group; anti-mPD-1 ( 3.0mg/kg, purchased from BioXcell, clone number: RMP1-14, product number: BE0146) group; fusion protein BY24.12 (2.5mg/kg) + anti-mPD-1 (3mg/kg) group.
  • mice were euthanized, serum and tumors were collected, and the serum was stored at -80°C.
  • the tumors were weighed and photographed, and then fixed into formal samples by formalin fixation and paraffin embedding (FFPE).
  • FFPE formalin fixation and paraffin embedding
  • the tumor growth inhibition rates (TGI%) of BY31.19 group, BY24.12 group, PD-1 group, BY24.12+anti-mPD-1 group were 15%, 17%, 24%, and 47% (in terms of tumor volume) have different degrees of inhibition.
  • the immunosuppressive protein BY24.12 combined with PD-1 antibody has a significantly better tumor suppression effect than BY24.12 and PD-1 when used alone, so there is a synergistic effect between the two.

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2025508267A (ja) * 2022-03-04 2025-03-21 パノロス バイオサイエンス インコーポレイテッド ＶＥＧＦ－Ｇｒａｂ及びＰＤ－１又はＰＤ－Ｌ１拮抗剤を含む併用投与用組成物

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL283522B2 (en) 2018-12-03 2025-03-01 Agensys Inc Pharmaceutical compositions containing anti-191P4D12 antibody drug conjugates and methods of using them
KR20220107257A (ko) * 2020-03-20 2022-08-02 레메젠 코, 리미티드 이중특이성 융합 단백질 및 그의 응용
WO2021238904A1 (zh) * 2020-05-25 2021-12-02 北京比洋生物技术有限公司 Fc-CD80融合蛋白和其缀合物以及它们的用途
CN116209466A (zh) * 2020-08-27 2023-06-02 上海君实生物医药科技股份有限公司 抗pd-1抗体在治疗鼻咽癌中的用途
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IL318220A (en) * 2022-07-25 2025-03-01 Agensys Inc Methods for treating patients with locally advanced or metastatic urinary tract cancer with antibody drug conjugates (ADCs) that bind 191P4D12 proteins in combination with pembrolizumab
TW202413427A (zh) * 2022-08-09 2024-04-01 大陸商上海濟煜醫藥科技有限公司 一種靶向ｐｄ－１和ｖｅｇｆ的抗體及其應用
JP2025534252A (ja) * 2022-09-14 2025-10-15 尋済生物科技（北京）有限公司 抗ｖｅｇｆａ抗体またはその抗原結合フラグメント、およびその使用
CN116102624B (zh) * 2023-03-07 2025-10-03 南京中医药大学 一种具有抑制血管生成作用的线性多肽及其环肽

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7531173B2 (en) 2005-02-02 2009-05-12 Regeneron Pharmaceuticals, Inc. Ophthalmic composition of a VEGF antagonist
WO2010136480A1 (en) * 2009-05-28 2010-12-02 Glaxo Group Limited Antigen-binding proteins
CN104195173A (zh) 2014-09-02 2014-12-10 北京比洋生物技术有限公司 具有双表达盒的谷氨酰胺合成酶高效表达载体
WO2017218977A2 (en) * 2016-06-17 2017-12-21 Genentech, Inc. Purification of multispecific antibodies
CN108623691A (zh) * 2017-03-17 2018-10-09 北京比洋生物技术有限公司 IgG样长效免疫融合蛋白及其应用
WO2018191438A1 (en) * 2017-04-11 2018-10-18 Inhibrx, Inc. Multispecific polypeptide constructs having constrained cd3 binding and methods of using the same
CN108884139A (zh) * 2015-11-02 2018-11-23 戊瑞治疗有限公司 Cd80胞外域多肽及其在癌症治疗中的用途

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI1916001T1 (sl) * 2002-03-04 2011-10-28 Imclone Llc Äśloveĺ ka protitelesa specifiäśna za kdr in njihova uporaba
BRPI0913366A8 (pt) * 2008-06-03 2017-07-11 Abbott Lab Imunoglobulinas de domínio variável duplo e seus usos
PE20120622A1 (es) * 2009-06-17 2012-05-26 Abbott Biotherapeutics Corp Anticuerpos anti-vegf y sus usos
UY33492A (es) * 2010-07-09 2012-01-31 Abbott Lab Inmunoglobulinas con dominio variable dual y usos de las mismas
EP2646007B1 (en) * 2010-12-02 2016-12-21 Neurotech USA, Inc. Cell lines that secrete anti-angiogenic antibody-scaffolds and soluble receptors and uses thereof
BR112014013205A2 (pt) * 2011-12-01 2020-10-27 Protevobio, Inc. proteína de fusão, seu uso e seu método de produção, composição farmacêutica, ácido nucleico, e kit
SG10201900002QA (en) * 2014-01-24 2019-02-27 Dana Farber Cancer Institue Inc Antibody molecules to pd-1 and uses thereof
WO2017025498A1 (en) * 2015-08-07 2017-02-16 Pieris Pharmaceuticals Gmbh Novel fusion polypeptide specific for lag-3 and pd-1
CN109069638B (zh) * 2016-03-24 2022-03-29 璟尚生物制药公司 用于癌症治疗的三特异性抑制剂
EP3596114A2 (en) * 2017-03-16 2020-01-22 Alpine Immune Sciences, Inc. Cd80 variant immunomodulatory proteins and uses thereof
CN109053895B (zh) * 2018-08-30 2020-06-09 中山康方生物医药有限公司 抗pd-1-抗vegfa的双功能抗体、其药物组合物及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7531173B2 (en) 2005-02-02 2009-05-12 Regeneron Pharmaceuticals, Inc. Ophthalmic composition of a VEGF antagonist
WO2010136480A1 (en) * 2009-05-28 2010-12-02 Glaxo Group Limited Antigen-binding proteins
CN104195173A (zh) 2014-09-02 2014-12-10 北京比洋生物技术有限公司 具有双表达盒的谷氨酰胺合成酶高效表达载体
CN108884139A (zh) * 2015-11-02 2018-11-23 戊瑞治疗有限公司 Cd80胞外域多肽及其在癌症治疗中的用途
WO2017218977A2 (en) * 2016-06-17 2017-12-21 Genentech, Inc. Purification of multispecific antibodies
CN108623691A (zh) * 2017-03-17 2018-10-09 北京比洋生物技术有限公司 IgG样长效免疫融合蛋白及其应用
WO2018191438A1 (en) * 2017-04-11 2018-10-18 Inhibrx, Inc. Multispecific polypeptide constructs having constrained cd3 binding and methods of using the same

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
AACR ANNUAL MEETING, 14 April 2018 (2018-04-14)
AL-LAZIKANO ET AL.: "Standard Modifications for the Canopy Structures of Immunoglobalins", J MOL. BIOL., vol. 273, 1997, pages 927 - 948
BIACORE ET AL.: "Analysis of Agcto-Ciglin Rheumami Using Surface Plasmons Resonance", GLYCO. J., vol. 17, 2000, pages 323 - 329
BUTTE, J ET AL.: "Programmed Death -1 Ligandl Interaction Specifications with the B7-1 Costimulatory Molecule to Gbit T Cell Responses", IMMUNITY, vol. 27, no. 1, July 2007 (2007-07-01), pages 111 - 122
HAILE ST ET AL.: "Soluable CD80 Restorres T Cell Activation and Overcomes Tumor Cell Programmed Death Ligand 1-Mediated Immune Suppression", J IMMUNOL., vol. 191, no. 5, 1 September 2013 (2013-09-01), pages 2829 - 36
HOLASH, J. ET AL.: "VEGF-Trap: a VEGF blocker with potent antitumor effects", PNAS, vol. 99, no. 17, 2002, pages 11393 - 11398, XP002611390, DOI: 10.1073/pnas.172398299
HORN LA ET AL.: "Soluble CD80 Protein Delays Tumor Growth and Promotes Tumor-Infiltratmg Lymphocytes", CANCER IMMUNOL RES., vol. 6, no. 1, 2018, pages 59 - 68, XP055642081, DOI: 10.1158/2326-6066.CIR-17-0026
HORN LALA ET AL.: "Soluble CD80 Protein DelayS Tumor Growth and Promoter Tumor-Infiltrazol", CANCERIMMUNOL RES., vol. 6, no. 1, January 2018 (2018-01-01), pages 59 - 68
HUI E: "T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition", SCIENCE, vol. 355, no. 6332, 2017, pages 1428 - 1433, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pubmed/?term=Enfu+Hui+Jeanne+Cheung>
IWAI Y. ET AL.: "Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade", PROC.NAT'L. ACAD. SCI. USA, vol. 99, 2002, pages 12293 - 7, XP055572034, DOI: 10.1073/pnas.192461099
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH CARE SERVICE, NATIONAL INSTITUTES OF HEALTH
KAMPHORSTAO ET AL.: "Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent", SCIENCE, vol. 355, no. 6332, 2017, pages 1423 - 1427, XP055427814, DOI: 10.1126/science.aaf0683
LASPERRE-PROST A ET AL.: "Immunomodulatory Activity of VEGF in Cancer", INT. REV. CELL MOL. BIOL., vol. 330, 2017, pages 295 - 342
MARTIN AC ET AL.: "Modeling of Antibody Hypervariable Loops: a Combined Algorithm", PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 9268 - 9272, XP000165667, DOI: 10.1073/pnas.86.23.9268
OSTRND-ROSENBERG S ET AL.: "Novel Strategies for Inhibiting PD-1 Pathway-Mediated Immune Suppression while Simultaneous Delivering Activity Signals to Tumor-Reactive T Cells", CANCER IMMUNOL IMMUNOTHER, vol. 64, no. 10, October 2015 (2015-10-01), pages 1287 - 93
See also references of EP3909986A4
YAO SZHU YCHEN L.: "Advances in targeting cell surface signaling molecules for immune modulation", NAT REV DRUG DISCOV, vol. 12, no. 2, 2013, pages 130 - 146
YAZAKIWU: "Methods in Molecular Biology", vol. 248, 2003, HUMANA PRESS, pages: 255 - 268
YU DC ET AL.: "Soluble vascular endothelial growth factor decision receptor FP3 exerts potent antiangiogenic effects", MOL.THER., vol. 20, no. 3, 2012, pages 938 - 947

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2025508267A (ja) * 2022-03-04 2025-03-21 パノロス バイオサイエンス インコーポレイテッド ＶＥＧＦ－Ｇｒａｂ及びＰＤ－１又はＰＤ－Ｌ１拮抗剤を含む併用投与用組成物

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