WO2019062642A1 - Protéine de fusion à double ciblage ciblant pd-1 ou pd-l1 et ciblant la famille du vegf et son utilisation - Google Patents

Protéine de fusion à double ciblage ciblant pd-1 ou pd-l1 et ciblant la famille du vegf et son utilisation Download PDF

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WO2019062642A1
WO2019062642A1 PCT/CN2018/106741 CN2018106741W WO2019062642A1 WO 2019062642 A1 WO2019062642 A1 WO 2019062642A1 CN 2018106741 W CN2018106741 W CN 2018106741W WO 2019062642 A1 WO2019062642 A1 WO 2019062642A1
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antibody
fusion protein
seq
subunit
light chain
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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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北京比洋生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention generally relates to the field of medical biotechnology.
  • the present invention relates to targeting programmed death-1 (PD-1) or programmed death-1 ligand (PD-L1) and targeting blood vessels a dual targeting fusion protein of the Vascular Endothelial Cell Growth Factor (VEGF) family, a polynucleotide encoding the dual targeting fusion protein, a vector comprising the polynucleotide, comprising the Host cells of a polynucleotide or vector, and the use of the dual targeting fusion protein in treating, preventing and/or diagnosing a disease associated with PD-1 or PD-L1 activity and VEGF family activity in an individual.
  • VEGF Vascular Endothelial Cell Growth Factor
  • Immune checkpoint is a type of inhibitory signaling molecule present in the immune system that prevents tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and is involved in maintaining tolerance to autoantigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264).
  • PD-1 Programmed death protein-1
  • PD-1 is an important immunological checkpoint protein and is currently an important target for tumor immunotherapy. PD-1 was first discovered in 1992, and its gene cloning and expression indicated that PD-1 activation can induce programmed cell death of T cells. PD-1 protein was found on activated T cells, B cells and myeloid cells. PD-1 is also inducibly expressed in macrophages, dendritic cells, and monocytes. There is no PD-1 expression on the resting lymphocyte surface.
  • PD-1 is a 55kDa type I transmembrane protein with a cytoplasmic region containing an immunoreceptor tyrosine inhibitory motif and homology to CD28 and CTLA-4.
  • Two cell surface glycoprotein ligands of PD-1 have been identified, Programmed Death Protein Ligand 1 (PD-L1) and Programmed Death Protein Ligand 2 (PD-L2).
  • Ligand expression of PD-1 has been found on many cancer cells, including human lung cancer, ovarian cancer, colon cancer, and various myeloma.
  • PD-1 ligands are highly expressed on the surface of various epithelial cancers, hematological cancers, and other malignant tumor cells.
  • PD-1 ligands such as PD-L1
  • PD-L1 Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD -L1 blockade, PNAS, 2002, 99(19): 12293-7).
  • PD-1 functions to limit T cell activation, inhibit T cell proliferation, and increase tolerance to antigen.
  • Upregulation of PD-1 expression on activated lymphocytes can lead to inhibition of acquired or innate immune responses, resulting in tumor infiltrating lymphocytes (including T lymphocytes), although they have tumor antigen specificity, Since the ligand of PD-1 on tumor cells binds to PD-1 on tumor infiltrating lymphocytes to produce a signal that inhibits the activation of tumor infiltrating lymphocytes, tumor cells can escape the killing of tumor cells by the immune system.
  • antibodies that inhibit the binding of PD-1 to PD-1 mainly include anti-PD-1 monoclonal antibodies and anti-PD-L1 monoclonal antibodies, but also products for PD-L2.
  • the more mature anti-PD-1 antibodies are the Nivolumab of BMS, and the Pembrolizumab of Merck.
  • Nawu monoclonal antibody (trade name) ) is a fully humanized IgG4 antibody molecule
  • pemizumab (trade name) ) is a humanized IgG4 antibody molecule.
  • the anti-PD-1 monoclonal antibody binds to PD-1 on T lymphocytes and inhibits the binding of PD-1 to its ligands PD-L1 and PD-L2, thereby promoting T lymphocyte activation, proliferation and immunity.
  • Activated cytokines such as IL-2, and relieve the inhibition of PD-1 on immune surveillance of T lymphocytes with anti-tumor activity.
  • NAV monoclonal antibodies currently approved by the US Food and Drug Administration include: melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, etc.; indications for pemimab include: head and neck cancer, non- Small cell lung cancer, melanoma, etc.
  • atezolizumab developed by Roche avelumab developed by Merck KGaA and Pfizer, and durvalumab developed by AstraZeneca also showed therapeutic effects on tumors.
  • anti-PD-1 antibodies and anti-PD-L1 antibodies have therapeutic effects on tumors, their average therapeutic efficiency is only about 20%, and the five-year survival rate of lung cancer is only 16%. A significant proportion of cancer patients still have no response to treatment with anti-PD-1 antibodies and anti-PD-L1 antibodies. Therefore, how to improve the effectiveness of cancer treatment is still a difficult problem that needs to be solved in the field of cancer treatment.
  • tumor angiogenesis is also an important cause of rapid tumor growth (Ferrara N and Alitalo K, Clinical applications of angiogenic growth factors and their inhibitors, Nat Med., 1999; 5(12): 1359-64) .
  • Tumor angiogenesis is a fairly complex process that is regulated positively and negatively by multiple factors.
  • the vascular endothelial growth factor family is one of the most potent positive regulators, which plays a role in stimulating neovascularization.
  • Vascular endothelial growth factor and vascular endothelial growth inhibitory factor exist in normal tissues at the same time and remain relatively balanced. This balance allows human blood vessels to be normally formed and differentiated. However, during tumor growth, the number of VEGF family molecules increases sharply, and the imbalance between regulation and angiogenesis inhibitors, thereby greatly promoting the proliferation and migration of vascular endothelial cells, improving vascular permeability, and inhibiting tumors. Apoptosis provides a good microenvironment for tumor growth and metastasis.
  • the VEGF family contains six closely related polypeptides, each of which is a highly conserved homodimeric glycoprotein, 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
  • VEGF-A is associated with microvessel density in some solid tumors
  • the concentration of VEGF-A in tissues is associated with the prognosis of solid tumors such as breast, lung, prostate and colon cancers. .
  • each VEGF family member is mediated by one or more of the cell surface VEGF receptor (VEGFR) family, including VEGFR1 (also known as Flt-1), VEGFR2 (also known as KDR). Flk-1), VEGFR3 (also known as Flt-4), etc., wherein VEGFR1 and 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 Flk-1
  • VEGFR3 also known as Flt-4
  • the main biological functions of the VEGF family include: (1) selective promotion of mitosis of vascular endothelial cells, stimulation of endothelial cell proliferation and promotion of angiogenesis; (2) improvement of permeability of blood vessels, especially microvessels, and extravasation of 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 to secrete collagenase and vascular endothelial cells.
  • VEGF family can induce gaps in the epithelial cells and open Window phenomenon can activate cytoplasmic vesicles and organelles of epithelial cells; VEGF family directly stimulates endothelial cells to release proteolytic enzymes, degrades matrix, releases more VEGF family molecules, accelerates tumor development, and extracellular protease activates extracellular Matrix binding and release of the VEGF family; VEGF family makes plasma proteins (including fiber) by increasing vascular permeability Release of retinoic acid, forming a cellulose network, providing a good matrix for tumor growth, development and metastasis; (5) VEGF family inhibits the body's immune response and promotes invasion and metastasis of malignant tumors (Lapeyre-Prost A et al, Immunomodulatory Activity of VE
  • PLGF placental growth factor
  • PLGF can promote trophoblast proliferation and differentiation in early pregnancy, induce endothelial cell proliferation and migration, resist endothelial cell apoptosis, increase vascular permeability, enhance the biological activity of low concentration VEGF, and participate in various tumor blood vessels. An important pro-angiogenic factor produced. Excessive PLGF expression leads to an increase in tumor growth and survival of blood vessels. PLGF was observed to be expressed in all vascular-rich tumors in primary tumors, whereas tumors with few vascularities only partially expressed PLGF. Therefore, PLGF can be used to explain the mechanism of tumor neovascularization, and inhibition of tumor growth can be achieved by inhibiting the biological activity of PLGF.
  • Bevacizumab (trade name: Avastin), developed by Genentech, is a recombinant human-mouse chimeric anti-VEGF antibody that blocks VEGFR from inactivation by blocking the binding of VEGF-A to VEGFR. This exerts an anti-angiogenic effect.
  • Bevacizumab is currently used for first-line treatment of metastatic colorectal cancer, and may be used for the treatment of metastatic lung cancer, breast cancer, pancreatic cancer, and kidney cancer in the future.
  • Bevacizumab is also one of the more successful antibody drugs developed.
  • Aflibercept developed by Sanofi-aventis and Regeneron, is a VEGF-Trap that fuses the second extracellular domain of VEGFR1 with the extracellular domain of VEGFR2 and the human IgG1 constant region.
  • a fusion protein that exerts an anti-tumor effect on a subset of tumor patients by inhibiting angiogenesis.
  • such alternative therapies are capable of targeting both the immunosuppressive protein PD-1 or PD-L1 and the VEGF family of molecules having immunosuppressive and pro-angiogenic effects, resulting in activation of the immune system and tumor blood vessels. Regression, thereby showing efficacy in patients who do not respond to monotherapy targeting PD-1 or PD-L1 or who are not responding to a single treatment targeting the VEGF family.
  • One approach to this type of alternative therapy is to co-administer two different biological products that target PD-1 or PD-L1 and target VEGF family molecules.
  • Co-administration requires the injection of two separate products or a single injection of a combination of two different proteins.
  • the two injections allowed flexibility in the amount of administration and administration time, it caused inconvenience and pain to the patient.
  • the combination preparation may provide some flexibility in the amount of administration, it is often difficult to find a formulation condition that allows the chemical and physical stability of the two proteins in solution because the molecular characteristics of the two proteins are different.
  • co-administration and combination therapy of two different drugs may increase the additional cost to the patient and/or payer.
  • alternative therapies for treating tumors and preferably such alternative therapies comprise dual targeting fusion proteins that target PD-1 or PD-L1 and target the VEGF family.
  • the present invention provides a dual targeting novel fusion protein that targets PD-1 or PD-L1 and targets the VEGF family, which is capable of inhibiting activation of the PD-1 pathway or the PD-L1 pathway and the VEGF family signaling pathway, And for treating, preventing, and/or diagnosing diseases associated with PD-1, PD-L1 activity, and VEGF family activity in an individual.
  • the present invention discloses a novel dual targeting fusion protein targeting PD-1 or PD-L1 and targeting the VEGF family, a polynucleotide encoding the dual targeting fusion protein, and a vector comprising the polynucleotide Use of a host cell comprising the polynucleotide or vector, and the dual targeting fusion protein for treating, preventing and/or diagnosing a disease associated with PD-1, PD-L1 activity and VEGF family activity in an individual .
  • the invention provides a dual targeting fusion protein that targets PD-1 or PD-L1 and targets the VEGF family, which inhibits binding of PD-1 to its ligand or inhibits PD -L1 binds to its receptor and inhibits the signaling pathway of the VEGF family, comprising (i) an anti-PD-1 antibody or an anti-PD-L1 antibody; and (ii) with said anti-PD-1 antibody or anti-PD-
  • the L1 antibody is operably linked to at least two VEGF family familiy inhibiting domains (hereinafter abbreviated as VID).
  • the dual targeting fusion protein of the invention comprises (i) an anti-PD-1 antibody or an anti-PD-L1 antibody; and (ii) two of said anti-PD-1 antibodies or anti-PD-L1 antibodies a VID operably linked to the C-terminus of each heavy chain in the heavy chain, optionally, said (i) and said (ii) being operably linked by a peptide linker, whereby two identical or different VIDs are each Their N-terminal amino acids are fused to the C-terminal amino acid of one of the anti-PD-1 antibodies or one of the heavy chains of the anti-PD-L1 antibody, optionally via a peptide linker, preferably, the VID comprises a VEGF family Part of the extracellular domain of the body.
  • the anti-PD-1 antibody contained in the dual targeting fusion protein may be any anti-PD-1 antibody as long as it is capable of inhibiting or reducing the binding of PD-1 to its ligand, including those known in the art.
  • Anti-PD-1 antibody and anti-PD-1 antibody developed in the future.
  • the anti-PD-1 antibody comprises an antibody selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/ All heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequences of 16, 17/18, 19/20, 21/22, 23/24, and 120/121
  • the anti-PD-1 antibody comprises SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/16, Paired heavy chain variable region sequence/light chain variable region sequences of 17/18, 19/20, 21/22, 23/24, and 120/121, or with the paired heavy chain variable region sequences/ a light chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; more preferably,
  • the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region of an anti-PD-1 antibody selected
  • the anti-PD-L1 antibody contained in the dual targeting fusion protein may be any anti-PD-L1 antibody as long as it is capable of inhibiting or reducing PD-L1 binding to its receptor (for example, with PD-1 or CD80 (B7-1)
  • the antibodies may be combined with either, including anti-PD-L1 antibodies known in the art and anti-PD-L1 antibodies developed in the future.
  • the anti-PD-L1 antibody in the fusion protein of the invention comprises a pair of heavy chain variable region sequences/light chain variable regions selected from the group consisting of SEQ ID NOs: 25/26, 27/28, and 29/30 All heavy chain CDRs and light chain CDRs contained in the sequence.
  • the anti-PD-L1 antibody comprises a pair of heavy chain variable region sequence/light chain variable region sequences selected from the group consisting of SEQ ID NOs: 25/26, 27/28 and 29/30, or The heavy chain variable region sequence/light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity More preferably, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab and durvalumab.
  • the anti-PD-1 antibody or anti-PD-L1 antibody is an IgG class antibody, in particular an IgG 1 subclass, an IgG 2 subclass, an IgG 4 subclass antibody.
  • the anti-PD-1 antibody or anti-PD-L1 antibody comprised in the fusion protein of the invention is an IgG 4 subclass antibody, in particular a human IgG 4 subclass antibody.
  • the IgG 4 subclass antibody comprises an amino acid substitution at position S228 (EU numbering) in the Fc region, in particular amino acid substitution S228P.
  • the heavy chain constant region amino acid sequence of an exemplary IgG 1 subclass anti-PD-1 antibody is shown in SEQ ID NO:33.
  • the heavy chain constant region amino acid sequence of an exemplary IgG 2 subclass anti-PD-1 antibody is shown in SEQ ID NO:34.
  • the heavy chain constant region amino acid sequence of an exemplary IgG 4 subclass anti-PD-1 antibody is shown in SEQ ID NO:35.
  • the anti-PD-1 antibody or anti-PD-L1 antibody comprises a variable region and a constant region of a full antibody.
  • the antibody light chain constant region type in the dual targeting fusion protein of the present invention may be a kappa type or a lambda type, preferably a kappa type.
  • the kappa type light chain constant region amino acid sequence of an exemplary anti-PD-1 antibody is shown in SEQ ID NO:31.
  • the lambda-type light chain constant region amino acid sequence of an exemplary anti-PD-1 antibody is shown in SEQ ID NO:32.
  • the VID contained in the dual targeting fusion protein comprises a portion of the extracellular domain of the receptor of the VEGF family.
  • the VID comprises immunoglobulin (Ig)-like domain 2 (Domain 2, abbreviated as D2) of VEGFR1 and Ig-like domain 3 (Domain 3, abbreviated as D3) of VEGFR2.
  • the VEGFR1-D2/VEGFR2-D3 has the amino acid sequence set forth in SEQ ID NO: 63 or at least 90%, 91%, 92%, 93 of the amino acid sequence of SEQ ID NO: 63 Amino acid sequence of %, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the VID comprises VEGFR1-D2 and Ig-like domain 4 (Domain 4, abbreviated D4) of VEGFR2-D3 and VEGFR2.
  • the VEGFR1-D2/VEGFR2-D3-D4 has the amino acid sequence set forth in SEQ ID NO: 64 or at least 90%, 91%, 92% of the amino acid sequence of SEQ ID NO: 64 Amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the VID comprises VEGFR1-D2.
  • the VEGFR1-D2 has the amino acid sequence set forth in SEQ ID NO: 65 or at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 65 Amino acid sequence of 95%, 96%, 97%, 98%, 99% or more identity.
  • the peptide linker joining the VID at the C-terminus of the heavy chain of the anti-PD-1 antibody or anti-PD-L1 antibody comprises one or more amino acids, preferably comprising a SEQ ID NO: 36 -62 peptide linker.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 73 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 75, hereinafter referred to as For the fusion protein BY24.3.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 77 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 79, hereinafter referred to as For the fusion protein BY24.7.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 81 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 83, hereinafter referred to as For the fusion protein BY24.4.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 85 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 87, hereinafter referred to as For the fusion protein BY24.5.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 89 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 91, hereinafter referred to as For the fusion protein BY24.6.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 93 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 95, hereinafter referred to as For the fusion protein BY24.8.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 97 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 99, hereinafter referred to as For the fusion protein BY24.9.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 101 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 103, hereinafter referred to as For the fusion protein BY24.10.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 105 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 107, hereinafter referred to as For the fusion protein BY24.11.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 109 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 111, hereinafter referred to as For the fusion protein BY24.12.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 113 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 115, hereinafter referred to as For the fusion protein BY24.13.
  • the fusion protein comprises the anti-PD-1 antibody light chain subunit of SEQ ID NO: 117 and the anti-PD-1 antibody heavy chain-VID fusion subunit of SEQ ID NO: 119, hereinafter referred to as For the fusion protein BY24.14.
  • the fusion protein comprises (i) an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab, and durvalumab, and (ii) each of the two heavy chains of the anti-PD-L1 antibody A VID molecule operably linked to the C-terminus of the strand.
  • the fusion protein specifically targets PD-1 or PD-L1 and VEGF family molecules, inhibiting signaling mediated by PD-1 or PD-L1 and VEGF family molecules.
  • the fusion protein of the present invention binds not only PD-1 or PD-L1 with high affinity at the N-terminus, but also binds a plurality of VEGF factors with high affinity at the C-terminus.
  • the structure of the fusion protein designed by the invention fully ensures the suitable physical spatial distance of the fusion protein to the two types of targets, and the fusion protein of this structure is specific to a molecule of PD-1 or PD-L1 and VEGF family molecules. Sexual binding does not affect the specific binding of the fusion protein to another molecule in the PD-1 or PD-L1 and VEGF family molecules.
  • the invention further provides a polynucleotide encoding a fusion protein of the invention, a vector comprising a polynucleotide encoding a fusion protein of the invention, preferably an expression vector, most preferably a glutamine synthetase expression vector having a double expression cassette.
  • the invention provides a host cell comprising a polynucleotide or vector of the invention.
  • the invention also provides a method for producing a fusion protein of the invention comprising the steps of (i) cultivating a host cell of the invention under conditions suitable for expression of a fusion protein of the invention, and (ii) recovering the fusion protein of the invention .
  • the invention provides a diagnostic kit and pharmaceutical composition comprising a fusion protein of the invention. Further, there is provided the use of a fusion protein, diagnostic kit or pharmaceutical composition of the invention for the treatment, prevention and/or diagnosis of a disease associated with PD-1 or PD-L1 activity and VEGF family activity, in particular
  • cancerous diseases eg solid tumors and soft tissue tumors
  • melanoma breast cancer, colon cancer, esophageal cancer, gastrointestinal tract Interstitial tumors (GIST), renal 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).
  • Figure 1 Schematic diagram showing the structure of a dual targeting fusion protein of the present invention targeting PD-1 or PD-L1 and targeting the VEGF family.
  • Figure 2 shows the results of the fusion protein of the present invention prepared and purified in Example 2 by SDS-PAGE and staining with Coomassie blue in the presence of a reducing agent (5 mM 1,4-dithiothreitol).
  • Lane 1 in Figure 2A Protein molecular weight standard marker
  • Lane 2 Fusion protein BY24.3
  • Lane 3 Fusion protein BY24.4
  • Lane 4 Fusion protein BY24.5
  • Lane 5 Fusion protein BY24.6
  • Lane 6 fusion protein BY24.8
  • lane 7 fusion protein BY24.9
  • lane 8 fusion protein BY24.10
  • lane 9 fusion protein BY24.11
  • lane 1 in Figure 2B protein molecular weight standard marker
  • lane 2 : fusion protein BY24.12
  • lane 3 fusion protein BY24.13
  • lane 4 fusion protein BY24.14
  • lane 5 antibody BY18.1
  • lane 6 protein 301-8
  • lane 7 fusion protein BY24.7.
  • Figure 3 shows the effect of the fusion protein BY24.3, antibody BY18.1 and protein 301-8 of the present invention on the body weight of experimental animals.
  • Figure 4 A schematic diagram showing the in vivo antitumor effect of the fusion protein BY24.3 of the present invention and the antibodies BY18.1 and 301-8.
  • the present invention provides fusion proteins and pharmaceutical compositions that block the PD-1 pathway or the PD-L1 pathway and the VEGF family signaling pathway at the immune checkpoint.
  • the invention also provides methods for producing the fusion protein, and the use of the fusion protein in treating, preventing, and/or diagnosing a disease associated with PD-1 or PD-L1 activity and VEGF family activity in an individual.
  • PD-1 pathway refers to any intracellular signaling pathway initiated by binding to PD-1, including but not limited to intracellular signaling pathways triggered by PD-1 binding to PD-L1, or PD-1 and The intracellular signaling pathway triggered by PD-L2 binding, or the intracellular signaling pathway triggered by the binding of PD-1 to both PD-L1 and PD-L2.
  • PD-L1 pathway refers to any intracellular signaling pathway initiated by binding to PD-L1, including but not limited to, intracellular signaling pathways triggered by PD-L1 binding to PD-1, or PD-L1 and Intracellular signaling pathway triggered by binding of CD80 (B7-1), or intracellular signaling pathway triggered by binding of PD-L1 to both PD-1 and CD80 (B7-1).
  • the term "specifically binds” means selective for binding of an antigen or molecule of interest and may be distinguished from unwanted or non-specific interactions.
  • the specific binding can be by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Analysis of agalacto- IgG in rheumatoid arthritis using surface plasmon resonance, Glyco J., 2000, 17, 323-329).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • affinity molecule X for its partner Y can generally dissociation constant (K D) is represented by the solution, the dissociation constant is the ratio of the dissociation rate constant and association rate constant (k off, respectively, and k on) of.
  • K D dissociation constant
  • association rate constant k off, respectively, and k on
  • Affinity can be measured by common methods known in the art.
  • One specific method for measuring affinity is surface plasmon resonance (SPR).
  • antibody is used herein in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), so long as they exhibit the desired antigen binding activity.
  • the antibody may be an intact antibody molecule or a functional fragment of an intact antibody molecule including, but not limited to, for example, Fab, F(ab') 2 .
  • the constant region of an antibody can be altered (eg, mutated) to modify antibody properties (eg, to increase or decrease one or more of the following properties: antibody glycosylation, number of cysteine residues, effector cell function, or complement function) .
  • full antibody full length antibody
  • complete antibody complete antibody
  • intact antibody refers to an antibody having a structure that is substantially similar in structure to the native antibody.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in an antibody molecule, which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the lesser of the two types of polypeptide chains present in an antibody molecule.
  • the kappa light chain and the lambda light chain refer to two major antibody light chain isoforms.
  • the “percent identity (%)" of the amino acid sequence means that the candidate sequence is aligned with the specific amino acid sequence shown in the present specification and, if necessary, the vacancy is introduced to achieve the maximum percent sequence identity, and no consideration is given.
  • a “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein that promotes secretion of the protein out of the cell.
  • the mature form of the extracellular protein has no signal sequence that is cleaved during the secretory process.
  • N-terminus refers to the last amino acid at the N-terminus
  • C-terminus refers to the last amino acid at the C-terminus
  • fusion refers to the direct attachment of two or more components by peptide bonds or by one or more peptide linkers.
  • fusion protein refers to a fusion polypeptide molecule comprising an antibody light chain subunit and an antibody heavy chain-VID fusion subunit, wherein the antibody light chain subunit is the smaller of the polypeptide chains present in the fusion protein, The antibody heavy chain-VID fusion subunit is the larger of the polypeptide chains present in the fusion protein.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom.
  • a host cell is any type of cellular system that can be used to produce a fusion protein of the invention.
  • Host cells include cultured cells, as well as transgenic animals, transgenic plants, or cultured plant tissues or cells within animal tissues.
  • mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and large mouse.
  • rodents eg, mice and large mouse.
  • the individual is a human.
  • treatment refers to the clinical intervention intended to alter the natural course of the disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis.
  • the fusion proteins of the invention are used to delay disease progression or to slow the progression of the disease.
  • anti-tumor effect refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid tumors and liquid tumors.
  • the present invention provides a dual targeting fusion protein that targets PD-1 or PD-L1 and targets the VEGF family, comprising (i) an anti-PD-1 antibody or an anti-PD-L1 antibody; and (ii) and the anti-antibody
  • the PD-1 antibody or the anti-PD-L1 antibody is operably linked to at least two VIDs, wherein the two components of the fusion protein are linked to each other directly or via a peptide linker.
  • the individual peptide chains of the component (i) anti-PD-1 antibody or anti-PD-L1 antibody in the fusion protein may be linked, for example, by a disulfide bond.
  • a fusion protein of the invention is a heterotetrameric glycoprotein consisting of two disulfide-bonded two antibody light chain subunits and two antibody heavy chain-VID fusion subunits. From the N-terminus to the C-terminus, each antibody heavy chain-VID fusion subunit has an antibody heavy chain followed by a VID wherein the antibody heavy chain and VID are linked directly by peptide bonds or by one or more peptide linkers.
  • the fusion proteins of the invention block the PD-1 pathway or the PD-L1 pathway at the immunological checkpoint and inhibit the VEGF family signaling pathway.
  • the immunological checkpoint PD-1 pathway blocked by this fusion protein is the signaling pathway mediated by PD-1 binding to its ligand.
  • the PD-L1 pathway blocked by this fusion protein is a signaling pathway mediated by the binding of PD-L1 to its receptor.
  • the fusion protein inhibits the VEGF family signaling pathway by signaling mediated by the binding of VEGF-A, -B, -C, -D, -E, and PLGF to receptors of the VEGF family (eg, VEGFR1, VEGFR2, and VEGFR3) way.
  • the fusion protein of the invention binds to PD-1 or PD-L1 at a dissociation constant (K D ) of 10 -8 M or less, for example, 10 -9 M to 10 -12 M;
  • the VEGF family specifically binds with a dissociation constant (K D ) of 10 -8 M or less, for example, 10 -9 M to 10 -12 M.
  • the anti-PD-1 antibody or anti-PD-L1 antibody contained in the fusion protein of the present invention is a heterotetrameric glycoprotein composed of two light-chain-bonded two light chains and two heavy chains.
  • the heavy chain of each anti-PD-1 antibody or anti-PD-L1 antibody has a variable region (VH), also referred to as a variable heavy chain domain or heavy chain.
  • the variable domains are followed by three constant domains (CH1, CH2 and CH3), also referred to as heavy chain constant regions.
  • the light chain of each anti-PD-1 antibody or anti-PD-L1 antibody has a variable region (VL), also known as a variable light chain domain or a light chain variable domain.
  • VL variable light chain domain
  • CL constant light chain
  • the anti-PD-1 antibody or anti-PD-L1 antibody consists essentially of two Fab molecules and one Fc domain joined by a hinge region of an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody contained in the fusion protein of the present invention can have a high affinity, for example, a K D of 10 -8 M or less, preferably 10 -9 M to 10 -12 M, Binding to PD-1 or PD-L1, respectively, and thereby blocking the signaling pathway mediated by PD-1 binding to ligand PD-L1/PD-L2 or blocking PD-L1 and receptor PD- 1/CD80 (B7-1) binds to the mediated signaling pathway.
  • Examples of the paired heavy chain variable region (VH) and light chain variable region (VL) of the anti-PD-1 antibody contained in the fusion protein of the present invention are provided herein below in Table 1A.
  • examples of the paired heavy chain variable region (VH) and light chain variable region (VL) of the anti-PD-L1 antibody contained in the fusion protein of the present invention are provided herein below in Table 1B.
  • the anti-PD-1 antibody or anti-PD-L1 antibody in the fusion protein of the invention comprises a sequence substantially identical to the amino acid sequence set forth in Table 1A or Table 1B, respectively, for example, with Table 1A or Table
  • the paired heavy chain variable region sequence/light chain variable region sequence shown in 1B has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or more sequences of sequence identity.
  • Table 1A Examples of heavy chain variable region and light chain variable region sequences of anti-PD-1 antibodies in fusion proteins
  • the anti-PD-1 antibody in the fusion protein of the invention comprises an antibody selected from the group consisting of SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/ All heavy chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequences of 15, 15/16, 17/18, 19/20, 21/22, 23/24, and 120/121 With light chain CDRs.
  • the anti-PD-L1 antibody in the fusion protein of the invention comprises a pair of heavy chain variable region sequences/light chain variable regions selected from the group consisting of SEQ ID NOs: 25/26, 27/28, and 29/30 All heavy chain CDRs and light chain CDRs contained in the sequence.
  • 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 particular heavy chain variable regions and/or light chains disclosed herein.
  • the CDRs in the amino acid sequence of the variable region are 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 of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins., J. Mol. Biol. 273:927 -948 (1997); and Martin AC et al, Modeling antibody hypervariable loops: a combined algorithm, Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).
  • the anti-PD-1 antibody or the anti-PD-L1 antibody in the fusion protein of the present invention may be classified into a kappa type or a lambda type based on the amino acid sequence of the light chain constant region thereof, and is preferably a kappa type.
  • amino acid sequence of the anti-PD-1 antibody light chain constant region in the fusion protein of the invention is provided herein below in Table 2.
  • the amino acid sequence of the anti-PD-1 antibody or the anti-PD-L1 antibody in the fusion protein of the present invention based on the heavy chain constant region thereof is preferably an IgG class antibody, particularly an IgG 1 subclass, an IgG 2 subclass, an IgG 4 subclass.
  • Antibodies, more particularly IgG 4 subclass antibodies are particularly IgG 4 subclass antibodies.
  • the IgG 4 subclass anti-PD-1 antibody or anti-PD-L1 antibody comprises an amino acid substitution preventing the occurrence of arm-exchange at the S228 position in the Fc region, in particular amino acid substitution S228P.
  • amino acid sequences of the heavy chain constant regions of the anti-PD-1 antibodies in the fusion proteins of the invention are provided in Table 3 below.
  • VID VEGF family
  • the "inhibiting VEGF family domain (VID)" in a fusion protein of the invention comprises a portion of the extracellular domain of VEGFR.
  • the VEGFR receptor is a tyrosine kinase receptor located on the cell surface, and its extracellular domain is composed of seven immunoglobulin (Ig)-like domains.
  • human VEGFR1 comprises seven Ig-like domains numbered 1, 2, 3, 4, 5, 6, and 7, with Ig-like domain 1 at the N-terminus of the extracellular domain and Ig-like domain 7 outside the extracellular domain. The C end 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 VID comprises at least one Ig-like domain of one or more VEGFRs selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3. In some aspects, the VID comprises at least 1, 2, 3, 4, 5, 6, but no more than 7 Ig-like domains of the VEGFR. In another aspect, the VID 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.
  • VIDs comprising at least one Ig-like domain of two or more VEGFRs are also contemplated herein.
  • the VID comprises at least one Ig-like domain from two or more VEGFRs selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3.
  • the VID of any combination of seven Ig-like domains comprising each VEGFR is contemplated herein.
  • a VID can comprise an Ig-like domain 2 of VEGFRl (eg, human VEGFRl) and an Ig-like domain 3 of VEGFR2 (eg, human VEGFR2).
  • the VID may comprise Ig-like domains 1-3 of VEGFR1 (eg, human VEGFR1), Ig-like domains 2-3 of VEGFR1 (eg, human VEGFR1), Ig-like structures of VEGFR2 (eg, human VEGFR2) Domains 1-3, Ig-like domain 2 of VEGFR1 (eg, human VEGFR1) and Ig-like domain 3-4 of VEGFR2 (eg, human VEGFR2), or Ig-like domain 2 and VEGFR3 of VEGFR1 (eg, human VEGFR1) (eg, Ig-like domain 3 of human VEGFR3).
  • VEGFR1 eg, human VEGFR1
  • Ig-like domains 2-3 of VEGFR1 eg, human VEGFR1
  • Ig-like structures of VEGFR2 eg, human VEGFR2
  • Domains 1-3 Ig-like domain 2 of VEGFR1 (eg, human VEGFR1) and Ig-like domain 3-4 of
  • Ig-like domains and other Ig-like domains that can be used as part of the VID can be found in U.S. Patent No. 5,751,173; Yu DC et al., 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 of which are hereby incorporated by reference in their entirety. reference.
  • the VID has any one of the amino acid sequences selected from SEQ ID NOs: 63-65 in Table 4 or at least 90%, 91%, 92 from the amino acid sequence set forth in SEQ ID NOs: 63-65. Amino acid sequence of %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the VID contained in the fusion protein of the present invention is capable of specifically binding to the VEGF family with high affinity, for example, a K D of 10 -8 M or less, preferably 10 -9 M to 10 -12 M, and thereby Binding of the VEGF family to cell surface VEGFR and subsequent signaling are inhibited.
  • the "peptide linker” optionally contained in the heavy chain C-terminus and the VID N-terminus of the anti-PD-1 antibody or anti-PD-L1 antibody in the fusion protein of the present invention is a peptide of one or more 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 comprising selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 36); AKTTPKLEEGEFSEARV (SEQ ID NO: 37); AKTTPKLGG (SEQ ID NO: 38); SAKTTPKLGG ( SEQ ID NO: 39); SAKTTP (SEQ ID NO: 40); RADAAP (SEQ ID NO: 41); RADAAPTVS (SEQ ID NO: 42); RADAAAAGGPGS (SEQ ID NO: 43); RADAAAA (SEQ ID NO: 44) SAKTTPKLEEGEFSEARV (SEQ ID NO: 45); ADAAP (SEQ ID NO: 46); DAAPTVSIFPP (SEQ ID NO: 47); TVAAP (SEQ ID NO: 48); TVAAPSVFIFPP (SEQ ID NO: 49); QPKAAP (SEQ) ID NO: 50); QPKAAPSVTLFPP (SEQ ID NO: 51); AKTTPP (SEQ ID NO: 36
  • the dual targeting fusion proteins of the invention can be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production.
  • a polynucleotide encoding an antibody light chain subunit of the dual targeting fusion protein and/or a polynucleotide encoding an antibody heavy chain-VID fusion subunit of the dual targeting fusion protein is isolated and inserted
  • One or more vectors for further cloning and/or expression in a host cell can be easily isolated and sequenced using conventional methods.
  • a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.
  • Expression vectors can be constructed using methods well known to those of skill 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 expression vector having a dual expression cassette is used.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques.
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising an expression vector of the invention.
  • the term "host cell” refers to any type of cellular system that can be engineered to produce a dual targeting fusion protein of the invention.
  • Host cells suitable for replicating and supporting the expression of the dual targeting fusion proteins of the invention are well known in the art. Such cells can be transfected or transduced with a particular expression vector, as desired, and a large number of cells containing the vector can be grown for inoculating large scale fermenters to obtain a sufficient amount of the dual targeting fusion protein of the invention for clinical use.
  • 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. Mammalian cell lines 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 (293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), Africa Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2) ), CHO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2/0.
  • SV40 transformed monkey kidney CV1 line COS-7
  • human embryonic kidney line (293 or 293F cells baby hamster kidney cells
  • BHK monkey kidney cells
  • CV1 African Green monkey kidney cells
  • HELA human cervical cancer cells
  • MDCK buffalo rat liver cells
  • W138 human liver cells
  • Hep G2 human liver cells
  • myeloma cell lines such as YO, NS0, P3X63, and Sp2/
  • a method of producing a dual targeting fusion protein of the invention comprising culturing a host cell as provided herein under conditions suitable for expression of the dual targeting fusion protein,
  • the host cell comprises a polynucleotide encoding the dual targeting fusion protein and the dual targeting fusion protein is recovered from a host cell (or host cell culture medium).
  • the dual targeting fusion protein prepared as described herein can be purified by known prior art 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, hydrophilicity, and the like, and these will be apparent to those skilled in the art.
  • the purity of the dual targeting fusion proteins of the invention can be determined by any of a variety of well known analytical methods, including gel electrophoresis, high performance liquid chromatography, and the like.
  • the physical/chemical properties and/or biological activities of the dual targeting fusion proteins provided herein can be identified, screened or characterized by a variety of assays known in the art.
  • compositions for example, pharmaceutical compositions comprising a dual targeting fusion protein as described herein formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).
  • compositions of the invention may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions for example, injectable solutions and infusible solutions
  • dispersions or suspensions for example, liposomes, and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • a common preferred composition is in the form of an injectable solution or an infusible solution.
  • a preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection.
  • the dual targeting fusion protein is administered by intravenous infusion or injection.
  • the dual targeting fusion protein is administered by intramuscular, intraperitoneal or subcutaneous injection.
  • parenteral administration and “parenteral administration” as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.
  • compositions should generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, dispersions, liposomes or lyophilized forms.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the dual-targeted fusion protein) in the required amount in a suitable solvent, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle containing base dispersion medium and other ingredients.
  • a coating agent such as lecithin or the like can be used.
  • the proper fluidity of the solution can be maintained by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the compositions of the compositions which delay the absorption, such as the monostearate and gelatin.
  • the dual targeting fusion proteins of the invention can be administered orally, for example, orally with an inert diluent or an edible carrier.
  • the dual targeting fusion proteins of the invention may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets or incorporated directly into the subject's diet.
  • the compound can be incorporated with excipients and in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, glutinous rice papers It is used in the form of a wafer or the like.
  • compositions can also be administered using medical devices known in the art.
  • compositions of the invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of a dual targeting fusion protein of the invention.
  • “Therapeutically effective amount” means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required.
  • the therapeutically effective amount can vary depending on various factors such as the disease state, the age, sex, and weight of the individual.
  • a therapeutically effective amount is any amount that is toxic or detrimental to a therapeutically beneficial effect.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%.
  • the ability of the dual targeting fusion proteins of the invention to inhibit measurable parameters e.g., tumor volume
  • prophylactically effective amount is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose is administered to the subject prior to the earlier stage of the disease or at an earlier stage of the disease.
  • Kits comprising the dual targeting fusion proteins described herein are also within the scope of the invention.
  • the kit may contain one or more additional elements including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
  • the dual targeting fusion proteins disclosed herein have diagnostic and therapeutic and prophylactic uses in vitro and in vivo.
  • these molecules can be administered to cultured cells in vitro or ex vivo or to a subject, eg, a human subject, to treat, prevent, and/or diagnose a variety of PD-1 activity, PD-L1 activity, and A disease associated with VEGF family activity, such as cancer.
  • the invention provides for the detection of a PD-1 or PD-L1 and VEGF family molecule in a biological sample, such as a serum, semen or urine or a tissue biopsy sample (eg, from a hyperproliferative or cancerous lesion) in vitro or in vivo.
  • Diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with a dual targeting fusion protein as described herein or administering the dual targeting to a subject under conditions that allow interaction to occur The fusion protein and (ii) detect the formation of a complex between the dual targeting fusion protein and the sample (and optionally, a control sample). Formation of the complex indicates the presence of PD-1 or PD-L1 and VEGF family molecules, and may indicate the suitability or need for the treatment and/or prevention described herein.
  • the PD-1 or PD-L1 and VEGF family molecules are detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval.
  • Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR-technology (eg, RT-PCR) or in vivo imaging techniques.
  • dual targeting fusion proteins used in in vivo and in vitro assays are labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound conjugates.
  • Suitable detectable materials include a variety of biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (eg, nuclear magnetic resonance) materials, and radioactive materials.
  • the level and/or distribution of PD-1 or PD-L1 and VEGF family molecules is determined in vivo, eg, in a non-invasive manner (eg, by using suitable imaging techniques (eg, positron emission tomography) Detection (PET) scan) detection of a detectable marker of a dual targeting fusion protein of the invention.
  • PET positron emission tomography
  • PET Detection
  • a detectable marker of a dual targeting fusion protein of the invention for example, by detecting a PET reagent (eg, 18 F-fluorodeoxyglucose (FDG)) in a detectable manner
  • FDG 18 F-fluorodeoxyglucose
  • the invention provides a diagnostic kit comprising the dual targeting fusion protein described herein and instructions for use.
  • the invention relates to the use of a dual targeting fusion protein for the treatment or prevention of a disease in need of enhancing an immune response and reducing angiogenesis in a subject, thereby inhibiting or reducing the growth or appearance of a related disease such as a cancerous tumor. , metastasis or recurrence.
  • a dual targeting fusion protein can be used alone to inhibit or prevent the growth of cancerous tumors.
  • the dual targeting fusion protein can be administered in combination with other cancer therapeutic/preventive agents.
  • the dual targeting fusion protein of the invention is administered in combination with one or more other drugs, the combination can be administered in any order or simultaneously.
  • the invention provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of a dual targeting fusion protein described herein.
  • the 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 a dual targeting fusion protein described herein.
  • cancers treated and/or prevented with a dual targeting fusion protein include, but are not limited to, solid tumors, hematological cancer (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma), and metastatic Lesion.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems, such as invasive lungs, breasts, ovaries, lymphoid, gastrointestinal (eg, colon), anal, genital, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (eg, brain, nerve or glial cells), head and neck, skin (eg, melanoma), nasopharynx (eg, 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 bowel cancer, and esophageal cancer. Cancer can be in early, intermediate or advanced stages or metastatic cancer.
  • the cancer is selected from the group consisting of 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 cancer, stomach cancer, hematological malignancies (eg, lymphoma).
  • GIST gastrointestinal stromal tumor
  • renal cancer eg, renal cell carcinoma
  • liver cancer eg, non-small cell lung cancer (NSCLC)
  • ovarian cancer pancreatic cancer
  • prostate cancer head and neck cancer
  • stomach cancer hematological malignancies
  • Example 1 Construction of a high-efficiency expression vector for glutamine synthetase containing a gene of interest
  • nucleotide sequences suitable for expression in Chinese hamster ovarian cancer cells were optimized and commissioned by Shanghai Jierui Biotechnology Co., Ltd. Engineering Co., Ltd. synthesized the nucleotide sequence.
  • the anti-PD-1 antibody produced after expression of the nucleotide sequence is referred to herein as antibody BY18.1.
  • the light chain (BY18.1L) nucleotide sequence of the anti-PD-1 antibody BY18.1 is shown in SEQ ID NO: 66; the light chain (BY18.1L) amino acid sequence of the anti-PD-1 antibody BY18.1 is SEQ ID NO: 67.
  • the heavy chain (BY18.1H) nucleotide sequence of the anti-PD-1 antibody BY18.1 is shown in SEQ ID NO: 68; the heavy chain (BY18.1H) amino acid sequence of the anti-PD-1 antibody BY18.1 is SEQ ID NO: 69.
  • the BY18.1L coding nucleotide was digested with XhoI-EcoRI, and the glutamine synthetase high expression vector with double expression cassette (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.) was used for XhoI.
  • .1L coding nucleotides of glutamine synthetase high expression vector with double expression cassette were digested with XbaI-SalI, and then ligated into the BY18.1H coding nucleotide double-digested by XbaI-SalI
  • the BY18.1L coding nucleotide can also be ligated into a glutamine synthetase high expression vector having a double expression cassette into which a BY18.1H coding nucleotide has been introduced, and the antibody BY18.1 is expressed and obtained.
  • the nucleotide sequence encoding the protein 301-8 is shown in SEQ ID NO: 70; the amino acid sequence of protein 301-8 is shown in SEQ ID NO: 71.
  • the nucleotide sequence encoding the protein 301-8 was digested with XhoI-EcoRI, and the glutamine synthetase high expression vector with double expression cassette was obtained (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.
  • the double-digested XhoI-EcoRI was used to ligate the XhoI-EcoRI double-digested protein 301-8-encoding nucleotide to the XhoI-EcoRI double-digested glutamine synthetase with double expression cassette.
  • the vector was efficiently expressed, and a glutamine synthetase high-efficiency expression vector having a double expression cassette into which the nucleotide sequence encoding the protein 301-8 was introduced was obtained. After being verified by sequencing, it was used for expression, and the expressed protein was named as protein 301-8.
  • the amino acid sequence of protein 301-8 is identical to the amino acid sequence of abecept disclosed in the prior art.
  • the heavy chain variable region and light chain variable region sequences of the anti-PD-1 antibody in Table 1A the light chain constant region sequence of the antibody in Table 2, the heavy chain constant region sequence of the antibody in Table 3, and the VID in Table 4
  • the sequence, and the peptide linker sequence of SEQ ID NO: 36-62 were optimized to be suitable for nucleotide sequences expressed in Chinese hamster ovarian cancer cells (CHO), and Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize SEQ ID NO: The polynucleotide sequence shown by the even number in 72-118.
  • the light chain subunit (BY24.3L) nucleotide sequence of the fusion protein BY24.3 ( ⁇ , IgG4) is shown in SEQ ID NO: 72; the light chain subunit of the fusion protein BY24.3 ( ⁇ , IgG4) (BY24) .3L)
  • the amino acid sequence is set forth in SEQ ID NO:73.
  • the heavy chain-VID fusion subunit (BY24.3H) nucleotide sequence of the fusion protein BY24.3 ( ⁇ , IgG4) is shown in SEQ ID NO: 74; the heavy chain of the fusion protein BY24.3 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.3H) amino acid sequence is set forth in SEQ ID NO:75.
  • the light chain subunit (BY24.7L) nucleotide sequence of the fusion protein BY24.7 ( ⁇ , IgG2) is shown in SEQ ID NO: 76; the light chain subunit of the fusion protein BY24.7 ( ⁇ , IgG2) (BY24) .7L)
  • the amino acid sequence is set forth in SEQ ID NO:77.
  • the heavy chain-VID fusion subunit (BY24.7H) nucleotide sequence of the fusion protein BY24.7 ( ⁇ , IgG2) is shown in SEQ ID NO: 78; the heavy chain of the fusion protein BY24.7 ( ⁇ , IgG2)
  • the VID fusion subunit (BY24.7H) amino acid sequence is set forth in SEQ ID NO:79.
  • the light chain subunit (BY24.4L) nucleotide sequence of the fusion protein BY24.4 ( ⁇ , IgG4) is shown in SEQ ID NO: 80; the light chain subunit of the fusion protein BY24.4 ( ⁇ , IgG4) (BY24) .4L)
  • the amino acid sequence is set forth in SEQ ID NO:81.
  • the heavy chain-VID fusion subunit (BY24.4H) nucleotide sequence of the fusion protein BY24.4 ( ⁇ , IgG4) is shown in SEQ ID NO: 82; the heavy chain of the fusion protein BY24.4 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.4H) amino acid sequence is set forth in SEQ ID NO:83.
  • the light chain subunit (BY24.5L) nucleotide sequence of the fusion protein BY24.5 ( ⁇ , IgG4) is shown in SEQ ID NO: 84; the light chain subunit of the fusion protein BY24.5 ( ⁇ , IgG4) (BY24) .5L)
  • the amino acid sequence is set forth in SEQ ID NO:85.
  • the heavy chain-VID fusion subunit (BY24.5H) nucleotide sequence of the fusion protein BY24.5 ( ⁇ , IgG4) is shown in SEQ ID NO: 86; the heavy chain of the fusion protein BY24.5 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.5H) amino acid sequence is set forth in SEQ ID NO:87.
  • the light chain subunit (BY24.6L) nucleotide sequence of the fusion protein BY24.6 ( ⁇ , IgG2) is shown in SEQ ID NO: 88; the light chain subunit of the fusion protein BY24.6 ( ⁇ , IgG2) (BY24) .6L)
  • the amino acid sequence is set forth in SEQ ID NO:89.
  • the heavy chain-VID fusion subunit (BY24.6H) nucleotide sequence of the fusion protein BY24.6 ( ⁇ , IgG2) is shown in SEQ ID NO: 90; the heavy chain of the fusion protein BY24.6 ( ⁇ , IgG2)
  • the VID fusion subunit (BY24.6H) amino acid sequence is set forth in SEQ ID NO:91.
  • the light chain subunit (BY24.8L) nucleotide sequence of the fusion protein BY24.8 ( ⁇ , IgG4) is shown in SEQ ID NO: 92; the light chain subunit of the fusion protein BY24.8 ( ⁇ , IgG4) (BY24) .8L)
  • the amino acid sequence is set forth in SEQ ID NO:93.
  • the heavy chain-VID fusion subunit (BY24.8H) nucleotide sequence of the fusion protein BY24.8 ( ⁇ , IgG4) is shown in SEQ ID NO: 94; the heavy chain of the fusion protein BY24.8 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.8H) amino acid sequence is set forth in SEQ ID NO:95.
  • the light chain subunit (BY24.9L) nucleotide sequence of the fusion protein BY24.9 ( ⁇ , IgG4) is shown in SEQ ID NO: 96; the light chain subunit of the fusion protein BY24.9 ( ⁇ , IgG4) (BY24) .9L)
  • the amino acid sequence is set forth in SEQ ID NO:97.
  • the heavy chain-VID fusion subunit (BY24.9H) nucleotide sequence of the fusion protein BY24.9 ( ⁇ , IgG4) is shown in SEQ ID NO: 98; the heavy chain of the fusion protein BY24.9 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.9H) amino acid sequence is set forth in SEQ ID NO:99.
  • the light chain subunit (BY24.10L) nucleotide sequence of the fusion protein BY24.10 ( ⁇ , IgG4) is shown in SEQ ID NO: 100; the light chain subunit of the fusion protein BY24.10 ( ⁇ , IgG4) (BY24) .10L)
  • the amino acid sequence is set forth in SEQ ID NO:101.
  • the heavy chain-VID fusion subunit (BY24.10H) nucleotide sequence of the fusion protein BY24.10 ( ⁇ , IgG4) is shown in SEQ ID NO: 102; the heavy chain of the fusion protein BY24.10 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.10H) amino acid sequence is set forth in SEQ ID NO:103.
  • the light chain subunit (BY24.11L) nucleotide sequence of the fusion protein BY24.11 ( ⁇ , IgG4) is shown in SEQ ID NO: 104; the light chain subunit of the fusion protein BY24.11 ( ⁇ , IgG4) (BY24)
  • the .11L) amino acid sequence is set forth in SEQ ID NO:105.
  • the heavy chain-VID fusion subunit (BY24.11H) nucleotide sequence of the fusion protein BY24.11 ( ⁇ , IgG4) is represented by SEQ ID NO: 106; the heavy chain of the fusion protein BY24.11 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.11H) amino acid sequence is set forth in SEQ ID NO:107.
  • the light chain subunit (BY24.12L) nucleotide sequence of the fusion protein BY24.12 ( ⁇ , IgG4) is shown in SEQ ID NO: 108; the light chain subunit of the fusion protein BY24.12 ( ⁇ , IgG4) (BY24)
  • the .12L) amino acid sequence is set forth in SEQ ID NO:109.
  • the heavy chain-VID fusion subunit (BY24.12H) nucleotide sequence of the fusion protein BY24.12 ( ⁇ , IgG4) is shown in SEQ ID NO: 110; the heavy chain of the fusion protein BY24.12 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.12H) amino acid sequence is set forth in SEQ ID NO:111.
  • the nucleotide sequence of the light chain subunit (BY24.13L) of the fusion protein BY24.13 ( ⁇ , IgG4) is shown in SEQ ID NO: 112; the light chain subunit of the fusion protein BY24.13 ( ⁇ , IgG4) (BY24) .13L)
  • the amino acid sequence is set forth in SEQ ID NO:113.
  • the heavy chain-VID fusion subunit (BY24.13H) nucleotide sequence of the fusion protein BY24.13 ( ⁇ , IgG4) is shown in SEQ ID NO: 114; the heavy chain of the fusion protein BY24.13 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.13H) amino acid sequence is set forth in SEQ ID NO:115.
  • the light chain subunit (BY24.14L) nucleotide sequence of the fusion protein BY24.14 ( ⁇ , IgG4) is shown in SEQ ID NO: 116; the light chain subunit of the fusion protein BY24.14 ( ⁇ , IgG4) (BY24)
  • the .14L) amino acid sequence is set forth in SEQ ID NO:117.
  • the heavy chain-VID fusion subunit (BY24.14H) nucleotide sequence of the fusion protein BY24.14 ( ⁇ , IgG4) is shown in SEQ ID NO: 118; the heavy chain of the fusion protein BY24.14 ( ⁇ , IgG4)
  • the VID fusion subunit (BY24.14H) amino acid sequence is set forth in SEQ ID NO:119.
  • BY24.3L, BY24.4L, LBY24.5L, BY24.6L, BY24.7L, BY24.8L, BY24.9L, BY24 were respectively digested by XhoI-EcoRI.
  • coding nucleotides are ligated to a high expression vector for glutamine synthetase with a double expression cassette (patent authorization number: CN104195173B, obtained from Beijing Biyang Bio Technology Co., Ltd.); BY24.3H, BY24.4H, LBY24.5H, BY24.6H, BY24.7H, BY24.8H, BY24.9H, BY24.10H, BY24.11H, by XbaI-SalI double digestion
  • the BY24.12H, BY24.13H and BY24.14H coding nucleotides were respectively cloned into a glutamine synthetase high-efficiency expression vector with a double expression cassette to which the corresponding fusion protein light chain subunit coding nucleotide has been ligated; or vice versa Also.
  • the recombinant vector was sequenced and verified for expression.
  • the expressed dual targeting fusion proteins were named as fusion protein BY24.3, BY24.4, BY24.5, BY24.6, BY24.7, BY24.8, BY24.9, BY24.10, BY24.11, BY24. .12, BY24.13 and BY24.14.
  • the recombinant expression vector 250 ug of the recombinant expression vector prepared in Example 1 and 500 ug of polyethylenimine (PEI) (Sigma, catalog number: 408727) were added to 1 ml of serum-free CD 293 medium. After mixing and allowing to stand at room temperature for 8 minutes, the PEI/DNA suspension was added dropwise to a shake flask in which 100 ml of the cell suspension was placed. Mix gently, place in a 5% CO 2 shaker at 37 ° C (120 rpm). The culture supernatant was collected after 5 days.
  • PEI polyethylenimine
  • transient expression produced antibody BY18.1 as a control and protein 301-8 as a control.
  • the fusion protein present in the culture supernatant collected in the above Example 2 (1) was purified using a HiTrap MabSelect SuRe 1 ml column (GE Healthcare Life Sciences product, catalog number: 11-0034-93) equilibrated with a pH 7.4 PBS solution. Briefly, a HiTrap MabSelect SuRe 1 ml column was equilibrated with a pH 7.4 PBS solution at a volume of 10 bed volumes at a flow rate of 0.5 ml/min; the culture supernatant collected in the above Example 2 (1) was filtered through a 0.45 ⁇ m filter.
  • the sample was loaded onto a HiTrap MabSelect SuRe 1 ml column equilibrated with a pH 7.4 PBS solution; after loading the supernatant, the column was first washed with a pH 7.4 PBS solution at a flow rate of 0.5 ml/min for 5-10 bed volumes, and This was followed by elution with 100 mM citrate buffer (pH 4.0) at a flow rate of 0.5 ml/min. The elution peak was collected and the protein of interest was present in the elution peak.
  • the purity and molecular weight of the fusion protein were analyzed by SDS-PAGE and staining with Coomassie blue in the presence of a reducing agent (5 mM 1,4-dithiothreitol). The result is shown in Figure 2.
  • the predicted values of the molecular weight theory and the actual measured values are shown in Table 5. Because of the glycosylation of proteins in eukaryotic expression systems, the actual measured molecular weight is slightly higher than the theoretical prediction.
  • Example 3 Detection of binding of the fusion protein of the present invention to human PD-1 and recombinant human VEGF-A using an ELISA method
  • the antigen PD-1 product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10377-H08H
  • antigen VEGF 165 product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11066-HNAH
  • PD-1 product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10377-H08H
  • antigen VEGF 165 product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11066-HNAH
  • the double-targeted fusion protein purified in the above Example 2 (2) was diluted to 5 ⁇ g/ml, and then subjected to 3-fold serial dilution, and a total of 9 gradients were diluted, and each concentration gradient was subjected to multi-well detection.
  • the diluted sample was added to 50 ⁇ l were PD-1 antigen VEGF 165 antigen-coated 96-well plates or, for 2 hours 37 °C. After washing 3 times, horseradish peroxidase-labeled goat anti-human secondary antibody (product of Beijing Zhongxiu Jinqiao Co., Ltd., product number: ZDR-5301) was added and incubated at 37 ° C for 1 hour.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • the ELISA results showed that the fusion proteins of the present invention were BY24.3, BY24.4, BY24.5, BY24.6, BY24.7, BY24.8, BY24.9, BY24, as well as the antibody BY18.1 as a control. 10.
  • BY24.11, BY24.12, BY24.13, and BY24.14 can specifically bind to PD-1; similarly to protein 301-8 as a control, the fusion proteins of the present invention are BY24.3, BY24.4, LBY24.5, BY24.6, BY24.7, BY24.8, BY24.9, BY24.10, BY24.11, BY24.12, BY24.13, and BY24.14 also specifically bind to VEGF-A.
  • the novel fusion proteins constructed by the present invention are capable of binding PD-1 with high affinity, wherein the fusion proteins are BY24.4, BY24.5, BY24.7, BY24.8, BY24.10, BY24. 11. BY24.12 and BY24.14 bind to PD-1 with a greater affinity than the antibody BY18.1, even the fusion protein BY24.8, which is about 10-fold more affinity than the antibody BY18.1.
  • the fusion protein BY24.3 and the antibody BY18.1 The affinity for PD-1 is basically the same; the novel fusion proteins constructed by the present invention are also capable of binding VEGF-A with high affinity, wherein the fusion protein BY24.3 shows high affinity binding to VEGF- similar to protein 301-8 as a control. A.
  • an anti-IgG antibody (GE Healthcare Life Sciences, catalog number: BR-1008-39) was covalently immobilized on a CM5 chip by amide coupling.
  • the CM5 chip was activated with 60 ⁇ l of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 60 ⁇ l of N-hydroxysuccinimide (NHS), followed by 5 ⁇ l of anti- IgG antibody plus 95 ⁇ l of dilution buffer HBST (0.1 M HEPES, 1.5 M NaCl, pH 7.4, plus 0.005% Tween 20) was filtered through a 0.2 um filter, and the anti-IgG antibody was covalently immobilized on the CM5 chip by amide coupling. On top, a capture system of approximately 9,000-14,000 resonance units (RU) is produced. The CM5 chip was blocked with 120 ⁇ l of ethanolamine.
  • the fusion protein of the present invention prepared in Example 2 the antibody BY18.1 and the protein 301-8 were each diluted to 5 ⁇ g/ml, and the dilution was injected at a flow rate of 10 ⁇ L/min for 2 minutes to prepare 1600 RU of Example 2.
  • the fusion protein of the present invention, antibody BY18.1 and protein 301-8 were non-covalently captured on the surface of the CM5 chip by the respective Fc regions.
  • the resulting complex was stabilized by cross-linking with EDC/NHS to avoid baseline drift during measurement and regeneration.
  • the antigen will be combined with PD-1 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10377-H08H), VEGF 165 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11066-HNAH), VEGF-B (Biovision product, catalog number: 4642-20) and PLGF-1 (Biovision product, catalog number: 4739-25) were formulated to the following concentration gradients: 7 nM, 22 nm, 66 nM, 200 nM, 600 nM. Binding was measured by injecting each concentration for 180 seconds at a flow rate of 30 ⁇ l/min with a dissociation time of 600 seconds.
  • the surface was regenerated by washing with a 3 M 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), and the affinity data shown in Table 7 below was obtained.
  • the fusion protein BY24.3 binds to VEGF-A, VEGF-B and PLGF-1 with high affinity, and the affinity reaches 9.59 ⁇ 10 -12 M, 1.23 ⁇ 10 -9 M and 1.82, respectively. ⁇ 10 -10 M, and the affinity (PDD) for PD-1 is substantially identical to the antibody BY18.1 as a control, and the fusion protein BY24.7 binds PD-1 with a greater affinity (KD) than the antibody BY18.1; fusion Protein BY24.3 showed high affinity binding to VEGF-A similar to protein 301-8 as a control; fusion protein BY24.3 also bound VEGF-B and PLGF-1 with high affinity.
  • Example 5 Inhibition of tumor growth by a fusion protein of the invention in a humanized B-hPD-1 mouse model
  • the tumor-bearing mice were randomly divided into groups of 6 and 4 groups, respectively: PBS solvent control group, protein 301-8 group (3.3 mg/kg), fusion protein BY24.3 Group (6.4 mg/kg) and antibody BY18.1 group (5 mg/kg), the dosage of each administration group was based on the dose of antibody BY18.1, the protein 301-8 group, the fusion protein BY24.3 group and the antibody.
  • the doses administered for the BY18.1 group are equivalent in molar amount.
  • the time of the first administration was set to day 0. All groups were administered intraperitoneally (ip), once every three days, six times in a row, and the experiment was terminated three days after the last administration.
  • Tumor volume and mouse body weight were measured twice a week, and mouse body weight and tumor volume were recorded.
  • the animals were euthanized, the tumor weighed stripping, pictures, calculate tumor growth inhibition rate (T umor G rowth I nhibition% ) and tumor weight inhibition rate (I nhibition R ate of T umor W eight%).
  • the formula used to calculate TGI% is: [1 - (mean of tumor volume change in the drug-administered group / mean change in tumor volume of the PBS solvent control group)] x 100%
  • the formula used to calculate IRTW% is: [1- (administration group) Tumor weight / PBS solvent control tumor weight)) x 100%.
  • the experiment was carried out in Beijing Baiao Saitu Gene Biotechnology Co., Ltd.
  • the mean tumor volume ⁇ standard error of the PBS solvent control group was 1386 ⁇ 170 mm 3
  • the mean tumor volume ⁇ standard error of the fusion protein BY24.3 and protein 301-8 groups were 452 ⁇ 69, 1023 ⁇ 256, respectively. They were 73.3% and 28.1%, respectively, and IRTW% were 74.6% and 25.7%, respectively.
  • the fusion protein BY24.3 was significantly different from the tumor volume of the PBS solvent control group (P ⁇ 0.05), indicating that the fusion protein BY24.3 has significant tumor suppressive effect. Protein 301-8 had a certain tumor suppressive effect, but there was no significant difference compared with the PBS solvent control group (P>0.05).
  • the antibody BY18.1 had a mean tumor volume ⁇ standard error of 739 ⁇ 128, a TGI% of 50.6%, and an IRTW% of 46.0%, which was significantly different from the tumor volume of the PBS solvent control group (P ⁇ 0.05).
  • the fusion protein BY24.3 showed excellent tumor inhibition, and there was a significant difference compared with the tumor inhibition effect of the antibody BY18.1 group and the protein 301-8 group as a drug control. The antitumor efficacy results of this experiment were further confirmed in the comparison of tumor weights in mice.
  • TGI Tumor growth inhibition rate
  • IRTW Tumor weight inhibition rate

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Abstract

L'invention concerne une protéine de fusion à double ciblage ciblant PD-1 ou PD-L1 et ciblant la famille du VEGF. Ladite protéine de fusion à double ciblage comprend (i) un anticorps anti-PD-1 ou un anticorps anti-PD-L1, et (ii) un VID lié de manière fonctionnelle à l'extrémité C-terminale de chacune de deux chaînes lourdes dudit anticorps anti-PD-1 ou dudit anticorps anti-PD-L1. L'invention concerne un polynucléotide codant pour la protéine de fusion à double ciblage, un vecteur comprenant le polynucléotide, une cellule hôte comprenant le polynucléotide ou le vecteur, et l'utilisation de la protéine de fusion à double ciblage dans le traitement, la prévention et/ou le diagnostic de maladies associées à l'activité PD-1, à l'activité PD-L1 et à l'activité de la famille du VEGF chez un individu.
PCT/CN2018/106741 2017-09-29 2018-09-20 Protéine de fusion à double ciblage ciblant pd-1 ou pd-l1 et ciblant la famille du vegf et son utilisation WO2019062642A1 (fr)

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Cited By (6)

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WO2020114355A1 (fr) * 2018-12-03 2020-06-11 Immuneonco Biopharmaceuticals (Shanghai) Co., Ltd Protéine de recombinaison ciblant pd-l1 et vegf
US20200299389A1 (en) * 2018-02-28 2020-09-24 Ap Biosciences, Inc. Bifunctional Proteins Combining Checkpoint Blockade for Targeted Therapy
US10981993B2 (en) * 2017-02-03 2021-04-20 Beijing Dongfang Biotech Co., Ltd. Anti-PD-1 monoclonal antibody and obtaining method therefor
US11008391B2 (en) 2015-08-11 2021-05-18 WuXi Biologics Ireland Limited Anti-PD-1 antibodies
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US11524053B2 (en) 2018-01-26 2022-12-13 The Regents Of The University Of California Methods and compositions for treatment of angiogenic disorders using anti-VEGF agents
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