WO2022111476A1 - ANTI-PD-L1-ANTI-VEGF-ANTI-TGF-β MULTISPECIFIC ANTIBODY, PHARMACEUTICAL COMPOSITION AND USE THEREOF - Google Patents

ANTI-PD-L1-ANTI-VEGF-ANTI-TGF-β MULTISPECIFIC ANTIBODY, PHARMACEUTICAL COMPOSITION AND USE THEREOF Download PDF

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WO2022111476A1
WO2022111476A1 PCT/CN2021/132464 CN2021132464W WO2022111476A1 WO 2022111476 A1 WO2022111476 A1 WO 2022111476A1 CN 2021132464 W CN2021132464 W CN 2021132464W WO 2022111476 A1 WO2022111476 A1 WO 2022111476A1
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seq
antibody
amino acid
vegf
tgf
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Chinese (zh)
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罗羿
陈俊颖
缪小牛
黄威峰
袁志军
王超
曾竣玮
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普米斯生物技术(珠海)有限公司
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Definitions

  • the invention belongs to the field of biomedicine, and relates to an anti-PD-L1-anti-VEGF-anti-TGF- ⁇ multispecific antibody, a pharmaceutical composition and uses thereof.
  • PD-L1 Programmed death 1 ligand 1
  • CD274 is a member of the B7 family and is a ligand of PD-1.
  • PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including an IgV-like domain, an IgC-like domain, a transmembrane hydrophobic domain and an intracellular domain consisting of 30 amino acids.
  • PD-L1 negatively regulates immune responses. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages and dendritic cells, etc.
  • PD-L1 In addition to lymphocytes, PD-L1 is also expressed in other tissues such as thymus, heart, placenta, etc. Endothelial cells, as well as various non-lymphoid lineages such as melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer, head and neck cancer, etc. (Akintunde Akinleye&Zoaib Rasool, Journal of Hematology&Oncology volume 12, Article number: 92 (2019)). PD-L1 is broad in regulating autoreactive T, B cells, and immune tolerance, and plays a role in peripheral tissue T and B cell responses. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.
  • VEGF Vascular endothelial growth factor
  • VPF vascular permeability factor
  • vasculotropin is a highly specific homodimer that promotes the growth of vascular endothelial cells. body protein.
  • VEGF family proteins include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor (PIGF), among which VEGF-A is involved in the early formation of blood vessels Play an important role.
  • VEGF-A vascular permeability factor
  • PIGF placental growth factor
  • VEGF vascular endothelial growth factor
  • VEGF is involved in the pathogenesis and progression of many angiogenesis-dependent diseases, including cancer, certain inflammatory diseases, and diabetic retinopathy. Therefore, VEGF is an important target in antitumor drug research.
  • the main receptors of VEGF proteins are VEGFR1, VEGFR2, VEGFR3, NRP1, NRP2 and NRP3.
  • VEGFA can bind to VEGFR1 and VEGFR2, activate endogenous kinase activation, and promote neogenesis.
  • Angiogenesis Blocking the binding of VEGF to the receptor can be applied to the treatment of various cancers, such as breast cancer, colon cancer, lung cancer, ovarian cancer, endometrial cancer, mesothelioma, cervical cancer, kidney cancer (Rakesh R. Ramjiawan, Arjan W. Griffioen, and Dan G. Duda, Angiogenesis. 2017 20(2):185–204.).
  • VEGF drugs listed or approved in the global market, covering 45 indications.
  • the representative product of VEGF-A antagonists in the anti-tumor direction is Roche's Avastin.
  • Bevacizumab The product was approved by the FDA in February 2004 for the treatment of metastatic colorectal cancer and advanced, metastatic or recurrent non-small cell lung cancer, and was approved for marketing in China in February 2010. A number of Avastin biosimilars have been approved for marketing abroad. Bevacizumab injection (trade name: Ankea) developed by Qilu Pharmaceuticals in China was also approved for marketing in China in December 2019.
  • Ranibizumab is a second generation humanized anti-VEGF recombinant murine monoclonal antibody fragment, Fab portion, obtained from the same parent murine antibody as Bevacizumab. It was approved by the US FDA on June 30, 2006 for the treatment of patients with age-related macular degeneration (AMD). Compared with bevacizumab, ranibizumab maintains a better affinity for VEGFA, can better inhibit angiogenesis, and has been developed for gastric cancer, rectal cancer and other indications.
  • Aflibercept is a recombinant protein fused to human IgG1-Fc after a partial extracellular fragment (Ig domain 2) of VEGFR1 and a partial extracellular fragment (Ig domain 3) of VEGFR2 are linked and combined.
  • VEGFR2 binds to VEGFA, thereby blocking angiogenesis in vascular epithelial cells.
  • Apacept is mainly indicated for the treatment of patients with neovascular (wet) age-related macular degeneration (AMD).
  • AMD age-related macular degeneration
  • the clinical treatment of advanced colorectal cancer is carried out (Caemen Stancan, etc, Rom J Morphol Embryol. 2018 59(2):455–467). Since apatacept possesses functional fragments of VEGFR1 and VEGFR2, it has the function of blocking VEGF and receptor binding similar to antibodies.
  • TGF- ⁇ is mainly expressed and secreted by the immune system (including TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3), and after binding to receptor TGF- ⁇ R (including RI/RII/RIII), it can regulate cell growth, proliferation, differentiation , migration and apoptosis, affect embryonic organ development, immunity, etc., and have important physiological functions. All three isoforms of TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 can bind to receptors on the cell surface. TGF- ⁇ RI does not directly bind TGF- ⁇ , and RIII can bind TGF- ⁇ , but its sugar modification is too complicated.
  • TGF- ⁇ RII has a very high affinity (about 5 pM) for TGF- ⁇ 1 and TGF- ⁇ 3, and a lower affinity (about 6 nM) for TGF- ⁇ 2.
  • TGF- ⁇ plays a very important and dual role in the occurrence and development of tumors.
  • TGF- ⁇ can regulate the expression of several apoptotic genes in the early stage of tumor to induce tumor cell apoptosis; in the later stage of tumor, most tumor cells Secretes a large amount of TGF- ⁇ , once the level of TGF- ⁇ is too high, it turns into a tumor-promoting factor: it can inhibit T and NK cells, promote regulatory T cells, promote tumor angiogenesis, and promote the transformation of epithelial cells to mesenchymal cells, etc.
  • TGF- ⁇ targeting drugs have also become an important direction for the development of anticancer drugs.
  • multifunctional antibodies targeting multiple targets are the direction of antibody drug development, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control.
  • the development of multispecific antibodies is difficult.
  • an anti-PD-L1-anti-VEGF-anti-TGF- ⁇ multispecific antibody (hereinafter also referred to as anti-PD-L1/VEGF/TGF- ⁇ multispecific antibody).
  • the inventors have surprisingly found that the anti-PD-L1-anti-VEGF-anti-TGF- ⁇ multispecific antibody of the present invention has high affinity for the three targets of PD-L1, VEGF and TGF- ⁇ , and can specifically block the The three targets are stable and safe, and have good antitumor activity.
  • the following invention is thus provided:
  • One aspect of the present invention pertains to a multispecific antibody comprising:
  • the first protein functional region is an anti-PD-L1 single domain antibody
  • the second protein functional region is a VEGF receptor, a VEGF receptor functional fragment, a fusion protein comprising a VEGF receptor functional fragment, or an anti-VEGF antibody,
  • the third protein functional domain is a TGF- ⁇ receptor, a TGF- ⁇ receptor functional fragment, a fusion protein comprising a TGF- ⁇ receptor functional fragment, or an anti-TGF- ⁇ antibody.
  • the multispecific antibody, wherein the VEGF is VEGFA.
  • the multispecific antibody wherein the VEGF receptor is VEGFR1 and/or VEGFR2.
  • the multispecific antibody wherein the VEGF receptor functional fragment is an extracellular fragment of VEGFR1 (eg, Ig domain 2) and/or VEGFR2 (eg, VEGFR2) with VEGF receptor function Extracellular fragment of Ig domain 3).
  • VEGFR1 eg, Ig domain 2
  • VEGFR2 eg, VEGFR2
  • VEGF receptor function Extracellular fragment of Ig domain 3
  • the multispecific antibody wherein the TGF- ⁇ is TGF- ⁇ 1 and/or TGF- ⁇ 3.
  • the multispecific antibody wherein the TGF- ⁇ receptor is TGF- ⁇ RII.
  • the multispecific antibody wherein the TGF- ⁇ receptor functional fragment is an extracellular fragment of TGF- ⁇ RII with TGF- ⁇ receptor function.
  • the multispecific antibody wherein the anti-PD-L1 single domain antibody, comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence such as SEQ ID HCDR1 shown in NO:26, HCDR2 shown in amino acid sequence as shown in SEQ ID NO:27 and HCDR3 shown in amino acid sequence as shown in SEQ ID NO:28;
  • amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:2.
  • variable regions of the light and heavy chains determine the binding of antigens; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs), of which the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3, the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3.
  • CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDRs contained by an antibody or antigen-binding fragment thereof of the invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.
  • the CDRs contained by an antibody or antigen-binding fragment thereof of the invention are preferably determined by the IMGT numbering system.
  • the IMGT numbering system see Ehrenmann F, Kaas Q, Lefranc M P.
  • the VEGF receptor in the multispecific antibody, is VEGFR1 and/or VEGFR2.
  • the multispecific antibody wherein the VEGF receptor functional fragment is a VEGFR1 extracellular fragment and/or a VEGFR2 extracellular fragment;
  • amino acid sequence of the VEGF receptor functional fragment is shown in SEQ ID NO:4.
  • the multispecific antibody wherein the anti-VEGF antibody, comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1 whose amino acid sequence is shown in SEQ ID NO:29, HCDR2 whose amino acid sequence is shown in SEQ ID NO:30, and HCDR3 whose amino acid sequence is shown in SEQ ID NO:31; and the The light chain variable region comprises LCDR1 whose amino acid sequence is as shown in SEQ ID NO:32, LCDR2 whose amino acid sequence is as shown in SEQ ID NO:33 and LCDR3 whose amino acid sequence is as shown in SEQ ID NO:34;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19;
  • the heavy chain constant region of the anti-VEGF antibody is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region thereof is a human Ig kappa chain C region;
  • the heavy chain constant region of the anti-VEGF antibody further comprises L234A mutation and L235A mutation (abbreviated as LALA mutation in the present invention) according to the EU numbering system; optionally, G237A mutation;
  • amino acid sequence of the heavy chain constant region of the anti-VEGF antibody is shown in SEQ ID NO: 17;
  • amino acid sequence of the light chain constant region of the anti-VEGF antibody is shown in SEQ ID NO:20.
  • the multispecific antibody wherein the TGF- ⁇ receptor is TGF- ⁇ RII.
  • the multispecific antibody wherein the TGF- ⁇ receptor functional fragment is an extracellular fragment of TGF- ⁇ RII;
  • amino acid sequence of the TGF- ⁇ receptor functional fragment is shown in SEQ ID NO:6.
  • the multispecific antibody wherein the first protein functional domain, the second protein functional domain and the third protein functional domain are independently 1, 2 or 3 copies .
  • the first protein functional domain, the second protein functional domain and the third protein functional domain are all 2 copies.
  • the first protein functional domain and the third protein functional domain are both 2 copies, and the second protein functional domain is 1 copy.
  • the multispecific antibody wherein, the first protein functional domain, the second protein functional domain and the third protein functional domain are directly connected or connected by connecting fragments;
  • amino acid sequence of the linker fragment is independently selected from SEQ ID NO:5 and SEQ ID NO:8.
  • the multispecific antibody further comprises a human IgG1 Fc segment
  • the Fc segment of the human IgG1 comprises L234A mutation and L235A mutation (abbreviated as LALA mutation in the present invention) according to the EU numbering system;
  • amino acid sequence of the Fc segment of the human IgG1 is shown in SEQ ID NO:3.
  • the multispecific antibody wherein the human IgG1 Fc segment is directly linked to or through two of the first protein functional domain, the second protein functional domain and the third protein functional domain connection fragment (linker) connection;
  • amino acid sequence of the linker fragment is independently selected from SEQ ID NO:5 and SEQ ID NO:8.
  • the multispecific antibody in order from N-terminal to C-terminal, comprises:
  • the first protein functional region the Fc segment of human IgG1, the second protein functional region and the third protein functional region;
  • the first protein functional region the Fc segment of human IgG1, the third protein functional region and the second protein functional region;
  • the first protein functional region, the second protein functional region, the Fc segment of human IgG1 and the third protein functional region are provided.
  • the multispecific antibody is a dimer formed by two identical peptide chains
  • amino acid sequence of the peptide chain is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.
  • the multispecific antibody is a dimer formed by two identical peptide chains, and from the N terminus to the C terminus, the peptide chains sequentially comprise:
  • the first protein functional region the Fc segment of human IgG1, the second protein functional region and the third protein functional region;
  • the first protein functional region the Fc segment of human IgG1, the third protein functional region and the second protein functional region;
  • amino acid sequence of the peptide chain is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.
  • the multispecific antibody is a dimer formed by two identical peptide chains through disulfide bonds (eg, 2 pairs or 3 pairs of disulfide bonds).
  • the multispecific antibody further comprises a human IgG1 heavy chain constant region
  • the human IgG1 heavy chain constant region further comprises a Knob-in-hole mutation
  • amino acid sequences of the two peptide chains of the human IgG1 heavy chain constant region are shown in SEQ ID NO: 12 and SEQ ID NO: 14, respectively.
  • the multispecific antibody which is a dimer formed by two different first peptide chains and second peptide chains, according to the N-terminus to the C-terminus,
  • the first peptide chain sequentially comprises: the first protein functional region, the human IgG1 heavy chain constant region and the third protein functional region; and the second peptide chain sequentially comprises: the first protein functional region, the human IgG1 heavy chain constant region and the second protein functional area;
  • amino acid sequence of the first peptide chain is shown in SEQ ID NO:11
  • amino acid sequence of the second peptide chain is shown in SEQ ID NO:13.
  • the multispecific antibody is formed by two different first peptide chains and second peptide chains through disulfide bonds (eg, 2 pairs or 3 pairs of disulfide bonds). dimer.
  • the multispecific antibody wherein:
  • the first protein functional region has two copies, wherein the two copies are respectively connected to the N-terminus of the two heavy chains of the anti-VEGF antibody, the N-terminus of the two light chains of the anti-VEGF antibody, or the two copies of the anti-VEGF antibody.
  • the C-terminus of the light chain, or one copy is attached to the N-terminus of one heavy chain of the anti-VEGF antibody and the other copy is attached to the N-terminus of the light chain bound to the other heavy chain of the anti-VEGF antibody; and the second There are two copies of the protein functional region, which are respectively connected to the C-terminus of the two heavy chains of the anti-VEGF antibody;
  • the second protein functional region has two copies, wherein the two copies are respectively connected to the N-terminus of the two heavy chains of the anti-VEGF antibody, the N-terminus of the two light chains of the anti-VEGF antibody, or the two copies of the anti-VEGF antibody.
  • the C-terminus of the light chain, or one copy is attached to the N-terminus of one heavy chain of the anti-VEGF antibody, and the other copy is attached to the N-terminus of the light chain bound to the other heavy chain of the anti-VEGF antibody; and the first There are two copies of the protein functional domain, which are respectively connected to the C-terminus of the two heavy chains of the anti-VEGF antibody.
  • the multispecific antibody is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein,
  • amino acid sequence of the first peptide chain is shown in SEQ ID NO: 15; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 18;
  • amino acid sequence of the first peptide chain is shown in SEQ ID NO: 21; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 22;
  • amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 24;
  • the amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 25.
  • the multispecific antibody is composed of two identical first peptide chains and two identical second peptide chains through disulfide bonds (eg, formed between two peptide chains) 2 pairs or 3 pairs of disulfide bonds) formed tetramers.
  • Another aspect of the present invention pertains to isolated nucleic acid molecules encoding the multispecific antibodies of any one of the present invention.
  • the present invention also relates to a vector comprising the isolated nucleic acid molecule of the present invention.
  • the present invention also relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a vector of the present invention.
  • Yet another aspect of the present invention relates to a method of preparing the multispecific antibody of any one of the present invention, comprising culturing a host cell of the present invention under suitable conditions, and recovering the polyspecific antibody from the cell culture Steps for specific antibodies.
  • Yet another aspect of the present invention relates to a conjugate comprising a multispecific antibody and a coupling moiety, wherein the multispecific antibody is the multispecific antibody of any one of the present invention, the conjugate is
  • the coupling moiety is a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • kits comprising the multispecific antibody of any one of the present invention, or the conjugate of the present invention
  • the kit further comprises a secondary antibody capable of specifically binding to the multispecific antibody; optionally, the secondary antibody further comprises a detectable label such as a radioisotope, fluorescent substance, luminescent substance , colored substances or enzymes.
  • a detectable label such as a radioisotope, fluorescent substance, luminescent substance , colored substances or enzymes.
  • Yet another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the multispecific antibody of any one of the present invention or the conjugate of the present invention; optionally, it further comprises pharmaceutically acceptable excipients .
  • Yet another aspect of the present invention relates to the use of the multispecific antibody of any one of the present invention or the conjugate of the present invention in the preparation of a medicament for preventing and/or treating malignant tumors; preferably, the malignant tumor is selected from Selected from lung cancer, endometrial cancer, mesothelioma, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer; preferably, the lung cancer for non-small cell lung cancer.
  • Yet another aspect of the present invention relates to a method of treating and/or preventing a malignant tumor, comprising administering to a subject in need thereof an effective amount of the multispecific antibody of any one of the present invention or the conjugate of the present invention
  • the malignant tumor is selected from the group consisting of lung cancer, endometrial cancer, mesothelioma, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, Esophageal cancer and head and neck cancer;
  • the lung cancer is non-small cell lung cancer.
  • the multispecific antibodies of the present invention or the conjugate of the present invention it is used for the treatment and/or prevention of malignant tumors; preferably, the malignant tumors are selected from lung cancer, endometrial cancer, Skin tumor, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer; preferably, the lung cancer is non-small cell lung cancer.
  • the method wherein the step of administering to a subject in need thereof an effective amount of the multispecific antibody of any one of the invention is before or after surgical treatment, and/or before or after radiation therapy.
  • the single administration dose of the multispecific antibody of the present invention is 0.1-100 mg per kilogram of body weight, preferably 4.8-24 mg or 1-10 mg; or, the single administration dose of the multispecific antibody of the present invention is each subject or 10-1000mg, preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg or 200mg;
  • it is administered every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
  • the mode of administration is intravenous drip or intravenous injection.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that elicits 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • antibody is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256(5517): 495), but can also be obtained by recombinant DNA technology (eg, see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody)
  • the antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • antigen-binding fragments of antibodies are diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but linkers that are too short are used to allow for both domains on the same chain Pairing between the domains forces the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993;90:6444 -6448 and Poljak RJet al., Structure 1994;2:1121-1123).
  • a fusion protein as described herein is a protein product of co-expression of two genes obtained by DNA recombination.
  • Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art (eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15).
  • isolated refers to artificially obtained from the natural state. If an "isolated” substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated or isolated
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus, etc.
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a disease-prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (eg, a tumor);
  • a therapeutically-effective amount refers to an amount sufficient to cure or at least partially prevent the development of a disease in a patient already suffering from the disease. The amount of disease and its complications. Determining such effective amounts is well within the skill of the art.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently and many more.
  • the amino acid sequence of the PD-L1 protein when referring to the amino acid sequence of the PD-L1 protein (Programmed death-ligand 1, NCBI Reference Sequence ID: NP_054862.1), it includes the full length of human PD-L1 protein, or the human PD-L1 protein.
  • PD-L1 protein shall include all such sequences, including the sequences shown as well as natural or artificial variants thereof. And, when describing the sequence fragment of PD-L1 protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.
  • PD-1 protein when referring to the amino acid sequence of PD-1 protein (NCBI Reference Sequence: NP_005009.2), it includes the full length of human PD-1 protein, or the extracellular fragment PD-1 of human PD-1 1ECD or a fragment comprising PD-1ECD; also includes a full-length fusion protein of PD-1 protein or a fusion protein of PD-1ECD, such as a fragment fused to a mouse or human IgG Fc protein fragment (mFc or hFc).
  • mutations or variations including but not limited to substitutions, deletions and/or additions
  • the term "addition", protein” shall include all such sequences, including natural or artificial variants thereof.
  • a sequence fragment of a PD-1 protein when described, it also includes the corresponding sequence fragment in its natural or artificial variant.
  • VEGFA protein when referring to the amino acid sequence of a VEGFA protein (NCBI Reference Sequence: NP_001165097.1), it includes the full length of human VEGFA protein, or a functional fragment of human VEGFA or a fragment comprising a functional fragment of VEGFA; also Include full-length fusion proteins of VEGFA protein or fusion proteins of functional fragments of VEGFA, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • Fc protein fragments fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • mutations or variations can be naturally generated or artificially introduced in the amino acid sequence of VEGFA protein without affecting its biological function.
  • addition protein
  • VEGFA VEGFA
  • addition protein shall include all such sequences, including natural or artificial variants thereof.
  • sequence fragment of a VEGFA protein when described, it also includes the corresponding sequence fragment in natural or artificial variants thereof.
  • TGF- ⁇ 1 protein when referring to the amino acid sequence of TGF- ⁇ 1 protein (NCBI Reference Sequence: NP_000651.3), it includes the full length of human TGF- ⁇ 1 protein, or a functional fragment of human TGF- ⁇ 1 or comprising TGF- ⁇ 1 - Fragments of ⁇ 1 functional fragments; also include full-length fusion proteins of TGF- ⁇ 1 protein or fusion proteins of TGF- ⁇ 1 functional fragments, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • mFc or hFc Fc protein fragments
  • the term "added), protein” shall include all such sequences, including natural or artificial variants thereof. Also, when a sequence fragment of a TGF-beta1 protein is described, it also includes the corresponding sequence fragment in its natural or artificial variant.
  • TGF- ⁇ 2 protein when referring to the amino acid sequence of TGF- ⁇ 2 protein (NCBI Reference Sequence: NP_003229.1), it includes the full length of human TGF- ⁇ 2 protein, or a functional fragment of human TGF- ⁇ 2 or comprising TGF- ⁇ - Fragments of ⁇ 2 functional fragments; also include full-length fusion proteins of TGF- ⁇ 2 protein or fusion proteins of TGF- ⁇ 2 functional fragments, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • mFc or hFc Fc protein fragments
  • TGF- ⁇ 3 protein when referring to the amino acid sequence of TGF- ⁇ 3 protein (NCBI Reference Sequence: NP_003230.1), it includes the full length of human TGF- ⁇ 3 protein, or a functional fragment of human TGF- ⁇ 3 or comprising TGF- ⁇ Fragments of ⁇ 3 functional fragments; also include full-length fusion proteins of TGF- ⁇ 3 protein or fusion proteins of TGF- ⁇ 3 functional fragments, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • mutations or variations can be naturally generated or artificially introduced in the amino acid sequence of TGF- ⁇ 3 protein without affecting its biological function.
  • additional protein shall include all such sequences, including natural or artificial variants thereof.
  • sequence fragment of a TGF-beta3 protein when described, it also includes the corresponding sequence fragment in its natural or artificial variant.
  • TGF- ⁇ RII protein when referring to the amino acid sequence of TGF- ⁇ RII protein (NCBI Reference Sequence: NP_003233.4), it includes the full length of human TGF- ⁇ RII protein, or the extracellular fragment of human TGF- ⁇ RII TGF- ⁇ RII ECD or a fragment comprising TGF- ⁇ RII ECD; also includes a full-length fusion protein of TGF- ⁇ RII protein or a fusion protein of TGF- ⁇ RII ECD, e.g., fused to a mouse or human IgG Fc protein fragment (mFc or hFc) fragment.
  • mFc or hFc human IgG Fc protein fragment
  • TGF- ⁇ RII protein in the amino acid sequence of TGF- ⁇ RII protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function.
  • additional protein shall include all such sequences, including natural or artificial variants thereof.
  • sequence fragment of a TGF-betaRII protein when described, it also includes the corresponding sequence fragment in its natural or artificial variant.
  • single domain antibody refers to cloning the variable region of the antibody heavy chain, and constructing a nanobody (VHH) composed of only one heavy chain variable region , which is the smallest fully functional antigen-binding fragment.
  • VHH single domain antibody
  • N1 light chain and heavy chain constant region 1
  • the "first” eg, the first protein functional region, the first peptide chain
  • the second eg, the second protein functional region, the second peptide chain
  • the first Three
  • the third protein functional region is for reference distinction or clarity of expression, and does not have a typical sequential meaning.
  • the present invention also relates to any one of the following items 1 to 10:
  • a multispecific antibody comprising:
  • a vector comprising the polynucleotide according to item 2.
  • a host cell comprising the vector described in item 3, or the polynucleotide described in item 2 integrated in its genome;
  • the host cell expresses the multispecific antibody of item 1.
  • a method of producing the multispecific antibody of item 1, comprising the steps of:
  • step (b) purifying and/or separating the culture obtained in step (a) to obtain the multispecific antibody.
  • conjugation moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or their combination.
  • the reagent, detection plate or kit is used for: detecting PD-L1, VEGF and/or TGF- ⁇ in the sample; wherein, the agent is used for treating or preventing the expression of PD-L1 (ie PD-L1 positive) tumors, VEGF-expressing tumors, and/or TGF- ⁇ -expressing tumors.
  • a pharmaceutical composition comprising:
  • a recombinant protein comprising: (i) the multispecific antibody of item 1; and (ii) an optional tag sequence to facilitate expression and/or purification.
  • the present invention also relates to any one of the following first to fourteenth aspects:
  • a multispecific antibody comprising:
  • the multispecific antibody includes 1-3 anti-PD-L1 single-domain antibodies, preferably, includes 1 or 2 anti-PD-L1 single-domain antibodies.
  • the PD-L1 single domain antibody can block the interaction between PD-1 and PD-L1.
  • amino acid sequence of the anti-PD-L1 single domain antibody is as shown in SEQ ID NO: 2, or has ⁇ 85% (preferably 90%, more preferably ⁇ 85% of the sequence shown in SEQ ID NO: 2) 95%) sequence identity.
  • the multispecific antibody includes 1-3 anti-VEGF antibody elements, preferably, includes 1 or 2 anti-VEGF antibody elements.
  • the anti-VEGF antibody element can bind to VEGF and block its activity.
  • amino acid sequence of the anti-VEGF antibody element is as shown in SEQ ID NO: 4, or has ⁇ 85% (preferably 90%, more preferably 95% with the sequence shown in SEQ ID NO: 4) %) sequence identity.
  • the multispecific antibody includes 1-3 extracellular domains of TGF- ⁇ RII, preferably 1 or 2 extracellular domains of TGF- ⁇ RII.
  • the TGF- ⁇ RII extracellular domain can bind to TGF- ⁇ ligand and block its downstream signaling pathway.
  • the amino acid sequence of the extracellular domain of TGF- ⁇ RII is as shown in SEQ ID NO: 6, or has ⁇ 85% (preferably 90%, more preferably ⁇ 85% of the sequence shown in SEQ ID NO: 6) 95%) sequence identity.
  • the multispecific antibody further comprises an Fc region derived from human immunoglobulin.
  • the Fc region of the multispecific antibody is selected from the group consisting of a CH1+CL1 domain, a human IgG domain or a combination thereof.
  • the Fc region is an engineered mutant; preferably, it is a LALA mutant; more preferably, it is a LALA mutant and contains a Knob-in-hole mutant.
  • the multispecific antibody comprises peptide chain I, and the peptide chain I includes the following elements: anti-PD-L1 nanobody VHH chain, Fc region of human immunoglobulin, anti-VEGF antibody elements, the TGF- ⁇ RII extracellular domain, and optional linking elements linking the above-mentioned elements;
  • each element can be arranged in different order from N-terminus to C-terminus of peptide chain I;
  • the multispecific antibody is a homodimer formed by the peptide chain I through the disulfide bond of the Fc region.
  • the sequence of the connecting element is (G 4 S) n , wherein n is a positive integer (eg 1, 2, 3, 4, 5 or 6), preferably, n is 2 or 4 .
  • amino acid sequence of the linking element is as shown in SEQ ID NO:6, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO:6 sequence identity.
  • the peptide chain I from N-terminal to C-terminal are respectively: anti-PD-L1 Nanobody VHH chain, Fc region of human immunoglobulin, connecting element, anti-VEGF antibody element, connecting element , and the TGF- ⁇ RII extracellular domain.
  • amino acid sequence of the peptide chain I is as shown in SEQ ID NO: 1, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 1 ) sequence identity.
  • the peptide chain I from N-terminal to C-terminal are respectively: anti-PD-L1 Nanobody VHH chain, Fc region of human immunoglobulin, connecting element, TGF- ⁇ RII extracellular domain, Linking elements, and anti-VEGF antibody elements.
  • amino acid sequence of the peptide chain I is shown in SEQ ID NO:7, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO:7 ) sequence identity.
  • the peptide chain I from the N-terminus to the C-terminus are: anti-PD-L1 Nanobody VHH chain, connecting element, TGF- ⁇ RII extracellular domain, connecting element, human immunoglobulin Fc regions, linking elements, and anti-VEGF antibody elements.
  • amino acid sequence of the peptide chain I is as shown in SEQ ID NO: 9, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 9 ) sequence identity.
  • the peptide chain I from N-terminal to C-terminal are respectively: anti-PD-L1 Nanobody VHH chain, connecting element, anti-VEGF antibody element, connecting element, Fc region of human immunoglobulin , the linking element, and the TGF- ⁇ RII extracellular domain.
  • amino acid sequence of the peptide chain I is shown in SEQ ID NO: 10, or has ⁇ 85% (preferably 90%, more preferably 95% of the sequence shown in SEQ ID NO: 10) ) sequence identity.
  • the multispecific antibody is a heterodimer composed of a peptide chain IIa and a peptide chain IIb, and the peptide chains IIa and IIb each independently include the following elements: anti-PD- L1 Nanobody VHH chain, human IgG1 knockout or hole mutant sequence, anti-VEGF antibody element, TGF- ⁇ RII extracellular domain, and optional linking elements linking the above elements;
  • each element can be arranged in different order from N-terminus to C-terminus of peptide chain IIa or peptide chain IIb;
  • the peptide chain IIa includes a human IgG1 knockout mutant sequence
  • the peptide chain IIb includes a human IgG1 hole mutant sequence
  • the multispecific antibody is composed of the peptide chain IIa and the peptide chain IIb.
  • the 132nd position based on the amino acid sequence shown in SEQ ID NO: 12 has the S132C mutation, and the 144th position has the T144W mutation.
  • the 127th position has a Y127C mutation
  • the 144th position has a T144S mutation
  • the 146th position has a L146A mutation mutation.
  • the peptide chain IIa from the N-terminus to the C-terminus is: anti-PD-L1 Nanobody VHH chain, human IgG1 knockout mutant sequence, connecting element, TGF- ⁇ RII extracellular domain, connecting element , and TGF- ⁇ RII extracellular domain; and the peptide chain IIb from the N-terminus to the C-terminus are: anti-PD-L1 nanobody VHH chain, human IgG1 hole mutation sequence, linking element, and anti-VEGF antibody element .
  • amino acid sequence of the peptide chain IIa is as shown in SEQ ID NO: 11, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 11 and the amino acid sequence of the peptide chain IIb is as shown in SEQ ID NO: 13, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 13 sequence identity.
  • the multispecific antibody includes peptide chain IIIa and peptide chain IIIb;
  • the peptide chain IIIa includes the following elements: the VH sequence of the anti-VEGF antibody, the constant region sequence of the human IgG1 heavy chain; the peptide chain IIIb includes the following elements: the VL sequence of the anti-VEGF antibody, the constant region of the human kappa light chain sequence;
  • the multispecific antibody is a heterotetramer formed by disulfide bonds between peptide chains IIIa and IIIb, and between peptide chains IIIb.
  • the human IgG1 heavy chain constant region sequence has LALA mutation.
  • amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 15, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 15 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 18, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 18 sequence identity.
  • the peptide chain IIIa from the N-terminus to the C-terminus is: the VH sequence of an anti-VEGF antibody, a human IgG1 heavy chain constant region with LALA mutation, a linking element, and an extracellular domain of TGF- ⁇ RII.
  • the peptide chain IIIb from the N-terminus to the C-terminus are respectively: the anti-PD-L1 nanobody VHH chain, the connecting element, the VL sequence of the anti-VEGF antibody, and the human kappa light chain constant region sequence.
  • the amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 21, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 21 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 22, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 22 sequence identity.
  • the peptide chain IIIa from the N-terminus to the C-terminus is: the VH sequence of an anti-VEGF antibody, a human IgG1 heavy chain constant region with LALA mutation, a linking element, and an anti-PD-L1 nanobody VHH and the peptide chain IIIb from N-terminal to C-terminal are: TGF- ⁇ RII extracellular domain, linking element, VL sequence of anti-VEGF antibody, and human kappa light chain constant region sequence.
  • amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 23, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 23 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 24, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 24 sequence identity.
  • the peptide chain IIIa from the N-terminus to the C-terminus is: the VH sequence of an anti-VEGF antibody, a human IgG1 heavy chain constant region with LALA mutation, a linking element, and an anti-PD-L1 nanobody VHH and the peptide chain IIIb from the N-terminus to the C-terminus is: the VL sequence of the anti-VEGF antibody, the human kappa light chain constant region sequence, the linking element, and the TGF- ⁇ RII extracellular domain.
  • amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 23, or has ⁇ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 23 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 25, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 25 sequence identity.
  • an isolated polynucleotide encoding the multispecific antibody of the first aspect of the present invention.
  • the ratio of the polynucleotides encoding the two peptide chains in the polynucleotides is 1:1.
  • a vector comprising the polynucleotide according to the second aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof; preferably, the expression vector includes a viral vector, such as Lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a host cell contains the vector according to the third aspect of the present invention, or the polynucleotide according to the second aspect of the present invention is integrated into its genome;
  • the host cell expresses the multispecific antibody according to the first aspect of the present invention.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of: Escherichia coli, yeast cells, and mammalian cells.
  • a method for producing a multispecific antibody as described in the first aspect of the present invention comprising the steps of:
  • step (b) purifying and/or separating the culture obtained in step (a) to obtain the multispecific antibody.
  • the purification can be carried out by protein A affinity column purification and separation to obtain the target antibody.
  • the purity of the purified and separated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, preferably 100%.
  • an immunoconjugate is provided, the immunoconjugate contains:
  • conjugation moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or their combination.
  • the radionuclide includes:
  • a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( taxanes), benzodiazepines, or benzodiazepine-containing drugs (eg, pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines) indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PPDs pyrrolo[1,4]benzodiazepines
  • indoline benzodiazepines indolinobenzodiazepines and oxazolidinobenzodiazepines
  • vinca alkaloids or combinations thereof.
  • the toxin is selected from the following group:
  • Auristatins eg, auristatin E, auristatin F, MMAE, and MMAF
  • chlortetracycline maytansoid, gamatoxin, gamatoxin A-chain, combretastatin, docarmicin, Lastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissin, Dihydroxyanthraxdione, Actinomycin, Diphtheria Toxin, Pseudomonas Exotoxin (PE) A, PE40, Acacia toxin, Acacia A chain, Capsule root toxin A chain, ⁇ - Sarcinus, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin,
  • the coupling moiety is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable Enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, prodrugs Activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin).
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: a multivalent (eg, bivalent) multispecific antibody according to the first aspect of the present invention.
  • the multivalent refers to the multispecific antibody according to the first aspect of the present invention comprising multiple repetitions in the amino acid sequence of the immunoconjugate.
  • the reagent, detection plate or kit is used for: detecting PD-L1, VEGF and/or TGF- ⁇ in the sample; wherein, the agent is used for treating or preventing the expression of PD-L1 (ie PD-L1 positive) tumors, VEGF-expressing tumors, and/or TGF- ⁇ -expressing tumors.
  • the coupling part of the immunoconjugate is a diagnostic isotope.
  • the reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agents .
  • the detection is in vivo detection or in vitro detection.
  • the detection includes flow detection and cellular immunofluorescence detection.
  • the agent is used to block the interaction between PD-1 and PD-L1, and at the same time block its activity by binding to VEGF, and can bind to TGF- ⁇ ligand and block its downstream signal path.
  • the tumors include but are not limited to: acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer , liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, or a combination thereof.
  • a pharmaceutical composition contains:
  • the coupling part of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
  • the pharmaceutical composition also contains other drugs for treating tumors, such as cytotoxic drugs.
  • the other drugs for treating tumors include paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, and pembrolizumab.
  • the pharmaceutical composition is used to treat tumors expressing PD-L1 protein (ie PD-L1 positive).
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare a medicine for preventing and treating tumors.
  • a ninth aspect of the present invention there is provided one or more uses of the multispecific antibody according to the first aspect of the present invention selected from the group consisting of:
  • the tumor is a tumor expressing PD-L1 protein (ie PD-L1 positive).
  • the use is non-diagnostic and non-therapeutic.
  • a recombinant protein having: (i) the multispecific antibody of the first aspect of the present invention; and (ii) optionally assisted expression and/or or purified tag sequences.
  • the tag sequence includes 6His tag, HA tag and Fc tag.
  • the recombinant protein specifically binds to PD-L1, VEGF and/or TGF- ⁇ molecules.
  • a method for detecting PD-L1, VEGF and/or TGF- ⁇ molecules in a sample comprising the steps of: (1) combining the sample with the method described in the first aspect of the present invention (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of PD-L1, VEGF and/or TGF- ⁇ molecules in the sample.
  • a method for treating a disease comprising: administering to a subject in need the multispecific antibody as described in the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the multispecific antibody as described in the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the multispecific antibody as described in the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the multispecific antibody as described in the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the multispecific antibody as described in
  • the subject includes mammals, preferably humans.
  • the detection reagent comprises the immunoconjugate and the detection method according to the sixth aspect of the present invention an acceptable carrier.
  • the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agent.
  • the dosage form of the detection reagent is liquid or powder (eg, water preparation, injection, freeze-dried powder, tablet, buccal preparation, aerosol preparation).
  • kits for detecting PD-L1, VEGF and/or TGF- ⁇ molecules contains the immunoconjugate according to the sixth aspect of the present invention or the The detection reagent described in the thirteenth aspect of the present invention, and the description.
  • the instruction manual describes that the kit is used to non-invasively detect the expression of PD-L1, VEGF and/or TGF- ⁇ molecules in the subject to be tested.
  • the kit is used for the detection of tumors expressing PD-L1 protein (ie, PD-L1 positive).
  • multispecific antibody of the present invention As used herein, the terms "multispecific antibody of the present invention”, “polyclonal antibody of the present invention”, “anti-PD-L1/VEGF/TGF-beta multispecific antibody” have the same meaning and refer to specific recognition and Multispecific antibody that binds PD-L1, VEGF and TGF-beta.
  • the second protein functional region when it is a VEGF receptor (eg VEGFR1 and/or VEGFR2) or its functional fragment, since it has the function of blocking the binding of VEGF to the receptor similar to that of an antibody, the fusion protein at this time is described in this paper.
  • the invention is also broadly referred to as a multispecific antibody.
  • the third protein functional domain when it is a TGF- ⁇ receptor (such as TGF- ⁇ RII) or its functional fragment, because it has the function of blocking the binding of TGF- ⁇ and the receptor similar to that of an antibody, this time
  • the fusion protein is also broadly referred to as a multispecific antibody in the present invention.
  • the multispecific antibodies of the present invention may also be referred to as trispecific antibodies.
  • the present invention provides an anti-PD-L1/VEGF/TGF- ⁇ multispecific antibody, which comprises: an anti-PD-L1 single domain antibody, an anti-VEGF antibody element and a TGF- ⁇ RII extracellular domain.
  • the anti-PD-L1/VEGF/TGF- ⁇ multispecific antibody of the present invention comprises peptide chain I, and the peptide chain I comprises the following elements: anti-PD-L1 nanobody VHH chain, human immune The Fc region of a globulin, an anti-VEGF antibody element, a TGF- ⁇ RII extracellular domain, and optionally a linking element linking the above elements;
  • each element can be arranged in different order from N-terminus to C-terminus of peptide chain I;
  • the multispecific antibody is a homodimer formed by the peptide chain I through the disulfide bond of the Fc region.
  • the anti-PD-L1/VEGF/TGF- ⁇ multispecific antibody of the present invention is a heterodimer composed of peptide chain IIa and peptide chain IIb, wherein said peptide chain IIa and IIb are heterodimers
  • Each independently comprises the following elements: an anti-PD-L1 Nanobody VHH chain, a human IgG1 knockout or hole mutant sequence, an anti-VEGF antibody element, a TGF- ⁇ RII extracellular domain, and an optional linking element linking the above elements;
  • each element can be arranged in different order from N-terminus to C-terminus of peptide chain IIa or peptide chain IIb;
  • the peptide chain IIa includes a human IgG1 knockout mutant sequence
  • the peptide chain IIb includes a human IgG1 hole mutant sequence
  • the multispecific antibody is composed of the peptide chain IIa and the peptide chain IIb.
  • the anti-PD-L1/VEGF/TGF- ⁇ multispecific antibody of the present invention comprises peptide chain IIIa and peptide chain IIIb;
  • the peptide chain IIIa includes the following elements: the VH sequence of the anti-VEGF antibody, the constant region sequence of the human IgG1 heavy chain; the peptide chain IIIb includes the following elements: the VL sequence of the anti-VEGF antibody, the constant region of the human kappa light chain sequence;
  • the multispecific antibody is a heterotetramer formed by disulfide bonds between peptide chains IIIa and IIIb, and between peptide chains IIIb.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable regions of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure .
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • FR framework region
  • the light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • a light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
  • affinity is theoretically defined by an equilibrium association between intact antibody and antigen.
  • the affinity of the polyclonal antibody of the present invention can be evaluated or determined by KD value (dissociation constant) (or other measurement methods), such as Bio-layer interferometry (BLI), using FortebioRed96 instrument to measure and determine.
  • KD value dissociation constant
  • BBI Bio-layer interferometry
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies of the present invention or fragments thereof to form the conjugate.
  • the present invention also includes cell surface markers or antigens that bind to the PD-L1/VEGF multispecific antibody or fragment thereof.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably and all refer to polypeptides that specifically bind to PD-L1, VEGF and/or TGF- ⁇ proteins, such as A protein or polypeptide having a heavy chain variable region. They may or may not contain the starting methionine.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a variable region-containing heavy chain, as long as the variable region is associated with the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), four FR amino acids
  • FRs framework regions
  • FRs framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Accordingly, the present invention includes those molecules having CDR-bearing antibody heavy chain variable regions, as long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a polyclonal antibody with PD-L1, VEGF and/or TGF- ⁇ protein binding activity.
  • the term also includes variant forms of polypeptides comprising the same CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
  • the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof.
  • the present invention also includes fragments of the single domain antibodies of the present invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • Polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; coding sequences and various additional coding sequences for the mature polypeptides; coding sequences (and optional additional coding sequences) for the mature polypeptides and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
  • Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents ( For example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind PD-L1, VEGF and/or TGF- ⁇ protein molecules, and thus can be used to treat tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above-mentioned single domain antibody (or its conjugate) of the present invention and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the antibody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
  • colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the PD-L1/VEGF/TGF- ⁇ multispecific antibody can be labeled with colloidal gold to obtain a colloidal gold-labeled antibody.
  • the present invention also relates to methods of detecting PD-L1, VEGF and/or TGF-beta proteins.
  • the method steps are roughly as follows: obtaining a cell and/or tissue sample; lysing the sample in a medium; detecting the level of PD-L1, VEGF and/or TGF-beta protein in the lysed sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample existing in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the present invention also provides a detection kit for detecting the level of PD-L1, VEGF and/or TGF- ⁇
  • the kit includes an antibody that recognizes PD-L1, VEGF and/or TGF- ⁇ protein, and is used for dissolving samples of Lysis medium, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the multispecific antibody of the present invention has a wide range of biological application value and clinical application value, and its application involves the diagnosis and treatment of diseases related to PD-L1, VEGF and/or TGF- ⁇ , basic medical research, biological research and many other fields.
  • a preferred application is for clinical diagnosis and targeted therapy against PD-L1, VEGF and/or TGF-beta, such as tumor therapy.
  • the present invention has achieved any one or more of the following technical effects (1) to (8):
  • the multispecific antibody of the present invention has high specificity against three antigens, PD-L1, VEGF and TGF- ⁇ .
  • the multispecific antibody of the present invention has strong affinity for binding to three antigens, PD-L1, VEGF and TGF- ⁇ .
  • the multispecific antibody of the present invention has a long half-life.
  • the multispecific antibody of the present invention neutralizes TGF- ⁇ and VEGF in the tumor microenvironment on the basis of inhibiting the PD-1/PD-L1 pathway, restores T cell activity, enhances immune response, and blocks tumor angiogenesis, Thereby, the effect of inhibiting the occurrence and development of tumors can be more effectively improved.
  • FIG. 1A to 1E Schematic diagrams of the structures of the multispecific antibodies PD-L1xTGF ⁇ xVEGF TriAb 1, PD-L1xTGF ⁇ xVEGF TriAb 2, PD-L1xTGF ⁇ xVEGF TriAb 3, PD-L1xTGF ⁇ xVEGF TriAb 4 and PD-L1xTGF ⁇ xVEGF TriAb 5, respectively. Among them, the meaning of each legend is as follows:
  • FIGS 2A to 2D Schematic diagrams of the structures of the multispecific antibodies PD-L1xTGF ⁇ xVEGF TriAb 6, PD-L1xTGF ⁇ xVEGF TriAb 7, PD-L1xTGF ⁇ xVEGF TriAb 8 and PD-L1xTGF ⁇ xVEGF TriAb 9, respectively. Among them, the meaning of each legend is as follows:
  • Figure 3A Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention to CHO-S cells overexpressing human PD-L1.
  • Figure 3B Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 of the present invention to CHO-S cells overexpressing human PD-L1.
  • Figure 4A Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention blocking the binding of VEGFA protein and VEGFR protein.
  • Figure 4B Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 of the present invention blocking the binding of VEGFA protein and VEGFR protein.
  • Figure 5A Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention to human TGF- ⁇ 1 protein.
  • Figure 5B Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention to human TGF- ⁇ 2 protein.
  • Figure 5C Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention to human TGF- ⁇ 3 protein.
  • Figure 5D Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 of the present invention to human TGF- ⁇ 1 protein.
  • Figure 6A SPR (Surface Plasmon Resonance) analysis of the simultaneous binding of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention to human PD-L1, TGF- ⁇ 1 and VEGFA proteins.
  • Figure 6B SPR analysis diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 of the present invention that simultaneously binds human PD-L1, TGF- ⁇ 1 and VEGFA proteins.
  • Figure 7A Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention blocking PD-1/PD-L1 signaling pathway.
  • Figure 7B Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 of the present invention blocking PD-1/PD-L1 signaling pathway.
  • Figure 8A Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention blocking the TGF- ⁇ signaling pathway.
  • Figure 8B Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 of the present invention blocking TGF- ⁇ signaling pathway.
  • Figure 9A Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 1-5 of the present invention blocking the VEGF signaling pathway.
  • Figure 9B Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 of the present invention blocking the VEGF signaling pathway.
  • Figure 10A Line graph of the inhibition of tumor growth in mice (inoculated with MDA-MB-231) by multispecific antibody PD-L1xTGF ⁇ xVEGF 6 inhibition at different doses.
  • Figure 10B Bar graph comparing tumor weights 34 days after mice were injected with different doses of PD-L1xTGF ⁇ xVEGF6 multispecific antibody.
  • Figure 10C Line graph of body weight change in mice vaccinated with MDA-MB-231 after injection of different doses of PD-L1xTGF ⁇ xVEGF6 multispecific antibody.
  • Figure 11A A line graph showing the inhibition of tumor growth in mice (inoculated with NCI-H1975) by multispecific antibody PD-L1xTGF ⁇ xVEGF 6 and PD-L1 monoclonal antibody Atezolizumab.
  • Figure 11B Line graph of body weight change in NCI-H1975-vaccinated mice following injection of PD-L1xTGF ⁇ xVEGF6 multispecific antibody or Atezolizumab.
  • 9 anti-PD-L1 x VEGF x TGF- ⁇ multispecific antibodies were constructed and named as PD-L1 x TGF ⁇ x VEGF TriAb 1 to PD-L1 x TGF ⁇ x VEGF TriAb 9, respectively. used in the following examples.
  • PD-L1xTGF ⁇ xVEGF TriAb 1 It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1A, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 1, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO:2) and human IgGl Fc amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO:3).
  • SEQ ID NO:4 VEGF-binding domain amino acid sequence of the anti-VEGF fusion protein Aflibercept was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • the N-terminus of the TGF- ⁇ RII extracellular domain was then linked to the C-terminus of the anti-VEGF fusion protein Aflibercept through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • PD-L1xTGF ⁇ xVEGF TriAb 2 It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1B, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 7, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO:2) and human IgGl Fc amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO:3).
  • SEQ ID NO:6 The N-terminus of the TGF- ⁇ RII ectodomain (SEQ ID NO:6) was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • the N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO: 4) of the anti-VEGF fusion protein Aflibercept is connected to the extracellular TGF- ⁇ RII through a flexible peptide of 11 amino acid residues (G4S) 2G (SEQ ID NO: 8). C-terminal of the domain (SEQ ID NO:6).
  • PD-L1xTGF ⁇ xVEGF TriAb 3 It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1C, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 9, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO: 2).
  • the N-terminus of the extracellular domain of TGF- ⁇ RII was linked to the C-terminus of an anti-PD-L1 Nanobody via a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5) , and linked to the human IgG1 Fc amino acid sequence at its C-terminus through a flexible peptide of 11 amino acid residues (G4S) 2G (SEQ ID NO: 8) (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 3).
  • the N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO:4) of the anti-VEGF fusion protein Aflibercept was linked to the C-terminus of Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • PD-L1xTGF ⁇ xVEGF TriAb 4 It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1D, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 10, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO: 2).
  • SEQ ID NO:4 The N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO:4) of the anti-VEGF fusion protein Aflibercept was linked to the anti-PD-L1 nanoparticle through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • PD-L1xTGF ⁇ xVEGF TriAb 5 It consists of 2 different polypeptide chains, the schematic diagram of which is shown in Figure 1E, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 11, which contains anti-PD-L1 Nanobody C - Ye-18-5 (SEQ ID NO: 2) and human IgG1 knob mutant amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO: 12).
  • SEQ ID NO: 6 The N-terminus of the TGF- ⁇ RII ectodomain (SEQ ID NO:6) was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 13, which comprises the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO: 2) and the amino acid sequence of human IgG1 hole mutation (introduced LALA mutation to reduce Fc function, SEQ ID NO: 14).
  • the N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO:4) of the anti-VEGF fusion protein Aflibercept was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • PD-L1xTGF ⁇ xVEGF TriAb 6 consists of 2 peptide chains #1 and 2 peptide chains #2, the schematic structure of which is shown in Figure 2A, and peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 15, which contains anti- Nanobody C-Ye-18-5 of PD-L1 (SEQ ID NO: 2).
  • SEQ ID NO: 16 The N-terminus of the VH amino acid sequence (SEQ ID NO: 16) derived from the anti-VEGF antibody Bevacizumab was linked to the anti-VEGF via a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • the C-terminus of the Nanobody of PD-L1 was directly linked at the C-terminus of the VH amino acid sequence derived from the human IgG1 amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17).
  • the N-terminus of the TGF- ⁇ RII extracellular domain (SEQ ID NO:6) was then linked to the C-terminus of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 18, which comprises the VL amino acid sequence (SEQ ID NO: 19) derived from the anti-VEGF antibody Bevacizumab, and a human kappa light chain constant at the C-terminus of the VL amino acid sequence Region (CL) amino acid sequence (SEQ ID NO: 20).
  • PD-L1xTGF ⁇ xVEGF TriAb 7 It is composed of 2 peptide chains #1 and 2 peptide chains #2, the schematic diagram of which is shown in Figure 2B, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 21, which is derived from The VH amino acid sequence (SEQ ID NO: 16) of the anti-VEGF antibody Bevacizumab was derived from the human IgGl amino acid sequence by direct linkage at the C-terminus of the VH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17).
  • TGF- ⁇ RII extracellular domain SEQ ID NO:6
  • G4S 21 amino acid residues
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:22, which comprises the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO:2).
  • the N-terminus of the VL amino acid sequence (SEQ ID NO 19) derived from the anti-VEGF antibody Bevacizumab was linked to the C of the anti-PD-L1 Nanobody through a flexible peptide of 21 amino acid residues (G4S) 4G (SEQ ID NO: 5)
  • the C-terminal of the VL amino acid sequence is directly linked to the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 20).
  • PD-L1xTGF ⁇ xVEGF TriAb 8 It consists of 2 peptide chains #1 and 2 peptide chains #2, its structural schematic diagram is shown in Figure 2C, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 23, which is derived from The VH amino acid sequence (SEQ ID NO: 16) of the anti-VEGF antibody Bevacizumab was derived from the human IgGl amino acid sequence by direct linkage at the C-terminus of the VH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17).
  • N-terminus of the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO:2) is then linked to the C of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). end.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:24, which contains the TGF-betaRII extracellular domain (SEQ ID NO:6).
  • the N-terminus of the VL amino acid sequence (SEQ ID NO 19) derived from the anti-VEGF antibody Bevacizumab was linked to the C-terminus of TGF- ⁇ RII by a flexible peptide of 21 amino acid residues (G4S) 4G (SEQ ID NO: 5), and the The C-terminus of the VL amino acid sequence is directly linked to the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 20).
  • PD-L1xTGF ⁇ xVEGF TriAb 9 consists of 2 peptide chains #1 and 2 peptide chains #2, the schematic diagram of which is shown in Figure 2D, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 23, which is derived from The VH amino acid sequence (SEQ ID NO: 16) of the anti-VEGF antibody Bevacizumab was derived from the human IgGl amino acid sequence by direct linkage at the C-terminus of the VH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17).
  • the N-terminus of the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO:2) is then linked to the C of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 25, which comprises the VL amino acid sequence (SEQ ID NO: 19) derived from the anti-VEGF antibody Bevacizumab, and a human kappa light chain constant at the C-terminus of the VL amino acid sequence Region (CL) amino acid sequence (SEQ ID NO: 20).
  • the N-terminus of the TGF- ⁇ RII ectodomain (SEQ ID NO:6) was linked to the C-terminus of CL by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
  • CHO-S cells overexpressing human PD-L1 (CHO-S-hPD-L1 cells) were generated by transfection of the pCHO1.0 vector (purchased from Invitrogen) of human PD-L1 cDNA (purchased from Sino Biological) cloned into MCS.
  • the expanded CHO-S-hPD-L1 cells were adjusted to a cell density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well was added to a 96-well flow plate, and centrifuged for later use.
  • Example 3 ELISA detection of multispecific antibody anti-blocking VEGF/VEGFR protein level binding
  • the human VEGFR2 protein was diluted to an appropriate concentration with ELISA coating solution, added to the ELISA plate, and coated overnight at 4°C. Block with 5% BSA for 1 hour at room temperature.
  • the samples to be tested were serially diluted and incubated with biotinylated human VEGFA protein for 1 hour at room temperature. Add the incubated samples to the blocked ELISA plate and react at room temperature for 2 hours.
  • Example 4 ELISA detection of multispecific antibody binding to human TGF- ⁇ protein
  • Human TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 proteins were diluted to appropriate concentrations with ELISA coating solution, added to ELISA plate, and coated overnight at 4°C. Block with 5% BSA for 1 hour at room temperature. The sample to be tested was diluted in gradient and added to the blocked ELISA plate, and the reaction was carried out at room temperature for 2 hours. Washed 3 times with PBS-T (1:2000Tween) washing solution, added diluted goat anti-human IgG Fc-HRP secondary antibody and incubated for 1 hour at room temperature. Wash 3 times with PBS-T washing solution, add ELISA TMB color developing solution, place at room temperature for 3 min, add ELISA stop solution, and read the absorbance value at 450nm.
  • Example 5 Validation of multispecific antibody binding to PD-L1, VEGF and TGF- ⁇ simultaneously
  • Example 6 Multispecific antibodies block PD-1/PD-L1 signaling pathway
  • the target cells PD-L1aAPC/CHO-K1 were seeded into 96-well cell culture white bottom plates at 4 ⁇ 10 4 cells/well, and cultured overnight for 16 h. The next day, 5 ⁇ 10 4 cells/well of effector cells PD-1NFAT-RE Luciferase/Jurkat were mixed with serially diluted multispecific antibodies and added to target cells for 6 hours of incubation.
  • the bio-glo luciferase assay system Promega G7940 was used for color development and the chemiluminescence signal was collected with a microplate reader.
  • TGF- ⁇ -SMAD Luciferase/HEK293 effector cells were seeded into 96-well cell culture plates at 4 ⁇ 10 4 cells/well, and cultured for 8 h.
  • the serially diluted multispecific antibody was mixed with a final concentration of 0.5 ng/ml TGF- ⁇ 1, incubated at room temperature for 30 min, and then effector cells were added for further incubation for 18 hours.
  • the bio-glo luciferase assay system (Promega G7940) was used for color development and the chemiluminescence signal was collected with a microplate reader.
  • Example 8 Multispecific antibodies block the VEGF signaling pathway
  • VEGFRII Luciferase/HEK293 effector cells were seeded into 96-well cell culture plates at a rate of 1 ⁇ 10 5 cells/well.
  • the serially diluted multispecific antibody was mixed with a final concentration of 100 ng/ml VEGFA, incubated at room temperature for 30 min, and then added to the effector cells for further incubation for 6 h.
  • the bio-glo luciferase assay system (Promega G7940) was used for color development and the chemiluminescence signal was collected with a microplate reader.
  • the thermal stability of different antibodies was detected by DSC (Differential scanning calorimetry, differential scanning calorimetry).
  • the sample was diluted to 1 mg/ml with PBS; 5000 ⁇ fluorescent color reagent Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100 ⁇ fluorescent color developer Sypro Orange.
  • Table 1 Tm values of PD-L1xTGF ⁇ xVEGF multispecific antibodies
  • Table 2 Tm values of PD-L1xTGF ⁇ xVEGF multispecific antibodies
  • the multispecific antibodies of the present invention such as PD-L1xTGF ⁇ xVEGF TriAb 1-5 and multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6-9 have better thermal stability, especially among the multispecific antibodies PD-L1xTGF ⁇ xVEGF TriAb
  • the Tm of 6-9 were all greater than 68°C, and the stability was good, similar to that of conventional monoclonal antibodies.
  • the long-term thermal stability of the multispecific antibody was evaluated by detecting the changes in the purity and biological activity of the multispecific antibody after being placed at 40°C for 30 days.
  • the purity of the target antibody after being placed at 40°C for 0, 7 and 14 days was determined by the method of SEC.
  • PD-L1xTGF ⁇ xVEGF TriAb 6 98.5% 98.0% 96.5% PD-L1xTGF ⁇ xVEGF TriAb 7 95.9% 91.8% 84.3% PD-L1xTGF ⁇ xVEGF TriAb 8 97.5% 94.8% 92.3% PD-L1xTGF ⁇ xVEGF TriAb 9 85.7% 85.6% 80.3%
  • each drug contained 6 SD rats (3 males and 3 males, 12/12 hours light/dark adjustment, temperature 24°C ⁇ 2°C, humidity 40%-70%, free water and diet) were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd.
  • SD rats were injected with PD-L1xTGF ⁇ xVEGF TriAb 1 or PD-L1xTGF ⁇ xVEGF TriAb 4 by a single tail vein injection at a dose of 10 mg/kg.
  • Blood collection time points 5min, 15min, 0.5h, 2h, 6h, 24h, 48h, 96h, 168h, 336h, and 504h after administration, blood was collected from the jugular vein of rats.
  • Whole blood samples were placed at 2°C-8°C for 30min, centrifuged at 12000rpm for 5min to collect serum, the obtained serum was then centrifuged at 2°C-8°C, 12000rpm for 5min, and stored at -80°C to detect free PD-L1xTGF ⁇ xVEGF TriAb 1 or PD-L1xTGF ⁇ xVEGF TriAb in serum by ELISA 4 molecules.
  • mice The pharmacokinetic properties of multispecific antibodies in mice were tested experimentally. Each drug contained 6 SD rats (3 males and 3 females, 12/12 hours light/dark adjustment, temperature 24°C ⁇ 2°C, humidity 40°C). %-70%, free water and diet) were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, SD rats were injected with PD-L1xTGF ⁇ xVEGF TriAb 6 or PD-L1xTGF ⁇ xVEGF TriAb 9 molecules in a single tail vein at a dose of 10 mg/kg.
  • Blood collection time points 5 min, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours and 504 hours after administration, blood was collected from the jugular vein of rats.
  • Whole blood samples were placed at 2°C-8°C for 30min, centrifuged at 12000rpm for 5min to collect serum, the obtained serum was centrifuged at 2°C-8°C, 12000rpm for 5min, and stored at -80°C.
  • Free PD-L1xTGF ⁇ xVEGF TriAb 6 or PD-L1xTGF ⁇ xVEGF in serum was detected by ELISA TriAb 9 molecule.
  • mice The results show that the half-life of the free state molecule of the multispecific antibody PD-L1xTGF ⁇ xVEGF TriAb 6 of the present invention is about 62.4 hours in mice, and the half-life of the free state molecule of PD-L1xTGF ⁇ xVEGF TriAb 9 is about 48.8 hours in mice.
  • Example 13 Tumor inhibitory activity study of multispecific antibody (MDA-MB-231)
  • human breast cancer MDA-MD-231 cells/NSG mice were injected into human PBMC model to determine the anti-tumor effect of multispecific antibodies.
  • Sufficient MDA-MD-231 cells purchased from Addexbio
  • cells were collected after trypsinization, washed three times with PBS, counted, and inoculated into 8-week-old females at 7 ⁇ 10 6 cells/mouse
  • the NSG severe immunodeficiency mice purchased from Shanghai Nsweeping Model Biotechnology Co., Ltd.
  • Subcutaneous tumor formation of NSG mice was observed every day, and 4 ⁇ 10 6 PBMCs were injected into each mouse through tail vein 20 days after inoculation.
  • the maximum wide axis W and the maximum long axis L of the subcutaneous tumor in the right abdomen of each animal were measured with a vernier caliper, and the weight of each mouse was weighed with an electronic balance.
  • the mice with too large and too small tumor volume were eliminated, and the NSG mice were divided into 3 groups according to the average tumor volume, with 6 mice in each group. According to Table 6 grouping and dosing schedule grouping and injecting the corresponding dose of PD-L1xTGF ⁇ xVEGF TriAb 6.
  • mice Mouse tumor volume and mouse body weight were measured twice a week. The body weight and tumor volume of the mice were last measured 34 days after inoculation with tumor cells, and the mice were euthanized; compared with the PBS group, PD-L1xTGF ⁇ xVEGF TriAb6 could dose-dependently inhibit the growth of tumors, 2.8mg/kg and 28mg/kg TGI at doses were 76% and 88%, respectively; and tumor volume was significantly different from the PBS group.
  • the results for mean tumor weight, as well as tumor growth inhibition rates were generally in agreement with the trend for tumor volume (Figure 10A, Figure 10B and Table 7).
  • Table 7 Effects of each treatment group on tumor volume and TGI after subcutaneous inoculation of MDA-MB-231 in NSG mice
  • mice in each group The state of the mice in each group was observed daily and no abnormality was found; the weight of the mice was weighed twice a week, and the weight of the mice in each group did not decrease significantly. At the end of the test, no obvious lesions were found in the liver, kidneys, lungs and other major organs of the mice in each group, indicating that the drugs in each group were at the doses used in this test. No obvious toxicity was observed in mice.
  • the multispecific antibodies of the present invention can dose-dependently inhibit the growth of MDA-MB-231 subcutaneous transplanted tumor with an effective dose of 2.8 mg/kg; under 2 doses (2.8 mg/kg) mg/kg and 28mg/kg) showed no obvious toxicity to mice.
  • human lung cancer NCI-H1975 cells/NSG mice were injected with human PBMC model to determine the anti-tumor effect of multispecific antibodies.
  • Enough NCI-H1975 cells (purchased from Addexbio) were cultured and expanded in vitro, cells were collected after trypsinization, washed three times with PBS, counted, and inoculated into 8-week-old female NSGs at a rate of 4 ⁇ 10 6 cells/mouse Severe immunodeficiency mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were subcutaneously placed on the right abdomen.
  • Subcutaneous tumor formation of NSG mice was observed daily, and 5 ⁇ 10 6 PBMCs were injected into each mouse through tail vein 7 days after inoculation.
  • PBMC peripheral blood mononuclear cells
  • the maximum width axis W and the maximum long axis L of the subcutaneous tumor in the right abdomen of each animal were measured using a vernier caliper, and the body weight of each mouse was weighed using an electronic balance.
  • the mice with too large and too small tumor volume were eliminated, and the NSG mice were divided into 3 groups according to the average tumor volume, with 6 mice in each group. Grouped according to the grouping dosing schedule in Table 8 and injected the corresponding dose of anti-PD-L1 antibody atezolizumab (Atezolizumab, Roche) or PD-L1xTGF ⁇ xVEGF TriAb 6. Dosages are administered in equimolar concentrations of each component.
  • Table 8 Experimental protocol for PD-L1xTGF ⁇ xVEGF TriAb 6 tumor suppressor activity
  • mice Mouse tumor volume and mouse body weight were measured 3 times a week. The mice were euthanized by the last measurement of body weight and tumor volume 20 days after tumor cell inoculation; the TGI of 10 mg/kg atezolizumab or 14 mg/kg PD-L1xTGF ⁇ xVEGF TriAb 6 were 67% and 90%, respectively ; And the tumor volume was significantly different compared with the PBS group. Compared with the atezolizumab group, the PD-L1xTGF ⁇ xVEGF TriAb 6 group showed better antitumor efficacy with statistical significance. The results for mean tumor weight, as well as tumor growth inhibition rates, were generally in agreement with the trend for tumor volume ( Figure 11A and Table 9).
  • mice in each group The state of the mice in each group was observed daily, and no abnormality was found; the weight of the mice was weighed twice a week, and the weight of the mice in each group did not decrease significantly. Body weight did not decrease (see Figure 11B); at the end of the test, no obvious lesions were found in the liver, kidneys, lungs and other major organs of the mice in each group after dissection, indicating that the drugs in each group were effective at the doses used in this test. No obvious toxicity was seen in mice.
  • the multispecific antibodies of the present invention such as PD-L1xTGF ⁇ xVEGF TriAb 6 can effectively inhibit the growth of NCI-H1975 subcutaneously transplanted tumor, and are significantly better than atezolizumab at the same molar concentration; at a dose of 14 mg/ml PD-L1xTGF ⁇ xVEGF TriAb 6 showed no obvious toxicity in mice.

Abstract

Provided is an anti-PD-L1-anti-VEGF-anti-TGF-β multispecific antibody, comprising: a first protein functional region anti-PD-L1 single domain antibody targeting PD-L1, a second protein functional region VEGF receptor targeting VEGF, a VEGF receptor functional fragment, a fusion protein or an anti-VEGF antibody comprising the VEGF receptor functional fragment, a third protein functional region TGF-β receptor targeting TGF-β, a TGF-β receptor functional fragment, and a fusion protein or an anti-TGF-β antibody comprising the TGF-β receptor functional fragment. The multispecific antibody has a strong affinity to an antigen, neutralizes TGF-β and VEGF in a tumor microenvironment on the basis of inhibiting a PD-1/PD-L1 pathway, restores T cell activity, enhances immune response, blocks tumor angiogenesis, and can inhibit tumor formation and development.

Description

抗PD-L1-抗VEGF-抗TGF-β多特异性抗体、其药物组合物及用途Anti-PD-L1-anti-VEGF-anti-TGF-β multispecific antibody, pharmaceutical composition and use thereof 技术领域technical field
本发明属于生物医药领域，涉及一种抗PD-L1-抗VEGF-抗TGF-β多特异性抗体、其药物组合物及用途。The invention belongs to the field of biomedicine, and relates to an anti-PD-L1-anti-VEGF-anti-TGF-β multispecific antibody, a pharmaceutical composition and uses thereof.
背景技术Background technique
程序性死亡因子1配体1(programmed death 1 ligand 1，PD-L1)又称CD274，为B7家族成员，是PD-1的配体。PD-L1属于I型跨膜蛋白，共290个氨基酸，包含1个IgV样区、1个IgC样区、1个跨膜疏水区和1个由30个氨基酸组成的胞内区。与其他B7家族分子不同的是，PD-L1具有负向调节免疫应答的作用。研究发现，PD-L1主要表达于活化的T细胞、B细胞、巨噬细胞和树突状细胞等，除淋巴细胞外，PD-L1也表达于其它多种组织如胸腺、心脏、胎盘等的内皮细胞，以及各类非淋巴系如黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌、头颈癌等(Akintunde Akinleye&Zoaib Rasool，Journal of Hematology&Oncology volume 12,Article number:92(2019))。PD-L1在调节自身反应性T、B细胞和免疫耐受方面具有一定广泛性，并且在外周组织T和B细胞应答起作用。PD-L1在肿瘤细胞上的高表达与癌症患者的不良预后相关。Programmed death 1 ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and is a ligand of PD-1. PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including an IgV-like domain, an IgC-like domain, a transmembrane hydrophobic domain and an intracellular domain consisting of 30 amino acids. Unlike other B7 family molecules, PD-L1 negatively regulates immune responses. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages and dendritic cells, etc. In addition to lymphocytes, PD-L1 is also expressed in other tissues such as thymus, heart, placenta, etc. Endothelial cells, as well as various non-lymphoid lineages such as melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer, head and neck cancer, etc. (Akintunde Akinleye&Zoaib Rasool, Journal of Hematology&Oncology volume 12, Article number: 92 (2019)). PD-L1 is broad in regulating autoreactive T, B cells, and immune tolerance, and plays a role in peripheral tissue T and B cell responses. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.
血管内皮生长因子(Vascular endothelial growth factor,VEGF)又称血管通透因子(vascular permeability factor,VPF)或血管调理素(vasculotropin)，是一种具有高度特异性的促血管内皮细胞生长的同型二聚体的蛋白。VEGF家族蛋白包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、VEGF-F和胎盘生长因子(Placental growth factor,PIGF)等，其中VEGF-A在血管早期的形成中发挥重要作用。1983年，senger等最先从豚鼠的肝癌细胞分离出来，具有增加微静脉、小静脉通透性，促进血管内皮细胞***、增殖以及诱导血管形成等作用。同时，VEGF参与许多血管生成依赖性疾病的发病及其进展，包括癌症、某些炎性疾病以及糖尿病视网膜病变等。因此，在抗肿瘤药物研究中，VEGF是一个重要的靶点。Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF) or vasculotropin, is a highly specific homodimer that promotes the growth of vascular endothelial cells. body protein. VEGF family proteins include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor (PIGF), among which VEGF-A is involved in the early formation of blood vessels Play an important role. In 1983, senger et al. first isolated liver cancer cells from guinea pigs, which can increase the permeability of venules and venules, promote the division and proliferation of vascular endothelial cells, and induce the formation of blood vessels. Meanwhile, VEGF is involved in the pathogenesis and progression of many angiogenesis-dependent diseases, including cancer, certain inflammatory diseases, and diabetic retinopathy. Therefore, VEGF is an important target in antitumor drug research.
VEGF蛋白主要受体有VEGFR1，VEGFR2，VEGFR3，NRP1，NRP2 NRP3， 然而，VEGF家族蛋白成员与VEGF受体的结合具有选择性，其中VEGFA可以VEGFR1，VEGFR2结合，激活内源性激酶活化，促进新生血管生成。阻断VEGF与受体的结合能够应用于多种癌症的治疗，例如乳腺癌、结肠癌、肺癌、卵巢癌、子宫内膜癌、间皮瘤、***、肾癌(Rakesh R.Ramjiawan,Arjan W.Griffioen,and Dan G.Duda,Angiogenesis.2017 20(2):185–204.)。The main receptors of VEGF proteins are VEGFR1, VEGFR2, VEGFR3, NRP1, NRP2 and NRP3. However, the binding of VEGF family protein members to VEGF receptors is selective, among which VEGFA can bind to VEGFR1 and VEGFR2, activate endogenous kinase activation, and promote neogenesis. Angiogenesis. Blocking the binding of VEGF to the receptor can be applied to the treatment of various cancers, such as breast cancer, colon cancer, lung cancer, ovarian cancer, endometrial cancer, mesothelioma, cervical cancer, kidney cancer (Rakesh R. Ramjiawan, Arjan W. Griffioen, and Dan G. Duda, Angiogenesis. 2017 20(2):185–204.).
目前在全球市场中，上市或通过审批的VEGF药物共有23种，并覆盖了45个适应症，其中应用于抗肿瘤方向的VEGF-A拮抗剂的代表性产品为罗氏的安维汀(Avastin)即贝伐珠单抗(Bevacizumab)。该产品于2004年2月经FDA批准上市，用于转移性结直肠癌及晚期、转移性或复发性非小细胞肺癌的治疗，并于2010年2月在国内获批上市。国外已有多个Avastin的生物类似药获批上市，国内由齐鲁制药开发的贝伐珠单抗注射液(商品名：安可达)也于2019年12月在国内获批上市。雷珠单抗是第二代人源化的抗VEGF重组鼠单克隆抗体片段Fab部分，其与贝伐单抗(Bevacizumab)是从相同亲本鼠抗体获得。美国FDA于2006年6月30日批准其用于治疗年龄相关性黄斑变性(AMD)的患者。雷珠单抗相对贝伐珠单抗保持更好的VEGFA的亲和力，能更好的抑制血管生成，已开展胃癌、直肠癌等适应症。阿帕西普(Aflibercept)是VEGFR1的部分胞外片段(Ig domain 2)与VEGFR2的部分胞外片段(Ig domain 3)连接组合后融合于人IgG1-Fc的重组蛋白，可以同时阻断VEGFR1与VEGFR2和VEGFA结合，从而阻断血管上皮细胞的血管生成。阿帕西普主要适用于治疗有新生血管性(湿性)年龄相关黄斑变性(AMD)患者。同时在临床上开展晚期肠癌治疗(Caemen Stancan,etc,Rom J Morphol Embryol.2018 59(2):455–467)。由于阿帕西普拥有VEGFR1和VEGFR2的功能片段，具有与抗体类似的阻断VEGF和受体结合的功能。At present, there are 23 VEGF drugs listed or approved in the global market, covering 45 indications. Among them, the representative product of VEGF-A antagonists in the anti-tumor direction is Roche's Avastin. Namely bevacizumab (Bevacizumab). The product was approved by the FDA in February 2004 for the treatment of metastatic colorectal cancer and advanced, metastatic or recurrent non-small cell lung cancer, and was approved for marketing in China in February 2010. A number of Avastin biosimilars have been approved for marketing abroad. Bevacizumab injection (trade name: Ankea) developed by Qilu Pharmaceuticals in China was also approved for marketing in China in December 2019. Ranibizumab is a second generation humanized anti-VEGF recombinant murine monoclonal antibody fragment, Fab portion, obtained from the same parent murine antibody as Bevacizumab. It was approved by the US FDA on June 30, 2006 for the treatment of patients with age-related macular degeneration (AMD). Compared with bevacizumab, ranibizumab maintains a better affinity for VEGFA, can better inhibit angiogenesis, and has been developed for gastric cancer, rectal cancer and other indications. Aflibercept is a recombinant protein fused to human IgG1-Fc after a partial extracellular fragment (Ig domain 2) of VEGFR1 and a partial extracellular fragment (Ig domain 3) of VEGFR2 are linked and combined. VEGFR2 binds to VEGFA, thereby blocking angiogenesis in vascular epithelial cells. Apacept is mainly indicated for the treatment of patients with neovascular (wet) age-related macular degeneration (AMD). At the same time, the clinical treatment of advanced colorectal cancer is carried out (Caemen Stancan, etc, Rom J Morphol Embryol. 2018 59(2):455–467). Since apatacept possesses functional fragments of VEGFR1 and VEGFR2, it has the function of blocking VEGF and receptor binding similar to antibodies.
TGF-β主要由免疫***表达并分泌(包括TGF-β1、TGF-β2、TGF-β3)，与受体TGF-βR(包括RI/RII/RIII)结合后，可调节细胞生长、增殖、分化、迁移和凋亡，影响胚胎器官发育、机体免疫等，具有重要的生理功能。TGF-β1、TGF-β2和TGF-β3三个亚型均可以结合细胞表面的受体。TGF-βRI不直接结合TGF-β，RIII可结合TGF-β，但其糖修饰过于复杂。TGF-βRII对TGF-β1和TGF-β3具有极高亲和力(约5pM)，对TGF-β2具有较低亲和力(约6nM)。TGF-β在肿瘤发生和发展中扮演着非常重要而且双重的角色，TGF-β在肿瘤早期可以调控几种凋亡基因的表达从而诱导 肿瘤细胞的凋亡；而在肿瘤后期，大多数肿瘤细胞分泌大量TGF-β，一旦TGF-β水平过高，则转变成一个肿瘤促进因子：可抑制T和NK细胞、促进调节性T细胞、促进肿瘤血管生成、促进上皮细胞向间充质细胞转化等，从而促进肿瘤转移和发展。已有报道，TGF-β信号通路相关基因的异常调控是PD-1抗体耐药的原因之一，因此，TGF-β靶向药也成为抗癌药研发的重要方向。TGF-β is mainly expressed and secreted by the immune system (including TGF-β1, TGF-β2, TGF-β3), and after binding to receptor TGF-βR (including RI/RII/RIII), it can regulate cell growth, proliferation, differentiation , migration and apoptosis, affect embryonic organ development, immunity, etc., and have important physiological functions. All three isoforms of TGF-β1, TGF-β2 and TGF-β3 can bind to receptors on the cell surface. TGF-βRI does not directly bind TGF-β, and RIII can bind TGF-β, but its sugar modification is too complicated. TGF-βRII has a very high affinity (about 5 pM) for TGF-β1 and TGF-β3, and a lower affinity (about 6 nM) for TGF-β2. TGF-β plays a very important and dual role in the occurrence and development of tumors. TGF-β can regulate the expression of several apoptotic genes in the early stage of tumor to induce tumor cell apoptosis; in the later stage of tumor, most tumor cells Secretes a large amount of TGF-β, once the level of TGF-β is too high, it turns into a tumor-promoting factor: it can inhibit T and NK cells, promote regulatory T cells, promote tumor angiogenesis, and promote the transformation of epithelial cells to mesenchymal cells, etc. , thereby promoting tumor metastasis and development. It has been reported that the abnormal regulation of genes related to the TGF-β signaling pathway is one of the reasons for the resistance of PD-1 antibodies. Therefore, TGF-β targeting drugs have also become an important direction for the development of anticancer drugs.
目前上市在售的抗体药物多为单克隆抗体，治疗性单克隆抗体已被用于治疗癌症、自身免疫病、炎症和其他疾病，多数是针对一个靶标的特异性。然而，病人接受单克隆抗体治疗可能产生耐药性或无应答。并且有些疾病在体内的影响因素是多方面的，包括不同的信号通路、不同的细胞因子和受体的调节机制等，单一靶点的免疫疗法似乎并不足以摧毁癌细胞。因此，需要通过组合不同的药物，或是使用多特异性抗体的多重靶向策略来实现。Most of the antibody drugs currently on the market are monoclonal antibodies. Therapeutic monoclonal antibodies have been used to treat cancer, autoimmune diseases, inflammation and other diseases, and most of them are specific for one target. However, patients receiving monoclonal antibody therapy may develop resistance or non-response. And some diseases have multiple factors in the body, including different signaling pathways, different cytokine and receptor regulatory mechanisms, etc. It seems that immunotherapy with a single target is not enough to destroy cancer cells. Therefore, it needs to be achieved by combining different drugs or using multiple targeting strategies using multispecific antibodies.
靶向多个靶点的多功能抗体虽然是抗体药物研发的方向，但面临诸多挑战，比如临床前评价模型、表达量低、稳定性差、工艺复杂、质控差异性大等问题，因此一直以来多特异性抗体的研发困难重重。Although multifunctional antibodies targeting multiple targets are the direction of antibody drug development, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control. The development of multispecific antibodies is difficult.
因此，本领域迫切需要开发一种特异性佳、疗效好且易于制备的抗肿瘤多特异性抗体。Therefore, there is an urgent need in the art to develop an anti-tumor multispecific antibody with good specificity, good curative effect and easy preparation.
发明内容SUMMARY OF THE INVENTION
本发明人深入的研究和创造性的劳动，开发了一种抗PD-L1-抗VEGF-抗TGF-β多特异性抗体(后文中也称为抗PD-L1/VEGF/TGF-β多特异性抗体、抗PD-L1xVEGFxTGF-β多特异性抗体或本发明的多特异性抗体)。本发明人惊奇地发现，本发明的抗PD-L1-抗VEGF-抗TGF-β多特异性抗体具有针对PD-L1、VEGF和TGF-β三靶点的高亲和力，能够同时特异性地阻断这三个靶点，稳定性和安全性良好，具有良好的抗肿瘤活性。由此提供了下述发明：Through in-depth research and creative work, the inventors developed an anti-PD-L1-anti-VEGF-anti-TGF-β multispecific antibody (hereinafter also referred to as anti-PD-L1/VEGF/TGF-β multispecific antibody). antibody, anti-PD-L1xVEGFxTGF-beta multispecific antibody or multispecific antibody of the invention). The inventors have surprisingly found that the anti-PD-L1-anti-VEGF-anti-TGF-β multispecific antibody of the present invention has high affinity for the three targets of PD-L1, VEGF and TGF-β, and can specifically block the The three targets are stable and safe, and have good antitumor activity. The following invention is thus provided:
本发明的一个方面涉及一种多特异性抗体，包括：One aspect of the present invention pertains to a multispecific antibody comprising:
靶向PD-L1的第一蛋白功能区，Targeting the first protein functional region of PD-L1,
靶向VEGF的第二蛋白功能区，和targeting the second protein domain of VEGF, and
靶向TGF-β的第三蛋白功能区；Targeting the third protein functional domain of TGF-β;
其中，in,
所述第一蛋白功能区为抗PD-L1单域抗体，The first protein functional region is an anti-PD-L1 single domain antibody,
所述第二蛋白功能区为VEGF受体、VEGF受体功能片段、包含VEGF受体功能片段的融合蛋白或者抗VEGF抗体，The second protein functional region is a VEGF receptor, a VEGF receptor functional fragment, a fusion protein comprising a VEGF receptor functional fragment, or an anti-VEGF antibody,
所述第三蛋白功能区为TGF-β受体、TGF-β受体功能片段、包含TGF-β受体功能片段的融合蛋白或者抗TGF-β抗体。The third protein functional domain is a TGF-β receptor, a TGF-β receptor functional fragment, a fusion protein comprising a TGF-β receptor functional fragment, or an anti-TGF-β antibody.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述VEGF是VEGFA。In some embodiments of the present invention, the multispecific antibody, wherein the VEGF is VEGFA.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述VEGF受体是VEGFR1和/或VEGFR2。In some embodiments of the present invention, the multispecific antibody, wherein the VEGF receptor is VEGFR1 and/or VEGFR2.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述VEGF受体功能片段是具有VEGF受体功能的VEGFR1的胞外片段(例如Ig domain 2)和/或VEGFR2(例如Ig domain 3)的胞外片段。In some embodiments of the present invention, the multispecific antibody, wherein the VEGF receptor functional fragment is an extracellular fragment of VEGFR1 (eg, Ig domain 2) and/or VEGFR2 (eg, VEGFR2) with VEGF receptor function Extracellular fragment of Ig domain 3).
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述TGF-β是TGF-β1和/或TGF-β3。In some embodiments of the present invention, the multispecific antibody, wherein the TGF-β is TGF-β1 and/or TGF-β3.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述TGF-β受体是TGF-βRII。In some embodiments of the present invention, the multispecific antibody, wherein the TGF-β receptor is TGF-βRII.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述TGF-β受体功能片段是具有TGF-β受体功能的TGF-βRII的胞外片段。In some embodiments of the present invention, the multispecific antibody, wherein the TGF-β receptor functional fragment is an extracellular fragment of TGF-βRII with TGF-β receptor function.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述抗PD-L1单域抗体，其包含重链可变区，所述重链可变区包含氨基酸序列如SEQ ID NO:26所示的HCDR1、氨基酸序列如SEQ ID NO:27所示的HCDR2和氨基酸序列如SEQ ID NO:28所示的HCDR3；In some embodiments of the present invention, the multispecific antibody, wherein the anti-PD-L1 single domain antibody, comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence such as SEQ ID HCDR1 shown in NO:26, HCDR2 shown in amino acid sequence as shown in SEQ ID NO:27 and HCDR3 shown in amino acid sequence as shown in SEQ ID NO:28;
优选地，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:2所示。Preferably, the amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:2.
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)，其中重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3。在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号***确定。在某些实施方式中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号***确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过 IMGT编号***确定。IMGT编号***定义，请参见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307。The variable regions of the light and heavy chains determine the binding of antigens; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs), of which the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3, the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3. In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by an antibody or antigen-binding fragment thereof of the invention are preferably determined by the Kabat, Chothia or IMGT numbering systems. In certain embodiments, the CDRs contained by an antibody or antigen-binding fragment thereof of the invention are preferably determined by the IMGT numbering system. For the definition of the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009;38(suppl_1):D301-D307.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述VEGF受体为VEGFR1和/或VEGFR2。In some embodiments of the present invention, in the multispecific antibody, the VEGF receptor is VEGFR1 and/or VEGFR2.
在本发明的一些实施方式中，所述的多特异性抗体，其中，VEGF受体功能片段为VEGFR1胞外片段和/或VEGFR2胞外片段；In some embodiments of the present invention, the multispecific antibody, wherein the VEGF receptor functional fragment is a VEGFR1 extracellular fragment and/or a VEGFR2 extracellular fragment;
优选地，所述VEGF受体功能片段的氨基酸序列如SEQ ID NO:4所示。Preferably, the amino acid sequence of the VEGF receptor functional fragment is shown in SEQ ID NO:4.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述抗VEGF抗体，包含重链可变区和轻链可变区，其中：In some embodiments of the present invention, the multispecific antibody, wherein the anti-VEGF antibody, comprises a heavy chain variable region and a light chain variable region, wherein:
所述重链可变区包含氨基酸序列如SEQ ID NO:29所示的HCDR1、氨基酸序列如SEQ ID NO:30所示的HCDR2和氨基酸序列如SEQ ID NO:31所示的HCDR3；并且所述轻链可变区包含氨基酸序列如SEQ ID NO:32所示的LCDR1、氨基酸序列如SEQ ID NO:33所示的LCDR2和氨基酸序列如SEQ ID NO:34所示的LCDR3；The heavy chain variable region comprises HCDR1 whose amino acid sequence is shown in SEQ ID NO:29, HCDR2 whose amino acid sequence is shown in SEQ ID NO:30, and HCDR3 whose amino acid sequence is shown in SEQ ID NO:31; and the The light chain variable region comprises LCDR1 whose amino acid sequence is as shown in SEQ ID NO:32, LCDR2 whose amino acid sequence is as shown in SEQ ID NO:33 and LCDR3 whose amino acid sequence is as shown in SEQ ID NO:34;
优选地，所述重链可变区的氨基酸序列如SEQ ID NO:16所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:19所示；Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19;
优选地，所述抗VEGF抗体的重链恒定区为人Ig gamma-1chain C region或人Ig gamma-4 chain C region，并且其轻链恒定区为人Ig kappa chain C region；Preferably, the heavy chain constant region of the anti-VEGF antibody is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region thereof is a human Ig kappa chain C region;
优选地，所述抗VEGF抗体的重链恒定区还包含按照EU编号***的L234A突变和L235A突变(本发明中简称为LALA突变)；可选地，还包含G237A突变；Preferably, the heavy chain constant region of the anti-VEGF antibody further comprises L234A mutation and L235A mutation (abbreviated as LALA mutation in the present invention) according to the EU numbering system; optionally, G237A mutation;
优选地，所述抗VEGF抗体的重链恒定区的氨基酸序列如SEQ ID NO:17所示；Preferably, the amino acid sequence of the heavy chain constant region of the anti-VEGF antibody is shown in SEQ ID NO: 17;
优选地，所述抗VEGF抗体的轻链恒定区的氨基酸序列如SEQ ID NO:20所示。Preferably, the amino acid sequence of the light chain constant region of the anti-VEGF antibody is shown in SEQ ID NO:20.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述TGF-β受体为TGF-βRII。In some embodiments of the present invention, the multispecific antibody, wherein the TGF-β receptor is TGF-βRII.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述TGF-β受体功能片段为TGF-βRII的胞外片段；In some embodiments of the present invention, the multispecific antibody, wherein the TGF-β receptor functional fragment is an extracellular fragment of TGF-βRII;
优选地，所述TGF-β受体功能片段的氨基酸序列如SEQ ID NO:6所示。Preferably, the amino acid sequence of the TGF-β receptor functional fragment is shown in SEQ ID NO:6.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述第一蛋白功能区、 第二蛋白功能区和第三蛋白功能区独立地为1个、2个或3个拷贝。In some embodiments of the present invention, the multispecific antibody, wherein the first protein functional domain, the second protein functional domain and the third protein functional domain are independently 1, 2 or 3 copies .
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述第一蛋白功能区、第二蛋白功能区和第三蛋白功能区均为2个拷贝。In some embodiments of the present invention, in the multispecific antibody, the first protein functional domain, the second protein functional domain and the third protein functional domain are all 2 copies.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述第一蛋白功能区和第三蛋白功能区均为2个拷贝，所述第二蛋白功能区为1个拷贝。In some embodiments of the present invention, in the multispecific antibody, the first protein functional domain and the third protein functional domain are both 2 copies, and the second protein functional domain is 1 copy.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述第一蛋白功能区、第二蛋白功能区和第三蛋白功能区为直接连接或通过连接片段连接；In some embodiments of the present invention, the multispecific antibody, wherein, the first protein functional domain, the second protein functional domain and the third protein functional domain are directly connected or connected by connecting fragments;
优选地，所述连接片段的氨基酸序列独立地选自SEQ ID NO:5和SEQ ID NO:8。Preferably, the amino acid sequence of the linker fragment is independently selected from SEQ ID NO:5 and SEQ ID NO:8.
在本发明的一些实施方式中，所述的多特异性抗体，其还包含人IgG1Fc段；In some embodiments of the present invention, the multispecific antibody further comprises a human IgG1 Fc segment;
优选地，所述人IgG1的Fc段包含按照EU编号***的L234A突变和L235A突变(本发明中简称为LALA突变)；Preferably, the Fc segment of the human IgG1 comprises L234A mutation and L235A mutation (abbreviated as LALA mutation in the present invention) according to the EU numbering system;
优选地，所述人IgG1的Fc段的氨基酸序列如SEQ ID NO:3所示。Preferably, the amino acid sequence of the Fc segment of the human IgG1 is shown in SEQ ID NO:3.
在本发明的一些实施方式中，所述的多特异性抗体，其中，所述人IgG1Fc段与第一蛋白功能区、第二蛋白功能区和第三蛋白功能区中的两个直接连接或通过连接片段(linker)连接；In some embodiments of the present invention, the multispecific antibody, wherein the human IgG1 Fc segment is directly linked to or through two of the first protein functional domain, the second protein functional domain and the third protein functional domain connection fragment (linker) connection;
优选地，所述连接片段的氨基酸序列独立地选自SEQ ID NO:5和SEQ ID NO:8。Preferably, the amino acid sequence of the linker fragment is independently selected from SEQ ID NO:5 and SEQ ID NO:8.
在本发明的一些实施方式中，所述的多特异性抗体，按照N末端至C末端，依次包含：In some embodiments of the present invention, the multispecific antibody, in order from N-terminal to C-terminal, comprises:
第一蛋白功能区、人IgG1的Fc段、第二蛋白功能区和第三蛋白功能区；The first protein functional region, the Fc segment of human IgG1, the second protein functional region and the third protein functional region;
第一蛋白功能区、人IgG1的Fc段、第三蛋白功能区和第二蛋白功能区；The first protein functional region, the Fc segment of human IgG1, the third protein functional region and the second protein functional region;
第一蛋白功能区、第三蛋白功能区、人IgG1的Fc段和第二蛋白功能区；或者The first protein functional region, the third protein functional region, the Fc segment of human IgG1 and the second protein functional region; or
第一蛋白功能区、第二蛋白功能区、人IgG1的Fc段和第三蛋白功能区。The first protein functional region, the second protein functional region, the Fc segment of human IgG1 and the third protein functional region.
在本发明的一些实施方式中，所述的多特异性抗体，其为由两条相同的肽链形成的二聚体；In some embodiments of the present invention, the multispecific antibody is a dimer formed by two identical peptide chains;
优选地，所述肽链的氨基酸序列如SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:10所示。Preferably, the amino acid sequence of the peptide chain is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.
在本发明的一些实施方式中，所述的多特异性抗体，其为由两条相同的肽链形成的二聚体，按照N末端至C末端，所述肽链依次包含：In some embodiments of the present invention, the multispecific antibody is a dimer formed by two identical peptide chains, and from the N terminus to the C terminus, the peptide chains sequentially comprise:
第一蛋白功能区、人IgG1的Fc段、第二蛋白功能区和第三蛋白功能区；The first protein functional region, the Fc segment of human IgG1, the second protein functional region and the third protein functional region;
第一蛋白功能区、人IgG1的Fc段、第三蛋白功能区和第二蛋白功能区；The first protein functional region, the Fc segment of human IgG1, the third protein functional region and the second protein functional region;
第一蛋白功能区、第三蛋白功能区、人IgG1的Fc段和第二蛋白功能区；或者The first protein functional region, the third protein functional region, the Fc segment of human IgG1 and the second protein functional region; or
第一蛋白功能区、第二蛋白功能区、人IgG1的Fc段和第三蛋白功能区；The first protein functional region, the second protein functional region, the Fc segment of human IgG1 and the third protein functional region;
优选地，所述肽链的氨基酸序列如SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:10所示。Preferably, the amino acid sequence of the peptide chain is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.
在本发明的一些实施方式中，所述的多特异性抗体，其为由两条相同的肽链通过二硫键(例如2对或3对二硫键)形成的二聚体。In some embodiments of the present invention, the multispecific antibody is a dimer formed by two identical peptide chains through disulfide bonds (eg, 2 pairs or 3 pairs of disulfide bonds).
在本发明的一些实施方式中，所述的多特异性抗体，其还包含人IgG1重链恒定区；In some embodiments of the present invention, the multispecific antibody further comprises a human IgG1 heavy chain constant region;
优选地，所述人IgG1重链恒定区还包含Knob-in-hole突变；Preferably, the human IgG1 heavy chain constant region further comprises a Knob-in-hole mutation;
优选地，所述人IgG1重链恒定区的两条肽链的氨基酸序列分别如SEQ ID NO:12和SEQ ID NO:14所示。Preferably, the amino acid sequences of the two peptide chains of the human IgG1 heavy chain constant region are shown in SEQ ID NO: 12 and SEQ ID NO: 14, respectively.
在本发明的一些实施方式中，所述的多特异性抗体，其为由两条不同的第一肽链和第二肽链形成的二聚体，按照N末端至C末端，In some embodiments of the present invention, the multispecific antibody, which is a dimer formed by two different first peptide chains and second peptide chains, according to the N-terminus to the C-terminus,
第一肽链依次包含：第一蛋白功能区、人IgG1重链恒定区和第三蛋白功能区；并且第二肽链依次包含：第一蛋白功能区、人IgG1重链恒定区和第二蛋白功能区；The first peptide chain sequentially comprises: the first protein functional region, the human IgG1 heavy chain constant region and the third protein functional region; and the second peptide chain sequentially comprises: the first protein functional region, the human IgG1 heavy chain constant region and the second protein functional area;
优选地，第一肽链的氨基酸序列如SEQ ID NO:11所示，并且第二肽链的氨基酸序列如SEQ ID NO:13所示。Preferably, the amino acid sequence of the first peptide chain is shown in SEQ ID NO:11, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO:13.
在本发明的一些实施方式中，所述的多特异性抗体，其为由两条不同的第一肽链和第二肽链通过二硫键(例如2对或3对二硫键)形成的二聚体。In some embodiments of the present invention, the multispecific antibody is formed by two different first peptide chains and second peptide chains through disulfide bonds (eg, 2 pairs or 3 pairs of disulfide bonds). dimer.
在本发明的一些实施方式中，所述的多特异性抗体，其中：In some embodiments of the invention, the multispecific antibody, wherein:
所述第一蛋白功能区为两个拷贝，其中，两个拷贝分别连接在抗VEGF抗体的两条重链的N末端、抗VEGF抗体的两条轻链的N末端或抗VEGF抗体的两条轻链的C末端，或者一个拷贝连接在抗VEGF抗体的一条重链的N末端，另一个拷贝连接在与抗VEGF抗体的另一条重链相结合的轻链的N末端；并且所述第二蛋白功能区为两个拷贝，分别连接在抗VEGF抗体的两条重链的C末端；The first protein functional region has two copies, wherein the two copies are respectively connected to the N-terminus of the two heavy chains of the anti-VEGF antibody, the N-terminus of the two light chains of the anti-VEGF antibody, or the two copies of the anti-VEGF antibody. the C-terminus of the light chain, or one copy is attached to the N-terminus of one heavy chain of the anti-VEGF antibody and the other copy is attached to the N-terminus of the light chain bound to the other heavy chain of the anti-VEGF antibody; and the second There are two copies of the protein functional region, which are respectively connected to the C-terminus of the two heavy chains of the anti-VEGF antibody;
或者，or,
所述第二蛋白功能区为两个拷贝，其中，两个拷贝分别连接在抗VEGF抗体的两条重链的N末端、抗VEGF抗体的两条轻链的N末端或抗VEGF抗体的两条轻链的C末端，或者一个拷贝连接在抗VEGF抗体的一条重链的N末端，另一个拷贝连接在与抗 VEGF抗体的另一条重链相结合的轻链的N末端；并且所述第一蛋白功能区为两个拷贝，分别连接在抗VEGF抗体的两条重链的C末端。The second protein functional region has two copies, wherein the two copies are respectively connected to the N-terminus of the two heavy chains of the anti-VEGF antibody, the N-terminus of the two light chains of the anti-VEGF antibody, or the two copies of the anti-VEGF antibody. the C-terminus of the light chain, or one copy is attached to the N-terminus of one heavy chain of the anti-VEGF antibody, and the other copy is attached to the N-terminus of the light chain bound to the other heavy chain of the anti-VEGF antibody; and the first There are two copies of the protein functional domain, which are respectively connected to the C-terminus of the two heavy chains of the anti-VEGF antibody.
在本发明的一些实施方式中，所述的多特异性抗体，其为由两条相同的第一肽链和两条相同的第二肽链形成的四聚体，其中，In some embodiments of the present invention, the multispecific antibody is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein,
所述第一肽链的氨基酸序列如SEQ ID NO:15所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:18所示；The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 15; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 18;
所述第一肽链的氨基酸序列如SEQ ID NO:21所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:22所示；The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 21; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 22;
所述第一肽链的氨基酸序列如SEQ ID NO:23所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:24所示；The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 24;
或者，or,
所述第一肽链的氨基酸序列如SEQ ID NO:23所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:25所示。The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 25.
在本发明的一些实施方式中，所述的多特异性抗体，其为由两条相同的第一肽链和两条相同的第二肽链通过二硫键(例如两条肽链之间形成2对或3对二硫键)形成的四聚体。In some embodiments of the present invention, the multispecific antibody is composed of two identical first peptide chains and two identical second peptide chains through disulfide bonds (eg, formed between two peptide chains) 2 pairs or 3 pairs of disulfide bonds) formed tetramers.
本发明的另一方面涉及分离的核酸分子，其编码本发明中任一项所述的多特异性抗体。Another aspect of the present invention pertains to isolated nucleic acid molecules encoding the multispecific antibodies of any one of the present invention.
本发明还涉及一种载体，其包含本发明的分离的核酸分子。The present invention also relates to a vector comprising the isolated nucleic acid molecule of the present invention.
本发明还涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。The present invention also relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a vector of the present invention.
本发明的再一方面涉及一种制备本发明中任一项所述的多特异性抗体的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述多特异性抗体的步骤。Yet another aspect of the present invention relates to a method of preparing the multispecific antibody of any one of the present invention, comprising culturing a host cell of the present invention under suitable conditions, and recovering the polyspecific antibody from the cell culture Steps for specific antibodies.
本发明的再一方面涉及一种偶联物，其包括多特异性抗体以及偶联部分，其中，所述多特异性抗体为本发明中任一项所述的多特异性抗体，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。Yet another aspect of the present invention relates to a conjugate comprising a multispecific antibody and a coupling moiety, wherein the multispecific antibody is the multispecific antibody of any one of the present invention, the conjugate is The coupling moiety is a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
本发明的再一方面涉及一种试剂盒，其包含本发明中任一项所述的多特异性抗体，或者包含本发明的偶联物；Yet another aspect of the present invention relates to a kit comprising the multispecific antibody of any one of the present invention, or the conjugate of the present invention;
优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述多特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。Preferably, the kit further comprises a secondary antibody capable of specifically binding to the multispecific antibody; optionally, the secondary antibody further comprises a detectable label such as a radioisotope, fluorescent substance, luminescent substance , colored substances or enzymes.
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的多特异性抗体或者包含本发明的偶联物；可选地，其还包括药学上可接受的辅料。Yet another aspect of the present invention relates to a pharmaceutical composition comprising the multispecific antibody of any one of the present invention or the conjugate of the present invention; optionally, it further comprises pharmaceutically acceptable excipients .
本发明的再一方面涉及本发明中任一项所述的多特异性抗体或者本发明的偶联物在制备预防和/或治疗恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤选自肺癌、子宫内膜癌、间皮瘤、***、直肠癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌；优选地，所述肺癌为非小细胞肺癌。Yet another aspect of the present invention relates to the use of the multispecific antibody of any one of the present invention or the conjugate of the present invention in the preparation of a medicament for preventing and/or treating malignant tumors; preferably, the malignant tumor is selected from Selected from lung cancer, endometrial cancer, mesothelioma, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer; preferably, the lung cancer for non-small cell lung cancer.
本发明的再一方面涉及一种治疗和/或预防恶性肿瘤的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的多特异性抗体或者本发明的偶联物的步骤；优选地，所述恶性肿瘤选自肺癌、子宫内膜癌、间皮瘤、***、直肠癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌；优选地，所述肺癌为非小细胞肺癌。Yet another aspect of the present invention relates to a method of treating and/or preventing a malignant tumor, comprising administering to a subject in need thereof an effective amount of the multispecific antibody of any one of the present invention or the conjugate of the present invention Preferably, the malignant tumor is selected from the group consisting of lung cancer, endometrial cancer, mesothelioma, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, Esophageal cancer and head and neck cancer; preferably, the lung cancer is non-small cell lung cancer.
根据本发明中任一项所述的多特异性抗体或者本发明的偶联物，其用于治疗和/或预防恶性肿瘤；优选地，所述恶性肿瘤选自肺癌、子宫内膜癌、间皮瘤、***、直肠癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌；优选地，所述肺癌为非小细胞肺癌。According to any one of the multispecific antibodies of the present invention or the conjugate of the present invention, it is used for the treatment and/or prevention of malignant tumors; preferably, the malignant tumors are selected from lung cancer, endometrial cancer, Skin tumor, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer; preferably, the lung cancer is non-small cell lung cancer.
在本发明的一些实施方式中，所述的方法，其中，给予有需求的受试者以有效量的本发明中任一项所述的多特异性抗体的步骤为在手术治疗之前或之后，和/或在放射治疗之前或之后。In some embodiments of the invention, the method, wherein the step of administering to a subject in need thereof an effective amount of the multispecific antibody of any one of the invention is before or after surgical treatment, and/or before or after radiation therapy.
在本发明的一些实施方式中，所述的方法，其中，In some embodiments of the invention, the method, wherein,
本发明的多特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选4.8-24mg或1-10mg；或者，本发明的多特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg；The single administration dose of the multispecific antibody of the present invention is 0.1-100 mg per kilogram of body weight, preferably 4.8-24 mg or 1-10 mg; or, the single administration dose of the multispecific antibody of the present invention is each subject or 10-1000mg, preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg or 200mg;
优选地，每3天、4天、5天、6天、10天、1周、2周或3周给药一次；Preferably, it is administered every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
优选地，给药方式为静脉滴注或静脉注射。Preferably, the mode of administration is intravenous drip or intravenous injection.
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。 As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that elicits 50% of the maximal effect.
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Bethesda M.d.,Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,(1987and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature 1989；342:878-883，或者IMGT编号***定义，见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307的定义。As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair, respectively, form the antibody binding site. The assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al. Human Nature 1989;342:878-883, or IMGT numbering system definition, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors , MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): Definition of D301-D307.
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克 隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2021132464-appb-000001
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。 As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (
Figure PCTCN2021132464-appb-000001
G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256(5517): 495), but can also be obtained by recombinant DNA technology (eg, see US Patent 4,816,567).
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522 525；Reichmann et al.,Nature,1988；332:323329；Presta,Curr.Op.Struct.Biol.1992；2:593-596；和Clark,Immunol.Today 2000；21:397 402。在一些情况下，抗体的抗原结合片段是双抗体(Diabodies)，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.et al.,Proc.Natl.Acad.Sci.USA 1993；90:6444-6448和Poljak R.J.et al.,Structure 1994；2:1121-1123)。 As used herein, the term "humanized antibody" refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody) The antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, eg, Jones et al., Nature 1986; 321:522 525; Reichmann et al., Nature, 1988; 332:323329; Presta, Curr. Op. Struct. Biol . 1992;2:593-596; and Clark, Immunol.Today 2000;21:397402. In some cases, antigen-binding fragments of antibodies are diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but linkers that are too short are used to allow for both domains on the same chain Pairing between the domains forces the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993;90:6444 -6448 and Poljak RJet al., Structure 1994;2:1121-1123).
如本文中所述的融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。现有技术中熟知生产和纯化抗体和抗原结合片段的方法(如冷泉港的抗体实验技术指南，5-8章和15章)。A fusion protein as described herein is a protein product of co-expression of two genes obtained by DNA recombination. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art (eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15).
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。As used herein, the term "isolated" or "isolated" refers to artificially obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of. The terms "isolated" or "isolated" do not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载 体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as λ phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication site.
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞等的动物细胞。As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
如本文中所使用的，术语“药学上可接受的辅料”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19 th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。 As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (See, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed . Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途的有效量将取决于待治疗的疾病的严重度、患者自己的免疫***的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a disease-prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (eg, a tumor); a therapeutically-effective amount refers to an amount sufficient to cure or at least partially prevent the development of a disease in a patient already suffering from the disease. The amount of disease and its complications. Determining such effective amounts is well within the skill of the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently and many more.
如本文中所使用的，当提及PD-L1蛋白(Programmed death-ligand 1,NCBI Reference Sequence ID:NP_054862.1)的氨基酸序列时，其包括人PD-L1蛋白的全长， 或者人PD-L1的胞外片段PD-L 1ECD或者包含PD-L 1ECD的片段；还包括PD-L1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD-L1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PD-L1蛋白”应包括所有此类序列，包括所示的序列以及其天然或人工的变体。并且，当描述PD-L1蛋白的序列片段时，其不仅包括的序列片段，还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of the PD-L1 protein (Programmed death-ligand 1, NCBI Reference Sequence ID: NP_054862.1), it includes the full length of human PD-L1 protein, or the human PD-L1 protein. The extracellular fragment PD-L1ECD of L1 or a fragment comprising PD-L1ECD; also includes a fusion protein of PD-L1ECD, such as a fragment fused to an Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that in the amino acid sequence of PD-L1 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "PD-L1 protein" shall include all such sequences, including the sequences shown as well as natural or artificial variants thereof. And, when describing the sequence fragment of PD-L1 protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.
如本文中所使用的，当提及PD-1蛋白(NCBI Reference Sequence:NP_005009.2)的氨基酸序列时，其包括人PD-1蛋白的全长，或者人PD-1的胞外片段PD-1ECD或者包含PD-1ECD的片段；还包括PD-1蛋白的全长的融合蛋白或PD-1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD-1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述PD-1蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of PD-1 protein (NCBI Reference Sequence: NP_005009.2), it includes the full length of human PD-1 protein, or the extracellular fragment PD-1 of human PD-1 1ECD or a fragment comprising PD-1ECD; also includes a full-length fusion protein of PD-1 protein or a fusion protein of PD-1ECD, such as a fragment fused to a mouse or human IgG Fc protein fragment (mFc or hFc). However, those skilled in the art understand that in the amino acid sequence of PD-1 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "addition", protein" shall include all such sequences, including natural or artificial variants thereof. Also, when a sequence fragment of a PD-1 protein is described, it also includes the corresponding sequence fragment in its natural or artificial variant.
如本文中所使用的，当提及VEGFA蛋白(NCBI Reference Sequence:NP_001165097.1)的氨基酸序列时，其包括人VEGFA蛋白的全长，或者人VEGFA的功能片段或者包含VEGFA功能片段的片段；还包括VEGFA蛋白的全长的融合蛋白或VEGFA功能片段的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在VEGFA蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述VEGFA蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of a VEGFA protein (NCBI Reference Sequence: NP_001165097.1), it includes the full length of human VEGFA protein, or a functional fragment of human VEGFA or a fragment comprising a functional fragment of VEGFA; also Include full-length fusion proteins of VEGFA protein or fusion proteins of functional fragments of VEGFA, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced in the amino acid sequence of VEGFA protein without affecting its biological function. Thus, in the present invention, the term "addition", protein" shall include all such sequences, including natural or artificial variants thereof. Also, when a sequence fragment of a VEGFA protein is described, it also includes the corresponding sequence fragment in natural or artificial variants thereof.
如本文中所使用的，当提及TGF-β1蛋白(NCBI Reference Sequence:NP_000651.3)的氨基酸序列时，其包括人TGF-β1蛋白的全长，或者人TGF-β1的功能片段或者包含TGF-β1功能片段的片段；还包括TGF-β1蛋白的全长的融合蛋白或TGF-β1功能片段的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在TGF-β1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术 语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述TGF-β1蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of TGF-β1 protein (NCBI Reference Sequence: NP_000651.3), it includes the full length of human TGF-β1 protein, or a functional fragment of human TGF-β1 or comprising TGF-β1 - Fragments of β1 functional fragments; also include full-length fusion proteins of TGF-β1 protein or fusion proteins of TGF-β1 functional fragments, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that in the amino acid sequence of TGF-β1 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "added), protein" shall include all such sequences, including natural or artificial variants thereof. Also, when a sequence fragment of a TGF-beta1 protein is described, it also includes the corresponding sequence fragment in its natural or artificial variant.
如本文中所使用的，当提及TGF-β2蛋白(NCBI Reference Sequence:NP_003229.1)的氨基酸序列时，其包括人TGF-β2蛋白的全长，或者人TGF-β2的功能片段或者包含TGF-β2功能片段的片段；还包括TGF-β2蛋白的全长的融合蛋白或TGF-β2功能片段的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在TGF-β2蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述TGF-β2蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of TGF-β2 protein (NCBI Reference Sequence: NP_003229.1), it includes the full length of human TGF-β2 protein, or a functional fragment of human TGF-β2 or comprising TGF-β - Fragments of β2 functional fragments; also include full-length fusion proteins of TGF-β2 protein or fusion proteins of TGF-β2 functional fragments, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that in the amino acid sequence of TGF-β2 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "addition", protein" shall include all such sequences, including natural or artificial variants thereof. Also, when a sequence fragment of a TGF-beta2 protein is described, it also includes the corresponding sequence fragment in its natural or artificial variant.
如本文中所使用的，当提及TGF-β3蛋白(NCBI Reference Sequence:NP_003230.1)的氨基酸序列时，其包括人TGF-β3蛋白的全长，或者人TGF-β3的功能片段或者包含TGF-β3功能片段的片段；还包括TGF-β3蛋白的全长的融合蛋白或TGF-β3功能片段的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在TGF-β3蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述TGF-β3蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of TGF-β3 protein (NCBI Reference Sequence: NP_003230.1), it includes the full length of human TGF-β3 protein, or a functional fragment of human TGF-β3 or comprising TGF-β Fragments of β3 functional fragments; also include full-length fusion proteins of TGF-β3 protein or fusion proteins of TGF-β3 functional fragments, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced in the amino acid sequence of TGF-β3 protein without affecting its biological function. Thus, in the present invention, the term "addition", protein" shall include all such sequences, including natural or artificial variants thereof. Also, when a sequence fragment of a TGF-beta3 protein is described, it also includes the corresponding sequence fragment in its natural or artificial variant.
如本文中所使用的，当提及TGF-βRII蛋白(NCBI Reference Sequence:NP_003233.4)的氨基酸序列时，其包括人TGF-βRII蛋白的全长，或者人TGF-βRII的胞外片段TGF-βRII ECD或者包含TGF-βRII ECD的片段；还包括TGF-βRII蛋白的全长的融合蛋白或TGF-βRII ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在TGF-βRII蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述TGF-βRII蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of TGF-βRII protein (NCBI Reference Sequence: NP_003233.4), it includes the full length of human TGF-βRII protein, or the extracellular fragment of human TGF-βRII TGF- βRII ECD or a fragment comprising TGF-βRII ECD; also includes a full-length fusion protein of TGF-βRII protein or a fusion protein of TGF-βRII ECD, e.g., fused to a mouse or human IgG Fc protein fragment (mFc or hFc) fragment. However, those skilled in the art understand that in the amino acid sequence of TGF-βRII protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "addition", protein" shall include all such sequences, including natural or artificial variants thereof. Also, when a sequence fragment of a TGF-betaRII protein is described, it also includes the corresponding sequence fragment in its natural or artificial variant.
本发明中，术语“单域抗体”、“VHH”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)，它是具有完整功能的最小的 抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)。In the present invention, the terms "single domain antibody", "VHH" and "nanobody" have the same meaning, refer to cloning the variable region of the antibody heavy chain, and constructing a nanobody (VHH) composed of only one heavy chain variable region , which is the smallest fully functional antigen-binding fragment. Usually, an antibody that naturally lacks light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a Nanobody (VHH) consisting of only one heavy chain variable region.
在本发明中，如果没有特别说明，所述“第一”(例如第一蛋白功能区、第一肽链)、“第二”(例如第二蛋白功能区、第二肽链)和“第三”(例如第三蛋白功能区)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。In the present invention, unless otherwise specified, the "first" (eg, the first protein functional region, the first peptide chain), "the second" (eg, the second protein functional region, the second peptide chain) and "the first" "Three" (eg, the third protein functional region) is for reference distinction or clarity of expression, and does not have a typical sequential meaning.
本发明还涉及如下1至10项中的任意一项：The present invention also relates to any one of the following items 1 to 10:
1.一种多特异性抗体，其包括：1. A multispecific antibody comprising:
(a)抗PD-L1单域抗体；(a) anti-PD-L1 single domain antibody;
(b)抗VEGF的抗体元件；和(b) an anti-VEGF antibody element; and
(c)TGF-βRII胞外结构域。(c) TGF-βRII extracellular domain.
2.一种分离的多核苷酸，其编码第1项所述的多特异性抗体。2. An isolated polynucleotide encoding the multispecific antibody of item 1.
3.一种载体，其含有第2项所述的多核苷酸。3. A vector comprising the polynucleotide according to item 2.
4.一种宿主细胞，其含有第3项所述的载体，或其基因组中整合有第2项所述的多核苷酸；4. A host cell comprising the vector described in item 3, or the polynucleotide described in item 2 integrated in its genome;
或者，所述宿主细胞表达第1项所述的多特异性抗体。Alternatively, the host cell expresses the multispecific antibody of item 1.
5.一种产生第1项所述多特异性抗体的方法，包括如下步骤：5. A method of producing the multispecific antibody of item 1, comprising the steps of:
(a)在合适的条件下，培养第4项所述的宿主细胞，从而获得含所述多特异性抗体的培养物；和(a) culturing the host cell of item 4 under suitable conditions, thereby obtaining a culture containing the multispecific antibody; and
(b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的多特异性抗体。(b) purifying and/or separating the culture obtained in step (a) to obtain the multispecific antibody.
6.一种免疫偶联物，其特征在于，所述免疫偶联物含有：6. An immunoconjugate, characterized in that, the immunoconjugate contains:
(a)第1项所述的多特异性抗体；和(a) the multispecific antibody of item 1; and
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。(b) a conjugation moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or their combination.
7.第1项所述的多特异性抗体或者第6项所述的免疫偶联物的用途，其用于制备药剂、试剂、检测板或试剂盒；7. Use of the multispecific antibody described in item 1 or the immunoconjugate described in item 6 for the preparation of a medicament, a reagent, a detection plate or a kit;
其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1、VEGF和/或TGF-β；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤、表达VEGF的肿瘤，和/或表达TGF-β的肿瘤。Wherein, the reagent, detection plate or kit is used for: detecting PD-L1, VEGF and/or TGF-β in the sample; wherein, the agent is used for treating or preventing the expression of PD-L1 (ie PD-L1 positive) tumors, VEGF-expressing tumors, and/or TGF-β-expressing tumors.
8.一种药物组合物，其含有：8. A pharmaceutical composition comprising:
(i)第1项所述的多特异性抗体，或第6项所述的免疫偶联物；和(i) the multispecific antibody of item 1, or the immunoconjugate of item 6; and
(ii)药学上可接受的载体。(ii) A pharmaceutically acceptable carrier.
9.第1项所述的多特异性抗体的一种或多种选自下组的用途：9. Use of one or more of the multispecific antibodies of item 1 selected from the group consisting of:
(i)用于检测人PD-L1、VEGF和/或TGF-β分子；(ii)用于流式检测；(iii)用于细胞免疫荧光检测；(iv)用于***；(v)用于肿瘤诊断；(vi)用于阻断PD-1和PD-L1的相互作用；(vii)用于通过结合VEGF从而阻断其活性；和(viii)用于与TGF-β结合并阻断其下游信号通路。(i) for the detection of human PD-L1, VEGF and/or TGF-β molecules; (ii) for flow detection; (iii) for cellular immunofluorescence detection; (iv) for tumor treatment; (v) For tumor diagnosis; (vi) for blocking the interaction of PD-1 and PD-L1; (vii) for blocking its activity by binding to VEGF; and (viii) for binding to TGF-β and blocking interrupt its downstream signaling pathway.
10.一种重组蛋白，其含有：(i)第1项所述的多特异性抗体；以及(ii)任选的协助表达和/或纯化的标签序列。10. A recombinant protein comprising: (i) the multispecific antibody of item 1; and (ii) an optional tag sequence to facilitate expression and/or purification.
本发明还涉及如下的第一方面至第十四方面中的任一方面：The present invention also relates to any one of the following first to fourteenth aspects:
在本发明的第一方面，提供了一种多特异性抗体，所述多特异性抗体包括：In a first aspect of the present invention, a multispecific antibody is provided, the multispecific antibody comprising:
(a)抗PD-L1单域抗体；(a) anti-PD-L1 single domain antibody;
(b)抗VEGF的抗体元件；和(b) an anti-VEGF antibody element; and
(c)TGF-βRII胞外结构域。(c) TGF-βRII extracellular domain.
在另一优选例中，所述的多特异性抗体包括1-3个抗PD-L1单域抗体，较佳地，包括1个或2个抗PD-L1单域抗体。In another preferred embodiment, the multispecific antibody includes 1-3 anti-PD-L1 single-domain antibodies, preferably, includes 1 or 2 anti-PD-L1 single-domain antibodies.
在另一优选例中，所述的PD-L1单域抗体可以阻断PD-1和PD-L1的相互作用。In another preferred embodiment, the PD-L1 single domain antibody can block the interaction between PD-1 and PD-L1.
在另一优选例中，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:2所示，或与SEQ ID NO:2所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred embodiment, the amino acid sequence of the anti-PD-L1 single domain antibody is as shown in SEQ ID NO: 2, or has ≥ 85% (preferably 90%, more preferably ≥ 85% of the sequence shown in SEQ ID NO: 2) 95%) sequence identity.
在另一优选例中，所述的多特异性抗体包括1-3个抗VEGF的抗体元件，较佳地，包括1个或2个抗VEGF的抗体元件。In another preferred embodiment, the multispecific antibody includes 1-3 anti-VEGF antibody elements, preferably, includes 1 or 2 anti-VEGF antibody elements.
在另一优选例中，所述的抗VEGF的抗体元件能够结合VEGF并阻断其活性。In another preferred embodiment, the anti-VEGF antibody element can bind to VEGF and block its activity.
在另一优选例中，所述抗VEGF的抗体元件的氨基酸序列如SEQ ID NO:4所示，或与SEQ ID NO:4所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred embodiment, the amino acid sequence of the anti-VEGF antibody element is as shown in SEQ ID NO: 4, or has ≥ 85% (preferably 90%, more preferably 95% with the sequence shown in SEQ ID NO: 4) %) sequence identity.
在另一优选例中，所述的多特异性抗体包括1-3个TGF-βRII胞外结构域，较佳地，包括1个或2个TGF-βRII胞外结构域。In another preferred embodiment, the multispecific antibody includes 1-3 extracellular domains of TGF-βRII, preferably 1 or 2 extracellular domains of TGF-βRII.
在另一优选例中，所述的TGF-βRII胞外结构域能够与TGF-β配体结合并阻断其 下游信号通路。In another preferred embodiment, the TGF-βRII extracellular domain can bind to TGF-β ligand and block its downstream signaling pathway.
在另一优选例中，所述TGF-βRII胞外结构域的氨基酸序列如SEQ ID NO:6所示，或与SEQ ID NO:6所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred embodiment, the amino acid sequence of the extracellular domain of TGF-βRII is as shown in SEQ ID NO: 6, or has ≥ 85% (preferably 90%, more preferably ≥ 85% of the sequence shown in SEQ ID NO: 6) 95%) sequence identity.
在另一优选例中，所述的多特异性抗体还包含源自人免疫球蛋白的Fc区。In another preferred embodiment, the multispecific antibody further comprises an Fc region derived from human immunoglobulin.
在另一优选例中，所述的人免疫球蛋白选自下组：IgG1、IgG2、IgG3、IgG4，或其组合；优选地为IgG1。In another preferred embodiment, the human immunoglobulin is selected from the following group: IgG1, IgG2, IgG3, IgG4, or a combination thereof; preferably IgG1.
在另一优选例中，所述多特异性抗体的Fc区选自下组：CH1+CL1结构域、人IgG结构域或其组合。In another preferred embodiment, the Fc region of the multispecific antibody is selected from the group consisting of a CH1+CL1 domain, a human IgG domain or a combination thereof.
在另一优选例中，所述Fc区是经过改造的突变型；优选地为LALA突变型；更加优选地为LALA突变型并且含有Knob-in-hole突变型。In another preferred embodiment, the Fc region is an engineered mutant; preferably, it is a LALA mutant; more preferably, it is a LALA mutant and contains a Knob-in-hole mutant.
在另一优选例中，所述的多特异性抗体包含肽链I，所述的肽链I中包含以下元件：抗PD-L1纳米抗体VHH链、人免疫球蛋白的Fc区、抗VEGF的抗体元件、TGF-βRII胞外结构域，和任选的连接上述元件的连接元件；In another preferred embodiment, the multispecific antibody comprises peptide chain I, and the peptide chain I includes the following elements: anti-PD-L1 nanobody VHH chain, Fc region of human immunoglobulin, anti-VEGF antibody elements, the TGF-βRII extracellular domain, and optional linking elements linking the above-mentioned elements;
其中，各元件可以以不同的顺序自肽链I的N端到C端排列；Wherein, each element can be arranged in different order from N-terminus to C-terminus of peptide chain I;
并且，所述的多特异性抗体是由肽链I通过所述Fc区的二硫键作用形成的同源二聚体。And, the multispecific antibody is a homodimer formed by the peptide chain I through the disulfide bond of the Fc region.
在另一优选例中，所述连接元件的序列为(G 4S) n，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n为2或4。 In another preferred example, the sequence of the connecting element is (G 4 S) n , wherein n is a positive integer ( eg 1, 2, 3, 4, 5 or 6), preferably, n is 2 or 4 .
在另一优选例中，所述连接元件的氨基酸序列如SEQ ID NO:6所示，或与SEQ ID NO:6所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred embodiment, the amino acid sequence of the linking element is as shown in SEQ ID NO:6, or has ≥85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO:6 sequence identity.
在另一优选例中，所述的肽链I自N端到C端分别为：抗PD-L1纳米抗体VHH链、人免疫球蛋白的Fc区、连接元件、抗VEGF的抗体元件、连接元件，和TGF-βRII胞外结构域。In another preferred embodiment, the peptide chain I from N-terminal to C-terminal are respectively: anti-PD-L1 Nanobody VHH chain, Fc region of human immunoglobulin, connecting element, anti-VEGF antibody element, connecting element , and the TGF-βRII extracellular domain.
在另一优选例中，所述的肽链I的氨基酸序列如SEQ ID NO:1所示，或与SEQ ID NO:1所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred example, the amino acid sequence of the peptide chain I is as shown in SEQ ID NO: 1, or has ≥ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 1 ) sequence identity.
在另一优选例中，所述的肽链I自N端到C端分别为：抗PD-L1纳米抗体VHH链、人免疫球蛋白的Fc区、连接元件、TGF-βRII胞外结构域、连接元件，和抗VEGF的抗体元件。In another preferred embodiment, the peptide chain I from N-terminal to C-terminal are respectively: anti-PD-L1 Nanobody VHH chain, Fc region of human immunoglobulin, connecting element, TGF-βRII extracellular domain, Linking elements, and anti-VEGF antibody elements.
在另一优选例中，所述的肽链I的氨基酸序列如SEQ ID NO:7所示，或与SEQ ID  NO:7所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred embodiment, the amino acid sequence of the peptide chain I is shown in SEQ ID NO:7, or has ≥85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO:7 ) sequence identity.
在另一优选例中，所述的肽链I自N端到C端分别为：抗PD-L1纳米抗体VHH链、连接元件、TGF-βRII胞外结构域、连接元件、人免疫球蛋白的Fc区、连接元件，和抗VEGF的抗体元件。In another preferred embodiment, the peptide chain I from the N-terminus to the C-terminus are: anti-PD-L1 Nanobody VHH chain, connecting element, TGF-βRII extracellular domain, connecting element, human immunoglobulin Fc regions, linking elements, and anti-VEGF antibody elements.
在另一优选例中，所述的肽链I的氨基酸序列如SEQ ID NO:9所示，或与SEQ ID NO:9所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred example, the amino acid sequence of the peptide chain I is as shown in SEQ ID NO: 9, or has ≥ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 9 ) sequence identity.
在另一优选例中，所述的肽链I自N端到C端分别为：抗PD-L1纳米抗体VHH链、连接元件、抗VEGF的抗体元件、连接元件、人免疫球蛋白的Fc区、连接元件，和TGF-βRII胞外结构域。In another preferred embodiment, the peptide chain I from N-terminal to C-terminal are respectively: anti-PD-L1 Nanobody VHH chain, connecting element, anti-VEGF antibody element, connecting element, Fc region of human immunoglobulin , the linking element, and the TGF-βRII extracellular domain.
在另一优选例中，所述的肽链I的氨基酸序列如SEQ ID NO:10所示，或与SEQ ID NO:10所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred example, the amino acid sequence of the peptide chain I is shown in SEQ ID NO: 10, or has ≥ 85% (preferably 90%, more preferably 95% of the sequence shown in SEQ ID NO: 10) ) sequence identity.
在另一优选例中，所述的多特异性抗体是由肽链IIa和肽链IIb构成的异源二聚体，所述的肽链IIa和IIb中各自独立地包括以下元件：抗PD-L1纳米抗体VHH链、人IgG1 knob突变或hole突变序列、抗VEGF的抗体元件、TGF-βRII胞外结构域，和任选的连接上述元件的连接元件；In another preferred embodiment, the multispecific antibody is a heterodimer composed of a peptide chain IIa and a peptide chain IIb, and the peptide chains IIa and IIb each independently include the following elements: anti-PD- L1 Nanobody VHH chain, human IgG1 knockout or hole mutant sequence, anti-VEGF antibody element, TGF-βRII extracellular domain, and optional linking elements linking the above elements;
其中，各元件可以以不同的顺序自肽链IIa或肽链IIb的N端到C端排列；Wherein, each element can be arranged in different order from N-terminus to C-terminus of peptide chain IIa or peptide chain IIb;
并且，所述的肽链IIa中包括人IgG1 knob突变序列；所述的肽链IIb中包括人IgG1 hole突变序列；并且，所述的多特异性抗体是由肽链IIa和肽链IIb通过所述人IgG恒定区的二硫键作用及杵-进入-臼结构(Knob–in-hole)而形成的异源二聚体。In addition, the peptide chain IIa includes a human IgG1 knockout mutant sequence; the peptide chain IIb includes a human IgG1 hole mutant sequence; and, the multispecific antibody is composed of the peptide chain IIa and the peptide chain IIb. The heterodimer formed by the disulfide bond interaction of the human IgG constant region and the knob-in-hole structure (Knob-in-hole).
在另一优选例中，所述人IgG1 knob突变的氨基酸序列中，基于SEQ ID NO:12所示的氨基酸序列的第132位具有S132C突变，并且第144位具有T144W突变。In another preferred embodiment, in the amino acid sequence of the human IgG1 knockout mutation, the 132nd position based on the amino acid sequence shown in SEQ ID NO: 12 has the S132C mutation, and the 144th position has the T144W mutation.
在另一优选例中，所述人IgG1 hole突变的氨基酸序列中，基于SEQ ID NO:14所示的氨基酸序列的第127位具有Y127C突变、第144位具有T144S突变，并且第146位具有L146A突变。In another preferred example, in the amino acid sequence of the human IgG1 hole mutation, based on the amino acid sequence shown in SEQ ID NO: 14, the 127th position has a Y127C mutation, the 144th position has a T144S mutation, and the 146th position has a L146A mutation mutation.
在另一优选例中，所述的肽链IIa自N端到C端分别为：抗PD-L1纳米抗体VHH链、人IgG1 knob突变序列、连接元件、TGF-βRII胞外结构域、连接元件，和TGF-βRII胞外结构域；并且所述的肽链IIb自N端到C端分别为：抗PD-L1纳米抗体VHH链、人IgG1 hole突变序列、连接元件，和抗VEGF的抗体元件。In another preferred embodiment, the peptide chain IIa from the N-terminus to the C-terminus is: anti-PD-L1 Nanobody VHH chain, human IgG1 knockout mutant sequence, connecting element, TGF-βRII extracellular domain, connecting element , and TGF-βRII extracellular domain; and the peptide chain IIb from the N-terminus to the C-terminus are: anti-PD-L1 nanobody VHH chain, human IgG1 hole mutation sequence, linking element, and anti-VEGF antibody element .
在另一优选例中，所述肽链IIa的氨基酸序列如SEQ ID NO:11所示，或与SEQ ID  NO:11所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性；并且所述肽链IIb的氨基酸序列如SEQ ID NO:13所示，或与SEQ ID NO:13所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred example, the amino acid sequence of the peptide chain IIa is as shown in SEQ ID NO: 11, or has ≥ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 11 and the amino acid sequence of the peptide chain IIb is as shown in SEQ ID NO: 13, or has ≥ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 13 sequence identity.
在另一优选例中，所述的多特异性抗体中包括肽链IIIa和肽链IIIb；In another preferred embodiment, the multispecific antibody includes peptide chain IIIa and peptide chain IIIb;
其中，所述肽链IIIa中包括以下元件：抗VEGF抗体的VH序列、人IgG1重链恒定区序列；所述肽链IIIb中包括以下元件：抗VEGF抗体的VL序列、人κ轻链恒定区序列；Wherein, the peptide chain IIIa includes the following elements: the VH sequence of the anti-VEGF antibody, the constant region sequence of the human IgG1 heavy chain; the peptide chain IIIb includes the following elements: the VL sequence of the anti-VEGF antibody, the constant region of the human kappa light chain sequence;
并且，所述的多特异性抗体是由肽链IIIa和肽链IIIb之间、以及肽链IIIb之间通过二硫键作用形成的异源四聚体。In addition, the multispecific antibody is a heterotetramer formed by disulfide bonds between peptide chains IIIa and IIIb, and between peptide chains IIIb.
在另一优选例中，所述的人IgG1重链恒定区序列具有LALA突变。In another preferred embodiment, the human IgG1 heavy chain constant region sequence has LALA mutation.
在另一优选例中，所述的肽链IIIa自N端到C端分别为：抗PD-L1纳米抗体VHH链、连接元件、抗VEGF抗体的VH序列、具有LALA突变的人IgG1重链恒定区、连接元件、TGF-βRII胞外结构域；并且所述的肽链IIIb自N端到C端分别为：抗VEGF抗体的VL序列、人κ轻链恒定区序列。In another preferred embodiment, the peptide chain IIIa from the N-terminus to the C-terminus is: anti-PD-L1 Nanobody VHH chain, linking element, VH sequence of anti-VEGF antibody, constant human IgG1 heavy chain with LALA mutation Region, linking element, TGF-βRII extracellular domain; and the peptide chain IIIb from N-terminal to C-terminal are: VL sequence of anti-VEGF antibody, human kappa light chain constant region sequence.
在另一优选例中，所述肽链IIIa的氨基酸序列如SEQ ID NO:15所示，或与SEQ ID NO:15所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性；并且所述肽链IIIb的氨基酸序列如SEQ ID NO:18所示，或与SEQ ID NO:18所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred example, the amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 15, or has ≥ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 15 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 18, or has ≥ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 18 sequence identity.
在另一优选例中，所述的肽链IIIa自N端到C端分别为：抗VEGF抗体的VH序列、具有LALA突变的人IgG1重链恒定区、连接元件、TGF-βRII胞外结构域；并且所述的肽链IIIb自N端到C端分别为：抗PD-L1纳米抗体VHH链、连接元件、抗VEGF抗体的VL序列，和人κ轻链恒定区序列。In another preferred embodiment, the peptide chain IIIa from the N-terminus to the C-terminus is: the VH sequence of an anti-VEGF antibody, a human IgG1 heavy chain constant region with LALA mutation, a linking element, and an extracellular domain of TGF-βRII. and the peptide chain IIIb from the N-terminus to the C-terminus are respectively: the anti-PD-L1 nanobody VHH chain, the connecting element, the VL sequence of the anti-VEGF antibody, and the human kappa light chain constant region sequence.
在另一优选例中，所述肽链IIIa的氨基酸序列如SEQ ID NO:21所示，或与SEQ ID NO:21所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性；并且所述肽链IIIb的氨基酸序列如SEQ ID NO:22所示，或与SEQ ID NO:22所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred embodiment, the amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 21, or has ≥ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 21 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 22, or has ≥ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 22 sequence identity.
在另一优选例中，所述的肽链IIIa自N端到C端分别为：抗VEGF抗体的VH序列、具有LALA突变的人IgG1重链恒定区、连接元件、抗PD-L1纳米抗体VHH链；并且所述的肽链IIIb自N端到C端分别为：TGF-βRII胞外结构域、连接元件、 抗VEGF抗体的VL序列，和人κ轻链恒定区序列。In another preferred embodiment, the peptide chain IIIa from the N-terminus to the C-terminus is: the VH sequence of an anti-VEGF antibody, a human IgG1 heavy chain constant region with LALA mutation, a linking element, and an anti-PD-L1 nanobody VHH and the peptide chain IIIb from N-terminal to C-terminal are: TGF-βRII extracellular domain, linking element, VL sequence of anti-VEGF antibody, and human kappa light chain constant region sequence.
在另一优选例中，所述肽链IIIa的氨基酸序列如SEQ ID NO:23所示，或与SEQ ID NO:23所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性；并且所述肽链IIIb的氨基酸序列如SEQ ID NO:24所示，或与SEQ ID NO:24所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred example, the amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 23, or has ≥ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 23 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 24, or has ≥ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 24 sequence identity.
在另一优选例中，所述的肽链IIIa自N端到C端分别为：抗VEGF抗体的VH序列、具有LALA突变的人IgG1重链恒定区、连接元件、抗PD-L1纳米抗体VHH链；并且所述的肽链IIIb自N端到C端分别为：抗VEGF抗体的VL序列、人κ轻链恒定区序列、连接元件、TGF-βRII胞外结构域。In another preferred embodiment, the peptide chain IIIa from the N-terminus to the C-terminus is: the VH sequence of an anti-VEGF antibody, a human IgG1 heavy chain constant region with LALA mutation, a linking element, and an anti-PD-L1 nanobody VHH and the peptide chain IIIb from the N-terminus to the C-terminus is: the VL sequence of the anti-VEGF antibody, the human kappa light chain constant region sequence, the linking element, and the TGF-βRII extracellular domain.
在另一优选例中，所述肽链IIIa的氨基酸序列如SEQ ID NO:23所示，或与SEQ ID NO:23所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性；并且所述肽链IIIb的氨基酸序列如SEQ ID NO:25所示，或与SEQ ID NO:25所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。In another preferred example, the amino acid sequence of the peptide chain IIIa is as shown in SEQ ID NO: 23, or has ≥ 85% (preferably 90%, more preferably 95%) of the sequence shown in SEQ ID NO: 23 and the amino acid sequence of the peptide chain IIIb is as shown in SEQ ID NO: 25, or has ≥ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 25 sequence identity.
在本发明的第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第一方面所述的多特异性抗体。In a second aspect of the present invention, there is provided an isolated polynucleotide encoding the multispecific antibody of the first aspect of the present invention.
在另一优选例中，当所述多特异性抗体为异二聚体时，所述多核苷酸中，分别编码所述的两条肽链的多核苷酸的比例为1:1。In another preferred example, when the multispecific antibody is a heterodimer, the ratio of the polynucleotides encoding the two peptide chains in the polynucleotides is 1:1.
在本发明的第三方面，提供了一种载体，所述载体含有如本发明第二方面所述的多核苷酸。In a third aspect of the present invention, there is provided a vector comprising the polynucleotide according to the second aspect of the present invention.
在另一优选例中，所述的载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移***、或其组合；优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof; preferably, the expression vector includes a viral vector, such as Lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
在本发明的第四方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第三方面所述的载体，或其基因组中整合有如本发明第二方面所述的多核苷酸；In the fourth aspect of the present invention, a host cell is provided, the host cell contains the vector according to the third aspect of the present invention, or the polynucleotide according to the second aspect of the present invention is integrated into its genome;
或者，所述的宿主细胞表达如本发明第一方面所述的多特异性抗体。Alternatively, the host cell expresses the multispecific antibody according to the first aspect of the present invention.
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。In another preferred embodiment, the host cells include prokaryotic cells or eukaryotic cells.
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。In another preferred embodiment, the host cell is selected from the group consisting of: Escherichia coli, yeast cells, and mammalian cells.
在本发明的第五方面，提供了一种产生如本发明第一方面所述多特异性抗体的方 法，包括步骤：In a fifth aspect of the present invention, there is provided a method for producing a multispecific antibody as described in the first aspect of the present invention, comprising the steps of:
(a)在合适的条件下，培养如本发明第四方面所述的宿主细胞，从而获得含所述多特异性抗体的培养物；和(a) culturing the host cell according to the fourth aspect of the invention under suitable conditions, thereby obtaining a culture containing the multispecific antibody; and
(b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的多特异性抗体。(b) purifying and/or separating the culture obtained in step (a) to obtain the multispecific antibody.
在另一优选例中，所述纯化可以通过蛋白A亲和柱纯化分离获得目标抗体。In another preferred embodiment, the purification can be carried out by protein A affinity column purification and separation to obtain the target antibody.
在另一优选例中，所述经过纯化分离后的目标抗体纯度大于95％，大于96％、大于97％、大于98％、大于99％，优选为100％。In another preferred embodiment, the purity of the purified and separated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, preferably 100%.
在本发明的第六方面，提供了一种免疫偶联物，所述免疫偶联物含有：In the sixth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate contains:
(a)如本发明第一方面所述的多特异性抗体；和(a) a multispecific antibody according to the first aspect of the invention; and
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。(b) a conjugation moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or their combination.
在另一优选例中，所述的放射性核素包括：In another preferred embodiment, the radionuclide includes:
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或(i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133Yb-169、Yb-177、或其组合。(ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.
在另一优选例中，所述偶联部分为药物或毒素。In another preferred embodiment, the coupling moiety is a drug or a toxin.
在另一优选例中，所述的药物为细胞毒性药物。In another preferred embodiment, the drug is a cytotoxic drug.
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。In another preferred embodiment, the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、 紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))、长春花生物碱(vinca alkaloids)、或其组合。Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( taxanes), benzodiazepines, or benzodiazepine-containing drugs (eg, pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines) indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
在另一优选例中，所述的毒素选自下组：In another preferred embodiment, the toxin is selected from the following group:
耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素、或其组合。Auristatins (eg, auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansoid, gamatoxin, gamatoxin A-chain, combretastatin, docarmicin, Lastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissin, Dihydroxyanthraxdione, Actinomycin, Diphtheria Toxin, Pseudomonas Exotoxin (PE) A, PE40, Acacia toxin, Acacia A chain, Capsule root toxin A chain, α - Sarcinus, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotontoxin, calicheamicin, Sapaonaria officinalis inhibitors, glucocorticoids, or combinations thereof.
在另一优选例中，所述偶联部分为可检测标记物。In another preferred embodiment, the coupling moiety is a detectable label.
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(如DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(如顺铂)。In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable Enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, prodrugs Activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin).
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第一方面所述的多特异性抗体。In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) multispecific antibody according to the first aspect of the present invention.
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第一方面所述的多特异性抗体。In another preferred embodiment, the multivalent refers to the multispecific antibody according to the first aspect of the present invention comprising multiple repetitions in the amino acid sequence of the immunoconjugate.
在本发明的第七方面，提供了如本发明第一方面所述的多特异性抗体，或如本发明第六方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；In the seventh aspect of the present invention, there is provided the use of the multispecific antibody according to the first aspect of the present invention, or the use of the immunoconjugate according to the sixth aspect of the present invention, for preparing medicaments, reagents, and detection plates or kit;
其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1、VEGF和/或TGF-β；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤、表达VEGF的肿瘤，和/或表达TGF-β的肿瘤。Wherein, the reagent, detection plate or kit is used for: detecting PD-L1, VEGF and/or TGF-β in the sample; wherein, the agent is used for treating or preventing the expression of PD-L1 (ie PD-L1 positive) tumors, VEGF-expressing tumors, and/or TGF-β-expressing tumors.
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。In another preferred embodiment, the coupling part of the immunoconjugate is a diagnostic isotope.
在另一优选例中，所述的试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。In another preferred embodiment, the reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agents .
在另一优选例中，所述检测样品中PD-L1、VEGF和/或TGF-β的试剂为(体内)检测PD-L1、VEGF和/或TGF-β分子的造影剂。In another preferred embodiment, the reagent for detecting PD-L1, VEGF and/or TGF-β in the sample is a contrast agent for (in vivo) detecting PD-L1, VEGF and/or TGF-β molecules.
在另一优选例中，所述的检测为体内检测或体外检测。In another preferred embodiment, the detection is in vivo detection or in vitro detection.
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。In another preferred embodiment, the detection includes flow detection and cellular immunofluorescence detection.
在另一优选例中，所述的药剂用于阻断PD-1和PD-L1的相互作用，同时通过结合VEGF从而阻断其活性，并且能够与TGF-β配体结合并阻断其下游信号通路。In another preferred embodiment, the agent is used to block the interaction between PD-1 and PD-L1, and at the same time block its activity by binding to VEGF, and can bind to TGF-β ligand and block its downstream signal path.
在另一优选例中，所述的肿瘤包括但不限于：急性髓细胞白血病、慢性粒细胞性白血病、多发性骨髓病、非霍奇金淋巴瘤、结直肠癌、乳腺癌、大肠癌、胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、***癌、***、淋巴癌、肾上腺肿瘤、***，或其组合。In another preferred example, the tumors include but are not limited to: acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer , liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, or a combination thereof.
在本发明的第八方面，提供了一种药物组合物，所述药物组合物含有：In the eighth aspect of the present invention, there is provided a pharmaceutical composition, the pharmaceutical composition contains:
(i)如本发明第一方面所述的多特异性抗体，或如本发明第六方面所述的免疫偶联物；和(i) a multispecific antibody according to the first aspect of the invention, or an immunoconjugate according to the sixth aspect of the invention; and
(ii)药学上可接受的载体。(ii) A pharmaceutically acceptable carrier.
在另一优选例中，所述的免疫偶联物的偶联部分为药物、毒素、和/或治疗用同位素。In another preferred embodiment, the coupling part of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
在另一优选例中，所述的药物组合物中还含有***的其他药物，如细胞毒性药物。In another preferred embodiment, the pharmaceutical composition also contains other drugs for treating tumors, such as cytotoxic drugs.
在另一优选例中，所述的***的其他药物包括紫杉醇、多柔比星、环磷酰胺、阿西替尼、乐伐替尼、派姆单抗。In another preferred example, the other drugs for treating tumors include paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, and pembrolizumab.
在另一优选例中，所述的药物组合物用于治疗表达PD-L1蛋白(即PD-L1阳性)的肿瘤。In another preferred embodiment, the pharmaceutical composition is used to treat tumors expressing PD-L1 protein (ie PD-L1 positive).
在另一优选例中，所述的药物组合物为注射剂型。In another preferred embodiment, the pharmaceutical composition is in the form of injection.
在另一优选例中，所述的药物组合物用于制备防治肿瘤的药物。In another preferred embodiment, the pharmaceutical composition is used to prepare a medicine for preventing and treating tumors.
在本发明的第九方面，提供了如本发明第一方面所述的多特异性抗体的一种或多种选自下组的用途，包括：In a ninth aspect of the present invention, there is provided one or more uses of the multispecific antibody according to the first aspect of the present invention selected from the group consisting of:
(i)用于检测人PD-L1、VEGF和/或TGF-β分子；(ii)用于流式检测；(iii) 用于细胞免疫荧光检测；(iv)用于***；(v)用于肿瘤诊断；(vi)用于阻断PD-1和PD-L1的相互作用；(vii)用于通过结合VEGF从而阻断其活性；和(viii)用于与TGF-β配体结合并阻断其下游信号通路。(i) for the detection of human PD-L1, VEGF and/or TGF-β molecules; (ii) for flow detection; (iii) for cellular immunofluorescence detection; (iv) for tumor treatment; (v) For tumor diagnosis; (vi) for blocking the interaction of PD-1 and PD-L1; (vii) for blocking the activity of VEGF by binding to it; and (viii) for binding to TGF-beta ligands and block its downstream signaling pathways.
在另一优选例中，所述的肿瘤为表达PD-L1蛋白(即PD-L1阳性)的肿瘤。In another preferred embodiment, the tumor is a tumor expressing PD-L1 protein (ie PD-L1 positive).
在另一优选例中，所述用途为非诊断的和非治疗的。In another preferred embodiment, the use is non-diagnostic and non-therapeutic.
在本发明的第十方面，提供了一种重组蛋白，所述的重组蛋白具有：(i)如本发明第一方面所述的多特异性抗体；以及(ii)任选的协助表达和/或纯化的标签序列。In a tenth aspect of the present invention, there is provided a recombinant protein having: (i) the multispecific antibody of the first aspect of the present invention; and (ii) optionally assisted expression and/or or purified tag sequences.
在另一优选例中，所述的标签序列包括6His标签、HA标签和Fc标签。In another preferred embodiment, the tag sequence includes 6His tag, HA tag and Fc tag.
在另一优选例中，所述的重组蛋白特异性结合于PD-L1、VEGF和/或TGF-β分子。In another preferred embodiment, the recombinant protein specifically binds to PD-L1, VEGF and/or TGF-β molecules.
在本发明的第十一方面，提供了一种检测样品中PD-L1、VEGF和/或TGF-β分子的方法，所述方法包括步骤：(1)将样品与如本发明第一方面所述的多特异性抗体接触；(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在PD-L1、VEGF和/或TGF-β分子。In an eleventh aspect of the present invention, there is provided a method for detecting PD-L1, VEGF and/or TGF-β molecules in a sample, the method comprising the steps of: (1) combining the sample with the method described in the first aspect of the present invention (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of PD-L1, VEGF and/or TGF-β molecules in the sample.
在本发明的第十二方面，提供了一种治疗疾病的方法，所述方法包括：给需要的对象施用如本发明第一方面所述的多特异性抗体、如本发明第六方面所述的免疫偶联物，或如本发明第八方面所述的药物组合物。In the twelfth aspect of the present invention, there is provided a method for treating a disease, the method comprising: administering to a subject in need the multispecific antibody as described in the first aspect of the present invention, as described in the sixth aspect of the present invention The immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention.
在另一优选例中，所述的对象包括哺乳动物，优选地是人。In another preferred embodiment, the subject includes mammals, preferably humans.
在本发明的第十三方面，提供了一种PD-L1、VEGF和/或TGF-β分子检测试剂，所述的检测试剂包含如本发明第六方面所述的免疫偶联物和检测学上可接受的载体。In the thirteenth aspect of the present invention, there is provided a PD-L1, VEGF and/or TGF-β molecular detection reagent, the detection reagent comprises the immunoconjugate and the detection method according to the sixth aspect of the present invention an acceptable carrier.
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。In another preferred embodiment, the coupling part of the immunoconjugate is a diagnostic isotope.
在另一优选例中，所述的检测学上可接受的载体为无毒的、惰性的水性载体介质。In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.
在另一优选例中，所述的检测试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agent.
在另一优选例中，所述的检测试剂用于体内检测。In another preferred embodiment, the detection reagent is used for in vivo detection.
在另一优选例中，所述的检测试剂的剂型为液态或粉状(如水剂，针剂，冻干粉，片剂，含服剂，吸雾剂)。In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (eg, water preparation, injection, freeze-dried powder, tablet, buccal preparation, aerosol preparation).
在本发明的第十四方面，提供了一种检测PD-L1、VEGF和/或TGF-β分子的试剂盒，所述试剂盒含有如本发明第六方面所述的免疫偶联物或如本发明第十三方面所 述的检测试剂，以及说明书。In the fourteenth aspect of the present invention, there is provided a kit for detecting PD-L1, VEGF and/or TGF-β molecules, the kit contains the immunoconjugate according to the sixth aspect of the present invention or the The detection reagent described in the thirteenth aspect of the present invention, and the description.
在另一优选例中，所述的说明书记载，所述的试剂盒用于非侵入性地检测待测对象的PD-L1、VEGF和/或TGF-β分子表达。In another preferred embodiment, the instruction manual describes that the kit is used to non-invasively detect the expression of PD-L1, VEGF and/or TGF-β molecules in the subject to be tested.
在另一优选例中，所述的试剂盒用于表达PD-L1蛋白(即PD-L1阳性)的肿瘤的检测。In another preferred embodiment, the kit is used for the detection of tumors expressing PD-L1 protein (ie, PD-L1 positive).
术语the term
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.
多特异性抗体multispecific antibody
如本文所用，术语“本发明的多特异性抗体”、“本发明的多抗”、“抗PD-L1/VEGF/TGF-β多特异性抗体”具有相同的含义，均指特异性识别和结合PD-L1、VEGF和TGF-β的多特异性抗体。对于第二蛋白功能区，当其为VEGF受体(例如VEGFR1和/或VEGFR2)或其功能片段时，由于具有与抗体类似的阻断VEGF和受体结合的功能，此时的融合蛋白在本发明中亦广义上地称为多特异性抗体。同理，对于第三蛋白功能区，当其为TGF-β受体(例如TGF-βRII)或其功能片段时，由于具有与抗体类似的阻断TGF-β和受体结合的功能，此时的融合蛋白在本发明中亦广义上地称为多特异性抗体。本发明的多特异性抗体也可以称为三特异性抗体。As used herein, the terms "multispecific antibody of the present invention", "polyclonal antibody of the present invention", "anti-PD-L1/VEGF/TGF-beta multispecific antibody" have the same meaning and refer to specific recognition and Multispecific antibody that binds PD-L1, VEGF and TGF-beta. As for the second protein functional region, when it is a VEGF receptor (eg VEGFR1 and/or VEGFR2) or its functional fragment, since it has the function of blocking the binding of VEGF to the receptor similar to that of an antibody, the fusion protein at this time is described in this paper. The invention is also broadly referred to as a multispecific antibody. Similarly, for the third protein functional domain, when it is a TGF-β receptor (such as TGF-βRII) or its functional fragment, because it has the function of blocking the binding of TGF-β and the receptor similar to that of an antibody, this time The fusion protein is also broadly referred to as a multispecific antibody in the present invention. The multispecific antibodies of the present invention may also be referred to as trispecific antibodies.
本发明提供了一种抗PD-L1/VEGF/TGF-β多特异性抗体，其包括：抗PD-L1单域抗体、抗VEGF的抗体元件和TGF-βRII胞外结构域。The present invention provides an anti-PD-L1/VEGF/TGF-β multispecific antibody, which comprises: an anti-PD-L1 single domain antibody, an anti-VEGF antibody element and a TGF-βRII extracellular domain.
在一个实施方式中，本发明的抗PD-L1/VEGF/TGF-β多特异性抗体包含肽链I，所述的肽链I中包含以下元件：抗PD-L1纳米抗体VHH链、人免疫球蛋白的Fc区、抗VEGF的抗体元件、TGF-βRII胞外结构域，和任选的连接上述元件的连接元件；In one embodiment, the anti-PD-L1/VEGF/TGF-β multispecific antibody of the present invention comprises peptide chain I, and the peptide chain I comprises the following elements: anti-PD-L1 nanobody VHH chain, human immune The Fc region of a globulin, an anti-VEGF antibody element, a TGF-βRII extracellular domain, and optionally a linking element linking the above elements;
其中，各元件可以以不同的顺序自肽链I的N端到C端排列；Wherein, each element can be arranged in different order from N-terminus to C-terminus of peptide chain I;
并且，所述的多特异性抗体是由肽链I通过所述Fc区的二硫键作用形成的同源二聚体。And, the multispecific antibody is a homodimer formed by the peptide chain I through the disulfide bond of the Fc region.
在另一个实施方式中，本发明的抗PD-L1/VEGF/TGF-β多特异性抗体是由肽链IIa和肽链IIb构成的异源二聚体，所述的肽链IIa和IIb中各自独立地包括以下元件：抗PD-L1纳米抗体VHH链、人IgG1 knob突变或hole突变序列、抗VEGF的抗体元件、TGF-βRII胞外结构域，和任选的连接上述元件的连接元件；In another embodiment, the anti-PD-L1/VEGF/TGF-β multispecific antibody of the present invention is a heterodimer composed of peptide chain IIa and peptide chain IIb, wherein said peptide chain IIa and IIb are heterodimers Each independently comprises the following elements: an anti-PD-L1 Nanobody VHH chain, a human IgG1 knockout or hole mutant sequence, an anti-VEGF antibody element, a TGF-βRII extracellular domain, and an optional linking element linking the above elements;
其中，各元件可以以不同的顺序自肽链IIa或肽链IIb的N端到C端排列；Wherein, each element can be arranged in different order from N-terminus to C-terminus of peptide chain IIa or peptide chain IIb;
并且，所述的肽链IIa中包括人IgG1 knob突变序列；所述的肽链IIb中包括人IgG1 hole突变序列；并且，所述的多特异性抗体是由肽链IIa和肽链IIb通过所述人IgG恒定区的二硫键作用及杵-进入-臼结构(Knob–in-hole)而形成的异源二聚体。In addition, the peptide chain IIa includes a human IgG1 knockout mutant sequence; the peptide chain IIb includes a human IgG1 hole mutant sequence; and, the multispecific antibody is composed of the peptide chain IIa and the peptide chain IIb. The heterodimer formed by the disulfide bond interaction of the human IgG constant region and the knob-in-hole structure (Knob-in-hole).
在另一个实施方式中，本发明的抗PD-L1/VEGF/TGF-β多特异性抗体包括肽链IIIa和肽链IIIb；In another embodiment, the anti-PD-L1/VEGF/TGF-β multispecific antibody of the present invention comprises peptide chain IIIa and peptide chain IIIb;
其中，所述肽链IIIa中包括以下元件：抗VEGF抗体的VH序列、人IgG1重链恒定区序列；所述肽链IIIb中包括以下元件：抗VEGF抗体的VL序列、人κ轻链恒定区序列；Wherein, the peptide chain IIIa includes the following elements: the VH sequence of the anti-VEGF antibody, the constant region sequence of the human IgG1 heavy chain; the peptide chain IIIb includes the following elements: the VL sequence of the anti-VEGF antibody, the constant region of the human kappa light chain sequence;
并且，所述的多特异性抗体是由肽链IIIa和肽链IIIb之间、以及肽链IIIb之间通过二硫键作用形成的异源四聚体。In addition, the multispecific antibody is a heterotetramer formed by disulfide bonds between peptide chains IIIa and IIIb, and between peptide chains IIIb.
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β-折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。As used herein, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are roughly in a β-sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a β-sheet structure . The CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
如本文所用，术语“框架区”(FR)指***CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可 变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。As used herein, the term "framework region" (FR) refers to amino acid sequences inserted between CDRs, ie, those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among immunoglobulins that differ within a single species . The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, a light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。Knowing the amino acid sequences of the CDRs, one skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.
如本文所用，术语″人框架区″是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
如本文所用，术语“亲和力”理论上通过完整抗体和抗原间的平衡缔合来定义。本发明多抗的亲和力可以通过KD值(解离常数)(或其它测定方式)进行评估或测定，例如生物膜层干涉技术(Bio-layer interferometry BLI)，使用FortebioRed96仪器测量确定。As used herein, the term "affinity" is theoretically defined by an equilibrium association between intact antibody and antigen. The affinity of the polyclonal antibody of the present invention can be evaluated or determined by KD value (dissociation constant) (or other measurement methods), such as Bio-layer interferometry (BLI), using FortebioRed96 instrument to measure and determine.
如本文所用，术语“接头”是指***免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。As used herein, the term "linker" refers to insertion into an immunoglobulin domain to provide sufficient mobility for the domains of the light and heavy chains to fold into an exchange of one or more amino acid residues of a dual variable region immunoglobulin base.
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的PD-L1/VEGF多特异性抗体或其片段结合的细胞表面标记物或抗原。As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies of the present invention or fragments thereof to form the conjugate. The present invention also includes cell surface markers or antigens that bind to the PD-L1/VEGF multispecific antibody or fragment thereof.
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。As used herein, the term "variable region" is used interchangeably with "complementarity determining region (CDR)".
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PD-L1、VEGF和/或TGF-β蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and all refer to polypeptides that specifically bind to PD-L1, VEGF and/or TGF-β proteins, such as A protein or polypeptide having a heavy chain variable region. They may or may not contain the starting methionine.
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联 物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。The present invention also provides other protein or fusion expression products with the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a variable region-containing heavy chain, as long as the variable region is associated with the heavy chain of an antibody of the invention The variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into four framework regions (FRs), four FR amino acids The sequence is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the β-sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Accordingly, the present invention includes those molecules having CDR-bearing antibody heavy chain variable regions, as long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein sex.
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。The present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. A polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein). These fragments, derivatives and analogs are well known to those skilled in the art in light of the teachings herein.
本发明抗体指具有PD-L1、VEGF和/或TGF-β蛋白结合活性的多抗。该术语还包括具有与本发明抗体相同功能的、包含相同CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的 功能。该术语还包括本发明抗体的活性片段和活性衍生物。The antibody of the present invention refers to a polyclonal antibody with PD-L1, VEGF and/or TGF-β protein binding activity. The term also includes variant forms of polypeptides comprising the same CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency The encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention.
本发明还提供了其他多肽，如包含单域抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。The invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single domain antibodies of the present invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。In the present invention, "conservative variants of the antibody of the present invention" means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 The amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
表ATable A
最初的残基initial residue 代表性的取代representative substitution 优选的取代Preferred substitution
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Lys；ArgGln; His; Lys; Arg GlnGln
Asp(D)Asp(D) GluGlu GluGlu
Cys(C)Cys(C) SerSer SerSer
Gln(Q)Gln(Q) AsnAsn AsnAsn
Glu(E)Glu(E) AspAsp AspAsp
Gly(G)Gly(G) Pro；AlaPro; Ala AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile(I) Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe LeuLeu
Leu(L)Leu(L) Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr LeuLeu
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) SerSer SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; Ala LeuLeu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be the coding or non-coding strand.
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。Polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; coding sequences and various additional coding sequences for the mature polypeptides; coding sequences (and optional additional coding sequences) for the mature polypeptides and non-coding sequences .
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. In particular, the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method. A feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。Once the relevant sequences have been obtained, recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. Biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生 物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。At present, DNA sequences encoding the proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。The present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。 Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 . If desired, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。The obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention. The medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒； 3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents ( For example, cisplatin) or any form of nanoparticles, etc.
药物组合物pharmaceutical composition
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
本发明的药物组合物可直接用于结合PD-L1、VEGF和/或TGF-β蛋白分子，因而可用于***。此外，还可同时使用其他治疗剂。The pharmaceutical composition of the present invention can be directly used to bind PD-L1, VEGF and/or TGF-β protein molecules, and thus can be used to treat tumors. In addition, other therapeutic agents may also be used concomitantly.
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。The pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above-mentioned single domain antibody (or its conjugate) of the present invention and a pharmaceutical an acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The drug formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. In addition, the polypeptides of the present invention may also be used with other therapeutic agents.
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
标记的抗体labeled antibody
在本发明的一个优选例中，所述抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。In a preferred embodiment of the present invention, the antibody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，PD-L1/VEGF/TGF-β多特异性抗体可以用胶体金标记，得到胶体金标记的抗体。Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred solution of the present invention, the PD-L1/VEGF/TGF-β multispecific antibody can be labeled with colloidal gold to obtain a colloidal gold-labeled antibody.
检测方法Detection method
本发明还涉及检测PD-L1、VEGF和/或TGF-β蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中PD-L1、VEGF和/或TGF-β蛋白的水平。The present invention also relates to methods of detecting PD-L1, VEGF and/or TGF-beta proteins. The method steps are roughly as follows: obtaining a cell and/or tissue sample; lysing the sample in a medium; detecting the level of PD-L1, VEGF and/or TGF-beta protein in the lysed sample.
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。In the detection method of the present invention, the sample to be used is not particularly limited, and a representative example is a cell-containing sample existing in a cell preservation solution.
试剂盒Reagent test kit
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。The present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer, and the like.
本发明还提供了用于检测PD-L1、VEGF和/或TGF-β水平的检测试剂盒，该试剂盒包括识别PD-L1、VEGF和/或TGF-β蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。The present invention also provides a detection kit for detecting the level of PD-L1, VEGF and/or TGF-β, the kit includes an antibody that recognizes PD-L1, VEGF and/or TGF-β protein, and is used for dissolving samples of Lysis medium, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit may be an in vitro diagnostic device.
应用application
如上所述，本发明的多特异性抗体有广泛生物应用价值和临床应用价值，其应用涉及到与PD-L1、VEGF和/或TGF-β相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对PD-L1、VEGF和/或TGF-β的临床诊断和靶向治疗，如肿瘤治疗。As mentioned above, the multispecific antibody of the present invention has a wide range of biological application value and clinical application value, and its application involves the diagnosis and treatment of diseases related to PD-L1, VEGF and/or TGF-β, basic medical research, biological research and many other fields. A preferred application is for clinical diagnosis and targeted therapy against PD-L1, VEGF and/or TGF-beta, such as tumor therapy.
发明的有益效果Beneficial Effects of Invention
本发明取得了如下技术效果(1)至(8)中的任意一项或者多项：The present invention has achieved any one or more of the following technical effects (1) to (8):
(1)本发明的多特异性抗体针对PD-L1、VEGF以及TGF-β三种抗原的特异性高。(1) The multispecific antibody of the present invention has high specificity against three antigens, PD-L1, VEGF and TGF-β.
(2)本发明的多特异性抗体结合PD-L1、VEGF以及TGF-β三种抗原的亲和力 强。(2) The multispecific antibody of the present invention has strong affinity for binding to three antigens, PD-L1, VEGF and TGF-β.
(3)本发明的多特异性抗体的生产简便。(3) The production of the multispecific antibody of the present invention is simple.
(4)本发明的多特异性抗体的稳定性特别是热稳定性良好。(4) The stability of the multispecific antibody of the present invention, especially the thermal stability, is good.
(5)本发明的多特异性抗体的半衰期长。(5) The multispecific antibody of the present invention has a long half-life.
(6)本发明的多特异性抗体的安全性良好。(6) The safety of the multispecific antibody of the present invention is good.
(7)本发明的多特异性抗体在抑制PD-1/PD-L1通路的基础上中和肿瘤微环境的TGF-β以及VEGF，恢复T细胞活性，增强免疫应答，阻断肿瘤血管生成，从而更有效地提高抑制肿瘤发生和发展的效果。(7) The multispecific antibody of the present invention neutralizes TGF-β and VEGF in the tumor microenvironment on the basis of inhibiting the PD-1/PD-L1 pathway, restores T cell activity, enhances immune response, and blocks tumor angiogenesis, Thereby, the effect of inhibiting the occurrence and development of tumors can be more effectively improved.
(8)本发明的多特异性抗体中的任意两个蛋白功能区之间或者三个蛋白功能区之间极可能存在协同作用。(8) It is very likely that there is synergy between any two protein functional domains or between three protein functional domains in the multispecific antibody of the present invention.
附图说明Description of drawings
图1A至图1E：分别是多特异性抗体PD-L1xTGFβxVEGF TriAb 1、PD-L1xTGFβxVEGF TriAb 2、PD-L1xTGFβxVEGF TriAb 3、PD-L1xTGFβxVEGF TriAb 4和PD-L1xTGFβxVEGF TriAb 5的结构示意图。其中，各图例代表的含义如下：1A to 1E: Schematic diagrams of the structures of the multispecific antibodies PD-L1xTGFβxVEGF TriAb 1, PD-L1xTGFβxVEGF TriAb 2, PD-L1xTGFβxVEGF TriAb 3, PD-L1xTGFβxVEGF TriAb 4 and PD-L1xTGFβxVEGF TriAb 5, respectively. Among them, the meaning of each legend is as follows:
Figure PCTCN2021132464-appb-000002
Figure PCTCN2021132464-appb-000002
图2A至图2D：分别是多特异性抗体PD-L1xTGFβxVEGF TriAb 6、PD-L1xTGFβxVEGF TriAb 7、PD-L1xTGFβxVEGF TriAb 8和PD-L1xTGFβxVEGF TriAb 9的结构示意图。其中，各图例代表的含义如下：Figures 2A to 2D: Schematic diagrams of the structures of the multispecific antibodies PD-L1xTGFβxVEGF TriAb 6, PD-L1xTGFβxVEGF TriAb 7, PD-L1xTGFβxVEGF TriAb 8 and PD-L1xTGFβxVEGF TriAb 9, respectively. Among them, the meaning of each legend is as follows:
Figure PCTCN2021132464-appb-000003
Figure PCTCN2021132464-appb-000003
图3A：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5与过表达人PD-L1的CHO-S细胞结合的四参数拟合曲线图。Figure 3A: Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention to CHO-S cells overexpressing human PD-L1.
图3B：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9与过表达人PD-L1的CHO-S细胞结合的四参数拟合曲线图。Figure 3B: Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 of the present invention to CHO-S cells overexpressing human PD-L1.
图4A：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5阻断VEGFA蛋白和VEGFR蛋白结合的四参数拟合曲线图。Figure 4A: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention blocking the binding of VEGFA protein and VEGFR protein.
图4B：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9阻断VEGFA蛋白和VEGFR蛋白结合的四参数拟合曲线图。Figure 4B: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 of the present invention blocking the binding of VEGFA protein and VEGFR protein.
图5A：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5与人TGF-β1蛋白结合的四参数拟合曲线图。Figure 5A: Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention to human TGF-β1 protein.
图5B：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5与人TGF-β2蛋白结合的四参数拟合曲线图。Figure 5B: Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention to human TGF-β2 protein.
图5C：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5与人TGF-β3蛋白结合的四参数拟合曲线图。Figure 5C: Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention to human TGF-β3 protein.
图5D：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9与人TGF-β1蛋白结合的四参数拟合曲线图。Figure 5D: Four-parameter fitting curve graph of the binding of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 of the present invention to human TGF-β1 protein.
图6A：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5同时结合人PD-L1、TGF-β1和VEGFA蛋白SPR(表面等离子体共振)分析图。Figure 6A: SPR (Surface Plasmon Resonance) analysis of the simultaneous binding of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention to human PD-L1, TGF-β1 and VEGFA proteins.
图6B：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9同时结合人PD-L1、TGF-β1和VEGFA蛋白SPR分析图。Figure 6B: SPR analysis diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 of the present invention that simultaneously binds human PD-L1, TGF-β1 and VEGFA proteins.
图7A：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5阻断PD-1/PD-L1信号通路的四参数拟合曲线图。Figure 7A: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention blocking PD-1/PD-L1 signaling pathway.
图7B：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9阻断PD-1/PD-L1信号通路的四参数拟合曲线图。Figure 7B: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 of the present invention blocking PD-1/PD-L1 signaling pathway.
图8A：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5阻断TGF-β信号通路的四参数拟合曲线图。Figure 8A: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention blocking the TGF-β signaling pathway.
图8B：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9阻断TGF-β信号通路的四参数拟合曲线图。Figure 8B: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 of the present invention blocking TGF-β signaling pathway.
图9A：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1-5阻断VEGF信号通路的四参数拟合曲线图。Figure 9A: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1-5 of the present invention blocking the VEGF signaling pathway.
图9B：本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9阻断VEGF信号通路的四参数拟合曲线图。Figure 9B: Four-parameter fitting curve diagram of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 of the present invention blocking the VEGF signaling pathway.
图10A：不同剂量的多特异性抗体PD-L1xTGFβxVEGF 6抑制抑制肿瘤在小鼠(接种MDA-MB-231)体内的生长折线图。Figure 10A: Line graph of the inhibition of tumor growth in mice (inoculated with MDA-MB-231) by multispecific antibody PD-L1xTGFβxVEGF 6 inhibition at different doses.
图10B：小鼠注射不同剂量的PD-L1xTGFβxVEGF 6多特异性抗体34天后肿瘤重量对比柱状图。Figure 10B: Bar graph comparing tumor weights 34 days after mice were injected with different doses of PD-L1xTGFβxVEGF6 multispecific antibody.
图10C：接种MDA-MB-231的小鼠注射不同剂量的PD-L1xTGFβxVEGF 6多特异性抗体后的体重变化折线图。Figure 10C: Line graph of body weight change in mice vaccinated with MDA-MB-231 after injection of different doses of PD-L1xTGFβxVEGF6 multispecific antibody.
图11A：多特异性抗体PD-L1xTGFβxVEGF 6与PD-L1单抗Atezolizumab抑制肿瘤在小鼠(接种NCI-H1975)体内的生长折线图。Figure 11A: A line graph showing the inhibition of tumor growth in mice (inoculated with NCI-H1975) by multispecific antibody PD-L1xTGFβxVEGF 6 and PD-L1 monoclonal antibody Atezolizumab.
图11B：接种NCI-H1975的小鼠注射PD-L1xTGFβxVEGF 6多特异性抗体或Atezolizumab后的体重变化折线图。Figure 11B: Line graph of body weight change in NCI-H1975-vaccinated mice following injection of PD-L1xTGFβxVEGF6 multispecific antibody or Atezolizumab.
表B：本发明涉及的序列Table B: Sequences involved in the present invention
Figure PCTCN2021132464-appb-000004
Figure PCTCN2021132464-appb-000004
Figure PCTCN2021132464-appb-000005
Figure PCTCN2021132464-appb-000005
Figure PCTCN2021132464-appb-000006
Figure PCTCN2021132464-appb-000006
Figure PCTCN2021132464-appb-000007
Figure PCTCN2021132464-appb-000007
Figure PCTCN2021132464-appb-000008
Figure PCTCN2021132464-appb-000008
Figure PCTCN2021132464-appb-000009
Figure PCTCN2021132464-appb-000009
Figure PCTCN2021132464-appb-000010
Figure PCTCN2021132464-appb-000010
Figure PCTCN2021132464-appb-000011
Figure PCTCN2021132464-appb-000011
Figure PCTCN2021132464-appb-000012
Figure PCTCN2021132464-appb-000012
Figure PCTCN2021132464-appb-000013
Figure PCTCN2021132464-appb-000013
Figure PCTCN2021132464-appb-000014
Figure PCTCN2021132464-appb-000014
Figure PCTCN2021132464-appb-000015
Figure PCTCN2021132464-appb-000015
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
实施例1：抗PD-L1×VEGF×TGF-β多特异性抗体的克隆和表达Example 1: Cloning and Expression of Anti-PD-L1×VEGF×TGF-β Multispecific Antibody
在本实施例中，构建了9种抗PD-L1 x VEGF x TGF-β多特异性抗体，分别命名为PD-L1xTGFβxVEGF TriAb 1至PD-L1xTGFβxVEGF TriAb 9。用于后面的实施例。In this example, 9 anti-PD-L1 x VEGF x TGF-β multispecific antibodies were constructed and named as PD-L1 x TGFβ x VEGF TriAb 1 to PD-L1 x TGFβ x VEGF TriAb 9, respectively. used in the following examples.
PD-L1xTGFβxVEGF TriAb 1：由2条相同的多肽链组成，其结构示意图如图1A所示，肽链具有SEQ ID NO:1所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)以及人IgG1Fc氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:3)。将抗VEGF融合蛋白Aflibercept的VEGF结合区域氨基酸序列(SEQ ID NO:4)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于Fc的C端。再将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于抗VEGF融合蛋白Aflibercept的C端。PD-L1xTGFβxVEGF TriAb 1: It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1A, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 1, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO:2) and human IgGl Fc amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO:3). The N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO:4) of the anti-VEGF fusion protein Aflibercept was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). The N-terminus of the TGF-βRII extracellular domain (SEQ ID NO:6) was then linked to the C-terminus of the anti-VEGF fusion protein Aflibercept through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
PD-L1xTGFβxVEGF TriAb 2：由2条相同的多肽链组成，其结构示意图如图1B所示，肽链具有SEQ ID NO:7所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)以及人IgG1Fc氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:3)。将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于Fc的C端。再将抗VEGF 融合蛋白Aflibercept的VEGF结合区域氨基酸序列(SEQ ID NO:4)的N端通过11个氨基酸残基(G4S)2G(SEQ ID NO:8)的柔性肽连接于TGF-βRII胞外结构域(SEQ ID NO:6)的C端。PD-L1xTGFβxVEGF TriAb 2: It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1B, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 7, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO:2) and human IgGl Fc amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO:3). The N-terminus of the TGF-βRII ectodomain (SEQ ID NO:6) was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). The N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO: 4) of the anti-VEGF fusion protein Aflibercept is connected to the extracellular TGF-βRII through a flexible peptide of 11 amino acid residues (G4S) 2G (SEQ ID NO: 8). C-terminal of the domain (SEQ ID NO:6).
PD-L1xTGFβxVEGF TriAb 3：由2条相同的多肽链组成，其结构示意图如图1C所示，肽链具有SEQ ID NO:9所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)。将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于抗PD-L1的纳米抗体的C端，并在其C端通过11个氨基酸残基(G4S)2G(SEQ ID NO:8)的柔性肽连接人IgG1Fc氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:3)。再将抗VEGF融合蛋白Aflibercept的VEGF结合区域氨基酸序列(SEQ ID NO:4)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于Fc的C端。PD-L1xTGFβxVEGF TriAb 3: It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1C, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 9, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO: 2). The N-terminus of the extracellular domain of TGF-βRII (SEQ ID NO:6) was linked to the C-terminus of an anti-PD-L1 Nanobody via a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5) , and linked to the human IgG1 Fc amino acid sequence at its C-terminus through a flexible peptide of 11 amino acid residues (G4S) 2G (SEQ ID NO: 8) (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 3). The N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO:4) of the anti-VEGF fusion protein Aflibercept was linked to the C-terminus of Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
PD-L1xTGFβxVEGF TriAb 4：由2条相同的多肽链组成，其结构示意图如图1D所示，肽链具有SEQ ID NO:10所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)。将抗VEGF融合蛋白Aflibercept的VEGF结合区域氨基酸序列(SEQ ID NO:4)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于抗PD-L1的纳米抗体的C端，并在其C端通过11个氨基酸残基(G4S)2G(SEQ ID NO:8)的柔性肽连接连接人IgG1Fc氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:3)。再将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于Fc的C端。PD-L1xTGFβxVEGF TriAb 4: It consists of 2 identical polypeptide chains, the schematic diagram of which is shown in Figure 1D, and the peptide chain has the amino acid sequence shown in SEQ ID NO: 10, which contains the anti-PD-L1 nanobody C-Ye -18-5 (SEQ ID NO: 2). The N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO:4) of the anti-VEGF fusion protein Aflibercept was linked to the anti-PD-L1 nanoparticle through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). The C-terminus of the antibody, and at its C-terminus, the human IgG1 Fc amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO:3 ). The N-terminus of the TGF-βRII extracellular domain (SEQ ID NO:6) was then linked to the C-terminus of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
PD-L1xTGFβxVEGF TriAb 5：由2条不同的多肽链组成，其结构示意图如图1E所示，肽链#1具有SEQ ID NO:11所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)以及人IgG1 knob突变氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:12)。将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于Fc的C端。再将另一个TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于上一个TGF-βRII的C端。肽链#2具有SEQ ID NO:13所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)以及人IgG1 hole突变氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:14)。将抗VEGF融合蛋白Aflibercept的VEGF结合区域氨基酸序列(SEQ ID NO:4)的 N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于Fc的C端。PD-L1xTGFβxVEGF TriAb 5: It consists of 2 different polypeptide chains, the schematic diagram of which is shown in Figure 1E, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 11, which contains anti-PD-L1 Nanobody C - Ye-18-5 (SEQ ID NO: 2) and human IgG1 knob mutant amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO: 12). The N-terminus of the TGF-βRII ectodomain (SEQ ID NO:6) was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). Then the N-terminus of another TGF-βRII extracellular domain (SEQ ID NO:6) is linked to the C of the previous TGF-βRII through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). end. Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 13, which comprises the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO: 2) and the amino acid sequence of human IgG1 hole mutation (introduced LALA mutation to reduce Fc function, SEQ ID NO: 14). The N-terminus of the VEGF-binding domain amino acid sequence (SEQ ID NO:4) of the anti-VEGF fusion protein Aflibercept was linked to the C-terminus of the Fc by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
PD-L1xTGFβxVEGF TriAb 6：由2条肽链#1和2条肽链#2组成，其结构示意图如图2A所示，肽链#1具有SEQ ID NO:15所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)。将衍生自抗VEGF抗体贝伐单抗(Bevacizumab)的VH氨基酸序列(SEQ ID NO:16)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于抗PD-L1的纳米抗体的C端，并在所述VH氨基酸序列C端直接连接衍生自人IgG1氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:17)。再将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接Fc的C端。肽链#2具有SEQ ID NO:18所示的氨基酸序列，其包含衍生自抗VEGF抗体Bevacizumab的VL氨基酸序列(SEQ ID NO:19)，以及在所述VL氨基酸序列C端的人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:20)。PD-L1xTGFβxVEGF TriAb 6: consists of 2 peptide chains # 1 and 2 peptide chains #2, the schematic structure of which is shown in Figure 2A, and peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 15, which contains anti- Nanobody C-Ye-18-5 of PD-L1 (SEQ ID NO: 2). The N-terminus of the VH amino acid sequence (SEQ ID NO: 16) derived from the anti-VEGF antibody Bevacizumab was linked to the anti-VEGF via a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). The C-terminus of the Nanobody of PD-L1 was directly linked at the C-terminus of the VH amino acid sequence derived from the human IgG1 amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17). The N-terminus of the TGF-βRII extracellular domain (SEQ ID NO:6) was then linked to the C-terminus of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 18, which comprises the VL amino acid sequence (SEQ ID NO: 19) derived from the anti-VEGF antibody Bevacizumab, and a human kappa light chain constant at the C-terminus of the VL amino acid sequence Region (CL) amino acid sequence (SEQ ID NO: 20).
PD-L1xTGFβxVEGF TriAb 7：由2条肽链#1和2条肽链#2组成，其结构示意图如图2B所示，肽链#1具有SEQ ID NO:21所示的氨基酸序列，其包衍生自抗VEGF抗体Bevacizumab的VH氨基酸序列(SEQ ID NO:16)，并在所述VH氨基酸序列C端直接连接衍生自人IgG1氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:17)。再将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接Fc的C端。肽链#2具有SEQ ID NO:22所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)。将衍生自抗VEGF抗体Bevacizumab的VL氨基酸序列(SEQ IDNO 19)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于抗PD-L1的纳米抗体的C端，并在所述VL氨基酸序列C端直接连接人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:20)。PD-L1xTGFβxVEGF TriAb 7: It is composed of 2 peptide chains # 1 and 2 peptide chains #2, the schematic diagram of which is shown in Figure 2B, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 21, which is derived from The VH amino acid sequence (SEQ ID NO: 16) of the anti-VEGF antibody Bevacizumab was derived from the human IgGl amino acid sequence by direct linkage at the C-terminus of the VH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17). The N-terminus of the TGF-βRII extracellular domain (SEQ ID NO:6) was then linked to the C-terminus of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:22, which comprises the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO:2). The N-terminus of the VL amino acid sequence (SEQ ID NO 19) derived from the anti-VEGF antibody Bevacizumab was linked to the C of the anti-PD-L1 Nanobody through a flexible peptide of 21 amino acid residues (G4S) 4G (SEQ ID NO: 5) The C-terminal of the VL amino acid sequence is directly linked to the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 20).
PD-L1xTGFβxVEGF TriAb 8：由2条肽链#1和2条肽链#2组成，其结构示意图如图2C所示，肽链#1具有SEQ ID NO:23所示的氨基酸序列，其包衍生自抗VEGF抗体Bevacizumab的VH氨基酸序列(SEQ ID NO:16)，并在所述VH氨基酸序列C端直接连接衍生自人IgG1氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:17)。再将抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接Fc的C端。肽链#2具有SEQ ID NO:24所示的氨基酸序列，其包含TGF-βRII胞外结构域(SEQ ID NO:6)。将衍生自抗VEGF抗体Bevacizumab的VL氨基酸序列(SEQ IDNO 19)的N端通过21个氨 基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于TGF-βRII的C端，并在所述VL氨基酸序列C端直接连接人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:20)。PD-L1xTGFβxVEGF TriAb 8: It consists of 2 peptide chains # 1 and 2 peptide chains #2, its structural schematic diagram is shown in Figure 2C, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 23, which is derived from The VH amino acid sequence (SEQ ID NO: 16) of the anti-VEGF antibody Bevacizumab was derived from the human IgGl amino acid sequence by direct linkage at the C-terminus of the VH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17). The N-terminus of the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO:2) is then linked to the C of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). end. Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:24, which contains the TGF-betaRII extracellular domain (SEQ ID NO:6). The N-terminus of the VL amino acid sequence (SEQ ID NO 19) derived from the anti-VEGF antibody Bevacizumab was linked to the C-terminus of TGF-βRII by a flexible peptide of 21 amino acid residues (G4S) 4G (SEQ ID NO: 5), and the The C-terminus of the VL amino acid sequence is directly linked to the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 20).
PD-L1xTGFβxVEGF TriAb 9：由2条肽链#1和2条肽链#2组成，其结构示意图如图2D所示，肽链#1具有SEQ ID NO:23所示的氨基酸序列，其包衍生自抗VEGF抗体Bevacizumab的VH氨基酸序列(SEQ ID NO:16)，并在所述VH氨基酸序列C端直接连接衍生自人IgG1氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:17)。再将抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:2)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接Fc的C端。肽链#2具有SEQ ID NO:25所示的氨基酸序列，其包含衍生自抗VEGF抗体Bevacizumab的VL氨基酸序列(SEQ ID NO:19)，以及在所述VL氨基酸序列C端的人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:20)。将TGF-βRII胞外结构域(SEQ ID NO:6)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:5)的柔性肽连接于CL的C端。PD-L1xTGFβxVEGF TriAb 9: consists of 2 peptide chains # 1 and 2 peptide chains #2, the schematic diagram of which is shown in Figure 2D, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 23, which is derived from The VH amino acid sequence (SEQ ID NO: 16) of the anti-VEGF antibody Bevacizumab was derived from the human IgGl amino acid sequence by direct linkage at the C-terminus of the VH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 17). The N-terminus of the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO:2) is then linked to the C of the Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5). end. Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 25, which comprises the VL amino acid sequence (SEQ ID NO: 19) derived from the anti-VEGF antibody Bevacizumab, and a human kappa light chain constant at the C-terminus of the VL amino acid sequence Region (CL) amino acid sequence (SEQ ID NO: 20). The N-terminus of the TGF-βRII ectodomain (SEQ ID NO:6) was linked to the C-terminus of CL by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO:5).
实施例2：多特异性抗体与PD-L1结合Example 2: Multispecific antibody binding to PD-L1
通过转染克隆到MCS的人PD-L1cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-L1的CHO-S细胞(CHO-S-hPD-L1细胞)。将扩大培养的CHO-S-hPD-L1细胞调整细胞密度至2×10 6细胞/ml，100μl/孔加入96孔流式板，离心备用。将多特异性抗体用PBS稀释，100nM开始2倍或3倍稀释，将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育60min，PBS清洗两次。100μl/孔加入用PBS稀释100倍的羊抗人IgG-Fc(PE)(购自Abcam)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Beckman)流式细胞仪上进行检测并计算对应的MFI。 CHO-S cells overexpressing human PD-L1 (CHO-S-hPD-L1 cells) were generated by transfection of the pCHO1.0 vector (purchased from Invitrogen) of human PD-L1 cDNA (purchased from Sino Biological) cloned into MCS. The expanded CHO-S-hPD-L1 cells were adjusted to a cell density of 2×10 6 cells/ml, 100 μl/well was added to a 96-well flow plate, and centrifuged for later use. Dilute the multispecific antibody with PBS, 100nM to start 2-fold or 3-fold dilution, add 100 μl/well of the above diluted sample to the above-mentioned 96-well flow plate with cells, incubate at 4°C for 60 min, and wash twice with PBS. 100 μl/well of goat anti-human IgG-Fc (PE) (purchased from Abcam) diluted 100 times with PBS was added, incubated at 4° C. for 30 min, and washed twice with PBS. 100 μl/well was added with PBS to resuspend the cells, and the cells were detected on a CytoFlex (Beckman) flow cytometer and the corresponding MFI was calculated.
实验结果如图3A、图3B所示。结果显示，本发明所有的多特异性抗体与CHO-hPD-L1细胞均有结合活性。The experimental results are shown in Figure 3A and Figure 3B. The results show that all the multispecific antibodies of the present invention have binding activity to CHO-hPD-L1 cells.
实施例3：ELISA检测多特异性抗体抗阻断VEGF/VEGFR蛋白水平结合Example 3: ELISA detection of multispecific antibody anti-blocking VEGF/VEGFR protein level binding
用ELISA包被液将人VEGFR2蛋白稀释至合适浓度，加入ELISA板，4℃包被过夜。5％BSA室温封闭1小时。将待测样品梯度稀释并与生物素化标记的人VEGFA蛋白室温共孵育1小时。将孵育好的样品加入封闭好的ELISA板，室温反应2小时。 PBS-T(1：2000Tween)洗液洗涤3次，加入稀释好的HRP标记链霉亲和素(SA-HRP)室温反应30分钟，PBS-T洗液洗涤3次，加入ELISA TMB显色液，室温放置3min,加入ELISA终止液，读取450nm处吸光度数值。The human VEGFR2 protein was diluted to an appropriate concentration with ELISA coating solution, added to the ELISA plate, and coated overnight at 4°C. Block with 5% BSA for 1 hour at room temperature. The samples to be tested were serially diluted and incubated with biotinylated human VEGFA protein for 1 hour at room temperature. Add the incubated samples to the blocked ELISA plate and react at room temperature for 2 hours. Wash 3 times with PBS-T (1:2000Tween) washing solution, add diluted HRP-labeled streptavidin (SA-HRP) for 30 minutes at room temperature, wash 3 times with PBS-T washing solution, add ELISA TMB color developing solution , placed at room temperature for 3min, added ELISA stop solution, read the absorbance value at 450nm.
结果如图4A和图4B所示。结果显示，本发明所有的多特异性抗体均能够完全阻断VEGF(例如VEGFA)和VEGFR蛋白的相互作用。The results are shown in Figures 4A and 4B. The results show that all the multispecific antibodies of the present invention can completely block the interaction between VEGF (eg VEGFA) and VEGFR protein.
实施例4：ELISA检测多特异性抗体抗与人TGF-β蛋白结合Example 4: ELISA detection of multispecific antibody binding to human TGF-β protein
用ELISA包被液分别将人TGF-β1、TGF-β2和TGF-β3蛋白稀释至合适浓度，加入ELISA板，4℃包被过夜。5％BSA室温封闭1小时。将待测样品梯度稀释后加入封闭好的ELISA板，室温反应2小时。PBS-T(1：2000Tween)洗液洗涤3次，加入稀释好的羊抗人IgG Fc-HRP二抗室温孵育1小时。PBS-T洗液洗涤3次，加入ELISA TMB显色液，室温放置3min，加入ELISA终止液，读取450nm处吸光度数值。Human TGF-β1, TGF-β2 and TGF-β3 proteins were diluted to appropriate concentrations with ELISA coating solution, added to ELISA plate, and coated overnight at 4°C. Block with 5% BSA for 1 hour at room temperature. The sample to be tested was diluted in gradient and added to the blocked ELISA plate, and the reaction was carried out at room temperature for 2 hours. Washed 3 times with PBS-T (1:2000Tween) washing solution, added diluted goat anti-human IgG Fc-HRP secondary antibody and incubated for 1 hour at room temperature. Wash 3 times with PBS-T washing solution, add ELISA TMB color developing solution, place at room temperature for 3 min, add ELISA stop solution, and read the absorbance value at 450nm.
结果如图5A至图5D所示。结果显示，本发明所有的多特异性抗体均具有结合TGF-β的活性。The results are shown in Figures 5A to 5D. The results show that all the multispecific antibodies of the present invention have the activity of binding to TGF-β.
实施例5：多特异性抗体同时结合PD-L1、VEGF以及TGF-β验证Example 5: Validation of multispecific antibody binding to PD-L1, VEGF and TGF-β simultaneously
ForteBio检测抗体和抗原结合按照现有的方法(Estep,P等人，基于高通量解决方案的抗体-抗原亲和力和表位结合的测量(High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs),2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，将多特异性抗体加载至AHC传感器上或者是将生物素化的人PD-L1蛋白加载至SA传感器上。再将传感器放入100nM的PD-L1抗原或是100nM的多特异性抗体溶液中作用，之后将传感器转移至PBS中解离。再将传感器放入100nM的人TGF-β1溶液中作用，之后将传感器转移至PBS中解离。最后将传感器放入100nM的人VEGFA溶液中作用，之后将传感器转移至PBS中解离。ForteBio detects antibody and antigen binding according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs), 2013.5(2):p.270-8). Briefly, the sensor was equilibrated off-line in assay buffer for 30 min, and then detected online for 60 s to establish a baseline. Multispecific antibodies were loaded onto the AHC sensor or biotinylated human PD-L1 protein was loaded onto the SA sensor. The sensor was then put into 100 nM PD-L1 antigen or 100 nM multispecific antibody solution, and then the sensor was transferred to PBS for dissociation. The sensor was then put into a 100 nM solution of human TGF-β1 for action, and then the sensor was transferred to PBS for dissociation. Finally, the sensor was put into 100 nM human VEGFA solution for action, and then the sensor was transferred to PBS for dissociation.
结果如图6A和图6B所示。结果显示，本发明所有的多特异性抗体均能够同时结合PD-L1、VEGF(例如VEGFA)以及TGF-β三种抗原。The results are shown in Figures 6A and 6B. The results show that all the multispecific antibodies of the present invention can simultaneously bind to three antigens, PD-L1, VEGF (eg VEGFA) and TGF-β.
实施例6：多特异性抗体阻断PD-1/PD-L1信号通路Example 6: Multispecific antibodies block PD-1/PD-L1 signaling pathway
将靶细胞PD-L1aAPC/CHO-K1按照4×10 4个/孔接种至96孔细胞培养白底板中，过夜培养16h。第二天，将5×10 4个/孔的效应细胞PD-1NFAT-RE Luciferase/Jurkat与梯度稀释后的多特异性抗体混合，并加入到靶细胞中共孵育6小时。使用Bio-glo luciferase assay system(Promega G7940)试剂盒显色后用酶标仪收集化学发光信号。 The target cells PD-L1aAPC/CHO-K1 were seeded into 96-well cell culture white bottom plates at 4×10 4 cells/well, and cultured overnight for 16 h. The next day, 5×10 4 cells/well of effector cells PD-1NFAT-RE Luciferase/Jurkat were mixed with serially diluted multispecific antibodies and added to target cells for 6 hours of incubation. The bio-glo luciferase assay system (Promega G7940) was used for color development and the chemiluminescence signal was collected with a microplate reader.
结果如图7A和图7B所示。结果显示，本发明所有的多特异性抗体均能够很好地阻断PD-1/PD-L1信号通路。The results are shown in Figures 7A and 7B. The results show that all the multispecific antibodies of the present invention can well block the PD-1/PD-L1 signaling pathway.
实施例7：多特异性抗体阻断TGF-β信号通路Example 7: Multispecific antibodies block the TGF-beta signaling pathway
将TGF-β-SMAD Luciferase/HEK293效应细胞按照4×10 4个/孔接种至96孔细胞培养板白底中，培养8h。将梯度稀释后的多特异性抗体与最终浓度为0.5ng/ml TGF-β1混合，室温孵育30min中后加入效应细胞继续孵育18小时。使用Bio-glo luciferase assay system(Promega G7940)试剂盒显色后用酶标仪收集化学发光信号。 TGF-β-SMAD Luciferase/HEK293 effector cells were seeded into 96-well cell culture plates at 4×10 4 cells/well, and cultured for 8 h. The serially diluted multispecific antibody was mixed with a final concentration of 0.5 ng/ml TGF-β1, incubated at room temperature for 30 min, and then effector cells were added for further incubation for 18 hours. The bio-glo luciferase assay system (Promega G7940) was used for color development and the chemiluminescence signal was collected with a microplate reader.
结果如图8A和图8B所示。结果显示，本发明所有的多特异性抗体均能够很好地阻断TGF-β信号通路。The results are shown in Figures 8A and 8B. The results show that all the multispecific antibodies of the present invention can well block the TGF-β signaling pathway.
实施例8：多特异性抗体阻断VEGF信号通路Example 8: Multispecific antibodies block the VEGF signaling pathway
将VEGFRII Luciferase/HEK293效应细胞按照1×10 5个/孔接种至96孔细胞培养板白底中。将梯度稀释后的多特异性抗体与最终浓度为100ng/ml VEGFA混合，室温孵育30min中后加入到效应细胞中继续孵育6h。使用Bio-glo luciferase assay system(Promega G7940)试剂盒显色后用酶标仪收集化学发光信号。 VEGFRII Luciferase/HEK293 effector cells were seeded into 96-well cell culture plates at a rate of 1×10 5 cells/well. The serially diluted multispecific antibody was mixed with a final concentration of 100 ng/ml VEGFA, incubated at room temperature for 30 min, and then added to the effector cells for further incubation for 6 h. The bio-glo luciferase assay system (Promega G7940) was used for color development and the chemiluminescence signal was collected with a microplate reader.
结果如图9A和图9B所示。结果显示，本发明所有的多特异性抗体均能够很好地阻断VEGF信号通路。The results are shown in Figures 9A and 9B. The results show that all the multispecific antibodies of the present invention can well block the VEGF signaling pathway.
实施例9：多特异性抗体的热稳定性Example 9: Thermostability of Multispecific Antibodies
利用DSC(Differential scanning calorimetry，差示扫描量热法)检测不同抗体的热稳定性。样品用PBS稀释到1mg/ml；将5000×荧光显色剂Cypro Orange(购于Bio-Rad)用超纯水稀释50倍得到100×荧光显色剂Sypro Orange。取50μl 1mg/ml的样品加入10μl 100×荧光显色剂Sypro Orange、40μl超纯水，混匀后，取30μl加入到96孔PCR板中，每个样品做3个复孔，放入PCR仪中，设置升温程序为：25℃恒温 5min，以0.5℃/min的速度升温至99℃。程序结束后在“Melt Curve”图中读取曲线的最低点的温度值，即为样品的Tm值。The thermal stability of different antibodies was detected by DSC (Differential scanning calorimetry, differential scanning calorimetry). The sample was diluted to 1 mg/ml with PBS; 5000× fluorescent color reagent Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100× fluorescent color developer Sypro Orange. Take 50μl of 1mg/ml sample and add 10μl of 100× fluorescent color reagent Sypro Orange and 40μl of ultrapure water. After mixing, take 30μl and add it to a 96-well PCR plate. Make 3 duplicate wells for each sample and put them into the PCR instrument. , set the heating program as follows: 25 °C constant temperature for 5 min, and the temperature is increased to 99 °C at a rate of 0.5 °C/min. After the program ends, read the temperature value at the lowest point of the curve in the "Melt Curve" graph, which is the Tm value of the sample.
结果如下表1和表2所示。The results are shown in Tables 1 and 2 below.
表1：PD-L1xTGFβxVEGF多特异性抗体的Tm值Table 1: Tm values of PD-L1xTGFβxVEGF multispecific antibodies
编号Numbering Tm1(℃)Tm1(℃)
PD-L1xTGFβxVEGF TriAb 1PD-L1xTGFβxVEGF TriAb 1 55.355.3
PD-L1xTGFβxVEGF TriAb 2PD-L1xTGFβxVEGF TriAb 2 55.855.8
PD-L1xTGFβxVEGF TriAb 3PD-L1xTGFβxVEGF TriAb 3 54.654.6
PD-L1xTGFβxVEGF TriAb 4PD-L1xTGFβxVEGF TriAb 4 5959
PD-L1xTGFβxVEGF TriAb 5PD-L1xTGFβxVEGF TriAb 5 N/AN/A
表2：PD-L1xTGFβxVEGF多特异性抗体的Tm值Table 2: Tm values of PD-L1xTGFβxVEGF multispecific antibodies
编号Numbering Tm1(℃)Tm1(℃)
PD-L1xTGFβxVEGF TriAb 6PD-L1xTGFβxVEGF TriAb 6 68.568.5
PD-L1xTGFβxVEGF TriAb 7PD-L1xTGFβxVEGF TriAb 7 68.168.1
PD-L1xTGFβxVEGF TriAb 8PD-L1xTGFβxVEGF TriAb 8 68.168.1
PD-L1xTGFβxVEGF TriAb 9PD-L1xTGFβxVEGF TriAb 9 68.568.5
结果显示，本发明的多特异性抗体例如PD-L1xTGFβxVEGF TriAb 1-5和多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9均具有较好的热稳定性，特别是其中多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9的Tm均大于68℃，稳定性较好，类似于常规单抗。The results show that the multispecific antibodies of the present invention such as PD-L1xTGFβxVEGF TriAb 1-5 and multispecific antibody PD-L1xTGFβxVEGF TriAb 6-9 have better thermal stability, especially among the multispecific antibodies PD-L1xTGFβxVEGF TriAb The Tm of 6-9 were all greater than 68°C, and the stability was good, similar to that of conventional monoclonal antibodies.
实施例10：多特异性抗体的加速稳定性实验测定Example 10: Accelerated Stability Assay of Multispecific Antibodies
本实验通过检测多特异性抗体在40℃放置30天之后的纯度及生物学活性的变化，从而评价该抗体的长期热稳定性。In this experiment, the long-term thermal stability of the multispecific antibody was evaluated by detecting the changes in the purity and biological activity of the multispecific antibody after being placed at 40°C for 30 days.
使用SEC的方法测定目的抗体在40℃放置0、7及14天后的纯度。The purity of the target antibody after being placed at 40°C for 0, 7 and 14 days was determined by the method of SEC.
实验结果如表3所示。The experimental results are shown in Table 3.
表3：加速稳定性样品单体比例Table 3: Accelerated Stability Sample Monomer Ratios
样品名 sample name 0天0 days 7天7 days 14天14 days
PD-L1xTGFβxVEGF TriAb 6PD-L1xTGFβxVEGF TriAb 6 98.5％98.5% 98.0％98.0% 96.5％96.5%
PD-L1xTGFβxVEGF TriAb 7PD-L1xTGFβxVEGF TriAb 7 95.9％95.9% 91.8％91.8% 84.3％84.3%
PD-L1xTGFβxVEGF TriAb 8PD-L1xTGFβxVEGF TriAb 8 97.5％97.5% 94.8％94.8% 92.3％92.3%
PD-L1xTGFβxVEGF TriAb 9PD-L1xTGFβxVEGF TriAb 9 85.7％85.7% 85.6％85.6% 80.3％80.3%
结果显示，本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6-9均具有较好的稳定性，其中PD-L1xTGFβxVEGF TriAb 6和PD-L1xTGFβxVEGF TriAb 8的单体纯度没有明显变化。The results showed that the multispecific antibodies PD-L1xTGFβxVEGF TriAb 6-9 of the present invention all had good stability, and the monomeric purity of PD-L1xTGFβxVEGF TriAb 6 and PD-L1xTGFβxVEGF TriAb 8 did not change significantly.
实施例11：多特异性抗体药代动力学评价Example 11: Multispecific Antibody Pharmacokinetic Evaluation
本实验检测了多特异性抗体在大鼠体内的药代动力学性质，每个药物6只SD大鼠(雌雄各3只，12/12小时光/暗调节，温度24℃±2℃，湿度40％-70％，自由进水饮食)购自浙江维通利华实验技术有限公司。实验当天对SD大鼠单次尾静脉注射PD-L1xTGFβxVEGF TriAb 1或PD-L1xTGFβxVEGF TriAb 4分子，注射剂量为10mg/kg。In this experiment, the pharmacokinetic properties of multispecific antibodies in rats were tested. Each drug contained 6 SD rats (3 males and 3 males, 12/12 hours light/dark adjustment, temperature 24°C ± 2°C, humidity 40%-70%, free water and diet) were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, SD rats were injected with PD-L1xTGFβxVEGF TriAb 1 or PD-L1xTGFβxVEGF TriAb 4 by a single tail vein injection at a dose of 10 mg/kg.
取血时间点：给药后5min、15min、0.5小时、2小时、6小时、24小时、48小时、96小时、168小时、336小时、504小时于大鼠颈静脉采血。全血样品2℃-8℃放置30min，12000rpm离心5min收集血清，所得血清再于2℃-8℃，12000rpm离心5min，-80℃保存ELISA检测血清中游离PD-L1xTGFβxVEGF TriAb 1或PD-L1xTGFβxVEGF TriAb 4分子。Blood collection time points: 5min, 15min, 0.5h, 2h, 6h, 24h, 48h, 96h, 168h, 336h, and 504h after administration, blood was collected from the jugular vein of rats. Whole blood samples were placed at 2℃-8℃ for 30min, centrifuged at 12000rpm for 5min to collect serum, the obtained serum was then centrifuged at 2℃-8℃, 12000rpm for 5min, and stored at -80℃ to detect free PD-L1xTGFβxVEGF TriAb 1 or PD-L1xTGFβxVEGF TriAb in serum by ELISA 4 molecules.
结果如表4所示。The results are shown in Table 4.
表4：多特异性抗体在SD大鼠中的T1/2Table 4: T1/2 of multispecific antibodies in SD rats
受试药物test drug 给药方式way of administration T1/2T1/2
PD-L1xTGFβxVEGF TriAb 1PD-L1xTGFβxVEGF TriAb 1 IVIV 110.6小时110.6 hours
PD-L1xTGFβxVEGF TriAb 4PD-L1xTGFβxVEGF TriAb 4 IVIV 86.9小时86.9 hours
结果显示，本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 1游离状态分子在SD大鼠体内半衰期约为110.6小时，PD-L1xTGFβxVEGF TriAb 4游离状态分子在SD大鼠体内半衰期约为86.9小时。The results show that the half-life of the multispecific antibody PD-L1xTGFβxVEGF TriAb 1 free state molecule of the present invention is about 110.6 hours in SD rats, and the half-life of the PD-L1xTGFβxVEGF TriAb 4 free state molecule in SD rats is about 86.9 hours.
实施例12：多特异性抗体药代动力学评价Example 12: Multispecific Antibody Pharmacokinetic Evaluation
实验检测了多特异性抗体在小鼠体内的药代动力学性质，每个药物6只SD大鼠(雌雄各3只，12/12小时光/暗调节，温度24℃±2℃，湿度40％-70％，自由进水饮食)购自浙江维通利华实验技术有限公司。实验当天对SD大鼠单次尾静脉注射PD-L1xTGFβxVEGF TriAb 6或PD-L1xTGFβxVEGF TriAb 9分子，注射剂量为10mg/kg。The pharmacokinetic properties of multispecific antibodies in mice were tested experimentally. Each drug contained 6 SD rats (3 males and 3 females, 12/12 hours light/dark adjustment, temperature 24°C ± 2°C, humidity 40°C). %-70%, free water and diet) were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, SD rats were injected with PD-L1xTGFβxVEGF TriAb 6 or PD-L1xTGFβxVEGF TriAb 9 molecules in a single tail vein at a dose of 10 mg/kg.
取血时间点：给药后5min、0.5小时、2小时、6小时、24小时、48小时、96小时、168小时、336小时、504小时于大鼠颈静脉采血。全血样品2℃-8℃放置30min，12000rpm离心5min收集血清，所得血清再于2℃-8℃，12000rpm离心5min，-80℃保存，ELISA检测血清中游离PD-L1xTGFβxVEGF TriAb 6或PD-L1xTGFβxVEGF TriAb 9分子。Blood collection time points: 5 min, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours and 504 hours after administration, blood was collected from the jugular vein of rats. Whole blood samples were placed at 2°C-8°C for 30min, centrifuged at 12000rpm for 5min to collect serum, the obtained serum was centrifuged at 2°C-8°C, 12000rpm for 5min, and stored at -80°C. Free PD-L1xTGFβxVEGF TriAb 6 or PD-L1xTGFβxVEGF in serum was detected by ELISA TriAb 9 molecule.
结果如表5所示。The results are shown in Table 5.
表5：三异性抗体在小鼠中的T1/2Table 5: T1/2 of triisoantibodies in mice
受试药物test drug 给药方式way of administration T1/2T1/2
PD-L1xTGFβxVEGF TriAb 6PD-L1xTGFβxVEGF TriAb 6 IVIV 62.4小时62.4 hours
PD-L1xTGFβxVEGF TriAb 9PD-L1xTGFβxVEGF TriAb 9 IVIV 48.8小时48.8 hours
结果显示，本发明的多特异性抗体PD-L1xTGFβxVEGF TriAb 6游离状态分子在小鼠体内半衰期约为62.4小时，PD-L1xTGFβxVEGF TriAb 9游离状态分子在小鼠体内半衰期约为48.8小时。The results show that the half-life of the free state molecule of the multispecific antibody PD-L1xTGFβxVEGF TriAb 6 of the present invention is about 62.4 hours in mice, and the half-life of the free state molecule of PD-L1xTGFβxVEGF TriAb 9 is about 48.8 hours in mice.
实施例13：多特异性抗体的肿瘤抑制活性研究(MDA-MB-231)Example 13: Tumor inhibitory activity study of multispecific antibody (MDA-MB-231)
本实验采用人乳腺癌MDA-MD-231细胞/NSG小鼠注射人PBMC模型测定多特异性抗体的抗肿瘤作用。体外培养扩增足够的MDA-MD-231细胞(购自Addexbio)，胰酶消化后收集细胞，用PBS清洗3遍后计数，按7×10 6细胞/小鼠的量接种到雌性8周龄的NSG重度免疫缺陷小鼠(购自上海南方模式生物科技股份有限公司)右侧腹部皮下。每日观察细胞在NSG小鼠皮下成瘤情况，接种20天后每只小鼠尾静脉注射4×10 6的PBMC。注射完PBMC过3天后，使用游标卡尺测量每只动物右侧腹部皮下肿瘤的最大宽轴W和最大长轴L，使用电子天平称量每只小鼠的体重。 In this experiment, human breast cancer MDA-MD-231 cells/NSG mice were injected into human PBMC model to determine the anti-tumor effect of multispecific antibodies. Sufficient MDA-MD-231 cells (purchased from Addexbio) were cultured and expanded in vitro, cells were collected after trypsinization, washed three times with PBS, counted, and inoculated into 8-week-old females at 7×10 6 cells/mouse The NSG severe immunodeficiency mice (purchased from Shanghai Nanfang Model Biotechnology Co., Ltd.) were subcutaneously on the right abdomen. Subcutaneous tumor formation of NSG mice was observed every day, and 4×10 6 PBMCs were injected into each mouse through tail vein 20 days after inoculation. Three days after the injection of PBMCs, the maximum wide axis W and the maximum long axis L of the subcutaneous tumor in the right abdomen of each animal were measured with a vernier caliper, and the weight of each mouse was weighed with an electronic balance.
按肿瘤体积T＝1/2×W×W×L计算每只小鼠右侧腹部皮下肿瘤体积。剔除瘤体积过大和过小的小鼠，按平均瘤体积将NSG小鼠平均分为3组，每组6只。按表6分组给 药方案分组并注射相应剂量的PD-L1xTGFβxVEGF TriAb 6。The subcutaneous tumor volume in the right abdomen of each mouse was calculated according to the tumor volume T=1/2×W×W×L. The mice with too large and too small tumor volume were eliminated, and the NSG mice were divided into 3 groups according to the average tumor volume, with 6 mice in each group. According to Table 6 grouping and dosing schedule grouping and injecting the corresponding dose of PD-L1xTGFβxVEGF TriAb 6.
表6：PD-L1xTGFβxVEGF TriAb 6肿瘤抑制活性实验方案Table 6: Experimental protocol for PD-L1xTGFβxVEGF TriAb 6 tumor suppressor activity
组别group 给药类别Administration category 给药剂量Dosage 给药频率 Dosing frequency
Group 1Group 1 PBSPBS —— 每周2次共8次8 times 2 times a week
Group 2Group 2 PD-L1xTGFβxVEGF TriAb 6PD-L1xTGFβxVEGF TriAb 6 28mg/kg28mg/kg 每周2次共8次8 times 2 times a week
Group 3Group 3 PD-L1xTGFβxVEGF TriAb 6PD-L1xTGFβxVEGF TriAb 6 2.8mg/kg2.8mg/kg 每周2次共8次8 times 2 times a week
每周2次测量小鼠肿瘤体积与小鼠体重。于接种肿瘤细胞34天后最后一次测量小鼠体重与肿瘤体积，对小鼠执行安乐死；与PBS组相比，PD-L1xTGFβxVEGF TriAb6能剂量依赖性地抑制肿瘤的生长，2.8mg/kg和28mg/kg剂量下的TGI分别为76％和88％；且肿瘤体积与PBS组相比具有显著性差异。肿瘤平均重量的结果，以及肿瘤生长抑制率与肿瘤体积的趋势基本一致(图10A、图10B和表7)。Mouse tumor volume and mouse body weight were measured twice a week. The body weight and tumor volume of the mice were last measured 34 days after inoculation with tumor cells, and the mice were euthanized; compared with the PBS group, PD-L1xTGFβxVEGF TriAb6 could dose-dependently inhibit the growth of tumors, 2.8mg/kg and 28mg/kg TGI at doses were 76% and 88%, respectively; and tumor volume was significantly different from the PBS group. The results for mean tumor weight, as well as tumor growth inhibition rates were generally in agreement with the trend for tumor volume (Figure 10A, Figure 10B and Table 7).
表7：各处理组对NSG小鼠皮下接种MDA-MB-231后的肿瘤体积和TGI的影响Table 7: Effects of each treatment group on tumor volume and TGI after subcutaneous inoculation of MDA-MB-231 in NSG mice
Figure PCTCN2021132464-appb-000016
Figure PCTCN2021132464-appb-000016
注：与PBS组相比，“***”P<0.001；“**”P<0.01。Note: Compared with the PBS group, "***"P<0.001; "**"P<0.01.
每日观察各组小鼠状态未发现异常；每周两次称量小鼠体重，各组小鼠体重未发生明显降低，实验结束时各剂量组小鼠平均体重相比刚开始给药治疗时的体重，未发生下降(见图10C)；试验结束时各组小鼠解剖观察肝脏、肾脏、肺脏等主要脏器亦未见明显病变，表明各组药物在本试验所采用的给药剂量下对小鼠未见明显毒性。The state of the mice in each group was observed daily and no abnormality was found; the weight of the mice was weighed twice a week, and the weight of the mice in each group did not decrease significantly. At the end of the test, no obvious lesions were found in the liver, kidneys, lungs and other major organs of the mice in each group, indicating that the drugs in each group were at the doses used in this test. No obvious toxicity was observed in mice.
综上，本发明的多特异性抗体例如PD-L1xTGFβxVEGF TriAb 6能够剂量依赖性地抑制MDA-MB-231皮下移植瘤的生长，起效剂量为2.8mg/kg；2个给药剂量下 (2.8mg/kg和28mg/kg)对小鼠未见明显毒性。To sum up, the multispecific antibodies of the present invention, such as PD-L1xTGFβxVEGF TriAb 6, can dose-dependently inhibit the growth of MDA-MB-231 subcutaneous transplanted tumor with an effective dose of 2.8 mg/kg; under 2 doses (2.8 mg/kg) mg/kg and 28mg/kg) showed no obvious toxicity to mice.
实施例14：多特异性抗体的肿瘤抑制活性研究(NCI-H1975)Example 14: Tumor inhibitory activity study of multispecific antibodies (NCI-H1975)
本实验采用人肺癌NCI-H1975细胞/NSG小鼠注射人PBMC模型测定多特异性抗体的抗肿瘤作用。体外培养扩增足够的NCI-H1975细胞(购自Addexbio)，胰酶消化后收集细胞，用PBS清洗3遍后计数，按4×10 6细胞/小鼠的量接种到雌性8周龄的NSG重度免疫缺陷小鼠(购自北京维通利华实验动物技术有限公司)右侧腹部皮下。每日观察细胞在NSG小鼠皮下成瘤情况，接种7天后每只小鼠尾静脉注射5×10 6PBMC。注射完PBMC过4天后，使用游标卡尺测量每只动物右侧腹部皮下肿瘤的最大宽轴W和最大长轴L，使用电子天平称量每只小鼠的体重。 In this experiment, human lung cancer NCI-H1975 cells/NSG mice were injected with human PBMC model to determine the anti-tumor effect of multispecific antibodies. Enough NCI-H1975 cells (purchased from Addexbio) were cultured and expanded in vitro, cells were collected after trypsinization, washed three times with PBS, counted, and inoculated into 8-week-old female NSGs at a rate of 4×10 6 cells/mouse Severe immunodeficiency mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were subcutaneously placed on the right abdomen. Subcutaneous tumor formation of NSG mice was observed daily, and 5×10 6 PBMCs were injected into each mouse through tail vein 7 days after inoculation. Four days after the injection of PBMC, the maximum width axis W and the maximum long axis L of the subcutaneous tumor in the right abdomen of each animal were measured using a vernier caliper, and the body weight of each mouse was weighed using an electronic balance.
按肿瘤体积T＝1/2×W×W×L计算每只小鼠右侧腹部皮下肿瘤体积。剔除瘤体积过大和过小的小鼠，按平均瘤体积将NSG小鼠平均分为3组，每组6只。按表8分组给药方案分组并注射相应剂量的抗PD-L1抗体阿特珠单抗(Atezolizumab，罗氏)或PD-L1xTGFβxVEGF TriAb 6。给药剂量按各组分等摩尔浓度剂量给药。The subcutaneous tumor volume in the right abdomen of each mouse was calculated according to the tumor volume T=1/2×W×W×L. The mice with too large and too small tumor volume were eliminated, and the NSG mice were divided into 3 groups according to the average tumor volume, with 6 mice in each group. Grouped according to the grouping dosing schedule in Table 8 and injected the corresponding dose of anti-PD-L1 antibody atezolizumab (Atezolizumab, Roche) or PD-L1xTGFβxVEGF TriAb 6. Dosages are administered in equimolar concentrations of each component.
表8：PD-L1xTGFβxVEGF TriAb 6肿瘤抑制活性实验方案Table 8: Experimental protocol for PD-L1xTGFβxVEGF TriAb 6 tumor suppressor activity
Figure PCTCN2021132464-appb-000017
Figure PCTCN2021132464-appb-000017
每周3次测量小鼠肿瘤体积与小鼠体重。于接种肿瘤细胞20天后最后一次测量小鼠体重与肿瘤体积，对小鼠执行安乐死；10mg/kg的阿特珠单抗或14mg/kg的PD-L1xTGFβxVEGF TriAb 6的TGI分别为67％和90％；且肿瘤体积与PBS组相比具有显著性差异。与阿特珠单抗组相比，PD-L1xTGFβxVEGF TriAb 6组显示出了更好的抗肿瘤药效，且具有统计学意义。肿瘤平均重量的结果，以及肿瘤生长抑制率与肿瘤体积的趋势基本一致(图11A和表9)。Mouse tumor volume and mouse body weight were measured 3 times a week. The mice were euthanized by the last measurement of body weight and tumor volume 20 days after tumor cell inoculation; the TGI of 10 mg/kg atezolizumab or 14 mg/kg PD-L1xTGFβxVEGF TriAb 6 were 67% and 90%, respectively ; And the tumor volume was significantly different compared with the PBS group. Compared with the atezolizumab group, the PD-L1xTGFβxVEGF TriAb 6 group showed better antitumor efficacy with statistical significance. The results for mean tumor weight, as well as tumor growth inhibition rates, were generally in agreement with the trend for tumor volume (Figure 11A and Table 9).
表9：各处理组对NSG小鼠皮下接种NCI-H1975后的肿瘤体积和TGI的影响Table 9: Effects of each treatment group on tumor volume and TGI after subcutaneous inoculation of NSG mice with NCI-H1975
Figure PCTCN2021132464-appb-000018
Figure PCTCN2021132464-appb-000018
Figure PCTCN2021132464-appb-000019
Figure PCTCN2021132464-appb-000019
注1：与PBS组相比，“***”P<0.001；“**”P<0.01。Note 1: Compared with the PBS group, "***"P<0.001; "**"P<0.01.
注2：与阿特珠单抗组相比，“###”P<0.001；“##”P<0.01。Note 2: Compared with atezolizumab group, "###" P<0.001; "##" P<0.01.
每日观察各组小鼠状态未发现异常；每周两次称量小鼠体重，各组小鼠体重未发生明显降低，实验结束时各剂量组小鼠体重相比刚开始给药治疗时的体重，未发生下降(见图11B)；试验结束时各组小鼠解剖观察肝脏、肾脏、肺脏等主要脏器亦未见明显病变，表明各组药物在本试验所采用的给药剂量下对小鼠未见明显毒性。The state of the mice in each group was observed daily, and no abnormality was found; the weight of the mice was weighed twice a week, and the weight of the mice in each group did not decrease significantly. Body weight did not decrease (see Figure 11B); at the end of the test, no obvious lesions were found in the liver, kidneys, lungs and other major organs of the mice in each group after dissection, indicating that the drugs in each group were effective at the doses used in this test. No obvious toxicity was seen in mice.
综上，本发明的多特异性抗体例如PD-L1xTGFβxVEGF TriAb 6可有效地抑制NCI-H1975皮下移植瘤的生长，且明显优于同摩尔浓度下的阿特珠单抗；在14mg/ml药剂量下PD-L1xTGFβxVEGF TriAb 6对小鼠未见明显毒性。In conclusion, the multispecific antibodies of the present invention such as PD-L1xTGFβxVEGF TriAb 6 can effectively inhibit the growth of NCI-H1975 subcutaneously transplanted tumor, and are significantly better than atezolizumab at the same molar concentration; at a dose of 14 mg/ml PD-L1xTGFβxVEGF TriAb 6 showed no obvious toxicity in mice.
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions of those details may be made within the scope of the present invention in light of all the teachings disclosed. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (37)

  1. 一种多特异性抗体，包括：A multispecific antibody that includes:
    靶向PD-L1的第一蛋白功能区，Targeting the first protein functional region of PD-L1,
    靶向VEGF的第二蛋白功能区，和targeting the second protein domain of VEGF, and
    靶向TGF-β的第三蛋白功能区；Targeting the third protein functional domain of TGF-β;
    其中，in,
    所述第一蛋白功能区为抗PD-L1单域抗体，The first protein functional region is an anti-PD-L1 single domain antibody,
    所述第二蛋白功能区为VEGF受体、VEGF受体功能片段、包含VEGF受体功能片段的融合蛋白或者抗VEGF抗体，The second protein functional region is a VEGF receptor, a VEGF receptor functional fragment, a fusion protein comprising a VEGF receptor functional fragment, or an anti-VEGF antibody,
    所述第三蛋白功能区为TGF-β受体、TGF-β受体功能片段、包含TGF-β受体功能片段的融合蛋白或者抗TGF-β抗体。The third protein functional domain is a TGF-β receptor, a TGF-β receptor functional fragment, a fusion protein comprising a TGF-β receptor functional fragment, or an anti-TGF-β antibody.
  2. 根据权利要求1所述的多特异性抗体，其中，所述抗PD-L1单域抗体，其包含重链可变区，所述重链可变区包含氨基酸序列如SEQ ID NO:26所示的HCDR1、氨基酸序列如SEQ ID NO:27所示的HCDR2和氨基酸序列如SEQ ID NO:28所示的HCDR3；The multispecific antibody according to claim 1, wherein the anti-PD-L1 single domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26 HCDR1, HCDR2 with amino acid sequence as shown in SEQ ID NO:27 and HCDR3 with amino acid sequence as shown in SEQ ID NO:28;
    优选地，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:2所示。Preferably, the amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:2.
  3. 根据权利要求1至2中任一权利要求所述的多特异性抗体，其中，所述VEGF受体为VEGFR1和/或VEGFR2。The multispecific antibody of any one of claims 1 to 2, wherein the VEGF receptor is VEGFR1 and/or VEGFR2.
  4. 根据权利要求1至3中任一权利要求所述的多特异性抗体，其中，VEGF受体功能片段为VEGFR1胞外片段和/或VEGFR2胞外片段；The multispecific antibody according to any one of claims 1 to 3, wherein the VEGF receptor functional fragment is a VEGFR1 extracellular fragment and/or a VEGFR2 extracellular fragment;
    优选地，所述VEGF受体功能片段的氨基酸序列如SEQ ID NO:4所示。Preferably, the amino acid sequence of the VEGF receptor functional fragment is shown in SEQ ID NO:4.
  5. 根据权利要求1至2中任一权利要求所述的多特异性抗体，其中，所述抗VEGF抗体，包含重链可变区和轻链可变区，其中：The multispecific antibody of any one of claims 1-2, wherein the anti-VEGF antibody comprises a heavy chain variable region and a light chain variable region, wherein:
    所述重链可变区包含氨基酸序列如SEQ ID NO:29所示的HCDR1、氨基酸序列 如SEQ ID NO:30所示的HCDR2和氨基酸序列如SEQ ID NO:31所示的HCDR3；并且所述轻链可变区包含氨基酸序列如SEQ ID NO:32所示的LCDR1、氨基酸序列如SEQ ID NO:33所示的LCDR2和氨基酸序列如SEQ ID NO:34所示的LCDR3；The heavy chain variable region comprises HCDR1 whose amino acid sequence is shown in SEQ ID NO:29, HCDR2 whose amino acid sequence is shown in SEQ ID NO:30, and HCDR3 whose amino acid sequence is shown in SEQ ID NO:31; and the The light chain variable region comprises LCDR1 whose amino acid sequence is as shown in SEQ ID NO:32, LCDR2 whose amino acid sequence is as shown in SEQ ID NO:33 and LCDR3 whose amino acid sequence is as shown in SEQ ID NO:34;
    优选地，所述重链可变区的氨基酸序列如SEQ ID NO:16所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:19所示；Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19;
    优选地，所述抗VEGF抗体的重链恒定区为人Ig gamma-1 chain C region或人Ig gamma-4 chain C region，并且其轻链恒定区为人Ig kappa chain C region；Preferably, the heavy chain constant region of the anti-VEGF antibody is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region thereof is a human Ig kappa chain C region;
    优选地，所述抗VEGF抗体的重链恒定区还包含按照EU编号***的L234A突变和L235A突变；可选地，还包含G237A突变；Preferably, the heavy chain constant region of the anti-VEGF antibody further comprises the L234A mutation and the L235A mutation according to the EU numbering system; optionally, the G237A mutation;
    优选地，所述抗VEGF抗体的重链恒定区的氨基酸序列如SEQ ID NO:17所示；Preferably, the amino acid sequence of the heavy chain constant region of the anti-VEGF antibody is shown in SEQ ID NO: 17;
    优选地，所述抗VEGF抗体的轻链恒定区的氨基酸序列如SEQ ID NO:20所示。Preferably, the amino acid sequence of the light chain constant region of the anti-VEGF antibody is shown in SEQ ID NO:20.
  6. 根据权利要求1至5中任一权利要求所述的多特异性抗体，其中，所述TGF-β受体为TGF-βRII。The multispecific antibody of any one of claims 1 to 5, wherein the TGF-beta receptor is TGF-betaRII.
  7. 根据权利要求1至6中任一权利要求所述的多特异性抗体，其中，所述TGF-β受体功能片段为TGF-βRII的胞外片段；The multispecific antibody according to any one of claims 1 to 6, wherein the TGF-β receptor functional fragment is an extracellular fragment of TGF-βRII;
    优选地，所述TGF-β受体功能片段的氨基酸序列如SEQ ID NO:6所示。Preferably, the amino acid sequence of the TGF-β receptor functional fragment is shown in SEQ ID NO:6.
  8. 根据权利要求1至7中任一权利要求所述的多特异性抗体，其中，所述第一蛋白功能区、第二蛋白功能区和第三蛋白功能区独立地为1个、2个或3个拷贝。The multispecific antibody of any one of claims 1 to 7, wherein the first, second, and third protein domains are independently 1, 2, or 3 a copy.
  9. 根据权利要求1至8中任一权利要求所述的多特异性抗体，其中，所述第一蛋白功能区、第二蛋白功能区和第三蛋白功能区为直接连接或通过连接片段连接；The multispecific antibody according to any one of claims 1 to 8, wherein the first protein functional domain, the second protein functional domain and the third protein functional domain are directly linked or linked by linking fragments;
    优选地，所述连接片段的氨基酸序列独立地选自SEQ ID NO:5和SEQ ID NO:8。Preferably, the amino acid sequence of the linker fragment is independently selected from SEQ ID NO:5 and SEQ ID NO:8.
  10. 根据权利要求1至9中任一权利要求所述的多特异性抗体，其还包含人IgG1Fc段；The multispecific antibody of any one of claims 1 to 9, further comprising a human IgGl Fc fragment;
    优选地，所述人IgG1的Fc段包含按照EU编号***的L234A突变和L235A突变；Preferably, the Fc fragment of the human IgG1 comprises the L234A mutation and the L235A mutation according to the EU numbering system;
    优选地，所述人IgG1的Fc段的氨基酸序列如SEQ ID NO:3所示。Preferably, the amino acid sequence of the Fc segment of the human IgG1 is shown in SEQ ID NO:3.
  11. 根据权利要求10所述的多特异性抗体，其中，所述人IgG1 Fc段与第一蛋白功能区、第二蛋白功能区和第三蛋白功能区中的两个直接连接或通过连接片段连接；The multispecific antibody of claim 10, wherein the human IgG1 Fc segment is directly linked to two of the first protein functional domain, the second protein functional domain and the third protein functional domain or is linked by a linker fragment;
    优选地，所述连接片段的氨基酸序列独立地选自SEQ ID NO:5和SEQ ID NO:8。Preferably, the amino acid sequence of the linker fragment is independently selected from SEQ ID NO:5 and SEQ ID NO:8.
  12. 根据权利要求1至11中任一权利要求所述的多特异性抗体，按照N末端至C末端，依次包含：The multispecific antibody according to any one of claims 1 to 11, comprising, in order from N-terminus to C-terminus:
    第一蛋白功能区、人IgG1的Fc段、第二蛋白功能区和第三蛋白功能区；The first protein functional region, the Fc segment of human IgG1, the second protein functional region and the third protein functional region;
    第一蛋白功能区、人IgG1的Fc段、第三蛋白功能区和第二蛋白功能区；The first protein functional region, the Fc segment of human IgG1, the third protein functional region and the second protein functional region;
    第一蛋白功能区、第三蛋白功能区、人IgG1的Fc段和第二蛋白功能区；或者The first protein functional region, the third protein functional region, the Fc segment of human IgG1 and the second protein functional region; or
    第一蛋白功能区、第二蛋白功能区、人IgG1的Fc段和第三蛋白功能区。The first protein functional region, the second protein functional region, the Fc segment of human IgG1 and the third protein functional region.
  13. 根据权利要求1至12中任一权利要求所述的多特异性抗体，其为由两条相同的肽链形成的二聚体；The multispecific antibody of any one of claims 1 to 12, which is a dimer formed from two identical peptide chains;
    优选地，所述肽链的氨基酸序列如SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:10所示。Preferably, the amino acid sequence of the peptide chain is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.
  14. 根据权利要求1至9中任一权利要求所述的多特异性抗体，其还包含人IgG1重链恒定区；The multispecific antibody of any one of claims 1 to 9, further comprising a human IgGl heavy chain constant region;
    优选地，所述人IgG1重链恒定区还包含Knob-in-hole突变；Preferably, the human IgG1 heavy chain constant region further comprises a Knob-in-hole mutation;
    优选地，所述人IgG1重链恒定区的两条肽链的氨基酸序列分别如SEQ ID NO:12和SEQ ID NO:14所示。Preferably, the amino acid sequences of the two peptide chains of the human IgG1 heavy chain constant region are shown in SEQ ID NO: 12 and SEQ ID NO: 14, respectively.
  15. 根据权利要求14所述的多特异性抗体，其为由两条不同的第一肽链和第二肽链形成的二聚体，按照N末端至C末端，The multispecific antibody according to claim 14, which is a dimer formed by two different first peptide chains and second peptide chains, according to N-terminal to C-terminal,
    第一肽链依次包含：第一蛋白功能区、人IgG1重链恒定区和第三蛋白功能区；并且第二肽链依次包含：第一蛋白功能区、人IgG1重链恒定区和第二蛋白功能区；The first peptide chain sequentially comprises: the first protein functional region, the human IgG1 heavy chain constant region and the third protein functional region; and the second peptide chain sequentially comprises: the first protein functional region, the human IgG1 heavy chain constant region and the second protein functional area;
    优选地，第一肽链的氨基酸序列如SEQ ID NO:11所示，并且第二肽链的氨基酸序 列如SEQ ID NO:13所示。Preferably, the amino acid sequence of the first peptide chain is shown in SEQ ID NO: 11, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 13.
  16. 根据权利要求1至2和5至9中任一权利要求所述的多特异性抗体，其中：The multispecific antibody of any one of claims 1 to 2 and 5 to 9, wherein:
    所述第一蛋白功能区为两个拷贝，其中，两个拷贝分别连接在抗VEGF抗体的两条重链的N末端、抗VEGF抗体的两条轻链的N末端或抗VEGF抗体的两条轻链的C末端，或者一个拷贝连接在抗VEGF抗体的一条重链的N末端，另一个拷贝连接在与抗VEGF抗体的另一条重链相结合的轻链的N末端；并且所述第二蛋白功能区为两个拷贝，分别连接在抗VEGF抗体的两条重链的C末端；The first protein functional region has two copies, wherein the two copies are respectively connected to the N-terminus of the two heavy chains of the anti-VEGF antibody, the N-terminus of the two light chains of the anti-VEGF antibody, or the two copies of the anti-VEGF antibody. the C-terminus of the light chain, or one copy is attached to the N-terminus of one heavy chain of the anti-VEGF antibody and the other copy is attached to the N-terminus of the light chain bound to the other heavy chain of the anti-VEGF antibody; and the second There are two copies of the protein functional region, which are respectively connected to the C-terminus of the two heavy chains of the anti-VEGF antibody;
    或者，or,
    所述第二蛋白功能区为两个拷贝，其中，两个拷贝分别连接在抗VEGF抗体的两条重链的N末端、抗VEGF抗体的两条轻链的N末端或抗VEGF抗体的两条轻链的C末端，或者一个拷贝连接在抗VEGF抗体的一条重链的N末端，另一个拷贝连接在与抗VEGF抗体的另一条重链相结合的轻链的N末端；并且所述第一蛋白功能区为两个拷贝，分别连接在抗VEGF抗体的两条重链的C末端。The second protein functional region has two copies, wherein the two copies are respectively connected to the N-terminus of the two heavy chains of the anti-VEGF antibody, the N-terminus of the two light chains of the anti-VEGF antibody, or the two copies of the anti-VEGF antibody. the C-terminus of the light chain, or one copy is attached to the N-terminus of one heavy chain of the anti-VEGF antibody, and the other copy is attached to the N-terminus of the light chain bound to the other heavy chain of the anti-VEGF antibody; and the first There are two copies of the protein functional region, which are respectively connected to the C-terminus of the two heavy chains of the anti-VEGF antibody.
  17. 根据权利要求16所述的多特异性抗体，其为由两条相同的第一肽链和两条相同的第二肽链形成的四聚体，其中，The multispecific antibody of claim 16, which is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein,
    所述第一肽链的氨基酸序列如SEQ ID NO:15所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:18所示；The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 15; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 18;
    所述第一肽链的氨基酸序列如SEQ ID NO:21所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:22所示；The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 21; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 22;
    所述第一肽链的氨基酸序列如SEQ ID NO:23所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:24所示；The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 24;
    或者，or,
    所述第一肽链的氨基酸序列如SEQ ID NO:23所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:25所示。The amino acid sequence of the first peptide chain is shown in SEQ ID NO: 23; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO: 25.
  18. 分离的核酸分子，其编码权利要求1至17中任一权利要求所述的多特异性抗体。An isolated nucleic acid molecule encoding the multispecific antibody of any one of claims 1-17.
  19. 一种载体，其包含权利要求18所述的分离的核酸分子。18. A vector comprising the isolated nucleic acid molecule of claim 18.
  20. 一种宿主细胞，其包含权利要求18所述的分离的核酸分子，或者权利要求19所述的载体。A host cell comprising the isolated nucleic acid molecule of claim 18, or the vector of claim 19.
  21. 制备权利要求1至17中任一权利要求所述的多特异性抗体的方法，其包括在合适的条件下培养权利要求20的宿主细胞，以及从细胞培养物中回收所述多特异性抗体的步骤。A method for preparing the multispecific antibody of any one of claims 1 to 17, comprising culturing the host cell of claim 20 under suitable conditions, and recovering the multispecific antibody from the cell culture. step.
  22. 偶联物，其包括多特异性抗体以及偶联部分，其中，所述多特异性抗体为权利要求1至17中任一权利要求所述的多特异性抗体，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。A conjugate comprising a multispecific antibody and a coupling moiety, wherein the multispecific antibody is the multispecific antibody of any one of claims 1 to 17, and the coupling moiety is detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  23. 试剂盒，其包含权利要求1至17中任一权利要求所述的多特异性抗体，或者包含权利要求22所述的偶联物；A kit comprising the multispecific antibody of any one of claims 1 to 17, or the conjugate of claim 22;
    优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述多特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。Preferably, the kit further comprises a secondary antibody capable of specifically binding to the multispecific antibody; optionally, the secondary antibody further comprises a detectable label such as a radioisotope, fluorescent substance, luminescent substance , colored substances or enzymes.
  24. 一种药物组合物，其包含权利要求1至17中任一权利要求所述的多特异性抗体或者包含权利要求22所述的偶联物；可选地，其还包括药学上可接受的辅料。A pharmaceutical composition comprising the multispecific antibody according to any one of claims 1 to 17 or the conjugate according to claim 22; optionally, it also comprises a pharmaceutically acceptable adjuvant .
  25. 权利要求1至17中任一权利要求所述的多特异性抗体或者权利要求22所述的偶联物在制备预防和/或治疗恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤选自肺癌、子宫内膜癌、间皮瘤、***、直肠癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌；优选地，所述肺癌为非小细胞肺癌。Use of the multispecific antibody according to any one of claims 1 to 17 or the conjugate according to claim 22 in the preparation of a medicament for preventing and/or treating malignant tumors; Selected from lung cancer, endometrial cancer, mesothelioma, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer; preferably, the lung cancer for non-small cell lung cancer.
  26. 一种治疗和/或预防恶性肿瘤的方法，包括给予有需求的受试者以有效量的权利要求1至17中任一权利要求所述的多特异性抗体或者权利要求22所述的偶联物的步骤； 优选地，所述恶性肿瘤选自肺癌、子宫内膜癌、间皮瘤、***、直肠癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌；优选地，所述肺癌为非小细胞肺癌。A method of treating and/or preventing malignant tumors, comprising administering to a subject in need an effective amount of the multispecific antibody of any one of claims 1 to 17 or the conjugate of claim 22 Preferably, the malignant tumor is selected from lung cancer, endometrial cancer, mesothelioma, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, Esophageal cancer and head and neck cancer; preferably, the lung cancer is non-small cell lung cancer.
  27. 根据权利要求1至17中任一权利要求所述的多特异性抗体或者权利要求22所述的偶联物，其用于治疗和/或预防恶性肿瘤；优选地，所述恶性肿瘤选自肺癌、子宫内膜癌、间皮瘤、***、直肠癌、黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌；优选地，所述肺癌为非小细胞肺癌。The multispecific antibody according to any one of claims 1 to 17 or the conjugate according to claim 22 for the treatment and/or prevention of malignant tumors; preferably, the malignant tumors are selected from lung cancer , endometrial cancer, mesothelioma, cervical cancer, rectal cancer, melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer; preferably, the lung cancer is non- Small Cell Lung Cancer.
  28. 一种多特异性抗体，其包括：A multispecific antibody comprising:
    (a)抗PD-L1单域抗体；(a) anti-PD-L1 single domain antibody;
    (b)抗VEGF的抗体元件；和(b) an anti-VEGF antibody element; and
    (c)TGF-βRII胞外结构域。(c) TGF-βRII extracellular domain.
  29. 一种分离的多核苷酸，其编码权利要求28所述的多特异性抗体。An isolated polynucleotide encoding the multispecific antibody of claim 28.
  30. 一种载体，其含有权利要求29所述的多核苷酸。A vector comprising the polynucleotide of claim 29.
  31. 一种宿主细胞，其含有权利要求30所述的载体，或其基因组中整合有权利要求29所述的多核苷酸；A host cell comprising the vector of claim 30, or the polynucleotide of claim 29 integrated in its genome;
    或者，所述宿主细胞表达权利要求28所述的多特异性抗体。Alternatively, the host cell expresses the multispecific antibody of claim 28.
  32. 一种产生权利要求28所述多特异性抗体的方法，包括如下步骤：A method of producing the multispecific antibody of claim 28, comprising the steps of:
    (a)在合适的条件下，培养权利要求31所述的宿主细胞，从而获得含所述多特异性抗体的培养物；和(a) culturing the host cell of claim 31 under suitable conditions, thereby obtaining a culture containing the multispecific antibody; and
    (b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的多特异性抗体。(b) purifying and/or separating the culture obtained in step (a) to obtain the multispecific antibody.
  33. 一种免疫偶联物，其特征在于，所述免疫偶联物含有：An immunoconjugate, characterized in that the immunoconjugate contains:
    (a)权利要求28所述的多特异性抗体；和(a) the multispecific antibody of claim 28; and
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。(b) a conjugation moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or their combination.
  34. 权利要求28所述的多特异性抗体或者权利要求33所述的免疫偶联物的用途，其用于制备药剂、试剂、检测板或试剂盒；Use of the multispecific antibody of claim 28 or the immunoconjugate of claim 33 for the preparation of a medicament, a reagent, a detection plate or a kit;
    其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1、VEGF和/或TGF-β；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤、表达VEGF的肿瘤，和/或表达TGF-β的肿瘤。Wherein, the reagent, detection plate or kit is used for: detecting PD-L1, VEGF and/or TGF-β in a sample; wherein, the agent is used for treating or preventing the expression of PD-L1 (ie PD-L1 positive) tumors, VEGF-expressing tumors, and/or TGF-β-expressing tumors.
  35. 一种药物组合物，其含有：A pharmaceutical composition comprising:
    (i)权利要求28所述的多特异性抗体，或权利要求33所述的免疫偶联物；和(i) the multispecific antibody of claim 28, or the immunoconjugate of claim 33; and
    (ii)药学上可接受的载体。(ii) A pharmaceutically acceptable carrier.
  36. 权利要求28所述的多特异性抗体的一种或多种选自下组的用途：Use of one or more of the multispecific antibodies of claim 28 selected from the group consisting of:
    (i)用于检测人PD-L1、VEGF和/或TGF-β分子；(ii)用于流式检测；(iii)用于细胞免疫荧光检测；(iv)用于***；(v)用于肿瘤诊断；(vi)用于阻断PD-1和PD-L1的相互作用；(vii)用于通过结合VEGF从而阻断其活性；和(viii)用于与TGF-β结合并阻断其下游信号通路。(i) for the detection of human PD-L1, VEGF and/or TGF-β molecules; (ii) for flow detection; (iii) for cellular immunofluorescence detection; (iv) for tumor treatment; (v) For tumor diagnosis; (vi) for blocking the interaction of PD-1 and PD-L1; (vii) for blocking its activity by binding to VEGF; and (viii) for binding to TGF-β and blocking interrupt its downstream signaling pathway.
  37. 一种重组蛋白，其含有：(i)权利要求28所述的多特异性抗体；以及(ii)任选的协助表达和/或纯化的标签序列。A recombinant protein comprising: (i) the multispecific antibody of claim 28; and (ii) an optional tag sequence to facilitate expression and/or purification.
PCT/CN2021/132464 2020-11-24 2021-11-23 ANTI-PD-L1-ANTI-VEGF-ANTI-TGF-β MULTISPECIFIC ANTIBODY, PHARMACEUTICAL COMPOSITION AND USE THEREOF WO2022111476A1 (en)

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