WO2023236991A1 - Anticorps trispécifique ciblant her2, pd-l1 et vegf - Google Patents

Anticorps trispécifique ciblant her2, pd-l1 et vegf Download PDF

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WO2023236991A1
WO2023236991A1 PCT/CN2023/098849 CN2023098849W WO2023236991A1 WO 2023236991 A1 WO2023236991 A1 WO 2023236991A1 CN 2023098849 W CN2023098849 W CN 2023098849W WO 2023236991 A1 WO2023236991 A1 WO 2023236991A1
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antibody
seq
terminus
sequence
vegf
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符智祥
刘婵娟
郎国竣
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三优生物医药(上海)有限公司
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a trispecific antibody targeting HER2, PD-L1 and VEGF and its preparation method and application.
  • HER2 also known as Neu, ErbB-2, CD340 or p185 belongs to the human epidermal growth factor receptor tyrosine kinase family. It has no extracellular ligands and can dimerize by itself or with other human epidermal growth factor receptors. Heterodimerization of ErbB family members activates intracellular signaling pathways and promotes cell proliferation (Wang, Z. (2017), "ErbB Receptors and Cancer", Methods Mol Biol 1652:3-35). In a variety of solid tumors, including breast cancer and gastric cancer, HER2 has gene amplification and increased protein expression levels, leading to an increase in the malignancy of tumor cells.
  • HER2 has become an important target for the treatment of HER2+ solid tumors.
  • a number of monoclonal antibody drugs targeting HER2 have been approved for marketing, including Trastuzumab jointly developed by Roche Pharmaceuticals and Genentech, Trastuzumab developed by Genentech, Pertuzumab and the HER2-targeted ADC drugs T-DM1 and DS-8201 (Oh, D.Y. and Y.J. Bang (2020), “HER2-targeted therapies-a role beyond breast cancer”, Nat Rev Clin Oncol 17(1):33-48).
  • a number of domestic and foreign biosimilar drugs targeting HER2 have also been approved for marketing.
  • monoclonal antibodies targeting HER2 often have low response rates and are prone to resistance. For example, 90% of patients will develop resistance to trastuzumab within 1 year.
  • VEGF Vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • the expression of VEGF is controlled by various factors such as hypoxia and inflammation. When the expression of VEGF is up-regulated, abnormal proliferation of blood vessels occurs.
  • VEGF is an important drug target in pathological angiogenesis-related diseases, such as solid tumors and eye diseases (including diabetic eye disease, age-related macular degeneration, etc.) (Apte, R.S. et al. (2019), "VEGF in Signaling and Disease: Beyond Discovery and Development”, Cell 176(6):1248-1264).
  • Programmed cell death 1 ligand 1 protein is the ligand of PD-1, an important immune checkpoint.
  • the combination of PD-1/PD-L1 can promote the apoptosis of T cells and inhibit the anti-tumor function of T cells.
  • Activity a variety of tumor cells express PD-L1 to evade the surveillance of the immune system (Jiang, Y. et al. (2020), "Progress and Challenges in Precise Treatment of Tumors With PD-1/PD-L1 Blockade", Front Immunol 11:339). Therefore, antibodies that block the interaction between PD-L1 and PD-1 can activate the adaptive immune system to fight tumors and have very broad adaptability in tumor treatment.
  • the currently prepared multispecific antibodies often have fewer antigen-binding sites, which often affects the ability of the antibody to bind to the antigen.
  • Sanofi recently developed a trispecific antibody against HER2/CD3/CD28 and found that the trispecific antibody inhibited the growth of breast cancer cells through CD4 immune cells.
  • this trispecific antibody has only one binding site for each target, whereas classic antibodies have two binding sites for each target. Therefore, the overall binding strength or affinity of this trispecific is lower than that of typical antibody drugs. Thousands of times, which affects the effectiveness of the trispecific antibody.
  • VHH domain from heavy chain antibodies has been proposed as a multispecific antibody building block due to its unique advantages such as small size (15 kD), easy manipulation, and good stability.
  • VHH and conventional antibody variable regions due to the structural differences between VHH and conventional antibody variable regions, in the design of antibody platforms based on VHH, we often encounter factors that are different from those when conventional variable regions are used as building blocks.
  • Lukas Pekar et al. proposed that the variable domains VH and VL of conventional IgG antibodies can be replaced with VHH to generate multispecific antibody molecules (Lukas Pekar et al. (2020), Biophysical and biochemical characterization of a VHH-based IgG -like bi-and trispecific antibody platform,mAbs 12(1),1812210).
  • This application solves the above problems.
  • This application constructs a trispecific antibody that simultaneously targets HER2, PD-L1 and VEGF, which uses the tumor microenvironment to deliver a precise combination of immunomodulatory signals, thereby effectively improving the treatment response rate and overcoming the treatment challenges faced by existing treatments. tolerance problem, and is safer and more effective than a combination therapy consisting of three monospecific antibodies.
  • the present invention provides a new type of trispecific antibody that simultaneously targets HER2, PD-L1 and VEGF.
  • Each arm of the antibody that specifically binds an antigen (referred to as the antigen arm) has the same characteristics as the homologous monoclonal antibody antigen arm. Similar affinities indicate that there is little interference between the arms of the constructed trispecific antibody. Therefore, the trispecific antibody simultaneously retains good antigen-binding specificity, selectivity and selectivity of each antigen-binding site. and good biological activity.
  • trispecific antibodies targeting HER2, PD-L1 and VEGF have multiple advantages: on the one hand, trispecific antibodies can synergistically exert ADCC, tumor cell proliferation inhibition, PD-1/PD-L1 Multiple anti-tumor activities such as blocking and VEGF neutralization can improve the therapeutic effect on HER2-positive tumors; on the other hand, it is expected to solve the problem of treatment tolerance in single drug or combination therapy.
  • the trispecific antibody molecule provided by the present invention is a symmetrical molecule containing 6 antigen-binding sites. Its symmetrical structure allows the antibody to be assembled in a manner similar to natural IgG molecules, avoiding chain mismatching that is common in multispecific antibody production, thereby improving assembly efficiency and yield, thus simplifying antibody production and purification. operation, improve efficiency and reduce costs.
  • the trispecific antibodies provided by the invention have good solubility, purity and thermal stability, which are key features for further downstream development.
  • the antibody molecule of the present invention has two identical antigen-binding sites for each target, so the binding ability of the corresponding natural divalent antibody to the target is basically retained, thereby avoiding the need for trispecific antibodies disclosed in the prior art. Low affinity problem.
  • the invention provides a trispecific antibody that simultaneously targets HER2, PD-L1 and VEGF, the antibody comprising two identical first polypeptides and two identical second polypeptides, wherein said Trispecific antibodies contain the following structure:
  • the first polypeptide contains from N-terminus to C-terminus: VHH1-(X)n-VH-CH1-Hinge-Fc-(Y)m-VHH2;
  • the second polypeptide includes from N-terminus to C-terminus: VL-CL;
  • the first polypeptide contains from N-terminus to C-terminus: VHH1-(X)n-VH-CH1-Hinge-Fc;
  • the second polypeptide includes from N-terminus to C-terminus: VHH2-(Y)m-VL-CL;
  • VHH1 represents the first VHH domain that binds to the first target
  • VHH2 represents the second VHH domain that binds to the second target
  • the combination of VH and VL binds to the third target
  • Fc represents the Fc domain of the immunoglobulin heavy chain
  • CH1 represents the CH1 domain of the immunoglobulin heavy chain
  • CL represents the CL domain of the immunoglobulin light chain.
  • the Fc domains of the two first polypeptides pair with each other and homodimerize, and VH-CH1 and VL-CL pair with each other to form Fab, thus forming a 4-mer structure similar to natural IgG immunoglobulin;
  • VHH1 and VHH2 bind to different targets.
  • CH1 and VH are derived from the same type of immunoglobulin molecule.
  • CH1, Hinge and Fc are derived from the same type of immunoglobulin molecule.
  • the heavy chain constant region domain comprising the CH1 domain and the Fc domain is derived from an immunoglobulin of the IgG type, in particular from a human IgG immunoglobulin, such as an IgG1, IgG2, IgG3 or IgG4 immunoglobulin. protein.
  • the heavy chain constant region domain comprising the CH1 domain and the Fc domain is derived from an immunoglobulin of the IgG1 type, in particular from human IgG1.
  • the IgG1 heavy chain constant region domain comprising the CH1 domain and the Fc domain comprises or is at least 90%, 91%, 92%, 93%, 94%, or consisting of an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical.
  • VL and CL are derived from the same type of immunoglobulin molecule.
  • CL is a kappa light chain constant region or a lambda light chain constant region.
  • X and Y may be the same or different.
  • X and Y are each independently a linker of 8-30 amino acids in length.
  • X and Y comprise or consist of the amino acid sequence shown in SEQ ID NO:5.
  • VH and VL are derived from monoclonal antibodies targeting HER2.
  • VH and VL are derived from trastuzumab.
  • VH includes or consists of the sequence shown in SEQ ID NO:2, and VL includes or consists of the sequence shown in SEQ ID NO:3.
  • VHH1 and VHH2 bind PD-L1 or VEGF, respectively.
  • the VHH1 is derived from an antibody targeting PD-L1
  • the VHH2 is derived from an antibody targeting VEGF.
  • the VHH1 is derived from the D21-4 antibody targeting PD-L1
  • the VHH2 is derived from the P30-10-26 antibody targeting VEGF.
  • the VHH1 comprises or consists of the sequence shown in SEQ ID NO: 1
  • the VHH2 comprises or consists of the sequence shown in SEQ ID NO: 4.
  • the VHH1 is derived from an antibody targeting VEGF
  • the VHH2 is derived from an antibody targeting PD-L1.
  • the VHH1 is derived from the P30-10-26 antibody targeting VEGF
  • the VHH2 is derived from the D21-4 antibody targeting PD-L1.
  • the VHH1 includes or consists of the sequence shown in SEQ ID NO:4, and the VHH2 includes or consists of the sequence shown in SEQ ID NO:1.
  • the VHH1 is derived from an antibody targeting PD-L1
  • the VHH2 is derived from an antibody targeting VEGF.
  • the VHH1 is derived from the D21-4 antibody targeting PD-L1
  • the VHH2 is derived from the P30-10-26 antibody targeting VEGF.
  • the VHH1 comprises or consists of the sequence shown in SEQ ID NO: 1
  • the VHH2 comprises or consists of the sequence shown in SEQ ID NO: 4.
  • the VHH1 is derived from an antibody targeting VEGF
  • the VHH2 is derived from an antibody targeting PD-L1.
  • the VHH1 is derived from the P30-10-26 antibody targeting VEGF
  • the VHH2 is derived from the D21-4 antibody targeting PD-L1.
  • the VHH1 includes or consists of the sequence shown in SEQ ID NO:4, and the VHH2 includes or consists of the sequence shown in SEQ ID NO:1.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF having the structure VHH1-(X)n-VH-CH1-Hinge-Fc-(Y)m-VHH2 2 first polypeptides and 2 second polypeptides with a structure of VL-CL, wherein VHH1 binds to PD-L1 and contains 3 CDRs as shown in SEQ ID NO: 12-14, and VHH2 binds to VEGF and contains as follows The 3 CDRs shown in SEQ ID NO:15-17, VH bind to the HER2 sequence, including the 3 heavy chain CDRs shown in SEQ ID NO:18-20, VL Binds to HER2 and contains 3 light chain CDRs as shown in SEQ ID NO: 21-23.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF having the structure VHH1-(X)n-VH-CH1-Hinge-Fc-(Y)m-VHH2 2 first polypeptides and 2 second polypeptides with the structure VL-CL, wherein VHH1 in the first polypeptide includes or consists of the sequence shown in SEQ ID NO:1, and VH includes the sequence shown in SEQ ID NO:1 The sequence shown in NO:2 or consists of it, VHH2 includes or consists of the sequence shown in SEQ ID NO:4, VL in the second polypeptide includes or consists of the sequence shown in SEQ ID NO:3 composition.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF, which has 2 first polypeptides of the structure VHH1-(X)n-VH-CH1-Hinge-Fc and two second polypeptides with the structure VHH2-(Y)m-VL-CL, in which VHH1 binds to PD-L1 and contains 3 CDRs as shown in SEQ ID NO:12-14, and VH binds to HER2 and contains as follows The 3 heavy chain CDRs shown in SEQ ID NO:18-20, the VHH2 in the second polypeptide binds to VEGF, including the 3 CDRs shown in SEQ ID NO:15-17, and the VL binds to HER2, including the SEQ ID The three light chain CDRs shown in NO:21-23.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF, which has 2 first polypeptides of the structure VHH1-(X)n-VH-CH1-Hinge-Fc and two second polypeptides with the structure VHH2-(Y)m-VL-CL, in which VHH1 binds to VEGF and contains 3 CDRs as shown in SEQ ID NO:15-17, and VH binds to HER2 and contains as shown in SEQ ID
  • the 3 heavy chain CDRs shown in NO:18-20, the VHH2 in the second polypeptide binds to PD-L1 and contains the 3 CDRs shown in SEQ ID NO:12-14, and the VL binds HER2 and contains the following SEQ ID The three light chain CDRs shown in NO:21-23.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF, which has 2 first polypeptides of the structure VHH1-(X)n-VH-CH1-Hinge-Fc and two second polypeptides with the structure VHH2-(Y)m-VL-CL, wherein VHH1 in the first polypeptide includes or consists of the sequence shown in SEQ ID NO: 1 or 4, and VH includes as shown
  • VHH2 in the second polypeptide includes or consists of the sequence shown in SEQ ID NO:4 or 1
  • VL includes or consists of the sequence shown in SEQ ID NO:3 sequence or consisting of.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF having the structure VHH1-(X)n-VH-CH1-Hinge-Fc-(Y)m-VHH2 2 first polypeptides and 2 second polypeptides with the structure VL-CL, wherein the first polypeptide includes or consists of the sequence shown in SEQ ID NO:6 or contains the same sequence as SEQ ID NO:6 A sequence that is at least 90% identical and includes the same CDRs, and the second polypeptide includes or consists of the sequence shown in SEQ ID NO:7 or includes a sequence that is at least 90% identical to SEQ ID NO:7 and includes the same CDRs. sequence.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF, which has 2 first polypeptides of the structure VHH1-(X)n-VH-CH1-Hinge-Fc and 2 second polypeptides with the structure VHH2-(Y)m-VL-CL, wherein the first polypeptide includes or consists of the sequence shown in SEQ ID NO:8 or contains the same sequence as SEQ ID NO:8 A sequence that is at least 90% identical and includes the same CDRs, and the second polypeptide includes or consists of the sequence shown in SEQ ID NO:9 or includes a sequence that is at least 90% identical to SEQ ID NO:9 and includes the same CDRs. sequence.
  • the invention provides a trispecific antibody targeting HER2, PD-L1 and VEGF, which has 2 first polypeptides of the structure VHH1-(X)n-VH-CH1-Hinge-Fc and 2 second polypeptides with the structure VHH2-(Y)m-VL-CL, wherein the first polypeptide includes or consists of the sequence shown in SEQ ID NO:10 or has the same sequence as SEQ ID NO:10 A sequence that is at least 90% identical and includes the same CDR, and the second polypeptide includes or consists of the sequence shown in SEQ ID NO:11 or contains the same sequence as SEQ ID NO:11 Sequences that are at least 90% identical and contain the same CDRs.
  • the invention provides a polynucleotide encoding an antibody molecule of the invention, a vector, preferably an expression vector, comprising said polynucleotide.
  • the invention provides host cells comprising polynucleotides or vectors of the invention.
  • the host cells can be prokaryotic cells and eukaryotic cells commonly used in the art.
  • the present invention provides a method for producing a trispecific antibody of the present invention, comprising step (i) culturing the third bispecific antibody of the present invention under conditions suitable for expressing the trispecific antibody of the first aspect of the present invention.
  • the host cell in three aspects, optionally, (ii) recovers the trispecific antibodies of the invention.
  • the present invention provides a pharmaceutical composition comprising the trispecific antibody of the present invention.
  • the pharmaceutical composition provided by the present invention also contains other therapeutic agents, and optional pharmaceutical excipients; preferably, the other therapeutic agents are selected from the group consisting of chemotherapeutic agents and cytotoxic agents.
  • the present invention provides uses of the trispecific antibodies and pharmaceutical compositions of the present invention for treating, preventing and/or diagnosing cancer.
  • the present invention provides the antibody described in the first aspect, the polynucleotide and vector described in the second aspect, the host cell described in the third aspect, and the pharmaceutical composition described in the fifth aspect. Use in medicines for the treatment, prevention and/or diagnosis of cancer.
  • the present invention provides a method for treating, preventing and/or diagnosing cancer, comprising administering an effective amount of the trispecific antibody of the present invention or the pharmaceutical composition of the present invention to a patient in need.
  • the cancer is, for example, breast, gastric, ovarian, gastroesophageal junction, bladder, small bowel and ampullary cancer, esophageal, lung and cervical cancer.
  • FIGS 1A-1C show the schematic structure of the trispecific antibodies described herein.
  • Figures 2A-2C are SEC-HPLC monomer detection patterns of trispecific antibodies.
  • Figure 2A shows the detection results of TsAb1
  • Figure 2B shows the detection results of TsAb2
  • Figure 2C shows the detection results of TsAb3.
  • Figures 3A-3C show the ELISA detection results of the binding activity of trispecific antibodies to recombinant human PD-L1 protein (Figure 3A), recombinant human HER2 protein ( Figure 3B) and recombinant human VEGF protein ( Figure 3C).
  • Figures 4A-4B show the FACS results of trispecific antibody binding to huPD-L1-CHO-S cells expressing PD-L1 ( Figure 4A) and SK-BR-3 cells expressing HER2 ( Figure 4B).
  • Figure 5 shows the results of trispecific antibodies blocking the interaction of PD-1 with PD-L1 on the surface of huPD-L1-CHO-S cells.
  • Figure 6A shows the results of the trispecific antibody reversing the inhibitory effect of PD-L1 on the PD-1 downstream signaling pathway
  • Figure 6B shows the results of the trispecific antibody inhibiting the VEGF/VEGFR downstream signaling pathway.
  • Figures 7A-7E show the ADCC activity mediated by trispecific antibodies.
  • Figure 7A is the detection result on SK-BR-3 cells.
  • Figure 7B is the detection result on BT-474 cells.
  • Figure 7C is the detection result on NCI- The detection results on N87 cells,
  • Figure 7D shows the PBMC killing detection results on SK-BR-3 cells, and
  • Figure 7E shows the PBMC killing detection results on NCI-N87 cells.
  • Figures 8A-8B show the results of trispecific antibodies inhibiting the proliferation of SK-BR-3 cells (Figure 8A) and HUVEC cells (Figure 8B).
  • Figures 9A-9B show the activity of trispecific antibodies in stimulating PBMC cells to secrete IFN ⁇ ( Figure 9A) and IL-2 ( Figure 9B) in the MLR.
  • Figures 10A-10C show the in vivo anti-tumor activity of trispecific antibodies in the huPD-L1 NCI-N87 mouse model.
  • antibody is used herein in its broadest sense to refer to a protein that contains an antigen-binding site.
  • immunoglobulin refers to a protein having the structure of a naturally occurring antibody.
  • IgG class immunoglobulins are approximately 150,000 dalton heterotetrameric glycoproteins composed of two disulfide-bonded light chains and two heavy chains. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3 ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL).
  • VH heavy chain variable region
  • CL light chain constant domain
  • an IgG immunoglobulin basically consists of two Fab molecules and two dimerized Fc regions connected by the immunoglobulin hinge region.
  • the heavy chain of an immunoglobulin can be assigned to one of five categories based on the type of its constant region, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), where one These classes can be further divided into subclasses such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1), and ⁇ 2 (IgA2).
  • the light chains of immunoglobulins can also be classified into one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen.
  • heavy chain antibodies such as those from the family Camelidae
  • a single VH domain may be sufficient to confer antigen binding specificity.
  • the VHH of natural heavy chain antibodies has the same structure as the heavy chain variable region of natural IgG antibodies, that is, it contains four conserved framework regions (FR) and three complementarity determining regions (CDR).
  • antigen binding site and “antigen binding domain” are used interchangeably to refer to the region of the antibody molecule that actually binds to the antigen.
  • the term “monospecific” antibody refers to an antibody that has one or more binding sites, each of which binds to the same epitope.
  • the term “multispecific” antibody refers to an antibody having at least two antigen-binding sites, each of the at least two antigen-binding sites being associated with a different epitope of the same antigen or with a different Different epitopes of the antigen bind.
  • valency in relation to antibodies refers to the total number of antigen-binding sites in the antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. Number of antigen binding sites.
  • a 6-valent antibody means that the antibody molecule contains a total of 6 antigen-binding sites regardless of whether the bound epitopes are the same.
  • the 6-valent antibody has three different antigen-binding specificities, There are two identical antigen-binding sites for each antigen-binding specificity.
  • Immunoglobulin heavy chain constant region domain refers to a constant region domain from, obtained from, or derived from an immunoglobulin heavy chain, including the heavy chain constant regions CH1, CH2 covalently linked sequentially from the N-terminus to the C-terminus. , CH3, and optionally the heavy chain constant region CH4.
  • the heavy chain constant regions CH1 and CH2 are connected through the heavy chain hinge region, but when appropriate, they can also be connected through a flexible connecting peptide.
  • the heavy chain constant region of the antibody molecule of the invention comprises CH1-Hinge-CH2-CH3.
  • the immunoglobulin heavy chain constant region domain can be selected based on the expected function of the antibody molecule.
  • the constant domain may be an IgA, IgD, IgE, IgG or IgM domain, in particular an immunoglobulin constant domain of a human IgG, e.g. a constant domain of a human IgG1, IgG2, IgG3 or IgG4, preferably of a human IgG1 constant domain.
  • the CH1 and Fc domains of an antibody can both be derived from IgG1.
  • the chain containing the Fc domain is the first polypeptide, also called the heavy chain, and the chain without the Fc domain is the second polypeptide, also called the light chain.
  • an antibody molecule of the invention consists of two heavy chains and two light chains.
  • a “complementarity determining region” or “CDR region” or “CDR” or “hypervariable region” is an antibody variable domain that is highly variable in sequence and forms a structurally defined loop (a “hypervariable loop") and /or a region containing antigen contact residues ("antigen contact sites").
  • CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs are numbered sequentially starting from the N-terminus and are generally referred to as CDR1, CDR2 and CDR3.
  • the CDR sequences in a defined VHH domain can be determined using protocols well known in the art.
  • Fc domain or "Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” contains two or three constant domains, namely a CH2 domain, a CH3 domain and an optional CH4 domain.
  • Fc domains useful in the antibodies of the invention include, but are not limited to, Fc domains of IgGl, IgG2, IgG3, or IgG4 having native or variant sequences.
  • the human IgG heavy chain Fc domain is usually defined as the segment from the amino acid residue at position Cys226 or Pro230 to the end of the carboxy group.
  • a complete antibody composition may include a population of antibodies in which all K447 residues have been eliminated, a population of antibodies in which no K447 residues have been eliminated, or a population of antibodies that are a mixture of antibodies with K447 residues and antibodies without K447 residues.
  • amino acid residue numbering in the Fc region or heavy chain constant region is according to, e.g., Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, Numbering is carried out according to the EU numbering system described in 1991 (also called the EU Index).
  • an Fc mutant comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc domain by one or more amino acid substitutions, deletions, or additions. In some embodiments, the Fc mutant is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a wild-type Fc domain and/or a parental Fc domain. source.
  • effector function refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with immunoglobulin isotype.
  • immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) , cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.
  • the antibody molecule of the invention may have altered effector functions relative to an antibody molecule with a wild-type Fc region, such as reduced or eliminated ADCC activity, etc.
  • antigen refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • any macromolecule including essentially all proteins or peptides, can be used as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • immune checkpoint refers to a class of inhibitory signaling molecules present in the immune system that avoid tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to self-antigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012,12(4):252-264). Research has found that one of the reasons why tumor cells can evade the immune system in the body and proliferate out of control is the use of inhibitory signaling pathways of immune checkpoints, thereby inhibiting the activity of T lymphocytes, making T lymphocytes unable to effectively exert their killing effect on tumors. (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation.
  • Immune checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, and TIM-3.
  • PD-1 programmed death 1
  • PD-L1 PD-L1
  • PD-L2 cytotoxic T lymphocyte antigen 4
  • LAG-3 LAG-3
  • TIM-3 TIM-3
  • VEGF Vascular endothelial growth factors
  • VEGF-A e.g., human VEGF-A protein under Accession No. UniProt NO.: P15692
  • VEGF-B e.g., human VEGF-A protein under Accession No. UniProt NO.: P15692
  • VEGF-B e.g., human VEGF-A protein under Accession No. UniProt NO.: P15692
  • VEGF-B VEGF-C
  • VEGF-D VEGF-D
  • VEGF-E placental growth factor
  • PIGF placental growth factor
  • Angiogenesis is a key process in the progression and metastasis of solid tumors, including gastric cancer. Tumors induce angiogenesis by secreting pro-angiogenic molecules such as VEGF-A.
  • anti-VEGF-A strategies such as anti-VEGF antibodies, have been proposed for the treatment of cancer and angiogenesis-related diseases.
  • PD-L1 refers to programmed cell death 1 ligand 1 protein (such as human PD-L1 protein under UniProtKB accession number Q9NZQ7). As an immune checkpoint molecule, PD-L1 is involved in mediating the activation threshold of T cells and limiting T effector cell responses. Therapeutic applications of anti-PD-L1 antibodies have been proposed in a variety of cancers. However, the therapeutic efficacy of PD-L1 depends to a certain extent on the selection of responder populations. In the antibodies of the invention, "antigen binding specificity for PD-L1" is provided by the VHH domain.
  • VHH is used herein to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, also known as a single variable domain fragment (sVD). Therefore, VHH differs from conventional VH of four-chain immunoglobulins in that it does not need to be paired with a light chain variable domain to form an antigen-binding site.
  • VHH molecules can be derived from antibodies produced in Camelidae species such as camels, alpacas, dromedaries, llamas and guanacos. Species other than camelids may also produce heavy chain antibodies that naturally lack light chains, and such VHHs are also within the scope of the invention. In some cases, for therapeutic applications of VHH, it is desirable to reduce its immunogenicity. Therefore, preferably, in one embodiment, the antibodies of the invention comprise a humanized VHH domain.
  • EC50 also known as “half effective concentration” refers to the concentration of a drug, antibody or agent that induces a response of 50% between baseline and maximum after a specified exposure time. In the context of this application, the unit of EC50 is "nM”.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • NK natural killer cells
  • macrophages, neutrophils, and eosinophils can also mediate ADCC effects.
  • eosinophils can kill certain parasites through ADCC.
  • flexible linker peptide or “linker” or “linker peptide” is used interchangeably and refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and/or serine ( S) and/or threonine residues (T), or from the hinge region of an immunoglobulin.
  • G glycine
  • S serine
  • T threonine residues
  • binding means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions.
  • the ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.
  • Percent identity (%) refers to a candidate sequence after aligning it with the specific amino acid sequence shown in this specification and introducing gaps if necessary to achieve the maximum percent sequence identity, and without taking into account any When conservative substitutions are included as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the specific amino acid sequence shown in this specification.
  • the invention contemplates variants of the antibody molecules of the invention that have a substantial degree of identity, e.g., at least 80% identity, with respect to the antibody molecules and their sequences specifically disclosed herein. , 85%, 90%, 95%, 97%, 98% or 99% or higher. The variants may contain conservative modifications.
  • conservative modifications include substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables for functionally similar amino acids. Such conservatively modified variants are in addition to and not exclusive of the polymorphic variants, interspecies homologs and alleles of the present invention.
  • the following 8 groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valerine amino acid (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine Acid (C), methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the term "conservative sequence modification” is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom.
  • a host cell is any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissue or animal tissue.
  • expression vector refers to a vector comprising a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, viruses and adeno-associated viruses).
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). mouse). In particular, individuals are people.
  • anti-tumor effect refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancer refers to physiological disorders in mammals that are typically characterized by unregulated cell growth.
  • cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy.
  • cancers suitable for treatment by the antibodies of the invention include breast cancer, gastric cancer, ovarian cancer, gastroesophageal junction cancer, bladder cancer, small bowel cancer and ampullary cancer, esophageal cancer, lung cancer and cervical cancer. . Includes those with metastatic forms of cancer.
  • the present invention provides, inter alia, multispecific antibodies useful in tumor/cancer treatment, and their therapeutic use in said tumors/cancers.
  • treatment refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis.
  • the antibody molecules of the invention are used to delay disease progression or to slow the progression of disease.
  • prevention includes the inhibition of the occurrence or progression of a disease or condition or symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for a preventive regimen.
  • prevention refers to the administration of a drug before signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • the term "effective amount” refers to an amount or dose of an antibody or composition of the invention that produces the desired effect in a patient in need of treatment or prophylaxis when administered to the patient in single or multiple doses.
  • the effective amount can be readily determined by the attending physician, who is one of ordinary skill in the art, by considering various factors such as: species of mammal; weight, age, and general health; the specific disease involved; the extent or severity of the disease; the individual The patient's response; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosage regimen chosen; and the use of any concomitant therapy.
  • therapeutically effective amount refers to an amount effective to achieve the desired therapeutic result, at required doses and for required periods of time.
  • the therapeutically effective amount of an antibody or antibody fragment or composition can vary depending on factors such as the disease state, the age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit the desired response in the individual.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%.
  • the ability of a compound to inhibit a measurable parameter eg, cancer
  • prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result, at required doses and for required periods of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • composition refers to a composition that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain additional ingredients that would be unacceptable toxicities to the subject to whom the composition is administered. ingredients.
  • the present invention provides a new type of symmetrical trispecific antibody molecule that simultaneously targets HER2, PD-L1 and VEGF and contains 6 antigen-binding sites, which contains the following structure:
  • the first polypeptide includes from N-terminus to C-terminus: VHH1-(X)n-VH-CH1-Hinge-Fc-(Y)m-VHH2;
  • the second polypeptide includes from N-terminus to C-terminus: VL-CL;
  • the first polypeptide includes from N-terminus to C-terminus: VHH1-(X)n-VH-CH1-Hinge-Fc;
  • the second polypeptide includes from N-terminus to C-terminus: VHH2-(Y)m-VL-CL.
  • Each component of the antibody molecule of the present invention is described below, namely (i) VHH antigen binding site; (ii) antibody heavy chain variable region/light chain variable region; (iii) immunoglobulin constant region domain; and (ii) antibody heavy chain variable region/light chain variable region; iv) Connectors.
  • the VHH antigen binding site in the antibody molecule of the present invention is a single heavy chain variable domain that can specifically bind the target antigen epitope with high affinity, for example, a heavy chain variable domain derived from a camelid heavy chain antibody, camelid humanized human VH domains, or humanized camelid antibody heavy chain variable domains, and their recombinant single domains.
  • the VHH antigen binding site of the antibody molecule of the invention is a humanized camelid antibody heavy chain variable domain.
  • Antibody proteins obtained from camelid species such as camels, alpacas, dromedaries, llamas and guanacos have been characterized in the prior art for their size, structure and antigenicity against human subjects.
  • Certain IgG antibodies from the Camelidae family of mammals in nature lack light chains and are therefore structurally distinct from the common four-chain antibody structure from other animals that has two heavy chains and two light chains. See PCT/EP 93/02214.
  • the heavy chain variable domain (i.e., VHH domain) of camelid heavy chain antibodies with high affinity for the target antigen can be obtained through genetic engineering methods. See U.S. Patent No. 5,759,808, issued June 2, 1998.
  • the amino acid sequence of Camelidae VHH can be recombinantly altered to obtain a sequence that more closely mimics the human sequence, ie, "humanized,” thereby reducing the antigenicity of Camelidae VHH to humans.
  • key elements derived from Camelidae VHH can also be transferred to the human VH domain to obtain a camelized human VH domain.
  • VHH Since its molecular weight is only one-tenth that of human IgG molecules and has a physical diameter of only a few nanometers, VHH is extremely thermally stable, stable to extreme pH and proteolytic digestion, and has low antigenicity.
  • the VHH-based antibody molecules of the present invention at least in one aspect, have good stability and manufacturability due to containing VHH as a building block.
  • the antibody molecule of the invention has immune checkpoint molecule binding specificity, and thereby inhibits the signaling pathway of the corresponding immune checkpoint molecule.
  • the antibody molecule of the invention has at least one immune checkpoint molecule-specific binding specificity. Binding specificity, e.g., in some embodiments, for PD-L1.
  • the antibody molecule of the invention has binding specificity for an angiogenic factor, and thereby inhibits the signaling pathway of the corresponding angiogenic factor.
  • the antibody molecule of the invention has at least one binding specificity for an angiogenic factor.
  • the binding specificity is for vascular endothelial growth factor (VEGF).
  • the antibody molecules of the invention have a tumor-associated antigen binding specificity and thereby target tumor cells expressing the antigen, e.g., the antibody molecules of the invention have at least one binding specificity for a tumor-associated antigen.
  • the antibody molecules of the invention have binding specificity for HER2.
  • the antibody molecules of the invention inhibit signaling pathways of immune checkpoint molecules, inhibit abnormal angiogenesis, and target tumor-associated antigens.
  • the antibody molecules of the invention have a first binding specificity for PD-L1 Sexual, targeting VEGF or VEGF a second binding specificity for the receptor, and a third binding specificity for the tumor-associated antigen HER2.
  • the antibody molecules of the invention comprise a VHH domain that specifically binds PD-L1 and VEGF.
  • the immunoglobulin domain that is part of the antibody molecule of the invention may be derived from any natural immunoglobulin molecule or derivative thereof, but is preferably derived from an IgG immunoglobulin, especially a human IgG1 immunoglobulin molecule.
  • the immunoglobulin heavy chain domain of the antibody molecule of the present invention includes VH, IgG heavy chain constant region CH1, IgG heavy chain hinge region, and IgG heavy chain constant region covalently connected sequentially from the N-terminus to the C-terminus. region CH2, and IgG heavy chain CH3 domain. More preferably, the antibody molecule is stably associated through disulfide bonds at the hinge regions of the two heavy chains (eg, EPKSCDKTHTCPPC) to facilitate the production performance of the antibody molecule.
  • the immunoglobulin domain can be fused to the VHH domain directly or through a linker.
  • the immunoglobulin heavy chain domain is connected to VHH at the N-terminus of VH through a linker, or is connected to VHH at the C-terminus of the CH3 domain through a linker, or the immunoglobulin light chain domain is connected to VL through a linker.
  • the N terminal is connected to VHH.
  • suitable CH1 and CL domains may be CH1 and CL domains from any natural immunoglobulin molecule or derivatives thereof.
  • the CH1 domain comprises an amino acid sequence from the CH1 region of an immunoglobulin IgG, particularly IgG1.
  • the light chain CL domain of the antibody molecule comprises an amino acid sequence derived from the CL region of an immunoglobulin kapa light chain or lambda light chain.
  • the Fc domain suitable for use in the antibody molecules of the invention can be any antibody Fc domain.
  • the Fc domain of an antibody of the invention may comprise two or three constant domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the Fc domain of the antibody of the present invention includes: CH2-CH3 from N-terminus to C-terminus, and more preferably includes: Hinge-CH2-CH3 from N-terminus to C-terminus.
  • the Fc domain of the antibody molecule is an Fc domain from an IgG, eg, an Fc domain from IgG1, IgG2 or IgG4, preferably an Fc domain from human IgG1.
  • antibody molecules of the invention may contain modifications in the Fc domain that alter effector function, depending on the intended use of the antibody molecule.
  • the linkers that can be used in the antibodies of the present invention are not particularly limited. Those skilled in the art can easily determine the available linker sequences based on the components to be connected and the location of the connection.
  • the linker is a flexible linker peptide of 5-50 amino acids, preferably a linker peptide comprising glycine (G) and/or serine (S) and/or threonine residues (T).
  • the linker is 5-50 amino acids in length, for example, 8, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers.
  • the linker comprises the amino acid sequence (G 4 S) n , wherein n is an integer equal to or greater than 1, for example, n is an integer 2, 3, 4, 5, 6, or 7.
  • the linker consists of the amino acid sequence (G 4 S) 3 .
  • the linker comprises the amino acid sequence TS( G4S )n, wherein n is an integer equal to or greater than 1, for example, n is an integer 2, 3, 4, 5, 6, or 7.
  • the linker is a hinge region from an immunoglobulin.
  • Linkers that can be used for the antibody molecules of the present invention can also be, for example, but not limited to, the following amino acid sequences: (Gly 3 Ser) 2, (Gly 4 Ser) 2, (Gly 3 Ser) 3 , (Gly 4 Ser) 3 ,(Gly 3 Ser) 4 ,(Gly 4 Ser) 4 ,(Gly 3 Ser) 5 ,(Gly 4 Ser) 5 ,(Gly 3 Ser) 6 ,(Gly 4 Ser) 6; GGG; DGGGS; TGEKP; GGRR ; EGKSSGSGSESKVD; KESGSSVSSEQLAQFRSLD; GGRRGGGS; LRQRDGGERP; LRQKDGGGSERP; and GSTGSSGKPGSGEGSTKG.
  • the three-dimensional structure of proteins and peptides can be simulated using computer programs or through phage display method to rationally design suitable flexible linker peptides.
  • the invention provides methods for producing the antibodies of the invention.
  • the polypeptide chains of the antibodies of the invention can be obtained, for example, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production, and assembled under appropriate conditions.
  • the polynucleotide encoding any polypeptide chain and/or polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • the polynucleotide can be readily isolated and sequenced using conventional methods.
  • polynucleotides encoding one or more polypeptide chains of an antibody of the invention are provided.
  • the invention provides a vector, preferably an expression vector, comprising one or more polynucleotides of the invention.
  • the invention provides a method for producing an antibody of the invention, said method comprising: culturing a host cell comprising a polypeptide chain encoding said antibody under conditions suitable for expression of said polypeptide chain; and The antibody is produced by assembling the polypeptide chain under conditions suitable for assembly of the polypeptide chain into the antibody.
  • Expression vectors can be constructed using methods well known to those skilled in the art.
  • Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • the invention also provides host cells comprising one or more polynucleotides of the invention.
  • host cells comprising expression vectors of the invention are provided.
  • Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used.
  • Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2/0, etc.
  • the host cell is a CHO, HEK293 or NSO cell.
  • Antibodies prepared by the methods described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. After purification, the purity of the antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. The physical/chemical properties and/or biological activities of the antibodies provided herein can be identified, screened or characterized by a variety of assays known in the art.
  • the invention provides compositions, eg, pharmaceutical compositions, comprising an antibody described herein formulated with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).
  • the antibodies of the invention are the only active ingredient in the pharmaceutical composition.
  • a pharmaceutical composition may comprise an antibody described herein together with more than one therapeutic agent.
  • the invention also provides pharmaceutical combinations comprising an antibody described herein and more than one therapeutic agent.
  • the therapeutic agent suitable for use in the pharmaceutical compositions and pharmaceutical combinations of the present invention may be a treatment selected from any one of the following categories (i)-(iv) Agents: (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and/or T cell activation and/or mobilization); (iii) reduce immunosuppression drugs; (iv) drugs with tumor-inhibiting effects.
  • drugs that enhance antigen presentation e.g., tumor antigen presentation
  • drugs that enhance effector cell responses e.g., B cell and/or T cell activation and/or mobilization
  • reduce immunosuppression drugs e.g., B cell and/or T cell activation and/or mobilization
  • compositions of the present invention may be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions eg, injectable solutions and infusible solutions
  • dispersions or suspensions e.g., liposomes, and suppositories.
  • liposomes e.g., liposomes, and suppositories.
  • suppositories e.g., suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions.
  • the pharmaceutical composition of the present invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of the antibody of the present invention.
  • a “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time.
  • the therapeutically effective amount may vary depending on a variety of factors such as disease state, age, gender and weight of the individual.
  • a therapeutically effective amount is any amount in which the toxic or harmful effects are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%.
  • prophylactically effective amount means an amount effective to achieve the desired prophylactic result, at the required dosage and for the required period of time. Typically, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • Kits containing the antibodies described herein are also within the scope of the invention.
  • a kit may contain one or more other elements, including, for example: instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other materials for administration to a subject.
  • antibodies according to the present invention are endowed with multiple antigen-binding sites and multiple antigen-binding specificities, it is particularly suitable for use as an anti-tumor, anti-angiogenesis, anti-inflammatory, and anti-autoimmune disease drug.
  • antibodies according to the invention are used in the treatment of cancer, such as breast, gastric, ovarian, gastroesophageal junction, bladder, small bowel and ampullary cancer, esophageal, lung and cervical cancer.
  • the antibodies of the invention can be used to treat HER2+ cancers.
  • the antibodies according to the invention are used for the treatment of angiogenesis-related diseases, for example, ophthalmic diseases involving neovascular abnormalities, for example, wet or neovascular age-related macular degeneration (AMD) and diabetic macular degeneration Edema (DME).
  • angiogenesis-related diseases for example, ophthalmic diseases involving neovascular abnormalities, for example, wet or neovascular age-related macular degeneration (AMD) and diabetic macular degeneration Edema (DME).
  • AMD neovascular age-related macular degeneration
  • DME diabetic macular degeneration Edema
  • the DNA coding sequences of human recombinant proteins PD-L1 (UniProtKB-Q9NZQ7), PD-1 (UniProtKB-Q15116), HER2 (UniProt NO-P04626) and VEGF (UniProtKB-P15692) were synthesized by Anhui General Biotechnology Co., Ltd. get. PCR amplified the target fragments and introduced a His tag at the C-terminus of the coding sequence through primers. The target fragments were constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) using homologous recombination.
  • pcDNA3.4 Invitrogen
  • the target fragments were constructed into eukaryotic expression vector pcDNA3.4 containing human IgG1 Fc fragment (hereinafter abbreviated as Fc) or mouse Fc fragment (hereinafter abbreviated as mFc) respectively through homologous recombination.
  • Fc human IgG1 Fc fragment
  • mFc mouse Fc fragment
  • the constructed recombinant protein expression vectors were transformed into Escherichia coli DH5 ⁇ competent cells, cultured at 37°C overnight, and then Use endotoxin-free plasmid extraction kit (OMEGA, D6950-01) for plasmid extraction to obtain the target plasmid for eukaryotic expression.
  • Recombinant human PD-L1-His, recombinant human PD-L1-mFc, recombinant human PD-1-His, recombinant human HER2-His, recombinant human VEGF-His, and recombinant human VEGF-Fc were all expressed through the Expi293 transient expression system (ThermoFisher , A14635) preparation, please refer to Expi293 TM Expression System USER GUIDE for the instant transfer method.
  • the cell suspension was collected and centrifuged at 15000 g for 10 min, and then the obtained expression supernatant was analyzed using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050) and MabSelect SuRe (Cytiva, 17543802). and purification, using gradient concentration imidazole solution and 100mM glycine hydrochloride (pH 3.0) to elute the target protein respectively. Each eluted protein was replaced into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096). After passing the SDS-PAGE identification and activity test, aliquot and freeze at -80°C.
  • the anti-human PD-L1 antibody D21-4 is derived from patent application WO2021083335A1 and is a self-developed alpaca-derived antibody. Its heavy chain variable domain sequence is shown in SEQ ID NO: 1.
  • the anti-human HER2 antibody Trastuzumab is derived from the patent application WO2003087131A2. Its heavy chain sequence is shown in SEQ ID NO:27, and its light chain sequence is shown in SEQ ID NO:7.
  • Anti-human VEGF antibody P30-10-26 is derived from patent application CN202110995278.7 and is a self-developed antibody.
  • the anti-PD-L1 antibody Avelumab is a commercially available antibody drug (purchased from Pfizer).
  • Anti-VEGF antibody Bevacizumab is derived from patent US6884879B1.
  • the complete control antibodies D21-4, P30-10-26, trastuzumab and bevacizumab were all expressed using the transient transfer system (ExpiCHO).
  • ExpiCHO transient transfer system
  • the transient transfer method please refer to the ExpiCHO TM Expression System Kit User Guide.
  • the cell suspension was centrifuged at high speed and the supernatant was collected.
  • the supernatant was filtered through a 0.22 ⁇ m filter membrane and purified by affinity chromatography using a Protein A/G column.
  • the obtained target protein was eluted with 100mM glycine hydrochloric acid (pH 3.0), concentrated, buffer exchanged, aliquoted, identified by SDS-PAGE and activity tested, and then stored in storage.
  • This example describes the structure of an exemplary anti-HER2/PD-L1/VEGF trispecific antibody (TsAb) and the construction of an expression vector.
  • the VHH domain (VHH PD-L1 ) of the anti-human PD-L1 antibody comes from antibody D21-4, and the amino acid sequence is shown in SEQ ID NO: 1; the amino acid sequence of the anti-HER2 antibody comes from trastuzumab, and its heavy chain
  • the amino acid sequences of the variable region and the light chain variable region are as shown in SEQ ID NO:2 and SEQ ID NO:3 respectively;
  • the anti-human VEGF antibody VHH domain (VHH VEGF ) is from P30-10-26, and the amino acid sequence is as SEQ ID NO:2 ID NO:4.
  • the sequence of the exemplary linker is GGGGSGGGGSGGGGS (SEQ ID NO: 5); the anti-VEGF monoclonal antibody Bevacizumab (Bevacizumab) has the light and heavy chain variable region amino acid sequences as SEQ ID NO: 24 and SEQ ID NO: 25 respectively. shown.
  • TsAb1 (referred to as TsAb1, hereinafter TsAb1 also refers to an antibody with this structure): contains two identical first polypeptides and two identical second polypeptides, and its structure is shown in Figure 1A.
  • the first polypeptide includes the VHH domain of the anti-human PD-L1 monoclonal antibody, the linker, the heavy chain variable region of trastuzumab, the human IgG1 heavy chain constant region, and the linker from the N terminus to the C terminus. and an anti-human VEGF monoclonal antibody VHH domain
  • the second polypeptide includes the light chain variable region and the kappa light chain constant region of trastuzumab from the N terminus to the C terminus.
  • the amino acid sequences of the exemplary TsAb1 first polypeptide and the second polypeptide are shown in SEQ ID NO: 6 and SEQ ID NO: 7 respectively.
  • TsAb2 contains two identical first polypeptides peptide and two identical second polypeptides, the structures of which are shown in Figure 1B.
  • the first polypeptide includes the VHH domain of the anti-human PD-L1 antibody, the linker, the heavy chain variable region of trastuzumab, and the human IgG1 heavy chain constant region from the N terminus to the C terminus;
  • the second polypeptide includes the VHH domain of the anti-human VEGF antibody, the linker, the light chain variable region of trastuzumab and the kappa light chain constant region from the N-terminus to the C-terminus.
  • the amino acid sequences of exemplary TsAb2 first polypeptide and second polypeptide are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • TsAb3 (abbreviated as TsAb3, hereinafter TsAb3 also refers to an antibody with this structure): contains two identical first polypeptides and two identical second polypeptides, and its structure is shown in Figure 1C.
  • the first polypeptide includes the VHH domain of the anti-human VEGF antibody, the linker, the heavy chain variable region of trastuzumab and the human IgG1 heavy chain constant region from the N-terminus to the C-terminus
  • the second polypeptide includes:
  • the polypeptide includes the VHH domain of the anti-PD-L1 antibody, the linker, the light chain variable region of trastuzumab and the kappa light chain constant region from the N terminus to the C terminus.
  • the amino acid sequences of the exemplary TsAb3 first polypeptide and the second polypeptide are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.
  • the coding sequences of each fragment were amplified by PCR, connected by overlap extension PCR, and then constructed into the modified eukaryotic expression vector plasmid pcDNA3.4 ( Invitrogen) to form a complete construct polypeptide expression vector.
  • the constructed vectors were transformed into E. coli DH5 ⁇ and cultured at 37°C overnight.
  • the endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used to extract the plasmid and obtain the endotoxin-free construct polypeptide expression plasmid for eukaryotic expression.
  • Table 1 shows the molecular composition/structure of three exemplary trispecific constructs obtained herein.
  • Example 2 The construct of Example 2 was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133). The specific operation was as follows: On the day of transfection, it was confirmed that the cell density was approximately 7 ⁇ 10 6 to 1 ⁇ 10 7 viable cells/mL. The survival rate is >98%. At this time, use fresh ExpiCHO expression medium preheated at 37°C to adjust the cell density to a final concentration of 6 ⁇ 10 cells/mL.
  • ExpiCHO TM Enhancer and ExpiCHO TM Feed were added to the culture medium, and the flask was placed on a 32°C shaker and 5% CO 2 to continue culturing. On day 5 after transfection, add the same volume of ExpiCHO TM Feed and mix the cell suspension gently while adding slowly.
  • the cell culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 min. The resulting supernatant was affinity purified with MabSelect SuRe (Cytiva, 17543802), and then eluted with 100 mM sodium acetate (pH 3.0). The target protein was then diluted with 1M Tris-HCl And, finally, the obtained protein was replaced into PBS buffer through ultrafiltration concentration tube (Millipore, UFC901096).
  • the concentration of the purified trispecific antibody obtained in Example 3.1 was measured using an ultra-trace spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300) by measuring the optical density at a wavelength of 280 nm, and the measured A280 reading was divided The value obtained by calculating the theoretical extinction coefficient of the antibody based on the amino acid sequence is used as the antibody concentration for subsequent research. After passing the quality inspection, aliquot and store at -80°C.
  • the monomer purity of the prepared trispecific antibody was detected by SEC-HPLC method.
  • the trispecific antibodies to be tested were diluted to 0.5mg/mL with mobile phase solution (150mmol/L phosphate buffer, pH 7.4). Detection was performed on an Agilent HPLC 1100 column (XBridge BEH SEC 3.5 ⁇ m, 7.8mm I.D. ⁇ 30cm, Waters), flow rate 0.8mL/min, injection volume 20 ⁇ L, and VWD detector wavelengths of 280nm and 214nm.
  • Differential scanning fluorescence can provide information about the structural stability of a protein based on the fluorescence change process in the protein map, detect the conformational changes of the protein, and obtain the melting temperature (Tm) of the protein.
  • the DSF method was used to detect the Tm value of the trispecific antibody.
  • the obtained antibodies TsAb1 and TsAb3 were prepared into 0.2 mg/mL PBS solutions respectively. Each test sample was added into a 96-well plate (Nunc) at 19 ⁇ L/well. Three parallel wells were set up and filled with PBS and trastuzumab. Antibody was used as a reference, and then 1 ⁇ L of SYPRO orange dye with a concentration of 100 ⁇ was added to each well, mixed and ready for use on the machine.
  • the sample thermal stability test uses ABI 7500 FAST RT-PCR instrument. The test type selects melting curve and adopts continuous mode. The scanning temperature range is 25 ⁇ 95°C, the heating rate is 1%, and the equilibrium is 5min at 25°C. Data is collected during the heating process. Select "ROX" as the reporter group, "None" as the quencher group, the reaction volume is 20 ⁇ L, and the temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the melting temperature Tm of the antibody.
  • TMB PurModics, TMBS-1000-01
  • 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM TM (GraphPad Software, San Diego, CA), and EC50 values were calculated.
  • the ELISA binding assay results are shown in Figure 3A and Table 3.
  • the binding ability of the trispecific antibodies TsAb1, TsAb2 and TsAb3 to PD-L1 protein is equivalent to or slightly weaker than that of the parent antibody D21-4.
  • a stably transfected cell line (huPD-L1-CHO-S) overexpressing human PD-L1 protein was obtained.
  • Collect cells in the exponential growth phase centrifuge at 300g to remove the supernatant, resuspend the cells in FACS buffer (PBS containing 1% BSA), count and adjust the cell suspension density to 2 ⁇ 10 6 viable cells/mL.
  • huPD-L1-CHO-S cells were added into a 96-well round bottom plate at 100 ⁇ L per well, and centrifuged at 300 g to remove the supernatant.
  • the incubated cell mixture was washed three times, 200 ⁇ L of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM TM (GraphPad Software, San Diego, CA), and EC50 values were calculated.
  • the FACS binding assay results are shown in Figure 4A and Table 3.
  • the binding abilities of the trispecific antibodies TsAb1 and TsAb3 to cell surface PD-L1 are comparable to those of the parent antibody D21-4, although the binding ability of the trispecific antibody TsAb2 to cell surface PD-L1
  • the binding ability is slightly weaker than the parent antibody D21-4, but it still retains good binding activity to PD-L1.
  • TMB PurModics, TMBS-1000-01
  • 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM TM (GraphPad Software, San Diego, CA), and EC50 values were calculated.
  • the ELISA binding assay results are shown in Figure 3B and Table 3.
  • the binding activity of the trispecific antibodies TsAb1, TsAb2 and TsAb3 to the HER2 protein is equivalent to or slightly weaker than the parent antibody Trastuzumab, indicating that the antibodies still retain good binding to the HER2 protein. active.
  • SK-BR-3 (ATCC, HTB-30) endogenously expressing HER2 in the exponential growth phase, centrifuge at 300g to remove the supernatant, and resuspend the cells in FACS buffer (PBS containing 1% BSA) , count and adjust the cell suspension density to 2 ⁇ 10 6 viable cells/mL. Subsequently, SK-BR-3 cells were added into a 96-well round-bottom plate at 100 ⁇ L per well, and centrifuged at 300g to remove the supernatant.
  • FACS buffer PBS containing 1% BSA
  • the FACS binding assay results are shown in Figure 4B and Table 3.
  • the binding activity of the trispecific antibody TsAb1 to cell surface HER2 is comparable to that of the parent antibody Trastuzumab.
  • the binding activity of the trispecific antibodies TsAb2 and TsAb3 to cell surface HER2 is slightly weaker than that of the parent antibody. , but still retains good binding activity to HER2.
  • TMB PurModics, TMBS-1000-01
  • 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM TM (GraphPad Software, San Diego, CA), and EC50 values were calculated.
  • the ELISA binding assay results are shown in Figure 3C and Table 3.
  • the trispecific antibodies TsAb1, TsAb2 and TsAb3 all retained good binding activity to VEGF.
  • Biacore T200 (Cytiva) instrument was used to detect the affinity of the obtained trispecific antibody to human recombinant proteins HER2-His, PD-L1-His and VEGF-His according to the manufacturer's instructions.
  • White HER2-His, PD-L1-His and VEGF-His were diluted to obtain solutions with concentrations of 30, 15, 7.5, 3.75, 1.875, and 0.938 nM/L respectively, and then the antigen solution was flowed through at a flow rate of 30 ⁇ L/min.
  • the chip was run with an association time of 120 s and a dissociation time of 180 s. After the dissociation is completed, use 10mM Gly-HCl (pH 2.0) to regenerate the chip for 20 seconds to completely remove the antibodies bound to the chip.
  • the experiment adopts multi-cycle operation, with the response signal taking the analysis time as the abscissa and the response value as the ordinate. After double-reference deduction, the obtained data were fitted by BIAcore T200 analysis software.
  • the fitting model used was a 1:1 Langmuir binding model to determine its binding and dissociation constants and other affinity indicators.
  • This example uses the FACS method to detect the blocking activity of trispecific antibodies on PD-1/PD-L1 interaction.
  • the specific method is as follows: collect huPD-L1-CHO-S cells, centrifuge at 300g to remove the supernatant, and use FACS to Resuspend in buffer, count and adjust the cell suspension density to 2 ⁇ 10 viable cells/mL. Add huPD-L1-CHO-S cells at 100 ⁇ L per well into a 96-well round bottom plate, centrifuge at 300 g to remove the supernatant, and then add different concentrations of trispecific antibody, control antibody D21-4, and human IgG1 isotype antibody (isotype) to the corresponding wells.
  • the blocking test results are shown in Figure 5 and Table 5.
  • the blocking activity of the trispecific antibodies TsAb1 and TsAb3 on the interaction between PD-1 and PD-L1 is equivalent to that of the parent antibody D21-4, and the blocking activity of TsAb2 on the interaction between PD-1 and PD-
  • the blocking activity of L1 interaction is slightly weaker than that of D21-4, but it still retains good blocking activity.
  • This example uses a luciferase reporter gene system to detect the impact of the anti-HER2/PD-L1/VEGF trispecific antibody obtained in this application on the PD-1/PD-L1 downstream signaling pathway.
  • the specific method is as follows: Take the logarithmic phase of PD -1-NF-AT-Jurkat cells (Jurkat cells Stably expressing PD-1 (UniProtKB-Q15116) and luciferase) and CD3L-PD-L1-CHO cells (stably expressing PD-L1 (UniProtKB-Q9NZQ7) and anti-CD3-scFv in CHO cells), the two Cells were mixed at a ratio of 1:5 to a density of 4 ⁇ 10 6 and 2 ⁇ 10 7 viable cells/mL, respectively.
  • the results of the luciferase reporter gene assay are shown in Figure 6A.
  • the trispecific antibody TsAb1 can reverse the inhibition of PD-1 downstream signaling pathways by PD-L1 binding and its function is equivalent to that of the parent antibody D21-4.
  • This example uses a luciferase reporter gene system to detect the activity of the anti-HER2/PD-L1/VEGF trispecific antibody obtained in this application on the VEGF/VEGFR2 downstream signaling pathway.
  • the specific method is as follows: take the logarithmic phase VEGFR2-NF-AT -HEK293 cells (HEK293T cells To stably express VEGFR2 (UniProtKB-P35968) and luciferase), the cell density was adjusted to 4 ⁇ 10 5 viable cells/mL and seeded at 50 ⁇ L/well into a 96-well transparent bottom white plate (Corning, 3610).
  • Use culture medium containing 60ng/mL VEGF-Fc to dilute the antibody to be tested in a gradient manner.
  • the results of the luciferase reporter gene system detection are shown in Figure 6B.
  • the trispecific antibodies TsAb1, TsAb2 and TsAb3 can inhibit the activation of the VEGFR2 downstream signaling pathway by VEGF and their functions are similar to those of the parental VEGF antibody P30-10-26 or bevacizumab. Resistance is similar.
  • Luciferase reporter gene system detects ADCC activity mediated by anti-HER2/PD-L1/VEGF trispecific antibodies
  • This example uses a luciferase reporter gene system to detect antibody-dependent cell-mediated cytotoxicity mediated by anti-HER2/PD-L1/VEGF trispecific antibodies.
  • the specific method is as follows: take the target cells (SK- BR-3, human breast ductal carcinoma BT474 or human gastric cancer NCI-N87 cells) and effector CD16a(V158)-NF-AT-Jurkat cells stably transfected with the CD16a(V158) sequence (UniProtKB-P08637) and containing Jurkat cells of pGL4.30 plasmid (Promega, E8481) of NF-AT-re nucleic acid sequence ), mix effector cells and target cells at a ratio of 1:10 and make their densities 4 ⁇ 10 6 and 4 ⁇ 10 5 viable cells/mL respectively.
  • FIGS 7A-7C show the ADCC activity results of the antibody on SK-BR-3, BT-474 and NCI-N87 cells respectively.
  • Both BT-474 and NCI-N87 cells can mediate ADCC activity comparable to that of the control antibody Trastuzumab.
  • NCI-N87 cells NCI-N87 cells overexpressing human PD-L1
  • culture medium containing 2% FBS to adjust the cell density to 2 ⁇ 10 5 cells
  • Viable cells/ml inoculate 50 ⁇ L/well into a 96-well flat-bottomed cell culture plate, and culture in a 37°C incubator overnight.
  • Use culture medium containing 2% FBS to gradually dilute the antibody to be tested and the control antibody.
  • PBMC Resuscitate PBMC in advance and incubate in a 37°C incubator for 2 hours. Gently aspirate the supernatant from the PBMC cell culture flask, centrifuge to remove the medium, resuspend the cells in complete medium containing 10% FBS and adjust the cell density to 4 ⁇ 10 6 viable cells/ml, inoculate 100 ⁇ L/well into the above 96-well cell culture plate.
  • the LDH detection kit (Takara, MK401) was used to detect the antibody-mediated killing of tumor cells by PBMC, and the killing results were displayed as the lysis rate of the target cells.
  • FIGS 7D-7E The results of the PBMC killing test are shown in Figures 7D-7E, where Figure 7D and Figure 7E are the ADCC activity results on SK-BR-3 and huPD-L1 NCI-N87 cells respectively. It can be seen that the trispecific antibody TsAb1 is effective in SK-BR ADCC activity mediated by -3 and huPD-L1 on NCI-N87 cells was comparable to the control antibody Trastuzumab.
  • the results of the proliferation inhibitory activity test are shown in Figure 8A.
  • the proliferation inhibitory activity of the trispecific antibody TsAb1 on SK-BR-3 is comparable to that of the control antibody Trastuzumab.
  • EBM-2 complete culture medium to revive human umbilical vein endothelial cells (HUVEC cells, ATCC: PCS-100-010) one week in advance.
  • HUVEC cells are passaged every 3 days.
  • HUVEC cells used for proliferation inhibition experiments should not be passaged more than 5 generations. .
  • the cell density was adjusted to 5 ⁇ 10 viable cells/ml using EBM-2 medium containing 0.5% FBS, seeded into a 96-well flat-bottomed cell culture plate at 50 ⁇ L/well and cultured overnight in a 37°C incubator.
  • EBM-2 basic medium containing 800ng/mL VEGF-Fc to dilute the antibody to be tested and the control antibody in a gradient manner and add 50 ⁇ L/well to a 96-well plate seeded with HUVEC cells. Gently tap to mix and incubate at 37°C. Culture in the incubator for 3 days. Add 20 ⁇ L MTS to each well of the 96-well plate, mix gently, and incubate in a 37°C incubator for 5 hours. After the 96-well plate is equilibrated to room temperature, read OD492 on a microplate reader.
  • the results of the HUVEC proliferation inhibition experiment are shown in Figure 8B.
  • the trispecific antibody TsAb1 can significantly inhibit VEGF-induced HUVEC cell proliferation, and its proliferation inhibition rate is comparable to the parental monoclonal antibody P30-10-26.
  • CD4+T cells were sorted from PBMC cells using Miltenyi CD4+T sorting kit (Miltenyi, 130-096-533).
  • Miltenyi CD14 MicroBeads kit (Miltenyi, 130-050-201) was used to sort CD14+ monocytes from PBMC cells.
  • RPMI 1640 complete medium rhGM-CSF and rhIL-4 were added to induce mononuclear cells. Nucleated cells form DC cells.
  • Use RPMI 1640 complete medium to perform a 4-fold gradient dilution of the test antibody and control antibody Avelumab.
  • the mixed lymphocyte experiment results are shown in Figures 9A and 9B.
  • the trispecific antibody TsAb1 can induce CD4+T cells to secrete IFN ⁇ and IL-2 in a concentration-dependent manner and is better than the marketed PD-L1 monoclonal antibody Avelumab, indicating that it can Release the inhibitory effect of PD-L1 on dendritic cells on T cells.
  • mice 8-week-old female NCG mice (purchased from Viton Lever) were selected, and the exogenous PD-L1-expressing human gastric cancer cell huPD-L1 NCI N87, which is responsive to trastuzumab, was cultured at 1 ⁇ 10 7 cells/ Only subcutaneous tumor loading was performed. After 7 days of tumor loading, the mice were randomly divided into 8/group according to tumor volume, resulting in a total of 7 groups. After grouping, each mouse was injected with 5 ⁇ 10 6 human PBMC cells through the tail vein to reconstitute the mouse immune system.
  • mice were injected intraperitoneally with equimolar doses of Trastuzumab (35nM/kg), Avelumab (35nM/kg), Bevacizumab (35nM/kg), Trastuzumab+Avelumab (35+35nM/kg), Avelumab+ Bevacizumab (35+35nM/kg) and high-dose (87.5nM/kg, or 17.8mpk) and low-dose (35nM/kg, or 7.1mpk) trispecific antibody TsAb1, with a dosing frequency of 2 times/week, in total Dosed 8 times.
  • Mice tumor volume and body weight were measured and recorded twice weekly. Calculate the tumor volume (unit: mm 3 ) according to the formula (length*width2)/ 2 .
  • the mice were sacrificed by cervical dislocation, the mouse tumor tissue was peeled off, and the tumors were measured and recorded.
  • the trispecific antibody TsAb1 can inhibit tumor growth more significantly and effectively at both high dose (87.5nM/kg) and low dose (35nM/kg), indicating that due to the trispecificity Antibody TsAb1 can recognize three targets (HER2/PD-L1/VEGF) at the same time, effectively promote/activate the anti-tumor mechanism based on each target, thereby obtaining efficient anti-tumor synergy, and thus plays an important role in the treatment of tumors. value.

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Abstract

La présente invention concerne un anticorps trispécifique ciblant HER2, PD-L1 et VEGF. L'anticorps trispécifique comprend deux premiers polypeptides identiques et deux seconds polypeptides identiques, 1) chaque premier polypeptide comprend VHH1-(X)n-VH-CH1-charnière-Fc-(Y)m-VHH2 de l'extrémité N-terminale à l'extrémité C-terminale et chaque second polypeptide comprend VL-CL de l'extrémité N-terminale à l'extrémité C-terminale, ou 2) chaque premier polypeptide comprend VHH1-(X)n-VH-CH1-charnière-Fc de l'extrémité N-terminale à l'extrémité C-terminale et chaque second polypeptide comprend VHH2-(Y)m-VL-CL de l'extrémité N-terminale à l'extrémité C-terminale ; VHH1 représente un premier domaine VHH qui se lie à un premier épitope, VHH2 représente un deuxième domaine VHH qui se lie à un deuxième épitope, et VH et VL sont combinés pour se lier à un troisième épitope ; x et y représentent des lieurs, n = 0 ou 1 et m = 0 ou 1 ; Fc représente le domaine Fc d'une chaîne lourde d'immunoglobuline, CH1 représente le domaine CH1 de la chaîne lourde d'immunoglobuline, et CL représente le domaine CL de la chaîne légère d'immunoglobuline ; les domaines Fc des deux premiers polypeptides sont appariés l'un avec l'autre et homodimérisés, et VH-CH1 et VL-CL sont appariés l'un avec l'autre pour former un Fab, formant ainsi une structure tétramère similaire à celle de l'immunoglobuline IgG naturelle ; et VHH1 et VHH2 se lient respectivement à différentes cibles choisies parmi PD-L1 ou VEGF, et VH et VL sont appariés pour se lier à HER2. De plus, la présente invention concerne en outre l'utilisation thérapeutique de l'anticorps trispécifique. De plus, la présente invention concerne en outre l'utilisation thérapeutique de l'anticorps trispécifique.
PCT/CN2023/098849 2022-06-10 2023-06-07 Anticorps trispécifique ciblant her2, pd-l1 et vegf WO2023236991A1 (fr)

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Citations (1)

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US20170043035A1 (en) * 2014-04-25 2017-02-16 The Trustees Of The University Of Pennsylvania Methods and compositions for treating metastatic breast cancer and other cancers in the brain

Patent Citations (1)

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US20170043035A1 (en) * 2014-04-25 2017-02-16 The Trustees Of The University Of Pennsylvania Methods and compositions for treating metastatic breast cancer and other cancers in the brain

Non-Patent Citations (2)

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BOGEN JAN P., CARRARA STEFANIA C., FIEBIG DAVID, GRZESCHIK JULIUS, HOCK BJÖRN, KOLMAR HARALD: "Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture", FRONTIERS IN IMMUNOLOGY, vol. 12, XP093021289, DOI: 10.3389/fimmu.2021.669496 *
CHEN, YILI ET AL.: "A Bispecific Antibody Targeting HER2 and PD-L1 Inhibits Tumor Growth with Superior Efficacy", J. BIOL. CHEM., vol. 297, no. 6, 31 December 2021 (2021-12-31), XP093060634, DOI: 10.1016/j.jbc.2021.101420 *

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