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WO2023241632A1 - 肿瘤微环境激活的抗ctla-4抗体的偶联体及其应用 - Google Patents

肿瘤微环境激活的抗ctla-4抗体的偶联体及其应用 Download PDF

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Publication number
WO2023241632A1
WO2023241632A1 PCT/CN2023/100258 CN2023100258W WO2023241632A1 WO 2023241632 A1 WO2023241632 A1 WO 2023241632A1 CN 2023100258 W CN2023100258 W CN 2023100258W WO 2023241632 A1 WO2023241632 A1 WO 2023241632A1
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alkylene
cancer
antibody
bond
conjugate
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PCT/CN2023/100258
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French (fr)
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刘辰
刘源
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亚飞（上海）生物医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—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 CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/54—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 organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/54—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 organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides

Definitions

  • the invention relates to an anti-CTLA-4 antibody conjugate activated by tumor microenvironment and its application.
  • Tumor immunotherapy is one of the breakthrough developments in the field of tumor treatment in recent years. In some patients, tumor immunotherapy makes long-term survival possible. Cancer immunotherapy includes tumor immune checkpoint antibodies, T cell bridging bispecific antibodies (bispecific T cell engager, bsTCE), chimeric antigen receptor cells (CAR-T), oncolytic viruses, etc.
  • CTLA-4 antibody is the first immune checkpoint inhibitor antibody approved by the FDA.
  • PD-1 antibody is another clinically effective immune checkpoint inhibitor.
  • CTLA-4 antibodies, PD-1 antibodies, and their combination therapy have achieved very superior effects in clinical trials for cancers including melanoma, non-small cell lung cancer, lymphoma, and liver cancer, and some of them have become first-line therapies.
  • immune checkpoint antibodies relieve the inhibitory signaling pathways of T cells, they often lead to non-specific activation of T cells in non-tumor target organs, resulting in immune-related adverse events (irAEs).
  • the response rate of immune checkpoint antibody monotherapy is often limited, usually only about 20-30%. People need to improve the response rate of patients through combined immunotherapy such as PD-1+CTLA-4 dual-target immunotherapy, but this This combination also significantly enhanced the effect of irAEs.
  • CTLA-4 combined with PD-1 in the treatment of malignant melanoma
  • the overall response rate was 58%, which was significantly higher than that of CTLA-4 alone (19%) or PD-1 alone (44%).
  • CTLA-4 is an inhibitory receptor expressed on the surface of T cells. Its mechanism is to competitively bind to B7.1 (CD80) and B7.2 (CD86) with CD28, causing T cells to not undergo excessive response after receiving the antigen presented by MHC. activation. When this inhibitory signal is relieved with CTLA-4 antibodies such as Ipilimumab, the T cells are exposed to MHC-antibody The activation and proliferation after stimulation by the original complex are increased, thereby improving the T cell-mediated tumor immunity effect. At the same time, recent studies have found that CTLA-4 is highly expressed on the surface of Treg cells.
  • CTLA-4 antibodies with Fc effector function can eliminate Treg cells within tumors through the ADCC effect mediated by IgG1 Fc, and relieve the microscopic effects of tumor suppression. environment, thereby enhancing anti-tumor immunity.
  • the present invention provides a tumor microenvironment-activated anti-CTLA-4 antibody conjugate, which has the following structure:
  • R5 represents an anti-CTLA-4 antibody in which one or more amino acid residues have been mutated to cysteine
  • cys represents the cysteine residue of R5;
  • S represents the S atom in the cysteine residue
  • R1 is a functional group that blocks R5 from binding to its antigen, ligand or receptor;
  • R2 is a connecting arm that can be hydrolytically cleaved by one or more proteases in the pathological microenvironment
  • R3 is the connecting arm, providing space for protease cleavage
  • L is a chemical bond activated in the acidic conditions of the pathological microenvironment
  • R4 is a group covalently connected to R5 through the sulfur atom on the cysteine in R5, which can restore, maintain or enhance R5 and its antigen, ligand and receptor after R1-R2-R3-L- is cleaved. body's ability to combine.
  • the invention provides a pharmaceutical composition comprising a conjugate as described herein.
  • the present invention provides a method for treating or preventing tumors, comprising providing a therapeutically or preventively effective amount of the conjugate according to any embodiment of this document to a subject in need thereof.
  • the present invention also provides the use of the conjugate described herein in the preparation of medicaments for treating or preventing tumors.
  • the invention also provides a conjugate as described in any embodiment herein for use in therapeutic or prophylactic treatment.
  • FIG. 1 Coupling efficiency of Ipi-L-3 with different compounds.
  • Figure 2 Enzyme-linked immunosorbent assay to characterize the binding ability of Ipi-L-3 TMEAbody.
  • Figure 4 Enzyme-linked immunosorbent assay to characterize the acid-activated properties of Ipi-L-3 TMEAbody.
  • FIG. 5 Ipi-L-3+CP23 TMEAbody can be activated by legumain.
  • FIG. 6 Enzyme-linked immunosorbent assay characterizes legumain-mediated activation of Ipi-L-3+CP23 TMEAbody.
  • FIG. 7 Schematic diagram of Ipi-L-3 TMEAbody activated by legumain or low pH environment.
  • Figure 8 (A) Enzyme-linked immunosorbent assay characterizes the coupling of Ipi-L-3 and R4-4; (B) Enzyme-linked immunosorbent assay characterizes the coupling of Ipi-L-3 and CP31.
  • Figure 9 Polyacrylamide gel electrophoresis pattern of TMEAbody after extreme low pH treatment.
  • FIG. 10 Enzyme-linked immunosorbent assay characterizes TMEAbody after treatment at extremely low pH conditions.
  • FIG. 11 Tumor penetration of different TMEAbodies.
  • Figure 12 In vivo efficacy of Ipi-L-3+CP23 TMEAbody in the hCTLA-4 gene knock-in MC38 mouse model.
  • FIG. 13 Ipi-L-3+CP23 increases plasma half-life and AUC.
  • FIG. 14 Ipi-L-3 TMEAbody shows lower peripheral T cell activation.
  • FIG. 16 Ipi-L-3 TMEAbody shows lower immunogenicity in mice.
  • Figure 17 Using FACS to analyze the number of Treg after treatment with Ipi-L-3+CP23 TMEAbody.
  • One of the purposes of the present invention is to provide a modified anti-CTLA-4 antibody conjugate, which is only activated in a pathological microenvironment (such as a tumor microenvironment or an inflammatory site) to release the binding to its ligand.
  • a pathological microenvironment such as a tumor microenvironment or an inflammatory site
  • anti-CTLA-4 antibodies with comparable or even improved potency, thus improving the targeting efficacy of biomolecules while overcoming drug resistance and reducing toxicity.
  • the modified anti-CTLA-4 antibody is a conjugate, which contains an anti-CTLA-4 antibody and a functional part covalently connected to the antibody, namely R1-R2-R3-L-R4.
  • antibody is used in the broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as It only needs to show the desired biological activity (Miller et al., (2003), Jour. of Immunology, 170: 4854-4861). In the context and figures, the abbreviation for antibody is replaced by "Ab”.
  • the basic antibody building block consists of a tetramer consisting of two pairs of identical polypeptide chains, each pair having one light chain and one heavy chain.
  • the N-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids that is primarily responsible for antigen recognition.
  • the C-terminal portion of each chain defines the constant region primarily responsible for effector functions.
  • the heavy chain variable region (VH) and the light chain variable region (VL) each contain three complementarity determining regions (CDR), including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3. These six CDRs form the antigen-binding site of the antibody.
  • the remaining amino acids of the variable region are relatively conserved and are called framework regions (FR).
  • VH and VL each contain four framework regions, called FR1, FR2, FR3 and FR4 respectively.
  • Antibodies can be derived from mice, humans or other substances, and can be humanized antibodies or chimeric antibodies.
  • the immunoglobulins disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.
  • Immunoglobulins can be from any species, such as human, mouse or rabbit.
  • Antibody fragments contain a portion of the entire length of an antibody, typically the antigen-binding or variable region of the antibody.
  • an antibody fragment is a functional fragment that retains the antigen-binding ability of an intact antibody, that is, an antibody fragment is an antigen-binding fragment of an antibody.
  • antibody fragments or functional fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; and single chain antibody molecules (scFv); and the like.
  • one or more (for example, 5 or less or 3 or less) amino acids at appropriate positions in the amino acid sequence of the anti-CTLA-4 antibody are mutated to cysteine, and then through this cysteine
  • the sulfhydryl group of the amino acid is covalently linked to the functional moiety (R1-R2-R3-L-R4) of the invention.
  • one or two amino acids of interest in an anti-CTLA-4 antibody of interest are mutated to cysteine for conjugation to a functional moiety.
  • the mutation location may exist in the complementarity-determining or non-complementarity-determining regions of the variable region.
  • the mutation is a substitution mutation.
  • the mutation occurs in the light chain variable region of the anti-CTLA-4 antibody.
  • mutants can be prepared and then tested for their binding activity to the corresponding antigen.
  • the amino acid residue at the mutated position is considered to be mutated to cysteine if the mutant retains 70% or more, preferably 80% or more, more preferably 90% or more of the binding activity compared to the wild-type antibody. for covalent attachment to functional moieties.
  • the conjugate produced by linking a mutant to R4 as described herein retains 80% or greater, preferably 90% or greater, more preferably 95% or greater binding activity, it is considered that the amino acid residue at that position can be covalently linked to the functional moiety as a cysteine.
  • a variety of anti-CTLA-4 antibodies can be used in the present invention.
  • a preferred anti-CTLA-4 antibody is Ipilimumab, the exemplary heavy chain and light chain amino acid sequences of which are shown in SEQ ID NO: 1 and 2, respectively.
  • one or more of G, A, S, T, L, I, F, E, K, D and Y in the CDR of the light chain variable region of the anti-CTLA-4 antibody more preferably G
  • One or more of A, S, T, L, I, K and Y, more preferably one or more of G, A, T, L, S and Y may be mutated to cysteine.
  • one or more of G, A, S, T, L, I, F, E, K, D and Y in the CDR of the heavy chain variable region of the anti-CTLA-4 antibody can be mutated to cysteine .
  • G, A, S, T, L, I, F in the non-CDR region of the variable region of the light chain or heavy chain one or more of E, K, D and Y, etc., preferably G, A, S, T, K, I, Y and L, more preferably one or more of G, A, T, Y and S , can be mutated to cysteine.
  • the S, T, L, I, F, E, K, One or more of the D, N, Q, R, and Y residues, etc. are mutated to cysteine.
  • substitution mutations can be introduced into a One or more of the following conserved sites: Gln3, Ser7, Ser25, Glu46, Thr69 and Asp73 in the non-complementary determining region (FR1, FR2 or FR3) of VH, and in the non-complementary determining region (FR1, FR2 or FR3) of VL ) Thr5, Tyr50, Arg62, Ser64, Ser66, Ser68, Thr73, Thr75, Ser77 and Asp83.
  • the above position numbers refer to SEQ ID NO: 1 and 2.
  • the mutation position on the heavy chain of anti-CTLA-4 antibody Ipilimumab is selected from: Gln3, Arg19, Leu20, Ser25, G1y26, Phe27, Thr28, Phe29, Ser30, Ser31, Tyr32, Thr33, Met34, His35, G1y44, Phe50, Ile51, Ser52, Tyr53, Asp54, Gly55, Asn56, Asn57, Lys58, Tyr59, Tyr60, Ala61, Asp62, Ser63, Lys65, Gly66, Thr69, Ser71, Arg72, Asp73, Asn74, Ser75, Lys76, Asn7 7, Thr99, Gly100, Trp101, Leu102, Gly103, Pro104, Leu105, Asp106 and Tyr107; preferably selected from: Ser30, Ser31, Tyr32, Thr33, Ile51, Asp54, Gly55, Asn56, Lys58, Tyr59, Tyr60, Al
  • the mutation position in the light chain of anti-CTLA-4 antibody Ipilimumab is selected from: Gln6, Arg24, Ala25, Ser26, Gln27, Ser28, Val29, Gly30, Ser31, Ser32, Tyr33, Leu34, Ala35, Tyr37, Ile49, Tyr50, Gly51, Ala52, Phe53, Ser54, Arg55, Ala56, Thr57, Gly58, Ile59, Pro60, Asp61, Arg62, Ser68, G1y69, Thr70, Gln90, Gln91, Tyr92, Gly93, Ser94, Ser95, Pro96, Trp 97.
  • Thr98, Phe99 and Gly100 preferably selected from the group consisting of: Ala25, Ser26, Ser28, Gly30, Ser31, Ser32, Leu34, Ala35, Gly51, Ala52, Ser54, Ala56, Thr57, Gly58, Ile59, Gly93, Ser94, Ser95, Thr98 and Gly100.
  • Mutagenesis, transfection, expression and purification of biomolecules can be performed by methods known in the art.
  • nucleic acids with anti-CTLA-4 antibodies mutated at selected positions can be directly synthesized, and then the nucleic acid molecules of different fragments obtained by enzymatic digestion can be ligated into an expression vector, and the expression vector can be transformed into bacteria or in eukaryotic host cells. Biomolecules containing cysteine mutations can undergo recombination in host cells get.
  • Bacterial or eukaryotic host cells suitable for use in the present invention can be host cells commonly used in the art, including but not limited to bacteria, yeast and mammalian cells.
  • Useful eukaryotic host cells include CHO cells, HEK293T cells or Pichia pastoris.
  • Expression vectors suitable for use in the present invention may be viral-based expression vectors known in the art, including but not limited to baculovirus, simian virus (SV40), retrovirus or vaccinia virus-based vectors.
  • Expression vectors containing appropriate regulatory elements and selectable markers can be used to generate mammalian cell lines that stably express the mutants.
  • the GS eukaryotic expression system Longza
  • DHFR eukaryotic expression system Invitrogen
  • Pichia pastoris expression system Invitrogen
  • Biomolecules of the invention can be purified by separation methods known in the art. These methods include, but are not limited to, affinity chromatography, DEAE ion exchange columns, gel filtration, and hydroxyapatite chromatography. For example, protein A or G columns can be used to separate antibody molecules from cell culture supernatants or cytoplasmic extracts.
  • biomolecules can be "engineered” so that they contain amino acid sequences that allow the biomolecules to be captured into the affinity matrix.
  • labels can be used to facilitate purification of polypeptides. Suitable markers include, but are not limited to, c-myc, hemagonium, poly-His (e.g., 6His) and FlagTM (Kodak). This tag can be inserted anywhere within the polypeptide, including the carboxyl terminus or the amino terminus.
  • the biomolecule conjugates of the present invention can also be purified by immunoaffinity chromatography.
  • Functional group moieties suitable for use in the present invention may be represented by the formula R1-R2-R3-L-R4.
  • R1, R2 and R3 are linked together by any suitable linkage, including but not limited to amide bond, ester bond, urethane bond, urea bond or hydrazone bond.
  • R3 and R4 are connected through L, and L is a hydrazone bond.
  • the amide bond can be represented by "-CO-NH-"
  • the ester bond can be represented by “-C(O)O-”
  • the urethane bond can be represented by "-NH-C(O)-O -”
  • the urea bond can be represented by "-NH-CO-NH-”
  • R1, R2, R3, L, R4 and R5 are covalently connected to each other.
  • R1, R2, R3, L, R4 and R5 are used as part of a molecule, they refer to groups; when used as raw materials for preparation, they refer to compound molecules, and at this time, their attachment positions (i.e., the position indicated by the wavy line below) is usually connected to H or OH or other corresponding groups well known in the art to satisfy the bond valence theory, and can also react with other parts to form the bonds described herein, including but not limited to The amide bond, ester bond, urethane bond, urea bond or hydrazone bond.
  • R1 is the protecting group of biomolecule R5. It can be selected from those that block biomolecules from interacting with their antigens, Any group bound by a ligand or receptor to prevent interference with the biomolecule by other molecules. For example, preventing a biomolecule from binding to its ligand or receptor before it reaches a pathological microenvironment such as a tumor or inflammatory microenvironment.
  • Suitable R1 is selected from: NR a R b -R-CO-, C 1-4 alkoxy-(C 1-4 alkoxy) n -carbonyl-C 1-4 alkylene-carbonyl-,
  • each R is independently C 1-4 alkyl; each n is independently an integer in the range of 1 to 30000, such as 1-15000, 1-5000, 1-2000, 1-150, 1-50, 1 -20 or 3-12; polyethylene glycol or peg m is a polyethylene glycol with a molecular weight of 44 to 132,000, such as a polyethylene glycol with a molecular weight of 1,000 to 50,000 or 10,000 to 30,000; m represents the molecular weight of the polyethylene glycol ;
  • the wavy line indicates the position where R1 is linked to R2;
  • R a is C 1-4 alkyl-O-[(CH 2 ) q -O] n -(CH 2 ) q -;
  • R b is C 1-4 alkyl -O-[(CH 2 ) q -O] n -(CH 2 ) q -C(O)-;
  • q is an integer from 1 to 4.
  • the molecular weight of R1 does not exceed
  • the NR a R b -R-CO- is:
  • each n is independently an integer from 1 to 1000.
  • the C 1-4 alkoxy-(C 1-4 alkoxy) n -carbonyl-C 1-4 alkylene-carbonyl- is:
  • n is an integer from 1 to 1000.
  • the R1 group is selected from:
  • R1 is preferably chosen so that the molecular weight of R1-R2-R3-L-R4 is higher than 200, preferably higher than 500, more preferably Higher than 1000, so that the molecular weight of the conjugate of the biomolecule is 5000 or higher, preferably 8000 or higher, more preferably 10000 or higher, thereby better preventing the biomolecule from interacting with its ligand before reaching the pathological microenvironment or Receptor binding.
  • R2 is a linker activated by hydrolysis by one or more proteolytic enzymes, proteases or peptidases in the pathological microenvironment.
  • proteolytic enzyme, protease or peptidase may be present in the disease Treat various proteolytic enzymes, proteases or peptidases in the microenvironment.
  • the protease may be a cysteine protease, asparagine protease, aspartic protease, glutamic acid protease, threonine protease, gelatinase, metalloprotease or asparagine peptide lyase; preferably, R2 is A peptide capable of being cleaved by at least one enzyme selected from the group consisting of: endoasparaginase, granzyme, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kinase Peptide-releasing enzyme, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, astrylase, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease
  • the present disclosure specifically relates to Legumain, which is mainly expressed and secreted by tumor cells in the tumor microenvironment.
  • Tumor-associated macrophages M2 type
  • monocytes IL-12
  • inflammatory macrophages M1 type
  • polypeptides are substrates for proteolytic enzymes and can be recognized and cleaved by proteolytic enzymes.
  • the R2 group of the present invention can be represented by -R2a-, -R2b-, -R2a-N-, -R2a-D-, -R2a-AAN-, -R2a-AAD- or -R2a-R2b-; wherein R2a is A peptide that can be cleaved at the amide bond by one or more proteolytic enzymes; R2b is a peptide that can form a carbamate with R3 through the amino group in the side chain, and the carbamate can be cleaved by one or more Cleaved by proteolytic enzymes; A is alanine; N is asparagine, and the amino group in its side chain forms carbamate with R3, and the carbamate can be cleaved by Legumain; D is aspartic acid, and its side chain The amino group in R3 forms a carbamate and can be cleaved by granzyme B.
  • R2a and R2b can be connected by forming an amide bond.
  • Granzyme B cleaves the bond between R2 and R3 (e.g. carbamate), the L in R3-L- is cleaved under acidic conditions, releasing the antibody with the R4 group.
  • a suitable polypeptide capable of being activated by a proteolytic enzyme may be a tripeptide. Any substrate peptide capable of being recognized and cleaved (activated) by proteolytic enzymes in pathological microenvironments known in the art can be used as R2 disclosed herein. Such peptides have the structure disclosed in WO 2016/026458, the entire contents of which are incorporated herein by reference. public.
  • the amino acid residue connected to R1 can be selected from Ala, Thr, Val and Ile, and the intermediate amino acid residue can be selected from Ala, Thr, Val and Asn,
  • the amino acid residue linked to R3 may be selected from Asn and Asp.
  • R2 is connected to R1 through the amino group of its amino acid residue through an amide bond, ester bond, urethane bond, urea bond or hydrazone bond, and through the carboxyl group of its amino acid residue through an amide bond, ester bond, The connection mode of urethane bond, urea bond or hydrazone bond is connected with R3.
  • R2 is a tripeptide selected from Ala-Ala-Asn and Ala-Ala-Asp.
  • Ala-Ala-Asn can be recognized and cleaved by Legumain, and Ala-Ala-Asp can be recognized and cleaved by granzymes.
  • the R3 group is a connecting arm, including but not limited to -HN-phenyl-R'-, where R' is a bond.
  • the NH group and R' are in the para position to the phenyl group.
  • the L group can be obtained by a chemical reaction between an R3 compound and an R4 compound.
  • the R4 group is a binding group that can restore, maintain, reduce or promote the binding ability of the anti-CTLA-4 antibody to its antigen, ligand or receptor after the R2 group and the R3 group are cleaved.
  • the resulting R4-s-Cys-R5 exhibits >60% affinity for the antigen, ligand or receptor of native R5.
  • R4 group can be represented by the following formula:
  • Rc is selected from C1-12 alkylene, C1-12 alkylene-O-C1-12 alkylene, C1-12 alkylene-C3-8 cycloalkyl, (C1-4 alkylene- O)p-C1-12 alkylene, C1-12 alkylenecarbonylamino-(C1-4 alkylene-O)p-C1-12 alkylene, -phenyl-C1-12 alkylene, C3-8 cycloalkyl, C1-12 alkylene-C3-8 cycloalkyl-C1-12 alkylene, C1-12 alkylene-NHCO-O-C1-12 alkylene, C1-12 alkylene Alkyl-COO- and C1-12 alkylene-phenyl-C1-12 alkylene; p is an integer from 1 to 10, preferably 1, 2, 3 or 4;
  • R 4-c is bond or -CO-
  • the L group is connected to the R4 group through R 4-c of the R4 group.
  • the R4 group is linked to the S atom of the cysteine of R5 via a maleimide.
  • Rc is C1-12 alkylene and R 4-c is -CO-; wherein the R4 group is connected to the L group through its carbonyl group.
  • the R4 group can be prepared from an R4 compound selected from:
  • the wavy line used in each indicated formula indicates the location where the part containing the wavy line is connected to other parts.
  • R5 represents an anti-CTLA4 antibody in which one or more amino acid residues are mutated to cysteine.
  • R5 is actually a biomolecule in which the thiol group of cysteine lacks a hydrogen atom. The lack of this sulfide hydrogen atom allows R5 to be considered as a group attached to R4 of the present invention.
  • anti-CTLA-4 antibodies can be activated in a variety of ways.
  • One The first way is to cleave R1-R2 from R3-L-R4-S-cys-R5 through proteolytic enzymes.
  • Another approach is to cleave R1-R3-L in the pathological microenvironment under acidic conditions, releasing R4-S-cys-R5.
  • the present invention found that the dual-activated (enzyme-activated and acid-activated) compounds of the present invention have higher activation efficiency in diseased tissues compared with single-activated compounds.
  • the conjugate of the present invention can be prepared by a method including reduction of mutated biomolecules by DTT, TCEP or other reducing agents; oxidation with Cu 2 SO 4 , dehydroascorbic acid or other oxidants; and then under liquid phase or solid phase conditions.
  • the oxidized biomolecule (R5) binds to R1-R2-R3-L-R4.
  • the final product can be collected in the liquid phase.
  • the present invention also includes functional parts, namely R1-R2-R3-L-R4; R2-R3-L-R4; R3-L-R4-S-cys-R5; R4-S -cys-R5 and mutated biomolecules; wherein R1, R2, R3, L, R4, R5 and their connection methods and mutated biomolecules are as defined in any part or any embodiment of the invention.
  • functional moieties are represented by CP3, CP7, CP13, CP23-CP27.
  • -S-cys- means that the sulfhydryl group of the cysteine introduced by R4 via mutation in R5 is covalently linked to R5.
  • R4-S-cys-R5 is a conjugate formed under the acidic conditions of pathological microenvironments.
  • conjugates, functional moieties, R2-R3-L-R4, R3-L-R4-S-cys-R5 and R4-S-cys-R5 as described herein can be synthesized by methods known in the art. For example, they can be prepared according to the method described in Example 1 of this application.
  • the invention also includes pharmaceutical compositions comprising a conjugate as described herein.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
  • the carrier can be any pharmaceutically acceptable carrier or excipient, which can vary depending on the dosage form and mode of administration.
  • Pharmaceutically acceptable carriers are generally safe and non-toxic and may contain any substance known to be used in the pharmaceutical industry in formulating pharmaceutical compositions, including fillers, diluents, coagulants, binders, lubricants, auxiliaries, Flow agents, stabilizers, colorants, wetting agents and disintegrants, etc.
  • Suitable pharmaceutically acceptable carriers include sugars, such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or dicalcium phosphate; starch, including corn starch, wheat starch, Rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; silicon dioxide, talc, stearic acid or other Salts, such as magnesium stearate or calcium stearate; and/or polyethylene glycol, etc.
  • a pharmaceutically acceptable carrier a major consideration is the mode of administration of the pharmaceutical dosage form. This is well known in the art.
  • compositions may contain a therapeutically or prophylactically effective amount of the conjugate.
  • Effective amount means an amount of an ingredient sufficient to produce the desired reaction. The specific effective amount depends on various factors, such as the specific disease to be treated, the patient's physical condition such as weight, age and sex, the duration of treatment, co-administered treatments, if any, and the specific formulation used .
  • an "effective amount” as used herein is a conventional amount of a biomolecule.
  • the therapeutically or prophylactically effective amount of conjugate included in the pharmaceutical composition of the present invention may be lower than the conventional amount of biomolecules but may produce better therapeutic or preventive effects because the biomolecules are less effective in reaching the pathology.
  • the microenvironment is protected by a protecting group before binding to its ligand or receptor.
  • the pharmaceutical composition of the present invention can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, etc., and can be administered by any suitable route to achieve the intended purpose.
  • it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally, or externally.
  • the dosage of the drug may depend on the patient's age, health and weight, concurrent treatments, and frequency of treatment.
  • the pharmaceutical compositions of the present invention may be administered to any subject in need thereof, such as a mammal, especially a human.
  • tumor cells or antigen-presenting cells (APCs) carrying tumor antigens partially or completely inhibit the host's immune killing of tumors by binding to T cells.
  • the conjugates of the invention are activated and released in pathological microenvironments by proteolytic enzymes, especially Legumain or granzymes, and/or under acidic conditions.
  • the conjugate of the present invention can effectively break through the individual's immune barrier, reach the pathological microenvironment, and then be activated and released in the pathological microenvironment. As a result, it can selectively promote the proliferation or killing of T cells, etc. in tumors or inflammatory microenvironments, thereby achieving low autoimmunity and high efficacy.
  • each conjugate or R4-S-cys-R5 or mutated biomolecule disclosed in the present invention can be used to treat tumors or inflammation, or can be used as an active ingredient in the preparation of drugs for treating tumors or inflammation.
  • the tumor or inflammation described herein is preferably a CTAL-4 mediated tumor or inflammation and may be any tumor or inflammation known to be treatable by an anti-CTAL4 antibody (especially an anti-CTLA4 antibody described herein, such as Ipilimumab), including but not Not limited to melanoma, cancers of the bladder, brain, breast, cervix, colon-rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testicle and blood, etc.
  • cancers include bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, Ovarian cancer, testicular cancer and blood cancer.
  • This method can be used in combination with any known radiotherapy or immunotherapy.
  • HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DMF 200mL
  • Para-aminobenzyl alcohol 4.1g, 0.03mol
  • N,N-diisopropylethylamine 8.7g, 0.06mol
  • Most of the DMF was removed by rotary evaporation.
  • Table 2 Functional group mass spectrometry information table of the present invention and its properties and yields
  • Example 2 Construction of a series of anti-hCTLA4TMEAbodies with different blocking groups and linkers
  • Ipilimumab was constructed through site-specific coupling with different compounds (including blocking groups and linkers) and Ipilimumab containing Y49C (Y49C is according to the Kabat numbering system of antibodies, which corresponds to the sequentially numbered Y50C) mutation on the light chain.
  • Y49C is according to the Kabat numbering system of antibodies, which corresponds to the sequentially numbered Y50C) mutation on the light chain.
  • Anti-human CTLA4-TMEAbody TAEAbody is the abbreviation of tumor microenvironment-activated antibody).
  • Ipilimumab antibody containing Y49C (herein abbreviated as Ipi-L-3) was reduced with dithiothreitol (DTT) at a molar ratio of 1:200 for 16 hours under the conditions of 50mM Tris (pH7.5) and 2mM EDTA at room temperature. It was then dialyzed into 50mM Tris (pH 7.5), 150mM NaCl buffer and then reoxidized with 1:100 molar ratio of dehydroascorbic acid (DHAA) for use with different compounds containing different hindering groups and different chemical linkers. CP12-CP18, CP23-CP30 coupling. The molar ratio of compound/antibody used for coupling was 1:20.
  • DTT dithiothreitol
  • the coupled TMEAbody was further purified by protein A column to remove excess compounds, and the coupling efficiency was evaluated by SDS-PAGE assay.
  • the results in Figure 1(A-F) show that Ipi-L-3 shows high coupling efficiency for these different compounds.
  • Example 4 Acid-mediated activation of Ipi-L-3+CP23, Ipi-L-3+CP24, Ipi-L-3+CP25 and Ipi-L-3+CP26 TMEAbodies
  • the coupled Ipi-L-3+CP23 TMEAbody was incubated with activated Legumain in pH 6.0, 50mM MES, 250mM NaCl buffer overnight at 37°C. Activation efficiency was assessed using Western blotting. Anti-human Fab HRP antibody (SIGMA, A0293) was used to detect the light chain of the antibody. As shown in Figure 5, 1 ⁇ g Ipi-L-3+CP23 TMEAbody can be activated by 1-5 ⁇ g Legumain.
  • Ipi-L-3 retains the binding activity of Ipi-L-3 after coupling with R4-4, which means that the structure after acid activation of Ipi-L-3+CP23 can be maintained. Identical binding activity to wild-type Ipilimumab antibody.
  • Ipi-L-3 In order to test the activity recovery after enzyme activation, we used CP31 to couple with Ipi-L-3, whose molecular structure is the same as that after enzyme-mediated activation. After conjugation with CP31, ELISA was used to evaluate the effect of small molecule fragments remaining on the antibody after activation on Ipi-L-3 binding activity. The results are shown in Figure 8(B). Ipi-L-3 retains the binding activity of Ipi-L-3 after coupling with CP31, which means that the structure after Legumain activates Ipi-L-3+CP23 can remain the same as the wild-type Similar binding activity to Ipilimumab antibodies.
  • Example 8 Evaluation of pharmacokinetics and tissue distribution of a series of TMEAbodies in mouse tumor models
  • TMEAbody In order to evaluate the distribution and activation of TMEAbody in vivo, the above series of 10mg/kg TMEAbody was injected into CT26 tumor-bearing Balb/c mice, and then at different time points, plasma and different tissues were taken for analysis of TMEAbody or activation. Concentration of TMEAbody. Homogenize the tissue in 2.5 volumes of PBS, add 1X Halt TM protease inhibitor Cocktail (100X) (Thermo Fisher, Cat. No. 78430), and collect the supernatant for ELISA detection. The concentration of TMEAbody was measured using 5 ⁇ g/ml anti-PEG antibody (Abcam, Cat. No.
  • A18829 as the secondary antibody, use Ipilimumab antibody at different concentrations to prepare a standard curve. The supernatants of plasma and tissue homogenates were diluted to appropriate folds for ELISA determination and the concentrations were converted using a standard curve.
  • the first parameter is the activation rate of tumors and other tissues. High specific activation rates in tumors rather than other tissues are preferred because of the lower peripheral toxicity that will result.
  • the activation rates of different TMEAbodies in different tissues are shown in Table 3.
  • Day D0 is the starting point of dosing, so ND means that there is no data in organs on this day, but only data in blood.
  • tumor penetration Another parameter we focused on was tumor penetration, as PEG increases the size of the antibody and may affect the tumor penetration of the antibody.
  • PEG increases the size of the antibody and may affect the tumor penetration of the antibody.
  • ratio of tumor AUC/blood AUC total concentration of activated vs. inactivated TMEAbody TMEABODY
  • PEG with a molecular weight greater than 20kD can significantly affect the penetration rate of antibodies.
  • Example 9 Therapeutic effect of Ipi-L-3 TMEAbody in the MC38 tumor model of hCTLA-4 gene knock-in mice
  • Ipi-L-3+CP23 TMEAbody showed a dose-dependent tumor growth inhibitory effect.
  • Ipi-L-3+CP23 TMEAbody showed the same complete response rate (CR) as WT Ipilimumab antibody.
  • Example 10 Ipi-L-3 TMEAbody exhibits prolonged serum half-life in human CTLA-4 knock-in mice
  • the antibody shows target antigen-mediated clearance in vivo.
  • TMEAbody to the target antigen
  • the clearance mediated by the target antigen will be inhibited, thereby prolonging its half-life.
  • hCTLA-4 knock-in mice (Shanghai Southern Model Organism) were injected with wild-type Ipilimumab antibody or Ipi-L-3+CP23 TMEAbody, plasma was collected at different time points, and anti-human Fc and anti-human Fab antibodies were used to pass the "sandwich" "Formula ELISA method to measure antibody concentration.
  • the Ipi-L-3+CP23 TMEAbody showed extended serum half-life and AUC compared to wild-type Ipilimumab antibody.
  • Example 11 Ipi-L-3 TMEAbody shows reduced peripheral T cell activation
  • Example 12 Ipi-L-3 TMEAbody induces lower GvHD effects in NSG mice implanted with human PBMCs
  • Ipilimumab and Ipi-L-3 TMEAbody were administered to NSG immunodeficient mice transplanted with human PBMC (Shanghai Southern Model Organism). 5 ⁇ 10 6 human PBMC cells were transplanted into NSG mice through the tail vein. After 10 days of reconstitution, 10 mg/kg Ipilimumab or Ipi-L-3 TMEAbody was administered twice a week. Animal weight was observed as an indication of graft versus host disease (GvHD effect).
  • Ipi-L-3 TMEAbody or wild-type Ipilimumab antibody was mixed with 1:1 incomplete Freund's adjuvant and injected intraperitoneally into the mice 14 days after the initial immunization. Plasma was collected 21 days after the initial immunization, and immunogenicity was analyzed by ELISA.
  • 0.5 ⁇ g/ml human CTLA-4-His protein (Sino Biological, Cat#11159-H08H) was coated on ELISA plates (BeaverBio, Cat#REF40301) overnight.
  • Example 14 Evaluation of the therapeutic effect of Ipi-L-3 TMEAbody on human CTLA-4 gene knock-in mouse MC38 tumor model by FACS
  • Ipi-L-3+CP23 TMEAbody and Ipilimumab antibodies were administered to human CTLA-4 gene knock-in C57 mice bearing MC38 tumors. middle. Seven days after administration, spleens and tumors were collected for FACS analysis of Treg subsets (CD4+CD25+FoxP3+) (Beckman Coulter, CytoFLEX). The data were then analyzed through GraphPad Prism 5 software.

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Abstract

本发明提供肿瘤微环境激活的抗CTLA-4抗体的偶联体及其应用。本发明的偶联体具有以下结构：R1-R2-R3-L-R4-S-cys-R5，其中，R1、R2、R3、L、R4、R5分别代表不同的功能基团、连接臂、化学键、CTLA-4抗体等结构。本发明的偶联体为双激活偶联体，能提高抗CTLA-4抗体的靶向效力，同时克服该抗体的耐药性、降低毒性。

Description

肿瘤微环境激活的抗CTLA-4抗体的偶联体及其应用 技术领域
本发明涉及一种肿瘤微环境激活的抗CTLA-4抗体的偶联体及其应用。
背景技术
肿瘤免疫疗法是近年来肿瘤治疗领域的突破性进展之一，在部分患者中，肿瘤免疫疗法使得长期生存成为可能。肿瘤免疫疗法包括肿瘤免疫检查点抗体，T细胞桥接双特异性抗体(bispecific T cell engager，bsTCE)，嵌合抗原受体细胞(CAR-T)，溶瘤病毒等等。其中，CTLA-4抗体是第一个被FDA批准的免疫检查点抑制剂抗体。PD-1抗体则是另外一个临床上疗效显著的免疫检查点抑制剂。CTLA-4抗体，PD-1抗体，以及两者的联合疗法在包括黑色素瘤、非小细胞肺癌、淋巴瘤和肝癌等癌症的临床试验中取得了非常优越的疗效，部分疗法已经成为一线疗法。
由于免疫检查点抗体解除了T细胞的抑制信号通路，因此往往导致T细胞在非肿瘤靶器官非特异激活，结果导致免疫相关毒性作用(immune related adverse events，irAEs)。而免疫检查点抗体单药治疗的响应率往往有限，通常只有20-30％左右，人们需要通过联合免疫治疗如PD-1+CTLA-4双靶点免疫治疗来提高患者的响应率，但是这种联合也显著增强了irAEs作用。比如，在CTLA-4联合PD-1治疗恶性黑色素瘤的临床研究中，总体响应率为58％，相较于单用CTLA-4(19％)或单用PD-1(44％)有显著的提高，但于此同时，伴随疗效的提高，临床上也观察到更为严重的治疗相关副作用(treatment related adverse events，TRAEs)，其中三到四级TRAEs提高到了55％，发生率相较于CTLA-4单用(27％)或PD-1单用(16％)显著上升。常见的irAEs包括脑垂体炎、肝炎、结肠炎、皮疹和腹泻等等。这些严重的irAEs甚至导致受试者发生死亡。
CTLA-4是T细胞表面表达的抑制性受体，其机制在于与CD28竞争性结合B7.1(CD80)和B7.2(CD86)，导致T细胞在接受MHC呈递的抗原后，不发生过度活化。当用CTLA-4抗体如Ipilimumab解除这种抑制信号后，T细胞受到MHC-抗 原复合物刺激后的活化和增殖增加，从而提高T细胞介导的肿瘤免疫效应。同时，最近的研究发现，CTLA-4在Treg细胞表面高表达，具有Fc效应功能的CTLA-4抗体，如Ipilimumab可以通过IgG1 Fc介导的ADCC效应来清除肿瘤内部Treg细胞，解除肿瘤抑制的微环境，从而起到增强抗肿瘤免疫的作用。
如上所述，无论是基于T细胞活化抑制信号的解除还是通过ADCC效应杀伤Treg细胞，两种效应都在正常器官中促进了自身免疫反应，从而导致irAEs的发生。因此，临床上亟需开发更安全的抗CTLA-4抗体，以将其免疫增强效应局限于肿瘤内部，而对正常组织不产生自身免疫毒性。
发明内容
基于上述技术问题，本发明提供了一种肿瘤微环境激活型抗CTLA-4抗体的偶联体，具有如下结构：
R1-R2-R3-L-R4-S-cys-R5
其中，
R5代表其一个或多个氨基酸残基被突变为半胱氨酸的抗CTLA-4抗体；
cys代表R5的半胱氨酸残基；
S代表半胱氨酸残基中的S原子；
R1为阻断R5与其抗原、配体或受体结合的功能基团；
R2为可被病理微环境中的一种或多种蛋白酶水解切割的连接臂；
R3为连接臂，为蛋白酶的切割提供空间；
L为在病理微环境的酸性条件激活的化学键；
R4是通过R5中的半胱氨酸上的硫原子共价连接至R5的基团，其在R1-R2-R3-L-被切割后能恢复、维持或者提高R5与其抗原、配体和受体的结合能力。
本发明提供了一种药物组合物，其包含如本文所述的偶联体。
本发明提供了一种治疗或预防肿瘤的方法，包括向有需要的受试者提供治疗或预防有效量的本文任一实施方案所述的偶联体。
本发明还提供本文所述的偶联体在制备治疗或预防肿瘤的药物中的用途。
本发明还提供用于治疗或预防治疗的本文任一实施方案所述的偶联体。
附图说明
图1：Ipi-L-3与不同化合物的偶联效率。
图2：酶联免疫吸附法表征Ipi-L-3 TMEAbody的结合能力。
图3：酸介导的Ipi-L-3 TMEAbody激活。
图4：酶联免疫吸附法表征Ipi-L-3 TMEAbody的酸激活特性。
图5：Ipi-L-3+CP23 TMEAbody可以被legumain激活。
图6：酶联免疫吸附法表征legumain介导的Ipi-L-3+CP23 TMEAbody的激活。
图7：Ipi-L-3 TMEAbody被legumain或低pH环境激活的示意图。
图8：(A)酶联免疫吸附法表征Ipi-L-3与R4-4的偶联；(B)酶联免疫吸附法表征Ipi-L-3与CP31的偶联。
图9：TMEAbody在极端低pH处理后的聚丙烯酰胺凝胶电泳图。
图10：酶联免疫吸附法表征极低pH条件处理后的TMEAbody。
图11：不同TMEAbody的肿瘤渗透率。
图12：Ipi-L-3+CP23 TMEAbody在hCTLA-4基因敲入的MC38小鼠模型中的体内药效。
图13：Ipi-L-3+CP23提升了血浆半衰期和AUC。
图14：Ipi-L-3 TMEAbody显示出较低的外周T细胞活化效应。
图15：Ipi-L-3+CP23在GvHD模型中产生较低的系统毒性。
图16：Ipi-L-3 TMEAbody在小鼠中显示了较低的免疫原性。
图17：使用FACS分析经Ipi-L-3+CP23 TMEAbody治疗后的Treg数量。
具体实施方式
应当被理解的是，在本发明公开的范围内，涉及的每个技术特征可以和在后面提到的技术特征，包括在实施例中所列举的技术特征相互结合，来形成解决技术问题的优选技术方案。此外，本发明所公开的内容不限于所描述的特定实施例，因此可以一个或多个变化。也应当理解的是，除非另有定义外，本文使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义具有相同的含义。尽管 与本文描述的那些类似或等同的其他任何方法和材料也可用于本发明的实践或测试，但本文所描述的是优选地方法和材料。本文提及的所有出版物均通过引用并入本文，以公开和描述与所引用的出版物相关的方法和/或材料。
必须注意的是，如本文和所附权利要求中所使用的，单数形式“一”，“一个”，“并且”和“该”包括复数指示物，除非上下文另有明确说明。还应注意的是，可以起草权利要求以排除任何可选要素。因此，本声明旨在作为使用与权利要求元素的叙述相关的“单独”，“仅”等专用术语或使用“否定”限制的先行基础。
本发明的目的之一是提供一种修饰过的抗CTLA-4抗体偶联体，其仅在病理微环境(例如在肿瘤微环境或者炎性部位)中被激活而释放出与其配体的结合力相当甚至提高的抗CTLA-4抗体，从而由此提高了生物分子的靶向效力，同时克服药物的耐药性并降低毒性。
在本发明中，修饰的抗CTLA-4抗体是一种偶联体，其包含抗CTLA-4抗体和与该抗体共价连接的功能部分，即R1-R2-R3-L-R4。
在本文公开的内容中，“抗体”按照最广义使用，并且涵盖了单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如双特异性抗体)和抗体片段，只要能表现出想要的生物学活性即可(Miller等，(2003)，Jour.of Immunology，170：4854-4861)。在上下文和附图中，抗体的缩写以“Ab”代替。
基本的抗体结构单元包含由两对相同的多肽链组成的四聚体，每对具有一条轻链和一条重链。每条链的N末端部分包括主要负责抗原识别的约100至110或更多氨基酸的可变区。每条链的C末端部分限定了主要负责效应子功能的恒定区。重链可变区(VH)和轻链可变区(VL)各自含有3个互补决定区(CDR)，包括HCDR1，HCDR2，HCDR3和LCDR1，LCDR2和LCDR3。这六个CDRs形成了抗体的抗原结合位点。可变区的剩余氨基酸是相对保守的并且被称为框架区(FR)。VH和VL各自含有4个框架区，分别称为FR1、FR2、FR3和FR4。
抗体可来源于鼠、人或其它物质，可以是人源化抗体或嵌合抗体。本发明公开的免疫球蛋白可以是任何类型(例如IgG，IgE，IgM，IgD和IgA)，类别(例如IgG1，IgG2，IgG3，IgG4，IgA1和IgA2)或免疫球蛋白分子的亚类。免疫球蛋白可以来自任何物种，如来源于人、鼠或兔。
抗体片段包含抗体全长的一部分，通常包含抗体的抗原结合区或可变区。优选 地，抗体的片段是一个功能性片段，即保留完整抗体的抗原结合能力，即抗体片段是抗体的抗原结合片段。抗体片段或功能片段的实例包括Fab，Fab′，F(ab′)2和Fv片段；双体；线性抗体；和单链抗体分子(scFv)；等等。
在本发明中，将抗CTLA-4抗体的氨基酸序列的合适位置中的一个或多个(例如5个或更少或3个或更少)氨基酸突变为半胱氨酸，然后通过该半胱氨酸的巯基与本发明的功能部分(R1-R2-R3-L-R4)共价连接。例如，将感兴趣的抗CTLA-4抗体中的感兴趣的一个或两个氨基酸突变为半胱氨酸，以缀合至功能部分。突变位置可以存在于可变区的互补决定区或非互补决定区中。优选地，该突变是取代突变。更优选地，突变发生在抗CTLA-4抗体的轻链可变区中。通常，可以制备突变体，然后测试其与相应抗原的结合活性。如果突变体与野生型抗体相比保留结合活性的70％或更多，优选80％或更多，更优选90％或更多，则认为突变位置的氨基酸残基可被突变为半胱氨酸以用于共价连接至功能部分。或者，在某些实施方案中，如果通过将突变体与本文所述的R4连接而产生的偶联体保留80％或更高，优选90％或更高，更优选95％或更高的结合活性，则认为该位置的氨基酸残基可以被为半胱氨酸以共价连接至功能部分。
各种抗CTLA-4抗体均可用于本发明。优选的抗CTLA-4抗体是Ipilimumab，其示例性的重链和轻链的氨基酸序列分别如SEQ ID NO：1和2所示。
通常，抗CTLA-4抗体的轻链可变区的CDR中的G，A，S，T，L，I，F，E，K，D和Y等中的一个或多个，更优选G，A，S，T，L，I，K和Y中的一个或多个，更优选G，A，T，L，S和Y中的一个或多个，可以突变为半胱氨酸。在一些实施例中，抗CTLA-4抗体重链可变区的CDR中的G，A，S，T，L，I，F，E，K，D和Y等中的一个或多个，更优选G，A，S，T，L，I，K和Y中的一个或多个，更优选G，A，T，L，S和Y中的一个或多个，可以突变为半胱氨酸。如果突变发生在抗CTLA-4抗体轻链或重链可变区的非CDR区，则轻链或重链的可变区的非CDR中的G，A，S，T，L，I，F，E，K，D和Y等中的一个或多个，优选G，A，S，T，K，I，Y和L，更优选G，A，T，Y和S中的一个或多个，可以突变为半胱氨酸。在一些实施例中，可以将抗CTLA-4轻链或重链可变区的非互补决定区中(例如在FR1，FR2或FR3)的S，T，L，I，F，E，K，D，N，Q，R和Y残基等中的一个或多个突变为半胱氨酸。在一些实施例中，可以将取代突变引入一 个或多个以下保守位点：VH的非互补决定区(FR1，FR2或FR3)中的Gln3，Ser7，Ser25，Glu46，Thr69和Asp73，以及在VL的非互补决定区(FR1，FR2或FR3)中的Thr5，Tyr50，Arg62，Ser64，Ser66，Ser68，Thr73，Thr75，Ser77和Asp83。上述位置编号参照SEQ ID NO：1和2。
在优选的实施方案中，抗CTLA-4抗体Ipilimumab重链上的突变位置选自：Gln3，Arg19，Leu20，Ser25，G1y26，Phe27，Thr28，Phe29，Ser30，Ser31，Tyr32，Thr33，Met34，His35，G1y44，Phe50，Ile51，Ser52，Tyr53，Asp54，Gly55，Asn56，Asn57，Lys58，Tyr59，Tyr60，Ala61，Asp62，Ser63，Lys65，Gly66，Thr69，Ser71，Arg72，Asp73，Asn74，Ser75，Lys76，Asn77，Thr99，Gly100，Trp101，Leu102，Gly103，Pro104，Leu105，Asp106和Tyr107；优选选自：Ser30，Ser31，Tyr32，Thr33，Ile51，Asp54，Gly55，Asn56，Lys58，Tyr59，Tyr60，Ala61，Asp62，Ser63，Lys65，Gly66，Gly100，Trp101，Leu102，Gly103，Pro104、Leu105，Asp106和Tyr107。
在优选的实施方案中，抗CTLA-4抗体Ipilimumab轻链中的突变位置选自：Gln6，Arg24，Ala25，Ser26，Gln27，Ser28，Val29，Gly30，Ser31，Ser32，Tyr33，Leu34，Ala35，Tyr37，Ile49，Tyr50，Gly51，Ala52，Phe53，Ser54，Arg55，Ala56，Thr57，Gly58，Ile59，Pro60，Asp61，Arg62，Ser68，G1y69，Thr70，Gln90，Gln91，Tyr92，Gly93，Ser94，Ser95，Pro96、Trp97、Thr98、Phe99和Gly100；优选选自：Ala25，Ser26，Ser28，Gly30，Ser31，Ser32，Leu34，Ala35，Gly51，Ala52，Ser54，Ala56，Thr57，Gly58，Ile59，Gly93，Ser94，Ser95，Thr98和Gly100。
上述位置编号参照SEQ ID NO：1和2。
已发现(CN 201880072546.4)，对抗CTLA-4抗体轻链可变区、重链可变区的FR以及CDR区的氨基酸残基进行突变后，获得的突变体仍然维持了较高的结合亲和力(为野生型抗体结合亲和力的60％以上)。本文将该申请公开的关于CTLA-4突变体的所有内容以引用的方式纳入本文。
生物分子的突变，转染，表达和纯化可以通过本领域已知的方法进行。例如，可以直接合成具有在选定的位置上发生了突变的抗CTLA-4抗体的核酸，然后可以将通过酶消化获得的不同片段的核酸分子连接到表达载体中，并将表达载体转化到细菌或真核宿主细胞中。含有半胱氨酸突变的生物分子可以通过宿主细胞中的重组 获得。
适用于本发明的细菌或真核宿主细胞可以是本领域常用的宿主细胞，包括但不限于细菌，酵母和哺乳动物细胞。有用的真核宿主细胞包括CHO细胞，HEK293T细胞或毕赤酵母(Pichia pastoris)。
适用于本发明的表达载体可以是本领域已知的基于病毒的表达载体，包括但不限于杆状病毒，猿猴病毒(SV40)，逆转录病毒或基于痘苗病毒的载体。含有合适调控元件和选择标记的表达载体可用于制备稳定表达突变体的哺乳动物细胞系。例如，GS真核表达系统(Lonza)，DHFR真核表达系统(Invitrogen)和巴斯德毕赤酵母表达系统(Invitrogen)可用于表达和制备。
可以通过本领域已知的分离方法纯化本发明的生物分子。这些方法包括但不限于亲和层析，DEAE离子交换柱，凝胶过滤和羟基磷灰石层析。例如，蛋白A柱或G柱可用于分离细胞培养物上清液或细胞质提取物中的抗体分子。在一些实施方案中，可以对生物分子进行“工程改造”以使它们含有允许生物分子被捕获到亲和基质的氨基酸序列。例如，可使用标记物促进多肽的纯化。合适的标记物包括但不限于c-myc，hemagonium，poly-His(例如，6His)和Flag TM(Kodak)。这种标记物可以插入多肽内的任何位置，包括羧基末端或氨基末端。还可以通过免疫亲和层析纯化本发明的生物分子偶联体。
适用于本发明的官能团部分可由式R1-R2-R3-L-R4表示。在官能团中，R1、R2和R3通过任何合适的连接方式连接在一起，包括但不限于酰胺键、酯键、氨基甲酸酯键、脲键或腙键。R3和R4通过L连接，L为腙键。在本发明中，酰胺键可以用“-CO-NH-”表示，酯键可以用“-C(O)O-”表示，氨基甲酸酯键可以用“-NH-C(O)-O-”表示，脲键可以用“-NH-CO-NH-”表示，腙键可以用由“-C(R’)＝N-NH-”表示，其中R’为H或C1-4烷基。本文中，如无特别说明，R1、R2、R3、L、R4和R5相互之间共价连接。此外，应理解的是，当R1、R2、R3、L、R4和R5作为分子中的一部分时，其指代基团；当作为制备原料时，其指代化合物分子，此时，其连接位置(即下文波浪线所示位置)上通常连接H或OH或本领域熟知的其它相应基团，以满足键价理论，同时也能与其它部分反应形成本文所述的连接键，包括但不限于所述酰胺键、酯键、氨基甲酸酯键、脲键或腙键。
本文中，R1是生物分子R5的保护基团。它可选自能阻止生物分子与其抗原、 配体或受体结合的任何基团，用以防止该生物分子受到其他分子的干扰。例如，在生物分子到达病理性微环境如肿瘤或炎症微环境之前阻止该生物分子与其配体或受体结合。合适的R1选自：NRaRb-R-CO-，C1-4烷氧基-(C1-4烷氧基)n-羰基-C1-4亚烷基-羰基-，
其中每个R独立地为C1-4烷基；每个n独立地为1至30000范围内的整数，例如1-15000、1-5000、1-2000、1-150、1-50、1-20或3-12；聚乙二醇或pegm是分子量为44至132000的聚乙二醇，例如分子量为1000至50000或10000至30000的聚乙二醇；m表示聚乙二醇的分子量；波浪线指示R1链接到R2的位置；Ra为C1-4烷基-O-[(CH2)q-O]n-(CH2)q-；Rb为C1-4烷基-O-[(CH2)q-O]n-(CH2)q-C(O)-；q为1-4的整数。优选地，R1的分子量不超过20kD。
在一些实施方案中，所述NRaRb-R-CO-为：
优选地，各n独立为1-1000的整数。
在一些实施方案中，所述C1-4烷氧基-(C1-4烷氧基)n-羰基-C1-4亚烷基-羰基-为：
优选地，n为1-1000的整数。
在一些实施例中，R1基团选自：
通常，如果突变位置存在于生物分子的功能结构域中，例如，在抗体的CDR中，则对R1的分子量没有特别限制，R1可以具有相对低的分子量。如果突变位置存在于生物分子的功能结构域之外，例如，在抗体的非CDR中，优选选择R1以使R1-R2-R3-L-R4的分子量高于200，优选高于500，更优选高于1000，以使生物分子的偶联体的分子量为5000或更高，优选8000或更高，更优选10000或更高，从而更好地防止生物分子在到达病理微环境之前与其配体或受体结合。
在本发明中，R2是一种被病理微环境中的一种或多种蛋白水解酶、蛋白酶或肽酶水解激活的连接臂。在本发明中，蛋白水解酶、蛋白酶或肽酶可以是存在于病 理微环境中的各种蛋白水解酶、蛋白酶或肽酶。例如，蛋白酶可以是半胱氨酸蛋白酶，天冬酰胺蛋白酶，天冬氨酸蛋白酶，谷氨酸蛋白酶，苏氨酸蛋白酶，明胶酶，金属蛋白酶或天冬酰胺肽裂解酶；优选地，R2是能够被选自以下酶中的至少一种切割的肽：天冬酰胺内切酶，粒酶，组织蛋白酶B，组织蛋白酶C，组织蛋白酶D，组织蛋白酶E，组织蛋白酶K，组织蛋白酶L，激肽释放酶，hK1，hK10，hK15，纤溶酶，胶原酶，IV型胶原酶，星形胶酶，因子Xa，胰凝乳蛋白酶样蛋白酶，胰蛋白酶样蛋白酶，弹性蛋白酶样蛋白酶，枯草杆菌蛋白酶样蛋白酶，猕猴桃蛋白酶，菠萝蛋白酶，钙蛋白酶，胱天蛋白酶，胱天蛋白酶-3，防御型半胱氨酸蛋白酶，木瓜蛋白酶，HIV-1蛋白酶，HSV蛋白酶，CMV蛋白酶，乳糜蛋白酶，胃蛋白酶，脂蛋白酶，血纤维蛋白溶酶，猪笼草蛋白酶，金属外肽酶，金属内肽酶，基质金属蛋白酶(MMP)，MMP1，MMP2，MMP3，MMP8，MMP9，MMP10，MMP11MMP13，MMP14，ADAM10，ADAM12，尿激酶纤溶酶原激活物(uPA)，神经激酶，前列腺特异性抗原(PSA，hK3)，白介素113转化酶，凝血酶，FAP(FAP-a)，穿膜肽酶，二肽基肽酶和二肽基肽酶IV(DPPIV/CD26)。在优选的实施例中，本发明公开的内容特别涉及Legumain，其在肿瘤微环境中主要由肿瘤细胞表达和分泌。通过Legumain的表达，肿瘤相关巨噬细胞(M2型)也不同于单核细胞和炎性巨噬细胞(M1型)。在本发明中，多肽是蛋白水解酶的底物，可以被蛋白水解酶识别和切割。
本发明的R2基团可以由-R2a-，-R2b-，-R2a-N-，-R2a-D-，-R2a-AAN-，-R2a-AAD-或-R2a-R2b-表示；其中R2a是能够被一种或多种蛋白水解酶在酰胺键上切割的肽；R2b是一种可以通过侧链中的氨基与R3形成氨基甲酸酯的肽，氨基甲酸酯可被一种或多种蛋白水解酶切割；A是丙氨酸；N是天冬酰胺，其侧链中的氨基与R3形成氨基甲酸酯，氨基甲酸酯可被Legumain切割；D是天冬氨酸，其侧链中的氨基与R3形成氨基甲酸酯，且可以被颗粒酶B裂解。R2a和R2b可以通过形成酰胺键而连接。在Legumain、颗粒酶B裂解R2和R3之间的键(例如氨基甲酸酯)之后，在酸性条件下R3-L-中的L被切割，从而释放出带有R4基团的抗体。在一些实施例中，能够被蛋白水解酶活化的合适的多肽可以是三肽。在本领域已知的病理微环境中能够被蛋白水解酶识别和切割(活化)的任何底物肽可以用作本文公开的R2。此类肽具有WO 2016/026458中公开的结构，其全部内容通过引用并入本 公开。在一些实施方案中，在适用于本发明的三肽结构中，与R1连接的氨基酸残基可以选自Ala、Thr、Val和Ile，中间氨基酸残基可以选自Ala、Thr、Val和Asn，与R3连接的氨基酸残基可选自Asn和Asp。通常，R2通过其氨基酸残基的氨基以酰胺键、酯键、氨基甲酸酯键、脲键或腙键的连接方式与R1连接，并通过其氨基酸残基的羧基以酰胺键、酯键、氨基甲酸酯键、脲键或腙键的连接方式与R3连接。在本发明的一些优选实施方案中，R2为选自Ala-Ala-Asn和Ala-Ala-Asp的三肽。Ala-Ala-Asn可以被Legumain识别和切割，Ala-Ala-Asp可以被颗粒酶识别和切割。
在本发明中，R3基团是连接臂，包括但不限于-HN-苯基-R’-，其中，R’为键。优选地，所述NH基团和R’位于苯基的对位。制备本发明的化合物时，可使用下式R3-1和R3-2所示的化合物制备获得本发明的R3连接臂：
在一些实施方案中，L基团是能够在病理学微环境中被酸性激活的化学键，具体为腙键(即-C(R’)＝N-NH-，其中R’为H或C1-4烷基)。例如，在一些实施例中，R1-R2-R3-L-R4的结构可以表示如下：
应理解，L基团可通过R3化合物与R4化合物之间的化学反应获得。
在本发明中，R4基团是在R2基团和R3基团裂解后能够恢复，维持，降低或促进抗CTLA-4抗体与其抗原，配体或受体的结合能力的结合基团。在一些实施方案中，在切割R2基团和R3基团之后，所得的R4-s-Cys-R5对天然R5的抗原，配体或受体表现出＞60％的亲和力。
合适的R4基团可以用下式表示：
其中，Rc选自C1-12亚烷基，C1-12亚烷基-O-C1-12亚烷基，C1-12亚烷基-C3-8环烷基，(C1-4亚烷基-O)p-C1-12亚烷基，C1-12亚烷基羰基氨基-(C1-4亚烷基-O)p-C1-12亚烷基，-苯基-C1-12亚烷基，C3-8环烷基，C1-12亚烷基-C3-8环烷基-C1-12亚烷基，C1-12亚烷基-NHCO-O-C1-12亚烷基，C1-12亚烷基-COO-和C1-12亚烷基-苯基-C1-12亚烷基；p为1-10的整数，优选为1、2、3或4；
R4-c为键或-CO-；
其中，L基团通过R4基团的R4-c与R4基团相连。
通常，R4基团通过马来酰亚胺与R5的半胱氨酸的S原子连接。
在优选的实施方案中，Rc为C1-12亚烷基，R4-c为-CO-；其中，R4基团通过其羰基与L基团连接。
在一些实施例中，R4基团可由选自以下的R4化合物制备得到：
应当理解的是，在本发明中，在每个指示的公式中使用的波浪线指示包含波浪线的部分与其他部分的连接位置。
如上所述，R5代表一个或多个氨基酸残基突变为半胱氨酸的抗CTLA4抗体。R5实际上是引入的半胱氨酸的巯基缺少氢原子的生物分子。缺少该硫基氢原子允许将R5视为连接至本发明R4的基团。
在本发明的一个或多个实施方案中，可以以多种方式激活抗CTLA-4抗体。一 种方式是通过蛋白水解酶将R1-R2从R3-L-R4-S-cys-R5上切割下来。另一种方法是在酸性条件下在病理微环境中裂解R1-R3-L，释放R4-S-cys-R5。本发明发现，本发明的双激活(酶激活和酸激活)化合物与单激活化合物相比，在患病组织中具有更高的激活效率。
本发明的偶联体可以通过包括通过DTT，TCEP或其他还原剂还原突变生物分子的方法来制备；用Cu2SO4，脱氢抗坏血酸或其他氧化剂氧化；然后在液相或固相条件下将氧化的生物分子(R5)与R1-R2-R3-L-R4结合。最终产物可以在液相中收集。
因此，除了偶联体之外，本发明还包含功能部分，即R1-R2-R3-L-R4；R2-R3-L-R4；R3-L-R4-S-cys-R5；R4-S-cys-R5和突变的生物分子；其中R1，R2，R3，L，R4，R5及其连接方式和突变的生物分子如本发明的任何部分或任何实施方案中所定义。
在一些实施方案中，功能部分由CP3、CP7、CP13、CP23-CP27显示。在本发明中，-S-cys-表示R4经由R5中的突变引入的半胱氨酸的巯基与R5共价连接。R4-S-cys-R5是在病理性微环境的酸性条件下形成的偶联体。
如本文所述的偶联体，功能部分，R2-R3-L-R4，R3-L-R4-S-cys-R5和R4-S-cys-R5可通过本领域已知的方法合成。例如，它们可以根据本申请的实施例1中描述的方法制备。
本发明还包括药物组合物，其包含如本文所述的偶联体。药物组合物可进一步包含药学上可接受的载体。载体可以是任何药学上可接受的载体或赋形剂，其可以根据剂型和给药方式而变化。药学上可接受的载体通常是安全且无毒的，并且可包含在制药工业中配制药物组合物中使用的任何已知物质，包括填料，稀释剂，凝结剂，粘合剂，润滑剂，助流剂，稳定剂，着色剂，润湿剂和崩解剂等。药学上可接受的合适的载体包括糖，如乳糖或蔗糖，甘露醇或山梨糖醇；纤维素制剂和/或磷酸钙，如磷酸三钙或磷酸氢钙；淀粉，包括玉米淀粉，小麦淀粉，大米淀粉，马铃薯淀粉，明胶，黄蓍胶，甲基纤维素，羟丙基甲基纤维素，羧甲基纤维素钠和/或聚乙烯吡咯烷酮；二氧化硅，滑石粉，硬脂酸或其盐，如硬脂酸镁或硬脂酸钙；和/或聚乙二醇等等。当选择药学上可接受的载体时，主要考虑的是药物剂型的给药方式。这在本领域中是众所周知的。
药物组合物可包含治疗或预防有效量的偶联体。“有效量”表示成分的量足以产生所需的反应。具体有效量取决于各种因素，例如待治疗的具体疾病，患者的身体状况，如体重，年龄和性别，治疗的持续时间，共同给药的治疗(如果有的话)，以及使用的具体配方。通常，如本文所述的“有效量”是常规量的生物分子。然而，在一些实施方案中，本发明药物组合物中包含的治疗或预防有效量的偶联体可以低于常规量的生物分子但可以产生更好的治疗或预防效果，因为生物分子在到达病理微环境与其配体或受体结合之前受保护基团保护。
本发明的药物组合物可以配制成各种合适的剂型，包括但不限于片剂，胶囊，注射剂等，并且可以通过任何合适的途径给药以达到预期目的。例如，它可以肠胃外，皮下，静脉内，肌肉内，腹膜内，透皮，口服，鞘内，颅内，鼻内或外给药。药物的剂量可取决于患者的年龄，健康状况和体重，并行进行的治疗，以及治疗的频率等。本发明的药物组合物可以施用于任何有此需要的受试者，例如作为哺乳动物，尤其是人类。
在肿瘤患者中，携带肿瘤抗原的肿瘤细胞或抗原呈递细胞(APC)通过与T细胞结合而部分或完全抑制宿主对肿瘤的免疫杀伤。然而，本发明的偶联体通过蛋白水解酶，尤其是Legumain或颗粒酶，和/或在酸性条件下，在病理微环境中被活化和释放。
因此，本发明的偶联体可以有效地突破个体的免疫屏障，到达病理微环境，然后在病理微环境中被激活和释放。结果，它可以选择性地促进肿瘤或炎症微环境中T细胞等的增殖或杀伤作用，从而实现低自身免疫性和高效力。
因此，本发明中公开的每种偶联体或R4-S-cys-R5或突变的生物分子可以用于治疗肿瘤或炎症，或者可以用作制备用于治疗肿瘤或炎症的药物的活性成分。本文所述的肿瘤或炎症优选为CTAL-4介导的肿瘤或炎症，可以是已知可由抗CTAL4抗体(尤其是本文所述的抗CTLA4抗体，如Ipilimumab)治疗的任何肿瘤或炎症，包括但不限于黑色素瘤，膀胱，脑，乳腺，子宫颈，结肠-直肠，食道，肾，肝，肺，鼻咽，胰腺，前列腺，皮肤，胃，子宫，卵巢，睾丸和血液中的癌症等。具体而言，癌症包括膀胱癌，脑癌，乳腺癌，宫颈癌，结直肠癌，食道癌，肾癌，肝癌，肺癌，鼻咽癌，胰腺癌，前列腺癌，皮肤癌，胃癌，子宫癌，卵巢癌，睾丸癌和血癌。
还包括用于治疗或预防肿瘤或炎症的方法，其包括向有此需要的受试者施用治 疗或预防有效量的如本文所述的偶联体或其药物组合物。该方法可以与任何已知的放射疗法或免疫疗法组合使用。
应当理解，本发明中使用的术语“包含”和“包括”或类似表达也意味着“由……组成”等。所有重量百分比或体积百分比的总和应等于100％。除非另有说明，实施例中使用的各种试剂和产品是商业产品。除非另有说明，否则实施例中提到的方法是根据常规技术实施的。以下实施例不旨在限制本发明的范围。
实施例1：功能基团CP1-CP30的合成
当R1、R2、R3、L和R4选自不同的基团时，合成的化合物如下表1-1和表1-2所示：
表1-1：化合物名称及用于形成化合物的R1、R2、R3和R4化合物

表1-2：化合物结构




当R2为Ala-Ala-Asn，R3为PABC(R3-1)时，合成路线图如下所示：
以CP5为例，合成路线如下所示：

1)Fmoc-Asn(Trt)-OH(20g，0.03mol)，
将2-(7-氮杂苯并三唑)-N，N，N′，N′-四甲基脲六氟磷酸盐(HATU)(15g，0.04mol)和DMF(200mL)加入到三口烧瓶中并搅拌30分钟。分别在0℃加入对氨基苯甲醇(4.1g，0.03mol)和N，N-二异丙基乙胺(8.7g，0.06mol)，然后在室温下搅拌3小时。通过旋转蒸发除去大部分DMF。将残余物溶于乙酸乙酯(200mL)中，依次用饱和氯化铵溶液和饱和氯化钠溶液洗涤，用无水硫酸钠干燥，然后过滤。蒸发除去溶剂，打浆粗产物，得到白色固体Fmoc-Asn(Trt)-PABC(21.3g；收率：90％)。
2)将Fmoc-Asn(Trt)-PABC(16.0g，22mmol)溶解在N，N-二甲基甲酰胺(80mL)中。加入哌啶(30mL)，然后在室温下搅拌2小时。减压蒸发除去溶剂。将残余物在真空烘箱中在高真空下干燥以除去少量哌啶，得到9.8g淡黄色固体NH2-Ann(Trt)-PABC，其可不经纯化用于下一步骤。
3)将Alloc-Ala-Ala-OH(5.0g，20.4mmol)，苯并三唑-N，N，N′，N′-四甲基 脲六氟磷酸盐(HBTU)(11.6g，30.6mmol)和DMF(50mL)加入到三口烧瓶中，在冰浴中搅拌30分钟。分别在0℃加入NH2-Asn(Trt)-PABC(9.8g，20.4mmol)和N，N-二异丙基乙胺(7.89g，61.2mmol)，然后在室温下搅拌过夜。减压蒸发除去溶剂。将残余物溶于乙酸乙酯(200mL)中，依次用饱和氯化铵溶液和饱和氯化钠溶液洗涤，用无水硫酸钠干燥，然后过滤。蒸发除去溶剂。将所得粗产物进行重结晶，得到白色固体Alloc-AAN(Trt)-PABC(13.0g；产率：90％)。
4)将Alloc-AAN(Trt)-PABC(10.0g，14.2mmol)溶解在二氯甲烷(100±15mL)中。加入三氟乙酸(20mL)，然后在室温下搅拌4小时。用水洗涤和分馏后，有机相用无水硫酸钠干燥。减压蒸发除去溶剂，通过高真空蒸发除去残留的三氟乙酸。粗品通过柱色谱法分离，得到Alloc-AAN-PABC(5.9g；收率：89％)。
5)将溶解在二氯甲烷(10mL)中的Alloc-AAN-PABC(467mg，1.01mmol)加入到三口烧瓶中。在冰浴中并在氮气保护下将二氯甲烷中的4-硝基苯基氯甲酸酯(406mg，2.02mmol)和二氯甲烷中的吡啶(160mg，2.03mmol)分别滴加到烧瓶中，室温下搅拌过夜。将1-(6-氨基己基)-1H-吡咯并2，5-二酮(235mg，1.2mmol)分批加入上述溶液中，并使其在室温下反应4小时。通过旋转蒸发干燥反应溶液。通过硅胶柱色谱法纯化得到的粗产物，得到白色固体S15-1(540mg；收率：80％)。
6)向单口烧瓶中依次加入DMF(10ml)，S15-1(208mg，0.31mmol)，乙酸(274mg，4.65mmol)，三苯基膦(72mg，0.062mmol)和三丁基氢化锡(1.17g，4.03mmol)。在用氮气置换烧瓶中的空气后，将混合物在室温下搅拌直至S15-1完全反应。反应完成后，减压蒸发除去DMF。粗产物通过硅胶柱色谱法分离纯化，得到S15-2(白色固体，116mg，收率：62％)。
7)将化合物1(940mg，0.18mmol)，苯并三唑-N，N，N′，N′四甲基脲六氟磷酸盐(HBTU)(95mg，0.25mmol)和DMF(10mL)加入三口烧瓶中，然后在冰浴中搅拌30分钟。然后在0℃分别加入化合物中间体2(108mg，0.18mmol)和N，N-二异丙基乙胺(70mg，0.54mmol)，然后在室温下搅拌过夜。减压蒸发除去溶剂。粗产物通过硅胶柱色谱法分离纯化，得到白色固体(85.5mg；收率：40％)，即为化合物CP5。
当R2具有Ala-Ala-Asp的氨基酸序列且R3是PABC时，合成路线如下所 示：
使用以上方法合成CP1，CP2，CP4，CP6，CP12，CP13，CP15，CP20-CP22，CP28-CP30。
当包含可酸活化的化学键时，合成方案如下所示：
如CP8所示，具体的合成过程如下所示：
1)R1-10(3.1g，0.01mol)，2-(7-氮杂苯并三唑)-N，N，N′，N′-四甲基脲六氟磷酸盐(HATU)(4.56g，0.012mol)和DMF(20mL)加入到三口烧瓶中并搅拌30分钟。在0℃分别加入R3-2(1.35g，0.01mol)和N，N-二异丙基乙胺(3.87g，0.03mol)，然后在室温下搅拌3小时。通过旋转蒸发除去DMF。将残 渣用硅胶柱色谱法精制，得到淡黄色油状物中间体1(2.5g；收率59％)。
2)依次称量中间体1(98mg，0.23mmol)和R4-18(42mg，0.23mmol)，并加入50ml单口烧瓶中。加入二氯甲烷(5mL)以溶解中间体1和R4-18，然后加入4A分子筛(81mg)。用氮气置换烧瓶中的空气后，使混合物在室温下反应过夜。反应溶液通过旋转蒸发干燥。粗产物通过硅胶柱色谱法纯化，得到白色固体(81mg；产率：60％)，其为化合物CP8。
用上述方法合成CP7，CP9-11，CP14，CP19。
以CP23为例，当R2的氨基酸序列为Ala-Ala-Asn且L为可酸激活的化学键时，合成方案如下：

1)中间体1的合成
取干燥洁净2L反应瓶，加入THF 500ml，称取Fmoc-Asn(Trt)-OH 80g，加入反应瓶中，搅拌溶解，加入DEPBT 46.6g，室温搅拌15分钟，加入PABC 16g，室温反应30分钟后，加入DIPEA 45ml，氮气换气保护，室温反应3小时，TLC监测反应毕(Fmoc-Asn(Trt)-OH反应完全)。减压蒸除反应液，加少量DMF(180ml)溶解，滴加至搅拌中的3L水中，析出淡黄色固体，水洗2-3次后，抽滤，收集固体，真空干燥，得类白色固体(收率90％以上)。
2)中间体2的合成
将THF 500ml，上步所得类白色固体依次加入到2L单口反应瓶中，搅拌溶解，冰盐浴降温至0-5℃，滴加哌啶100ml，滴加完毕，逐渐恢复至室温反应1h，TLC监测反应完毕。减压蒸除溶剂，加少量DMF溶解，滴加至搅拌中得2L水中，机械搅拌30min，抽滤，重复水洗2-3次，抽滤，滤饼加甲基叔丁基醚800ml，搅拌30min，抽滤，滤饼加PE∶EA＝10∶1洗2次，抽滤，收集滤饼，真空干燥后，得类白色固体80g，纯度70％。
3)中间体3的合成
在干燥洁净的250ml单口反应瓶中依次加入50ml THF，5.04g Boc-Ala-Ala-OH，3.89g DEPBT，室温反应10min，加入2.6g NH2-Asn(Trt)-PABC，氮气换气保护，室温反应15min，滴加DIPEA 3.5ml，氮气换气保护，室温反应3小时，减压蒸除溶剂，加水打浆2-3次，抽滤，得浅黄色固体，3.7g，过柱纯化，得产品2.0g，纯度94.8％，收率26.6％。
4)中间体4的合成
称取Boc-Ala-Ala-Asn(Trt)-PABC 1.8g加入100ml单口反应瓶中，加入TFA28.5ml，滴加水1.5ml，室温反应30min，TLC监测反应毕，减压蒸除溶剂，加甲基叔丁基醚打浆，抽滤，得固体，加二氧六环∶水＝1∶1溶液溶解，加1N氢氧化钠调节pH值至13，室温搅拌40min，减压蒸除溶剂，硅胶拌样过柱，得产品450mg。收率47.5％。
5)中间体5的合成
依次向500ml三口反应瓶中加入100ml二氧六环，9.8g NH2-AAN-PABC，3.28g Na2CO3(配制成饱和水溶液滴加)，冰盐浴降温至0-5℃，称取Fmoc-C 16.6g，加10ml二氧六环溶解，滴加至上述反应液中，逐渐升温至室温反应30min(析出固体，补加THF继续反应)，至反应终止，减压蒸除溶剂，加水打浆，抽滤，得类白色固体，加甲基叔丁基醚打浆，抽滤，得类白色固体，真空干燥至恒重，得产品15.5g，纯度80％，收率：80％。
6)中间体6的合成
将中间体5(5g，8.3mol)溶于DMF(40ml)，在室温搅拌情况下，加入Dess-Martin试剂(5.3g，12.5mmol)，继续搅拌2小时。反应液先后用Na2S2O4饱和溶液和NaHCO3饱和溶液淬灭，过滤，滤液减压浓缩，然后固体用水洗得到中间体6(4.7g棕色固体，收率：94％)。
7)中间体7的合成
将中间体6(566mg，0.94mmol)溶于DMF(10ML)，在搅拌情况下加入化合物2(280mg，0.94mmol)，氮气保护下，室温搅拌过夜。反应液用中压制备得到中间体7(719mg白色固体，收率：100％)。
8)中间体8的合成
将中间体7(280mg，0.37mmol)溶于DMF(10ml)，在搅拌情况下加入DBU(5ul)，室温搅拌3h，然后再加入DBU(5ul)，继续搅拌2h。反应液用高压制备得到中间体8(85mg白色固体，收率：42％)。
9)终产物CP23的合成
将化合物SCM-5K(1.45g，0.29mmol)溶于DMF(20ml)，在搅拌情况下加入中间体8(190mg，0.35mmol)，室温搅拌过夜。反应液减压浓缩，用高压液相纯化得到产物CP23(1.12g白色固体，收率：77％)。
使用以上方法合成CP3，CP5，CP16-CP18，CP24-CP27。
通过质谱(MS)验证化合物CP1-CP30，其分子量示于表2，其与基于其结构计算的分子量一致。
表2：本发明的功能基团质谱信息表及其性状收率

实施例2：构建具有不同封闭基团和接头的一系列抗hCTLA4TMEAbody
与不同化合物(包括阻碍基团和连接子)与轻链上包含Y49C(Y49C是按照抗体的Kabat编号系统，该编号对应按顺序依次编号的Y50C)突变的Ipilimumab通过位点特异偶联构建一系列抗人CTLA4-TMEAbody(TMEAbody是肿瘤微环境激活抗体的缩写)。含有Y49C的Ipilimumab抗体(在此缩写为Ipi-L-3)在室温50mM Tris(pH7.5)，2mM EDTA条件下，以摩尔比为1∶200的二硫苏糖醇(DTT)还原16h，然后透析至50mM Tris(pH7.5)，150mM NaCl缓冲液中，然后用1∶100摩尔比的脱氢抗坏血酸(DHAA)进行再氧化，用于与包含不同阻碍基团和不同化学接头的不同化合物CP12-CP18、CP23-CP30偶联。用于偶联的化合物/抗体的摩尔比为1∶20。
通过蛋白A柱进一步纯化偶联的TMEAbody以除去过量的化合物，并通过SDS-PAGE测定评估偶联效率。图1(A-F)的结果表明，Ipi-L-3对这些不同的化合物显示出高的偶联效率。
实施例3：Ipi-L-3 TMEAbody的ELISA表征
为了表征构建的TMEAbody与人CTLA-4蛋白的结合特性，0.5μg/ml带His标签的人CTLA-4蛋白(义翘生物，Cat#11159-H08H)包被在Maxisorp ELISA板(Nunc)上4℃过夜孵育。然后将板用PBST洗三次并用2％BSA在室温下封闭2小时。ELISA板用PBST洗三次之后，用系列浓度的偶联后的TMEAbody，TMEAbody偶联之前的(半胱氨酸突变形式)抗体和对照野生型Ipilimumab抗体孵育一小时。然后将板用PBST洗涤3次，并与HRP(Invitrogen，Cat#A18829)缀合的抗人Fc抗体以1∶5000的稀释度在室温下孵育0.5小时。用PBST洗涤3次后，将板用四甲基联苯胺(TMB，索莱宝，目录号PR1200)和ELISA终止缓冲液(索莱宝，目 录号C1058)显影。然后通过ELISA酶标仪(伯腾，ELx800 Absorbance Microplate Reader)测量450nm处的吸光度。然后通过GraphPad Prism 5软件分析数据。ELISA结果如图2(A-F)所示。这些结果表明TMEA抗体与CTLA-4蛋白的结合能力在不同水平上被不同的封闭基团封闭。通常，封闭效率随封闭基团的大小而增加。
实施例4：酸介导的Ipi-L-3+CP23，Ipi-L-3+CP24，Ipi-L-3+CP25和Ipi-L-3+CP26TMEAbody的激活
化合物CP23，CP24，CP25和CP26含有在酸性pH下不稳定的腙键。我们将与这些化合物偶联的Ipi-L-3 TMEAbody透析至pH4.5、pH5.0、pH6.0柠檬酸缓冲液，并通过SDS-PAGE监测酸介导的裂解。如图3(A-B)所示，所有带有这些化合物的TMEAbody均显示出不同程度的裂解，裂解程度取决于pH值的大小，pH越低，裂解效率越高。
然后用实施例2中的方法进行ELISA测定以评价结合活性的恢复。结果总结在图4(A-D)中。低pH介导的裂解导致相应的活性恢复，其活性恢复水平与裂解水平成正比。
实施例5：Legumain介导的Ipi-L-3+CP23 TMEAbody激活
将偶联的Ipi-L-3+CP23 TMEAbody与活化的Legumain在pH6.0、50mM MES、250mM NaCl缓冲液中于37℃孵育过夜。使用蛋白质印迹法评估活化效率。使用抗人Fab HRP抗体(SIGMA，A0293)来检测抗体的轻链。如图5所示，1μg Ipi-L-3+CP23 TMEAbody可以被1-5μg Legumain激活。
然后将上述ELISA测定测试Legumain消化后的结合能力恢复。结果如图6所示。结果显示，不同程度的Legumain裂解导致相应的活性恢复。
实施例6：酸激活的ELISA表征以及酶激活的ELISA表征
Ipi-L-3与CP23偶联的TMEAbody的Legumain和酸激活示意图如图7所示，为了测试酸激活之后的活性恢复情况，我们用R4-4与Ipi-L-3偶联，分子结构与酸介导的激活后的相同。
与R4-4偶联之后，用ELISA评价激活之后剩余在抗体上的小分子片段对Ipi- L-3结合活性的影响。结果展示在图8(A)中，Ipi-L-3与R4-4偶联之后保持了Ipi-L-3的结合活性，意味着在酸激活Ipi-L-3+CP23之后的结构可以保持与野生型Ipilimumab抗体相同的结合活性。
为了测试酶激活之后的活性恢复情况，我们用CP31与Ipi-L-3偶联，分子结构与酶介导的激活后的相同。与CP31偶联之后，用ELISA评价激活之后剩余在抗体上的小分子片段对Ipi-L-3结合活性的影响。结果展示在图8(B)中，Ipi-L-3与CP31偶联之后保持了Ipi-L-3的结合活性，意味着在Legumain激活Ipi-L-3+CP23之后的结构可以保持与野生型Ipilimumab抗体相同的结合活性。
实施例7：Ipi-L-3+CP23的快速酸激活及活性恢复评估
为了建立Ipi-L-3 CP23 TMEAbody的快速酸激活方法，用HCl调整偶联后的Ipi-L-3+CP23 TMEAbody pH值，使其分别为pH2.0、pH2.5和pH3.5，并分别处理1h3h和5h。同时利用SDS-PAGE和ELISA评估切割效率和活性的恢复。
结果如图9和图10(A-D)所示。结果显示，在极低的pH值条件下，例如在pH2.0和pH2.5，该TMEAbody可被快速切割。然而，ELISA结果显示极低的pH会导致抗体活性的显著丧失，这可能是由于极低pH破坏了抗体的结构。此外，野生型的Ipilimumab抗体在pH3.5、pH2.5和pH2.0的条件下也会导致活性的明显丧失。
实施例8：评价一系列TMEAbody在小鼠肿瘤模型中的药代动力学和组织分布
为了评估TMEAbody在体内的分布与激活，将上述系列10mg/kg TMEAbody注射到CT26荷瘤Balb/c小鼠体内，之后在不同的时间点，取血浆和不同的组织用于分析TMEAbody或激活后的TMEAbody的浓度。组织匀浆在2.5体积的PBS中，加入1XHaltTM蛋白酶抑制剂Cocktail(100X)(赛默飞世尔，货号78430)，收集上清，用于ELISA检测。TMEAbody浓度的测定采用5μg/ml的抗PEG抗体(Abcam，货号ab51257)捕获TMEAbody，捕获的TMEAbody用抗人Fc抗体(Invitrogen，A18829，1∶50 000稀释)检测。用系列浓度的不同TMEAbody绘制标准曲线。血浆和组织匀浆的上清稀释至合适的倍数用于ELISA测定并且用标准曲线换算浓度。为了测定 活性TMEAbody的浓度，用0.5μg/ml人CTLA-4蛋白(Sino Biological，11159-H08H)包被并且用系列浓度的野生型Ipilimumab作为一抗，用1∶5000抗-人Fc抗体(Invitrogen，货号A18829)作为二抗，利用不同浓度的Ipilimumab抗体制作标准曲线。血浆和组织匀浆的上清稀释至合适的倍数用于ELISA测定并且用标准曲线换算浓度。
我们关注TMEAbody的PK和组织分布数据中两个重要参数。第一个参数是肿瘤和其他组织的激活率。优选肿瘤中而不是其他组织中的高特异性激活率，因为其将产生的较低的外周毒性。不同TMEAbody在不同组织中的激活率展示在表3中。
表3：TMEAbody在不同组织中不同时间(天)的激活效率


D0天是给药起始点，因此ND表示该日无器官中的数据，而仅有血液中的数据。
如表3中的结果所示，Ipi-L-3+CP23/CP24/CP25/CP26/CP28/CP30表现出不同程度的肿瘤优先激活，激活效率随阻断基团的增加而降低。Legumain和酸激活并存的连接子(CP23、CP24、CP25、CP26)与仅是Legumain(CP28、CP30)激活的连接子相比表现出更高的激活效率。
我们关注的另外一个参数是肿瘤渗透率，因为PEG会增加抗体的尺寸，可能会影响抗体的肿瘤渗透率。我们使用肿瘤AUC/血液AUC(激活与未激活的TMEAbody总浓度TMEABODY)比值来表示渗透率。如图11所示，分子量大于20kD的PEG会显著影响抗体的渗透率。
实施例9：Ipi-L-3 TMEAbody在hCTLA-4基因敲入小鼠的MC38肿瘤模型中的治疗作用
为评价Ipi-L-3 TMEAbody治疗小鼠肿瘤的体内疗效，将Ipi-L-3+CP23 TMEAbody、野生型Ipilimumab及对照人IgG给药至人CTLA-4基因敲入的C57BL/6小鼠(上海南方模式生物)MC38结肠腺癌肿瘤模型中。在人CTLA-4基因敲入的C57BL/6小鼠左下腹象限皮下注射2E6MC38细胞。肿瘤生长7天后，按照相同平均肿瘤体积将动物分组。每周两次给动物注射指定剂量的对照人IgG，野生型Ipilimumab或Ipi-L-3+CP23 TMEAbody(不含PEG分子量的抗体浓度，n＝6)，并监测每只动物的肿瘤体积。
结果如图12(A-E)所示，Ipi-L-3+CP23 TMEAbody显示出剂量依赖性的肿瘤生长抑制效应。剂量为0.5mg/kg时，Ipi-L-3+CP23 TMEAbody显示出与WT Ipilimumab抗体相同的完全缓解率(CR)。
实施例10：Ipi-L-3 TMEAbody在人CTLA-4基因敲入小鼠中显示出延长的血清半衰期
抗体在体内显示出靶标抗原介导的清除，我们推测TMEAbody由于与靶标抗原的结合力下降，由靶标抗原介导的清除作用将受到抑制，从而延长其半衰期。将hCTLA-4敲入小鼠(上海南方模式生物)注射给予野生型Ipilimumab抗体或Ipi-L-3+CP23 TMEAbody，在不同时间点收集血浆，并使用抗人Fc和抗人Fab抗体通过“三明治”式ELISA方法测定抗体浓度。如图13所示，与野生型Ipilimumab抗体相比，Ipi-L-3+CP23 TMEAbody显示出延长的血清半衰期和AUC。
实施例11：Ipi-L-3 TMEAbody显示出降低的外周T细胞活化效应
为了评估野生型Ipilimumab抗体和Ipi-L-3+CP23 TMEAbody对外周CD4+T细胞的激活作用，分别以1mg/kg、3mg/kg、10mg/kg的剂量给hCTLA-4敲入小鼠(上海南方模式生物)注射野生型Ipilimumab，Ipi-L-3+CP23 TMEAbody或PBS。在指定的时间点，收集血液并监测CD4+T细胞的Ki67作为增殖标志物。如图14(A-C)所示，Ipi-L-3+CP23 TMEAbody在3mg/kg和10mg/kg剂量下均未刺激CD4+T细胞增殖，表明TMEAbody由于其活性被封闭导致其外周血T细胞活化效应低，提示其具有更低的免疫相关毒性。
实施例12：Ipi-L-3 TMEAbody在植入人PBMC的NSG小鼠中引起较低的GvHD效应
为了进一步评价Ipilimumab与Ipi-L-3 TMEAbody的免疫毒性，野生型的Ipilimumab与Ipi-L-3+CP23 TMEAbody施用到人PBMC移植的NSG免疫缺陷小鼠中(上海南方模式生物)。将5×106的人PBMC细胞通过尾静脉移植入NSG小鼠中，在重建10天之后，按照10mg/kg Ipilimumab或者Ipi-L-3 TMEAbody给药，一周两次。观察动物体重作为移植物抗宿主病(GvHD效应)的指征。从图15可以看出，野生型Ipilimumab显著加重GvHD，导致体重急剧下降，而Ipi-L-3+CP23TMEAbody没有明显加重GvHD，提示Ipi-L-3+CP23降低了健康组织中毒性T细胞的激活。
实施例13：Ipi-L-3 TMEAbody在小鼠中引起较低的免疫原性
为评价Ipi-L-3 TMEAbody的免疫原性，将Ipi-L-3+CP23 TMEAbody和WT CTLA-4抗体注射至Balb/c小鼠进行免疫，设PBS作为阴性对照。初次免疫采用50μg Ipi-L-3+CP23 TMEAbody或野生型Ipilimumab抗体与1∶1完全的弗氏佐剂(CFA)混合，经小鼠腹腔注射，每组5只小鼠。为了进一步加强免疫，初次免疫后14天，将25μg Ipi-L-3 TMEAbody或野生型Ipilimumab抗体与1∶1不完全弗氏佐剂混合，经小鼠腹腔注入。初次免疫后21天收集血浆，ELISA分析免疫原性。0.5μg/ml人CTLA-4-His蛋白(Sino Biological，Cat#11159-H08H)在ELISA平板(BeaverBio，Cat#REF40301)上包被过夜。1％BSA封闭后，将一系列浓度的血清与0.1078μg/ml野生型Ipilimumab抗体混合，然后将100μl的混合物加入到人CTLA-4-his蛋白固定的96孔ELISA板孔中。以过氧化物酶偶联的山羊抗人IgG(Fab特异性)抗体(Sigma，Cat#A0293)1∶5000作为二抗，用四甲基联苯胺(TMB，索莱宝，目录号PR1200)和ELISA终止缓冲液对板进行显色。用ELISA平板阅读器(Biotek，ELx800 Absorbance Microplate reader)测定450nm处的吸光度。数据通过GraphPad Prism 5软件进行分析。
ELISA格式见附图16(A)，结果如图16(B)所示。这些结果表明Ipi-L-3TMEAbody比野生型Ipilimumab抗体相比具有更低的诱导产生中和性抗药物抗体(nADA)的风险。
实施例14：通过FACS评价Ipi-L-3 TMEAbody对人CTLA-4基因敲入小鼠MC38肿瘤模型的治疗效果
为了评估Ipi-L-3 TMEAbody治疗后的小鼠肿瘤和外周的Treg细胞耗竭效果，将Ipi-L-3+CP23 TMEAbody和Ipilimumab抗体施用于携带MC38肿瘤的人CTLA-4基因敲入C57小鼠中。给药7天后，收集脾脏和肿瘤，用于Treg亚群(CD4+CD25+FoxP3+)的FACS分析(贝克曼库尔特，CytoFLEX)。然后通过GraphPad Prism 5软件分析数据。
结果如图17(A-B)所示。这些结果表明，Ipi-L-3 TMEAbody降低了肿瘤中而非脾脏中Treg细胞的百分比，提示其在肿瘤局部激活发挥抗肿瘤疗效，而在外周组织中激活较低。

Claims (10)

  1. 一种抗CTLA-4抗体的偶联体，具有如下结构：
    R1-R2-R3-L-R4-S-cys-R5
    其中，
    R5代表其一个或多个氨基酸残基被突变为半胱氨酸的抗CTLA-4抗体；
    cys代表所述半胱氨酸残基；
    S代表所述半胱氨酸残基中的S原子；
    R1为阻断R5与其抗原、配体或受体结合的功能基团；
    R2为可被病理微环境中的一种或多种蛋白酶水解切割的连接臂；
    R3为为蛋白酶的切割提供空间的连接臂；
    L为在病理微环境的酸性条件被激活的化学键；优选地，L为腙键；
    R4是通过所述半胱氨酸残基的硫原子共价连接至R5的基团，其在R1-R2-R3-L-被切割后能恢复、维持或者提高R5与其抗原、配体和受体的结合能力。
  2. 如权利要求1所述的偶联体，其特征在于，R1选自：NRaRb-R-CO-，C1-4烷氧基-(C1-4烷氧基)n-羰基-C1-4亚烷基-羰基-，
    其中：
    各R独立为C1-4烷基；
    各n独立为1至30000范围内的整数；
    pegm是分子量为44至132000的聚乙二醇，其中m表示聚乙二醇的分子量；
    Ra为C1-4烷基-O-[(CH2)q-O]n-(CH2)q-；
    Rb为C1-4烷基-O-[(CH2)q-O]n-(CH2)q-C(O)-；
    q为1-4的整数；
    波浪线指示R1连接到R2的位置；
    优选地，所述NRaRb-R-CO-为：
    所述C1-4烷氧基-(C1-4烷氧基)n-羰基-C1-4亚烷基-羰基-为：
    优选地，R1选自：

    更优选地，R1为所述R1-7、R1-15、R1-16、R1-17、R1-20或R1-21。
  3. 如权利要求1所述的偶联体，其特征在于，R2为三肽，其中，该三肽中与R1连接的氨基酸残基选自Ala、Thr、Val和Ile，中间氨基酸残基选自Ala、Thr、Val和Asn，与R3连接的氨基酸残基选自Asn和Asp；优选地，R2为Ala-Ala-Asn或Ala-Ala-Asp；
    其中，R2通过其氨基酸残基的氨基以酰胺键、酯键、氨基甲酸酯键、脲键或腙键的连接方式与R1连接，并通过其氨基酸残基的羧基以酰胺键、酯键、氨基甲酸酯键、脲键或腙键的连接方式与R3连接。
  4. 如权利要求1所述的偶联体，其特征在于，R3为-HN-苯基-R’-，其中，R’为键；优选地，所述NH基团和R’位于苯基的对位。
  5. 如权利要求1所述的偶联体，其特征在于，所述R1-R2-R3-L-R4的结构表示为：
    其中，R’为H或C1-4烷基。
  6. 如权利要求1所述的偶联体，其特征在于，R4如下式所示：
    其中，Rc选自C1-12亚烷基，C1-12亚烷基-O-C1-12亚烷基，C1-12亚烷基-C3-8环烷基，(C1-4亚烷基-O)p-C1-12亚烷基，C1-12亚烷基羰基氨基-(C1-4亚烷基-O)p-C1-12亚烷基，-苯基-C1-12亚烷基，C3-8环烷基，C1-12亚烷基-C3-8环烷基-C1-12亚烷基，C1-12亚烷基-NHCO-O-C1-12亚烷基，C1-12亚烷基-COO-和C1-12亚烷基-苯基-C1-12亚烷基；p为1-10的整数，优选为1、2、3或4；
    R4-c为键或-CO-；
    其中，L基团通过R4基团的R4-c与R4基团相连；R4基团通过马来酰亚胺与R5的半胱氨酸的S原子连接；
    优选地，Rc为C1-12亚烷基，R4-c为-CO-。
  7. 如权利要求1所述的偶联体，其特征在于，所述抗CTLA-4抗体为Ipilimumab；
    优选地，所述Ipilimumab重链上的突变位置选自：Gln3，Arg19，Leu20，Ser25，Gly26，Phe27，Thr28，Phe29，Ser30，Ser31，Tyr32，Thr33，Met34，His35，Gly44，Phe50，Ile51，Ser52，Tyr53，Asp54，Gly55，Asn56，Asn57，Lys58，Tyr59，Tyr60，Ala61，Asp62，Ser63，Lys65，Gly66，Thr69，Ser71，Arg72，Asp73，Asn74，Ser75，Lys76，Asn77，Thr99，Gly100，Trp101，Leu102，Gly103，Pro104，Leu105，Asp106和Tyr107；优选选自：Ser30，Ser31，Tyr32，Thr33，Ile51，Asp54，Gly55，Asn56，Lys58，Tyr59，Tyr60，Ala61，Asp62，Ser63，Lys65，Gly66，Gly100，Trp101，Leu102，Gly103，Pro104、Leu105，Asp106和Tyr107；
    优选地，所述Ipilimumab轻链中的突变位置选自：Gln6，Arg24，Ala25，Ser26，Gln27，Ser28，Val29，Gly30，Ser31，Ser32，Tyr33，Leu34，Ala35，Tyr37，Ile49，Tyr50，Gly51，Ala52，Phe53，Ser54，Arg55，Ala56，Thr57，Gly58，Ile59，Pro60，Asp61，Arg62，Ser68，Gly69，Thr70，Gln90，Gln91， Tyr92，Gly93，Ser94，Ser95，Pro96、Trp97、Thr98、Phe99和Gly100；优选选自：Ala25，Ser26，Ser28，Gly30，Ser31，Ser32，Leu34，Ala35，Gly51，Ala52，Ser54，Ala56，Thr57，Gly58，Ile59，Gly93，Ser94，Ser95，Thr98和Gly100；
    上述位置编号参照SEQ ID NO：1和2；
    优选地，所述Ipilimumab轻链Tyr50被突变成C。
  8. 如权利要求1所述的偶联体，其特征在于，所述R1-R2-R3-L-R4选自：

  9. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-8中任一项所述的偶联体和药学上可接受的载体。
  10. 权利要求1-8中任一项所述的偶联体在制备治疗或预防肿瘤或炎症的药物中的用途；优选地，所述肿瘤或炎症为能用抗CTLA-4抗体治疗或预防的肿瘤或炎症；更优选地，所述肿瘤选自：膀胱癌，脑癌，乳腺癌，宫颈癌，结直肠癌，食道癌，肾癌，肝癌，肺癌，鼻咽癌，胰腺癌，前列腺癌，皮肤癌，胃癌，子宫癌，卵巢癌，睾丸癌和血癌。
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