WO2020063880A1 - 一种多肽组合物 - Google Patents

一种多肽组合物 Download PDF

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Publication number
WO2020063880A1
WO2020063880A1 PCT/CN2019/108588 CN2019108588W WO2020063880A1 WO 2020063880 A1 WO2020063880 A1 WO 2020063880A1 CN 2019108588 W CN2019108588 W CN 2019108588W WO 2020063880 A1 WO2020063880 A1 WO 2020063880A1
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Prior art keywords
fragment
toxin
polypeptide
intein
composition
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PCT/CN2019/108588
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English (en)
French (fr)
Inventor
朱建伟
王静
韩雷
陈俊升
谢跃庆
江华
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Shanghai Jiao Tong University
Jecho Laboratories Inc
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Shanghai Jiao Tong University
Jecho Laboratories Inc
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Priority to EP19867591.0A priority Critical patent/EP3858868B1/en
Priority to US17/280,636 priority patent/US12305202B2/en
Priority to CN201980064610.9A priority patent/CN112888710B/zh
Publication of WO2020063880A1 publication Critical patent/WO2020063880A1/zh
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02036—NAD(+)--diphthamide ADP-ribosyltransferase (2.4.2.36)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present application relates to the field of immunotherapy, in particular to a polypeptide composition, and also to a kit containing the polypeptide composition and its use in treating tumors.
  • Immunotoxin refers to a therapeutic chimeric protein composed of a cell targeting portion and a toxin portion. It has the function of specifically targeting and killing diseased cells, and can be prepared by chemical coupling or genetic recombination.
  • the targeting portion of immunotoxins can deliver toxin drugs to tumor cells, improve specific killing effects, and reduce the effect on normal cells. toxicity.
  • the cell-targeting portion can be an antibody.
  • the cell-targeting portion can specifically target tumor cells and is responsible for specifically binding to the cells and being endocytosed by the cells.
  • the toxin part enters the cytoplasm through the cell-targeting part, and interferes with the cell process in the cytoplasm to directly induce cell death, or modify the cell membrane, induce apoptotic proteins and indirectly cause cell death, play a function of inhibiting tumor cell growth and lead to cell death.
  • the application provides a polypeptide composition comprising a first polypeptide and a second polypeptide.
  • the application also provides a method for preparing the composition, a kit comprising the first polypeptide and a second polypeptide, and uses and methods of the composition and the kit in treating tumors.
  • the present application provides a composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first toxin fragment and a first intein fragment, and the second polypeptide Comprising a second toxin fragment and a second intein fragment; wherein the first polypeptide is different from the second polypeptide; neither the first toxin fragment nor the second toxin fragment is biologically toxic; and the The first polypeptide and the second polypeptide can form the first toxin fragment and the second toxin fragment through the interaction of the first intein fragment and the second intein fragment. Biotoxic toxin.
  • the first toxin fragment is different from the second toxin fragment.
  • the first toxin fragment and the second toxin fragment are derived from the same toxin.
  • the first toxin fragment comprises a first fragment of the toxic active region of the toxin
  • the second toxin fragment comprises a second fragment of the toxic active region of the toxin
  • the toxic active region is A fragment and a second fragment of the toxic active region constitute the complete toxic active region of the toxin.
  • the second toxin fragment does not include a translocation region of the toxin or a fragment thereof.
  • the first toxin fragment further comprises a translocation region of the toxin or a fragment thereof.
  • the first toxin fragment does not include the entire toxic active region of the toxin.
  • the second toxin fragment does not include the entire toxic active region of the toxin.
  • the toxin is selected from the group consisting of a bacterial toxin, a human toxin, and a phytotoxin. In certain embodiments, the toxin is selected from the group consisting of Pseudomonas aeruginosa exotoxin and diphtheria toxin. In certain embodiments, the toxin is selected from the group consisting of ricin, saponin, and phytoxin. In some embodiments, the toxin is a truncated PE38 of Pseudomonas aeruginosa exotoxin, which comprises the amino acid sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 16.
  • the first intein fragment is different from the second intein fragment. In certain embodiments, the first intein fragment and the second intein fragment are derived from the same intein. In certain embodiments, the intein is a cleaved intein. In certain embodiments, the fragmented intein is selected from the group consisting of SsPDnaB, SspDnaE, and NpuDnaE.
  • the C-terminus of the first toxin fragment is directly or indirectly connected to the N-terminus of the first intein fragment.
  • the amino acid residues at the C-terminus of the first toxin fragment are derived from a random coiled region of the toxin.
  • the N-terminus of the second toxin fragment is directly or indirectly connected to the C-terminus of the second intein fragment.
  • the amino acid residues at the N-terminus of the second toxin fragment are derived from the random coiled regions of the toxin.
  • the amino acid residues at positions 1-3 of the N-terminus of the second toxin fragment are sequentially CFN.
  • the first intein fragment comprises an amino acid sequence as shown in SEQ ID NO: 2.
  • the second intein fragment comprises an amino acid sequence as shown in SEQ ID NO: 3.
  • the first toxin fragment comprises an amino acid sequence as shown in SEQ ID NO: 4.
  • the second toxin fragment comprises an amino acid sequence as shown in SEQ ID NO: 5.
  • the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 6.
  • the second polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 7 and SEQ ID NO: 15.
  • the N-terminus of the first toxin fragment is directly or indirectly connected to the C-terminus of the first intein fragment.
  • the amino acid residues at the N-terminus of the first toxin fragment are derived from a random coiled region of the toxin.
  • the C-terminus of the second toxin fragment is directly or indirectly connected to the N-terminus of the second intein fragment.
  • the amino acid residues at the C-terminus of the second toxin fragment are derived from a random coiled region of the toxin.
  • the amino acid residues at positions 1-3 of the N-terminus of the first toxin fragment are sequentially CFN.
  • the first intein fragment comprises an N-terminal protein region of the fragmented intein; and the second intein fragment comprises a C-terminal protein of the fragmented intein region.
  • the first intein fragment comprises a C-terminal protein region of the fragmented intein; and the second intein fragment comprises an N-terminal protein of the fragmented intein region.
  • the N-terminal protein region is an N-terminal protein region of NpuDnaE.
  • the C-terminal protein region is a C-terminal protein region of NpuDnaE.
  • said interaction of said first intein fragment and said second intein fragment comprises a protein reaction of said first and intein fragment Splicing effect.
  • the first polypeptide and / or the second polypeptide further include a targeting moiety that targets a tumor-specific antigen.
  • the first polypeptide comprises a first targeting moiety, and the first targeting moiety is located at the N-terminus of the first toxin fragment.
  • the second polypeptide comprises a second targeting moiety, and the second targeting moiety is located at the N-terminus of the second toxin fragment.
  • the tumor-specific antigen is selected from the group consisting of HER2, PD-L1, EGFR, mesothelin, and Lewis Y.
  • the first targeting moiety and / or the second targeting moiety comprises an antibody or an antigen-binding fragment or variant thereof.
  • the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab) 2, dAb, an isolated complementarity determining region CDR, Fv, and scFv.
  • the variant of the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) one or more amino acids are substituted, deleted or added to the antibody or the antigen-binding fragment thereof A protein or polypeptide; and b) a protein or polypeptide having at least 90% sequence homology with said antibody or said antigen-binding fragment thereof.
  • the first targeting moiety and / or the second targeting moiety is a ScFv.
  • the targeting moiety comprises an amino acid sequence as shown in SEQ ID NO: 8.
  • the present application provides a method for preparing a composition, comprising the following steps: 1) providing the first polypeptide; 2) providing the second polypeptide; 3) providing the first polypeptide A peptide is mixed with the second polypeptide to obtain the composition.
  • the molar ratio of the first polypeptide to the second polypeptide in the composition is 10: 1-1: 10.
  • the preparation method further comprises adding a reducing agent.
  • the reducing agent is selected from the group consisting of DTT and ⁇ mercaptoethanol.
  • the concentration of the reducing agent in the composition is 0.001-10000 nM.
  • the reducing agent in the preparation method, is added simultaneously with or after the mixing. In some embodiments, in the preparation method, it further comprises incubating the composition after adding the reducing agent. In some embodiments, in the preparation method, wherein the incubation temperature is 1 ° C-50 ° C. In some embodiments, in the preparation method, wherein the incubation time is 2-120 minutes.
  • the present application provides a vector comprising a nucleic acid encoding the first polypeptide and / or comprising a nucleic acid encoding the second polypeptide.
  • the application provides a cell that expresses the first polypeptide, and / or that expresses the second polypeptide.
  • the present application provides a kit comprising the first polypeptide according to 1); and the second polypeptide according to 2). In certain embodiments, in the kit, wherein the first polypeptide and the second polypeptide are not mixed with each other in the kit. In certain embodiments, in the kit, wherein the first polypeptide and the second polypeptide are located in different containers.
  • kits wherein the kit further comprises a reducing agent.
  • the reducing agent is selected from the group consisting of DTT and ⁇ -mercaptoethanol.
  • the kit wherein the reducing agent is contained in a separate container.
  • the kit in the kit, it comprises the composition.
  • the present application provides use of the composition, the kit, the carrier, or the cell in the manufacture of a medicament for treating a disease, which includes a tumor.
  • the tumor is selected from the group consisting of breast cancer, melanoma, ovarian cancer, colon cancer, mesothelioma, adenoma, pancreatic cancer, and bladder cancer.
  • the present application provides a composition, a kit, a carrier, or a cell for treating a tumor.
  • the present application provides a method for treating a tumor, which comprises administering the composition, the kit, the vector, or the cell.
  • Figure 1 shows the structure of the composition of the present application
  • Figure 2 shows the components of the scFvPM1, scFvPnIn and IcPc expression vectors of the present application
  • Figure 3 shows the effect of the mutant of the immunotoxin of the present application on the activity of SKOV3 cells
  • Figure 4 shows the situation of cleavage intein-mediated trans-splicing at different DTT concentrations
  • Figure 5 shows the situation of cleavage intein-mediated trans-splicing at different temperatures
  • Figure 6 shows cleavage intein-mediated trans-splicing at different reaction times
  • Figure 7 shows the affinity of the intact immunotoxin and the divided immunotoxin for SKOV3 cell surface antigens
  • Figure 8 shows the uptake of intact immunotoxins and divided immunotoxins by SKOV3 cells
  • Figure 9 shows the effects of intact immunotoxins and post-segmented immunotoxins on the viability of SKOV3 cells
  • Figure 10 shows the effects of intact immunotoxins and segmented immunotoxins on MCF7 cell viability
  • Figure 11 shows the effects of intact immunotoxins and post-segmented immunotoxins on the viability of CHO cells
  • Figure 12 shows the intein-mediated trans-splicing of the immunotoxin in the cell culture medium after segmentation
  • Figure 13 shows the apoptosis of SKOV3 cells induced by intact immunotoxin and immunotoxin after segmentation
  • Figure 14 shows the composition of the first polypeptide and the second polypeptide in the composition
  • Figure 15 shows the constituent structures of the first polypeptide and the second polypeptide in the composition.
  • the term "recombinant immunotoxin (RIT)” is generally a therapeutic chimeric protein composed of a cell targeting portion and a toxin portion, which has the function of specifically targeting and killing diseased cells.
  • the cell targeting portion binds to a specific antigen on the surface of the target cell, and the cell targeting portion is responsible for specifically binding the target cell and being endocytosed by the target cell.
  • the toxin part can enter the cytoplasm through the cell-targeting part, which can directly induce cell death by interfering with the cell process in the cytoplasm, or the toxin part can modify the cell membrane and induce apoptotic proteins to cause cell death indirectly, exerting inhibition of the growth of diseased cells and leading to cell death Functions.
  • toxin generally refers to any substance that is harmful to the growth and proliferation of cells and can play a role in reducing, inhibiting or destroying cells or malignant tumors. These substances are usually some large molecules that interfere with other macromolecules in the organism Acting protein.
  • Toxins can include bacterial toxins, plant toxins, and human toxins according to their source.
  • bacterial toxins can include Pseudomonas aeruginosa exotoxin (PE) and diphtheria toxin (DT), and phytotoxins can include ricin and acacia toxin.
  • toxins can function by inhibiting protein synthesis through enzymatic hydrolysis.
  • a toxin may include the following functional regions: a cell-binding region, a translocation region, and a toxically active region.
  • cell-binding region generally refers to a functional region of a toxin that can enrich the toxin on the surface of a target cell.
  • translocation region generally refers to a functional region of a toxin that allows the toxin to cross the membrane to the cytoplasmic region.
  • the term "toxic active region” generally refers to a functional region of a toxin capable of inactivating some important cellular processes and killing the cells.
  • polypeptide generally refers to a compound formed by ⁇ -amino acids linked together by peptide bonds, which is also an intermediate product of proteolysis.
  • a compound formed by the dehydration condensation of two amino acid molecules is called a dipeptide.
  • tripeptides, tetrapeptides, and pentapeptides there are tripeptides, tetrapeptides, and pentapeptides.
  • a compound usually formed by dehydration condensation of three or more amino acid molecules can be referred to as a polypeptide.
  • biotoxicity generally refers to cytotoxicity, which can be a simple cell killing event caused by a cell or a chemical substance, and does not depend on a cell death mechanism of apoptosis or necrosis.
  • the term "antibody” generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein, which consists of two identical light chains and two identical heavy chains.
  • each L chain is connected to the H chain through a covalent disulfide bond, and two H chains are connected to each other through one or more disulfide bonds. The number of disulfide bonds depends on the same type of H chain type.
  • Each H and L chain also has regularly spaced intrachain disulfide bonds.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three (for each ⁇ and ⁇ chain) or four (for ⁇ and ⁇ isotypes) constant domains (CH).
  • VH variable domain
  • the term "antigen-binding fragment” generally refers to a part of an intact antibody, for example, the antigen-binding fragment may be an antigen-binding region and / or a variable region of the intact antibody.
  • Antigen-binding fragments can be obtained by chemical methods and / or genetic engineering methods.
  • the chemical method is to generate an antigen-binding fragment by breaking the disulfide bond in the hinge region, or using a protease, including pepsin and papain, to digest the antibody to generate the antigen-binding fragment.
  • the genetic engineering method refers to the use of recombinant DNA and protein engineering technology to process and reassemble the genes encoding antibodies according to different needs, and then transfect the appropriate antibody cells expressed by appropriate recipient cells.
  • intein generally refers to an insertion sequence located in a host protein.
  • the intein gene is not an independent gene and needs to be inserted into an exidein gene in order to replicate and transcribe. It can be excised from the precursor protein and the exon peptides on both sides can be linked to become a mature protein.
  • the nucleotide sequence corresponding to the intein is chimeric in the nucleic acid sequence corresponding to the host protein, exists in the same open reading frame as the host protein gene, and is simultaneously transcribed and translated with the host protein gene. When the translation forms a protein precursor The intein is then excised from the host protein to form a mature, active protein.
  • intein According to the existing form of intein, it can be divided into a whole intein and a broken intein.
  • the two splicing regions of the whole intein coexist on the same polypeptide fragment, and the two splicing regions of the broken intein are split.
  • the two splicing regions exist on different polypeptide fragments, so they can also be called isolated inteins.
  • protein trans-splicing generally refers to a protein splicing reaction mediated by a fragmented intein.
  • the N-terminal fragment (In) and the C-terminal fragment (Ic) of the fragmented intein recognize each other and bind with non-covalent bonds. After binding, the structure is correctly folded and the active center is reconstructed
  • the cleavage-type intein completes the protein splicing reaction according to the typical protein splicing pathway, and connects the protein exteins on both sides with natural peptide bonds.
  • the term "antigen” generally refers to a substance that can induce an immune response in the body, that is, it can be specifically recognized and bound by the antigen receptor (TCR / BCR) on the surface of T / B lymphocytes to activate T / B cells Substances that make them proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro. Therefore, the antigenic substance has two important characteristics: immunogenicity and immunoreactivity.
  • Immunogenicity refers to the ability of an antigen to induce a specific immune response in the body to produce antibodies and / or sensitize lymphocytes; immunoreactivity refers to the ability to react with the corresponding immune effector substance (antibody or sensitized lymphocytes) in vivo and in vivo The ability to specifically bind the response.
  • tumor-specific antigen generally refers to a neoantigen on the surface of certain tumor cells, which is hardly present on normal cells, and is also called a unique tumor antigen.
  • tumor generally refers to a new organism formed by a cell of a local tissue that has lost normal regulation of its growth at the gene level under the action of various carcinogenic factors, resulting in clonal abnormal proliferation. (neogrowth), because this new organism is mostly a place-like block-shaped process, also known as neoplasm.
  • the term "monoclonal antibody” generally refers to a group of substantially homogeneous antibodies, that is, the individual antibodies contained in the group are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is not interpreted as requiring a single determinant modifier "monoclonal" on the antigen by any special method Produce antibodies.
  • the monoclonal antibodies can be prepared by hybridoma technology or produced using recombinant DNA methods in bacteria, eukaryotes or plant cells. They can also be obtained from phage antibody libraries using, for example, Clackson etal., Nature, 352 : 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991).
  • single chain antibody generally refers to a molecule composed of an antibody heavy chain variable region and a light chain variable region linked by a short peptide linker.
  • chimeric antibody generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, and The rest of the strand is homologous to the corresponding sequence in another species.
  • the variable regions of the light and heavy chains are derived from the variable region of an antibody of one animal species (e.g., mouse, rat, etc.), while the constant portion is homologous to the antibody sequence of another species (e.g., human) .
  • non-human B cells or hybridoma cells can be used to generate variable regions, while the constant regions combined with them are derived from humans.
  • variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined with it.
  • the constant region of the chimeric antibody can be derived from humans, the probability of the chimeric antibody to elicit an immune response at the time of injection will be lower than the use of antibodies with non-human constant regions.
  • humanized antibody generally refers to the use of genetic engineering techniques to reduce the immunogenicity of antibodies, immunoglobulin-binding proteins, and polypeptides derived from non-human species (such as mice or rats) to humans. A modified antibody that still retains the antigen-binding properties of the original antibody.
  • CDR grafting (Jones et al., Nature 321: 522 (1986)) and its variants can be used; including “reshaping” (Verhoeyen, et al., 1988 Science 239: 1534-1536; Riechmann , et al., 1988 Nature 332: 323-337; Tempest, et al., Bio / Technol 1991 9: 266-271), "hypermerization” (Queen, et al., 1989 Proc Natl Acad) Sci USA 86: 10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88: 2869-2873; Co, et al., 1992 J Immunol 148: 1149-1154) and "veneering", (Mark, et al., "Derivation of thermally active humanized and veneered anti-CD18antibodies.” In: MetcalfW, Dalton BJ, eds.Cellular d
  • the term "fully human antibody” generally means that all of the antibody (including the constant region parts of the antibody, the CH and CL regions) are encoded by genes of human origin. All-human antibodies can greatly reduce the immune side effects caused by heterologous antibodies.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells.
  • the vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for expression of DNA and RNA transcription and / or translation.
  • the vector also includes a vector having a plurality of the aforementioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing suitable cells containing the vector, the vector can produce a desired expression product.
  • the term "cell” generally refers to an individual cell, cell line, or cell culture that can or already contains a plasmid or vector including a nucleic acid molecule described herein, or is capable of expressing a polypeptide or polypeptide fragment described herein. .
  • the cell may include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny cells may not be exactly the same morphologically or genomically as the original parental cells, but they only need to be able to express the polypeptides or polypeptide fragments described herein.
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cell can be a prokaryotic cell (such as E. coli) or a eukaryotic cell.
  • identity generally refers to the percentage of the number of identical amino acid residues of the candidate sequence compared to a particular peptide or polypeptide sequence to the number of all amino acid residues.
  • the term “comprising” generally means the meaning of including, summing up, containing, or including. In some cases, it also means “as”, “consisting of”.
  • the present application provides a composition.
  • the composition may be a polypeptide composition.
  • the composition may include a first polypeptide and a second polypeptide, wherein the first polypeptide may include a first toxin fragment and a first polypeptide.
  • the second polypeptide may include a second toxin fragment and a second intein fragment; wherein the first polypeptide may be different from the second polypeptide; both the first toxin fragment and the second toxin fragment may have no biological toxicity
  • the first polypeptide and the second polypeptide can form a biotoxic toxin by the interaction of the first intein fragment and the second intein fragment with the first toxin fragment and the second toxin fragment.
  • first toxin fragment generally refers to a non-biotoxic toxin fragment, which may not contain the entire toxin. Under certain conditions, the first toxin fragment can form a biotoxic toxin with other toxin fragments.
  • second toxin fragment generally refers to a toxin fragment that is not biologically toxic and may not contain the entire toxin. Under certain conditions, the second toxin fragment can form a biotoxic toxin with other toxin fragments. For example, under certain conditions, the second toxin fragment may form a biotoxic toxin with the first toxin fragment.
  • first intein fragment generally refers to a partial fragment of an intein, which may not include a complete intein fragment. Under certain conditions, the first intein fragment can interact with other intein fragments, so that the first toxin fragment and the second toxin fragment form a biotoxic toxin.
  • the term "second intein fragment” generally refers to a partial fragment of an intein, which may not include a complete intein fragment. Under certain conditions, the second intein fragment can interact with other intein fragments, so that the first toxin fragment and the second toxin fragment form a biotoxic toxin. For example, under certain conditions, the second intein fragment can interact with the first intein fragment, so that the first toxin fragment and the second toxin fragment form a biotoxic toxin.
  • first polypeptide generally refers to a polypeptide comprising a first toxin fragment and a first intein fragment without being biotoxic.
  • first intein fragment in the first polypeptide can interact with the intein fragment in other polypeptides, so that the first toxin fragment in the first polypeptide and the toxin fragment in other polypeptides form Biotoxic toxins.
  • second polypeptide generally refers to a polypeptide comprising a second toxin fragment and a second intein fragment that are not biotoxic.
  • the second intein fragment in the second polypeptide can interact with the intein fragment in other polypeptides, so that the second toxin fragment in the second polypeptide and the toxin fragment in other polypeptides form Biotoxic toxins.
  • the second intein fragment in the second polypeptide may interact with the first intein fragment in the first polypeptide, so that the second toxin fragment and the first toxin fragment in the second polypeptide The first toxin fragment in a polypeptide forms a biotoxic toxin.
  • the composition may include a first polypeptide and a second polypeptide, the first polypeptide may include the first toxin fragment and the first intein fragment, and the second poly
  • the peptide may include the second toxin fragment and the second intein fragment, and the amino acid sequences of the first polypeptide and the second polypeptide may be different, for example, the amino acid sequences of the first polypeptide and the second polypeptide Less than 99%, less than 98%, less than 95%, 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or Even smaller, both the first toxin fragment and the second toxin fragment may be non-biotoxic, and the first polypeptide and the second polypeptide may also be non-biotoxic. Under certain conditions, the first intein fragment and the second intein fragment can interact, so that the first toxin fragment and the second toxin fragment can form a biotoxic toxin.
  • the first toxin fragment may be different from the second toxin fragment.
  • the amino acid sequences of the first toxin fragment and the second toxin fragment may be different.
  • the amino acid sequences of the first toxin fragment and the second toxin fragment are less than 99%, less than 98%, and less than 95% , 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less.
  • the first toxin fragment and the second toxin fragment may be derived from the same toxin.
  • the first toxin fragment and the second toxin fragment may be intercepted from different sequence positions of the same toxin.
  • the first toxin fragment may include a first fragment of the toxic active region of the toxin
  • the second toxin fragment may include a second fragment of the toxic active region of the toxin
  • the first fragment of the toxic active region and the second fragment of the toxic active region constitute The complete toxic active region of the toxin
  • first fragment of toxic active region generally refers to a partial fragment of the toxic active region of the toxin.
  • the first fragment of the toxic active region does not contain the complete toxin active region of the toxin.
  • the toxic active region A fragment can form a complete toxic active region with other toxic active region fragments.
  • the term "second fragment of the toxic active region” generally refers to a partial fragment of the toxic active region of the toxin.
  • the second fragment of the toxic active region does not include the complete toxin active region of the toxin.
  • the toxic active region The two fragments can form a complete toxic active region with other fragments of the toxic active region.
  • the second fragment of the toxic active region may form a complete toxic active region with the first fragment of the toxic active region.
  • the toxin may comprise a toxic active region.
  • the first toxin fragment may include a first fragment of a toxic active region of a toxin
  • the second toxin fragment may include a second fragment of a toxic active region of the same toxin
  • neither the first toxin fragment nor the second toxin fragment includes the described The complete toxic active area of the toxin.
  • the first fragment of the toxic active region and the second fragment of the toxic active region may constitute a complete toxic active region of the toxin.
  • the toxin may also contain a translocation region.
  • the second toxin fragment may not include a translocation region or a fragment thereof, and the first toxin fragment may further include a translocation region or a fragment thereof.
  • the second toxin fragment may not contain the complete translocation region of the toxin, or the second toxin fragment may not contain the translocation region fragment of the toxin.
  • the first toxin fragment may further include a complete translocation region of the toxin, or the first toxin fragment may further include a translocation region fragment of the toxin.
  • the first toxin fragment may comprise a first fragment of a toxic active region of the toxin and a complete translocation region of the toxin
  • the second toxin fragment may comprise a second fragment of a toxic active region of the same toxin
  • the first toxin fragment may comprise a first fragment of the toxic active region of the toxin and a fragment of the translocation region of the toxin
  • the second toxin fragment may comprise a second fragment of the toxic active region of the same toxin.
  • the toxin may be selected from the group consisting of a bacterial toxin, a human toxin, and a phytotoxin.
  • the toxin may be a bacterial toxin, a human toxin, a phytotoxin, or a combination thereof.
  • the toxin may be selected from the group consisting of Pseudomonas aeruginosa exotoxin and diphtheria toxin.
  • the toxin may be Pseudomonas aeruginosa exotoxin, diphtheria toxin, or a combination thereof.
  • the toxin may be selected from the group consisting of ricin, saponin, and toxin.
  • the toxin may be ricin, saporin, phytoxin, or a combination thereof.
  • the toxin may be a truncated PE38 of Pseudomonas aeruginosa exotoxin (PE), and the truncated PE38 may include the amino acid shown in any one of SEQ ID NO: 1 and SEQ ID NO: 16 sequence.
  • PE Pseudomonas aeruginosa exotoxin
  • the term "truncated PE38” generally refers to a fragment after the cell binding region has been truncated by PE.
  • the complete PE contains three functional regions, namely the cell-binding region (Ia region, 1-252aa), the translocation region (region II, 253-364aa), and the toxic active region (region III, 400-613aa).
  • the translocation region can be The toxin transmembrane reaches the functional region of the cytoplasmic region, and the toxic active region has ADP ribosylation function, which is a key functional region that inactivates the elongation factor eEF2 and kills cells.
  • PE truncated PE38 truncates the cell-binding region (Ia region, 1-252aa), so the truncated PE38 includes only the translocation region (region II, 253-364aa) and the toxic active region (region III, 400-613aa) ).
  • the first intein fragment may be different from the second intein fragment, and the first intein fragment and the second intein fragment may be derived from the same intein
  • the intein may be a cleaved intein.
  • the amino acid sequences of the first intein fragment and the second intein fragment may be different.
  • the amino acid sequences of the first and second intein fragments are less than 99% and less than 98% in identity. %, Less than 95%, 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less.
  • the first intein fragment and the second intein fragment may be partial fragments of the same fragmented intein, and the first and second intein fragments may be derived from the same fragmented intein. Different sequence positions.
  • the fragmented intein can be selected from the group consisting of SsP DnaB, Ssp DnaE, and Npu DnaE.
  • the fragmented intein can be SsP DnaB, Ssp DnaE, Npu DnaE, or a combination thereof.
  • the C-terminus of the first toxin fragment may be directly or indirectly connected to the N-terminus of the first intein fragment, and the amino acid residue at the C-terminus of the first toxin fragment It may be derived from the random coiled region of the toxin.
  • the N-terminus of the second toxin fragment may be directly or indirectly connected to the C-terminus of the second intein fragment. Amino acid residues are derived from the random coiled regions of the toxin.
  • amino acid residue at the C-terminus generally refers to an amino acid residue at the end of a polypeptide that can carry a free ⁇ -carboxyl group.
  • N-terminal amino acid residue generally refers to an amino acid residue of an ⁇ -amino group that can be carried at the end of a polypeptide chain.
  • random coiled region generally refers to a region of a peptide chain that has a relatively irregular arrangement of loops or coiled structures. Random coiling is the removal of ⁇ -helix, ⁇ -fold, ⁇ - Common secondary structure of proteins outside the corner.
  • the toxin is segmented in a random curl region to obtain a first toxin fragment and a second toxin fragment, and the C-terminus of the first toxin fragment and the N of the second toxin fragment are corresponding at the segmentation site of the random curl region. end.
  • the C-terminus of the first toxin fragment and the N-terminus of the first intein fragment may be directly connected, or may be indirectly connected through a linker or other peptide chain; in the second polypeptide, The N-terminus of the second toxin fragment is directly connected to the C-terminus of the second intein fragment, or may be indirectly connected through other peptide chains.
  • the amino acid residues at positions 1-3 of the N-terminus of the second toxin fragment may be CFN in sequence.
  • the first amino acid residue at the N-terminus of the second toxin fragment may be cysteine (C)
  • the second amino acid residue at the N-terminus of the second toxin fragment may be phenylalanine (F)
  • the amino acid residue at position 3 at the N-terminus of the second toxin fragment may be aspartic acid (N).
  • the first intein fragment may include an N-terminal protein region of the fragmented intein; and the second intein fragment includes a C-terminal protein region of the fragmented intein .
  • N-terminal protein region generally refers to a region in a polypeptide chain near the N-terminus.
  • the N-terminal protein region of the fragmented intein may be the N-terminal protein region of NpuDnaE.
  • C-terminal protein region generally refers to a region in a polypeptide chain near the C-terminus.
  • the C-terminal protein region of the fragmented intein may be the C-terminal protein region of NpuDnaE.
  • the composition may include a first polypeptide and a second polypeptide
  • the first polypeptide may include a first toxin fragment and a first intein fragment
  • the second polypeptide may include a first polypeptide Ditoxin fragment and second intein fragment.
  • the first toxin fragment may comprise a first fragment and a translocation region of the toxic active region of the toxin
  • the second toxin fragment may comprise a second fragment of the toxic active region of the toxin.
  • the first polypeptide may include a translocation region of the toxin, a first fragment of the toxin active region, and a first intein fragment, a translocation region of the toxin, and a first fragment of the toxin active region. It can be directly or indirectly linked to the first intein fragment.
  • the second polypeptide may sequentially contain a second intein fragment and a second fragment of the toxic active region of the toxin, and the second intein fragment and the second fragment of the toxic active region of the toxin may be between or Indirect connection.
  • the three amino acid residues at the N-terminus of the second toxin fragment can be site-directed so that the amino acid residues at positions 1-3 of the N-terminus are sequentially CFN.
  • the composition may include a first polypeptide and a second polypeptide
  • the first polypeptide (as shown in FIG. 1A) includes a first toxin fragment (referred to as Pn) and a first intein.
  • Fragment (abbreviated as In)
  • the second polypeptide (as shown in FIG. 1B) includes a second toxin fragment (abbreviated as Pc) and a second intein fragment (abbreviated as Ic).
  • the N-terminus of In is fused to the C-terminus of Pn.
  • the N-terminus of Pc is fused to the C-terminus of Ic.
  • the toxin may be a truncated PE38 of Pseudomonas aeruginosa exotoxin (PE), the fragmented intein is NpuDnaE, and NpuDnaE may be truncated into an N-terminal protein region and a C-terminal protein region.
  • the N-terminal protein region is the N-terminal protein region of NpuDnaE
  • the C-terminal protein region is the C-terminal protein region of NpuDnaE
  • the N-terminal protein region constitutes the first intein fragment
  • the C-terminal protein region constitutes the second intein fragment.
  • the toxic active region (region III, 400-613aa) of PE38 includes a random coil region, an alpha helix region, and a lamellar region.
  • the first toxin fragment and the first Ditoxin fragments can be ligated and restored to their original conformation.
  • the truncated PE38 can be segmented in the random coil region, rather than in the alpha helix region or sheet region.
  • three amino acid residues at the N-terminus of the second toxin fragment may be site-directed to make the The amino acid residues are in turn CFN.
  • the toxin makes the composition have a killing effect on tumor cells.
  • the composition includes a first polypeptide and a second polypeptide
  • the first intein fragment may include an amino acid sequence as shown in SEQ ID NO: 2
  • the second intein fragment may include According to the amino acid sequence shown in SEQ ID NO: 3
  • the first toxin fragment may include the amino acid sequence shown in SEQ ID NO: 4
  • the second toxin fragment may include the amino acid shown in SEQ ID NO: 5
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 6
  • the second polypeptide may include the amino acid shown in any one of SEQ ID NO: 7 and SEQ ID NO: 15 sequence.
  • the reducing property of the tumor microenvironment can promote the protein trans-splicing reaction of the fragmented intein, so that when the composition is on the surface of a tumor cell, the first intein fragment and the second intein fragment Protein trans-splicing occurs between the intein fragments, so that the first toxin fragment and the second toxin fragment can form a biotoxic toxin. After the toxin is taken up by the tumor cells, it can play a role in killing the diseased cells.
  • the N-terminus of the first toxin fragment may be directly or indirectly connected to the C-terminus of the first intein fragment, and the amino acid residue at the N-terminus of the first toxin fragment It may be derived from the random coiled region of the toxin.
  • the C-terminus of the second toxin fragment may be directly or indirectly connected to the N-terminus of the second intein fragment.
  • Amino acid residues are derived from the random coiled regions of the toxin.
  • the toxin is segmented in a random coil region to obtain a first toxin fragment and a second toxin fragment.
  • the segmentation site of the random coil corresponds to the N-terminus of the first toxin fragment and the C of the second toxin fragment.
  • the N-terminus of the obtained first toxin fragment is directly or indirectly connected to the C-terminus of the first intein fragment
  • the C-terminus of the second toxin fragment is directly or indirectly connected to the N-terminus of the second intein fragment.
  • the amino acid residues at positions 1-3 of the N-terminus of the first toxin fragment are sequentially CFN.
  • the amino acid residue at the N-terminus of the first toxin fragment may be cysteine (C)
  • the amino acid residue at the N-terminus of the first toxin fragment may be phenylalanine (F)
  • the amino acid residue at the N-terminus of the first toxin fragment may be aspartic acid (N).
  • the first intein fragment may include a C-terminal protein region of the fragmented intein; and the second intein fragment may include an N-terminal protein of the fragmented intein region.
  • the N-terminus of the first toxin fragment in the first polypeptide, may be directly or indirectly linked to the C-terminus of the first intein fragment, and in the second polypeptide, the C of the second toxin fragment The terminus may be directly or indirectly linked to the N-terminus of the second intein fragment.
  • the composition may include a first polypeptide and a second polypeptide
  • the first polypeptide may include a first toxin fragment and a first intein fragment
  • the second polypeptide may include a first polypeptide A ditoxin fragment and a second intein fragment
  • the first toxin fragment may comprise a first fragment and a translocation region of the toxic active region of the toxin
  • the second toxin fragment may comprise a second fragment of the toxic active region of the toxin.
  • the first polypeptide may include a first intein fragment, a first fragment of the toxin active region, and a translocation region of the toxin, and a first intein fragment and the toxin active region first.
  • the fragment and the translocation region of the toxin can be directly or indirectly linked.
  • the second polypeptide may sequentially contain a second fragment of the toxin's toxic active region and a second intein fragment, and the second fragment of the toxin's toxic active region and the second intein fragment may be between or Indirect connection.
  • the three amino acid residues at the N-terminus of the first toxin fragment can be subjected to site-directed mutation, so that the amino acid residues at positions 1-3 of the N-terminus are sequentially CFN.
  • the interaction of the first intein fragment and the second intein fragment may include protein trans-splicing of the first and second intein fragments .
  • the protein trans-splicing reaction may be a protein splicing reaction mediated by a fragmented intein.
  • the first polypeptide and / or the second polypeptide may further include a targeting portion, which may target a tumor-specific antigen.
  • the term "targeting moiety” generally refers to a moiety that specifically binds and / or recognizes a tumor antigen.
  • the first polypeptide and the second polypeptide may each include a targeting moiety, which may specifically bind and / or recognize a tumor-specific antigen.
  • the first polypeptide can include a targeting moiety that can specifically bind and / or recognize a tumor-specific antigen.
  • the second polypeptide can include a targeting moiety that can specifically bind and / or recognize a tumor-specific antigen.
  • the targeting portion may include an antibody or an antigen-binding fragment or variant thereof.
  • the antibody may be selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
  • the tumor-specific antigen may be selected from the group: HER2, PD-L1, EGFR, mesothelin, and Lewis Y.
  • the targeting moiety may be a monoclonal antibody that specifically binds and / or recognizes HER2, PD-L1, EGFR, mesothelin, and / or Lewis Y.
  • the targeting moiety may be a single chain antibody that specifically binds and / or recognizes HER2, PD-L1, EGFR, mesothelin, and / or Lewis Y.
  • the targeting moiety may be a chimeric antibody that specifically binds and / or recognizes HER2, PD-L1, EGFR, mesothelin, and / or Lewis Y.
  • the targeting moiety may be a humanized antibody that specifically binds and / or recognizes HER2, PD-L1, EGFR, mesothelin, and / or Lewis Y.
  • the targeting moiety may be a fully human antibody that specifically binds and / or recognizes HER2, PD-L1, EGFR, mesothelin, and / or Lewis Y.
  • the antigen-binding fragment may be selected from the group consisting of Fab, Fab ', F (ab) 2 , dAb, an isolated complementarity determining region CDR, Fv, and scFv.
  • the targeting moiety may be a Fab, Fab ', F (ab) 2 , dAb, an isolated complementarity determining region CDR, Fv, scFv, or a combination thereof.
  • Fab generally refers to an antigen-binding fragment composed of a complete light chain, a heavy chain variable region (VH), and a first constant domain (CH1) of the heavy chain.
  • VH heavy chain variable region
  • CH1 first constant domain
  • Fab ' generally refers to an antigen-binding fragment having several additional residues at the carboxy terminus of the CH1 domain compared to a Fab fragment.
  • Fab' includes one or more halves from the hinge region of an antibody. Cystine.
  • F (ab) 2 generally refers to an antigen-binding fragment obtained from a pair of Fab fragments linked by cysteine.
  • Fv generally refers to an antigen-binding fragment consisting of the VL and VH domains of one arm of an antibody.
  • complementarity determining region CDR generally refers to the three hypervariable regions (HVR) of the light chain variable region (VL) and VH, which can form precise epitopes with the spatial structure due to their spatial structure. Complementary, so the hypervariable area is also called complementarity determining area.
  • the variant of the antibody or antigen-binding fragment thereof may be one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, or more ) Amino acid protein or polypeptide.
  • the variant of the antibody or antigen-binding fragment thereof may be at least 90% (e.g., at least 90%, at least 91%, at least 92%) with the antibody or the antigen-binding fragment thereof, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more) proteins or polypeptides of sequence homology.
  • the first targeting moiety and / or the second targeting moiety may be a ScFv.
  • the first targeting moiety may be a ScFv.
  • the second targeting moiety may be ScFv.
  • both the first targeting moiety and the second targeting moiety may be ScFv.
  • the targeting portion may include an amino acid sequence as shown in SEQ ID NO: 8.
  • the first polypeptide may include a first targeting portion, and the first targeting portion may be located at the N-terminus of the first toxin fragment.
  • the first targeting moiety may be directly linked to the N-terminal amino acid residue of the first toxin fragment; or the first targeting moiety may be indirectly linked to the N-terminal amino acid residue of the first toxin fragment, and the indirect linkage may be through insertion linkage Peptide attachment or insertion into other peptide chains.
  • the second polypeptide may include a second targeting moiety, and the second targeting moiety may be located at the N-terminus of the second toxin fragment.
  • the second targeting moiety may be directly linked to the N-terminal amino acid residue of the second toxin fragment; or the second targeting moiety may be indirectly linked to the N-terminal amino acid residue of the second toxin fragment, and the indirect linkage may be through insertion Peptide attachment or insertion into other peptide chains.
  • the composition may include a first polypeptide and a second polypeptide, and from the N-terminus to the C-terminus, the first polypeptide may sequentially contain a translocation region of a toxin and a toxic active region of the toxin.
  • the first fragment and the first intein fragment, the translocation region of the toxin and the toxin active region of the toxin can be directly or indirectly connected to each other; from the N-terminus to the C-terminus, the second The peptide comprises a second intein fragment and a second fragment of the toxic active region of the toxin in turn, and the second intein fragment and the second fragment of the toxic active region of the toxin may be directly or indirectly linked.
  • the first polypeptide may further include a first targeting moiety
  • the second polypeptide may further include a second targeting moiety.
  • the first polypeptide may include a first targeting moiety, a translocation region of the toxin, a first fragment of the toxin's toxic active region, and a first intein fragment in sequence.
  • the targeting moiety, the translocation region of the toxin, the first fragment of the toxin's toxic active region and the first intein fragment can be directly or indirectly connected; from the N-terminus to the C-terminus, the second polypeptide contains the second The peptide fragment and the second fragment of the toxic active region of the toxin and the second targeting portion, the second targeting portion, the second intein fragment, and the second fragment of the toxic active region of the toxin may be directly or indirectly connected.
  • the first intein fragment interacts with the second intein fragment, making the first target of the first polypeptide Immunity is formed by linking a portion to a first toxin fragment (containing a translocation region of the toxin and a first fragment of the toxic active region) and a second targeting portion of the second polypeptide and a second toxin fragment (containing a second fragment of the toxic active region) to form an immunity Toxin, from the N-terminus to the C-terminus, the immunotoxin comprises a first targeting moiety, a translocation region of the toxin, a first fragment of the toxic active region, a second fragment of the toxic active region, and a second targeting moiety, in order, the first targeting moiety
  • the toxin translocation region, the first fragment of the toxic active region, and the second fragment of the toxic active region may be directly or indirectly connected.
  • the first polypeptide may further include a first targeting moiety, and the second polypeptide may not include a targeting moiety.
  • the first The peptide contains a first targeting moiety, a translocation region of the toxin, a first fragment of the toxic active region of the toxin, and a first intein fragment.
  • the first targeting moiety, the translocation region of the toxin, and the toxic active region of the toxin are first.
  • the fragment and the first intein fragment can be directly or indirectly connected; from the N-terminus to the C-terminus, the second polypeptide contains the second intein fragment and the second active fragment of the toxin in order, and the second intein The fragment is linked indirectly to the second fragment of the toxic active region of the toxin.
  • the first intein fragment interacts with the second intein fragment, making the first target of the first polypeptide
  • the first toxin fragment including the translocation region of the toxin, the first fragment of the toxic active region of the toxin
  • the second toxin fragment of the second polypeptide including the second fragment of the toxic active region
  • the immunotoxin contains the first targeting moiety, the translocation region of the toxin, the first fragment of the toxic active region and the second segment of the toxic active region, the first targeting moiety, the translocation region of the toxin, and the toxin in order.
  • the first fragment of the toxic active region and the second fragment of the toxic active region are directly or indirectly connected.
  • the composition may include a first polypeptide and a second polypeptide, and from the N-terminus to the C-terminus, the first polypeptide may sequentially include a first intein fragment and a toxic activity of a toxin.
  • the first fragment and the translocation region of the toxin, the first intein fragment, the toxin active region of the toxin and the translocation region of the toxin can be directly or indirectly connected; from the N-terminus to the C-terminus, the second polypeptide can be The second fragment of the toxic active region of the toxin and the second intein fragment are sequentially contained, and the second fragment of the toxic active region of the toxin and the second intein fragment may be connected to each other or indirectly.
  • the first polypeptide may further include a first targeting moiety
  • the second polypeptide may further include a second targeting moiety.
  • the first polypeptide may comprise a first intein fragment, a first fragment of a toxin-active region, a translocation region of the toxin, and a first targeting moiety.
  • the targeting moiety, the first intein fragment, the first fragment of the toxic active region of the toxin and the translocation region of the toxin can be directly or indirectly connected; from the N-terminus to the C-terminus, the second polypeptide contains the second targeting Part, the second fragment of the toxic active region of the toxin and the second intein fragment, and the second targeting moiety, the second fragment of the toxic active region of the toxin, and the second intein fragment may be directly or indirectly connected.
  • the first intein fragment interacts with the second intein fragment, making the second target of the second polypeptide Targeting moiety and a second toxin fragment (containing a second fragment of the toxic active region) and a first targeting moiety of the first polypeptide
  • the immunotoxin contains a second targeting moiety, the toxin's toxic active region, and Two fragments, the first fragment of the toxic active region of the toxin, the translocation region of the toxin, and the second targeting moiety, the second targeting moiety, the second fragment of the toxic active region of the toxin, the first fragment of the toxic active region of the toxin, and the The indexed regions are directly or indirectly connected.
  • the first polypeptide may not include a targeting moiety, and the second polypeptide may further include a second targeting moiety.
  • the first The peptide may comprise the first intein fragment, the first fragment of the toxin active region, and the translocation region of the toxin in sequence.
  • the first intein fragment, the first fragment of the toxin active region, and the translocation region of the toxin may be directly or Indirect linkage; from the N-terminus to the C-terminus, the second polypeptide may in turn contain a second targeting moiety, a second fragment of the toxic active region of the toxin, and a second intein fragment, and the second targeting moiety and the second intein The fragment is directly or indirectly connected to the second fragment of the toxic active region of the toxin.
  • the first intein fragment interacts with the second intein fragment, making the second target of the second polypeptide
  • An immunotoxin is formed by linking a portion with a second toxin fragment (containing a second fragment of the toxic active region of the toxin) and a first toxin fragment of the first polypeptide (containing the first fragment of the toxic active region of the toxin and the translocation region of the toxin); From the N-terminus to the C-terminus, the immunotoxin contains a second targeting moiety, a second fragment of the toxin's toxic active region, a first fragment of the toxin's toxic active region, and a translocation region of the toxin.
  • the second targeting moiety, the toxicity of the toxin The second fragment of the active region, the first fragment of the toxic active region of the toxin, and the translocation region of the toxin are connected indirectly.
  • the composition may include a first polypeptide and a second polypeptide
  • the first polypeptide of the present application may include a first targeting moiety
  • the second polypeptide may not include a target.
  • the first targeting part may be ScFv targeting HER2, and the toxin uses truncated PE38.
  • the first polypeptide (as shown in FIG. 2B), from the N-terminus to the C-terminus, a ScFv antigen-binding fragment that targets HER2, a first toxin fragment (Pn), and a first intein fragment (In) may be included in this order.
  • the first polypeptide is named ScFvPnIn.
  • a second intein fragment (Ic) and a second toxin fragment (Pc) are sequentially included, and the second polypeptide is named IcPc .
  • T7 is the promoter of the E. coli expression vector.
  • the C-terminal amino acid residue of the first toxin fragment is derived from the random coiled region of the truncated PE38, and the amino acid residue at the N terminal of the second toxin fragment is derived from the random coiled region of the truncated PE38.
  • the present application provides a method for preparing the composition, which includes the following steps: 1) providing the first polypeptide; 2) providing the second polypeptide; 3) adding the first polypeptide A polypeptide is mixed with the second polypeptide to obtain the composition.
  • steps 1, 2 and 3 may occur simultaneously or not at the same time. For the situations where steps 1, 2 and 3 do not occur at the same time, step 1 may occur before step 2, or step 2 occurs before step 1. , Or steps 1 and 2 occur simultaneously.
  • the molar ratio of the first polypeptide to the second polypeptide in the composition may be 10: 1-1: 10 (for example, 10: 1-1: 10; 9: 1-1: 9; 8: 1-1: 8; 7: 1-1: 7; 6: 1-1: 6; 5: 1-1: 5; 4: 1-1: 4; 3: 1-1: 3; 2: 1-1: 2).
  • the composition obtained according to the above preparation method may include the first polypeptide and the second polypeptide, and the first polypeptide and the second polypeptide may not be biologically toxic.
  • the present application provides a method for preparing the toxin.
  • the method includes the following steps: 1) providing the first polypeptide; 2) providing the second polypeptide; A peptide is mixed with the second polypeptide to obtain the toxin.
  • the mixing in step 3) further includes adding a reducing agent to the mixture.
  • the first intein fragment in the first polypeptide and the second intein fragment in the second polypeptide can undergo trans-splicing, so that the The first toxin fragment and the second toxin fragment in the second polypeptide form a biotoxic toxin.
  • the preparation method may further include adding a reducing agent.
  • the step of adding the reducing agent may occur simultaneously or not at the same time as the above steps 1, 2 and 3.
  • the term "reducing agent" generally refers to a substance that loses or deviates from an electron in a redox reaction.
  • the composition after adding a reducing agent, the composition may include a biotoxic toxin and / or a biotoxic immunotoxin.
  • the reducing agent may be selected from the group consisting of DTT and ⁇ -mercaptoethanol.
  • the reducing agent may be DTT and ⁇ mercaptoethanol.
  • the reducing agent may be DTT.
  • the reducing agent may be beta mercaptoethanol.
  • the concentration of the reducing agent in the composition is 0.001-10000nM, for example, 0.001-10000nM; 0.002-5000nM; 0.003-2000nM; 0.005-1000nM; 0.01-500nM; 0.015-200nM; 0.02-100nM ; 0.05-80 nM; 0.1-50 nM; 0.5-20 nM; or 0.1-10 nM.
  • the reducing agent may be added at the same time as the mixing in step 3 or after the mixing.
  • the preparation method may further include incubating the composition after adding the reducing agent.
  • incubation generally refers to leaving a mixed sample at a certain temperature.
  • the mixture containing the first polypeptide, the second polypeptide, and the reducing agent may be allowed to stand at a certain temperature for a period of time.
  • the incubation temperature may be 1 ° -50 ° C, for example, 1 ° -50 ° C, 4 ° -50 ° C, 4 ° -45 ° C, 4 ° -40 ° C, 4 ° -37 ° C, 8 °C -37 °C, 13 °C -37 °C, 17 °C -37 °C, 17 °C -35 °C, 17 °C -30 °C, 17 °C -25 °C, 17 °C -23 °C or 20 °C -23 °C.
  • the incubation time may be 2-120 minutes, such as 2-120 minutes, 2-100 minutes, 2-80 minutes, 3-80 minutes, 4-80 minutes, 5-80 minutes, 10- 80 minutes, 15-80 minutes, 20-80 minutes, 20-80 minutes, 40-80 minutes, 50-80 minutes, 50-70 minutes, or 50-60 minutes.
  • a mixture containing the first polypeptide, the second polypeptide, and the reducing agent can be left to stand for 2-120 minutes, 2-100 minutes, 2-80 minutes, 4-80 minutes, 5-80 minutes, 10-80 minutes, 15-80 minutes, 20-80 minutes, 20-80 minutes, 40-80 minutes, 50-80 minutes, or 50-70 minutes.
  • the mixture containing the first polypeptide, the second polypeptide, and the reducing agent is left to stand for 2-120 minutes, 2-100 minutes, 2-80 minutes, 4-80 minutes, 5 -80 minutes, 10-80 minutes, 15-80 minutes, 20-80 minutes, 20-80 minutes, 40-80 minutes, 50-80 minutes, or 50-70 minutes.
  • the mixture containing the first polypeptide, the second polypeptide, and the reducing agent is allowed to stand for 2-120 minutes, 2-100 minutes, 2-80 minutes, 4-80 minutes, 5 -80 minutes, 10-80 minutes, 15-80 minutes, 20-80 minutes, 20-80 minutes, 40-80 minutes, 50-80 minutes, or 50-70 minutes.
  • the mixture containing the first polypeptide, the second polypeptide, and the reducing agent is left to stand for 2-120 minutes, 2-100 minutes, 2-80 minutes, 4-80 minutes, 5 -80 minutes, 10-80 minutes, 15-80 minutes, 20-80 minutes, 20-80 minutes, 40-80 minutes, 50-80 minutes, or 50-70 minutes.
  • the mixture containing the first polypeptide, the second polypeptide and the reducing agent is allowed to stand for 2-120 minutes, 2-100 minutes, 2-80 minutes, 4-80 minutes, 5 -80 minutes, 10-80 minutes, 15-80 minutes, 20-80 minutes, 20-80 minutes, 40-80 minutes, 50-80 minutes, or 50-70 minutes.
  • the composition described in this application may have one or more of the following properties: 1) The composition may be divided into a non-toxic first polypeptide and a second polypeptide, which are respectively administered to a desired target To restore toxicity (such as in the tumor microenvironment) can improve the safety of medication and reduce the non-specific toxicity of the composition to normal cells; 2) the composition can be divided into two parts, the first polypeptide and the second polypeptide, and has Higher flexibility and adjustability, for example, different administration methods, dosages, and different combinations of administration methods can be used to achieve better therapeutic effects; The polypeptide is linked to the second polypeptide to prepare an active substance (such as toxicity).
  • the preparation method has universal applicability and can be applied to various types of compositions, such as immunotoxins or protein drugs; 4)
  • the composition is divided After forming the non-toxic first polypeptide and the second polypeptide into two parts, the prokaryotic or eukaryotic cells can be used to express the divided two parts, and the expressed two parts have no toxic effect on the host cell.
  • the prokaryotic or eukaryotic cells can be used to express the divided two parts, and the expressed two parts have no toxic effect on the host cell.
  • immune toxin Connected mode is the toxin composition, expressed in eukaryotic cells intact antibody antibody facilitate assembly; 5) for different tumor-associated antigens, the composition may contain different antibody targeting moiety, it can be improved target specific composition.
  • the present application provides a vector which may comprise a nucleic acid encoding a first polypeptide, and / or which may comprise a nucleic acid encoding a second polypeptide.
  • the vector may comprise a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide.
  • the vector may comprise a nucleic acid encoding a first polypeptide.
  • the vector may comprise a nucleic acid encoding a second polypeptide.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in an appropriate host cell and under appropriate conditions.
  • the vector may also contain expression control elements that allow the coding region to be properly expressed in an appropriate host.
  • control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5 'non-transcribed sequences and 5' and 3 'non-translated sequences respectively involved in transcription and translation initiation, such as TATA box, plus Cap sequences, CAAT sequences, etc.
  • the 5 ' non-transcribed expression control sequence may comprise a promoter region, and the promoter region may comprise a promoter sequence for transcriptionally controlling a functionally linked nucleic acid.
  • the expression control sequence may further include an enhancer sequence or an upstream activator sequence.
  • suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), of which the A certain part can be fused with a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not contain additional introns.
  • the vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the application provides a cell that can express a first polypeptide, and / or that can express a second polypeptide.
  • the application provides a cell that can express a first polypeptide and a second polypeptide.
  • the application provides a cell that can express a first polypeptide.
  • the application provides a cell that can express a second polypeptide.
  • each or each cell may comprise one or one of the nucleic acid molecules or vectors described herein.
  • each or each cell may comprise multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors as described herein.
  • the vectors described herein can be introduced into the cells, such as E. coli and the like.
  • the vectors described herein can be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application provides a kit, which may include a first polypeptide and a second polypeptide.
  • the first polypeptide and the second polypeptide may not be mixed with each other in the kit, that is, the first polypeptide and the second polypeptide are placed in the kit separately from each other and will not be mixed with each other.
  • the first polypeptide and the second polypeptide may be located in different containers. For example, the first polypeptide is placed in one container and the second polypeptide is placed in another container.
  • the two containers described above are independent of each other so that the second polypeptide and the second polypeptide are not mixed with each other.
  • the kit may further include a reducing agent.
  • the kit may include a first polypeptide, a second polypeptide, and a reducing agent.
  • the reducing agent may be selected from the group: DTT and ⁇ -mercaptoethanol.
  • the kit may comprise a first polypeptide, a second polypeptide, and DTT.
  • the kit may comprise a first polypeptide, a second polypeptide, and beta mercaptoethanol.
  • the kit may comprise a first polypeptide, a second polypeptide, beta mercaptoethanol, and DTT.
  • the reducing agent may be contained in a separate container.
  • the container containing the reducing agent and the container containing other components are independent of each other, that is, the container containing the first polypeptide and the second polypeptide can no longer hold
  • the reducing agent, reducing agent, and other components are placed separately.
  • kits may comprise the composition, the composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide may comprise a first toxin fragment and a first intein fragment
  • the second polypeptide may comprise a second toxin fragment and a second intein fragment, and before the first intein fragment interacts with the second intein fragment, the first in the composition Neither the polypeptide nor the second polypeptide is biologically toxic.
  • kits of the present application may be contained in separate containers (ie, kits having separate portions) or provided in a single container.
  • the kit of the present application may further include the composition and / or instructions for performing the method. Instructions can be provided as a paper or electronic user manual.
  • the manual may contain instructions for explaining the results obtained when the method described above is performed using the kit of the present application or the composition is used.
  • the application provides a use of the composition, the kit, the vector, and / or the cell in the manufacture of a medicament for treating a disease, which may include a tumor.
  • the tumor may be selected from the group consisting of breast cancer, melanoma, ovarian cancer, colon cancer, mesothelioma, adenoma, pancreatic cancer, and bladder cancer.
  • the medicament can be used to treat a tumor-containing disease, such as treating breast cancer, melanoma, ovarian cancer, colon cancer, mesothelioma, adenoma, pancreatic cancer, and / or bladder cancer.
  • the pharmaceutical composition of the present application can inhibit or delay the development or progression of a tumor-containing disease, can reduce the size of a tumor (even substantially eliminate the tumor), and / or can reduce and / or stabilize the disease state.
  • the application provides the composition, the kit, the vector, and / or the cell, which are capable of treating a tumor.
  • the treatment of a tumor refers to inhibiting the growth of the tumor, reducing the size of the tumor (even substantially eliminating the tumor), and / or reducing and / or stabilizing the disease state.
  • the application provides a method for treating a tumor, comprising administering the composition, the kit, the vector, and / or the cell.
  • the mode of administration includes oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, and / Or via subcutaneous depot.
  • the application also provides a method of administering the composition, which may include the step of administering the first polypeptide in admixture with the second polypeptide and a reducing agent. Under in vitro conditions, the first polypeptide and the second polypeptide may form the toxin or the immunotoxin having biotoxicity under reducing conditions, for example, may have a killing effect on the cells, or Has an apoptotic effect.
  • the application also provides a method of treating a tumor, comprising administering the first polypeptide and the second polypeptide to a subject.
  • the first polypeptide and the second polypeptide can form the toxin or the immunotoxin with biotoxicity under the conditions of tumor cell microenvironment, for example, it can kill the tumor cells Effect, or produce an apoptotic effect.
  • the application time of different parts of the composition may be different.
  • different portions may be administered at intervals of 1 second, 1 minute, 1 hour, 10 hours, 1 day or more.
  • different parts of the composition can be administered simultaneously.
  • Different parts of the composition described in this application can be applied in the same or different ways, or the same or different dosages can be used.
  • the administration of each part of the composition of the present application is flexible and adjustable as long as the desired therapeutic effect can be achieved.
  • site-directed mutagenesis of the immunotoxin scFvPE38 is required.
  • the amino acid sequence of the immunotoxin scFvPE38 is shown in SEQ ID NO: 9.
  • Site-directed mutagenesis was used to mutate the three amino acids downstream of the selected site.
  • the mutated immunotoxin and its vector were named scFvPM1, scFvPM2, and scFvPM3, respectively.
  • the vectors scFvPM1, scFvPM2, and scFvPM3 were used to express the mutated immunotoxin and purify the protein of interest.
  • the amino acid sequences of the obtained mutants scFvPM1, scFvPM2, and scFvPM3 were as follows: : 16), SEQ ID NO: 11 and SEQ ID NO: 12.
  • the mutants scFvPM1, scFvPM2, and scFvPM3 were used to treat HER2 antigen-positive SKOV3 cells, and the effects of different mutants on cell viability were examined. As shown in Figure 3, the scFvPM1 mutant has higher cytotoxicity than other mutants, so the corresponding scFvPM1 was selected.
  • the mutation site of the toxin is the cleavage site of the toxin. This mutation site is also the insertion site of the cleavage intein, and the three amino acid mutation sequences corresponding to the selected site are "CFN" sequences.
  • scFvPM1 is divided into two parts, scFvPn and Pc.
  • the amino acid sequence of the corresponding scFvPn is shown in SEQ ID NO: 13
  • the amino acid sequence of Pc is shown in SEQ ID NO: 14.
  • ScFvPn is fused to the N-terminus of the fragmented intein Npu DnaE (referred to as In), the C-terminus of NpuDnaE (referred to as Ic) and Pc are fused.
  • the amino acid sequence is shown in SEQ ID NO: 3.
  • the target fragment is synthesized by PCR (polymerase chain reaction), and start and stop codons are introduced at both ends of the target fragment, as well as restriction enzyme sites such as NdeI / HindIII.
  • the target fragment was ligated to the E. coli expression vector pET28a containing the T7 promoter by enzymatic digestion, and the target vectors pET-scFvPnIn (as shown in FIG. 2B) and pET-IcPc (as shown in FIG. 2C) were obtained.
  • the dissolved inclusion bodies were added dropwise to a 50-fold volume of renaturation solution (100 mM Tris-HCl, 500 mM arginine, 1 mM ethylenediamine tetraacetic acid disodium, 1 mM reduced glutathione, 0.1 mM oxidized glutathione Glycine, pH 9.0-9.5).
  • renaturation solution 100 mM Tris-HCl, 500 mM arginine, 1 mM ethylenediamine tetraacetic acid disodium, 1 mM reduced glutathione, 0.1 mM oxidized glutathione Glycine, pH 9.0-9.5.
  • the diluted renatured protein solution was affinity purified using a CaptoL and His-trap column, respectively.
  • the scFvPnIn was purified by a CaptoL column and eluted with citric acid buffer, and then the sample was immediately neutralized with 1M tris-Hcl buffer at pH 9.0.
  • IcPc was purified by His-trap column and eluted with different imidazole concentration buffers. The eluted fractions were collected and analyzed for protein purity by SDS-PAGE.
  • the protein was concentrated by centrifugation with MILLIPORE Amicon Ultra (10MWCO) ultrafiltration centrifuge tube, replaced with PBS buffer, and frozen at -20 ° C or -80 ° C.
  • the amino acid sequence of the expressed scFvPnIn is shown in SEQ ID NO: 6, and the amino acid sequence of IcPc is shown in SEQ ID NO: 15.
  • Fragmented inteins mediate protein trans-splicing reactions in two parts of the immunotoxin.
  • the scFvPnIn and IcPc proteins purified in Example 2 were mixed at a molar ratio of 1: 1, and 1.0 mM DTT, 2.5 mM DTT, 5.0 mM DTT, 7.5 mM DTT, and 10.0 mM DTT were added at the same time. After incubation for 30 minutes, the results are shown in Figure 4.
  • N refers to the sample added with scFvPnIn only
  • C refers to the sample added with IcPc only
  • 1.0 refers to the addition of 1.0mM DTT, 2.5 mM DTT, 5.0 mM DTT, 7.5 mM DTT, and 10.0 mM DTT samples. It was found that when 1.0 mM DTT was added, scFvPnIn and IcPc quickly trans-spliced, and obvious bands appeared at 85KD (see arrow in Figure 4). ).
  • Example 2 The two scFvPnIn and IcPc proteins purified in Example 2 were mixed at a molar ratio of 1: 1, 1.0 mM DTT was added at the same time, and the mixture was incubated at 4 ° C, 17 ° C, and 37 ° C for 30 minutes. The results are shown in the figure. 5 shown. “N” refers to the sample with only scFvPnIn added, and “C” refers to the sample with only IcPc added. Samples "4", "7", and “37” correspond to samples placed at 4 ° C, 17 ° C, and 37 ° C, respectively.
  • the two scFvPnIn and IcPc proteins purified in Example 2 were mixed at a molar ratio of 1: 1, 1.0 mM DTT was added at the same time, and then the above mixture was placed at 37 ° C and left to stand for 0 min, 1 min, 5 min, 10 min, 30min, 60min, and 120min, the results are shown in Fig. 6, and obvious bands appeared at 85KD (see arrow in Fig. 6).
  • N refers to the sample with only scFvPnIn added
  • C refers to the sample with only IcPc added
  • samples "0", “1”, “5", “10”, “30”, “60” and “120” Refers to the samples at rest for 0min, 1min, 5min, 10min, 30min, 60min, and 120min, respectively. It was found that a trans-splicing reaction could occur at 5 minutes, generating a complete immunotoxin. The reaction reached the plateau phase at 60 min. The thiol compound needs to be removed at the end of the reaction.
  • Flow cytometry was used to analyze the antigen affinity of the immunotoxin.
  • the subjects examined were the intact immunotoxin and the N part of the immunotoxin after segmentation.
  • the tumor cells with high antigen expression SKOV3 were treated with the whole immunotoxin and the N part of the immunotoxin after segmentation.
  • Nuclei were treated with 4,6- Dipyridin-2-phenylindole (DAPI) staining, the cell slide was removed, placed on a glass slide, observed and photographed under a confocal laser microscope.
  • N refers to the cases of individual scFvPnIn, individual IcPc, and mixed scFvPnIn and IcPc (adding reducing agent), respectively.
  • Both scFvPM1 and scFvPnIn can be labeled with a green fluorescent secondary antibody, and IcPc is connected to red fluorescent protein. Therefore, both the I-cPc and the immunotoxin generated by the reaction in part C can show red fluorescence.
  • SKOV3 cells take up more intact immunotoxins and scFvPnIn of the immunotoxin N part after the segmentation, but there is no obvious uptake of IcPc of the immunotoxin C part after the segmentation.
  • a green and red fluorescence co-localized region is shown inside, indicating that the two-part reaction produces a new intact immunotoxin.
  • control sample refers to PBS solution, scFvPnIn alone, IcPc alone, mixed scFvPnIn and IcPc (without reducing agent added) The case of mixed scFvPnIn and IcPc (with the addition of reducing agent DTT) and DTT alone.
  • Samples "0”, “1”, “5 “”, “10”, “30”, and “60” refer to the samples when the cell culture solution was left to stand for 0 min, 1 min, 5 min, 10 min, 30 min, and 60 min after the addition.
  • scFvPnIn and IcPc undergo a trans-splicing reaction to generate the complete immunotoxin scFvPM1.
  • immunotoxins are also highly cytotoxic to MCF7 cells with low expression of HER2, but the divided immunotoxins are not toxic to MCF7. After the two-stage toxins are reconstituted, some activities are restored but due to the low expression of the antigen, MCF7 is less toxic.
  • Immunotoxin scFvPM1 binds to cell elongation factor eEF2 after entering the cell, which makes it inactivated by ADP ribosylation, inhibits cellular protein synthesis, and causes apoptosis.
  • the effects of intact immunotoxins and post-fragmented immunotoxins on apoptosis induction are now examined.
  • Cells were plated in a 12-well plate at 2 ⁇ 10 5 / well. After overnight culture, cells were treated with immunotoxin for 48 hours in each group, the cell surface culture material was washed away, the cells were digested with trypsin, and cell suspensions were prepared. Cells were treated with propidium iodide (PI) for 10 min.
  • PI propidium iodide
  • AnnexinV can selectively bind to phosphatidylserine on the surface of apoptotic cells.
  • PI cannot pass through the living cell membrane, but can pass through the damaged cell membrane to stain the nucleus. Therefore, early apoptotic cells and late apoptotic cells can be divided by AnnexinV-FITC and PI mark. As shown in Fig.
  • N refers to scFvPnIn alone, IcPc alone, mixed scFvPnIn and IcPc (added with reducing agent DTT ), The case of DTT and PBS alone.

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Abstract

提供了一种组合物，其包含第一多肽和第二多肽，其中，所述第一多肽包含第一毒素片段和第一内含肽片段，所述第二多肽包含第二毒素片段和第二内含肽片段；其中所述第一多肽与所述第二多肽不同；所述第一毒素片段和所述第二毒素片段均无生物毒性；且所述第一多肽和所述第二多肽可通过所述第一内含肽片段与所述第二内含肽片段的相互作用而使所述第一毒素片段与所述第二毒素片段形成有生物毒性的毒素。还提供了该组合物的制备方法、包含所述第一多肽和第二多肽的试剂盒，以及所述组合物、试剂盒在治疗肿瘤中的用途和方法。

Description

一种多肽组合物 技术领域
本申请涉及免疫治疗领域，具体涉及一种多肽组合物，还涉及包含多肽组合物的试剂盒以及其在治疗肿瘤方面的用途。
背景技术
免疫毒素是指由细胞靶向部分和毒素部分组成的治疗性嵌合蛋白，具有特异性靶向病变细胞并杀伤病变细胞的功能，可以通过化学偶联或者基因重组的方法制得。
研究显示，恶性肿瘤会过度表达许多肿瘤相关抗原，为靶向治疗提供相应的靶点，免疫毒素的靶向部分可以将毒素药物定向递送至肿瘤细胞，提高特异性杀伤作用，降低对正常细胞的毒性。细胞靶向部分可以是抗体，细胞靶向部分能够特异性地靶向肿瘤细胞，负责特异性结合细胞并被细胞内吞。毒素部分通过细胞靶向部分进入胞质，在胞质中干扰细胞过程直接诱导细胞死亡，或者修饰细胞膜，诱导凋亡蛋白而间接引起细胞死亡，发挥抑制肿瘤细胞增长的功能并导致细胞死亡。
然而，正常组织亦会不同程度地表达肿瘤相关抗原，因此，免疫毒素对正常组织细胞仍然存在非特异性毒性，存在用药安全性问题。
发明内容
本申请提供一种多肽组合物，其包含第一多肽和第二多肽。本申请还提供了该组合物的制备方法、包含所述第一多肽和第二多肽的试剂盒，以及所述组合物、试剂盒在治疗肿瘤中的用途和方法。
一方面，本申请提供一种组合物，其包含第一多肽和第二多肽，其中，所述第一多肽包含第一毒素片段和第一内含肽片段，所述第二多肽包含第二毒素片段和第二内含肽片段；其中所述第一多肽与所述第二多肽不同；所述第一毒素片段和所述第二毒素片段均无生物毒性；且所述第一多肽和所述第二多肽可通过所述第一内含肽片段与所述第二内含肽片段的相互作用而使所述第一毒素片段与所述第二毒素片段形成有生物毒性的毒素。在某些实施方式中，所述第一毒素片段与所述第二毒素片段不同。在某些实施方式中，所述第一毒素片段和所述第二毒素片段源自相同的毒素。
在某些实施方式中，所述第一毒素片段包含所述毒素的毒性活性区第一片段，所述第二毒素片段包含所述毒素的毒性活性区第二片段，且所述毒性活性区第一片段和所述毒性活性区第二片段构成所述毒素的完整毒性活性区。在某些实施方式中，所述第二毒素片段不包含 所述毒素的转位区或其片段。在某些实施方式中，所述第一毒素片段还包含所述毒素的转位区或其片段。在某些实施方式中，所述第一毒素片段不包含所述毒素的完整毒性活性区。在某些实施方式中，所述第二毒素片段不包含所述毒素的完整毒性活性区。
在某些实施方式中，所所述毒素选自以下组：细菌毒素、人源毒素和植物毒素。在某些实施方式中，所述毒素选自以下组：绿脓杆菌外毒素和白喉毒素。在某些实施方式中，所述毒素选自以下组：蓖麻毒素、皂草素和白树毒素。在某些实施方式中，所述毒素为绿脓杆菌外毒素的截短体PE38，其包含如SEQ ID NO:1和SEQ ID NO:16中任一项所示的氨基酸序列。
在某些实施方式中，所述第一内含肽片段与所述第二内含肽片段不同。在某些实施方式中，所述第一内含肽片段和所述第二内含肽片段源自相同的内含肽。在某些实施方式中，所述内含肽为断裂型内含肽。在某些实施方式中，所述断裂型内含肽选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。
在某些实施方式中，所述第一多肽中，所述第一毒素片段的C端与所述第一内含肽片段的N端直接或间接相连。在某些实施方式中，所述第一毒素片段C端的氨基酸残基源自所述毒素的无规卷曲区。在某些实施方式中，所述第二多肽中，所述第二毒素片段的N端与所述第二内含肽片段的C端直接或间接相连。在某些实施方式中，所述第二毒素片段N端的氨基酸残基源自所述毒素的无规卷曲区。在某些实施方式中，所述第二毒素片段N端第1-3位的氨基酸残基依次为CFN。
在某些实施方式中，所述第一内含肽片段包含如SEQ ID NO:2所示的氨基酸序列。在某些实施方式中，所述第二内含肽片段包含如SEQ ID NO:3所示的氨基酸序列。在某些实施方式中，所述第一毒素片段包含如SEQ ID NO:4所示的氨基酸序列。在某些实施方式中，所述第二毒素片段包含如SEQ ID NO:5所示的氨基酸序列。在某些实施方式中，所述第一多肽包含如SEQ ID NO:6所示的氨基酸序列。在某些实施方式中，所述第二多肽包含如SEQ ID NO:7和SEQ ID NO:15中任一项所示的氨基酸序列。
在某些实施方式中，所述第一多肽中，所述第一毒素片段的N端与所述第一内含肽片段的C端直接或间接相连。在某些实施方式中，所述第一毒素片段N端的氨基酸残基源自所述毒素的无规卷曲区。在某些实施方式中，所述第二多肽中，所述第二毒素片段的C端与所述第二内含肽片段的N端直接或间接相连。在某些实施方式中，所述第二毒素片段C端的氨基酸残基源自所述毒素的无规卷曲区。在某些实施方式中，所述第一毒素片段N端第1-3位的氨基酸残基依次为CFN。
在某些实施方式中，所述第一内含肽片段包含所述断裂型内含肽的N端蛋白质区域；且所述第二内含肽片段包含所述断裂型内含肽的C端蛋白质区域。
在某些实施方式中，所述第一内含肽片段包含所述断裂型内含肽的C端蛋白质区域；且所述第二内含肽片段包含所述断裂型内含肽的N端蛋白质区域。
在某些实施方式中，所述N端蛋白质区域为Npu DnaE的N端蛋白质区域。
在某些实施方式中，所述C端蛋白质区域为Npu DnaE的C端蛋白质区域。
在某些实施方式中，所述第一内含肽片段与所述第二内含肽片段的所述相互作用包含所述第一内含肽片段和所述第二内含肽片段的蛋白质反式剪接作用。
在某些实施方式中，所述第一多肽和/或所述第二多肽还包括靶向部分，所述靶向部分靶向肿瘤特异性抗原。在某些实施方式中，所述第一多肽包含第一靶向部分，且所述第一靶向部分位于所述第一毒素片段的N端。在某些实施方式中，所述第二多肽包含第二靶向部分，且所述第二靶向部分位于所述第二毒素片段的N端。
在某些实施方式中，所述肿瘤特异性抗原选自以下组：HER2、PD-L1、EGFR、mesothelin和Lewis Y。在某些实施方式中，所述第一靶向部分和/或所述第二靶向部分包含抗体或其抗原结合片段或变体。在某些实施方式中，所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。在某些实施方式中，所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，dAb，分离的互补决定区CDR，Fv和scFv。
在某些实施方式中，所述抗体或其抗原结合片段的所述变体选自下组：a)在所述抗体或所述其抗原结合片段中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽；和b)与所述抗体或所述其抗原结合片段具有至少90％序列同源性的蛋白质或多肽。
在某些实施方式中，所述第一靶向部分和/或所述第二靶向部分为ScFv。在某些实施方式中，所述靶向部分包含如SEQ ID NO:8所示的氨基酸序列。
另一方面，本申请提供一种组合物的制备方法，其包括以下步骤：1)提供所述的第一多肽；2)提供所述的第二多肽；3)将所述第一多肽与所述第二多肽混合以得到所述组合物。在某些实施方式中，在所述的制备方法中，其中所述组合物中所述第一多肽与所述第二多肽的摩尔比为10:1-1:10。
在某些实施方式中，在所述的制备方法中，其还包括加入还原剂。在某些实施方式中，在所述的制备方法中，其中所述还原剂选自以下组：DTT和β巯基乙醇。在某些实施方式中，在所述的制备方法中，其中所述组合物中所述还原剂的浓度为0.001-10000nM。
在某些实施方式中，在所述的制备方法中，其中在所述混合的同时或所述混合之后加入 所述还原剂。在某些实施方式中，在所述的制备方法中，其还包括在加入所述还原剂后孵育所述组合物。在某些实施方式中，在所述的制备方法中，其中所述孵育的温度为1℃-50℃。在某些实施方式中，在所述的制备方法中，其中所述孵育的时间为2-120分钟。
另一方面，本申请提供一种载体，其包含编码所述的第一多肽的核酸，和/或，其包含编码所述的第二多肽的核酸。
另一方面，本申请提供一种细胞，其表达所述的第一多肽，和/或，其表达所述的第二多肽。
另一方面，本申请提供一种试剂盒，其包含1)所述的第一多肽；以及2)所述的第二多肽。在某些实施方式中，在所述的试剂盒中，其中所述第一多肽和所述第二多肽在所述试剂盒中彼此不混合。在某些实施方式中，在所述的试剂盒中，其中所述第一多肽和所述第二多肽位于不同的容器内。
在某些实施方式中，在所述的试剂盒中，其中所述试剂盒还包含还原剂。在某些实施方式中，在所述的试剂盒中，其中所述还原剂选自以下组：DTT和β巯基乙醇。在某些实施方式中，在所述的试剂盒中，其中所述还原剂包含于独立的容器内。
在某些实施方式中，在所述的试剂盒中，其包含所述的组合物。
另一方面，本申请提供一种所述的组合物、所述的试剂盒、所述的载体，或所述的细胞在制备治疗疾病的药物中的用途，所述疾病包含肿瘤。在某些实施方式中，所述的肿瘤选自以下组：乳腺癌、黑色素瘤、卵巢癌、结肠癌、间皮瘤、腺体瘤、胰腺癌和膀胱癌。
另一方面，本申请提供一种所述的组合物、所述的试剂盒、所述的载体，或所述的细胞，其治疗肿瘤。
另一方面，本申请提供一种治疗肿瘤的方法，其包括施用所述的组合物、所述的试剂盒、所述的载体，或所述的细胞。
本领域技术人员能够从下文的详细描述中容易地洞察到本公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的 示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请组合物的结构；
图2显示的是本申请scFvPM1、scFvPnIn和IcPc的表达载体的组成元件；
图3显示的是本申请免疫毒素的突变体对SKOV3细胞活性的影响；
图4显示的是不同DTT浓度下断裂型内含肽介导的反式剪接的情况；
图5显示的不同温度下断裂型内含肽介导的反式剪接的情况；
图6显示的是不同反应时间下断裂型内含肽介导的反式剪接的；
图7显示的是完整免疫毒素和分割后免疫毒素对SKOV3细胞表面抗原亲和力；
图8显示的是SKOV3细胞对完整免疫毒素和分割后免疫毒素的摄取；
图9显示的是完整免疫毒素和分割后免疫毒素对SKOV3细胞活力的影响；
图10显示的是完整免疫毒素和分割后免疫毒素对MCF7细胞活力的影响；
图11显示的是完整免疫毒素和分割后免疫毒素对CHO细胞活力的影响；
图12显示的是分割后免疫毒素在细胞培养液中发生内含肽介导的反式剪接的情况；
图13显示的是完整免疫毒素和分割后免疫毒素诱导SKOV3细胞凋亡的情况；
图14显示的是组合物中的第一多肽和第二多肽的组成结构；
图15显示的是组合物中的第一多肽和第二多肽的组成结构。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“免疫毒素(recombinant immunotoxin，RIT)”通常是由细胞靶向部分和毒素部分组成的治疗性嵌合蛋白，其具有特异性靶向病变细胞并杀伤病变细胞的功能。通过细胞靶向部分与靶细胞表面特异性受体抗原结合，细胞靶向部分负责特异性结合靶细胞并被靶细胞内吞。毒素部分可以通过细胞靶向部分进入胞质，在胞质中干扰细胞过程直接诱导细胞死亡，或者毒素部分可以修饰细胞膜，诱导凋亡蛋白而间接引起细胞死亡，发挥抑制病变细胞增长并导致细胞死亡的功能。
在本申请中，术语“毒素”通常是指对细胞的生长和增殖有害并可以发挥作用以减少、抑制或摧毁细胞或恶性肿瘤的任何物质，这些物质通常是一些会干扰生物体中其他大分子作用的蛋白质。毒素按照其来源，可以包括细菌毒素、植物毒素以及人源毒素。例如，细菌毒素可包括绿脓杆菌外毒素(PE)和白喉毒素(DT)，植物毒素可包括蓖麻毒素和相思豆毒素。 在某些情形中，毒素可以通过酶解作用抑制蛋白合成来发挥功能。通常，毒素可以包括以下功能区：细胞结合区、转位区和毒性活性区。
在本申请中，术语“细胞结合区”通常是指可将毒素富集于靶细胞表面的毒素的功能区。
在本申请中，术语“转位区”通常是指可使毒素跨膜到达胞质区的毒素的功能区。
在本申请中，术语“毒性活性区”通常是指能够使一些重要的细胞过程失活并杀死细胞的毒素的功能区。
在本申请中，术语“多肽”通常是指α-氨基酸以肽键连接在一起而形成的化合物，它也是蛋白质水解的中间产物。由两个氨基酸分子脱水缩合而成的化合物叫做二肽，同理类推还有三肽、四肽、五肽等。通常由三个或三个以上氨基酸分子脱水缩合而成的化合物可以称为多肽。
在本申请中，术语“生物毒性”通常是指细胞毒性，可以是由细胞或化学物质引起的单纯细胞杀伤事件，不依赖于凋亡或坏死的细胞死亡机理。
在本申请中，术语“抗体”通常是指能够特异性识别和/或中和特定抗原的多肽分子。基本的四链抗体单元是异四聚体糖蛋白，其由两条相同的轻链和两条相同的重链构成。在IgG的情况中，每个L链通过一个共价二硫键连接到H链，而两条H链通过一个或多个二硫键彼此相连，二硫键的数目取决于H链的同种型。每个H和L链还具有规律间隔的链内二硫键。每条H链在N-末端具有可变结构域(VH)，接着是三个(对于每条α和γ链)或四个(对于μ和ε同种型)恒定结构域(CH)。
在本申请中，术语“抗原结合片段”通常是指完整抗体的一部分，例如，抗原结合片段可以是完整抗体的抗原结合区和/或可变区。抗原结合片段可以通过化学方法和/或基因工程的方法获得。化学方法是通过打断铰链区的二硫键产生抗原结合片段，或者采用蛋白酶，包括胃蛋白酶和木瓜蛋白酶，消化抗体后产生抗原结合片段。基因工程方法是指利用重组DNA及蛋白质工程技术对编码抗体的基因按不同需要进行加工改造和重新装配，经转染适当的受体细胞所表达的抗体分子。
在本申请中，术语“内含肽”通常是指是位于宿主蛋白质中的一段插入序列。内含肽基因不是一个独立的基因，需要插入于外显肽基因才能复制转录，可从前体蛋白中切除并将两侧外显肽连接起来成为成熟蛋白质。内含肽对应的核苷酸序列嵌合在宿主蛋白对应的核酸序列之中，与宿主蛋白基因存在于同一开放阅读框架内，并与宿主蛋白质基因进行同步转录和翻译，当翻译形成蛋白质前体后，内含肽从宿主蛋白质中切除，从而形成成熟的具有活性的蛋白。根据内含肽的存在形式，可以分为整体内含肽和断裂型内含肽，整体内含肽的两个剪接 区域共同存在于同一多肽片段上，断裂型内含肽的两个剪接区域分裂成两份或者更多片段，两个剪接区域存在于不同的多肽片段上，所以又可以称为分离内含肽。
在本申请中，术语“蛋白质反式剪接(protein trans-splicing)”通常是指由断裂型内含肽介导的蛋白质剪接反应。在这种类型的剪接过程中，首先是断裂型内含肽的N端片段(In)和C端片段(Ic)相互识别并以非共价键结合，结合后正确折叠其结构，重建活性中心的断裂型内含肽按照典型的蛋白质剪接途径完成蛋白质剪接反应，以天然肽键将两侧的蛋白质外显肽连接。
在本申请中，术语“抗原”通常是指能诱导机体发生免疫应答的物质，即能被T/B淋巴细胞表面的抗原受体(TCR/BCR)特异性识别与结合，活化T/B细胞，使之增殖分化，产生免疫应答产物(致敏淋巴细胞或抗体)，并能与相应产物在体内外发生特异性结合的物质。因此，抗原物质具备两个重要特性：免疫原性和免疫反应性。免疫原性即指抗原诱导机体发生特异性免疫应答，产生抗体和/或致敏淋巴细胞的能力；免疫反应性是指能与相应的免疫效应物质(抗体或致敏淋巴细胞)在体内外发事特异性结合反应的能力。
在本申请中，术语“肿瘤特异性抗原”通常是指某种肿瘤细胞表面而几乎不存在于正常细胞上的新抗原，又称为独特肿瘤抗原。
在本申请中，术语“肿瘤”通常是指机体在各种致癌因素作用下，局部组织的某一个细胞在基因水平上失去对其生长的正常调控，导致其克隆性异常增生而形成的新生物(neogrowth)，因为这种新生物多呈占位性块状突起，也称赘生物(neoplasm)。
在本申请中，术语“单克隆抗体”通常是指一群基本同源的抗体，即包含在该群的各个抗体除了可能的以微量存在的天然发生的突变之外是相同的。单克隆抗体是高度特异性的，直接针对单个抗原性位点。此外，与包括针对不同决定簇(表位)的不同抗体的多克隆抗体制备物相反，每个单克隆抗体针对抗原上的单一决定簇修饰语“单克隆”不是被解释为需要通过任何特殊方法产生抗体。例如，所述单克隆抗体可以通过杂交瘤技术制备或者通过使用重组DNA方法在细菌、真核动物或植物细胞中产生单克隆抗体也可以得自噬菌体抗体文库，使用例如Clackson etal.,Nature,352:624-628(1991)和Marks et al.,Mol.Biol.，222:581-597(1991)所述的技术进行。
在本申请中，术语“单链抗体(scFv)”通常是指由抗体重链可变区和轻链可变区通过短肽连接子(linker)连接而成的分子。
在本申请中，术语“嵌合抗体”通常是指这样的抗体，其中每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别，而该链的其余 区段则与另一物种中的相应序列同源。例如，轻链和重链的可变区均来自一个动物物种(如小鼠、大鼠等)的抗体的可变区，而恒定部分则与来自另一物种(如人)的抗体序列同源。例如，为获得嵌合抗体，可利用非人源的B细胞或杂交瘤细胞产生可变区，而与其组合的恒定区则来自人。所述可变区具有易于制备的优点，并且其特异性不受与其组合的恒定区的来源的影响。同时，由于嵌合抗体的恒定区可来源于人类，因此嵌合在注射时抗体引发免疫应答的可能性会低于使用恒定区为非人来源的抗体。
在本申请中，术语“人源化抗体”通常是指使用遗传工程技术将衍生自非人物种(例如小鼠或大鼠)的抗体、免疫球蛋白结合蛋白和多肽对人体的免疫原性降低，同时仍保留原始抗体的抗原结合特性的改造抗体。例如，可以使用CDR移植(Jones et al.,Nature 321:522(1986))及其变体；包括“重塑”(reshaping)，(Verhoeyen,et al.,1988 Science 239:1534-1536；Riechmann,et al.,1988 Nature 332:323-337；Tempest,et al.,Bio/Technol 1991 9:266-271)，“高度加成”(hyperchimerization)，(Queen,et al.,1989 Proc Natl Acad Sci USA 86:10029-10033；Co,et al.,1991 Proc Natl Acad Sci USA 88:2869-2873；Co,et al.,1992 J Immunol 148:1149-1154)和“贴面”(veneering)，(Mark,et al.,“Derivation of therapeutically active humanized and veneered anti-CD18antibodies.”In:Metcalf B W,Dalton B J,eds.Cellular adhesion:molecular definition to therapeutic potential.New York:Plenum Press,1994:291-312)等技术手段，对非人源的结合域进行人源化。如果其他区域，例如铰链区和恒定区结构域也源自非人来源，则这些区域也可以被人源化。
在本申请中，术语“全人源抗体”通常是指抗体所有全部(包括抗体的恒定区部分，CH和CL区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述多肽或多肽片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲 本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的多肽或多肽片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞。
在本申请中，术语“同一性”通常是指候选序列与特定肽或多肽序列相比，相同的氨基酸残基的数目占所有的氨基酸残基的数目的百分比。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
组合物及其制备方法
一方面，本申请提供一种组合物，组合物可以是一种多肽组合物，组合物可以包含第一多肽和第二多肽，其中，第一多肽可包含第一毒素片段和第一内含肽片段，第二多肽可包含第二毒素片段和第二内含肽片段；其中第一多肽可以与第二多肽不同；第一毒素片段和第二毒素片段可以均无生物毒性；且第一多肽和第二多肽可通过第一内含肽片段与第二内含肽片段的相互作用而使第一毒素片段与第二毒素片段形成有生物毒性的毒素。
在本申请中，术语“第一毒素片段”通常是指没有生物毒性的毒素片段，它可以不包含完整的毒素。在一定条件下，第一毒素片段可以和其他毒素片段形成有生物毒性的毒素。
在本申请中，术语“第二毒素片段”通常是指没有生物毒性的毒素片段，它可以不包含完整的毒素。在一定条件下，第二毒素片段可以和其他毒素片段形成有生物毒性的毒素。例如，在一定条件下，所述第二毒素片段可以和所述第一毒素片段形成有生物毒性的毒素。
在本申请中，术语“第一内含肽片段”通常是指内含肽的部分片段，它可以不包含完整的内含肽片段。在一定条件下，第一内含肽片段可以和其他内含肽片段发生相互作用，使得所述第一毒素片段和所述第二毒素片段形成有生物毒性的毒素。
在本申请中，术语“第二内含肽片段”通常是指内含肽的部分片段，它可以不包含完整的内含肽片段。在一定条件下，第二内含肽片段可以和其他内含肽片段发生相互作用，使得所述第一毒素片段和所述第二毒素片段形成有生物毒性的毒素。例如，在一定条件下，第二内含肽片段可以和第一内含肽片段发生相互作用，使得所述第一毒素片段和所述第二毒素片段形成有生物毒性的毒素。
在本申请中，术语“第一多肽”通常是指没有生物毒性的包含第一毒素片段和第一内含肽片段的多肽。在一定条件下，第一多肽中的第一内含肽片段可以和其他多肽中的内含肽片段发生相互作用，使得第一多肽中的第一毒素片段和其他多肽中的毒素片段形成有生物毒性的毒素。
在本申请中，术语“第二多肽”通常是指没有生物毒性的包含第二毒素片段和第二内含肽片段的多肽。在一定条件下，第二多肽中的第二内含肽片段可以和其他多肽中的内含肽片段发生相互作用，使得第二多肽中的第二毒素片段和其他多肽中的毒素片段形成有生物毒性的毒素。例如，在一定条件下，第二多肽中的第二内含肽片段可以和第一多肽中的第一内含肽片段发生相互作用，使得第二多肽中的第二毒素片段和第一多肽中的第一毒素片段形成有生物毒性的毒素。
在本申请中，所述组合物可以包含第一多肽和第二多肽，所述第一多肽可以包含所述第一毒素片段和所述第一内含肽片段，所述第二多肽可以包含所述第二毒素片段和所述第二内含肽片段，第一多肽和第二多肽的氨基酸序列可以是不同的，例如，第一多肽和第二多肽的氨基酸序列的同一性小于99％、小于98％、小于95％、90％、小于80％、小于70％、小于60％、小于50％、小于40％、小于30％、小于20％、小于10％或更小，所述第一毒素片段和所述第二毒素片段可以均无生物毒性，所述第一多肽和第二多肽也可以均无生物毒性。在一定条件下，第一内含肽片段和第二内含肽片段可以相互作用，使得第一毒素片段与第二毒素片段可以形成有生物毒性的毒素。
在本申请中，第一毒素片段可以与第二毒素片段不同。在本申请中，第一毒素片段和第二毒素片段的氨基酸序列可以是不同的，例如，第一毒素片段和第二毒素片段的氨基酸序列的同一性小于99％、小于98％、小于95％、90％、小于80％、小于70％、小于60％、小于50％、小于40％、小于30％、小于20％、小于10％或更小。在某些情形中，第一毒素片段和第二毒素片段可以源自相同的毒素。第一毒素片段和第二毒素片段可以截取自相同的毒素的不同序列位置。
在本申请中，第一毒素片段可以包含毒素的毒性活性区第一片段，第二毒素片段可以包含毒素的毒性活性区第二片段，且毒性活性区第一片段和毒性活性区第二片段构成所述毒素的完整毒性活性区。
在本申请中，术语“毒性活性区第一片段”通常是指毒素的毒性活性区的部分片段，毒性活性区第一片段不包含毒素的完整毒素活性区，在一定条件下，毒性活性区第一片段可以和其他的毒性活性区的片段形成完整的毒性活性区。
在本申请中，术语“毒性活性区第二片段”通常是指毒素的毒性活性区的部分片段，毒性活性区第二片段不包含毒素的完整毒素活性区，在一定条件下，毒性活性区第二片段可以和其他的毒性活性区的片段形成完整的毒性活性区。例如，在一定条件下，毒性活性区第二片段可以和毒性活性区第一片段形成完整的毒性活性区。
在本申请中，毒素可以包含毒性活性区。在本申请中，第一毒素片段可以包含毒素的毒性活性区第一片段，第二毒素片段可以包含相同毒素的毒性活性区第二片段，第一毒素片段和第二毒素片段都不包含所述毒素的完整的毒性活性区。在一定条件下，毒性活性区第一片段和毒性活性区第二片段可以构成所述毒素的完整的毒性活性区。
在本申请中，毒素还可以包含转位区。在本申请中，第二毒素片段可以不包含毒素的转位区或其片段，第一毒素片段还可以包含所述毒素的转位区或其片段。第二毒素片段可以不包含毒素完整的转位区，或者第二毒素片段可以不包含毒素的转位区片段。第一毒素片段还可以包含所述毒素完整的转位区，或者第一毒素片段还可以包含所述毒素的转位区片段。例如，第一毒素片段可以包含毒素的毒性活性区第一片段和毒素完整的转位区，第二毒素片段可以包含相同的毒素的毒性活性区第二片段。例如，第一毒素片段可以包含毒素的毒性活性区第一片段和毒素的转位区片段，第二毒素片段可以包含相同的毒素的毒性活性区第二片段。
在本申请中，所述毒素可以选自以下组：细菌毒素、人源毒素和植物毒素。在本申请中，所述毒素可以是细菌毒素、人源毒素、植物毒素或其组合。在本申请中，所述毒素可以选自以下组：绿脓杆菌外毒素和白喉毒素。例如，毒素可以是绿脓杆菌外毒素、白喉毒素或其组合。在本申请中，所述毒素可以选自以下组：蓖麻毒素、皂草素和白树毒素。例如，毒素可以是蓖麻毒素、皂草素、白树毒素或其组合。
在本申请中，所述毒素可以为绿脓杆菌外毒素(PE)的截短体PE38，截短体PE38可包含如SEQ ID NO:1和SEQ ID NO:16中任一项所示的氨基酸序列。
在本申请中，术语“截短体PE38”通常是指由PE截去了细胞结合区之后的片段。完整的PE含有三个功能区，即细胞结合区(Ia区，1-252aa)、转位区(II区，253-364aa)和毒性活性区(III区，400-613aa)，转位区可使毒素跨膜达到胞质区的功能区，毒性活性区具有ADP核糖基化功能，是使延长因子eEF2失活、杀伤细胞的关键功能区。PE的截短体PE38截去了细胞结合区(Ia区，1-252aa)，因此截短体PE38仅包括转位区(II区，253-364aa)和毒性活性区(III区，400-613aa)。
在本申请中，所述第一内含肽片段可以与所述第二内含肽片段不同，所述第一内含肽片段和所述第二内含肽片段可以源自相同的内含肽，所述内含肽可以为断裂型内含肽。例如，第一内含肽片段和第二内含肽片段的氨基酸序列可以是不同的，例如，第一内含肽片段和第二内含肽片段的氨基酸序列的同一性小于99％、小于98％、小于95％、90％、小于80％、小于70％、小于60％、小于50％、小于40％、小于30％、小于20％、小于10％、或更小。第一内含肽片段和第二内含肽片段可以是相同的断裂型内含肽的部分片段，且第一内含肽片段和 第二内含肽片段可以源自相同断裂型内含肽的不同序列位置。
在本申请中，所述断裂型内含肽可以选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。例如，所述断裂型内含肽可以是SsP DnaB、Ssp DnaE、Npu DnaE或其组合。
如图14所示，在本申请中，在第一多肽中，第一毒素片段的C端可以与第一内含肽片段的N端直接或间接相连，第一毒素片段C端的氨基酸残基可以源自所述毒素的无规卷曲区，在第二多肽中，第二毒素片段的N端可以与第二内含肽片段的C端直接或间接相连，所述第二毒素片段N端的氨基酸残基源自所述毒素的无规卷曲区。
在本申请中，C端和N端是多肽链的两个末端，术语“C端的氨基酸残基”通常是指在多肽的末端，可以携带游离的α-羧基的氨基酸残基。
在本申请中，术语“N端的氨基酸残基”通常是指在多肽链的末端，可以携带的α-氨基的氨基酸残基。
在本申请中，术语“无规卷曲区”通常是指在肽链中，相对没有规律性的排布的环或者卷曲结构的区域，无规卷曲是除α-螺旋、β-折叠、β-转角之外的蛋白质常见的二级结构。
例如，将所述毒素在无规卷曲区进行分割，得到第一毒素片段和第二毒素片段，在无规卷曲区的分割位点处对应第一毒素片段的C端和第二毒素片段的N端。
在第一多肽中，第一毒素片段的C端与第一内含肽片段的N端二者之间可以直接相连，或者可以通过连接子或其他肽链间接相连；在第二多肽中，第二毒素片段的N端与第二内含肽片段的C端直接，或者可以通过其他肽链间接相连。
在本申请中，所述第二毒素片段N端第1-3位的氨基酸残基可以依次为CFN。在本申请中，第二毒素片段N端的第1位的氨基酸残基可以为半胱氨酸(C)，第二毒素片段N端的第2位的氨基酸残基可以为苯丙氨酸(F)，第二毒素片段N端的第3位的氨基酸残基可以为天冬氨酸(N)。
在本申请中，所述第一内含肽片段可以包含所述断裂型内含肽的N端蛋白质区域；且所述第二内含肽片段包含所述断裂型内含肽的C端蛋白质区域。
在本申请中，术语“N端蛋白质区域”通常是指在多肽链中，靠近N端的区域。在某些情形中，所述断裂型内含肽的N端蛋白质区域可以为Npu DnaE的N端蛋白质区域。
在本申请中，术语“C端蛋白质区域”通常是指在多肽链中，靠近C端的区域。在某些情形中，所述断裂型内含肽的C端蛋白质区域可以为Npu DnaE的C端蛋白质区域。
如图14所示，在本申请中，组合物可以包含第一多肽和第二多肽，第一多肽可以包含第一毒素片段和第一内含肽片段，第二多肽可以包含第二毒素片段和第二内含肽片段。第一毒 素片段可以包含毒素的毒性活性区第一片段和转位区，第二毒素片段可以包含毒素的毒性活性区第二片段。从N端到C端，第一多肽可以依次包含毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段，毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段之间可以直接或间接连接。从N端到C端，第二多肽可以依次包含第二内含肽片段和毒素的毒性活性区第二片段，第二内含肽片段和毒素的毒性活性区第二片段之间可以之间或间接连接。其中第二毒素片段的N端的三个氨基酸残基进行可以定点突变，使其N端第1-3位的氨基酸残基依次为CFN。
在本申请中，如图1所示，组合物可以包含第一多肽和第二多肽，第一多肽(如图1A)包含第一毒素片段(简称作Pn)和第一内含肽片段(简称作In)，第二多肽(如图1B)包含第二毒素片段(简称作Pc)和第二内含肽片段(简称作Ic)，In的N端融合在Pn的C端，Pc的N端融合在Ic的C端。为了提高断裂型内含肽进行反式剪接反应的效率，对Pc的N端的氨基酸进行定点突变，使得Pc的N端第1-3位的氨基酸残基依次为CFN。当所述组合物处于还原性的条件下时，例如处于肿瘤的微环境中，In和Ic发生内含肽反式剪接反应(如图1D)，Pn和Pc连接形成具有生物毒性的毒素(如图1C)，对肿瘤细胞具有杀伤作用。
在本申请中，所述毒素可以为绿脓杆菌外毒素(PE)的截短体PE38，所述断裂型内含肽为Npu DnaE，Npu DnaE可以截成N端蛋白质区域和C端蛋白质区域，N端蛋白质区域为Npu DnaE的N端蛋白质区域，C端蛋白质区域为Npu DnaE的C端蛋白质区域，N端蛋白质区域构成第一内含肽片段，C端蛋白质区域构成第二内含肽片段。在本申请中，PE38的毒性活性区(III区，400-613aa)包括无规卷曲区、α螺旋区和片层区，为了在内含肽发生反式剪接反应之后，第一毒素片段和第二毒素片段能够进行连接并恢复原有构象，可以将截短体PE38在无规卷曲区进行分割，而不是在α螺旋区或片层区。通过使用Npu DnaE将PE38在无规卷曲区进行分割，得到无生物毒性的第一毒素片段和第二毒素片段。在本申请中，为了提高所述断裂型内含肽介导的反式剪接反应，可以对第二毒素片段的N端的三个氨基酸残基进行定点突变，使其N端第1-3位的氨基酸残基依次为CFN。定点突变后，有利于第一内含肽片段和第二内含肽片段的蛋白质发生相互作用，从而使得第一毒素片段的C端与所述第二毒素片段的N端连接形成有生物毒性的毒素，使得组合物对肿瘤细胞具有杀伤作用。
在本申请中，组合物包含第一多肽和第二多肽，所述第一内含肽片段可以包含如SEQ ID NO:2所示的氨基酸序列，所述第二内含肽片段可以包含如SEQ ID NO:3所示的氨基酸序列，所述第一毒素片段可以包含如SEQ ID NO:4所示的氨基酸序列，所述第二毒素片段可以包含如SEQ ID NO:5所示的氨基酸序列，所述第一多肽可以包含如SEQ ID NO:6所示的氨基酸序 列，所述第二多肽可以包含如SEQ ID NO:7和SEQ ID NO:15中任一项所示的氨基酸序列。在本申请中，肿瘤微环境的还原性可以促进所述断裂型内含肽的蛋白质反式剪接反应，从而使得所述组合物在肿瘤细胞表面时，第一内含肽片段和所述第二内含肽片段之间发生蛋白质反式剪接作用，使得第一毒素片段与所述第二毒素片段可以形成有生物毒性的毒素，肿瘤细胞摄取毒素后，可发挥杀伤病变细胞的功能。
如图15所示，在本申请中，在第一多肽中，第一毒素片段的N端可以与第一内含肽片段的C端直接或间接相连，第一毒素片段N端的氨基酸残基可以源自所述毒素的无规卷曲区，在第二多肽中，第二毒素片段的C端可以与第二内含肽片段的N端直接或间接相连，所述第二毒素片段C端的氨基酸残基源自所述毒素的无规卷曲区。例如，将所述毒素在无规卷曲区进行分割，得到第一毒素片段和第二毒素片段，在无规卷曲区的分割位点处对应第一毒素片段的N端和第二毒素片段的C端，得到的第一毒素片段的N端与第一内含肽片段的C端直接或间接相连，第二毒素片段的C端与第二内含肽片段的N端直接或间接相连。
在本申请中，所述第一毒素片段N端第1-3位的氨基酸残基依次为CFN。在本申请中，第一毒素片段N端的第1位的氨基酸残基可以为半胱氨酸(C)，第一毒素片段N端的第2位的氨基酸残基可以为苯丙氨酸(F)，第一毒素片段N端的第3位的氨基酸残基可以为天冬氨酸(N)。
在本申请中，所述第一内含肽片段可以包含所述断裂型内含肽的C端蛋白质区域；且所述第二内含肽片段可以包含所述断裂型内含肽的N端蛋白质区域。在本申请中，在第一多肽中，第一毒素片段的N端可以与所述第一内含肽片段的C端直接或间接连接，在第二多肽中，第二毒素片段的C端可以与所述第二内含肽片段的N端直接或间接相连。
如图15所示，在本申请中，组合物可以包含第一多肽和第二多肽，第一多肽可以包含第一毒素片段和第一内含肽片段，第二多肽可以包含第二毒素片段和第二内含肽片段，第一毒素片段可以包含毒素的毒性活性区第一片段和转位区，第二毒素片段可以包含毒素的毒性活性区第二片段。从N端到C端，第一多肽可以依次包含第一内含肽片段、毒素的毒性活性区第一片段和毒素的转位区，第一内含肽片段、毒素的毒性活性区第一片段和毒素的转位区可以直接或间接连接。从N端到C端，第二多肽可以依次包含毒素的毒性活性区第二片段和第二内含肽片段，毒素的毒性活性区第二片段和第二内含肽片段之间可以之间或间接连接。其中第一毒素片段的N端的三个氨基酸残基可以进行定点突变，使其N端第1-3位的氨基酸残基依次为CFN。
在本申请中，所述第一内含肽片段与所述第二内含肽片段的所述相互作用可以包含所述 第一内含肽片段和第二内含肽片段的蛋白质反式剪接作用。在本申请中，蛋白质反式剪接反应可以是由断裂型内含肽介导的蛋白质剪接反应。
在本申请中，第一多肽和/或第二多肽还可以包括靶向部分，所述靶向部分可以靶向肿瘤特异性抗原。
在本申请中，术语“靶向部分”通常是指特异性结合和/或识别肿瘤抗原的部分。例如，第一多肽和第二多肽可以分别包括靶向部分，所述靶向部分可以特异性结合和/或识别肿瘤特异性抗原。例如，所述第一多肽可以包括靶向部分，所述靶向部分可以特异性结合和/或识别肿瘤特异性抗原。例如，所述第二多肽可以包括靶向部分，所述靶向部分可以特异性结合和/或识别肿瘤特异性抗原。在本申请中，所述靶向部分可以包含抗体或其抗原结合片段或变体。所述抗体可以选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。在本申请中，肿瘤特异性抗原可以选自以下组：HER2、PD-L1、EGFR、mesothelin和Lewis Y。例如，靶向部分可以是特异性结合和/或识别HER2、PD-L1、EGFR、mesothelin和/或Lewis Y的单克隆抗体。例如，靶向部分可以是特异性结合和/或识别HER2、PD-L1、EGFR、mesothelin和/或Lewis Y的单链抗体。例如，靶向部分可以是特异性结合和/或识别HER2、PD-L1、EGFR、mesothelin和/或Lewis Y的嵌合抗体。例如，靶向部分可以是特异性结合和/或识别HER2、PD-L1、EGFR、mesothelin和/或Lewis Y的人源化抗体。例如，靶向部分可以是特异性结合和/或识别HER2、PD-L1、EGFR、mesothelin和/或Lewis Y的全人源抗体。
在本申请中，所述抗原结合片段可以选自下组：Fab，Fab’，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。例如，靶向部分可以是Fab、Fab’、F(ab) 2、dAb、分离的互补决定区CDR、Fv、scFv或其组合。
在本申请中，术语“Fab”通常是指由完整的轻链、重链可变区(VH)和重链的第一恒定结构域(CH1)组成的抗原结合片段。每个Fab片段相对于抗原结合是单价的，即它具有单一的抗原结合位点。
在本申请中，术语“Fab'”通常是指与Fab片段相比在CH1结构域的羧基末端具有几个额外的残基的抗原结合片段，Fab'包括来自抗体铰链区的一个或多个半胱氨酸。
在本申请中，术语“F(ab) 2”通常是指由半胱氨酸相连接的成对的Fab片段所得到的抗原结合片段。
在本申请中，术语“Fv”通常是指由抗体单臂的VL和VH结构域组成的抗原结合片段。
在本申请中，术语“互补决定区CDR”通常是指轻链可变区(VL)与VH的3个高变区(HVR)，该部位因在空间结构上可与抗原决定簇形成精密的互补，故高变区又称互补性决 定区。
在本申请中，所述抗体或其抗原结合片段的所述变体可以是在所述抗体或所述其抗原结合片段中经过取代、缺失或添加一个或多个(例如，至少1个，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个，至少15个，至少20个，至少30个，或更多)氨基酸的蛋白质或多肽。
在本申请中，所述抗体或其抗原结合片段的所述变体可以是与所述抗体或所述其抗原结合片段具有至少90％(例如，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或更多)序列同源性的蛋白质或多肽。
在本申请中，所述第一靶向部分和/或所述第二靶向部分可以是ScFv。例如，所述第一靶向部分可以为ScFv。例如，所述第二靶向部分可以是ScFv。例如，所述第一靶向部分和所述第二靶向部分都可以是ScFv。在本申请中，所述靶向部分可以包含如SEQ ID NO:8所示的氨基酸序列。
在本申请中，第一多肽可以包含第一靶向部分，且所述第一靶向部分可以位于所述第一毒素片段的N端。例如，第一靶向部分可以与第一毒素片段的N端氨基酸残基直接连接；或者第一靶向部分可以与第一毒素片段的N端氨基酸残基间接连接，间接连接可以是通过插入连接肽连接或者插入其他肽链连接。
在本申请中，第二多肽可以包含第二靶向部分，且所述第二靶向部分可以位于所述第二毒素片段的N端。例如，第二靶向部分可以与第二毒素片段的N端的氨基酸残基直接连接；或者第二靶向部分可以与第二毒素片段的N端的氨基酸残基间接连接，间接连接可以是通过插入连接肽连接或者插入其他肽链连接。
如图14所示，在本申请中，组合物可以包含第一多肽和第二多肽，从N端到C端，第一多肽可以依次包含毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段，毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段之间可以直接或间接连接；从N端到C端，第二多肽依次包含第二内含肽片段和毒素的毒性活性区第二片段，第二内含肽片段和毒素的毒性活性区第二片段之间可以直接或间接连接。
对于如图14所示的组合物，在某些情况下，第一多肽还可以包含第一靶向部分，第二多肽还可以包含第二靶向部分。在某些情形中，从N端到C端，第一多肽可以依次包含第一靶向部分、毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段，第一靶向部分、毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段之间可以直接或间接连接；从N端到C端，第二多肽依次包含第二内含肽片段、和毒素的毒性活性区第二片段和第二靶向部 分，第二靶向部分、第二内含肽片段和毒素的毒性活性区第二片段之间可以直接或间接连接。在一定条件下，如在组合物中加入还原剂时，或者组合物处于肿瘤的微环境时，第一内含肽片段与第二内含肽片段相互作用，使得第一多肽的第一靶向部分和第一毒素片段(包含毒素的转位区和毒性活性区第一片段)以及第二多肽的第二靶向部分和第二毒素片段(包含毒性活性区第二片段)连接形成免疫毒素，从N端到C端，免疫毒素依次包含第一靶向部分、毒素的转位区、毒性活性区第一片段、毒性活性区第二片段和第二靶向部分，第一靶向部分、毒素的转位区、毒性活性区第一片段、毒性活性区第二片段之间可以直接或间接连接。
对于如图14所示的组合物，在某些情况下，第一多肽还可以包含第一靶向部分，第二多肽可以不包含靶向部分，从N端到C端，第一多肽依次包含第一靶向部分、毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段，第一靶向部分、毒素的转位区、毒素的毒性活性区第一片段和第一内含肽片段之间可以直接或间接连接；从N端到C端，第二多肽依次包含第二内含肽片段和毒素的毒性活性区第二片段，第二内含肽片段和毒素的毒性活性区第二片段之间或间接连接。在一定条件下，如在组合物中加入还原剂时，或者组合物处于肿瘤的微环境时，第一内含肽片段与第二内含肽片段相互作用，使得第一多肽的第一靶向部分和第一毒素片段(包括毒素的转位区、毒素的毒性活性区第一片段)和第二多肽的第二毒素片段(包含毒性活性区第二片段)连接形成免疫毒素，从N端到C端，免疫毒素依次包含第一靶向部分、毒素的转位区、毒素的毒性活性区第一片段和毒性活性区第二片段，第一靶向部分、毒素的转位区、毒素的毒性活性区第一片段和毒性活性区第二片段之间直接或间接连接。
如图15所示，在本申请中，组合物可以包含第一多肽和第二多肽，从N端到C端，第一多肽可以依次包含第一内含肽片段、毒素的毒性活性区第一片段和毒素的转位区，第一内含肽片段、毒素的毒性活性区第一片段和毒素的转位区可以直接或间接连接；从N端到C端，第二多肽可以依次包含毒素的毒性活性区第二片段和第二内含肽片段，毒素的毒性活性区第二片段和第二内含肽片段之间可以之间或间接连接。
对于如图15所示的组合物，在某些情况下，第一多肽还可以包含第一靶向部分，第二多肽还可以包含第二靶向部分。在某些情形中，从N端到C端，第一多肽可以依次包含第一内含肽片段、毒素的毒性活性区第一片段、毒素的转位区和第一靶向部分，第一靶向部分、第一内含肽片段、毒素的毒性活性区第一片段和毒素的转位区之间可以直接或间接连接；从N端到C端，第二多肽依次包含第二靶向部分、毒素的毒性活性区第二片段和第二内含肽片段，第二靶向部分、毒素的毒性活性区第二片段和第二内含肽片段之间可以直接或间接连接。在一定条件下，如在组合物中加入还原剂时，或者组合物处于肿瘤的微环境时，第一内含肽片 段与第二内含肽片段相互作用，使得第二多肽的第二靶向部分和第二毒素片段(包含毒性活性区第二片段)和第一多肽的第一靶向部分
和第一毒素片段(包含毒素的毒性活性区第一片段和毒素的转位区)连接形成免疫毒素，从N端到C端，免疫毒素依次包含第二靶向部分、毒素的毒性活性区第二片段、毒素的毒性活性区第一片段、毒素的转位区和第二靶向部分，第二靶向部分、毒素的毒性活性区第二片段、毒素的毒性活性区第一片段和毒素的转位区直接或间接连接。
对于如图15所示的组合物，在某些情况下，第一多肽可以不包含靶向部分，第二多肽还可以包含第二靶向部分，从N端到C端，第一多肽可以依次包含第一内含肽片段、毒素的毒性活性区第一片段和毒素的转位区，第一内含肽片段、毒素的毒性活性区第一片段和毒素的转位区可以直接或间接连接；从N端到C端，第二多肽可以依次包含第二靶向部分、毒素的毒性活性区第二片段和第二内含肽片段，第二靶向部分、第二内含肽片段和毒素的毒性活性区第二片段之间直接或间接连接。在一定条件下，如在组合物中加入还原剂时，或者组合物处于肿瘤的微环境时，第一内含肽片段与第二内含肽片段相互作用，使得第二多肽的第二靶向部分和第二毒素片段(包含毒素的毒性活性区第二片段)和第一多肽的第一毒素片段(包含毒素的毒性活性区第一片段和毒素的转位区)连接形成免疫毒素，从N端到C端，免疫毒素依次包含第二靶向部分、毒素的毒性活性区第二片段、毒素的毒性活性区第一片段和毒素的转位区，第二靶向部分、毒素的毒性活性区第二片段、毒素的毒性活性区第一片段和毒素的转位区之间之间或间接连接。
例如，在本申请中，如图2所示，组合物可以包含第一多肽和第二多肽，本申请的第一多肽可以包含第一靶向部分，第二多肽可以不包含靶向部分，所述第一靶向部分可以为靶向HER2的ScFv，毒素采用截短体PE38。在第一多肽(如图2B所示)中，从N端到C端，依次可以包括靶向HER2的ScFv抗原结合片段、第一毒素片段(Pn)和第一内含肽片段(In)，所述第一多肽命名为ScFvPnIn。在第二多肽(如图2C所示)中，从N端到C端，依次包括第二内含肽片段(Ic)和第二毒素片段(Pc)，所述第二多肽命名为IcPc。其中T7为大肠杆菌表达载体的启动子。所述第一毒素片段的C端的氨基酸残基源自所述截短体PE38的无规卷曲区，第二毒素片段N端的氨基酸残基源自所述截短体PE38的无规卷曲区。为了提高断裂型内含肽进行反式剪接反应的效率，对Pc的N端的氨基酸进行定点突变，使得Pc的N端第1-3位的氨基酸残基依次为CFN。利用ScFv靶向HER2的作用，ScFvPnIn可以靶向到达肿瘤细胞表面时，利用肿瘤微环境的还原性，ScFvPnIn中的In和IcPc中的Ic发生内含肽反式剪接反应，Pn和Pc重新连接，得到免疫毒素ScFvPM1(如图2A所示)，对肿瘤细胞具有 杀伤作用。所述免疫毒素ScFvPM1的氨基酸序列如SEQ ID NO:10所示。
另一方面，本申请提供一种所述组合物的制备方法，制备方法包括以下步骤：1)提供所述第一多肽；2)提供所述第二多肽；3)将所述第一多肽与所述第二多肽混合以得到所述组合物。在本申请中，上述步骤1、2和3可以同时发生或者不同时发生，对于步骤1、2和3不同时发生的情形，步骤1可以发生在步骤2之前，或者步骤2发生在步骤1之前，或者步骤1和2同时发生。在本申请中，组合物中第一多肽与所述第二多肽的摩尔比可以为10:1-1:10(例如，10:1-1:10；9:1-1:9；8:1-1:8；7:1-1:7；6:1-1:6；5:1-1:5；4:1-1:4；3:1-1:3；2:1-1:2)。根据上述制备方法得到的组合物可以包含所述第一多肽和所述第二多肽，且所述第一多肽和第二多肽可以没有生物毒性。
另一方面，本申请提供一种所述毒素的制备方法，制备方法包括以下步骤：1)提供所述第一多肽；2)提供所述第二多肽；3)将所述第一多肽与所述第二多肽混合以得到所述毒素。
在本申请中，无生物毒性的所述第一多肽和无生物毒性的所述第二多肽经所述混合后可以得到具备生物毒性的所述毒素。在本申请所述的制备方法中，步骤3)所述的混合还包含加入还原剂混合。在本申请中，加入还原剂后，所述第一多肽中的第一内含肽片段和第二多肽中的第二内含肽片段可以发生反式剪接作用，使得第一多肽中的第一毒素片段和第二多肽中的第二毒素片段形成有生物毒性的毒素。
在本申请中，所述制备方法还可以包括加入还原剂。在本申请中，加入还原剂的步骤可以与上述步骤1、2和3同时发生或者不同时发生。在本申请中，术语“还原剂”通常是指在氧化还原反应中，失去电子或有电子偏离的物质。在本申请中，加入还原剂之后，所述第一多肽中的第一内含肽片段和第二多肽中的第二内含肽片段可以发生反式剪接作用，使得第一多肽中的第一毒素片段和第二多肽中的第二毒素片段形成有生物毒性的毒素。在本申请中，加入还原剂后，所述组合物中可以包含具备生物毒性的毒素和/或具备生物毒性的免疫毒素。
在本申请中，所述还原剂可以选自以下组：DTT和β巯基乙醇。例如，所述还原剂可以是DTT和β巯基乙醇。例如，所述还原剂可以是DTT。例如，所述还原剂可以是β巯基乙醇。
在本申请中，所述组合物中所述还原剂的浓度为0.001-10000nM，例如，0.001-10000nM；0.002-5000nM；0.003-2000nM；0.005-1000nM；0.01-500nM；0.015-200nM；0.02-100nM；0.05-80nM；0.1-50nM；0.5-20nM；或0.1-10nM。所述还原剂可以在步骤3中的所述混合的同时或所述混合之后加入。
在本申请中，制备方法还可以包括在加入所述还原剂后孵育所述组合物。在本申请中， 术语“孵育”通常是在一定温度下，将混合的样品静置。在申请中，在加入还原剂后，可以将包含第一多肽、第二多肽和还原剂的混合物，在一定的温度下，静置一段时间。
在本申请中，所述孵育的温度可以为1℃-50℃，例如1℃-50℃、4℃-50℃、4℃-45℃、4℃-40℃、4℃-37℃、8℃-37℃、13℃-37℃、17℃-37℃、17℃-35℃、17℃-30℃、17℃-25℃、17℃-23℃或20℃-23℃。在本申请中，所述孵育的时间可以为2-120分钟，例如2-120分钟、2-100分钟、2-80分钟、3-80分钟、4-80分钟、5-80分钟、10-80分钟、15-80分钟、20-80分钟、20-80分钟、40-80分钟、50-80分钟、50-70分钟或50-60分钟。例如，在1℃-50℃温度下，可以将包含第一多肽、第二多肽和还原剂的混合物静置2-120分钟、2-100分钟、2-80分钟、4-80分钟、5-80分钟、10-80分钟、15-80分钟、20-80分钟、20-80分钟、40-80分钟、50-80分钟或50-70分钟。例如，在4℃-45℃温度下，将包含第一多肽、第二多肽和还原剂的混合物静置2-120分钟、2-100分钟、2-80分钟、4-80分钟、5-80分钟、10-80分钟、15-80分钟、20-80分钟、20-80分钟、40-80分钟、50-80分钟或50-70分钟。例如，在4℃-37℃温度下，将包含第一多肽、第二多肽和还原剂的混合物静置2-120分钟、2-100分钟、2-80分钟、4-80分钟、5-80分钟、10-80分钟、15-80分钟、20-80分钟、20-80分钟、40-80分钟、50-80分钟或50-70分钟。例如，在17℃-37℃温度下，将包含第一多肽、第二多肽和还原剂的混合物静置2-120分钟、2-100分钟、2-80分钟、4-80分钟、5-80分钟、10-80分钟、15-80分钟、20-80分钟、20-80分钟、40-80分钟、50-80分钟或50-70分钟。例如，在4℃-17℃温度下，将包含第一多肽、第二多肽和还原剂的混合物静置2-120分钟、2-100分钟、2-80分钟、4-80分钟、5-80分钟、10-80分钟、15-80分钟、20-80分钟、20-80分钟、40-80分钟、50-80分钟或50-70分钟。
本申请所述的组合物可以具有以下性质中的一种或多种：1)可以将组合物分为无毒性的第一多肽和第二多肽分别用药，使其在所期望的靶点处(如肿瘤微环境下)恢复毒性，能够提高用药安全性，降低组合物对正常细胞的非特异性毒性；2)可以将组合物分成第一多肽和第二多肽两部分给药，具有较高的灵活性和可调节性，例如可以采用不同的给药方式、给药剂量和不同的给药组合方法，以达到更好的治疗效果；3)通过内含肽使组合物的第一多肽和第二多肽连接从而制备有活性的物质(如毒性)，制备方法具有普遍适用性，可以应用于多种类型的组合物，例如免疫毒素或者蛋白药物；4)当组合物被分割成无毒性的第一多肽和第二多肽两部分后，可以采用原核或真核细胞分别表达被分割成的两部分，且被表达的两部分对宿主细胞无毒性作用，例如，当免疫毒素的组成方式是完整抗体连接毒素时，用真核细胞表达有利于抗体的组装；5)针对不同的肿瘤相关抗原，组合物可以包含不同的抗体靶向部分， 可以提高组合物的靶向特异性。
载体、细胞、试剂盒和用途
另一方面，本申请提供一种载体，其可以包含编码第一多肽的核酸，和/或，其可以包含编码第二多肽的核酸。例如，所述载体可以包含编码第一多肽的核酸和编码第二多肽的核酸。例如，所述载体可以包含编码第一多肽的核酸。例如，所述载体可以包含编码第二多肽的核酸。
所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。另一方面，本申请提供一种细胞，其可以表达第一多肽，和/或，其可以表达第二多肽。例如，本申请提供一种细胞，其可以表达第一多肽和第二多肽。例如，本申请提供一种细胞，其可以表达第一多肽。例如，本申请提供一种细胞，其可以表达第二多肽。在某些实施方式中，每种或每个细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述细胞中，例如大肠杆菌等。可通过本领域已知的方法将本申请所述的载体引入所述细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请提供一种试剂盒，其可以包含第一多肽以及第二多肽。在本申请中，第一多肽和第二多肽可以在所述试剂盒中彼此不混合，即第一多肽和第二多肽是相互分离的放置在试剂盒中，彼此不会混合。在本申请中，第一多肽和第二多肽可以位于不同的容器内， 例如，第一多肽被放置在一个容器中，第二多肽被放置在另外一个容器中，上述两个容器相互独立，从而使得第二多肽和第二多肽彼此不混合。
在本申请中，试剂盒还可以包含还原剂。在本申请中，所述试剂盒可以包含第一多肽、第二多肽和还原剂。在本申请中，还原剂可以选自以下组：DTT和β巯基乙醇。例如，所述试剂盒可以包含第一多肽、第二多肽和DTT。例如，所述试剂盒可以包含第一多肽、第二多肽和β巯基乙醇。例如，所述试剂盒可以包含第一多肽、第二多肽、β巯基乙醇和DTT。
在本申请中，所述还原剂可以包含于独立的容器中。例如，容纳还原剂的容器和容纳其他组分(如第一多肽和第二多肽)的容器是相互独立的，也就是说，容纳第一多肽和第二多肽的容器不能再容纳还原剂，还原剂和其他组分(如第一多肽和第二多肽)是分离的放置的。
在本申请中，试剂盒可以包含所述组合物，所述组合物包含第一多肽和第二多肽，其中，所述第一多肽可包含第一毒素片段和第一内含肽片段，所述第二多肽可包含第二毒素片段和第二内含肽片段，所述第一内含肽片段与所述第二内含肽片段相互作用之前，所述组合物中的第一多肽和第二多肽均无生物毒性。
本申请试剂盒的组分可包含在分开的容器中(即具有分开部分的试剂盒)，或在单个容器内提供。此外，本申请的试剂盒还可包含所述组合物和/或实施所述方法的说明书。说明书可通过纸或电子形式的用户手册提供。例如，手册可包含用于解释当使用本申请的试剂盒实施上述方法或使用所述组合物时得到的结果的说明。
另一方面，本申请提供了一种所述组合物、所述试剂盒、所述载体和/或所述细胞在制备治疗疾病的药物中的用途，所述疾病可以包含肿瘤。在本申请中，所述肿瘤可以选自以下组：乳腺癌、黑色素瘤、卵巢癌、结肠癌、间皮瘤、腺体瘤、胰腺癌和膀胱癌。在本申请中，所述药物可以用于治疗包含肿瘤的疾病，例如治疗乳腺癌、黑色素瘤、卵巢癌、结肠癌、间皮瘤、腺体瘤、胰腺癌和/或膀胱癌。例如，本申请的药物组合物可以抑制或延缓包含肿瘤的疾病的发展或进展，可以减小肿瘤的大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
另一方面，本申请提供了所述组合物、所述试剂盒、所述载体和/或所述细胞，其能够治疗肿瘤。在本申请中，所述治疗肿瘤是指抑制肿瘤生长，可以减小肿瘤的大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
另一方面，本申请提供了一种治疗肿瘤的方法，其包括施用所述的组合物、所述试剂盒、所述载体和/或所述细胞。在本申请中，所述施用的方式包括口服给药、静脉内给药、肌肉内给药、在肿瘤部位的原位给药、吸入、直肠给药、阴道给药、经皮给药和/或通过皮下储存库给药。本申请还提供施用所述组合物的方法，其可以包括以下的步骤：将所述第一多肽与所 述第二多肽和还原剂混合施用。在体外条件下，所述第一多肽和所述第二多肽可以在还原性条件下形成具备生物毒性的所述毒素或所述免疫毒素，例如，可以对所述细胞产生杀伤作用，或者产生凋亡作用。
本申请还提供一种治疗肿瘤的方法，其包括向受试者施用所述第一多肽和所述第二多肽。在体内条件下，所述第一多肽和所述第二多肽可以在肿瘤细胞微环境条件下形成具备生物毒性的所述毒素或所述免疫毒素，例如，可以对所述肿瘤细胞产生杀伤作用，或者产生凋亡作用。
在本申请中，所述组合物的不同部分的施用时间可以不同。例如，不同部分可以间隔1秒、1分钟、1小时、10小时、1天或更多时间施用。在某些情形中，所述组合物的不同部分可以同时施用。本申请所述的组合物的不同部分可以采用相同或不同的施用方式，或者，也可以采用相同或不同的施用剂量。本申请的组合物的各部分的给药具有灵活性和可调节性，只要能达到所需的治疗效果即可。
实施例
实施例1表达载体构建
为了筛选合适的毒素断裂位点，首先需要对免疫毒素scFvPE38进行定点突变，所述免疫毒素scFvPE38的氨基酸序列如SEQ ID NO:9所示。采用定点突变的方式对待选位点的下游三个氨基酸进行突变，突变后的免疫毒素及其载体分别命名为scFvPM1、scFvPM2和scFvPM3。采用载体scFvPM1、scFvPM2和scFvPM3表达突变后的免疫毒素并纯化目的蛋白，得到的突变体scFvPM1、scFvPM2和scFvPM3的氨基酸序列分别如SEQ ID NO:10(其中截短体PM1的氨基酸序列如SEQ ID NO:16所示)、SEQ ID NO:11和SEQ ID NO:12所示。
用突变体scFvPM1、scFvPM2和scFvPM3处理HER2抗原阳性的SKOV3细胞，考察不同突变体对细胞活性的影响，如图3所示，scFvPM1突变体相对其他突变体具有更高的细胞毒性，因而选择scFvPM1对应的突变位点作为毒素的断裂位点，该突变位点也是断裂型内含肽的插入位点，对应于选定位点的下游三个氨基酸突变序列为“CFN”序列。
在选定的位点处将scFvPM1分割成两部分序列scFvPn和Pc，对应的scFvPn的氨基酸序列如SEQ ID NO:13所示，Pc的氨基酸序列SEQ ID NO:14所示。将scFvPn与断裂型内含肽Npu DnaE的N端(简称作In)融合，Npu DnaE的C端(简称作Ic)和Pc融合，对应的In的氨基酸序列如SEQ ID NO:2所示，Ic的氨基酸序列如SEQ ID NO:3。通过PCR(聚合酶链式反应)合成目的片段，并在目的片段两端分别引入起始密码子和终止密码子，以及限制性内切酶位点比如NdeI/HindIII等。将目的片段通过酶切连接的方式分别连接至含有T7启动子 的大肠杆菌表达载体pET28a上，获得目的载体pET-scFvPnIn(如图2B所示)和pET-IcPc(如图2C所示)。
实施例2蛋白的表达和纯化
将pET-scFvPnIn和pET-IcPc载体质粒分别转化大肠杆菌感受态BL21(DE3)，挑单克隆摇菌，LB培养液加入100μg/ml氨苄抗生素，37℃培养至细胞生长对数期(OD600＝0.6)，加终浓度1mmol/l诱导剂IPTG(异丙基硫代半乳糖苷)诱导蛋白表达。37℃诱导4h，5000rpm5-10min离心收集菌体，PBS缓冲液洗涤一次，5000rpm 5-10min离心收集菌体，高压均质机裂解菌体，离心收集包涵体沉淀，用含有1M盐酸胍的PBS缓冲液洗涤包涵体沉淀两次，然后用含有6M盐酸胍的PBS缓冲液溶解包涵体沉淀。将溶解后包涵体滴加至50倍体积的复性液中(100mM Tris-HCl，500mM精氨酸，1mM乙二胺四乙酸二钠，1mM还原型谷胱甘肽，0.1mM氧化型谷胱甘肽，pH 9.0-9.5)。
稀释复性后蛋白溶液分别用capto L和His-trap柱亲和纯化。scFvPnIn通过capto L柱纯化，柠檬酸缓冲液洗脱，随后用PH9.0的1M tris-Hcl缓冲液立即中和样品。IcPc通过His-trap柱纯化，不同咪唑浓度缓冲液洗脱，收集洗脱组分，用SDS-PAGE分析蛋白纯度。利用MILLIPORE Amicon Ultra(10MWCO)超滤离心管离心浓缩蛋白，并替换为PBS缓冲液，于-20℃或-80℃冻存。表达得到的scFvPnIn的氨基酸序列如SEQ ID NO:6所示，IcPc的氨基酸序列如SEQ ID NO:15所示。
实施例3体外反式剪接反应
断裂型内含肽介导免疫毒素两部分进行蛋白质反式剪接反应。将实施例2中纯化所得的scFvPnIn和IcPc两部分蛋白按照摩尔比1：1进行混合，同时分别加入1.0mM DTT、2.5mM DTT、5.0mM DTT、7.5mM DTT和10.0mM DTT，在37℃条件下孵育30min，结果如图4所示。“N”是指仅加入scFvPnIn的样品，“C”是指仅加入IcPc的样品，“1.0”、“2.5”、“5.0”、“7.5”和“10.0”分别是指加入1.0mM DTT、2.5mM DTT、5.0mM DTT、7.5mM DTT和10.0mM DTT的样品，结果发现，当加入1.0mM DTT时，scFvPnIn和IcPc快速发生反式剪接反应，在85KD处有明显条带出现(参见图4箭头)。
将实施例2中纯化所得的scFvPnIn和IcPc两部分蛋白按照摩尔比1：1进行混合，同时加入1.0mM DTT，然后将上述混合物分别置于4℃、17℃和37℃孵育30min，结果如图5所示。“N”是指仅加入scFvPnIn的样品，“C”是指仅加入IcPc的样品，样品“4”、“7”和“37”分别对应于置于4℃、17℃和37℃的样品，结果发现，静置于4℃时反式剪接反应即 可发生，17℃和37℃反应效率较高，在85KD处有明显条带出现(参见图5箭头)，说明产生了完整的免疫毒素。
将实施例2中纯化所得的scFvPnIn和IcPc两部分蛋白按照摩尔比1：1进行混合，同时加入1.0mM DTT，然后将上述混合物置于37℃下，分别静置0min、1min、5min、10min、30min、60min和120min，结果如图6所示，在85KD处有明显条带出现(参见图6箭头)。“N”是指仅加入scFvPnIn的样品，“C”是指仅加入IcPc的样品，样品“0”、“1”、“5”、“10”、“30”、“60”和“120”分别是指静置0min、1min、5min、10min、30min、60min和120min时的样品。结果发现，5min时反式剪接反应即可发生，生成完整的免疫毒素。在60min时反应到达平台期。反应结束需要去除巯基化合物，可以通过加入蛋白上样缓冲液(60mM Tris-HCl，pH6.8；2％SDS；0.1％溴酚兰；25％甘油，14.4mMβ-巯基乙醇)，以达到终止反应的目的。反应终止取样品进行SDS-PAGE检测。
实施例4免疫毒素抗原亲和力分析
采用流式细胞仪分析免疫毒素的抗原亲和力，考察对象是完整免疫毒素以及分割后免疫毒素的N部分，抗原高表达的肿瘤细胞SKOV3分别用完整免疫毒素和分割后免疫毒素N部分处理，冰上孵育1h后，PBS清洗，然后加抗毒素的抗体(rabbit anti-ETA)处理细胞，冰上孵育1h后，PBS清洗，再用荧光标记的二抗(Alexa488-anti rabbit IgG)处理，冰上孵育1h后，PBS清洗，上流式细胞仪分析，此外还设置PBS为“对照样品”，Herceptin为阳性对照。如图7所示，样品“1”、“2”、“3”和“4”分别是指“scFvPnIn”、“scFvPM1”、“Herceptin”和“对照样品”，测试结果发现，完整免疫毒素scFvPM1和分割后免疫毒素的N部分scFvPnIn对SKOV3细胞均具有明显的抗原亲和力。
实施例5免疫毒素入胞分析
通过共聚焦激光显微镜观察肿瘤细胞SKOV3对完整免疫毒素和分割后免疫毒素的摄取。无菌玻片至于12孔细胞培养板各孔，以5*10 4/ml密度铺细胞，培养过夜后加毒素处理，完整免疫毒素和分割后免疫毒素的两部分分别处理细胞4h，洗去细胞表面未结合免疫毒素，用4％多聚甲醛固定细胞，0.2％TritonX-100透化细胞，用3％BSA封闭后分别用抗毒素的一抗和荧光标记的二抗处理，细胞核用4，6-联脒-2-苯基吲哚(DAPI)染色，取出细胞爬片，倒置于载玻片上，于共聚焦激光显微镜下观察并拍摄照片。如图8所示，“N”、“C”和“N+C”分别是指单独的scFvPnIn、单独的IcPc和混合的scFvPnIn和IcPc(加入还原剂)的情况。scFvPM1和scFvPnIn均可以用绿色荧光二抗标记，IcPc连接红色荧光蛋白，因此，单独的C 部分IcPc以及反应生成的免疫毒素均可以显示红色荧光。从图8可以看出，SKOV3细胞对完整免疫毒素摄取较多，对分割后免疫毒素N部分scFvPnIn也有明显摄取，但是分割后免疫毒素C部分IcPc无明显摄取，scFvPnIn和IcPc两部分合用时，细胞内显示出绿色和红色荧光共定位的区域，表明两部分反应生成新的完整免疫毒素。
实施例6免疫毒素细胞毒性分析
将免疫毒素scFvPM1分割后无毒性的scFvPnIn和IcPc后，分别以单用与合用的方式处理SKOV3细胞，处理72h后采用CCK8试剂盒检测细胞活力，在图9-图11中，“对照样品”、“N”、“C”、“N+C”、“N+C+DTT”和“DTT”分别是指PBS溶液、单独的scFvPnIn、单独的IcPc、混合的scFvPnIn和IcPc(未加入还原剂)、混合的scFvPnIn和IcPc(加入还原剂DTT)和单独的DTT的情况。如图9所示，单独的scFvPnIn和IcPc对细胞活力无影响，scFvPnIn和IcPc两者混合使用时，还原性条件下可以降低细胞活性，并且单独的还原剂对细胞活性无影响，说明合用时生成完整的免疫毒素并且恢复了免疫毒素的细胞杀伤作用。取SKOV3细胞的培养液进行Western blot检测，如图12所示，“N”是指仅加入scFvPnIn的样品，“C”是指仅加入IcPc的样品，样品“0”、“1”、“5”、“10”、“30”和“60”分别是指加样后细胞培养液静置0min、1min、5min、10min、30min和60min时的样品。scFvPnIn和IcPc发生反式剪接反应生成完整的免疫毒素scFvPM1。
如图10所示，完整免疫毒素对HER2低表达的MCF7细胞也存在较强的细胞毒性，但是分割后的免疫毒素对MCF7无毒性，两段毒素重组后恢复部分活性但是由于抗原低表达，对MCF7毒性较低。
如图11所示，对于无HER2抗原表达的CHO细胞，无论是完整免疫毒素还是分割后免疫毒素对其均无细胞毒性，表明免疫毒素的靶向作用只对表达抗原的肿瘤细胞具有选择性杀伤作用。
实施例7免疫毒素诱导细胞凋亡
免疫毒素scFvPM1入胞后与细胞延长因子eEF2结合，使其发生ADP核糖基化而失活，抑制细胞蛋白合成，引起细胞凋亡。现考察完整免疫毒素以及分割后免疫毒素合用时对细胞凋亡的诱导作用。细胞铺12孔板，2×10 5/孔，培养过夜后，加各组免疫毒素处理细胞48h，洗去细胞表面培养物质，加胰酶消化细胞，制备细胞悬液，然后分别用AnnexinV-FITC和碘化丙啶(PI)处理细胞10min，AnnexinV结合缓冲液重悬细胞，上流式细胞仪分析。AnnexinV可以选择性结合凋亡细胞表面的磷脂酰丝氨酸，PI不能通过活细胞膜，但却能穿过破损的细 胞膜而对核染色，因此，早期凋亡和晚期凋亡细胞可以分别被AnnexinV-FITC和PI标记。如图13所示，“N”、“C”、“N+C+DTT”、“DTT”和“PBS”分别是指单独的scFvPnIn、单独的IcPc、混合的scFvPnIn和IcPc(加入还原剂DTT)、单独的DTT和PBS的情况。测试结果发现，完整免疫毒素scFvPM1以及分割后免疫毒素合用并添加DTT(混合的scFvPnIn和IcPc(加入还原剂DTT))时，可以明显诱导细胞凋亡作用，而单独使用分割后免疫毒素scFvPnIn或IcPc，检测到较少凋亡细胞，证明分割后免疫毒素两部分无毒性。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本文所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (69)

  1. 组合物，其包含第一多肽和第二多肽，其中，
    所述第一多肽包含第一毒素片段和第一内含肽片段，所述第二多肽包含第二毒素片段和第二内含肽片段；其中所述第一多肽与所述第二多肽不同；
    所述第一毒素片段和所述第二毒素片段均无生物毒性；
    且所述第一多肽和所述第二多肽可通过所述第一内含肽片段与所述第二内含肽片段的相互作用而使所述第一毒素片段与所述第二毒素片段形成有生物毒性的毒素。
  2. 根据权利要求1所述的组合物，其中，所述第一毒素片段与所述第二毒素片段不同。
  3. 根据权利要求1-2中任一项所述的组合物，其中所述第一毒素片段和所述第二毒素片段源自相同的毒素。
  4. 根据权利要求3所述的组合物，其中所述第一毒素片段包含所述毒素的毒性活性区第一片段，所述第二毒素片段包含所述毒素的毒性活性区第二片段，且所述毒性活性区第一片段和所述毒性活性区第二片段构成所述毒素的完整毒性活性区。
  5. 根据权利要求1-4中任一项所述的组合物，其中所述第二毒素片段不包含所述毒素的转位区或其片段。
  6. 根据权利要求1-5中任一项所述的组合物，其中所述第一毒素片段还包含所述毒素的转位区或其片段。
  7. 根据权利要求1-6中任一项所述的组合物，其中所述第一毒素片段不包含所述毒素的完整毒性活性区。
  8. 根据权利要求1-7中任一项所述的组合物，其中所述第二毒素片段不包含所述毒素的完整毒性活性区。
  9. 根据权利要求1-8中任一项所述的组合物，其中所述毒素选自以下组：细菌毒素、人源毒素和植物毒素。
  10. 根据权利要求1-9中任一项所述的组合物，其中所述毒素选自以下组：绿脓杆菌外毒素和白喉毒素。
  11. 根据权利要求1-9中任一项所述的组合物，其中所述毒素选自以下组：蓖麻毒素、皂草素和白树毒素。
  12. 根据权利要求1-11中任一项所述的组合物，其中所述毒素为绿脓杆菌外毒素的截短体PE38，其包含如SEQ ID NO:1和SEQ ID NO:16中任一项所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的组合物，其中，所述第一内含肽片段与所述第二内含肽片段不同。
  14. 根据权利要求1-13中任一项所述的组合物，其中所述第一内含肽片段和所述第二内含肽片段源自相同的内含肽。
  15. 根据权利要求14所述的组合物，其中所述内含肽为断裂型内含肽。
  16. 根据权利要求15所述的组合物，其中所述断裂型内含肽选自以下组：SsP DnaB、Ssp DnaE和Npu DnaE。
  17. 根据权利要求1-16中任一项所述的组合物，其中所述第一多肽中，所述第一毒素片段的C端与所述第一内含肽片段的N端直接或间接相连。
  18. 根据权利要求17所述的组合物，其中所述第一毒素片段C端的氨基酸残基源自所述毒素的无规卷曲区。
  19. 根据权利要求17-18中任一项所述的组合物，其中所述第二多肽中，所述第二毒素片段的N端与所述第二内含肽片段的C端直接或间接相连。
  20. 根据权利要求19所述的组合物，其中所述第二毒素片段N端的氨基酸残基源自所述毒素的无规卷曲区。
  21. 根据权利要求17-20中任一项所述的组合物，其中所述第二毒素片段N端第1-3位的氨基酸残基依次为CFN。
  22. 根据权利要求17-21中任一项所述的组合物，其中所述第一内含肽片段包含如SEQ ID NO:2所示的氨基酸序列。
  23. 根据权利要求17-22中任一项所述的组合物，其中所述第二内含肽片段包含如SEQ ID NO:3所示的氨基酸序列。
  24. 根据权利要求17-23中任一项所述的组合物，其中所述第一毒素片段包含如SEQ ID NO:4所示的氨基酸序列。
  25. 根据权利要求17-24中任一项所述的组合物，其中所述第二毒素片段包含如SEQ ID NO:5所示的氨基酸序列。
  26. 根据权利要求17-25中任一项所述的组合物，其中所述第一多肽包含如SEQ ID NO:6所示的氨基酸序列。
  27. 根据权利要求17-26中任一项所述的组合物，其中所述第二多肽包含如SEQ ID NO:7、SEQ ID NO:7和SEQ ID NO:15中任一项所示的氨基酸序列。
  28. 根据权利要求1-16中任一项所述的组合物，其中所述第一多肽中，所述第一毒素片段的N端与所述第一内含肽片段的C端直接或间接相连。
  29. 根据权利要求28所述的组合物，其中所述第一毒素片段N端的氨基酸残基源自所述 毒素的无规卷曲区。
  30. 根据权利要求28-29中任一项所述的组合物，其中所述第二多肽中，所述第二毒素片段的C端与所述第二内含肽片段的N端直接或间接相连。
  31. 根据权利要求30所述的组合物，其中所述第二毒素片段C端的氨基酸残基源自所述毒素的无规卷曲区。
  32. 根据权利要求28-31中任一项所述的组合物，其中所述第一毒素片段N端第1-3位的氨基酸残基依次为CFN。
  33. 根据权利要求17-27中任一项所述的组合物，所述第一内含肽片段包含所述断裂型内含肽的N端蛋白质区域；且所述第二内含肽片段包含所述断裂型内含肽的C端蛋白质区域。
  34. 根据权利要求28-32中任一项所述的组合物，所述第一内含肽片段包含所述断裂型内含肽的C端蛋白质区域；且所述第二内含肽片段包含所述断裂型内含肽的N端蛋白质区域。
  35. 根据权利要求33-34中任一项所述的组合物，其中所述N端蛋白质区域为Npu DnaE的N端蛋白质区域。
  36. 根据权利要求33-35中任一项所述的组合物，所述C端蛋白质区域为Npu DnaE的C端蛋白质区域。
  37. 根据权利要求1-36中任一项所述的组合物，其中所述第一内含肽片段与所述第二内含肽片段的所述相互作用包含所述第一内含肽片段和所述第二内含肽片段的蛋白质反式剪接作用。
  38. 根据权利要求1-28中任一项所述的组合物，其中所述第一多肽和/或所述第二多肽还包括靶向部分，所述靶向部分靶向肿瘤特异性抗原。
  39. 根据权利要求38所述的组合物，其中所述第一多肽包含第一靶向部分，且所述第一靶向部分位于所述第一毒素片段的N端。
  40. 根据权利要求38-39中任一项所述的组合物，其中所述第二多肽包含第二靶向部分，且所述第二靶向部分位于所述第二毒素片段的N端。
  41. 根据权利要求1-40中任一项所述的组合物，其中所述肿瘤特异性抗原选自以下组：HER2、PD-L1、EGFR、mesothelin和Lewis Y。
  42. 根据权利要求39-41中任一项所述的组合物，其中所述第一靶向部分和/或所述第二靶向部分包含抗体或其抗原结合片段或变体。
  43. 根据权利要求42所述的组合物，其中所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。
  44. 根据权利要求42-43中任一项所述的组合物，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab) 2，dAb，分离的互补决定区CDR，Fv和scFv。
  45. 根据权利要求42-44中任一项所述的组合物，其中所述抗体或其抗原结合片段的所述变体选自下组：
    a)在所述抗体或所述其抗原结合片段中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽；和
    b)与所述抗体或所述其抗原结合片段具有至少90％序列同源性的蛋白质或多肽。
  46. 根据权利要求42-44中任一项所述的组合物，其中所述第一靶向部分和/或所述第二靶向部分为ScFv。
  47. 根据权利要求38-46中任一项所述的组合物，其中所述靶向部分包含如SEQ ID NO:8所示的氨基酸序列。
  48. 一种组合物的制备方法，其包括以下步骤：
    1)提供权利要求1-46中任一项所述的第一多肽；
    2)提供权利要求1-46中任一项所述的第二多肽；
    3)将所述第一多肽与所述第二多肽混合以得到所述组合物。
  49. 根据权利要求48所述的制备方法，其中所述组合物中所述第一多肽与所述第二多肽的摩尔比为10:1-1:10。
  50. 根据权利要求48-49中任一项所述的制备方法，其还包括加入还原剂。
  51. 根据权利要求50所述的制备方法，其中所述还原剂选自以下组：DTT和β巯基乙醇。
  52. 根据权利要求50-51中任一项所述的制备方法，其中所述组合物中所述还原剂的浓度为0.001-10000nM。
  53. 根据权利要求50-52中任一项所述的制备方法，其中在所述混合的同时或所述混合之后加入所述还原剂。
  54. 根据权利要求50-53中任一项所述的制备方法，其还包括在加入所述还原剂后孵育所述组合物。
  55. 根据权利要求54所述的制备方法，其中所述孵育的温度为1℃-50℃。
  56. 根据权利要求54-55中任一项所述的制备方法，其中所述孵育的时间为2-120分钟。
  57. 载体，其包含编码根据权利要求1-46中任一项所述的第一多肽的核酸，和/或，其包 含编码根据权利要求1-46中任一项所述的第二多肽的核酸。
  58. 细胞，其表达根据权利要求1-46中任一项所述的第一多肽，和/或，其表达根据权利要求1-46中任一项所述的第二多肽。
  59. 试剂盒，其包含1)权利要求1-46中任一项所述的第一多肽；以及
    2)权利要求1-46中任一项所述的第二多肽。
  60. 根据权利要求59所述的试剂盒，其中所述第一多肽和所述第二多肽在所述试剂盒中彼此不混合。
  61. 根据权利要求60所述的试剂盒，其中所述第一多肽和所述第二多肽位于不同的容器内。
  62. 根据权利要求59-61中任一项所述的试剂盒，其中所述试剂盒还包含还原剂。
  63. 根据权利要求62所述的试剂盒，其中所述还原剂选自以下组：DTT和β巯基乙醇。
  64. 根据权利要求62-63中任一项所述的试剂盒，其中所述还原剂包含于独立的容器内。
  65. 根据权利要求59-64中任一项所述的试剂盒，其包含根据权利要求1-54中任一项所述的组合物。
  66. 权利要求1-46中任一项所述的组合物、权利要求59-65中任一项所述的试剂盒、权利要求57所述的载体，或权利要求58所述的细胞在制备治疗疾病的药物中的用途，所述疾病包含肿瘤。
  67. 根据权利要求66所述的用途，所述的肿瘤选自以下组：乳腺癌、黑色素瘤、卵巢癌、结肠癌、间皮瘤、腺体瘤、胰腺癌和膀胱癌。
  68. 权利要求1-46中任一项所述的组合物、权利要求59-65中任一项所述的试剂盒、权利要求57所述的载体，或权利要求58所述的细胞，其治疗肿瘤。
  69. 治疗肿瘤的方法，其包括施用权利要求1-46中任一项所述的组合物、权利要求59-65中任一项所述的试剂盒、权利要求57所述的载体，或权利要求58所述的细胞。
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