WO2024083226A1 - Protéine de fusion d'anticorps et préparation et utilisation de celle-ci - Google Patents
Protéine de fusion d'anticorps et préparation et utilisation de celle-ci Download PDFInfo
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Definitions
- the present invention belongs to the field of biomedicine, and specifically relates to an activatable multifunctional antibody fusion protein that targets tumor-specific antigens and has the biological effects of cytokines.
- the present invention also relates to the preparation and application of the antibody fusion protein.
- Antibody-cytokine fusion protein is a very promising tumor immunotherapy product that reduces the peripheral immunotoxicity of cytokines through antibody targeting on the one hand, and prolongs the half-life of cytokines and enhances the immunoregulatory effect of cytokines and antibodies on the other hand by fusing with antibodies (Xue, Hsu, Fu, & Peng, 2021).
- Cytokine interleukin-2 (IL-2) is essential for the survival and expansion of T cells, especially natural killer CD8+T cells and NK cells.
- High-dose recombinant human IL-2 aldesleukin (trade name Proleukin) was approved by the U.S.
- the activatable antibody-cytokine fusion protein retains the advantages of the antibody-cytokine fusion protein.
- the binding site of IL-2 and the receptor is blocked by a shielding peptide, and the active IL-2 cytokine is released through the cleavage of metalloproteinases specifically expressed in the tumor microenvironment, which can simultaneously achieve the purposes of extending the half-life of IL-2, tumor-targeted release, and reducing peripheral toxicity.
- Hsu et al. reported (Hsu et al., 2021) that IL-2 prodrug specifically activates CD8+T cells in the tumor microenvironment, enhances tumor killing, and shows good safety.
- antibody-coupled IL-2 prodrug products include Werewolf's cleavable IL2 (US11352403, WO2019222295), XILIO's shielded IL2 cytokine (WO2021202675), and Askgenen's cytokine prodrug (WO2019173832).
- the present invention designs an activatable antibody fusion protein having the structure shown in Figure 1A.
- the present invention provides an activatable antibody fusion protein, which is described here by taking the CLDN18.2 activatable antibody fusion protein as an example. It is mainly composed of an anti-CLDN18.2 antibody and an IL-2/IL-2R ⁇ complex, and IL-2 and IL-2R ⁇ are coupled through a cleavable connecting peptide to make it an activatable antibody fusion protein; at the same time, the Fc-coupled sugar chain is modified through genetic engineering and cell engineering methods, further improving the antibody-dependent cell activity of the fusion protein.
- This article describes CLDN18.2 activatable antibody fusion proteins in the form of multifunctional fusion proteins H7E12-2-Pro-IL2 (H7E12-2 antibody, a multifunctional fusion protein of an IL-12/IL-2R ⁇ conjugate plus a peptide chain cleavable by MMP14), 432-Pro-IL2 (432 antibody, a multifunctional fusion protein of an IL-12/IL-2R ⁇ conjugate plus a peptide chain cleavable by MMP14), 362-Pro-IL2 (362 antibody, a multifunctional fusion protein of an IL-12/IL-2R ⁇ conjugate plus a peptide chain cleavable by MMP14), and Hit2.2-Pro-IL2 (Hit2.2 antibody , IL-12/IL-2R ⁇ conjugate plus a multifunctional fusion protein of an MMP14-cleavable peptide chain) is used as an example, using this architectural design in combination with a CLDN18.2 target antibody, a multifunctional fusion protein targeting Claudin18.
- the present invention provides an activatable antibody fusion protein, characterized in that it comprises an antibody portion that specifically binds to a target, an immunoglobulin Fc portion, a shielding portion and a cytokine portion, wherein the shielding portion is fused to the immunoglobulin Fc portion via a connecting peptide L1, and the cytokine portion is fused to the shielding portion via a cleavable connecting peptide L2.
- the activatable antibody fusion protein increases the targeting of the cytokine by combining the cytokine with the antibody targeting part/Fc part, and prolongs the half-life of the cytokine by fusion with the Fc part; at the same time, the shielding part fuses with the cytokine through a cleavable linker and inhibits the activity of the cytokine, which reduces the activity of the cytokine that is harmful to normal tissues, and when it reaches the tumor, the protease specifically expressed in the tumor tissue cuts the cleavable linker and releases the cytokine from the shielding part into the tumor microenvironment, as shown in the schematic diagram in Figure 1A.
- the activatable antibody fusion protein comprises, from the N-terminus to the C-terminus: an antibody portion that specifically binds to a target, an immunoglobulin Fc portion, a connecting peptide L1 connecting the Fc fragment and the immunoglobulin Fc portion, a shielding portion connected to the connecting peptide L1,
- the cleavable linker peptide L2 linked to the cytokine, and the cytokine, are shown schematically in FIG1B.
- the target is a tumor-specific antigen
- the tumor-specific antigen is selected from one or more of the following groups: Claudin18.2, CA125, AFP, CEA, EGFR, HER2, B7H3, B7H6, MUC1, MUC16, GPC3, CD24, CD20.
- the tumor-specific antigen is CLDN18.2, HER2 or CD20. More preferably, the tumor-specific antigen is CLDN18.2.
- the cytokine is selected from one or more of the following groups: interleukin-2 (IL-2), interferon alpha (IFN ⁇ ), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN ⁇ ), interleukin-7 (IL-7), interleukin-12 (IL-12), and interleukin-21 (IL-21).
- IL-2 interleukin-2
- IFN ⁇ interferon alpha
- GM-CSF granulocyte-macrophage colony stimulating factor
- IFN ⁇ interferon gamma
- IL-7 interleukin-7
- IL-12 interleukin-12
- IL-21 interleukin-21
- the cytokine is an IL-2 wild polypeptide or mutant or truncation, preferably an IL-2 wild polypeptide or an IL-2 truncation or an IL-2 mutant.
- the amino acid sequence of the cytokine is shown in SEQ ID NO: 27 or SEQ ID NO: 74 or SEQ ID NO: 86.
- the shielding moiety is a receptor of the cytokine or a binding fragment thereof, or an antibody or a binding fragment thereof that specifically binds to the cytokine, which can inhibit the activity of the cytokine by binding to the cytokine.
- the shielding moiety inhibits the binding of IL-2 cytokine to IL-2R ⁇ and/or IL-2R ⁇ on immune cells to inhibit the activity of the cytokine.
- the shielding portion is selected from: IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ or a mutant or truncation thereof.
- the shielding portion is IL-2R ⁇ .
- the amino acid sequence of the shielding portion is as shown in SEQ ID NO: 29 or SEQ ID NO: 81 or SEQ ID NO: 82 or SEQ ID NO: 83 or SEQ ID NO: 84 or SEQ ID NO: 85.
- the portion of the antibody that specifically binds to a target is selected from the group consisting of Fab, Fab', F(ab') 2 , Fv, dsFv, diabody, Fd, and Fd' fragments.
- the antibody portion that specifically binds to the target forms an antibody structure with the immunoglobulin Fc portion comprising a heavy chain and a light chain, wherein: the amino acid sequence of the light chain is selected from the amino acid sequences shown in SEQ ID NOs: 3, 7, 11 and 15; and/or the amino acid sequence of the heavy chain is selected from the amino acid sequences shown in SEQ ID NOs: 31, 37, 35, 33 and 9, 13, 17, 19, 21, 49, 51, 53, 55, 57, 59, 61, 63, 69, 75, 77, 79.
- the amino acid sequence of the light chain is as shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:5; or the amino acid sequence of the light chain is as shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:31; or the amino acid sequence of the light chain is as shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9; or the amino acid sequence of the light chain is as shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:21; or the amino acid sequence of the light chain is as shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:37; or the amino acid sequence of the light chain is as shown in SEQ ID NO: NO:11, the amino acid sequence of the heavy chain is shown in SEQ ID NO:13; or the amino acid sequence of the light chain is shown in SEQ ID NO:
- the immunoglobulin Fc portion is selected from the constant region amino acid sequence of IgG1, IgG2, IgG3, and IgG4.
- the immunoglobulin Fc portion is selected from The amino acid sequence of the constant region of IgG1 is shown in SEQ ID NO:39.
- the immunoglobulin Fc portion comprises one or more amino acid substitutions selected from the group consisting of S239D, S298A, I332E and A330L, preferably S239D and I332E or S239D, I332E and A330L, the amino acid numbering being according to the EU numbering system.
- the activatable antibody fusion protein of the present invention is defucosylated. In some embodiments, the activatable antibody fusion protein of the present invention lacks fucosylation at Asn 297 in the Fc portion of the immunoglobulin, such as lacking G0F, G1F, G2F, G0F-GN, etc.
- the connecting peptide L1 is selected from a flexible connecting peptide comprising glycine (G) and serine (S) residues, preferably comprising (GGGGS) n repeats, wherein n is selected from an integer of 1-6, more preferably as shown in the amino acid sequence of SEQ ID NO:23.
- the cleavable linker peptide L2 is cleaved by a tumor-associated protease, thereby releasing the active cytokine.
- the protease is selected from matrix metallopeptidase-1 (MMP1), MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP19, MMP20, MMP21, uPA, FAPa or cathepsin B.
- MMP1 matrix metallopeptidase-1
- MMP2 MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP19, MMP20, MMP21, uPA, FAPa or cathepsin B.
- sequences known to be specifically cleaved by MMP14 include, for example, IFARS-L, LARA-LK, LGPSH-Y, LPPLG-F, LQIGH-L, NSPMS-L, PKLAA-I, PTPRS-Y, RKLAF-L, RPLN-LS, RRPVA-Y, SSPLN-Y, SVPSA-I, SYPRA-Y, TMLLA-L, VGPAF-L, RPRS-LL, KIPSA-L, AHPSA-L, SPRN-LR, YGPRA- I, YPAG-LR, KAPAH-L, SQPMA-Y, HTVRG-L, LKVMN-Y, NPLG-IR, PRS-LKS, SSPLA-L, RLPYP-L, FIPFP-F, TPYG-LV, IGALA-L, RPRG-LT, IGPQF-L, VVPNN-L, SAVG-LR, VR
- the protease is selected from caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase-cleaved caspase 11, and caspase 12.
- the cleavable connecting peptide L2 is cleaved by matrix metallopeptidase 14.
- the amino acid sequence of the connecting peptide L2 is shown in SEQ ID NO: 25 or SEQ ID NO: 71 or SEQ ID NO: 72 or SEQ ID NO: 73.
- the fusion protein includes, from N-terminus to C-terminus, an antibody portion that specifically binds to a target, such as an immunoglobulin Fc portion as shown in SEQ ID NO:39, a connecting peptide L1 as shown in SEQ ID NO:23, a shielding portion as shown in any one of SEQ ID NO:29, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:85, a connecting peptide L2 as shown in any one of SEQ ID NO:25, SEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73, and a cytokine portion as shown in SEQ ID NO:27, SEQ ID NO:74, or SEQ ID NO:86.
- the fusion protein includes, from N-terminus to C-terminus, an antibody portion that specifically binds to a target, and a sequence selected from any one of SEQ ID NO:1 or 87
- the fusion protein is selected from the antibody fusion proteins shown in Table 1. In a specific embodiment, the fusion protein is the antibody fusion protein ICP-068 or ICP-415 of the present invention.
- the present invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding an activatable antibody fusion protein according to the first aspect of the present invention.
- the nucleic acid molecule comprises a polynucleotide sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 32, 34, 36 or 38.
- the present invention provides a host cell comprising the nucleic acid molecule according to the second aspect of the present invention.
- the host cell has an altered glycosylation mechanism so that fucose residues are not attached to N-oligosaccharide chains or such attachment is minimized.
- the host cell lacks relevant fucosyltransferase activity or fucose transport activity.
- the fucosyltransferase is ⁇ 1,6-fucosyltransferase (FUT8).
- the fucose transporter is GDP-fucose transporter (FUCT1).
- the host cell is selected from CHO cells, COS cells, HeLa cells, HEK cells, such as HEK 293 cells.
- the present invention provides a method for producing the activatable antibody fusion protein of the first aspect of the present invention, comprising culturing the host cell of the third aspect of the present invention to express the fusion protein, and isolating the expressed fusion protein.
- the present invention provides an activatable antibody fusion protein product produced by the method according to the fourth aspect of the present invention, characterized in that the fucosylation level of Asn 297 at position Fc region of the immunoglobulin is reduced, preferably, the activatable antibody fusion protein with fucosylation modification at Asn 297 at position Fc region of the immunoglobulin accounts for 10% or less of the total amount of all activatable antibody fusion proteins, and the amino acid numbering is according to the EU system numbering.
- the non-fucosylated activatable antibody fusion protein has enhanced antibody-dependent cellular cytotoxicity compared to a fucosylated control fusion protein.
- the present invention provides the use of the activatable antibody fusion protein, nucleic acid molecule, and activatable antibody fusion protein product of the present invention in the preparation of a drug or reagent for diagnosing, treating, or preventing a tumor or autoimmune disease.
- the tumor is a tumor associated with CLDN18.2, a tumor associated with HER2, or a tumor associated with CD20.
- the tumor is a solid tumor, such as gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, breast cancer; blood cancer, such as lymphoma (such as non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, diffuse large B-cell non-Hodgkin's lymphoma, follicular lymphoma, etc.), leukemia (such as chronic lymphocytic leukemia, etc.).
- lymphoma such as non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, diffuse large B-cell non-Hodgkin's lymphoma, follicular lymphoma, etc.
- leukemia such as chronic lymphocytic leukemia, etc.
- the autoimmune diseases include, for example, rheumatoid arthritis, autoimmune hemolytic anemia, pure red cell aplasia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura, Evans syndrome, vasculitis (such as granulomatosis with polyangiitis, etc.), bullous skin diseases (such as pemphigus, pemphigoid, etc.).
- the activatable antibody fusion protein of the present invention targets tumor-specific antigens, thereby carrying cytokines to the target tumor location, and activating the activity of cytokines in the tumor microenvironment through enzymes specifically expressed by the tumor, thereby achieving effective targeting.
- the coupling of Fc and cytokines increases the half-life of the cytokines.
- the present invention uses an activatable antibody fusion protein containing immunoglobulin Fc, which not only has the antigen targeting effect of the antibody, but also retains the effector function of the Fc part, including antibody-dependent cell-mediated cytotoxicity (ADCC).
- ADCC antibody-dependent cell-mediated cytotoxicity
- CLDN18.2 activatable antibody fusion protein will be used as an example for explanation.
- the examples listed are for illustrative purposes only and do not limit the scope of the present invention.
- Those skilled in the art will appreciate that the concept of the present invention is also applicable to various tumor-specific antigens and cytokines without being limited by their specific sequences.
- the activatable antibody fusion protein of the present invention is mainly composed of an anti-CLDN18.2 antibody and an IL-2/IL-2R ⁇ complex, and IL-2 and IL-2R ⁇ are coupled through a cleavable connecting peptide to make it an activatable antibody fusion protein; at the same time, the Fc-coupled sugar chain is modified through genetic engineering and cell engineering methods to further improve the antibody-dependent cell activity of the fusion protein.
- the activatable IL-2 is fused to the Fc of the CLDN18.2 antibody through a linker sequence 1 to obtain an activatable antibody fusion protein CLDN18.2-Pro-IL2 targeting CLDN18.2.
- the activatable antibody fusion protein targeting CLDN18.2 comprises, starting from the N-terminus: the binding region of the antibody targeting CLDN18.2, the Fc fragment of the antibody, the linker sequence 1 connecting the Fc fragment and the IL-2 receptor, the interleukin-2 receptor subunit ⁇ (IL-2R ⁇ ) connected to the first linker, the cleavable linker sequence 2 connected to IL-2, and the interleukin-2 (IL-2) wild type or IL-2 mutant protein.
- This article describes CLDN18.2 activatable antibody fusion proteins in the form of multifunctional fusion proteins H7E12-2-Pro-IL2 (H7E12-2 antibody, a multifunctional fusion protein of an IL-12/IL-2R ⁇ conjugate plus a peptide chain cleavable by MMP14), 432-Pro-IL2 (432 antibody, a multifunctional fusion protein of an IL-12/IL-2R ⁇ conjugate plus a peptide chain cleavable by MMP14), 362-Pro-IL2 (362 antibody, a multifunctional fusion protein of an IL-12/IL-2R ⁇ conjugate plus a peptide chain cleavable by MMP14), and Hit2.2-Pro-IL2 (Hit2.2 antibody , IL-12/IL-2R ⁇ conjugate plus a multifunctional fusion protein of an MMP14-cleavable peptide chain) is used as an example, using this architectural design in combination with a CLDN18.2 target antibody, a multifunctional fusion protein targeting Claudin18.
- antibodies e.g., monoclonal antibodies
- antigen-binding fragments thereof that specifically bind to CLDN18.2.
- monoclonal anti-CLDN18.2 antibodies that specifically bind to CLDN18.2, wherein the anti-CLDN18.2 antibodies include variants of parent antibodies.
- antibodies that specifically bind to CLDN18.2 e.g., human CLDN18.2.
- CLDN18.2 refers to any CLDN18.2 receptor known to those skilled in the art.
- the CLDN18.2 may be from a mammal, such as CLDN18.2 may be from a human or a cynomolgus monkey.
- the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.
- Sequence "identity” or “identity” has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule ((Gribskov & Devereux, 1991; Griffin & Griffin, 1994; Heijne, 1987; Smith, 1994).
- an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody, but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and/or one or more antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody.
- an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived.
- Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.
- Antibodies include antibody fragments.
- antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (Welschof & Krauss, 2003).
- the fragment may include multiple chains linked together, for example, by disulfide bonds and/or by peptide linkers.
- Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
- Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (eg, by replacing the corresponding region), acquires immunospecific binding (ie, exhibits at least or at least about 10 7 -10 8 M -1
- "Functional fragments" or “analogs of anti-CLDN18.2 antibodies” are fragments or analogs that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction.
- functional fragments are generally synonymous with "antibody fragments", and in the case of antibodies, can refer to fragments that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab') 2 , etc.
- the "Fv” fragment consists of a dimer (VH-VL dimer) formed by non-covalent binding of the variable domain of a heavy chain and the variable domain of a light chain.
- VH-VL dimer dimer
- the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH-VL dimer, as in the case of an intact antibody.
- the six CDRs together confer target binding specificity to the intact antibody.
- a single variable domain or half of an Fv that includes only three target-specific CDRs
- monoclonal antibody refers to a colony of identical antibodies, representing that each individual antibody molecule in a monoclonal antibody colony is identical to other antibody molecules. This characteristic is contrary to the characteristic of a polyclonal colony of antibodies, which comprises antibodies with a variety of different sequences.
- Monoclonal antibodies can be prepared by many well-known methods. For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by merging with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal colony of host cells in vitro by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding antibodies.
- a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies produced synthetically with the same domains.
- telomere binding As used herein, "specific binding” or “immunospecifically binds” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen.
- the antigen may be an isolated antigen or present in a tumor cell.
- an antibody that immunospecifically binds (or specifically binds) to an antigen binds to the antigen with an affinity constant Ka of about 1 ⁇ 10 7 M -1 or 1 ⁇ 10 8 M -1 or greater (or a dissociation constant (Kd) of 1 ⁇ 10 -7 M or 1 ⁇ 10 -8 M or less).
- the affinity constant may be determined by standard kinetic methods for antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art.
- SPR surface plasmon resonance
- ITC isothermal titration calorimetry
- Instruments and methods for measuring the rate are known and commercially available.
- nucleic acid molecule refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by a phosphodiester bond.
- nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
- expression refers to the process of producing a polypeptide by transcription and translation of a polynucleotide.
- the expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
- host cell is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid.
- the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
- vector is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
- Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation.
- Vectors also include those containing nucleic acids encoding polypeptides.
- Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression/display of polypeptides encoded by nucleic acids.
- Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
- vectors also include “viral vectors” or “viral vectors.”
- Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) the exogenous genes into cells.
- expression vector includes vectors capable of expressing DNA, which is operably linked to regulatory sequences such as promoter regions that are capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc.
- Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
- expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA.
- Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and/or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.
- activatable antibody fusion protein refers to a protein in which a complete antibody and a cytokine are fused via a connecting peptide to form a fusion protein, and the cytokine is fused to its receptor via a cleavable linker.
- the binding of the cytokine receptor to the cytokine reduces the binding activity of the activatable cytokine to normal tissues, and on the other hand, the cleavage of the cleavable linker by metalloproteinases in tumor tissues releases the cytokine from the cytokine receptor.
- Claudin is an important member of the tight junction protein family and plays an important role in the connection between cells and cells and between cells and matrix.
- Claudin18 is encoded by the gene CLDN18 and is a four-transmembrane protein with two extracellular regions. It is expressed in tissues such as the stomach, pancreas and lung. Claudin18 is considered to be a diagnostic marker and therapeutic target (Krause et al., 2008).
- CLDN18.2 The Claudin18 family includes two variants, Claudin18.1 (CLDN18.1) and Claudin18.2 (CLDN18.2).
- CLDN18.2 is widely present in gastric tumor tissues; it has recently been found to be expressed in pancreatic cancer, esophageal cancer, and lung cancer (Jovov et al., 2007; Karanjawala et al., 2008).
- In normal cells due to the tight connection between cells, antibodies cannot contact Claudin18.2 expressed by cells; in tumor cells, due to cytopathic changes, the connection between cells becomes loose, thereby exposing CLDN18.2 to the outside, and antibodies can bind to these exposed CLDN18.2 molecules. Therefore, CLDN18.2 has become an ideal drug target molecule (Klamp et al., 2011).
- the existing CLDN18.2 antibody Zolbetuximab (IMAB362) is a human-mouse chimeric human IgG1 antibody that has entered clinical research.
- HER2 Human Epidermal Growth Factor Receptor-2 Receptor 2, HER2
- HER2 Human Epidermal Growth Factor Receptor-2 Receptor 2
- Dimerization of the HER2 receptor leads to autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor and initiates multiple signal transduction pathways, leading to cell proliferation and carcinogenesis.
- Amplification or overexpression of HER2 occurs in nearly 15-30% of breast cancer and 10-30% of gastric cancer/gastroesophageal cancer, and serves as a prognostic and predictive biomarker.
- Overexpression of HER2 has also been seen in other cancers, such as ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer.
- CD20 is a transmembrane protein expressed only on B cells. Although the biological role of this antigen has not been fully determined, its unique expression characteristics make it a good target for cancer immunotherapy.
- the anti-CD20 monoclonal antibody rituximab was approved in 1997 for the treatment of non-Hodgkin's lymphoma.
- IL-2 The cytokine interleukin-2 (IL-2) is essential for the survival and expansion of T cells, especially natural killer CD8+T cells and NK cells.
- IL-2 "wild" polypeptide refers to a form of IL-2 that is otherwise identical to IL-2 "mutant”, but has wild-type IL-2 amino acids at each amino acid position of the IL-2 mutant.
- IL-2 truncate refers to a form of IL-2 obtained by truncating one or more amino acids from the C- and/or N-terminus of wild-type or mutant IL-2
- IL-2 mutant refers to an IL-2 "wild" polypeptide that is otherwise identical to IL-2, having an amino acid different from the wild type at each amino acid position.
- IL-2 is mainly secreted by activated CD4+ helper T cells, and promotes the downstream JAK1/JAK3-STAT5 signaling pathway by binding to the IL-2R ⁇ dimer receptors on CD8+ T cells or NK cells, thereby promoting the survival of T cells and NK cells; in addition, IL-2 is also necessary for the maintenance of regulatory T cells (Treg).
- Treg expresses high-affinity IL-2R ⁇ trimer receptors, whose affinity for IL-2 is Kd ⁇ 10-11 M, the affinity of IL-2R ⁇ dimer receptor for IL-2 is Kd ⁇ 10-9 M, and the affinity of IL-2R ⁇ monomer for IL-2 is Kd ⁇ 10-8 M (Hernandez et al., 2022).
- IL-2R ⁇ also known as CD25, is one of the receptors of IL-2. It participates in the regulation of immune tolerance by controlling the activity of regulatory T cells (Tregs), while Tregs inhibit the activation and expansion of autoreactive T cells.
- IL-2R ⁇ and IL-2 have relatively low affinity, with a dissociation constant Kd of about 10nM.
- the extracellular domain of IL-2Ra is similar to a bent arm, with D1 and D2 located at the N-terminal and C-terminal domains forming a 90° angle. D1 and D2 are connected by 42 amino acid residues, and the C-terminal of the D2 domain is connected to the transmembrane domain by 54 amino acid residues.
- IL-2R ⁇ truncation refers to the same as the IL-2 "wild" polypeptide in other aspects, but with different lengths of amino acids truncated at the C-terminus.
- immune cells refer to cells involved in or associated with immune responses, including lymphocytes, dendritic cells, monocytes/macrophages, granulocytes, mast cells, etc.
- the immune cells include T lymphocytes, B lymphocytes, NK cells, etc.
- the "defucosylation" used in this article refers to the natural lack of fucose transporters or fucosyltransferases in host cells, or the knockout or reduction of fucose transporters or fucosyltransferases by gene editing methods, so that the human IgG antibody expressed by the host cell, or the N-glycan at the Asn-297 site of the IgG Fc segment contained therein, loses or significantly reduces the ability of core fucosylation, thereby enhancing its affinity for Fc ⁇ receptors and ADCC activity.
- pharmaceutical composition refers to a pharmaceutically acceptable composition, which includes, for example, one or more, such as two, three, four, five, six, seven, eight, or more, of the therapeutic agents described herein, formulated together with a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible, etc.
- the carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).
- treating an individual suffering from a disease or condition means that the symptoms of the individual are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and/or cure. Prevention refers to preventing potential diseases and/or preventing symptoms from worsening or disease development. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided and any composition provided herein.
- therapeutic effect refers to the effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.
- FIG1 shows the structure of an activatable antibody fusion protein, wherein:
- B) shows a schematic diagram of the structure of the activatable-cytokine fusion protein.
- FIG2 shows the SDS-PAGE image of the CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:
- A) shows the SDS-PAGE image of activatable IL2 fusion protein and CLDN18.2 antibody
- F shows the SDS-PAGE image of antibody fusion protein with enhanced ADCC of the novel linker peptide.
- FIG3 shows the SEC-HPLC graph of the CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:
- A) shows the SEC-HPLC graph of activatable IL2 fusion protein and CLDN18.2 antibody
- E shows the SEC-HPLC graph of antibody fusion protein with enhanced ADCC of novel linker peptide.
- FIG4 shows the glycoforms of the CLDN18.2-Pro-IL-2 antibody fusion protein in fucose knockout host cells, wherein:
- A) shows the glycoform analysis of the fusion protein ICP-130 expressed by H7E12-Pro-IL2 in CHO-K1-host cells knocked out for ⁇ 1,6-fucosyltransferase (FUT8);
- B) shows the glycoform analysis of the fusion protein ICP-155 expressed by H7E12-Pro-IL2 in SLC35C1 knockout (FUCT1 deletion) CHO-K1-GFT - (CHOK1-AF) host cells.
- FIG5 shows the detection of the binding ability of CLDN18.2-Pro-IL-2 antibody fusion protein to IL-2 receptor.
- FIG. 6 shows the reducing SDS-PAGE image of the in vitro MMP14 cleavage of the CLDN18.2-Pro-IL-2 antibody fusion protein.
- FIG7 shows the effect of antibody fusion protein mutants on NK-92 cell proliferation, wherein:
- A) shows the effects of active and inactive forms of ICP-302 and ICP-303 on NK-92 cell proliferation
- FIG8 shows the effect of antibody fusion protein mutants on CTLL-2 cell proliferation, wherein:
- A) shows the effects of active and inactive forms of ICP-302 and ICP-303 on the proliferation of CTLL-2 cells
- C) shows the effect of active and inactive forms of ICP-198 on the proliferation of CTLL-2 cells.
- FIG9 shows the effects of active and inactive forms of CLDN18.2-Pro-IL-2 antibody fusion protein on IFN- ⁇ release by PBMC-derived T cells, wherein:
- A) shows the effects of active and inactive forms of ICP-070 on IFN- ⁇ release by PBMC-derived T cells
- FIG10 shows the detection of ADCC activity of primary NK cells mediated by active and inactive forms of the defucosylating CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:
- A) shows the comparison of ADCC activity between ICP-087 and its monoclonal antibody ICP-038
- FIG11 shows the ADCC reporter cell line activity detection mediated by ADCC enhanced CLDN18.2-Pro-IL-2 antibody fusion protein, wherein:
- A) shows the comparison of ADCC activities of ICP-070 (wild-type Fc) and its corresponding ADCC-enhanced ICP-087 (defucose), ICP-155 (defucose) and ICP-154 (S239D, I332E) fusion proteins;
- C) shows the ADCC activity of ICP-415 (wild-type Fc) and its corresponding ADCC mutation-enhanced ICP-501, ICP-502 and ICP-503 and ICP-198 (defucosylated) antibody fusion proteins in CD16/V158 cells;
- D) shows the ADCC activity of ICP-415 (wild-type Fc) and its corresponding ADCC mutation-enhanced ICP-501, ICP-502 and ICP-503 and ICP-198 (defucosylated) antibody fusion proteins in CD16/F158 cells;
- FIG12 shows the detection of the effects of active and inactive forms of CLDN18.2-Pro-IL-2 antibody fusion protein on STAT5 phosphorylation signals in NK-92 cells, wherein:
- A) shows the effects of active and inactive forms of ICP-070 on STAT5 phosphorylation signals in NK-92 cells
- FIG. 13 shows the tumor suppressor activity test of ICP-024 and control drug ICP-015 in mouse colon cancer model MC-38-hCLDN18.2-A11 cells.
- FIG14 shows the individual tumor growth curves of mice in each drug administration group in the drug efficacy experiment described in FIG13, wherein:
- A) shows the growth curve of individual tumors in mice of the control group
- FIG. 15 shows the detection of tumor suppressor activity of ICP-414 and other drug administration groups in MC-38-hCLDN18.2 cells, a mouse colon cancer model, wherein:
- A) shows the growth curve of individual tumors in each group of mice
- C) shows the individual tumor growth curve of mice in the ICP-414 administration group.
- FIG16 shows the detection of tumor suppressor activity of ICP-415 and other drug administration groups in MC-38-hCLDN18.2 cells, a mouse colon cancer model, wherein:
- A) shows the growth curve of individual tumors in each group of mice
- C) shows the individual tumor growth curve of mice in the ICP-4415 administration group.
- FIG. 17 shows the tumor suppressor activity test of ICP-416 and other drug administration groups in the mouse colon cancer model MC-38-hCLDN18.2 cells, wherein:
- A) shows the growth curve of individual tumors in each group of mice
- C) shows the individual tumor growth curve of mice in the ICP-416 administration group.
- FIG. 18 shows the tumor suppressor activity test of ICP-024 and control drug ICP-015 in mouse colon cancer model CT-26-hCLDN18.2 cells.
- FIG. 19 shows the individual tumor growth curves of mice in each drug administration group in the drug efficacy experiment described in FIG. 18 , wherein:
- A) shows the growth curve of individual tumors in mice of the control group
- FIG20 shows the flow cytometry determination of the absolute count of peripheral blood lymphocytes in mice at the end point of the efficacy experiment described in FIG18, wherein:
- A) shows the absolute numerical changes of peripheral blood CD45+ cells in each drug administration group
- FIG. 21 shows the tumor suppressor activity test of different CLDN18.2 antibody fusion proteins and the control drug ICP-069 in the mouse colon cancer model MC-38-hCLDN18.2-A11 cells.
- FIG. 22 shows the individual tumor growth curves of mice in each drug administration group in the drug efficacy experiment described in FIG. 21 above, wherein:
- A) shows the growth curve of individual tumors in mice of the control group
- E shows the individual tumor growth curve of mice in the ICP-024 administration group
- F shows the tumor growth curve of individual mice in the ICP-087 administration group.
- FIG. 23 depicts the tumor restimulation and immune memory formation experiments in mice with ICP-024 and ICP-087 tumor regression in the efficacy experiments described in FIG. 22 .
- FIG. 24 shows the individual tumor growth curves of mice in each administration group in the tumor restimulation experiment of ICP-024 and ICP-087 tumor regression mice described in FIG. 23 , wherein:
- A) shows the growth curve of individual tumors in mice of the control group
- C) shows the individual tumor growth curve of mice in the ICP-087 administration group.
- FIG. 25 shows that compared with Fc-IL-2, the CLDN18.2-Pro-IL-2 antibody fusion protein did not cause capillary leakage and had good peripheral safety.
- Figure 26 shows SDS-PAGE images of different antibody fusion proteins, wherein:
- A) shows the SDS-PAGE image of ICP-269 antibody fusion protein
- Figure 27 shows SEC-HPLC graphs of different antibody fusion proteins, wherein:
- A) shows the SEC-HPLC graph of ICP-269 antibody fusion protein
- FIG28 shows the SDS-PAGE diagram of different antibody fusion proteins cleaved by MMP14 in vitro, wherein:
- FIG29 shows the binding ability of different antibody fusion proteins to their target antigens, wherein:
- A) shows the binding of ICP-269 to Her2 high-expressing cells BT474;
- FIG30 shows the effects of different antibody fusion proteins on T cell proliferation, wherein:
- A) shows the effects of ICP-269 and ICP-270 on NK-92 cell proliferation
- IMAB362 Ganymed Pharmaceuticals, see NW_004504382.1.
- Trastuzumab, trade name ( ) is a recombinant DNA-derived humanized monoclonal antibody developed by Roche in Switzerland, containing a human IgG1 subtype framework, and the complementary determining region is derived from mouse anti-p185HER2 antibody, which can specifically act on the extracellular site IV subregion of human epidermal growth factor receptor-2 (HER2), competitively blocking the binding of human epidermal growth factor to HER2, thereby inhibiting the growth of tumor cells.
- HER2 human epidermal growth factor receptor-2
- the product was first approved for marketing by the US FDA on September 25, 1998, and was imported into China in 2002.
- the US patent for trastuzumab expired in June 2019.
- the applicant of the present invention refers to the anti-HER2 monoclonal antibody data and sequences disclosed in Carter, P. and L. Presta, et al. (1992). "Humanization of an anti p185HER2 antibody for human cancer therapy.” Proc. Natl. Acad. Sci. USA 89 (10): 4285-9., and constructed the light and heavy chain variable region and constant region genes of the anti-HER2 humanized antibody, and constructed the Trastuzumab antibody by itself.
- Rituximab also known as RITUXAN, is a human Mouse chimeric monoclonal antibody, composed of human IgG1 kappa constant region and mouse CD20 antibody variable region, can specifically bind to the transmembrane protein CD20 on the surface of B cells, and kill CD20-positive B lymphocytes through two pathways: antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
- ADCC antibody-dependent cell-mediated cytotoxicity
- CDC complement-dependent cytotoxicity
- Rituximab was originally developed by Roche Pharma (Schweiz) Ltd., approved for marketing by the U.S. FDA in 1997, and launched in China in 2000.
- the Chinese patent for rituximab injection expired in 2013.
- the applicant of the present invention constructed the Rituximab antibody by himself with reference to US5736137 .
- the activatable antibody fusion protein increases the targeting of the cytokine interleukin-2 (IL-2) by combining it with the antibody, and prolongs the half-life of the cytokine by fusion with the antibody Fc.
- the interleukin-2 receptor subunit ⁇ (IL-2R ⁇ ) is fused with IL2 through a cleavable linker and inhibits the activity of IL-2, which reduces the binding activity of interleukin-2 (IL-2) to normal tissues, and when it reaches the tumor, the metalloproteinase in the tumor tissue cuts the cleavable linker and releases IL-2 from the IL-2 receptor to subjects in need.
- the present invention includes a method for reducing the binding activity of activatable interleukin-2 (IL-2) to normal tissues and targeting cancer cells, comprising administering an effective amount of an activatable interleukin-2 (IL-2) fusion protein comprising: interleukin-2 (IL-2) wild type or mutant protein or truncation; a cleavable linker connected to IL-2; an interleukin-2 receptor binding region (IL-2 ⁇ or IL-2 ⁇ receptor) or mutant protein or truncation connected to a cleavable linker; and a half-life extender connected to IL-2 or IL-2 receptor, wherein cleavage of the cleavable linker releases IL-2 from the IL-2 receptor to a subject in need thereof.
- an activatable interleukin-2 (IL-2) fusion protein comprising: interleukin-2 (IL-2) wild type or mutant protein or truncation; a cleavable linker connected to IL-2; an interleukin-2 receptor
- the activatable IL2 is fused to the Fc of the CLDN18.2 antibody through a linker 1 to obtain an activatable antibody fusion protein CLDN18.2-Pro-IL2 targeting CLDN18.2.
- the activatable antibody fusion protein targeting CLDN18.2 comprises, starting from the N-terminus, the binding region of the antibody targeting CLDN18.2, the Fc fragment of the antibody, a linker 1 connecting the Fc fragment and the IL-2 receptor, an interleukin-2 receptor subunit ⁇ (IL-2R ⁇ ) or a mutant or truncated IL-2R ⁇ connected to the linker 1, a cleavable linker 2 connected to IL-2, and an interleukin-2 (IL-2) wild type or IL-2 mutant protein or IL-2 truncation.
- the heavy chain HC of the anti-CLDN18.2 humanized monoclonal antibody H7E12-2 is sequentially fused with linker 1, interleukin-2 receptor subunit ⁇ , cleavable sequence 2, and interleukin-2 (IL-2), and the corresponding amino acid sequence is SEQ ID NO: 17, and is co-expressed with the light chain amino acid sequence SEQ ID NO: 15 to obtain the antibody fusion protein H7E12-2-Pro-IL2, protein number ICP-070.
- the heavy chain HC of Zolbetuximab (362) (gene NW_004504382.1) is fused with linker 1, interleukin-2 receptor subunit ⁇ , cleavable sequence 2, and interleukin-2 (IL-2) in sequence, and the corresponding amino acid sequence SEQ ID NO: 13 and the light chain amino acid sequence SEQ ID NO: 11 are co-expressed to obtain the antibody fusion protein 362-Pro-IL2, protein number ICP-069.
- the heavy chain HC of human CLDN18.2 antibody 432 is fused with linker 1, interleukin-2 receptor subunit ⁇ , cleavable sequence 2, and interleukin-2 (IL-2) in sequence, and the corresponding amino acid sequence SEQ ID NO: 9 and the light chain amino acid sequence SEQ ID NO: 7 are co-expressed to obtain the antibody fusion protein 432-Pro-IL2, with protein number ICP-068.
- linker 1 interleukin-2 receptor subunit ⁇
- cleavable sequence 2 cleavable sequence 2
- IL-2 interleukin-2
- the heavy chain HC of the human CLDN18.2 antibody Hit2.2 is fused with the linker 1, the interleukin-2 receptor subunit ⁇ , the cleavable sequence 2, and the interleukin-2 (IL-2) in sequence, and the corresponding amino acid sequence SEQ ID NO: 5 and the light chain amino acid sequence SEQ ID NO: 3 are co-expressed to obtain the antibody fusion protein Hit2.2-Pro-IL2, with the protein number ICP-024.
- the human CLDN18.2 antibody 432 heavy chain HC is fused with linker 1, interleukin-2 receptor subunit ⁇ mutant 1 or mutant 2 or mutant 3, linker 3 or linker 4 linker 5, and interleukin-2 (IL-2) in sequence, wherein the amino acid sequence of interleukin-2 receptor subunit ⁇ mutant 1 or mutant 2 or mutant 3 is as shown in SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83; the amino acid sequence of linker 3 or linker 4 linker 5 is as shown in SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73.
- the activatable antibody fusion protein molecules ICP-302, ICP-303, ICP-414, ICP-415, ICP-416, and ICP-417 were obtained, with corresponding heavy chain amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59.
- Interleukin-2 receptor subunit ⁇ (IL-2R ⁇ ) (SEQ ID NO: 27) was connected to the Fc region of the antibody via a first linker sequence (SEQ ID NO: 23), and then interleukin-2 (IL-2) was connected to the interleukin-2 receptor binding region via a second cleavable linker (SEQ ID NO: 25) to prepare a fusion protein ICP-015 (SEQ ID NO: 1). These connections were achieved by DNA homologous recombination.
- IL2 binds to IL-2R ⁇ , and the toxicity in the heart, lungs, kidneys or central nervous system is reduced; on the other hand, the cleavable linker linked to IL2 is cleaved by the upregulated protease MMP14 in the tumor microenvironment and released from its interleukin-2 receptor binding region, thereby concentrating in the tumor site; at the same time, the antibody Fc region can prolong the half-life of IL-2.
- the fusion protein comprises: (1) target protein binding sequence; (2) human IgG1 Fc segment; 3) linker 1; (4) human IL-2R ⁇ or mutant protein; (5) cleavable linker 2, linker 3 or linker 4 or or linker 5; (6) human IL-2; or (1) human IgG1 Fc segment; (2) linker 1; (3) human IL-2R ⁇ ; (4) cleavable linker 2; (5) human IL-2.
- the IL2 fusion protein ICP-015 and various activatable antibody fusion proteins shown in the table below are obtained. Their sequences and expression host cells are shown in Table 1.
- Table 1 Summary of antibody and fusion protein sequences and host cells
- CHO-S cells were cultured at 37°C, 8% CO 2 , and 100 rpm to a cell density of 6 ⁇ 10 6 cells/mL.
- the constructed vectors were transfected into the above cells using liposomes, and the transfection plasmid concentration was 1 mg/ml.
- the liposome concentration was determined according to the ExpiCHOTM Expression System kit, and cultured at 32°C, 5% CO 2 , and 100 rpm for 7-10 days. Feed was performed once 18-22 hours after transfection and between the 5th day.
- the above culture product was centrifuged, filtered through a 0.22 ⁇ m filter membrane, and the culture supernatant was collected.
- the antibody or fusion protein was purified using Protein A and an ion column.
- the specific operation steps of ProteinA and ion column purification are as follows: after high-speed centrifugation of the cell culture fluid, the supernatant is taken and affinity chromatography is performed using Cytiva's ProteinA chromatography column.
- the equilibrium buffer used for chromatography is 1 ⁇ PBS (pH7.4). After the cell supernatant is loaded and combined, it is washed with PBS until the ultraviolet returns to the baseline, and then the target protein is eluted with 0.1M glycine (pH3.0) elution buffer, and the pH is adjusted to neutral with Tris for storage.
- the pH of the product obtained by affinity chromatography is adjusted to 1-2 pH units lower or higher than pI, and appropriately diluted to control the sample conductivity below 5ms/cm.
- appropriate corresponding pH buffers such as phosphate buffer, acetate buffer and other conditions
- conventional ion exchange chromatography methods in the art such as anion exchange or cation exchange are used to perform NaCl gradient elution under corresponding pH conditions, and the collection tube where the target protein is located is selected according to UV280 absorption and stored together.
- the purified eluate was ultrafiltered and exchanged into a buffer solution.
- the protein purity and content were detected by SDS-PAGE gel electrophoresis, as shown in FIG2 .
- the purity of the fusion protein was further determined by SEC-HPLC. The results showed that the purity of the target antibody or fusion protein was above 90% after one-step purification, which was relatively high, as shown in Figure 3A-E and Table 2.
- Example 3 Preparation and glycoform identification of ADCC-enhanced CLDN18.2-Pro-IL-2 activatable antibody fusion protein
- H7E12-2-Pro-IL2 in FUT8 knockout CHO-K1/FUT8- cells was completed by Nanjing Pengbo Biotechnology Co., Ltd.
- the cell line was passaged in Expi-CHOS expression medium for production, with a passage density of 0.2-0.3x 10 6 /mL and a passage cycle of 2-3 days.
- the cells were diluted to 2x 10 6 /mL the day before transfection. On the day of transfection, the cell density should be around 6x 10 6 /mL and the viability should be greater than 95%.
- the transfection was performed according to the instructions of the transfection kit.
- the plasmids used were 7E12-2-pro-IL2 heavy chain and light chain, with a dosage of 100 ⁇ g, and the light chain and heavy chain were 100 ⁇ g.
- the molar concentration ratio of the chains was 1:1.
- Enhancer and Expichos Feed medium were added and the temperature was lowered to 32°C for culture.
- Expichos Feed medium was added.
- the cell viability was monitored. When the cell viability was less than 70%, the cells were harvested and the protein was purified, numbered ICP-130. The protein expression and purity are shown in Figures 2, 3 and Table 2.
- the expressed fusion protein ICP-130 was quantitatively analyzed for glycoforms by fluorescence labeling chromatography and mass spectrometry, and the results are shown in Figure 4 and Table 3. The results showed that the main glycoforms G0F and G1F modified by fucose in the Fc segment were below the detection limit.
- H7E12-2-Pro-IL2 and 432-Pro-IL2 in SLC35C1 knockout (FUCT1 deletion) CHO-K1/GFT - (CHOK1-AF) cells was performed by Beijing Huafang Tianshi Biopharmaceutical Co., Ltd.
- the cell lines were cultured in the transient transfection medium Trans pro CD01 for passaging.
- Transient transfection was performed using PEI, and the total transfection volume was 200mL.
- the cell density was adjusted to 5x10 6 /mL, and then the transfection complex was prepared.
- the above calculated plasmid and PEI were mixed and reacted for 10min, and then added to the prepared cells.
- sodium butyrate and 0.1 g/L sodium dextran sulfate were added, and the temperature was lowered to 32°C for culture.
- 3% MaxFeed TM 403A1 (Dongning Biotech)
- 0.3% MaxFeed TM 403B1 MaxFeed TM 403B1
- the expressed fusion protein ICP-155 was quantitatively analyzed for glycoforms by fluorescence labeling chromatography and mass spectrometry, and the results are shown in Figure 4B and Table 4. The results showed that the main glycoforms G0F and G1F modified by fucose in the Fc segment were below the detection limit.
- Example 4 Determination of the binding ability of CLDN18.2-Pro-IL-2 antibody fusion protein to IL-2R ⁇ and IL-2R ⁇
- HEK -Blue TM IL-2 cells purchased from Invivogen overexpressed human CD25 (IL-2R ⁇ ), CD122 (IL-2R ⁇ ), and CD132 (IL-2R ⁇ ) genes, so low-affinity IL-2R ⁇ receptors, medium-affinity IL-2R ⁇ receptors, and high-affinity IL-2R ⁇ receptors can be expressed on the cell membrane.
- 8x 104 cells were incubated with different CLDN18.2-Pro-IL-2 fusion proteins at room temperature for 30 minutes, with a starting concentration of 20nM, 5-fold dilution, and 9 concentration gradients.
- the cells were washed once with flow buffer, PE-labeled anti-human IgG Fc secondary antibody was added, and incubated at room temperature for 30 minutes. After the incubation, the cells were washed once with flow buffer, and the mean fluorescence intensity was detected using a NovoCyte Quanteon flow cytometer (Agilent). The data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was obtained by fitting the data using nonlinear S-curve regression, and the EC 50 value was calculated from it. The results are shown in Figure 5 and Table 5.
- ICP-159 is a fusion protein of human IgG1 Fc and IL-2 expressed in series.
- As a positive control for binding to IL-2 receptor, its binding strength on HEK-Blue TM IL-2 cells was EC50 0.74 nM.
- Different CLDN18.2-Pro-IL-2 fusion proteins have IL-2-fold IL-2R binding. ⁇ shielding does not bind to the IL-2 receptor expressed on HEK-Blue TM IL-2 cells at all, proving that the shielding effect is good and achieves the effect of inhibiting the binding of IL-2 to the receptor.
- Example 5 In vitro MMP14 cleavage of CLDN18.2-Pro-IL-2 antibody fusion protein
- Example 6 Detection of NK-92 and CTLL-2 cell proliferation activity of CLDN18.2-Pro-IL-2 antibody fusion protein
- the human NK cell line NK-92 (CRL-2407, ATCC) and the mouse T lymphocyte line CTLL-2 (purchased from the Institute of Biophysics, Chinese Academy of Sciences) are both IL-2-dependent cell lines.
- the cell proliferation-promoting activity of the MMP14-cleaved (+) or uncleaved (-) fusion protein was evaluated using the in vitro cultured IL-2-starved NK-92 and CTLL-2 cell line models. 3000 cells/well were added to a 96-well plate. The starting concentration of the active and inactive forms of the fusion protein was 26 nM, and 9 concentration points were diluted 5-fold. A PBS negative control was set.
- the flat-bottomed 96-well plate was taken out and equilibrated to room temperature, and 30 ⁇ L of CTG-Glo reagent (Promega, Madison, WI) was added. The cells were shaken and mixed for full lysis for 10 minutes. After standing at room temperature for 10 minutes, 50 ⁇ L of the supernatant was transferred to a flat-bottomed 384-well plate and the fluorescence signal was detected in an Envision multi-function microplate reader (Perkin Elmer, Waltham, MA). Data were analyzed using GraphPad Prism 7.0 software, and nonlinear S-curve regression was used to fit the data to obtain dose-effect curves, from which EC 50 values were calculated.
- the fusion proteins ICP-068, ICP-106, ICP-070 and ICP-087 were expressed as ICP-068+, ICP-106+, ICP-07+ and ICP-087+ after MMP cleavage, respectively; the uncleaved ICP-068, ICP-106, ICP-070 and ICP-087 were expressed as ICP-068-, ICP-106-, ICP-070- and ICP-087-, respectively.
- the results showed that CLDN18.2 monoclonal antibodies H7E12-2 and 432 had no in vitro activity in promoting the proliferation of NK-92 and CTLL-2.
- the EC50 of fusion proteins ICP-068+, ICP-106+, ICP-07+ and ICP-087+ in NK-92 cells were 17.7 ⁇ 5.2nM, 20.7 ⁇ 8.2nM, 7.5 ⁇ 1.7nM, 12.9 ⁇ 3.1nM, respectively, which was comparable to the EC50 activity of human recombinant IL-2 of 13.5 ⁇ 1.5; the EC50 of uncleaved ICP-068-, ICP-106-, ICP-070- and ICP-087- in NK-92 cells were 419.0 ⁇ 78.9nM, 157.0 ⁇ 39.7nM, 296.3 ⁇ 57.0nM, 154.7 ⁇ 26.8nM, respectively.
- the EC50 of fusion proteins ICP-068+, ICP-106+, ICP-070+ and ICP-087+ were 12.0 ⁇ 2.1nM, 8.1 ⁇ 1.9nM, 6.1 ⁇ 2.0nM, 6.2 ⁇ 1.9nM, respectively, which was comparable to the EC50 activity of human recombinant IL-2 of 10.3 ⁇ 4.7;
- the EC50 of uncleaved ICP-068-, ICP-106-, ICP-070- and ICP-087- in CTLL-2 cells were 201.3 ⁇ 66.3nM, 59.0 ⁇ 17.0nM, 77.7 ⁇ 1.5nM, 68.5 ⁇ 26.5nM, respectively.
- the EC 50 fold changes of the inactive and active forms of the fusion proteins ICP-068, ICP-106, ICP-070 and ICP-087 in CTLL-2 cells were 17.4 ⁇ 4.8 times, 7.2 ⁇ 0.4 times, 15.7 ⁇ 4.6 times and 9.0 ⁇ 0.6 times, respectively.
- the activity of the cytokine IL-2 in the shielded form of CLDN18.2-Pro-IL-2 fusion protein was significantly reduced in CTLL-2 mouse T cells. After MMP14 cleavage, IL-2 can be effectively released to promote the expansion of T cells.
- the fusion proteins composed of different IL-2Ra mutants or IL-2 mutants, such as ICP-302, ICP-303, ICP-414, ICP-415, ICP-416, and ICP-417, were also used to detect the proliferation activity of NK-92 and CTLL-2 cells, as shown in Figures 7, 8, and Table 7.
- Table 7 Summary of EC50 and fold changes of antibody fusion protein mutants on proliferation of NK-92 cells and CTLL-2 cells
- Example 7 Detection of IFN- ⁇ release from primary T cells using CLDN18.2-Pro-IL-2 antibody fusion protein
- IL-2 is necessary for maintaining the survival of primary T cells, so the effect of CLDN18.2-Pro-IL-2 fusion protein on the homeostasis and IFN- ⁇ release of primary T cells was tested.
- PBMC cells were plated at 2x10 5 cells/well, and different concentrations of fusion protein were added, starting with a concentration of 36 nM, and 4-fold gradient dilutions were made to 8 concentration points. After incubation at 37°C in a cell culture incubator for 5 days, the supernatant was collected and used for the detection of IFN- ⁇ content using a human IFN- ⁇ ELISA kit. The results are shown in Figure 9 and Table 8.
- Table 8 Summary of CLDN18.2-Pro-IL-2 fusion protein promoting the release of IFN- ⁇ cytokines by primary T cells
- ICP-068+, ICP-106+, ICP-07+ and ICP-087+ all promoted the concentration of The IFN- ⁇ cytokine release was dependent on the degree gradient, and the ability to stimulate PBMC-derived primary T cells to release IFN- ⁇ was significantly enhanced compared with human recombinant IL-2.
- the background activity of ICP-087- and ICP-106- in the uncut state was slightly enhanced compared with ICP-070- and ICP-106-.
- IFN- ⁇ mediates T cell killing of tumor cells through multiple pathways.
- the results showed that the inactivated form of CLDN18.2 fusion protein can effectively reduce T cell proliferation, activation and cytokine release when it is not cleaved by the MMP14 enzyme in the tumor microenvironment, and has the effect of reducing peripheral toxicity; at the same time, the active form of CLDN18.2 fusion protein after MMP14 cleavage can maintain the steady-state proliferation of T cells, promote T cell activation and the release of cytokines such as IFN- ⁇ . Its ability to release IFN- ⁇ is stronger than that of recombinant IL-2, showing its potent tumor killing effect.
- Example 8 Detection of ADCC activity of primary NK cells mediated by defucosylated CLDN18.2-Pro-IL-2 antibody fusion protein
- ADCC is caused by the Fab end of the antibody binding to the antigen epitope of the tumor cell, and its Fc end binding to the FC ⁇ R on the surface of the natural killer cell (NK cell).
- the NK cell is activated to release cytotoxic substances such as perforin and granzyme, which mediate the killing of target cells by NK cells and cause apoptosis of target cells.
- PBMCs peripheral blood mononuclear cells
- 293T-hCLDN18.2 cell number KC-0986, purchased from Kangyuan Bochuang
- the target cells 293T-hCLDN18.2 were labeled with 1.6 ⁇ M CFSE (Carboxyfluorescein Diacetate Succinimidyl Ester, 565082, BD), and labeled for 10 minutes at room temperature in the dark.
- the cells were washed twice with 5 volumes of pre-cooled serum-free medium, and the labeled cells were resuspended in ADCC culture medium and counted. 5 x 10 4 cells were added to each well of a 96-well U-shaped plate, and then ICP-068, ICP-106, ICP-070 and ICP-087 active and inactive fusion proteins were added for gradient dilution.
- ADCC activity is an important immunoregulatory mechanism for CLDN18.2 antibody to exert its tumor-killing effect.
- the ADCC of the fucose-removed CLDN18.2-Pro-IL-2 fusion protein is significantly improved compared with the Fc wild-type monoclonal antibody, indicating that the fusion protein structure does not affect the function of the Fc end of the CLDN18.2 antibody; at the same time, the fusion protein produced by the host cells in which FUT8 or FUCT1 is knocked out in the present invention effectively improves the ADCC activity of CLDN18.2-Pro-IL-2, and kills tumor cells with high expression of CLDN18.2 in a targeted manner, providing a theoretical basis for its clinical application.
- the CLDN18.2-Pro-IL-2 fusion protein of the present invention can release IL-2 after enzymatic cleavage, which can effectively activate the function of NK cells in the tumor microenvironment and further enhance the ADCC function of the CLDN18.2 antibody. Therefore, the fusion protein design of the present invention improves the ADCC activity of the CLDN18.2 antibody through two pathways, and has more significant tumor killing activity than the monoclonal antibody drugs currently in the clinical development stage.
- Example 9 Activity detection of Jurkat-NFAT-Luc2-CD16a-V158 ADCC reporter cell line mediated by CLDN18.2-Pro-IL-2 antibody fusion protein
- Antibody-dependent cell-mediated cytotoxicity is the main mechanism of action for many antibody drugs to kill tumor cells.
- Fc ⁇ RIIIA CD16a
- Fc ⁇ RIIIA CD16a
- NK cells NK cells on tumor cells.
- Jurkat-NFAT-Luc2-CD16a-V158 produced by Kangyuan Broadcom
- target cells 293T-hCLDN18.2 purchased from Kangyuan Bochuang, cell number KC-0986
- the effector cells overexpressed high-affinity Fc ⁇ RIIIA CD16a-V158 in the extracellular region and were co-incubated with target cells and different concentrations of CLDN18.2-Pro-IL-2 fusion protein (starting at 86.9 nM, 4-fold dilution, 9 concentration points).
- the Fc end of the fusion protein bound to the high-affinity CD16a-V158 in the extracellular region to activate the NFAT-luc2 luciferase reporter system.
- the ADCC activity of the ADCC-enhanced CLDN18.2-Pro-IL-2 fusion protein can be detected by detecting the content of luciferase using a Plus multifunctional microplate reader (BMG LABTECH).
- the data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was obtained by fitting the data using nonlinear S-curve regression, and the EC50 value was calculated from this.
- the experimental results are shown in Figure 11 and Table 10.
- Table 10 Summary of EC50 of Jurkat-NFAT-Luc2-CD16a-V158 ADCC reporter cell line mediated by CLDN18.2-Pro-IL-2 antibody fusion protein
- the ADCC activity EC values of ICP-070 (Fc wild type), ICP-153 (ADCC enhanced by amino acid mutation), ICP-087 (N297 defucosylated, 292T-FUT8-), and ICP-155 (N297 defucosylated, CHO-K1-AF) were significantly higher than those of ICP-070 (Fc wild type), ICP-153 (ADCC enhanced by amino acid mutation), ICP-087 (N297 defucosylated, 292T-FUT8-), and ICP-155 (N297 defucosylated, CHO-K1-AF).
- ADCC enhancing mutations were performed to obtain ICP-501 (S239D and I332E mutations), ICP-502 (S239D, A330L and I332E mutations) and ICP-503 (F243L, R292P, Y300L, V305I and P396L mutations), and ADCC reporter cell line activity was detected using Jurkat-NFAT-Luc2-CD16a-V158 and Jurkat-NFAT-Luc2-CD16a-F158. The results are shown in Figure 11C, Figure 11D and Table 11.
- the ADCC activity Ec50 of ICP-501, ICP-502 and ICP-503 in the CD16a-V158 subtype were 4.1pM, 4.1pM and 4.9pM, respectively, and compared with the unmutated ICP-415, the ADCC effect was increased by 11.0 times, 11.0 times and 9.2 times, respectively; while the ADCC activity Ec50 in the CD16a-V158 subtype was 15pM, 9.3pM and 25pM, respectively, and compared with the mutated ICP-415, the ADCC effect was increased by 16.5 times, 26.5 times and 9.9 times, respectively.
- Table 11 Summary of the results of Jurkat-NFAT-Luc2-CD16a-V158 and Jurkat-NFAT-Luc2-CD16a-F158 ADCC reporter cell lines mediated by CLDN18.2-Pro-IL-2 antibody fusion protein
- Example 10 CLDN18.2-Pro-IL-2 antibody fusion protein mediated NK-92 cell STAT5 phosphorylation detection
- Active IL-2 activates the downstream JAK1/JAK3-STAT5 signaling pathway and promotes STAT5 phosphorylation (p-STAT5) by binding to IL-2R ⁇ dimers on the surface of NK cells or CD8+T cells, or IL-2R ⁇ trimers on the surface of Treg cells. Therefore, the ability of cleaved and uncleaved CLDN18.2-Pro-IL-2 fusion proteins to promote STAT5 phosphorylation in NK-92 cells was compared. 2.22 x 10 5 /mL NK-92 cells were resuspended in MEM basal medium and transferred to 96 96-well cell culture plate, 90 ⁇ L per well.
- the starting concentration of the antibody was 3 nM, and it was diluted 3 times with MEM basal medium gradient series, with a total of 10 concentration points. 10 ⁇ L of antibody solution was transferred to a 96-well plate and incubated in a 37°C incubator for 15 minutes.
- NK-92 cells were washed twice with PBS buffer, then 100 ⁇ L of 90% cold methanol was added, fixed at 4°C for 30 minutes, washed twice with PBS buffer, and then Alexa Fluor 647 labeled antibody diluted according to the pSTAT5 antibody manual was added, incubated at room temperature for half an hour, washed twice with PBS buffer, and the phosphorylation level of STAT5 was detected using a NovoCyte Quanteon flow cytometer (Agilent). The experimental results are shown in Figures 12A-D and Table 12.
- Table 12 Summary of EC50 of CLDN18.2-Pro-IL-2 antibody fusion protein promoting STAT5 phosphorylation in NK-92 cells
- the EC50 of ICP-068+, ICP-106+, ICP-70+ and ICP-087+ after enzyme cleavage for promoting STAT5 phosphorylation in NK-92 cells were 0.15nM, 0.08nM, 0.07nM and 0.07nM, respectively, which was comparable to the activity of recombinant IL-2 ( EC50 was 0.11nM).
- the inactive forms of ICP-068-, ICP-070-, ICP-087- and ICP-106- that were not cleaved had no ability to promote STAT5 phosphorylation ( Figures 12A-12D).
- Example 11 Efficacy determination of CLDN18.2-Pro-IL-2 antibody fusion protein in MC-38-hCLDN18.2 and CT-26-hCLDN18.2 mouse colon cancer cell models
- the inventors used activatable antibody fusion protein Hit2.2-Pro-IL2 (protein number ICP-024), fusion protein Fc-Pro-IL2, (protein number ICP-015), and antibody-mediated Hit2.2-Pro-IL2 to Three groups of Hit2.1 (protein number ICP-025) were enrolled at the same time, and the efficacy was tested in MC-38-hCLDN18.2-A11 and CT-26-hCLDN18.2 mouse colon cancer cell models at equimolar doses.
- MC-38-hCLDN18.2-A11 purchased from Nanjing Bowang mouse colon cancer cells in the logarithmic growth phase were inoculated into 6-8 week old female C57BL/6 mice (Weitong Lihua) with an inoculation volume of 5 ⁇ 10 5 /mouse and an inoculation volume of 0.1 mL.
- intraperitoneal administration was started, and the administration cycle was BIW (twice a week), and the drug was supplied 5 times with a dosage of 0.26 nmol/mouse.
- routine monitoring included the effects of tumor growth and treatment on the normal behavior of animals, including the activity of experimental animals, food and water intake, weight gain or loss (weight measured twice a week), eyes, hair and other abnormalities. Clinical symptoms observed during the experiment were recorded in the original data.
- the average tumor volume of the control group exceeded 2000 mm 3, which was set as the experimental endpoint.
- spleen, draining lymph nodes and tumor tissues were collected for immune cell infiltration analysis.
- tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the major diameter and b represents the minor diameter).
- TGI% (1-T/C) ⁇ 100%.
- T and C are the tumor weights (TW) of the treatment group and the PBS control group at a specific time point, respectively).
- TGI% (1-T/C) ⁇ 100%.
- T and C are the tumor weights (TW) of the treatment group and the PBS control group at a specific time point, respectively).
- TW tumor weights
- the tumor growth curves of individual mice are shown in Figures 14A-D, among which 4/8 (50%) mice in the ICP-024 administration group achieved complete response (CR, complete response) ( Figure 14B), showing the excellent anti-tumor ability of ICP-024 alone in the MC-3-8hCLDN18.2 mouse colon cancer model.
- ICP-414, ICP-415 and ICP-416 had a significantly higher efficacy.
- the results were significant, showing that the antibody fusion protein mutant had significant anti-tumor ability in the MC38-hCLDN18.2 mouse colon cancer model.
- CT-26-hCLDN18.2 was used to compare the tumor inhibition rate of ICP-024 with the control test drugs ICP-015 and ICP-025.
- CT-26-hCLDN18.2 purchased from Nanjing Bowang
- mouse colon cancer cells in the logarithmic growth phase were inoculated into 6-8 week old BALb/c female mice (Weitong Lihua), with an inoculation amount of 5 ⁇ 10 5 /mouse and an inoculation volume of 0.1mL.
- the administration cycle was BIW (twice a week) x 5 times, and the dosage was 0.26nmol/mouse.
- the experimental grouping, administration, tumor measurement and TGI calculation were the same as those of the above MC-38-hCLDN18.2-A11.
- the average tumor volume of the control group exceeded 2000mm 3 , which was set as the experimental endpoint.
- peripheral blood was collected for absolute immune cell count analysis to detect its potential peripheral toxicity.
- the tumor growth curve is shown in FIG18 .
- the tumor inhibition rate TGI of ICP-024 was 97.99% (p ⁇ 0.05, Oneway ANOVA), while the TGI of the control drug ICP-015 and the control monoclonal antibody ICP-025 were 56.12% and -33.81%, respectively, which were not significant compared with the PBS control group.
- mice The tumor growth curves of individual mice are shown in FIG19A-D .
- 5/8 (62.5%) mice achieved complete tumor clearance (CR, complete response)
- 2/8 (25%) mice had tumors less than 70 mm 3
- only 1 (12.5%) mouse had tumor progression (PD, progressive disease), indicating that ICP-024 alone has excellent anti-tumor ability in the CT-26-hCLDN18.2 mouse colon cancer model.
- Peripheral blood immune cell count analysis is shown in Figures 20A-D.
- the potential peripheral immune toxicity of each dosing group at the efficacy endpoint after repeated dosing was detected.
- the results of the absolute count of peripheral blood immune cells showed that compared with the control group, ICP-015 promoted the expansion of peripheral CD45+ immune cells, CD3+T cells and CD8+T cells, while ICP-024 showed good peripheral safety and did not cause excessive activation and expansion of peripheral blood immune cells.
- Example 12 CLDN18.2-Pro-IL-2 antibody fusion protein induces immune memory formation in the MC-38-hCLDN18.2 mouse colon cancer cell model
- the CLDN18.2-Pro-IL-2 fusion protein (ICP-069) with the reference antibody Zolbetuximab as the backbone was compared with the MC-38-hCLDN18.2-A11 (purchased from Nanjing Bowang) mouse colon.
- the MC-38-hCLDN18.2-A11 tumor modeling and grouping, and tumor size detection are the same as those in Example 11 above. When the tumor size reaches 60-80 mm 3 , intraperitoneal administration is started.
- the administration cycle is BIW (twice a week) for a total of 5 times, and the dosage is 260 nmol/mouse.
- the tumor inhibition rate TGI of ICP-024 was 93.75% (p ⁇ 0.0001, Oneway ANOVA)
- the tumor inhibition rate TGI of ICP-068 was 100.94% (p ⁇ 0.0001, Oneway ANOVA)
- the tumor inhibition rate TGI of ICP-069 was 67.38% (p ⁇ 0.001, Oneway ANOVA)
- the tumor inhibition rate TGI of ICP-070 was 59.58% (p ⁇ 0.01, Oneway ANOVA)
- the tumor inhibition rate TGI of ICP-087 was 108.84% (p ⁇ 0.0001, Oneway ANOVA).
- mice in the ICP-024 group achieved complete tumor regression
- 3/8 (37.5%) mice in the ICP-068 group achieved complete tumor regression
- 2/8 (25%) mice in the ICP-069 group achieved complete tumor regression
- no mouse in the ICP-070 group achieved complete tumor regression
- 8/8 (100%) mice in the ICP-087 group achieved complete tumor regression.
- mice with completely regressed tumors continued to be routinely fed and tumor monitored for 6 weeks, and then the contralateral side was re-inoculated with 5x 105 MC-38-hCLDN18.2-A11 (purchased from Nanjing Bowang) cells/mouse.
- 5x 105 MC-38-hCLDN18.2-A11 purchasedd from Nanjing Bowang
- 8 wild-type mice were inoculated as tumor control.
- the tumor growth was measured every week, and the observation was terminated when the average tumor value of the control group mice was about 1200mm3 .
- Figure 23 compared with the control group, there was no contralateral tumor progression in the ICP-024 group, and one mouse in the ICP-087 group had tumor progression.
- the contralateral tumor growth curves of individual mice are shown in Figures 24A-C.
- the tumor incidence of the contralateral inoculation of ICP-024 was 0% (0/70); the contralateral tumor incidence of ICP-087 was 12.5% (1/8), and only one mouse had contralateral tumor growth.
- Example 13 Toxicity study of CLDN18.2-Pro-IL-2 antibody fusion protein in MC-38-hCLDN18.2 mouse colon cancer cell model
- the main toxicity of IL-2 is capillary leakage and multiple organ failure.
- MC-38-hCLDN18.2 tumor-bearing mice to study the toxicity of CLDN18.2-Pro-IL-2.
- Mouse tumor cell inoculation and tumor measurement were as described in Example 12. When the tumor occurred and reached 500- 800 mm 3 , a single intraperitoneal administration was performed, and the dosage was 0.26 nmol/mouse. 96 hours after administration, the mouse lungs were collected and weighed, and after drying at 37°C for 48 hours, they were weighed again, and the difference was the net weight.
- connection method of Trastuzumab-Pro-IL-2 to IL2 and its shielding peptide is as described in Example 1, and its heavy chain and light chain sequences are shown in SEQ ID NO:42 and SEQ ID NO:41, and it is named ICP-269.
- the CHO-S cell density is adjusted to 6 ⁇ 10 6 cells/mL, and liposomes are used for transfection.
- the plasmid concentration is 1 mg/ml and cultured at 32°C, 5% CO 2 , and 100 rpm for 7-10 days. Feeds are added 18-22 hours after transfection and between the 5th day.
- the clear liquid is collected by ultrafiltration, and the protein is purified by Protein A, ion exchange column or molecular sieve.
- the collection tube where the target protein is located is selected according to UV280 absorption and stored together.
- the purified protein is ultrafiltered and exchanged into the target buffer.
- the binding ability of ICP-269 was detected using BT474 cells that highly express Her2.
- the binding constant Ec50 was 4.8nM, which is comparable to the trastuzumab binding constant Ec50 3.9nM ( Figure 29A, Table 13). This result indicates that after trastuzumab is made into an antibody fusion protein, it does not affect the binding ability of the antibody end.
- ICP-269 was digested with MMP14, and the digested proteins were electrophoresed under non-reducing and reducing conditions, as shown in Figures 28A and 28B.
- the digested proteins were tested for IL-2 activity using CTLL-2 and NK-92 cell proliferation assays ( Figures 30A and 30B).
- Trastuzumab-Pro-IL-2 has the characteristics of an activatable antibody fusion protein ( FIG. 30B , Table 16).
- connection method of Rituximab-Pro-IL-2 to IL2 and its shielding peptide is as described in Example 1, and its sequence is shown in SEQ ID NO:44 and SEQ ID NO:43, and it is named ICP-270.
- ICP-270 Its expression, purification and characterization are as described in Example 13.
- the purification results were detected by SDS-PAGE gel electrophoresis and SEC-HPLC high-performance liquid chromatography.
- the protein purity and content are shown in Figures 26B and 27B.
- the results show that ICP-270 has a high purity after one-step purification by protein A, with a purity greater than 95%.
- the binding capacity of ICP-270 was detected by using the CD20-highly expressing cell REC-1. Its binding constant Ec50 was 7.0 nM, which was equivalent to the rituximab binding constant Ec50 2.4 nM ( Figure 29B, Table 14). This result shows that the binding capacity of the antibody end of rituximab is not affected after it is made into an antibody fusion protein. Table 14 shows the binding constants of ICP-270 and CD20-highly expressing cell REC-1.
- ICP-270 was digested by MMP14, and the digested protein was run on non-reduced and reduced protein gels, as shown in Figures 28A and 28B.
- the digested protein was tested for IL-2 activity using CTLL-2 and NK-92 cell proliferation experiments ( Figures 30A and 30B).
- the EC50 of the fusion protein ICP-270+ that is, the digested ICP-270 in NK-92 cells was 9.15 ⁇ 1.2nM, which was equivalent to the EC50 activity of human recombinant IL-2 of 10.45 ⁇ 0.78; the uncut ICP-270-, its EC50 in NK-92 cells was 408 ⁇ 142.84nM, and the activity was reduced by 45.5 ⁇ 21.92
- the EC50 of the fusion protein ICP-270 in the inactive form and active form of CTLL-2 were 208 ⁇ 15.56 and 17 ⁇ 2.83, respectively, with a fold change of 12.5 ⁇ 3.54 times.
- Table 15 Summary of EC50 and fold change of ICP-269 and ICP-270 on NK-92 cell proliferation
- Table 16 Summary of EC50 and fold change of ICP-269 and ICP-270 on CTLL-2 cell proliferation
- the activated antibody fusion protein releases active IL-2 through the cleavable connecting peptide, thereby specifically increasing the phosphorylation of STAT5 in T cells or NK cells in the tumor microenvironment, promoting the proliferation of NK and T cells, and the release of cytokine IFN- ⁇ , achieving the effect of tumor killing.
- the CLDN18.2-Pro-IL-2, Trastuzumab-Pro-IL-2 and Rituximab-Pro-IL-2 fusion proteins provided by the present invention play a direct tumor killing role.
- the ADCC-enhanced CLDN18.2-Pro-IL-2 fusion protein provided by the present invention effectively improves the ADCC activity of the fusion protein, further improves its ADCC activity, and thereby produces antibody-mediated cell killing of CLDN18.2-positive tumor cells.
- the CLDN18.2-Pro-IL-2, Trastuzumab-Pro-IL-2 and Rituximab-Pro-IL-2 fusion proteins provided by the present invention, the ADCC-enhanced Fc end and the IL-2 cytokine end released and activated by enzymatic cleavage, through the joint action of NK cells locally infiltrating the tumor microenvironment, further increase the function of the antibody fusion protein and achieve a synergistic effect.
- the present invention provides a generally applicable antibody fusion protein form, which can promote the killing of target cells by immune cells through various molecular mechanisms, while effectively reducing the peripheral toxicity of IL-2 cytokines.
- the antibody fusion protein drug provided by the present invention has good tumor treatment effect and safety, and is a highly efficient and low-toxic antibody cytokine fusion protein product.
- NKTR-214 an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models. Clin Cancer Res, 22(3), 680-690. doi:10.1158/1078-0432. CCR-15-1631;
- a cytokine receptor-masked IL2 prodrug selectively activates tumor-infiltrating lymphocytes for potent antitumor therapy. Nat Commun, 12(1), 2768. doi:10.1038/s41467-021-22980-w;
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Abstract
L'invention concerne une protéine de fusion d'anticorps activable, ladite protéine de fusion comprenant une partie anticorps se liant spécifiquement à une cible, une partie Fc d'immunoglobuline, une partie de masquage et une partie cytokine. La présente invention concerne en outre un procédé de préparation de la protéine de fusion et son utilisation dans le traitement et/ou la prévention de tumeurs.
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