fusion protein containing the tgf-¿receptor and its pharmaceutical use

专利摘要:
the present invention provides a fg protein containing tgf-ß receptor and the pharmaceutical use thereof. furthermore, the present invention provides a bifunctional fusion protein comprising the targeting fraction of the pd-l1 antibody and the extracellular domain tgf-ßrii and a pharmaceutical composition comprising the fusion protein containing the tgf-ß receptor and the use thereof in preparation of a cancer medicine.
公开号:BR112019023184A2
申请号:R112019023184
申请日:2018-05-11
公开日:2020-05-19
发明作者:Gu Jinming；Tao Weikang；Luo Xiao
申请人:Jiangsu Hengrui Medicine Co；Shanghai hengrui pharmaceutical co ltd；
IPC主号:

专利说明:

Invention Patent Descriptive Report for: FUSION PROTEIN CONTAINING THE TGF-B RECEPTOR AND THE PHARMACEUTICAL USE OF THE SAME
FIELD OF INVENTION
[001] The present invention relates to the field of tumor immunotherapy drugs. In particular, the invention relates to fusion proteins for the treatment of cancer, involving a fusion protein comprising a target molecule and an immunomodulatory factor (such as TGF-pRII). More specifically, the present invention relates to a fusion protein formed by a molecule targeting the PD-L1 antibody and an immunomodulatory factor (such as TGF-pRII), a pharmaceutical composition comprising it and the use of it as a drug against cancer.
BACKGROUND OF THE INVENTION
[002] In the treatment of cancer, people have already recognized the high toxicity caused by chemotherapy and the negative effects that can lead to the generation of cancer cells resistant to drugs. Even if the treatment targeted overexpressed or activated proteins that are associated with tumor survival, cancer cells would still depend on mutations to reduce or escape dependence on the pathways that are targeted by targeted therapy, and also
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2/76 would survive in other ways. Tumor immunotherapy has received a lot of attention in recent years and is the focus of cancer treatment. It is difficult to develop resistance to drugs and this is the great advantage of this therapy. Based on theory and the immunological method, tumor immunotherapy mainly increases the immunogenicity of tumor cells and the sensitivity to effector cell death, stimulates and enhances the anti-tumor immune response and injects the immune cells and effector molecules into the host to coordinate the immune system to kill tumor cells and inhibit tumor growth.
[003] Programmed death protein 1 (PD-1) is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells and myeloid cells that have two ligands, PD-L1 (programmed death ligand 1) and PD-L2. PD-L1 interacts with PD-1 in T cells and plays an important role in the negative regulation of immune responses. Expression of the PD-L1 protein can be detected in many human tumor tissues. The tumor microenvironment can induce the expression of PD-L1 in tumor cells. PD-L1 expression is beneficial for the occurrence and growth of tumors and induces apoptosis
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3/76 of anti-tumor T cells. The inhibitor of the PD-1 / PD-L1 pathway can block the binding of PD-1 to PD-L1, block the negative regulatory signal and restore T cell activity to improve the immune response. Therefore, immunoregulation directed towards PD-1 / PD-L1 is important for tumor suppression.
[004] The transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily that regulates cell growth and differentiation. TGF-β transmits the signal through a heterotetrameric receptor complex that comprises two type I and two type II transmembrane serine / threonine kinase receptors.
[005] TGF-β is a multifunctional cytokine that has suppressive or tumor-promoting effects in a cell-dependent or dependent or background manner. The tumor suppressive effect of TGF-β signaling is due to the ability to induce the expression of multiple genes. When an epigenetic mutation or modification occurs during tumor development, cancer cells gradually tolerate inhibition of TGF-β signaling, which leads to the development of tumors.
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[006] Studies have found that blocking the TGF-β signaling pathway can reduce tumor metastasis. The dominant negative truncated Smad2 / 3 mutant was used to inhibit the TGF-β signaling pathway of the breast tumor cell line and it was found that the metastatic capacity of the tumor cells was inhibited. Colon cancer microsatellite instability studies found that inactive TGF-βΗΙΙ mutations reduced metastases and increased postoperative survival. However, in general, the use of inhibitors of the TGF-β signaling pathway alone has a weak effect on clinical treatment, which may be related to the high expression of TGF-β mainly in tumor cells and to the bioavailability of the inhibitors of the pathway. signaling.
[007] Therefore, inhibiting the PD-1 / PD-L1 pathway based on the blocking and neutralization of TGF-β in the tumor microenvironment can restore T cell activity, increase the immune response and improve the inhibition effect of the development of the occurrence of tumors. An PD-L1 antibody is provided by the applicant's previous PCT application PCT / CN2016 / 104320.
[008] Until now, TGF-β antibody / receptor fusion proteins disclosed in documents have been described
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W02006074451A2, W02ΟΟ9152610A1, WO2011109789Α2, WO2Ο13164694Α1, WO2Ο14164427Α1, WO2Ο15077540Α2, WO9309228A1, WO9409815A1, W0201502015511. However, some fusion proteins still have problems with instability or low expression. It is still necessary to develop products with better performance in production and clinical practice. The present invention provides a technical solution that benefits production and has a more stable performance.
SUMMARY OF THE INVENTION
[009] The present invention provides a TGF-β receptor-containing fusion protein, comprising a targeting fraction and a TGF-β receptor fraction, wherein the TGF-β receptor fraction is a truncated form at the N-terminus of the extracellular domain of TGF-βΗΙΙ.
[010] In a preferred embodiment of the present invention, where the truncated form at the N-terminus of the extracellular domain of the TGF-βΗΙΙ involves a deletion of 26 or less contiguous amino acids at the N-terminus of the extracellular terminal of the extracellular domain of the TGF-βΗΙΙ, preferably a deletion of 14 to 26 contiguous amino acids, more preferably a deletion of 14 to 21 contiguous amino acids,
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6/76 more preferably a deletion of 14 to 21 contiguous amino acids; As non-limiting examples, the N-terminal truncated form of the extracellular domain of TGF-pRII comprises the sequence of SEQ ID NO: 14 or SEQ ID NO: 15.
[011] In a preferred embodiment of the present invention, the sequence of the TGF-pRII extracellular domain is shown as SEQ ID NO: 13.
[012] In a preferred embodiment of the present invention, where the targeting fraction is a cell-specific targeting fraction; preferably, the targeting fraction is a specific targeting fraction for cancer cells.
[013] In a preferred embodiment of the present invention, in which the specific targeting fraction of cancer cells is selected from the group consisting of an antibody or antigen-binding fragment thereof, a growth factor, a hormone, a peptide, a receptor and a cytokine.
[014] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is selected from the group consisting of an antibody
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7/76 complete, a chimeric antibody, Fab ', Fab, F (ab') 2, a single domain antibody (DAB), Fv, scFv, a small antibody a bispecific antibody and a tri-specific antibody or a mixture of themselves.
[015] In a preferred embodiment of the present invention, wherein the antibody or antigen-binding fragment thereof binds to one or more of the following polypeptides or proteins selected from the group consisting of HER2, HER3, immunological checkpoint molecule , CD33, VEGF, VEGFR VEGFR -2, CD152, TNF, IL-1, IL-5, IL-17, IL-6R, IL-1, IL-2R, BLYS, PCSK9, EGER, c-Met, CD2, CD3, CDlla, CD19, CD30, CD38, CD20, CD52, CD60, CD80, CD86, TNF-α, IL-12, IL-17, IL-23, IL-6, IL-Ιβ, RSVF, IgE, RANK, BLyS, α4β7, PD-1, CCR4, SLAMF7, GD2, CD21, CD79b, IL20Ra, CD22, CD79a, CD72, IGF-1R and RANKL; preferably in which the antibody or antigen binding fragment thereof binds to the immunological checkpoint molecule.
[016] In a preferred embodiment of the present invention, wherein the antibody is an anti-PD-Ll antibody; preferably, the anti-PD-Ll antibody is selected from the group consisting of: MSB0010718C, MEDI4736, BMS-936559 and
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MPDL3280A; or the anti-PD-Ll antibody comprises one or more CDRs selected from the group consisting of those below or mutants:
HCDR1: SYWMH SEQ ID NO: 1 HCDR2: IR XiPNSG X2TSYNEKFKN SEQ 1 ID NO: 2 HCDR3: GGSSYDYFDY SEQ ID NO: 3 LCDR1: RASESVSIHGTHLMH SEQ ID NO: 4 LCDR2: AASNLES SEQ ID NO: 5 LCDR3: QQSFEDPLT SEQ ID NO: 6; where Xi is H or G, preferably G; X ₂ is G or F,
preferably F.
[017] In a preferred embodiment of the present invention, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or functional fragment thereof, a humanized antibody or a functional fragment thereof, or a human antibody or a functional fragment of it.
[018] In a preferred embodiment of the present invention, wherein the humanized antibody comprises a variable region of the heavy chain of SEQ ID NO: 7, preferably
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9/76 comprises a heavy chain variable region of SEQ ID NO: 9.
[019] In a preferred embodiment of the present invention, wherein the humanized antibody further comprises a heavy chain of SEQ ID NO: 11.
[020] In a preferred embodiment of the present invention, wherein the humanized antibody comprises a variable region of the light chain of SEQ ID NO: 8 or 10 or the mutant thereof.
[021] In a preferred embodiment of the present invention, wherein the humanized antibody comprises a light chain of SEQ ID NO: 12.
[022] In a preferred embodiment of the present invention, wherein the fusion protein comprising the TGF-β receptor is as shown in general formula (I):
Ab-L-TGF-βΗΙΙ ECD (I) where TGF-βΗΙΙ ECD is a truncated form of the extracellular domain of TGF-βΗΙΙ;
Ab is an antibody;
L is a ligand.
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[023] In a preferred embodiment of the present invention, where the linker is (G4S) _X G, where x is 3 to 6, preferably it is 4 to 5.
[024] The present invention further provides a pharmaceutical composition, comprising a therapeutically effective amount of a TGF-β receptor-containing fusion protein as described above, and one or more pharmaceutically acceptable carriers, diluents or excipients.
[025] The present invention further provides a DNA molecule encoding the fusion protein comprising the TGF-β receptor as described above.
[026] The present invention further provides an expression vector, comprising the DNA molecule as described above.
[027] The present invention further provides a host cell transformed with the expression vector as described above, wherein the host cell is selected from the group consisting of a bacterial, yeast and mammalian cell; preferably a mammalian cell.
[028] The present invention further provides a use of the fusion protein containing the TGF-β receptor, as described above, or the pharmaceutical composition thereof for the preparation
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11/76 of a drug for the treatment of tumors; preferably for the preparation of a medicament for the treatment of a tumor mediated by PD-L1; more preferably a cancer that expresses PD-L1.
[029] The present invention further provides a method for treating or preventing a tumor comprising administering to a patient in need of a therapeutically effective amount of the TGF-β receptor-containing fusion protein, as described above.
[030] The present invention further provides a TGF-pRII truncated extracellular domain, wherein the TGF-pRII truncated extracellular domain involves a deletion of 26 or less contiguous amino acids at the N-terminus of SEQ ID NO: 13, preferably a deletion of 14 to 26 contiguous amino acids at the N-terminus, more preferably a deletion of 14 to 21 contiguous amino acids at the N-terminus; non-limiting examples of the truncated extracellular TGF-pRII domain comprise the sequence shown as SEQ ID NO: 14 or SEQ ID NO: 15.
[031] The present invention further provides a pharmaceutical composition, comprising a therapeutically amount
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12/76 effective truncated extracellular TGF-pRII domain of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.
[032] The present invention further provides a use of the truncated extracellular TGF-pRII domain of the present invention or a pharmaceutical composition thereof for the preparation of a medicament for the treatment or for the inhibition of diseases or disorders associated with the proliferation or metastasis of cancer cells.
[033] The present invention further provides a method for treating or preventing a tumor comprising administering to a patient in need of a therapeutically effective amount of the truncated extracellular TGF-pRII domain of the present invention or the pharmaceutical composition thereof. .
[034] The tumor or cancer described in this disclosure is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, stomach cancer, prostate cancer, kidney cancer, cancer cervix, myeloma, lymphoma, leukemia, thyroid, endometrial cancer, uterine cancer, bladder cancer, cancer
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13/76 neuroendocrine, head and neck cancer, liver cancer, nasopharynx cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, skin cancer squamous cell, protuberant dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.
DESCRIPTION OF THE DRAWINGS
[035] Figure 1: Schematic diagram of the fusion protein structure.
[036] Figure 2: Results showing the binding of the fusion protein to human TGF-βΙ in vitro.
[037] Figure 3: Results showing the binding of the fusion protein to human TGF-βΙ in vitro.
[038] Figure 4: Results showing the binding of the fusion protein to human PD-L1 in vitro.
[039] Figure 5: Result showing the detection of blockade of the PD-1 / PD-L1 pathway by the in vitro fusion protein.
[040] Figure 6: The fusion protein inhibits Τ6Γβ-induced pSMAD3 reporter activity in a dose-dependent manner.
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[041] Figure 7: All samples of fusion proteins increase IFN-γ cytokine secretion by activated T lymphocytes.
[042] Figure 8: Effect of the fusion protein on the tumor weight of tumor-bearing mice.
DETAILED DESCRIPTION OF THE INVENTION
[043] In order to make the invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise stated elsewhere, all scientific technical terms used herein have the same meanings as those commonly understood by a specialist in the technique to which the present disclosure belongs.
[044] As used herein, the one-letter code and the three-letter code for amino acids are those described in J. Biol. Chem, 243, (1968) p3558.
[045] As used herein, antibody refers to immunoglobulin, a four peptide chain structure formed by two identical heavy chains and two identical light chains connected by inter-chain disulfide bond. Different regions in the heavy chain of
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15/76 immunoglobulins exhibit different compositions and sequences of amino acids, so they have different antigenicity. Therefore, immunoglobulins can be divided into five categories, also called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE; the corresponding heavy chains are μ chain, δ chain, Y chain, a chain, and chain, respectively. According to the amino acid composition of the hinge region and the number and location of the heavy chain disulfide bonds, immunoglobulins can be divided into different subcategories, for example, IgG can be divided into IgGl, IgG2, IgG3 and IgG4. The light chain can be divided into a k or λ chain, based on different constant regions. Each category of Ig among these five categories involves k or λ chains.
[046] In the present invention, the antibody light chain mentioned herein further comprises a light chain constant region, which comprises a human or murine κ, λ chain or a variant thereof.
[047] In the present invention, the antibody heavy chain mentioned herein further comprises a constant region of the chain
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16/76 heavy, comprising IgGl, IgG2, IgG3, human or murine IgG4 or a variant thereof.
[048] At the N-terminus of the heavy and light chains of the antibody about 110 amino acids vary widely, what is known as the variable region (Fv region); the amino acid sequence at the C-terminus is relatively stable, which is known as the constant region. The variable region comprises three hypervariable regions (HVR) and four FR regions (FR) with relatively conserved sequence. Three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each variable region of the light chain (LCVR) and each variable region of the heavy chain (HCVR) is composed of three CDR regions and four FR regions, arranged from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Three CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; three heavy chain CDR regions refer to HCDR1, HCDR2 and HCDR3. The number and location of the amino acid residues of the CDR region in the LCVR and HCVR regions of the antibody or antigen binding fragment here in accordance with the known Kabat numbering criteria
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17/76 (LCDR1-3, HCDR2-3) or with the numbering criteria of Kabat and Chothia (HCDR1).
[049] The antibody of the present invention comprises complete antibody selected from the group consisting of murine antibody, chimeric antibody and humanized antibody, preferably it is humanized antibody.
[050] The term murine antibody in the present invention refers to the human anti-PD-Ll monoclonal antibody prepared according to the knowledge and skills in the field. During preparation, the test subject was injected with PD-L1 antigen and then the antibody expressing the hybridoma having the desired sequence or functional characteristics was isolated. In a preferred embodiment of the present invention, the murine PD-L1 antibody or antigen-binding fragment thereof, further comprises the light chain constant region of the κ, λ chain or a variant thereof, or comprises the constant region of the chain IgG1, IgG2, murine IgG3 or a variant thereof.
[051] The term chimeric antibody is an antibody formed by the fusion of the variable region of a murine antibody with the constant region of the human antibody, in order to alleviate
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18/76 the immune response induced by the murine antibody. To establish a chimeric antibody, a first specific hybridoma-secreting murine monoclonal antibody is first established, variable region genes are then cloned from murine hybridoma cells, and then human antibody constant region genes are cloned as desired, genes from the murine variable region are linked to human genes from the constant region to form a chimeric gene that can be inserted into a human vector, and finally, the chimeric antibody molecule is expressed in an eukaryotic or prokaryotic industrial system. In a preferred embodiment of the present invention, the light chain of the chimeric antibody PD-L1 further comprises the light chain constant regions derived from the human κ, λ chain or a variant thereof. The heavy chain of the chimeric antibody PD-L1 further comprises the heavy chain constant regions derived from human IgG1, IgG2, IgG3, IgG4 or a variant thereof. The human antibody constant regions can be selected from the IgG1, IgG2, IgG3, IgG4 heavy chain constant regions or a variant thereof, preferably comprising the IgG2 heavy chain constant region
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19/76 or human IgG4 or IgG4 without ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation.
[052] The term humanized antibody, also known as CDR-grafted antibody, refers to an antibody generated by murine CDR sequences grafted onto the structure of the human antibody variable region, that is, antibody generated from different types of structure sequences of human germline antibody. The humanized antibody achieves the disadvantageously strong anti-antibody response induced by the chimeric antibody that carries a large number of murine components. Such structural sequences can be obtained from a public DNA database covering the germline antibody gene sequences or published references. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available on the web at www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, et al.1991 Sequences of Proteins of Immunological Interest, 5th Ed. To avoid the decrease in activity caused by the reduction
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20/76 of immunogenicity, the structure of the variable region of the human antibody is subjected to a minimum reverse mutation to maintain activity. The humanized antibody of the present invention also comprises a humanized antibody which is further obtained by displaying phages for the purpose of CDR affinity maturation.
[053] The terms human antibody and antibody from human are used interchangeably to mean that one or more variable and constant regions are derived from a human immunoglobulin sequence. In a preferred embodiment, all variable and constant regions are derived from human immunoglobulin sequences, that is, fully human derived antibodies or fully human antibodies. These antibodies can be obtained in many ways, including phage display technology; isolation of B cells from human PBMC, spleen or lymph nodes; construction of a library of human native single-stranded phage antibodies; or by immunizing transgenic mice that express light and heavy chains of human antibodies; and screening for antibodies so obtained.
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[054] As used herein, antigen binding fragment or functional fragment refers to the Fab fragment, Fab 'fragment, F (ab') 2 fragment with antigen binding activity, as well as Fv fragment, scFv fragment that binds to the Human PD-L1. The Fv fragment is the minimum antibody fragment that involves all the antigen binding sites, the Fv fragment comprises a variable region of the heavy chain and a variable region of the light chain, but without a constant region. Generally, the Fv antibody further comprises a polypeptide linker between the VH and VL domains to form a necessary structure for binding to the antigen. In addition, different ligands can be used to connect the variable regions of two antibodies to form a polypeptide, called single chain antibody or single chain Fv (scFv). As used herein, the term PD-L1 binding means the ability to interact with human PD-L1. As used herein, the term antigen-binding site of the present invention refers to the three-dimensional discontinuous sites on the antigen, recognized by the antibody or antigen-binding fragment of the present invention.
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[055] As used herein, the term ADCC, namely antibody-dependent cell-mediated cytotoxicity, refers to cells that express Fc receptors that directly kill target cells coated by an antibody, recognizing the antibody's Fc segment. The ADCC effector function of the antibody can be reduced or eliminated by modifying the Fc segment in IgG. Amodification refers to mutations in the antibody heavy chain constant region, such as the selected mutations of N297A, L234A, L235A in IgGl; IgG2 / 4 chimera; or F234A / L235A mutations in IgG4.
[056] The mutation in the mutant sequence of the present invention includes, but is not limited to, reverse mutation, conservative modification or conservative replacement or replacement. Conservative modification or conservative substitution or replacement in the present disclosure refers to the substitution of amino acids in a protein with other amino acids with similar characteristics (for example, charge, side chain size, hydrophobicity / hydrophilicity, structure conformation and rigidity, etc.), so that changes can often be made without changing the biological activity of the
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23/76 protein. Those skilled in the art recognize that, in general, the substitution of a single amino acid in the non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benj amin / Cummings Pub. Co., p. 224 (4th Ed)). In addition, structurally or functionally similar amino acid substitutions are less likely to disrupt biological activity.
[057] The mutant sequence, as used in the present invention, means that the nucleotide sequence and the amino acid sequence of the present invention are subject to substitution, insertion or deletion, thus, the obtained nucleotide sequence and the amino acid sequence share identity. percent variable with the nucleotide sequence and amino acid sequence of the present invention.
[058] As used herein, identity indicates the degree of similarity between two nucleic acids or two amino acid sequences. The sequence identity in the present invention is at least 85%, 90% or 95%, preferably at least 95%. Representative examples include, but are not limited to, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
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99%, 100%. The comparison of the sequences and the determination of the percentage of identity between the two sequences can be performed using the standard configurations of the BLASTN / BLASTP algorithm, available on the website of the National Center for Biotechnology Institute.
[059] The PD-L1 antibody or antigen-binding protein thereof of the present invention can include any of the anti-PD-L1 antibodies or antigen-binding fragments thereof described in the art. The anti-PD-Ll antibody may be a commercially available PD-L1 antibody or has been disclosed in the literature, including, but not limited to, the PD-L1 antibody BMS-936559, MPDL3280A, MEDI4736, MSB0010718C (see US2014341917, US20130034559, US8779108 ) and the like. The antibody can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody. The antibody fragment includes Fab fragment, Fab 'fragment, F (ab') 2 fragment with antigen binding activity and Fv fragment and scFv fragment that bind to the antigen.
[060] As an exemplary PD-L1 antibody preparation process of the present invention, see PCT / CN2016 / 104 32 0, the
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25/76 PD-L1 antibody comprises CDRs from variable regions of the heavy chain, as described below:
HCDR1: SYWMH SEQ ID NO: 1
HCDR2: RI XiPNSG X ₂ TSYNEKFKN SEQ ID NO: 2
HCDR3: GGSSYDYFDY SEQ ID NO: 3.
[061] In an alternative mode, Xi is selected from
H or G; and X2 is selected from G or F.
[062] In another embodiment, an exemplary PD-L1 antibody of the invention further comprises the sequence of CDRs of a variable region of the light chain, as described below:
LCDR1: RASESVSIHGTHLMH SEQ ID NO: 4
LCDR2: AASNLES SEQ ID NO: 5
LCDR3: QQSFEDPLT SEQ ID NO: 6.
[063] In another embodiment, the CDR regions above are humanized by CDR grafting, and the FR of the humanized light chain models are IGKV7-3 * 01 and hjk2.1, the FR of the humanized heavy chain models are IGHV1-46 * O1 and hjh6.1, and the humanized variable region sequences are as follows: humanized heavy chain variable region:
OVOLyOSGAEVKKPGASVKFSCKASGYTFTSY ^ MHiVVROAPGQGLEIVMGRIX ^ NSGX2TSYNEKFKWFTA / 77 Dr5757 ¹ KW £ ^, L55Z-RS ^, ££> r.4 ^ TyC4j GGSSYDYFDYTVGGGT
SEQ ID NO: 7 humanized light chain variable region:
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DIVLTQSPASLA VSPGQRA TJTCRK ^ N ^ GTHUVtiiWYOOKPGOPPKLLIYKAS ^
IttGVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSYEDPlXFGQGTKLEIK
SEQ ID NO: 8
[064] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic fraction represents the FR sequence and the underlined fraction represents the CDR sequence.
[065] In another embodiment, the design of the reverse mutation in the humanized antibody of the present invention was carried out, see the table as follows:
Table 1
[066] NOTE: For example, Y91F indicates a reverse mutation from Y to F at position 91, according to the Kabat numbering system.
[067] Graft indicates that the murine antibody CDR was implanted in the FR sequences of the human germline.
[068] The new humanized antibody can be obtained by various combinations of heavy chain and light chain mutations shown in the table above.
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[069] In another aspect of the invention, a modality for the construction of a humanized clone is provided, as follows:
[070] The primers were designed and the fragments of the VH / VK gene of each humanized antibody were constructed by PCR and then inserted into the pHr expression vector (with signal peptide and constant region gene fragment (CH1-Fc / CL )) to perform homologous recombination in order to construct a complete antibody expression vector: VH-CHl-Fc-pHr / VK-CL-pHr.
1. Design of the Initiator:
[071] The DNAWorks online software (v3.2.2) (http://helixweb.nih.gov/dnaworks/) was used to design the various primers for VH / VK synthesis containing gene fragments necessary for recombination: Signal peptide 5'-30bp + VH / VK + 30bp CH1 / CL-3 '.
2. Splicing of fragments:
[072] In accordance with the TaKaRa Company's Primer STAR GXL DNA polymerase operating instructions, using the primers designed above, gene fragments containing VH / VK necessary for recombination by two-step PCR amplification were obtained.
3. Construction of the pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC / CL)) and
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28/76 enzymatic digestion:
[073] The pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC / CL)) was designed and constructed using some special restriction endonuclease, such as BsmBI, which recognizes the distinguishing characteristic between the sequence and the restriction site. BsmBI digested the vector, and then the digested fragments were extracted using gel and stored for use.
4. Recombinant construction of the VH-CHl-Fc-pHr / VK-CL-pHr expression vector
[074] VH / VK containing gene fragments necessary for recombination and the pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC / CL)) digested with BsmBI were added to the competent DH5H cells in proportion 3: 1, incubated at 0 ° C on ice for 30min, hatched with heat at 42 ° C for 90s, combined with 5 volumes of LB medium, incubated at 37 ° C for 45min, sown in an LB-Amp plate and grown at 37 ° C overnight. The single clone was chosen for sequencing and a clone of interest was obtained.
5. The plasmid was constructed according to the design of the present example, then the purified protein and affinity
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29/76 of the obtained protein were measured by the detection described in Example SPR.
6. Finally, the affinity of the humanized mutant reverse mutation antibodies or hybridoma with human PD-Ll-his was measured by BIACORE, the humanized reverse mutation sites obtained and the combination of sequences by screening are as follows:
heavy chain variable region:
OVOLVQSGAEVKKPGASVKVSCKASGYTFTSYWMFIJWROAPGOGLEWMGRIGP ^ SGFTSYNEKFKN / VTMTRDTSTSTEYMELSSLRSEDTA FYYCARGGSSYDYFDYWG QGTTVTVSS
SEQIDNC): 9 where CDR2 is a sequence where Xi of SEQ ID NO: 7 is G and X ₂ is F.
light chain variable region:
DIVL TQSPASLA VSPGQRA 77TCRASESVSIHGTHLMH WYQQKPGQPPKLLIYAASN LESGVPARFSGSGSGTDFTLTINPFEAEDTANYYCQQSFEDPLYFGQGTKLEIK
SEQ1DNO: 10
[075] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic fraction represents the FR sequence and the underlined fraction represents the CDR sequence.
[076] In another aspect of the present invention, a modality is provided for the construction and expression of a human IgG4 anti-PD-Ll antibody, and a PD-L1 antibody used for the construction of the fusion protein is also provided. The PD-L1 antibody
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30/76 can also be used as a control molecule in the test examples of the present invention.
Since PD-L1 is also expressed in activated T cells, therefore, the use of IgGl constant regions of the wild type can cause Fc-mediated effects, such as ADCC and CDC, which could result in the reduction of activated T cells. The present invention selects the mutated IgG4 to obtain antibodies without ADCC and CDC. The clone obtained by affinity maturation was converted to the IgG4 type, and the central region of the IgG4 hinge contained the S228P mutation, and the F234A and L235A mutations were introduced (mAbs 4: 3, 310-318; May / June 2012) . At the same time, to prevent breakages from occurring at the C-terminus of the antibody heavy chain when the linker peptide (which is used to bind the TGF-pRII extracellular domain), the last K amino acid of the PD-L1 antibody heavy chain -LI was further mutated to A in order to increase the stability of the fusion protein. The PD-L1 antibody sequence of the present invention used to construct fusion proteins is as follows:
[077] PD-L1 antibody heavy chain: IgG4 (AA) (S228P)
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OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGOGLEWMGRI GPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYD YFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK
VDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVYSLQCL
SEQ ID NO: 11
PD-L1 antibody light chain:
DIVLTOSPASLAVSPGORATITCRASESVSIHGTHLMHWYOQKPGOPPKLLIYA
ASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCOOSFEDPLTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTYTKS
SEQ ID NO: 12
[078] NOTE: The underlined fraction is a variable region sequence of the heavy or light chain of the antibody, or the nucleotide sequence encoding it; the remaining fraction is the sequence of the antibody constant region and the nucleotide sequence encoding it.
[079] As used herein, a fusion protein described in the present invention is a protein product obtained by the concomitant expression of two genes using DNA recombination technology. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in
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Antibodies, A Laboratory Manual, Cold Spring Harbor, chapters 5 through 8 and 15. For example, mice can be immunized with human PD-L1 or fragments thereof, and the resulting antibodies can be renatured, purified and sequenced for the sequences amino acids using conventional methods well known in the art. Antigen-binding fragments can also be prepared by conventional methods. The antibody or antigen binding fragments of the present invention are designed to engraft non-human antibody derived CDRs into one or more human FRs. When aligning against the germline database of the IMGT human antibody variable region using the MOE software, the germline sequences of the human structure can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt. cines. f r or from The Immunoglobulin Facts Book, 2001, ISBN 012441351.
[080] Projected antibodies or antigen-binding fragments of the present invention can be prepared and purified using known methods. For example, cDNA sequences that encode a heavy chain and a light chain can be cloned and manipulated into an expression vector
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GS. The engineered immunoglobulin expression vector can then be stably transfected into CHO cells. As a more recommended method known in the art, the mammalian expression system will result in glycosylation of the antibody, typically at highly conserved N-terminal sites in the Fc region. Stable clones can be obtained by expression of an antibody that specifically binds to human PD-L1. Positive clones can be expanded in serum-free culture to produce antibodies in bioreactors. The culture medium, in which the antibody was secreted, can be purified by conventional techniques. For example, the medium can be loaded onto a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer. The column is washed to remove non-specific binding components. The bound antibody is eluted by a pH gradient and the antibody fractions are detected by SDS-PAGE and then collected. The antibody can be filtered and concentrated using standard techniques. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion or ion exchange. The product can be frozen immediately, for example at -70 ° C, or it can be lyophilized.
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[081] The immunomodulatory molecule of the present invention can be used to attenuate the immune tolerance of cancer cells. The present invention uses a truncated form of the extracellular domain of TGF-βΗΙΙ as the immunomodulatory molecule in the fusion protein. The TGF-β II receptor (TGF-βΗΙΙ) binds the TGF-βΙ and 3 ligands with high affinity. The Τ6Ε-βΗΙΙ / Τ6Ε-β complex recruits TGF-βΗΙ to form a signal transduction complex (Won et al, Cancer Res. 1999; 59: 1273-7). The TGF-βΗΙΙ extracellular domain is a 136-amino acid residue peptide from the TGF-βΗΙΙ extracellular amino acid, a particular example of which is shown in SEQ ID NO: 13. Other variants of about 136 amino acids in length and derived from human extracellular domain of TGF-βΗΙΙ, which are capable of binding to TGF-βΙ and 3, also belong to the extracellular domain of TGF-βΗΙΙ of the invention. The present invention has found that the structure and function of the contiguous truncated form at the N-terminus of the TGF-βΗΙΙ extracellular domain is more stable than that of the non-truncated molecule. A fusion protein comprising the N-terminal truncated form of the extracellular domain of TGF-βΗΙΙ (a polypeptide shown as aa.1-136 of SEQ ID NO: 13) is susceptible to cleavage. In
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35/76 in particular, a truncated form comprising a deletion of at most 26 amino acids at its N-terminus is more stable, preferably a truncation of 14 to 26 amino acids, more preferably a truncation of 14 to 21 amino acids at the N-terminus with a level of higher expression, more preferably, a truncation of 19 or 21 contiguous amino acids at the N-terminus.
[082] Administration and treatment, as applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of a pharmaceutical, therapeutic, diagnostic agent or exogenous composition to the animal, human, subject, cell, tissue, organ or biological fluid. Administration and treatment may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. The treatment of a cell includes the contact of a reagent with the cell, as well as the contact of a reagent with a fluid, where the fluid is in contact with the cell. Administration and treatment also means in vitro and ex vivo treatments, for example, of a cell by a reagent, diagnostic compound, binding compound or by another cell.
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Treatment, as it applies to human beings, veterinary beings or research beings, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
[083] Treating means administering a therapeutic agent, such as a composition containing any of the binding compounds of the present invention, internally or externally to a patient with one or more symptoms of disease for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population to be treated, to induce regression or to prevent the progression of such symptoms from a clinically measurable level. The amount of a therapeutic agent that is effective in alleviating any specific symptom of the disease (also called a therapeutically effective amount) can vary according to factors such as the condition of the disease, the age and weight of the patient and the ability of the drug to obtain a desired response in the patient. If a symptom of the disease has been relieved it can be assessed by any clinical measure normally used by doctors or other specialized health professionals to
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37/76 assess the severity or status of symptom progression. While one embodiment of the present invention (for example, a treatment method or article of manufacture) may not be effective in alleviating the symptoms of the target disease in all patients, it should alleviate the symptoms of the target disease in a statistically significant number of patients , as determined by any statistical test known in the art as the Student's t test, the chi-square test, the U test according to Mann and Whitney, the Kruskal-Wallis test (H test), Jonckheere-Terpstra test and the Wilcoxon test.
[084] Conservative modifications or conservative substitution refer to the substitution of amino acids in a protein with other amino acids that have similar characteristics (for example, charge, side chain size, hydrophobicity / hydrophilicity, structure conformation and rigidity, etc.), so that changes can be made frequently without altering the biological activity of the protein. Those skilled in the art recognize that, in general, the replacement of a single amino acid in the nonessential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular
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Biology of the Gene, The Benj amin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, structurally or functionally similar amino acid substitutions are less likely to disrupt biological activity.
[085] Effective amount covers an amount sufficient to improve or prevent a symptom or sign of the medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a specific patient or veterinary subject may vary depending on factors such as the condition being treated, the patient's general health condition, the route and dose of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing protocol that avoids significant side effects or toxic effects.
[086] Exogenous refers to substances that are produced outside of an organism, cell or human body, depending on the context. Endogenous refers to substances produced within a cell, organism or human body, depending on the context.
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[087] Homology refers to the sequence similarity between the two polynucleotide sequences or between the two polypeptide sequences. The molecules are considered homologous in one position, when this position in both sequences to be compared is occupied by the same base subunit or amino acid monomer, for example, when a position in each of the two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of corresponding or homologous positions shared by two sequences divided by the number of all positions to be compared and then multiplied by 100. For example, in an ideal alignment, if 6 out of 10 positions in two sequences are corresponding or homologous, then the two sequences share 60% homology. The comparison is usually made when two strings are aligned to provide the maximum percentage of homology.
[088] Immune checkpoint molecules include the stimulating immune checkpoint molecule and the inhibitory immune checkpoint molecule, and exemplary molecules include CD27, CD28, CD40, CD40L, CD122, 0X40, OX40L, GITR, IGOS , A2AR, B7-H3, B7-H4, BTLA,
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CTLA-4, IDO, KIR (killer cell immunoglobulin-like receptor), LAG3, PD-1, PD-L1, PD-L2, TIM-3, VISTA, etc.
[089] As used herein, the expressions cell, cell line and cell culture are used interchangeably and all of these designations include their progeny. Thus, the words transformant and transformed cell include the cells and primary cultures subject to it, regardless of the number of passages. It should also be understood that the entire progeny may not be exactly identical in terms of DNA content, due to intentional or unintended mutations. Mutant progeny that have the same biological function or activity as that of originally transformed cells are obtained by screening and must be included in the invention. Where the distinctive designations are intended will be clearly understood from the context.
[090] As used herein, the polymerase chain reaction or PCR refers to a procedure or technique in which small amounts of specific segments of nucleic acid, RNA and / or DNA are amplified as described in, for example in U.S. Pat. No. 4,683,195. Sequence information is usually required at the ends or
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41/76 beyond the region of interest, so that oligonucleotide primers can be designed; the sequence of these primers will be identical or similar to the opposite strand of the template to be amplified. The 5 'terminal nucleotides of the two primers coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See, generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used in the present invention is considered an example, but not the only one, of the polymerase reaction method for amplifying a nucleic acid test sample. The method comprises the use of nucleic acids known as primers and the nucleic acid polymerases to amplify or generate a specific segment of nucleic acid.
[091] Optionally or optionally means that the event or situation described subsequently may, but not necessarily, and the description includes the cases in which the event
Petition 870190112847, of 11/04/2019, p. 57/109 or circumstance occurs or not. For example, optionally containing 1 to 3 variable regions of the antibody heavy chain means that the variable region of the antibody heavy chain with the specific sequence may be present, but not necessarily.
[092] Pharmaceutical composition refers to a mixture comprising one or more compounds according to the present invention or physiologically / pharmaceutically acceptable salts or prodrugs with other chemical components, said chemical components are such as physiologically / pharmaceutically carriers and excipients acceptable. The pharmaceutical composition aims to promote administration by an organism, facilitating the absorption of the active ingredient and, thus, exerting a biological effect.
EXAMPLES AND TEST EXAMPLES
[093] In the following, the present disclosure will be described in detail with reference to the examples. However, the scope of the present invention is not limited to these.
[094] In the examples of the present invention, where specific conditions are not described, experiments are
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43/76 generally conducted under conventional conditions or under the conditions proposed by the manufacturers of materials or products. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Modern Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Where the source of the reagents is not specifically indicated, the reagents are conventional reagents available on the market.
EXAMPLES
Example 1: Cloning and trap expression of the PD-Ll / TGF-β fusion protein
[095] The TGF-βΗΙΙ extracellular domain (full or truncated form of SEQ ID NO: 13) is used as the fraction of the immunomodulatory molecule in the fusion protein, and the PD-L1 antibody is used as a targeting fraction of fusion protein to form a PD-L1 antibody fusion protein / TGF-βΗΙΙ extracellular domain (PD-L1 / TGF-β trap). Studies have found that the truncated form of the TGF-βΗΙΙ extracellular domain is relatively stable, the truncated form especially involves the exclusion of less than 26 amino acids at its N-terminus, preferably a deletion of 14 to 26
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44/76 amino acids, more preferably a deletion of 14 to 21 contiguous amino acids, which exhibits greater expression and stable structure; more preferably a deletion of 14, 19 or 21 contiguous amino acids. The sequences of the non-limiting examples of the TGF-pRII extracellular domain and its truncated form of the invention are as follows:
[096] TGF-pRII extracellular domain sequence: EGD (1-136)
1PPHVQKSVNNDMIVTDNNGAVKFPQ.LCKFCDV.RFSTCDNQKSCMSNCSIT S1CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCTMKEKKK PGETFFMCSCSSDECNDNIIFPEE
SEQ ID NO: 13
[097] Sequence of the truncated TGF-pRII extracellular domain involving a deletion of 19 contiguous N-terminal amino acids: EGD (20-136)
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDE NJTLETVCHDPKLPYHDFlLEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNnF SEEYNTSNPD
SEQ ID NO: 14
[098] Sequence of the truncated TGF-pRII extracellular domain involving a deletion of 21 contiguous amino acids at the N: EGD terminal (22-136):
VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENIT LETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNnFSE EYNTSNPD
SEQ ID NO: 15
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[099] Sequence of the truncated TGF-pRII extracellular domain involving a deletion of 14 contiguous amino acids at the N-terminal: ECD (15-136):
VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW RKNDENITLETVCHDPKLPYHDFILEDAASPKC1MKEKKKPGETFFMCSCSSDEC NDNIIFSEEYNTSNPD
SEOIDNO: 16
[0100] The C-terminal amino acid of the PD-L1 antibody heavy chain of the present invention was linked by the ligand (G ₄ S) _x G to the TGF-pRII extracellular domain with varying lengths by the homologous recombination technique and was conventionally expressed by expression system 293 together with the light chain and the obtained fusion proteins are shown in Table 2:
Table 2: PD-Ll antibody fusion protein / TGF-pRII extracellular domain
ProteinFusion in description of the sequence the number ofamino acidscontiguous deleted inTerminal N Protein in Ab- (G ₄ S) ₄ G -ECD (1-136) No deletion
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fusion 1Proteinfusion 2 in Ab- (G ₄ S) 3G-ECD 15-136) 14 Proteinfusion 3 in Ab- (G ₄ S) N19A) 3G-ECD 15-136, 14 Proteinfusion 4 in Ab- (G ₄ S) 3G-ECD 20-136) 19 Proteinfusion 5 in Ab- (G ₄ S) 3G-ECD 22-136) 21 Proteinfusion 6 in Ab- (G ₄ S) 3G-ECD 27-136) 26 Proteinfusion 7 in Ab- (G4S ₄ g-ecd (15-136) 14 Proteinfusion 8 in Ab- (G4SN19A) ₄ g-ecd (15-136, 14 Proteinfusion 9 in Ab- (G4S ₄ g-ecd (20-136) 19 Proteinfusion 10 in Ab- (G4S) ₄ G-ECD (22-136) 21 Proteinfusion 11 in Ab- (G4S) ₄ G-ECD (27-136) 26
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Proteinfusion 12 in Ab- (G ₄ S ) sG-ECD (15-136) 14 Proteinfusion 13 in Ab- (G ₄ S N19A) ) sG-ECD (15-136, 14 Proteinfusion 14 in Ab- (g ₄ s ) sG-ECD (20-136) 19 Proteinfusion 15 in Ab- (g ₄ s ) sG-ECD (22-136) 21 Proteinfusion 16 in Ab- (g ₄ s ) sG-ECD (27-136) 26 Proteinfusion 17 in Ab- ( G ₄ S) eG-ECD ( 27-136) 26
[0101] NOTE: Ab represents the PD-L1 antibody of the present invention, ECD (n-136) in the Sequence Description represents the complete or truncated form of the TGF-pRII extracellular domain, n represents the initial number of amino acids after testing the truncation of the extracellular TGF-pRII domain. The structure of the fusion protein of the present invention is shown in Figure 1; NI 9A indicates that the amino acid at position 19 of the extracellular domain of TGF-pRII has been transformed into A.
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[0102] The nucleotide sequence encoding the PD-L1 antibody, the nucleotide sequence encoding the extracellular domain of TGF-βΗΙΙ, and the nucleotide sequence of the ligand protein fragment ((G4S) _X G) are obtained by the technique conventional. The C-terminal nucleotide of the PD-L1 antibody was linked via the protein binding to the N-terminal nucleotide of the extracellular TGF-βΗΙΙ domain of different length by the homologous recombination technique and then cloned into the Phr-BsmbI vector. The recombinant PD-Ll / TGF-β trap was expressed in 293 cells and purified as described in Example 2. The purified protein can be used in the experiments of the following examples.
Example 2: Purification of the PD-Ll / TGF-β trap fusion protein
[0103] The cell culture medium was centrifuged at high speed and the supernatant was collected, and the first purification step was performed by affinity chromatography. The chromatographic medium is Protein A or a derived filling material that interacts with Fc, such as GE's Mabselect. The equilibration buffer was 1 x PBS (137 mmol / L NaCl, 2.7 mmol / L KC1, 10 mmol / L Na2HPO4, 2 mmol / L KH2PO4, pH
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7.4). After equilibrating 5 column volumes, the cell supernatant was charged for ligation and the flow rate was controlled so that the sample could remain in the column for ± 1 minute. After loading the sample, the column was washed with 1 x PBS (pH 7.4) until the A280 reading was reduced to the baseline. Then, the column was washed with 0.1 M glycine elution buffer (pH 3.0) and the eluted peak was collected according to the A280 ultraviolet absorption peak, and the eluted sample collected was neutralized with 1 M Tris (pH 8.5).
[0104] The neutralized eluted sample was concentrated by ultrafiltration and then subjected to size exclusion chromatography, the buffer was 1 * PBS and the column was XK26 / 60 Superdex 200 (GE). The flow rate was controlled at 4 ml / min, the loading volume was less than 5 ml and the peak protein target was pooled according to the A280 ultraviolet absorption. The purity of the collected protein was greater than 95%, as identified by SEC-HPLC, and was verified by LC-MS. The verified sample was divided into aliquots for use. The PD-Ll / TGF-β trap was obtained.
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[0105] The performance and benefits of the present invention are verified by biochemical test methods, as indicated below.
Evaluation of biological activity in vitro
Test Example 1: ELISA detection in vitro of the binding of the PD-Ll / TGF-β trap to TGF-βΙ
[0106] The detection process and the results are shown below:
The. The 96-well plates were coated with 100 μΐ / well of human TGF-βΙ (8915LC, GST) at a concentration of 1 pg / ml at 4 ° C overnight.
B. Washing 3 times with 250 μΐ of 1 χ PBST, 250 μΐ of 5% milk in PBS was added to block at 37 ° C for 2 hours.
ç. Washing 3 times with 250μ1 of PBST 1 χ, gradient dilution of the PD-Ll / TGF-β trap, TGF-β trap and positive control were added and incubated for 1 hour at 37 ° C.
d. Wash 3 times with 250 μΐ 1 χ PBST.
and. 100 μΐ of anti-human Fc-HRP antibody (1: 4000) was added to each well and incubated for 40 minutes at 37 ° C.
f. 100 μΐ of TMB was added to each well, incubated for 10 minutes at room temperature, and the reaction was
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51/76 interrupted by the addition of 100 μΐ of 1 M H2SO4.
g. The absorbance at 450 nm was measured in a microplate reader and the data were analyzed by Graphpad Prism5.
[0107] The results of binding the fusion protein to human TGF-βΙ in vitro are shown in Figures 2 and 3. The ELISA showed that fusion protein 1 in Table 2 did not retain human TGF-βΙ binding activity. Mass spectrometry analysis showed that fusion protein 1 (ie, the non-truncated form of the extracellular domain of TGF-βΗΙΙ (1-136)) was unstable and it was easy to break the heavy chain of TGF-βΗΙΙ, and the positive control had the same defect. Fusion proteins comprising the N-terminal truncated form of the ΤΰΕβΗΙΙ extracellular domain, such as fusion proteins 7, 9, 10, 12-15, are specific for binding to human TGF-βΙ.
Test Example 2: ELISA detection in vitro of the binding of the PD-Ll / TGF-β trap to the PD-LI
[0108] Antigen used for detection: PD-Ll-His
FTVTVPKDLYVVEYGSNMTTECKFPVEKQLDLAALIVYWEMEDKNnQFVHGEE DLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYK RITVKWAPYNK1NQRILVVDPVTSEHELTCQAEGYPKAEV1WTSSDHQVLSGK TTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPP NEREQKLISEEDLHHHHHH
SEQ ID NO: 17
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[0109] The detection process and the results are shown below:
The. 96-well plates were coated with 100 μΐ / well of human PD-Ll-His (SEQ ID NO: 17) at a concentration of 5 pg / ml at 4 ° C overnight.
B. Washing 3 times with 250 μΐ of 1 χ PBST, 250 μΐ of 5% milk in PBS was added to block at 37 ° C for 2 hours.
ç. Washing 3 times with 250μ1 of PBST 1 χ, gradient dilution of the PD-Ll / TGF-β trap, PD-L1 antibody as a positive control were added, and incubated for 1 hour at 37 ° C.
d. Wash 3 times with 250μ1 of 1 χ PBST.
and. 100μ1 of anti-human Fc-HRP antibody (1: 4000) was added to each well and incubated for 40 minutes at 37 ° C.
f. 100 μΐ of TMB added to each well, incubated for 10 minutes at room temperature, and the reaction was stopped by adding 100 μΐ of 1 M H2SO4.
g. The absorbance at 450 nm was measured in a microplate reader and the data were analyzed by Graphpad Prism5.
[0110] The results of binding the fusion protein of the present invention to human PD-L1 in vitro are shown in
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FIG. 4. The ELISA showed that all fusion proteins maintained the human PD-L1 binding activity.
Test Example 3: Detection of PD-1 / PD-L1 block in vitro
1. Purpose of the test:
[0111] To investigate the blocking effect of the PD-Ll / TGF-β trap on the PD-1 / PD-L1 signaling pathway, a cell-based antibody blocking experiment was carried out on cells containing the PD receptor molecules -1 and human PD-L1 that were built by Promaga, respectively.
2. Test samples
[0112] ® PD-L1 Antibody: SEQ ID NO: 11, SEQ ID NO:
12;
[0113] (D Control 1 (20T-Fc): ECD (20-136) -Fc, a fusion protein comprising the truncated ECF (20-136) TGF-βΗΙΙ extracellular domain fragment and Fc
[0114]
The sequence is as follows:
GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSÍTSICEKPQEVCVAVWRKNDENJT LETVCHDPKLPYHDFILEDAASPKCÍMKEKKKPGETFFMCSCSSDECNDNIIFSE
EYNTSNPDAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMÍSRTPEVTCVVV D Y W VSQEDPE VQFN VDG LT LV VHN AKTKPREEQFNSTYRV VSV VLHQD W LNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLG
SEQ ID NO: 18;
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[0115] (D Control 2 (22T-Fc): ECD (22-136) -Fc, a fusion protein that comprises the truncated ECD (22-136) TGF-pRII extracellular domain fragment and Fc
[0116] The sequence is as follows:
VKFPQLCKFCDVRFSTCDNQKSCMSNCSJTS1CEKPQEVCVAVWRKNDENITLET VCHDPKLPYHDFBLEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYN TSNPDAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKT1SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SD1AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLG
SEQ ID NO: 19:
[0117] ( ) Fusion protein 9, 15, fusion protein;
[0118] @ Human IgG: blank control, human immunoglobulin obtained from mixed normal human serum by purification using a conventional affinity chromatography method, such as protein A;
[0119] @ Positive control (M7824, prepared according to WO2015118175 as the reference patent): PD-Ll antibody fusion protein / TGF-pRII extracellular domain;
Antibody light chain amino acid sequence
PD-L1:
QSALTQPASVSGSPGQSIT1SCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVS NRPSGVSNRFSGSKSGNTASLllSGLQAEDEADYYCSSYTSSSTRVFGTGTKVTV LGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAG VETTKPSKQSNNK.YAASSYI.SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO: 20
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[0120] PD-L1 antibody heavy chain H chain amino acid sequence / TGF-pRII extracellular domain (1-136):
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPS GGITFYADWKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTV DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTFITCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKS VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCV
AVWRKNDENITLETVCHDPKLPYHDFILEDAASPKC1MKEKKKPGETFFMCSCS SDECNDNIIFSEEYNTSNPD
SEQ ID NO: 21.
3. Testing process
[0121] CHO / PD-L1 cells (CS187108, Promega) were digested and resuspended in complete F-12 Nutrient Mix medium (Ham). Cell density was adjusted to 4 x 10 ⁵ / ml using complete medium according to the cell count results. The cell suspension was transferred to the loading tank, added to the 96-well plate at 100 pL / well using a multichannel pipette and incubated at 37 ° C, 5% CO ₂ incubator for 20-24 h; the Jurkat / DP-1 cell suspension (CS187102, Promega) was prepared the next day, and the cells were resuspended according to the results of the
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56/76 cell count using assay medium, and cell density was adjusted to 1.25 x 10 ⁶ / ml; Cell culture plates comprising CHO / PD-L1 cells were removed from the incubator, 95pL of the culture solution was removed per well using a multichannel pipette, and the gradient diluted fusion protein, PD-L1 antibody and positive control (M7824 ) were added respectively to 40 pL / well. Then, the Jurkat / DP-1 cell suspension was transferred to a loading tank, added to the cell culture plate at 40 pL / well, and incubated at 37 ° C, 5% CO2 for 5-6 h . During the protein incubation, the Bio-Gio TM reagent was removed and allowed to return to room temperature. The cell culture plates were removed and placed at room temperature for 5 to 10 minutes. Then, 40 pL of Bio-Glo ™ reagent was added to each well, incubated in a safety cabinet for 5 to 10 minutes, and the chemiluminescence signal value was read using a multifunctional microplate reader.
4. Results:
[0122] As shown in Fig. 5, as well as the positive control molecule, fusion protein 9 of the present invention
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57/76 was able to effectively block the binding of Jurkat cells that express PD-1 to CH0 / PD-L1 cells, and had an effect on a dose dependent, along with the concentration of the drug. Fusion protein 15 has the same blocking ability as fusion protein 9.
Test Example 4: Binding affinity and in vitro kinetic detection by Biacore
[0123] The affinity of the test molecule with the human or murine TGF-βΙ protein or with the human PD-L1 protein was determined by Biacore T200 (GE). The experimental procedure was as follows:
[0124] A certain amount of PD-Ll / TGF-β trap was captured with protein A chip and then human or murine TGF-βΙ (8915LC, CST) or human PD-L1 (Bell Biological) flowed through the surface of the chip. The reaction signal was detected in real time using Biacore to obtain the association and dissociation curves. The biochip was then washed and regenerated with glycine-hydrochloric acid (pH 1.5, GE). The buffer solution used in the experiment was the HBS-EP (GE) buffer. The experimental data were adjusted to the Langmuir model (1:
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1) using BIAevaluation software version 4.1 (GE), and the affinity values were obtained and as shown in Table 3.
Table 3: Affinity of the fusion proteins of the invention with human TGF-βΙ or PD-L1 in vitro
Fusion protein * in Affinity sample ka(1 / Ms) kd(1 / s) KE (M) Proteinfusion 9 in Human TGF-βΙ 1.73E7 7.28E-4 4, 22E-11 Proteinfusion 15 in2.69E7 6.08E-4 2, 26E-11 Proteinfusion 9 in Murine TGF-βΙ 4.33E7 1.33E-3 3, 07E-11 Proteinfusion 15 in3.57E7 1.22E-3 3, 42E-11 Proteinfusion 9 in PD-L1human 1.97E6 1.24E-4 6, 31E-11 Proteinfusion 15 in2.00E6 1.24E-4 6, 10E-11
* The shape of the fusion protein is shown in Table 2.
[0125] The fusion protein binding activity is shown in Table 3. The results indicate that the fusion proteins 9 and 15 of the present invention have an extremely high affinity for human, murine TGF-βΙ and human PD-L1.
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Test Example 5: SMAD3 reporter gene inhibition assay
1. Purpose of the test:
[0126] In this experiment, the Smad3 linker (SBE) with the luciferase reporter gene was expressed in HepG2 cells to study the inhibitory effect of the PD-Ll / TGF-β trap on TGF-βΙ-induced Smad3 activation and the PD-L1 A / TGF-β trap activity in vitro was evaluated according to the IC50 value.
2. Test sample: fusion protein 9, positive control (M7824)
3. Testing process
[0127] HepG2 cells were cultured in complete MEM medium (GE, SH30243.01) containing 10% FBS and passed every 3 days. On the first day of the experiment, 25,000 cells per well were inoculated into 96-well plates (Corning, 3903) and cultured at 37 ° C, 5% CO2 for 24 hours. The next day, the medium in the cell culture plates was discarded and 100 ng of plasmid 3TP-Lux was transfected per well. The cells were further cultured at 37 ° C, 5% CO2 for 24 hours. Six hours before adding the test sample, the complete medium in the 96-well plate was discarded and 80 pL of incomplete medium (MEM
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60/76 + 0.5% FBS) were added to each well. After 6 hours, 10 pL of human TGF-βΙ solution (R&D, 240-B-010) prepared in incomplete medium (final concentration of 2 ng / mL) and 10 pL of the test sample (the final concentration is 500, 50, 5, 0.5, 0, 05, 0, 005, 0, 0005 and 0 nM) were added, the human TGF-βΙ solvent was used as a control, and the cells were cultured at 37 ° C, 5% CO2 for another 18 h. Then, 100 pL of luciferase substrate prepared by the ONE-GloTM Luciferase Assay system (Promega, E6110) was added to each well, and incubated at room temperature for 10 minutes in the dark, and then the luminescent signal value was read using a Victors multi-plate reader (Perkin Elmer). The IC50 value of the test sample was obtained through calculation using the Graphpad Prism 5.0 data software.
[0128] Figure 6 shows that fusion protein 9 inhibits S6Εβ-induced pSMAD3 activity in a dose-dependent manner and has comparable efficacy and IC 50 (concentration required to inhibit 50% of maximum activity) to that of the M7824 positive control. The results of the PD-L1 antibody test showed that it had no inhibitory effect (IC50> 500 nM).
Test Example 6: In vitro detection of IFNy secretion by
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PBMC due to stimulation of tuberculin (TB)
1. Purpose of the test
[0129] To investigate the activation of T lymphocytes by the PD-Ll / TGF-β trap, human peripheral blood mononuclear cells (PBMC) were collected and purified, and ΙΓΝγ secretion was detected after stimulation with tuberculin (TB) for 5 days.
2. Test sample
[0130] ® human IgG; @ PD-L1 antibody; (3) Fusion protein 9 (Ϊ) Control 1 (20T-Fc): ECD (20-136) -Fc; @ PD-L1 Antibody + Control 1 (20T-Fc).
3. Testing process:
[0131] Fifteen ml of purified fresh PBMC, about 3 x 10 ⁷ cells, and 20 ml of tuberculin were added to them, and cultured in an incubator for 5 days at 37 ° C, 5% CO2 · On day 6, cultured cells were collected and centrifuged, washed once with PBS and again suspended in fresh medium with a density adjusted to 1 x 10 ⁶ cells / ml, 90μ1 of the resuspended cells were added to the 96-well plate. Ten (10) pL / well of different concentrations of antibodies were added separately to the corresponding wells of the cell culture plate above 96 wells
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62/76 μΐ of PBS were added to the control and blank groups, respectively. Then, the cell culture plate was incubated in an incubator for three days at 37 ° C, 5% CO2. The cell culture plate was removed and the supernatant was removed from each well after centrifugation (4000 rpm, 10 min). After a 10-fold dilution, IFN-γ secretion was detected by ELISA (human IFN-γ detection kit, Xinbosheng, EHC 102g. 96), according to the reagent instructions for specific operations. As shown in the figure, the PD-Ll / TGF-β trap fusion protein samples were able to increase IFN-γ cytokine secretion by activated T lymphocytes and had a drug dose concentration effect.
Table 4
Antibody EC50(nM) Maximum IFNy secretion (pg / ml) Minimum IFNy secretion (pg / ml) Multiple (IFNy secretion) AntibodyPD-L1 0.05 2684 737 3, 6 Protein fromfusion 9 0.12 3422 638 5, 4 Control 1 > 50 780 490 1, 6
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(20T-Fc) LI Antibody+ control 1 0.054 2879 746 3, 9 Human IgG > 50 375 298 1.2 Control inWhite / 536 536 1
4. Result
[0132] As shown in Figure 7 and Table 4, fusion protein 9 was able to improve the activated T lymphocyte for dose-dependent IFN-γ secretion and had a stronger activation effect than PD-L1 and 20T-FC antibody.
Pharmacokinetic evaluation
Test Example 7:
[0133] Three female SD rats were purchased from Jiesijie Experimental Animal Co., Ltd. and maintained in a light-dark cycle every 12 hours (temperature is 24 ± 3 ° C, relative humidity is 50 60%), the rats had free access to water and diet. On the day of the experiment, the SD rats were injected with the fusion protein in the tail vein at a dose of 6 mg / kg and an injection volume of 5 ml / kg.
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[0134] Blood was collected at time points: 15 min, 7 hours (the first day), 24 hours (day 2), 3 day, 4 day, 6 day, 8 day, 10th day, and 15 ° day after the administration, 200 pL of blood (equivalent to 100 pi of serum) were collected from the deep vein of the rats. The blood sample was placed at room temperature for 30 minutes to allow agglutination and then centrifuged at 10,000 g for 10 minutes at 4 ° C. The supernatant was taken and stored at -80 ° C immediately. The concentration of the fusion protein in the serum was measured by ELISA.
[0135] The measurement process is shown below:
The. The 96-well plates were coated with 100 µl / well of human PD-Ll-His at a concentration of 2 pg / ml, overnight at 4 ° C.
B. Washing 4 times with 250 pl of 1 * PBST, 250 µl of 5% milk in PBS was added to block at 37 ° C for 3 hours.
ç. Washing 4 times with 250 pl of 1 * PBST, 100 µl of the gradient diluted serum sample was added and incubated at 37 ° C for 1 hour, with fusion protein 9 as a positive control.
d. Wash 5 times with 250 pl 1 χ PBST.
and. 100 µl / well of biotinylated human anti-TGF-pRII antibody (R&D) was added and incubated for 1 hour at 37 ° C.
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f. Wash 5 times with 250 μΐ 1 χ PBST.
g. 100 μΐ / well of TMB were added, incubated for minutes at room temperature and the reaction was stopped by adding 100 μΐ of 1 M H2sO4.
H. The absorbance at 450 nm was measured in a microplate reader and the data were analyzed by Graphpad Prism5.
Table 5: Tl / 2 of fusion protein in rats
Test drug Way ofadministration T1 / 2(Mean ± SD, h) ProteinFusion 9 IV (6 mg / kg) 236 ± 10
[0136] The results of the PK analysis indicated that the half-life of fusion protein 9 of the present invention in rats was about 236 h (9.8 days), see table 5.
Evaluation of biological activity in vivo
Test Example 8: Effect of the PD-Ll / TGF-β trap on the murine subcutaneous xenograft of human breast cancer MDA-MB-231
[0137] The murine strain used in this experiment was a female NOD / SCID mouse (Cavens). The mononuclear cells of the human peripheral blood used in the experiment were extracted from the newly collected blood, and the method of extraction was as follows: The venous blood anticoagulated with heparin was
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66/76 mixed with PBS containing 2% BBS in the same volume and after mixing, 25 ml of diluted blood was added slowly to a centrifuge tube containing 15 ml of lymphocyte separation solution and centrifuged at 1200 g for 10 minutes in room temperature. The lymphocyte layer was pipetted to another centrifuge tube, the cells were washed by PBS and centrifuged at 300 g for 8 minutes at room temperature. After repeating once, the cells were resuspended in RPMI-1640 medium containing 10% FBS, and the cells were added to a 6-well plate pre-coated with CD3 antibody (OKT3, 40 ng / ml) at 2 χ 10 ⁶ cells / well (2 ml) and then placed in an incubator at 37 ° C for 4 days.
[0138] Test sample:
[0139] (T) blank control: PBS; 9-4.8mpk fusion protein; (3) 9-2 4mpk fusion protein; (4) PD-L1 -4mpk antibody; @ PD-L1 antibody -20mpk; @ -4mpk PD-L1 antibody + control 1 (20T-Fc) -2.14mpk; @control 1 (20T-Fc) -2.14mpk.
[0140] MDA-MB-231 cells were resuspended in serum-free RPMI-1640 medium, and mixed with an equal volume of Matrigel, 100 μΐ (2.3 χ 10 ⁶ ) were inoculated subcutaneously in the right flank of the mice NOD / SCID. Eleven
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67/76 days later, animals with large or smaller tumor size were excluded, mice were randomized into groups, with 9 animals in each group. 5 χ 10 ⁵ stimulated PBMCs (60 μΐ) were injected into the tumor tissues and the other PBMCs were cultured even more without stimulation. A week later, 5 χ 10 ⁶ PBMC (100 μΐ) were injected intraperitoneally into mice with tumors, as the first injection cycle. During the experimental period, for 2 and a half rounds, 5 injections of PBMC were provided. On the day of the first intratumoral injection, intraperitoneal administration was performed, three times a week, for a total of 14 administrations. The administration regimens are shown in Table 6. Tumor volume and weight were measured twice a week. The experimental results were shown in Table 7.
Table 6: Grouping and administering tests
Group Administration dose (T) Blank control: PBS 0 @ 9-4.8mpk fusion protein 4.8 mg / kg (3) 9-24mpk fusion protein 24mg / kg (4) PD-Ll-4mpk Antibody 4mg / kg
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© Antibody PD-L1 -2 0mpk 20mg / kg © Antibody PD-L1 -4mpk + control1-2.14mpk 4 mg / kg +2.14 mg / kg © Control l-2.14mpk 2.14mg / kg
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Table 7: Effect of fusion protein 9 on the murine subcutaneous xenograft of MDA-MB-231
Group Day 0 Day 25 Day 32 Day 33 Average Average % TGI Average % TGI Average P (vsPBS) (V mm ³ ) (V mm ³ ) (V mm ³ ) (TW g) (TW) (T) Blank control: PBS 62.5 ± 2.9 623.4 ± 43.3 - 941.1 + 54.9 - 0.859 + 0.063 - @ 9-4.8mpk fusion protein 62.6 ± 3.5 414.6 + 17.1 *** 37.24% 618.9 + 28.7 *** 36.68% 0.454 + 0.025 *** 2.06E-05 (|) 9-24mpk fusion protein 62.7 ± 3.3 329.8 + 22.5 *** 52.38% 495.3 + 42.6 *** 50.76% 0.367 + 0.026 *** 2.20E-06 (í) Anti bodyPD-L1 -4mpk 63.1 ± 3.5 454.4 ± 40.8 * 30.24% 722.8 + 65.8 * 24.91% 0.592 + 0.052 ** 0.0050
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(D An ti corpoPD-L1 -20mpk 62.6 ± 3.3 466.4 ± 17.2 ** 28.01% 741.8 ± 32.9 ** 22.70% 0.650 ± 0.033 ** 0.0100 @ AntibodyPD-L1-4mpk + control1-2.14mpk 62.6 ± 3.3 447.5 ± 29.6 ** 31.38% 669.2 ± 45.3 ** 30.96% 0.566 ± 0.039 ** 0.0012 © control1-2.14mpk 60.7 ± 3.3 601.5 ± 30.9 3.58% 861.7 ± 34.2 8, 83% 0.652 ± 0.041 * 0.0178
NOTE: Day 0: time of the first administration; * p <0.05 ** p <0.01 *** p <0.001, compared with
PBS by Student's t test.
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[0141] The results were shown in Figure 8, antibody fusion protein 9 (4.8, 24 mg / kg) can significantly inhibit the growth of the murine subcutaneous xenograft of human breast cancer MDA-MB-231. There was a dose-dependent relationship between high and low doses, and this was higher for the reference drug's PD-L1 antibody (4.20 mg / kg), TGF-pRII 20T-FC control molecule (2.14 mg / kg). kg) and the combination group (antibody PD-L1 -4 mg / kg + 20 T-FC -2.14 mg / kg) in equivalent molar dose, respectively. Each dose of fusion protein 9 maintained an ideal anti-tumor effect since the 14th day after administration; when compared to the 20mpk PD-L1 antibody, high-dose fusion protein 9 has an obvious advantage (p <0.05). On the 25th day after administration, the anti-tumor effect of each antibody reached the ideal level. The inhibition rates achieved by the fusion protein 9 group combined with the PDL-1 antibody (in high and low doses) were 37.24%, 52.38%, 30.24%, 28.01% and 31.38 %, respectively. On the 32nd day after administration, the antitumor effect of fusion protein 9 was still very significant. The TGI% of the low and high dose group was 36.68% and 50.76%, respectively, and the tumor volume was
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72/76 statistically different when compared to the control group (p <0.05).
Test Example 9: Physical stability of the PD-Ll / TGF-β trap
[0142] This test example was used to detect the stability of fusion protein 9 and fusion protein 15.
[0143] DSC (differential scanning calorimetry) was used to detect the thermal stability of different antibodies and the stability in different buffer systems was compared. Buffer systems comprise 10 mM acetate / 135 mM NaCl (pH 5.5) and 10 mM acetate / 9% trehalose (pH 5.5).
[0144] The sample was dissolved in the corresponding buffers and the concentration was controlled to about 50 mg / ml. Detection was performed by MicroCal * VP-Capillary DSC (Malvern). Before testing, each blank sample and buffer was degassed for 1 to 2 minutes using a vacuum degassing device. Each well of the plate received 400 μΐ of sample or blank buffer (the load quantity was 300 μΐ). Finally, two pairs of well plates received Decon 90 at 14% and ddH ₂ O, respectively, and were ready for
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73/7 6 wash. The sample was loaded onto the plate, and then the plate was sealed with a plastic cover. The scan started at a temperature of 25 ° C and ended at 100 ° C, and the scan rate is 60 ° C / h. The results are shown in Table 8, indicating that fusion protein 9 and fusion protein 15 show good thermal stability in these two test systems.
Table 8
Sample Plug Tm-start(° C) TM (° C) Fusion protein 9 10 mM acetate /135 mM NaCl 57.99 66, 33 10 mM acetate /9% trehalose 58, 64 67, 83 Fusion protein 15 10 mM acetate /135 mM NaCl 57.33 66, 17 10 mM acetate /9% trehalose 57.41 67.44
[0145] Periodic stability at a certain concentration was investigated by monitoring purity via SEC-HPLC, under exemplary conditions, for example, the sample concentration
Petition 870190112847, of 11/04/2019, p. 89/109 was controlled at about 50 mg / ml. The stability of different antibodies in 10 mM acetate / 135 mM NaCl (pH 5.5) was compared under conditions such as 5 cycles of freezing and thawing at -80 ° C and storage at 40 ° C for one month. The HPLC column of the Xbridge BEH SEC 200A protein (Waters) was used for detection. The result was shown in Table 9 as follows, these two fusion proteins showed good stability.
Table 9
Temperature protein fromfusion 9 (A%) protein fromfusion 15 (A%) 40 ° C 3.39% 1.8% -80 ° Cfreeze-thaw 1.44% 1.39%
[0146] NOTE: The% indicates the rate of change.
Test Example 10: Chemical stability of the fusion protein
[0147] Deamidation is a common chemical modification that will influence the stability of the antibody at a later stage. In particular, super-deamidation of amino acids in the CDR regions should be avoided or the reduction of mutations in these regions is preferable. 1600 pg of antibody to be tested were dissolved in 200 μΐ of 10 mM acetate / 135 mM NaCl (pH 5.5) and placed in an incubator at 40 ° C. The samples
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75/76 were collected on days 0, 14 and 28 for the enzymatic hydrolysis assay. 100 pg of each sample collected at different times were dissolved in 100 pL of 0.2 M His-HCl solution, 8 M Gua-HCl, pH 6.0; 3 pi of 0.1 g / mL of DTT were added and then the sample was incubated in a water bath at 50 ° C for 1 hour. Then, the sample was ultrafiltered twice with 0.02M His-HCl (pH 6.0) and digested overnight at 37 ° C in a water bath, adding 3 pL of
0.25 mg / ml trypsin. Deamidation modification was examined using an Agilent 6530 Q-TOF LC-MS and the results are shown in Table 10 below.
Table 10
Sample Jailheavy Modification site Day 0 Day 14 Day 28 Protein from Jail N314 2.38% 2.28% 2.45% fusion 9 heavy N324 0.20% 3.60% 7.88% Protein from Jail N314 2.87% 2.86% 2.87% fusion 15 heavy N324 0.00% 3.61% 7.93%
[0148] NOTE: N represents the detectable modified asparagine and the number represents the position in the N-terminal light or heavy chain. The percentage content represents the
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Ί6 / Ί6 proportion of modification of deamidation detected by LC-MS in relation to the signal of all peptides at that site.
[0149] The results of mass spectrometry showed that the two fusion proteins do not have obvious deamidation modification sites, suggesting that the fusion proteins have good chemical stability.

权利要求:
Claims (25)
[1]
1. Fusion protein characterized by the fact that it contains TGF-β receptor, comprising a targeting fraction and a TGF-β receptor fraction, in which the TGF-β receptor fraction is a truncated form at the N-terminus of the extracellular domain TGF-βΗΙΙ.
[2]
2. Fusion protein containing TGF-β receptor, according to claim 1, characterized by the fact that the truncated form at the N-terminus of the extracellular domain of TGF-βΗΙΙ involves a deletion of 26 or less contiguous amino acids at the N-terminus of the terminal extracellular TGF-βΗΙΙ, preferably, a deletion of 14 to 26 contiguous amino acids, more preferably, a deletion of 14 to 21 contiguous amino acids, more preferably, a deletion of 14, 19 or 21 contiguous amino acids.
[3]
3. Fusion protein containing TGF-β receptor, according to claim 1 or claim 2, characterized by the fact that the sequence of the extracellular domain of TGF-βΗΙΙ is shown in SEQ ID NO: 13; more preferably, comprising the sequence of SEQ ID NO: 14, 15 or 16.
[4]
Fusion protein containing the TGF-β receptor according to any one of claims 1 to 3, characterized by
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2/8 the fact that the targeting fraction is a specific targeting fraction for the cell; preferably the targeting fraction is a targeting fraction specific to cancer cells.
[5]
5. Fusion protein containing TGF-β receptor, according to claim 4, characterized by the fact that the specific targeting fraction for cancer cells is selected from the group consisting of an antibody or antigen-binding fragment thereof, a growth factor, a hormone, a peptide, a receptor and a cytokine.
[6]
6. Fusion protein containing TGF-β receptor, according to claim 5, characterized by the fact that the antibody or antigen-binding fragment thereof is selected from the group consisting of a complete antibody, a chimeric antibody, Fab ', Fab, F (ab') 2, a single domain antibody, Fv, scFv, a small antibody, a bi-specific antibody and a tri-specific antibody or a mixture thereof.
[7]
Fusion protein containing the TGF-β receptor according to claim 5 or claim 6, characterized in that the antibody or antigen-binding fragment thereof binds to one or more of the following polypeptides or
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3/8 proteins selected from the group consisting of HER2, HER3, immunological checkpoint molecule, CD33, VEGF, VEGFR, VEGFR-2, CD152, TNF, IL-1, IL-5, IL-17, IL-6R , IL-1, IL-2R, BLYS, PCSK9, EGFR, c-Met, CD2, CD3, CD11, CD19, CD30, CD38, CD20, CD52, CD60, CD80, CD86, TNF-α, IL-12, IL -17, IL-23, IL-6, IL-Ιβ, RSVF, IgE, RANK, BLyS, α4β7, PD-1, CCR4, SLAMF7, GD2, CD21, CD79b, IL20Ra, CD22, CD79a, CD72, IGF-1R and RANKL; preferably, wherein the antibody or antigen-binding fragment thereof binds to the immunological checkpoint molecule.
[8]
Fusion protein containing the TGF-β receptor according to any one of claims 5 to 7, characterized in that the antibody is an anti-PD-Ll antibody; preferably, the anti-PD-Ll antibody is selected from the group consisting of MSB0010718C, MEDI4736, BMS-936559 and MPDL3280A; or the anti-PD-Ll antibody comprises one or more CDR (s) selected from the group consisting of:
HCDR1 of SEQ ID NO: 1 or its mutant;
HCDR2 of SEQ ID NO: 2 or the mutant thereof;
HCDR3 of SEQ ID NO: 3 or the mutant thereof;
LCDR1 of SEQ ID NO: 4 or the mutant thereof;
LCDR2 of SEQ ID NO: 5 or the mutant thereof; and
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LCDR3 of SEQ ID NO: 6 or the mutant thereof.
[9]
Fusion protein containing TGF-β receptor according to any one of claims 5 to 8, characterized in that the antibody or antigen-binding fragment thereof is a chimeric antibody or a functional fragment thereof, an antibody humanized or a functional fragment thereof or a human antibody or a functional fragment thereof.
[10]
Fusion protein containing TGF-β receptor according to claim 9, characterized in that the humanized antibody comprises a heavy chain variable region of SEQ ID NO: 7, preferably comprises a heavy chain variable region of SEQ ID NO: 9.
[11]
11. Fusion protein containing TGF-β receptor according to claim 9, characterized by the fact that the humanized antibody further comprises a heavy chain of SEQ ID NO: 11.
[12]
12. Fusion protein containing TGF-β receptor, according to claim 9, characterized by the fact that the humanized antibody comprises a variable region of the light chain of SEQ ID NO: 8 or SEQ ID NO: 10 or the mutants thereof.
[13]
13. Fusion protein containing TGF-β receptor, according to
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5/8 with claim 9, characterized in that the humanized antibody comprises a light chain of SEQ ID NO: 12.
[14]
14. TGF-β receptor-containing fusion protein according to any one of claims 1 to 13, characterized in that the fusion protein comprising the TGF-β receptor is shown as a general formula (D:
Ab-L-TGF-βΗΙΙ ECD (I) where TGF-βΗΙΙ ECD is a truncated form of the extracellular domain of TGF-βΗΙΙ;
Ab is an antibody;
L is a ligand.
[15]
15. Fusion protein containing the TGF-β receptor according to claim 14, characterized by the fact that the linker is (G4S) _X G, where x is 3 to 6, preferably 4 to 5 .
[16]
16. Pharmaceutical composition characterized by the fact that it comprises a therapeutically effective amount of the fusion protein containing the TGF-β receptor, according to any one of claims 1 to 15, and one or more pharmaceutically acceptable carriers, diluents or excipients.
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[17]
17. DNA molecule characterized by the fact that it encodes the fusion protein containing the TGF-β receptor, according to any one of claims 1 to 15.
[18]
18. Expression vector characterized by the fact that it comprises the DNA molecule, as defined in claim 17.
[19]
19. Host cell comprising the expression vector, as defined in claim 18, characterized by the fact that the host cell is selected from the group consisting of a bacterial, yeast and mammalian cell; preferably a mammalian cell.
[20]
20. Use of the TGF-β receptor-containing fusion protein, as defined in any of claims 1 to 15, or the pharmaceutical composition, as defined in claim 16, characterized by the fact that it is for the preparation of a medicament for the treatment of tumor; preferably, for the preparation of a medicament for the treatment of a tumor mediated by PD-L1; more preferably a cancer that expresses PD-L1.
[21]
21. Method characterized by the fact that it is for the treatment or prevention of tumor, comprising the
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Administering to a patient in need of a therapeutically effective amount of the TGF-β receptor-containing fusion protein according to any one of claims 1 to 15, or the pharmaceutical composition according to claim 16.
[22]
22. TGF-βΗΙΙ truncated extracellular domain characterized by the fact that the TGF-βΗΙΙ truncated extracellular domain involves a deletion of 26 or less contiguous amino acids at the N-terminus of SEQ ID NO: 13, preferably a deletion of 14 to 26 contiguous amino acids at the N-terminus, more preferably, a deletion of 14 to 21 contiguous amino acids at the N-terminus; more preferably, the truncated extracellular TGF-βular domain comprises the selected sequence of SEQ ID NO: 14 or SEQ ID NO: 15.
[23]
23. Pharmaceutical composition characterized by the fact that it comprises a therapeutically effective amount of the truncated extracellular domain of TGF-βΗΙΙ according to claim 22, and one or more pharmaceutically acceptable carriers, diluents or excipients.
[24]
24. Use of the TGF-βΗΙΙ truncated extracellular domain, as defined in claim 22, or the pharmaceutical composition, as defined in claim
Petition 870190112847, of 11/04/2019, p. 99/109
8/8
23, characterized by the fact that it is for the preparation of a medication for the treatment or for the inhibition of diseases or disorders associated with the proliferation or metastasis of cancer cells.
[25]
25. Method for the treatment or prevention of a tumor characterized by the fact that it comprises administering to a patient in need of a therapeutically effective amount of the truncated extracellular domain of TGF-pRII, as defined in claim 22 or the pharmaceutical composition as defined in claim 23.

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同族专利:

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公开号 | 公开日

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WO2018205985A1|2018-11-15|

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AU2018264455A1|2019-11-14|

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CA3061791A1|2019-10-29|

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US20200157180A1|2020-05-21|

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MX2019013023A|2019-12-18|

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CN110050000A|2019-07-23|

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EP3623389A1|2020-03-18|

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JP2020519289A|2020-07-02|

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KR20200004801A|2020-01-14|

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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CN201710334292|2017-05-12|

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PCT/CN2018/086451|WO2018205985A1|2017-05-12|2018-05-11|FUSION PROTEIN CONTAINING TGF-β RECEPTOR AND MEDICINAL USES THEREOF|

---