WO2018205985A1 - 含有TGF-β受体的融合蛋白及其医药用途 - Google Patents

含有TGF-β受体的融合蛋白及其医药用途 Download PDF

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WO2018205985A1
WO2018205985A1 PCT/CN2018/086451 CN2018086451W WO2018205985A1 WO 2018205985 A1 WO2018205985 A1 WO 2018205985A1 CN 2018086451 W CN2018086451 W CN 2018086451W WO 2018205985 A1 WO2018205985 A1 WO 2018205985A1
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tgf
antibody
fusion protein
seq
βrii
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PCT/CN2018/086451
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English (en)
French (fr)
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顾津明
罗肖
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to JP2019562379A priority Critical patent/JP7264827B2/ja
Priority to BR112019023184A priority patent/BR112019023184A2/pt
Priority to AU2018264455A priority patent/AU2018264455A1/en
Priority to EP18798641.9A priority patent/EP3623389A4/en
Priority to CA3061791A priority patent/CA3061791A1/en
Priority to CN201880004344.6A priority patent/CN110050000B/zh
Priority to KR1020197032251A priority patent/KR102629503B1/ko
Priority to MX2019013023A priority patent/MX2019013023A/es
Priority to US16/610,585 priority patent/US11274142B2/en
Publication of WO2018205985A1 publication Critical patent/WO2018205985A1/zh

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    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the invention relates to the field of tumor immunotherapy drugs.
  • the invention relates to fusion proteins for use in the treatment of cancer, including fusion proteins comprising a targeting molecule and an immunomodulatory factor such as TGF-[beta]RII. More specifically, it relates to a fusion protein of a targeting molecule PD-L1 antibody and an immunomodulatory factor such as TGF- ⁇ RII, a pharmaceutical composition comprising the same, and its use as an anticancer drug.
  • Tumor immunotherapy mainly enhances the immunogenicity of tumor cells and the sensitivity to effector cell killing through immunological principles and methods, stimulates and enhances the body's anti-tumor immune response, and injects immune cells and effector molecules into the host to coordinate with the body.
  • the immune system kills tumors and inhibits tumor growth.
  • PD-1 Programmed death 1
  • B cells and myeloid cells which have two ligands, programmed death ligand 1, PD-L1 and PD-L2.
  • PD-L1 interacts with receptor PD-1 on T cells and plays an important role in the negative regulation of immune responses.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells.
  • the expression of PD-L1 is beneficial to the occurrence and growth of tumors, and induces anti-tumor. Apoptosis of T cells.
  • the PD-1/PD-L1 pathway inhibitor can block the binding of PD-1 to PD-L1, block the negative regulatory signal, and restore the T cell to activate the immune response. Therefore, PD-1/PD- Immunoregulation of L1 as a target has important implications for tumor suppression.
  • TGF- ⁇ Transforming growth factor- ⁇ belongs to the TGF- ⁇ superfamily that regulates cell growth and differentiation.
  • TGF- ⁇ transmits a signal through a heterotetrameric receptor complex composed of two type I and two type II transmembrane serine/threonine kinase receptors.
  • TGF- ⁇ is a multifunctional cytokine that exerts tumor suppressor or tumor-promoting effects in a cell or background-dependent manner.
  • the tumor suppressive effect of TGF- ⁇ signaling stems from its ability to induce expression of multiple genes.
  • cancer cells gradually endure the inhibition of TGF- ⁇ signaling, which ultimately leads to the development of tumors.
  • TGF- ⁇ signaling pathway can reduce tumor metastasis.
  • the truncated Smad2/3 dystrophic mutant was used to inhibit the TGF- ⁇ signaling pathway of breast tumor cell lines, and it was found that the metastatic ability of tumor cells was inhibited.
  • Microsatellite instability studies of colon cancer found that TGF- ⁇ RII inactive mutations reduced metastasis and increased postoperative survival.
  • inhibitors that inhibit TGF- ⁇ signaling pathway are only effective in clinical treatment, and may be related to the high expression of TGF- ⁇ mainly in tumor cells and the bioavailability of signaling pathway inhibitors.
  • a PD-L1 antibody is provided by the Applicant's prior PCT application PCT/CN2016/104320.
  • the present invention provides a fusion protein comprising a TGF- ⁇ receptor comprising a targeting moiety and a TGF- ⁇ receptor moiety, wherein the TGF- ⁇ receptor moiety is an N-terminal truncation of the extracellular domain of TGF- ⁇ RII form.
  • the N-terminal truncated form of the extracellular domain of TGF- ⁇ RII is selected from the group consisting of a deletion of 26 or less contiguous amino acids on the N-terminus of the extracellular domain of TGF- ⁇ RII, preferably 14-26 consecutive Deletions of amino acids, more preferably deletions of 14-21 contiguous amino acids; most preferably deletions comprising 14-21 contiguous amino acids, non-limiting examples include the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.
  • sequence of the extracellular domain of TGF- ⁇ RII is set forth in SEQ ID NO: 13.
  • the targeting moiety is a cell-specific targeting moiety; preferably, the targeting moiety is a cancer cell-specific targeting moiety.
  • the cancer cell-specific targeting moiety is selected from the group consisting of an antibody or antigen-binding fragment thereof, a growth factor, a hormone, a peptide, a receptor or a cytokine.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of a full length antibody, a chimeric antibody, Fab', Fab, F(ab') 2 , single domain antibody (DAB), Fv , scFv, small antibodies, bispecific antibodies, trispecific antibodies or mixtures thereof.
  • the antibody or antigen-binding fragment thereof binds to one or more of the following polypeptides or proteins: HER2, HER3, immunoassay 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, CD11a, CD19, CD30, CD38 , CD20, CD52, CD60, CD80, CD86, TNF- ⁇ , IL-12, IL-17, IL-23, IL-6, IL-1 ⁇ , RSVF, IgE, RANK, BLyS, ⁇ 4 ⁇ 7, PD-1, CCR4 , SLAMF7, GD2, CD21, CD79b, IL20R ⁇ , CD22, CD79a, CD72, IGF-1R and RANKL; preferably the antibody or antigen
  • the antibody is a PD-L1 antibody; preferably, the PD-L1 antibody is selected from the group consisting of: MSB0010718C, MEDI4736, BMS-936559, MPDL3280A, PD-L1 antibody, or comprises An antibody having one or more CDR sequences selected from the following CDR region sequences or CDR region sequences:
  • X 1 is selected from H or G, preferably G;
  • X 2 is selected from G or F, preferably F.
  • the antibody or antigen-binding fragment thereof is a chimeric antibody or a functional fragment thereof, a humanized antibody or a functional fragment thereof, or a human antibody or a functional fragment thereof.
  • the heavy chain variable region of the humanized antibody has the sequence set forth in SEQ ID NO: 7, preferably having the sequence set forth in SEQ ID NO:9.
  • the humanized antibody further comprises the heavy chain of SEQ ID NO:11.
  • the humanized antibody comprises the mutated sequence of the antibody light chain variable region sequence or the light chain variable region of SEQ ID NO: 8 or 10.
  • the humanized antibody comprises the light chain of the sequence SEQ ID NO: 12.
  • the TGF-beta receptor-containing fusion protein is represented by the general formula (I):
  • TGF- ⁇ RII ECD is a truncated form of the extracellular domain of TGF- ⁇ RII
  • Ab is an antibody
  • L is the linker sequence
  • the linking sequence L is (G 4 S) x G, wherein x is 3-6, preferably 4-5.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a TGF-beta receptor-containing fusion protein as described above, together with one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the invention further provides a DNA molecule encoding a fusion protein comprising a TGF-beta receptor as described above.
  • the invention further provides an expression vector comprising a DNA molecule as described above.
  • the invention further provides a host cell transformed with an expression vector as described above, the host cell being selected from the group consisting of a bacterial, a yeast, and a mammalian cell; preferably a mammalian cell.
  • the present invention further provides the use of the TGF-beta receptor-containing fusion protein or a pharmaceutical composition comprising the same, in the preparation of a medicament for tumor therapy; preferably in the preparation for the treatment of PD-L1 mediated Use in a drug for tumors; more preferably a cancer expressing PD-L1.
  • the invention further provides a method of treating and preventing a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a TGF-beta receptor-containing fusion protein of the invention or a pharmaceutical composition comprising the same.
  • the invention further provides a truncated extracellular region of TGF- ⁇ RII, wherein the truncation of the extracellular region of TGF- ⁇ RII is selected from the deletion of 26 or fewer consecutive amino acids at the N-terminus of SEQ ID NO: 13.
  • a deletion of 14-26 contiguous amino acids at the N-terminus more preferably a deletion of 14-21 contiguous amino acids at the N-terminus; most preferably a deletion comprising 14-21 contiguous amino acids, non-limiting examples comprising SEQ ID NO: 14 or SEQ ID NO:
  • SEQ ID NO: 14 or SEQ ID NO: The sequence shown in 15.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the truncated extracellular region of TGF- ⁇ RII of the invention, and one or more pharmaceutically acceptable carriers, diluents or excipients .
  • the invention further provides the use of a truncated TGF- ⁇ RII extracellular region of the invention or a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment or inhibition of a disease or condition in which cancer cells proliferate or metastasize.
  • the invention further provides a method of treating and preventing a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a truncated TGF-[beta]RII extracellular region of the invention or a pharmaceutical composition comprising the same.
  • the tumor or cancer described in the present disclosure is selected from the group consisting of a tumor or cancer: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, myeloma, lymphoma, leukemia, thyroid, endometrium, uterus, Bladder, neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, squamous cutaneous fibrosarcoma, Meck Cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • a tumor or cancer colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, myeloma, lymphoma, leukemia, thyroid, endometrium, uterus
  • Figure 1 Schematic diagram of the structure of the fusion protein.
  • Figure 2 Results of in vitro binding of fusion protein to human TGF- ⁇ 1.
  • Figure 3 Results of in vitro binding of fusion protein to human TGF- ⁇ 1.
  • Figure 4 Results of in vitro binding of fusion protein to human PD-L1.
  • Figure 5 In vitro detection of PD-1/PD-L1 pathway blocking results by fusion protein.
  • FIG. 6 Fusion protein inhibits TGF ⁇ -induced pSMAD3 reporter activity in a dose-dependent manner.
  • Figure 8 Effect of fusion protein on tumor weight of tumor-bearing mice.
  • the "antibody” as used in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising IgG1, IgG2, IgG3, IgG4 or a variant thereof of human or murine origin.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) in number and position, or conform to the numbering rules of kabat and chothia. (HCDR1).
  • the antibody of the present invention includes a full length antibody selected from a murine antibody, a chimeric antibody, and a humanized antibody, preferably a humanized antibody.
  • murine antibody is in the present invention a monoclonal antibody to human PD-L1 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the PD-L1 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional property is isolated.
  • the murine PD-L1 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, a lambda chain or a variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or variants thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the mouse hybridoma cell, and the mouse variable region gene is cloned as needed, and the mouse variable region is cloned.
  • the gene is ligated into a human vector after being ligated into a human chimeric gene, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the PD-L1 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC after amino acid mutation (antibody-dependent) Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into human antibody variable region frameworks, ie different types of human germline An antibody produced in an antibody framework sequence. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc.
  • humanized antibodies of the invention also include humanized antibodies that are further affinity matured by phage display.
  • variable and constant regions are derived from a human immunoglobulin sequence.
  • One of the preferred ways is that all of the variable and constant regions are derived from human immunoglobulin sequences, ie, "fully human antibodies” or “fully human antibodies.”
  • These antibodies can be obtained in a variety of ways, including isolation of B cells from human PBMC, spleen, lymph node tissue by phage display technology, construction of a natural single-stranded phage human antibody library, or by immunization of transgenic mice expressing human antibody light and heavy chains. , screening for antibodies obtained.
  • the "antibody or antigen-binding" or “functional fragment” thereof as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab')2 fragment, and an Fv fragment ScFv fragment which binds to an antibody.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (sFv).
  • binding to PD-L1 means that it can interact with human PD-L1.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • the "ADCC” described in the present invention is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody. Target cells.
  • the ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc segment on IgG.
  • the modification refers to mutations in the heavy chain constant region of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, F234A/L235A mutation of IgG4.
  • “Mutation” in the "mutation sequence” of the present invention includes, but is not limited to, “backmutation”, “conservative modification” or “conservative substitution or substitution”.
  • “Conservatively modified” or “conservative substitution or substitution” as used in the present disclosure refers to other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.)
  • the amino acids allow frequent changes without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a 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 Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • mutant sequence means that the nucleotide sequence and the amino acid sequence of the present invention are different from the nucleotide sequence and the amino acid sequence of the present invention by appropriately modifying a nucleotide sequence and an amino acid sequence, such as substitution, insertion or deletion. Nucleotide sequence and amino acid sequence of percent sequence identity.
  • Identity refers to sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the sequence identity in the present invention may be at least 85%, 90% or 95%, preferably at least 95%.
  • Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%.
  • Sequence comparisons and percent identity determination between the two sequences can be performed by default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • the "PD-L1 antibody or antigen-binding protein thereof" of the present invention may include any of the anti-PD-L1 antibodies or antigen-binding fragments thereof described in the art.
  • the anti-PD-L1 antibody may be a PD-L1 antibody which is commercially available or has been disclosed in the literature. Including, but not limited to, PD-L1 antibody BMS-936559, MPDL3280A, MEDI4736, MSB0010718C (see US2014341917, US20130034559, US8779108) and the like.
  • the antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • Antibody fragments include Fab fragments having antigen binding activity, Fab' fragments, F(ab') 2 fragments, and Fv fragments and ScFv fragments that bind to antibodies.
  • HCDR1 SYWMH SEQ ID NO: 1
  • HCDR2 RI X 1 PNSG X 2 TSYNEKFKN SEQ ID NO: 2
  • HCDR3 GGSSYDYFDY SEQ ID NO: 3
  • X 1 is selected from H or G and X 2 is selected from G or F.
  • an exemplary PD-L1 antibody of the invention further comprises a CDR sequence of a light chain variable region as described below:
  • LCDR1 is selected from the group consisting of: RASESVSIHGTHLMH SEQ ID NO: 4
  • LCDR2 is selected from: AASNLES SEQ ID NO: 5
  • LCDR3 is selected from the group consisting of: QQSFEDPLT SEQ ID NO: 6;
  • the present invention utilizes a CDR grafting strategy for antibody humanization of the above CDR regions, and the humanized light chain template of the humanized framework is IGKV7-3*01 and hjk2.1, humanized weight
  • the strand templates are IGHV1-46*01 and hjh6.1, and the humanized variable region sequences are as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • transplantation refers to the implantation of a murine antibody CDR into a human germline FR region sequence.
  • an embodiment of a humanized clone construction is provided, as follows:
  • the primers were designed to construct the VH/VK gene fragment of each humanized antibody, and then homologously recombined with the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) to construct the full-length antibody expression vector VH- CH1-FC-pHr/VK-CL-pHr.
  • Primer design use the online software DNAWorks(v3.2.2) (http://helixweb.nih.gov/dnaworks/) to design multiple primers to synthesize VH/VK gene fragments containing recombinant: 5'-30bp signal peptide + VH/VK+30bp CH1/CL-3'.
  • the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragments) was constructed using a number of specific restriction enzymes, such as BsmBI, to distinguish the recognition sequence from the restriction site.
  • the vector was digested with BsmBI, and the gel was recovered for use.
  • VH/VK containing recombinant gene fragment and BsmBI digestion and expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) were added to DH5H competent cells in a ratio of 3:1, ice at 0 °C Bath for 30 min, heat shock at 42 °C for 90 s, add 5 volumes of LB medium, incubate for 45 min at 37 ° C, coat LB-Amp plate, incubate overnight at 37 ° C, pick monoclonal and send to sequencing to obtain each clone.
  • the plasmid was constructed according to the design of the present example, and then the purified protein was expressed, and the obtained protein affinity was determined by the test example SPR.
  • CDR2 is SEQ ID NO: 7 and X 1 is G, and X 2 is F.
  • the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic is FR sequence in the sequence; the underlined is the CDR sequence.
  • the PD-L1 antibody can also be used as a test control molecule in the present invention.
  • the use of wild-type IgG1 constant regions causes Fc-mediated effects such as ADCC and CDC, resulting in the reduction of activated T cells.
  • the present invention selects mutant IgG4 to obtain antibodies without ADCC and CDC.
  • the clone obtained by affinity maturation was converted to the IgG4 type, and the core hinge region of IgG4 contained the S228P mutation. Further introduction of F234A and L235A mutations (mAbs 4:3, 310-318; May/June 2012).
  • the PD-L1 antibody sequence of the present invention for fusion protein construction is as follows:
  • the underlined portion is the variable region sequence of the antibody heavy or light chain, or the nucleotide sequence encoding the same; the unlined portion is the antibody constant region sequence and its corresponding coding nucleotide sequence.
  • the fusion protein described in the present invention is a protein product co-expressed by two genes obtained by DNA recombination.
  • Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as Cold Spring Harbor Antibody Technical Guide, Chapters 5-8 and 15.
  • a mouse can be immunized with human PD-L1 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human PD-L1. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • the "immunomodulatory molecule" of the present invention can be used to attenuate the immune tolerance of cancer cells.
  • the present invention employs a truncated form of the extracellular domain of TGF-[beta]RII as part of the immunomodulatory molecule in the fusion protein.
  • TGF- ⁇ receptor II TGF- ⁇ RII
  • the TGF- ⁇ RII/TGF- ⁇ complex recruits TGF- ⁇ RI to form a signal transduction complex (Won et al, Cancer Res. 1999; 59: 1273-7).
  • the extracellular domain of TGF- ⁇ RII is a 136 amino acid residue peptide from the N-terminus of TGF- ⁇ RII extracellular, an exemplary example of which is shown in SEQ ID NO: 13.
  • Other variants of about 136 amino acids in length, and which are capable of binding to TGF- ⁇ 1 and 3, which are derived from the human extracellular region of TGF- ⁇ RII, are likewise within the scope of the extracellular domain of TGF- ⁇ RII of the invention.
  • the present inventors have found that the structure and function of the N-terminal continuous truncated form of the TGF- ⁇ RII extracellular domain are more stable than the untruncated molecule.
  • a fusion protein comprising the TGF-[beta]RII extracellular domain N-terminal untruncated (1-136 polypeptide set forth in SEQ ID NO: 13) is susceptible to cleavage.
  • it is more stable after truncation of 26 or less amino acids at its N-terminus, preferably truncation of 14-26 amino acids, more preferably N-terminal 14-21 amino acid truncated forms, with higher expression, most Preferably, the N-terminal 19 or 21 contiguous amino acids are truncated.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, for example a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount" effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. While embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in ameliorating the symptoms of each target disease, any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a 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 Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared x 100. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.
  • Immunity checkpoint molecules include stimulatory immunoassay molecule and inhibitory immunoassay molecule, and exemplary molecules include CD27, CD28, CD40, CD40L, CD122, OX40, OX40L, GITR, ICOS, A2AR, B7. -H3, B7-H4, BTLA, CTLA-4, IDO, KIR (Killer-cell Immunoglobulin-like Receptor), LAG3, PD-1, PD-L1, PD-L2, TIM-3, VISTA, and the like.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may 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, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • Example 1 Fusion protein PD-L1/TGF- ⁇ trap cloning and expression
  • the TGF- ⁇ RII extracellular domain (full length or truncated form of SEQ ID NO: 13) is used as a portion of the immunomodulatory molecule in the fusion protein, and the PD-L1 antibody is used as a targeting portion of the fusion protein to form a PD-L1 antibody/ TGF- ⁇ RII extracellular domain fusion protein (PD-L1/TGF- ⁇ trap).
  • PD-L1/TGF- ⁇ trap PD-L1/TGF- ⁇ trap
  • the N-terminal truncation is in the form of 14-21 contiguous amino acids, having a higher expression level and a stable structure, more preferably a N-terminal truncation of 14, 19 or 21 contiguous amino acids.
  • sequences of the non-limiting examples of the TGF- ⁇ RII extracellular domain of the invention and its truncated forms are as follows:
  • TGF- ⁇ RII extracellular domain sequence ECD (1-136)
  • the TGF- ⁇ RII extracellular domain sequence has a 19 amino acid truncation or deletion at the N-terminus: ECD (20-136)
  • the TGF- ⁇ RII extracellular domain sequence has a 21 amino acid truncation or deletion at the N-terminus: ECD (22-136)
  • the TGF- ⁇ RII extracellular domain sequence has a 14 amino acid truncation or deletion at the N-terminus: ECD (15-136)
  • Table 2 PD-L1 antibody/TGF- ⁇ RII extracellular domain fusion protein
  • Fusion protein Sequence description N-terminal consecutive amino acid deletions Fusion protein 1 Ab-(G 4 S) 4 G-ECD (1-136) Not missing Fusion protein 2 Ab-(G 4 S) 3 G-ECD (15-136) 14 Fusion protein 3 Ab-(G 4 S) 3 G-ECD (15-136, N19A) 14 Fusion protein 4 Ab-(G 4 S) 3 G-ECD (20-136) 19 Fusion protein 5 Ab-(G 4 S) 3 G-ECD (22-136) twenty one Fusion protein 6 Ab-(G 4 S) 3 G-ECD (27-136) 26 Fusion protein 7 Ab-(G4S) 4 G-ECD (15-136) 14 Fusion protein 8 Ab-(G4S) 4 G-ECD (15-136, N19A) 14 Fusion protein 9 Ab-(G4S) 4 G-ECD (20-136) 19 Fusion protein 10 Ab-(G4S) 4 G-ECD (22-136) twenty one Fusion protein 11 Ab-(G4S) 4 G-ECD (27-136) 26 Fusion protein 12 Ab-(G 4 S) 5 G-EC
  • Ab is the PD-L1 antibody of the present invention.
  • ECD (n-136) is a full-length or truncated form of the extracellular domain of TGF- ⁇ RII
  • n is a truncated amino acid of the extracellular domain of TGF- ⁇ RII.
  • the starting number of digits The structure of the fusion protein of the present invention is shown in Figure 1; N19A indicates that the amino acid mutation at position 19 of the extracellular region of TGF- ⁇ RII is A.
  • the nucleotide sequence encoding the PD-L1 antibody, the nucleotide sequence encoding the extracellular region of TGF- ⁇ RII, and the nucleotide sequence of the adaptor protein fragment ((G 4 S) x G) are obtained by means of a conventional technique in the art.
  • the C-terminal nucleotide of the PD-L1 antibody was ligated to the Phr-BsmbI vector by a N-terminal nucleotide of the extracellular region of TGF- ⁇ RII by a linker protein using a homologous recombination technique.
  • Recombinant PD-L1/TGF- ⁇ trap was expressed in 293 cells and purified by Example 2.
  • the purified protein can be used in the experiments of the following examples.
  • the cell culture medium was centrifuged at a high speed, and the supernatant was collected, and the first step of purification was carried out by affinity chromatography.
  • the chromatographic medium is Protein A or a derivatized filler that interacts with Fc, such as Ma's Mabselect.
  • the equilibration buffer was 1 ⁇ PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na 2 HPO 4 , 2 mmol/L KH 2 PO 4 , pH 7.4), and after equilibrating 5 column volumes, the cells were placed on the cells. Clear sample loading, flow rate control is the sample retention time on the column ⁇ 1min.
  • the column was washed with 1 x PBS (pH 7.4) until the A280 UV absorption was reduced to the baseline.
  • the column was then washed with 0.1 M glycine (pH 3.0) elution buffer, and the eluted peak was collected according to the A280 ultraviolet absorption peak, and the collected eluted sample was neutralized with 1 M Tris (pH 8.5).
  • the above-mentioned neutralized eluted sample was subjected to ultrafiltration concentration and subjected to size exclusion chromatography, the buffer was 1 ⁇ PBS, the column was XK26/60 Superdex 200 (GE), the flow rate was controlled at 4 ml/min, and the sample volume was less than 5 ml, combined with the target protein peak according to A280 UV absorption.
  • the collected protein was identified by SEC-HPLC to be more than 95% pure, and was identified by LC-MS as correct. PD-L1/TGF- ⁇ trap was obtained.
  • Test Example 1 ELISA for detection of PD-L1/TGF- ⁇ trap in vitro binding to TGF- ⁇ 1:
  • 96-well plates were coated with 100 ⁇ l/well of human TGF- ⁇ 1 (8915LC, CST) at a concentration of 1 ⁇ g/ml, and overnight at 4 °C.
  • Test Example 2 ELISA assay for PD-L1/TGF- ⁇ trap in vitro binding to PD-L1 assay:
  • 96-well plates were coated with 100 ⁇ l/well of human PD-L1-His (SEQ ID NO: 17) at a concentration of 5 ⁇ g/ml, overnight at 4 °C.
  • FIG. 1 The results of in vitro binding of the fusion protein of the present invention to human PD-L1 are shown in FIG.
  • the ELISA showed that the fusion protein retained the binding activity to human PD-L1.
  • Test Example 3 In vitro detection of PD-1/PD-L1 pathway blockade assay
  • 1PD-L1 antibody SEQ ID NO: 11, SEQ ID NO: 12;
  • 5-human IgG blank control, human immunoglobulin obtained by purification using a conventional affinity chromatography method such as ProteinA from mixed normal human serum;
  • CHO/PD-L1 cells (CS187108, Promega) were used for digestion and resuspended in F-12 Nuutent Mixture (Ham) complete medium.
  • the cell density was adjusted to 4 ⁇ 10 5 /mL using complete medium according to the cell count results.
  • the suspension was transferred to a sample tank, and added to a 96-well plate at 100 ⁇ L/well using a multi-channel pipette, placed at 37 ° C, and cultured in a 5% CO 2 incubator for 20 to 24 hours; the next day, Jurkat/PD- was prepared.
  • Bio-Glo TM Reagent remove its temperature returned to room temperature.
  • the cell culture plate was taken out, placed at room temperature for 5-10 min, then 40 ⁇ L Bio-Glo TM Reagent was added to each well, and incubated in a safety cabinet for 5-10 min, and the chemiluminescence signal value was read using a multi-function microplate reader.
  • the fusion protein 9 of the present invention was able to effectively block the binding of Jurkat cells expressing PD-1 molecules to CHO/PD-L1 cells, and had a dose-dependent effect of drug concentration, as did the positive molecules.
  • Fusion protein 15 has the same level of blocking ability as fusion protein 9.
  • Test Example 4 Biacore assay for in vitro binding affinity and kinetics
  • the affinity of the test molecule to human or murine TGF- ⁇ 1 or human PD-L1 protein was determined by Biacore T200 (GE). The experimental procedure is described as follows:
  • a certain amount of PD-L1/TGF- ⁇ trap was affinity-captured with Protein A chip, and then passed through human or mouse TGF- ⁇ 1 (8915LC, CST) or human PD-L1 (Sino Biological) on the surface of the chip, using Biacore real-time.
  • the reaction signal was detected to obtain binding and dissociation curves, and then the biochip was washed and regenerated with glycine-hydrochloric acid (pH 1.5, GE).
  • the buffer solution used in the experiment was HBS-EP Buffer (GE).
  • the experimental data were fitted with the (1:1) Langmuir model using BIAevaluation version 4.1 software (GE) to give the affinity values as shown in Table 3.
  • Table 3 In vitro affinity of fusion proteins of the invention with TGF- ⁇ 1 or human PD-L1
  • 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 extremely high affinity for human, mouse TGF- ⁇ 1 and human PD-L1.
  • Test Example 5 SMAD3 reporter gene inhibition assay
  • Smad3 binding element SBE
  • luciferase reporter gene was expressed in HepG2 cells to study the inhibitory effect of PD-L1/TGF- ⁇ trap on TGF- ⁇ 1-induced Smad3 activation, and PD-L1/TGF- ⁇ trap was evaluated according to IC50 size. In vitro activity.
  • Test sample fusion protein 9, positive control (M7824)
  • HepG2 cells were cultured in MEM complete medium (GE, SH30243.01) containing 10% FBS and passaged every 3 days.
  • MEM complete medium GE, SH30243.01
  • 96-well plates (Corning, 3903) were seeded at a density of 25,000 cells per well, and cultured at 37 ° C under 5% CO 2 for 24 hours.
  • the medium in the cell culture plates was discarded, and 100 ng of 3TP-Lux plasmid was transfected per well.
  • the cells were further cultured for 24 hours at 37 ° C under 5% CO 2 .
  • the complete medium in the 96-well plate was discarded, and 80 ⁇ L of incomplete medium (MEM + 0.5% FBS) was added to each well.
  • luciferase substrate ONE-GloTM Luciferase Assay system promega, E6110 was added to each well, and allowed to stand at room temperature for 10 minutes in the dark, and then the luminescent signal value was read using a Victor3 multi-plate reader (Perkin Elmer). .
  • the IC50 value of the sample to be tested was calculated using the data processing software Graphpad Prism 5.0.
  • Figure 6 shows that fusion protein 9 inhibits TGF ⁇ -induced pSMAD3 reporter activity in a dose-dependent manner and has comparable efficacy and IC50 (concentration required to inhibit 50% of maximum activity) to positive control M7824.
  • IC50 concentration required to inhibit 50% of maximum activity
  • Test Example 6 In vitro detection of tuberculosis (TB) stimulation of PBMC release of IFN ⁇
  • PBMC peripheral blood mononuclear cells
  • Test samples 1 Human IgG; 2PD-L1 antibody; 3 fusion protein 9; 4 control 1 (20T-Fc): ECD (20-136)-Fc; 5PD-L1 antibody + control 1 (20T-Fc).
  • the purified PBMC was freshly isolated, 15 mL of about 3 ⁇ 10 7 cells, and 20 ⁇ L of tuberculin was added thereto, and cultured in a 37 ° C, 5% CO 2 incubator for 5 days. On the sixth day, the cultured cells were collected and centrifuged, washed once with PBS, resuspended in fresh medium, adjusted to a density of 1 ⁇ 10 6 cells per ml, and seeded into 96-well cell culture plates at 90 ⁇ L per well. Different concentrations of antibodies were separately added to the corresponding wells of the above 96-well cell culture plate, 10 ⁇ L per well, and 10 ⁇ L of PBS was added to the control group and the blank group, respectively.
  • the cell culture plates were incubated at 37 ° C for 3 days in a 5% CO 2 incubator.
  • the cell culture plate was taken out, and the supernatant was taken out per well by centrifugation (4000 rpm, 10 min), and after 10-fold dilution, the level of IFN- ⁇ was measured by an ELISA method (human IFN- ⁇ detection kit, Xinbosheng, EHC 102g.96).
  • ELISA method human IFN- ⁇ detection kit, Xinbosheng, EHC 102g.96.
  • PD-L1/TGF- ⁇ trap fusion protein samples were able to enhance the secretion of cytokine IFN- ⁇ by activated T lymphocytes, and there was a drug concentration dose effect.
  • the fusion protein 9 was able to dose-dependently enhance the activated T lymphocyte secreting cytokine IFN- ⁇ and had a stronger activation than the PD-L1 antibody, 20T-FC.
  • SD rats Three SD rats were used for experimentation, female, 12/12 hour light/dark adjustment, temperature 24 ⁇ 3 °C constant temperature, humidity 50-60%, free access to drinking water. Purchased from Jiesijie Experimental Animal Co., Ltd. On the day of the experiment, SD rats were injected with fusion protein in the tail vein at a dose of 6 mg/kg and an injection volume of 5 ml/kg.
  • the time of blood collection was: 15 min, 7 h, 24 h (day 2) after the first day of administration, the third day, the fourth day, the sixth day, the eighth day, the tenth day, the fifteenth day, in the rat Blood was taken from the fundus vein, 200 ⁇ l each time (equivalent to taking 100 ⁇ l of serum); the collected blood samples were allowed to stand at room temperature for half an hour until agglutination, and then centrifuged at 10000 x g for 10 minutes at 4 °C. The supernatant was collected and immediately stored at -80 ° C. The concentration of the fusion protein in the serum was measured by ELISA.
  • 96-well plates were coated with 100 ⁇ l/well at a concentration of 2 ⁇ g/ml of human PD-L1-His at 4 ° C overnight.
  • Test Example 8 Effect of PD-L1/TGF- ⁇ trap on subcutaneous transplantation of human breast cancer MDA-MB-231 mice
  • the mouse strain used in this experiment was a NOD/SCID female mouse (Cavens).
  • the human peripheral blood mononuclear cells used in the experiment were extracted from freshly collected blood.
  • the extraction method was as follows: Heparin anticoagulated venous blood Mix with the same volume of 2% FBS in PBS, mix and mix 25ml of diluted blood slowly into a centrifuge tube containing 15ml lymphocyte separation solution, centrifuge at 1200g for 10 minutes at room temperature, pipette the lymphocyte layer to another centrifugation Tube, add PBS to wash the cells, centrifuge at 300g for 8 minutes at room temperature, repeat once, resuspend the cells with RPMI-1640 medium containing 10% FBS, and add the cells to the previously coated CD3 antibody (OKT3, 40ng/ml) In a 6-well plate, 2 ⁇ 10 6 cells (2 ml) per well were placed in a 37 ° C incubator for 4 days.
  • MDA-MB-231 cells were resuspended in serum-free RPMI-1640 medium, mixed with an equal volume of matrigel, 100 ⁇ l (2.3 ⁇ 10 6 ) was inoculated subcutaneously into the right flank of NOD/SCID mice, and tumor volume was removed 11 days later. After oversized animals, mice were randomized into groups of 9 each. 5 ⁇ 10 5 stimulated PBMCs (60 ⁇ l) were injected into the tumor tissues, and the remaining PBMCs were stopped and continued to culture. After 1 week, 5 ⁇ 10 6 PBMCs (100 ⁇ l) were intraperitoneally injected into the tumor-bearing mice. Considered the first round of injection.
  • Group Dosage 1 blank control PBS 0 2 fusion protein 9-4.8mpk 4.8mg/kg 3 fusion protein 9-24mpk 24mg/kg 4PD-L1 antibody - 4mpk 4mg/kg 5PD-L1 antibody-20mpk 20mg/kg 6PD-L1 antibody - 4mpk + control 1-2.14mpk 4mg/kg+2.14mg/kg 7 control 1-2.14mpk 2.14mg/kg
  • Antibody fusion protein 9 (4.8, 24 mg/kg) can significantly inhibit the growth of subcutaneous xenografts in human breast cancer MDA-MB-231 mice. The dose-dependent relationship between high and low doses is superior to each other.
  • Molecular dose of reference drug PD-L1 antibody (4, 20 mg/kg), TGF- ⁇ RII control molecule 20T-FC (2.14 mg/kg) and combination group (PD-L1 antibody-4 mg/kg+20T-FC- 2.14 mg/kg).
  • Each dose of fusion protein 9 maintained its ideal anti-tumor effect from the 14th day after administration, and its high dose was superior to PD-L1 antibody -20mpk (p ⁇ 0.05); 25d after administration, The anti-tumor effect of each antibody was the best.
  • the inhibition rates of fusion protein 9 and PDL-1 antibody were 37.24%, 52.38%, 30.24%, 28.01%, and 31.38%, respectively, and 32 days after administration.
  • the anti-tumor effect of fusion protein 9 was still very obvious.
  • the %TGI of the low-dose group was 36.68% and 50.76%, respectively, and the tumor volume was statistically different compared with the control group (p ⁇ 0.05).
  • Test Example 9 Physical stability of PD-L1/TGF- ⁇ trap
  • This test example was used to detect the stability of fusion protein fusion protein 9 and fusion protein 15.
  • DSC Different scanning calorimetry
  • the sample was replaced with the corresponding buffer, and the sample concentration was controlled at about 50 mg/ml, and detection was performed using a MicroCal* VP-Capillary DSC (Malvern).
  • each sample and the blank buffer were degassed by a vacuum degasser for 1 to 2 min.
  • the last two pairs of orifice plates were respectively filled with 14% Decon 90 and ddH 2 O for cleaning.
  • a plastic soft cover was placed.
  • the scanning temperature starts from 25 ° C to the end of 100 ° C and the scanning rate is 60 ° C / h.
  • Table 8 The specific results are shown in Table 8 below. Both the fusion protein 9 and the fusion protein 15 exhibited good thermal stability in both test systems.
  • the sample purity was monitored by SEC-HPLC to investigate the periodic stability under certain conditions.
  • the sample concentration was controlled at about 50 mg/ml, and compared at 10 mM acetate/135 mM NaCl (pH 5.5) at, for example, -80 °C. Repeated freezing and thawing for 5 times and 40 ° C for one month of stability.
  • the column was detected using an Xbridge protein BEH SEC 200A (Waters) HPLC column. The test results are shown in Table 9 below, and both fusion proteins showed good stability.
  • Fusion protein 9 ( ⁇ %) Fusion protein 15 ( ⁇ %) 40 ° C 3.39% 1.8% -80 °C freeze-thaw 1.44% 1.39%
  • ⁇ % indicates the rate of change.
  • Test Example 10 Chemical stability of fusion proteins
  • Deamidation modification is a common chemical modification in antibodies that may affect late stability.
  • partial amino acid deamidation modification of CDR regions is generally chosen to avoid or reduce mutations. 1600 ⁇ g of the antibody to be tested was dissolved in 200 ⁇ l of 10 mM acetate/135 mM NaCl (pH 5.5) and stored in a 40 ° C incubator; samples were taken at 0, 14, and 28 days, respectively, for enzymatic hydrolysis experiments.
  • N represents the detection of modified asparagine, and the number represents the position at which the light chain or heavy chain N-terminus begins to count.
  • the percent content represents the ratio of deamidation modification detected by LC-MS to the signal of all peptides at that site.

Abstract

提供了含有TGF-β受体的融合蛋白及其医药用途。进一步提供了包含所述PD-L1抗体靶向部分和TGF-βRII胞外区的双功能融合蛋白，以及包含所述含有TGF-β受体融合蛋白的药物组合物，以及其制备抗癌药物的用途。

Description

含有TGF-β受体的融合蛋白及其医药用途 技术领域
本发明涉及肿瘤免疫治疗药物领域。具体地说，本发明涉及用于癌症治疗的融合蛋白，包括包含靶向分子和免疫调节因子如TGF-βRII的融合蛋白。更具体地涉及靶向分子PD-L1抗体和免疫调节因子如TGF-βRII的融合蛋白，包含其的药物组合物，以及其作为抗癌药物的应用。
背景技术
在肿瘤治疗中，人们早已认识到化疗所带来的高毒性及可导致耐药性癌细胞产生的负面影响。即使是靶向性针对与肿瘤生存生长相关的过度表达或激活的蛋白的治疗手段，仍会有癌细胞通过变异来减少或逃脱对靶向性治疗所针对通路的依赖，并利用其它的通路继续生存。肿瘤免疫治疗近年来备受关注，是肿瘤治疗领域的焦点，较难产生耐药性是该疗法的突出优势。肿瘤免疫治疗主要通过免疫学原理和方法，提高肿瘤细胞的免疫原性和对效应细胞杀伤的敏感性，激发和增强机体抗肿瘤免疫应答，并应用免疫细胞和效应分子输注宿主体内，协同机体免疫系统杀伤肿瘤、抑制肿瘤生长。
程序性死亡受体1(programmed death 1，PD-1)为CD28超家族成员。PD-1表达于活化的T细胞，B细胞及髓系细胞，其有两个配体，即程序性死亡配体-1(programmed death ligand 1，PD-L1)和PD-L2。PD-L1与T细胞上的受体PD-1相互作用，在免疫应答的负调控方面发挥着重要作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡。PD-1/PD-L1通路抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，从而增强免疫应答，因此，以PD-1/PD-L1为靶点的免疫调节对肿瘤抑制有重要的意义。
转化生长因子-β(transforming growth factor-β，TGF-β)属于调节细胞生长和分化的TGF-β超家族。TGF-β通过异源四聚体受体复合物传递信号，这个受体复合物是由两个I型和两个II型的跨膜丝氨酸/苏氨酸激酶受体组成。
TGF-β是一种多功能的细胞因子，以细胞或背景依赖的方式发挥肿瘤抑制或肿瘤促进的作用。TGF-β信号的肿瘤抑制作用源于其诱导多个基因表达的能力。当肿瘤发展过程中引入突变或表观遗传修饰时，癌细胞逐渐耐受TGF-β信号的抑制作用，最终导致肿瘤的发展。
研究发现阻断TGF-β信号传导通路能够减少肿瘤的转移。运用截短的Smad2/3显负性突变体抑制乳腺肿瘤细胞系的TGF-β信号通路，结果发现肿瘤细胞的转移 能力被抑制。结肠癌的微卫星不稳定性研究发现，TGF-βRII无活性的突变，使转移减少，增加了患者术后的存活率。但总体而言，抑制TGF-β信号通路的抑制剂单用在临床治疗中效果微弱，可能跟TGF-β主要在肿瘤细胞内异常性高表达及信号通路抑制剂的生物利用度有关。
因此，在靶向中和肿瘤微环境的TGF-β基础上抑制PD-1/PD-L1通路，可以使T细胞恢复活性，增强免疫应答，更有效地提高抑治肿瘤发生和发展的效果。本申请人在先的PCT申请PCT/CN2016/104320提供了一种PD-L1抗体。
目前已有抗体/TGF-β受体融合蛋白公开，如WO2006074451A2、WO2009152610A1、WO2011109789A2、WO2013164694A1、WO2014164427A1、WO2015077540A2、WO9309228A1、WO9409815A1、WO2015077540A2、WO2015118175A2。但一些融合蛋白仍存在不稳定或表达量不高的问题。在实际生产和临床应用上仍需进一步开发具有更优性能的产品。本发明提供一种更利于生产，性能更稳定的技术方案。
发明内容
本发明提供一种含有TGF-β受体的融合蛋白，包含靶向部分和TGF-β受体部分，其中，所述的TGF-β受体部分为TGF-βRII胞外区的N端截短形式。
在本发明一个优选的实施方案中，TGF-βRII胞外区的N端截短形式选自在TGF-βRII胞外区的N端上有26个以下连续氨基酸的缺失，优选14-26个连续氨基酸的缺失，更优选14-21个连续氨基酸的缺失；最优选包含14-21个连续氨基酸的缺失，非限制性实施例包括SEQ ID NO：14或SEQ ID NO：15所示的序列。
在本发明一个优选的实施方案中，TGF-βRII胞外区的序列如SEQ ID NO：13所示。
在本发明一个优选的实施方案中，所述的靶向部分为细胞特异性靶向部分；优选的，所述的靶向部分为癌细胞特异性靶向部分。
在本发明一个优选的实施方案中，所述的癌细胞特异性靶向部分选自抗体或其抗原结合片段、生长因子、激素、肽、受体或细胞因子。
在本发明一个优选的实施方案中，所述的抗体或其抗原结合片段选自全长抗体、嵌合抗体、Fab’、Fab、F(ab’) 2、单结构域抗体(DAB)、Fv、scFv、小抗体、双特异性抗体、三特异性抗体或其混合物。
在本发明一个优选的实施方案中，所述的抗体或其抗原结合片段结合选自以下一种或多种多肽或蛋白：HER2、HER3、免疫检验点分子、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、CD11a、CD19、CD30、CD38、CD20、CD52、CD60、CD80、CD86、TNF-α、IL-12、IL-17、IL-23、IL-6、IL-1β、RSVF、IgE、RANK、BLyS、α4β7、PD-1、CCR4、SLAMF7、GD2、 CD21、CD79b、IL20Rα、CD22、CD79a、CD72、IGF-1R和RANKL；优选所述的抗体或其抗原结合片段结合免疫检验点分子。
在本发明一个优选的实施方案中，所述的抗体为PD-L1抗体；优选的，所述的PD-L1抗体选自：MSB0010718C、MEDI4736、BMS-936559、MPDL3280A的PD-L1抗体，或包含1个或多个选自以下的CDR区序列或CDR区序列的突变序列的抗体：
Figure PCTCN2018086451-appb-000001
其中X 1选自H或G，优选为G；X 2选自G或F，优选为F。
在本发明一个优选的实施方案中，所述的抗体或其抗原结合片段为嵌合抗体或其功能片段、人源化抗体或其功能片段、或人抗体或其或其功能片段。
在本发明一个优选的实施方案中，所述人源化抗体的重链可变区具有如SEQ ID NO：7所示的序列，优选具有如SEQ ID NO：9所示的序列。
在本发明一个优选的实施方案中，所述的人源化抗体进一步包括序列SEQ ID NO：11所示的重链。
在本发明一个优选的实施方案中，所述的人源化抗体包含SEQ ID NO：8或10所示的抗体轻链可变区序列或轻链可变区的突变序列。
在本发明一个优选的实施方案中，所述的人源化抗体包含序列SEQ ID NO：12所示的轻链。
在本发明一个优选的实施方案中，所述的含有TGF-β受体的融合蛋白如通式(I)所示：
Ab-L-TGF-βRII ECD         (I)
其中TGF-βRII ECD为TGF-βRII胞外区的截短形式；
Ab为抗体；
L为连接序列。
在本发明一个优选的实施方案中，所述的连接序列L为(G 4S) xG，其中x为3-6，优选为4-5。
本发明进一步提供一种药物组合物，其含有治疗有效量的如上所述的含有TGF-β受体的融合蛋白，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明进一步提供一种编码如上所述的含有TGF-β受体的融合蛋白的DNA分子。
本发明进一步提供一种含有如上所述的DNA分子的表达载体。
本发明进一步提供一种用如上所述的表达载体转化的宿主细胞，所述宿主细胞选自细菌、酵母菌和哺乳动物细胞；优选哺乳动物细胞。
本发明进一步提供一种所述的含有TGF-β受体的融合蛋白或包含其的药物组合物，在制备用于肿瘤治疗的药物中的用途；优选在制备用于治疗PD-L1介导的肿瘤的药物中的用途；更优选为表达PD-L1的癌症。
本发明进一步提供一种治疗和预防肿瘤的方法，包括给予所需患者治疗有效量的本发明所述的含有TGF-β受体的融合蛋白或包含其的药物组合物。
本发明进一步提供一种截短的TGF-βRII胞外区，其中所述的TGF-βRII胞外区的截短选自在SEQ ID NO：13的N端上有26个以下连续氨基酸的缺失，优选N端14-26个连续氨基酸的缺失，更优选N端14-21个连续氨基酸的缺失；最优选包含14-21个连续氨基酸的缺失，非限制性实施例包括SEQ ID NO：14或SEQ ID NO：15所示的序列。
本发明进一步提供一种药物组合物，其含有治疗有效量的本发明所述的截短的TGF-βRII胞外区，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明进一步提供一种本发明所述的截短的TGF-βRII胞外区或包含其的药物组合物，在制备用于治疗、抑制癌细胞增殖或转移的疾病或病症的药物中的用途。
本发明进一步提供一种治疗和预防肿瘤的方法，包括给予所需患者治疗有效量的本发明所述的截短的TGF-βRII胞外区或包含其的药物组合物。
本公开中所述肿瘤或癌症选自以下部位的肿瘤或癌症：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、骨髓瘤、淋巴瘤、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合征。
附图说明
图1：融合蛋白结构示意图。
图2：融合蛋白体外结合人源TGF-β1的结果。
图3：融合蛋白体外结合人源TGF-β1的结果。
图4：融合蛋白体外结合人源PD-L1的结果。
图5：融合蛋白体外检测PD-1/PD-L1通路阻断实验结果。
图6：融合蛋白以剂量依赖性形式抑制TGFβ诱导的pSMAD3报告物活性。
图7：融合蛋白样品均能够增强激活的T淋巴细胞分泌细胞因子IFN-γ。
图8：融合蛋白对荷瘤小鼠瘤重的影响。
具体实施方式
一、术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDE2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
本发明的抗体包括选自鼠源抗体、嵌合抗体、人源化抗体的全长抗体，优选人源化抗体。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人PD-L1的单克隆抗体。制备时用PD-L1抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源PD-L1抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗 体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的PD-L1嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的PD-L1嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
术语“人抗体”与“人源抗体”可以互换使用，是指有一或多个可变区和恒定区来源于人免疫球蛋白序列。其中一个优选的方式是所有的可变区和恒定区均来自于人免疫球蛋白序列，即“完全人源抗体”或“全人抗体”。这些抗体可以通过多种方式制备获得，包括通过噬菌体展示技术，从人PBMC、脾脏、淋巴结组织分离B细胞，构建天然单链噬菌体人抗体库，或者通过免疫可表达人抗体轻重链的转基因小鼠，筛选获得的抗体。
本发明中所述的“抗体或其抗原结合”或“功能片段”，指具有抗原结合活性的Fab片段，Fab‘片段，F(ab’)2片段，以及与抗体结合的Fv片段ScFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本发明的术语“与PD-L1结合”，指能与人PD-L1相互作用。本发明的术语“抗原结合位点”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A；IgG2/4嵌合体，IgG4的F234A/L235A突变。
本发明所述的“突变序列”中的“突变”包括但不限于“回复突变”、“保守修饰”或“保守置换或取代”。本公开中所述的“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
本发明所述的“突变序列”是指对本发明的核苷酸序列和氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本发明的核苷酸序列和氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和氨基酸序列。
本发明中所述“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。本发明中的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
本发明所述的“PD-L1抗体或其抗原结合蛋白”可包括本领域中所述的任何抗PD-L1抗体或其抗原结合片段。抗PD-L1抗体可以是市售可得的或已通过文献公开的PD-L1抗体。包括但不限于，如PD-L1抗体BMS-936559，MPDL3280A，MEDI4736，MSB0010718C(参见US2014341917、US20130034559、US8779108)等。抗体可以是单克隆抗体、嵌合抗体、人源化抗体，或人抗体。抗体片段包括具有抗原结合活性的Fab片段，Fab‘片段，F(ab’) 2片段，以及与抗体结合的Fv片段和ScFv片段。
本发明示例性的PD-L1抗体制备过程参见(PCT/CN2016/104320)包括如下所述的重链可变区的CDR序列：
HCDR1：          SYWMH                      SEQ ID NO：1
HCDR2：          RI X 1PNSG X 2TSYNEKFKN      SEQ ID NO：2
HCDR3：          GGSSYDYFDY                 SEQ ID NO：3
在可选的实施方式中，X 1选自H或G，X 2选自G或F。
在另一个实施方式中，本发明示例性的PD-L1抗体进一步包括如下所述的轻链可变区的CDR序列：
LCDR1选自：           RASESVSIHGTHLMH            SEQ ID NO：4
LCDR2选自：           AASNLES                    SEQ ID NO：5
LCDR3选自：           QQSFEDPLT                  SEQ ID NO：6；
在另一个实施方式中，本发明对上述的CDR区采用CDR移植策略进行抗体人源化，人源化构架的人源化轻链模板为IGKV7-3*01和hjk2.1，人源化重链模板为IGHV1-46*01和hjh6.1，人源化可变区序列如下：
人源化重链可变区
Figure PCTCN2018086451-appb-000002
SEQ ID NO：7
人源化轻链可变区
Figure PCTCN2018086451-appb-000003
SEQ ID NO：8
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
在另一个实施方式中，对本发明人源化抗体的回复突变设计，见下表1：
表1
Figure PCTCN2018086451-appb-000004
注：如Y91F表示依照Kabat编号系统，将91位Y突变回F。
“植入”代表鼠抗体CDR植入人种系FR区序列。
表中各种轻重链的突变组合可得到新的人源化抗体。
本发明的另一方面，提供一种人源化克隆构建的实施例，如下：
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pHr/VK-CL-pHr。
1、引物设计：利用在线软件DNAWorks(v3.2.2)(http：//helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH/VK+30bp CH1/CL-3’。
2、片段拼接：按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。
3、表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)构建及酶切
利用一些特殊的限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)。BsmBI酶切载体，切胶回收备用。
4、重组构建表达载体VH-CH1-FC-pHr/VK-CL-pHr
VH/VK含重组所需基因片段与BsmBI酶切回收表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)按3∶1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
5、根据本实施例的设计方案构建质粒，然后表达纯化蛋白，用检测例SPR测定所得蛋白亲和力。
6、最终用BIACORE测试人源化回复突变体与人源PD-L1-his或杂交瘤抗体的亲和力，筛选得到的人源化回复突变位点的选择及序列组合如下：
重链可变区：
Figure PCTCN2018086451-appb-000005
SEQ ID NO：9
其中，CDR2为SEQ ID NO：7的X 1为G，X 2为F。
轻链可变区：
Figure PCTCN2018086451-appb-000006
SEQ ID NO：10
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列；下划线为CDR序列。
本发明的另一方面，提供一种构建和表达抗PD-L1人源IgG4类型的实施例，并进一步用于融合蛋白构建的PD-L1抗体。该PD-L1抗体也可用作本发明中的测试例对照分子。
由于PD-L1在活化T细胞中也有表达，如果采用野生型IgG1恒定区会引起Fc介导的效应作用，比如ADCC和CDC，从而导致活化T细胞的消减。本发明选择突变IgG4以得到无ADCC和CDC的抗体。将亲合力成熟得到的克隆转换成IgG4类型，IgG4的核心铰链区包含S228P突变。并进一步引入F234A和L235A突变(mAbs 4：3，310-318；May/June 2012)。同时，为防止抗体重链C末端在引入连接肽连接TGF-βRII胞外区时发生断裂，又将PD-L1抗体重链的最后一位K突 变成A，以增加融合蛋白的稳定性。本发明用于融合蛋白构建的的PD-L1抗体序列如下：
PD-L1抗体重链序列：IgG4(AA)(S228P)
Figure PCTCN2018086451-appb-000007
SEQ ID NO：11
PD-L1抗体轻链序列
Figure PCTCN2018086451-appb-000008
SEQ ID NO：12
注：划线部分为抗体重链或轻链的可变区序列，或编码其的核苷酸序列；未划线部分为抗体恒定区序列及其相应的编码核苷酸序列。
本发明中所述的融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，小鼠可以用人PD-L1或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http：//imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人PD-L1特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体 可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本发明所述的“免疫调节分子”可用于削弱癌细胞的免疫耐受性。本发明采用TGF-βRII胞外结构域的截短形式作为融合蛋白中免疫调节分子部分。“TGF-β受体II(TGF-βRII)”以高亲和力结合配体TGF-β1和3。TGF-β RII/TGF-β复合物招募TGF-β RI以形成信号转导复合物(Won等，Cancer Res.1999；59：1273-7)。TGF-βRII的胞外结构域是TGF-βRII细胞外自N端开始的一段长136个氨基酸残基的肽段，其中一个示例性的例子如SEQ ID NO：13所示。其他长度约为136个氨基酸，并且来源于人的具有TGF-βRII的胞外区的能够与TGF-β1和3相结合的变体同样同样属于本发明TGF-βRII的胞外结构域的范围。本发明研究发现，TGF-βRII胞外结构域N端连续截短形式的结构和功能较未截短分子稳定。含TGF-βRII胞外结构域N端未截短(SEQ ID NO：13所示的1-136多肽)形式的融合蛋白容易断裂。尤其是在其N端作26个以下氨基酸的截短后更为稳定，优选14-26个氨基酸的截短，更优选N端14-21个氨基酸截短形式，具有更高的表达量，最优选截短N端19或21个连续氨基酸。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本发明的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频 繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“免疫检验点(immune checkpoint)分子”包括刺激性免疫检验点分子和抑制性免疫检验点分子，示例性分子包括CD27、CD28、CD40、CD40L、CD122、OX40、OX40L、GITR、ICOS、A2AR、B7-H3、B7-H4、BTLA、CTLA-4、IDO、KIR(Killer-cell Immunoglobulin-like Receptor)、LAG3、PD-1、PD-L1、PD-L2、TIM-3、VISTA等。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51：263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩 增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
二、实施例与测试例
以下结合实施例进一步描述本发明，但这些实施例并非限制着本发明的范围。
本发明实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1：融合蛋白PD-L1/TGF-βtrap克隆和表达
采用TGF-βRII胞外结构域(SEQ ID NO：13的全长或截短形式)作为融合蛋白中免疫调节分子部分，将PD-L1抗体作为融合蛋白的靶向部分，形成PD-L1抗体/TGF-βRII胞外区融合蛋白(PD-L1/TGF-βtrap)。研究发现，TGF-βRII胞外结构域截短形式的结构和功能较为稳定，尤其是在其N端作26个以下氨基酸的截短后更为稳定，优选14-26个氨基酸的截短，更优选N端截短14-21个连续氨基酸的形式，具有更高的表达量和稳定的结构，更优选N端截短14、19或21个连续氨基酸的形式。本发明TGF-βRII胞外结构域及其截短形式的非限制性实施例序列如下：
TGF-βRII胞外结构域序列：ECD(1-136)
Figure PCTCN2018086451-appb-000009
SEQ ID NO：13
TGF-βRII胞外结构域序列在N端有19个氨基酸的截短或缺失：ECD(20-136)
Figure PCTCN2018086451-appb-000010
SEQ ID NO：14
TGF-βRII胞外结构域序列在N端有21个氨基酸的截短或缺失：ECD(22-136)
Figure PCTCN2018086451-appb-000011
SEQ ID NO：15
TGF-βRII胞外结构域序列在N端有14个氨基酸的截短或缺失：ECD(15-136)
Figure PCTCN2018086451-appb-000012
SEQ ID NO：16
利用同源重组技术将本发明PD-L1抗体的重链C末端氨基酸通过(G 4S) xG连接不同长度TGF-βRII胞外区，与轻链一起，通过293表达系统进行常规表达，得到如表2所示的融合蛋白：
表2：PD-L1抗体/TGF-βRII胞外区融合蛋白
融合蛋白例 序列描述 N端连续氨基酸缺失数
融合蛋白1 Ab-(G 4S) 4G-ECD(1-136) 未缺失
融合蛋白2 Ab-(G 4S) 3G-ECD(15-136) 14
融合蛋白3 Ab-(G 4S) 3G-ECD(15-136，N19A) 14
融合蛋白4 Ab-(G 4S) 3G-ECD(20-136) 19
融合蛋白5 Ab-(G 4S) 3G-ECD(22-136) 21
融合蛋白6 Ab-(G 4S) 3G-ECD(27-136) 26
融合蛋白7 Ab-(G4S) 4G-ECD(15-136) 14
融合蛋白8 Ab-(G4S) 4G-ECD(15-136，N19A) 14
融合蛋白9 Ab-(G4S) 4G-ECD(20-136) 19
融合蛋白10 Ab-(G4S) 4G-ECD(22-136) 21
融合蛋白11 Ab-(G4S) 4G-ECD(27-136) 26
融合蛋白12 Ab-(G 4S) 5G-ECD(15-136) 14
融合蛋白13 Ab-(G 4S) 5G-ECD(15-136，N19A) 14
融合蛋白14 Ab-(G 4S) 5G-ECD(20-136) 19
融合蛋白15 Ab-(G 4S) 5G-ECD(22-136) 21
融合蛋白16 Ab-(G 4S) 5G-ECD(27-136) 26
融合蛋白17 Ab-(G 4S) 6G-ECD(27-136) 26
注：Ab为本发明所述PD-L1抗体，序列描述中ECD(n-136)为TGF-βRII胞外区的全长或截短形式，n为TGF-βRII胞外区截短后的氨基酸起始位数。本发明融合蛋白结构如图1所示；N19A表示表示TGF-βRII胞外区的第19位氨基酸突变为A。
编码PD-L1抗体的核苷酸序列、编码TGF-βRII胞外区的核苷酸序列、接头蛋白片段((G 4S) xG)的核苷酸序列通过所属领域常规技术手段获得。利用同源重组技术将PD-L1抗体的C末端核苷酸通过接头蛋白连接不同长度TGF-βRII胞外区的N末端核苷酸，克隆到Phr-BsmbI载体上。重组的PD-L1/TGF-βtrap在293细胞表达，通过实施例2进行纯化。纯化的蛋白可用于下述各实施例实验中。
实施例2：PD-L1/TGF-βtrap融合蛋白纯化
细胞培养液高速离心后收集上清，利用亲合层析进行第一步纯化。层析介质为与Fc相互作用的Protein A或者衍生填料，如GE的Mabselect。平衡缓冲液为1×PBS(137mmol/L NaCl，2.7mmol/L KCl，10mmol/L Na 2HPO 4，2mmol/L KH 2PO 4，pH7.4)，平衡5倍柱体积后，将细胞上清上样结合，流速控制为样品在柱上保留时间≥1min。上样结束后，用1×PBS(pH7.4)冲洗柱子，直至A280紫外吸收降至基线。然后用0.1M甘氨酸(pH3.0)的洗脱缓冲液冲洗层析柱，根据A280紫外吸收峰收集洗脱峰，收集的洗脱样品用1M Tris(pH8.5)中和。
将上述中和后的洗脱样品超滤浓缩后进行体积排阻层析，缓冲液为1×PBS，层析柱为XK26/60 Superdex200(GE)，流速控制在4ml/min，上样体积小于5ml，根据A280紫外吸收合并目的蛋白峰。收集的蛋白经SEC-HPLC鉴定纯度大于95％，经LC-MS鉴定为正确后分装备用。得到PD-L1/TGF-βtrap。
以下用生化测试方法验证本发明性能及有益效果。
体外活性生物学评价
测试例1：ELISA检测PD-L1/TGF-βtrap体外结合TGF-β1实验：
检测流程描述如下：
a.以1μg/ml浓度的人源TGF-β1(8915LC，CST)为抗原按100μl/孔包被96孔板，4℃过夜。
b.250μl 1×PBST洗涤3次，加入250μl 5％牛奶PBS，37℃封闭2小时。
c.250μl 1×PBST洗涤3次，加入梯度稀释的PD-L1/TGF-β trap，TGF-β trap和阳性对照，37℃孵育1小时。
d.250μl 1×PBST洗涤3次。
e.每孔加入100μl Anti-human FC antibody-HRP(1∶4000)，37℃孵育40分钟。
f.每孔加入100μl TMB，室温孵育10分钟后加入100μl 1M H 2SO 4终止反应。
g.酶标仪上检测450nm测吸收值，Graphpad Prism5分析数据。
融合蛋白体外结合人源TGF-β1的结果如图2、图3所示。ELISA显示表2中融合蛋白1未保留对人源TGF-β1的结合活性。质谱分析结果显示融合蛋白1(即TGF-βRII胞外区非截短形式(1-136))不稳定，容易在重链TGF-βRII部分断裂， 阳性对照同样存在相同情况，而融合蛋白7、9、10、12-15等TGFβRII胞外区N端截短形式的融合蛋白对于结合至人源TGF-β1是具有特异性的。
测试例2：ELISA检测PD-L1/TGF-βtrap体外结合PD-L1实验：
检测用抗原：PD-L1-His
Figure PCTCN2018086451-appb-000013
SEQ ID NO：17
检测流程描述如下：
a.以5μg/ml浓度的人源PD-L1-His(SEQ ID NO：17)为抗原按100μl/孔包被96孔板，4℃过夜。
b.250μl 1×PBST洗涤3次，加入250μl 5％牛奶PBS 37℃封闭2小时。
c.250μl 1×PBST洗涤3次，加入梯度稀释的PD-L1/TGF-β trap，PD-L1抗体为阳性对照，37℃孵育1小时。
d.250μl 1×PBST洗涤3次。
e.每孔加入100μl抗人FC抗体-HRP(1∶4000)，37℃孵育40分钟。
f.每孔加入100μl TMB，室温孵育10分钟后加入100μl 1M H 2SO 4终止反应。
g.酶标仪上检测450nm测吸收值，Graphpad Prism5分析数据。
本发明融合蛋白体外结合人源PD-L1的结果如图4所示。ELISA显示融合蛋白均保留了对人源PD-L1的结合活性。
测试例3：体外检测PD-1/PD-L1通路阻断实验
1、测试目的：
为了研究PD-L1/TGF-βtrap对PD-1/PD-L1信号通路的阻断作用，采用来自Promaga公司构建的分别带有人源PD-1和PD-L1受体分子的细胞，进行基于细胞水平上的抗体阻断实验。
2、测试样品：
①PD-L1抗体：SEQ ID NO：11，SEQ ID NO：12；
②对照1(20T-Fc)：ECD(20-136)-Fc，TGF-βRII胞外区截短片段ECD(20-136)与Fc的融合蛋白
序列如下：
Figure PCTCN2018086451-appb-000014
Figure PCTCN2018086451-appb-000015
SEQ ID NO：18；
③对照2(22T-Fc)：ECD(22-136)-Fc，TGF-βRII胞外区截短片段ECD(22-136)与Fc的融合蛋白
序列如下：
Figure PCTCN2018086451-appb-000016
SEQ ID NO：19；
④融合蛋白9，融合蛋白15；
⑤人IgG：空白对照，从混合的正常人血清中，利用传统的亲和层析方法如ProteinA纯化获得的人免疫球蛋白；
⑥阳性对照(M7824，参考专利WO2015118175制备)：PD-L1抗体/TGF-βRII胞外区融合蛋白
PD-L1抗体轻链的氨基酸序列
Figure PCTCN2018086451-appb-000017
SEQ ID NO：20
PD-L1抗体重链/TGF-βRII胞外区(1-136)的H链的氨基酸序列：
Figure PCTCN2018086451-appb-000018
Figure PCTCN2018086451-appb-000019
SEQ ID NO：21。
3、测试过程
取CHO/PD-L1细胞(CS187108，Promega)，消化并用F-12Nutrient Mixture(Ham)完全培养基重悬细胞，根据细胞计数结果使用完全培养基调整细胞密度至4×10 5/mL，将细胞悬液转移至加样槽中，使用多道移液器以100μL/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养20～24h；第二天制备Jurkat/PD-1(CS187102，Promega)细胞悬液，根据细胞计数结果使用分析培养基重悬细胞，并调整细胞密度至1.25×10 6/mL；将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出，使用多道移液器每孔取出95μL培养液，按照40μL/孔加入梯度稀释的融合蛋白，以及PD-L1抗体及阳性对照(M7824)，然后将Jurkat/PD-1细胞悬液转移至加样槽中，以40μL/孔加入到细胞培养板中，置于37℃，5％CO 2培养箱培养5～6h。在蛋白孵育期间，将Bio-Glo TM Reagent取出使其温度恢复至室温。取出细胞培养板，置于室温放置5～10min，然后每孔加入40μL Bio-Glo TM Reagent，置于安全柜中孵育5～10min，使用多功能酶标仪读取化学发光信号值。
4、结果
如图5所示，本发明融合蛋白9同阳性分子一样均能够有效地阻断表达有PD-1分子的Jurkat细胞同CHO/PD-L1细胞结合，并且有药物浓度剂量依赖效应。融合蛋白15与融合蛋白9有相同水平的阻断能力。
测试例4：Biacore检测体外结合亲和力和动力学实验
通过Biacore T200(GE)测定待测分子与人或鼠源TGF-β1或人源PD-L1蛋白的亲和力，实验过程描述如下：
用Protein A芯片亲和捕获一定量的PD-L1/TGF-βtrap，然后于芯片表面流经人或鼠源TGF-β1(8915LC，CST)或人源PD-L1(Sino Biological)，利用Biacore实时检测反应信号，从而获得结合和解离曲线，然后用甘氨酸-盐酸(pH 1.5，GE)将生物芯片洗净再生。实验中用到的缓冲溶液为HBS-EP Buffer(GE)。实验得到的数据用BIAevaluation version 4.1软件(GE)以(1∶1)Langmuir模型进行拟合，得出如表3所示的亲和力数值。
表3：本发明融合蛋白与TGF-β1或人源PD-L1的体外亲和力
Figure PCTCN2018086451-appb-000020
Figure PCTCN2018086451-appb-000021
*对应序号的融合蛋白形式见表2。
融合蛋白结合活性见表3，结果表明，本发明融合蛋白9和15均对人，小鼠TGF-β1以及人PD-L1具极高的亲和力。
测试例5：SMAD3报告基因抑制实验
1、测试目的
该实验通过HepG2细胞表达带荧光素酶报告基因的Smad3结合原件(SBE)来研究PD-L1/TGF-βtrap对TGF-β1诱导Smad3活化的抑制作用，根据IC50大小评价PD-L1/TGF-βtrap的体外活性。
2、测试样品：融合蛋白9、阳性对照(M7824)
3、测试过程
HepG2细胞使用含有10％FBS的MEM完全培养基(GE，SH30243.01)培养，每3天传代一次。实验第一天以每孔25,000个细胞的密度接种于96孔板(Corning，3903)，在37℃、5％CO 2条件下培养24小时。第二天，弃去细胞培养板中的培养基，每孔转染100ng 3TP-Lux质粒。细胞在37℃、5％CO 2条件下继续培养24小时。加入待测样品前6小时，弃去96孔板中完全培养基，每孔加入80μL不完全培养基(MEM+0.5％FBS)。6小时后再加入10μL使用不完全培养基配制的人TGF-β1(R&D，240-B-010)溶液，终浓度为2ng/mL和10μL待测样品，终浓度为500、50、5、0.5、0.05、0.005、0.0005和0nM，以人TGF-β1溶剂为对照，细胞在37℃、5％CO 2条件下继续培养18h。然后每孔加入100μL配制好的萤光素酶底物ONE-GloTM Luciferase Assay system(promega，E6110)，室温避光放置10分钟，然后使用Victor3多功能酶标仪(Perkin Elmer)读取发光信号值。待测样品的IC50值使用数据处理软件Graphpad Prism5.0计算得到。
图6显示融合蛋白9以剂量依赖性形式抑制TGFβ诱导的pSMAD3报告物活性，且与阳性对照M7824具有相当的功效和IC50(抑制最大活性的50％所需的浓度)。PD-L1抗体的测试结果显示其不具有抑制作用(IC50＞500nM)。
测试例6：体外检测结核杆菌素(TB)刺激PBMC释放IFNγ实验
1、测试目的
为了研究PD-L1/TGF-βtrap对T淋巴细胞的激活作用，收集和纯化人外周血单核细胞(PBMC)，采用结核杆菌素(TB)体外刺激5天，检测IFNγ细胞因子的分泌水平。
2、测试样品：①Human IgG；②PD-L1抗体；③融合蛋白9；④对照1(20T-Fc)：ECD(20-136)-Fc；⑤PD-L1抗体+对照1(20T-Fc)。
3、测试过程
新鲜分离纯化的PBMC，15mL约3×10 7个，加入20μL结核菌素，37℃、5％CO 2培养箱培养5天。第6天，收集上述培养的细胞离心，用PBS洗一次，重悬至新鲜的培养基中，调整密度为1×10 6个每毫升，接种至96孔细胞培养板，每孔90μL。将不同浓度的抗体分别加入上述96孔细胞培养板的对应孔中，每孔10μL，对照组和空白组分别加入10μL PBS。细胞培养板置于37℃，5％CO 2培养箱孵育3天。取出细胞培养板，离心(4000rpm，10min)每孔取上清，10倍稀释后，采用ELISA的方法(人IFN-γ检测试剂盒，欣博盛，EHC102g.96)检测IFN-γ的水平。具体操作参考试剂说明书。结果如图所示，PD-L1/TGF-βtrap融合蛋白样品均能够增强激活的T淋巴细胞分泌细胞因子IFN-γ，并且有药物浓度剂量效应。
表4
Figure PCTCN2018086451-appb-000022
4、结果
如图7、表4所示，融合蛋白9能够剂量依赖地增强激活的T淋巴细胞分泌细胞因子IFN-γ，并且具有比PD-L1抗体，20T-FC更强的激活作用。
药代动力学评价
测试例7：
实验用SD大鼠3只，雌性，12/12小时光/暗调节，温度24±3℃恒温，湿度50-60％，自由进食饮水。购自杰思捷实验动物有限公司。实验当天对SD大鼠分别尾静脉注射融合蛋白，给药剂量为6mg/kg，注射体积为5ml/kg。
取血时间点为：第1天给药后15min、7h、24h(第2天)，第3天，第4天，第6天，第8天，第10天，第15天，于大鼠眼底静脉取血，每次200μl(相当于取血清100μl)；收集的血样在室温下置放半小时至凝集，然后4℃下10000xg离心10分钟。收集上清，立即放置-80℃贮存。用ELISA检测血清中的融合蛋白浓度。
检测流程描述如下：
a.以2μg/ml浓度的人源PD-L1-His为抗原按100μl/孔包被96孔板，4℃过夜。
b.250μl 1×PBST洗涤4次，加入250μl 5％牛奶PBS，37℃封闭3小时。
c.250μl 1×PBST洗涤4次，加入100μl梯度稀释的待测血清，以融合蛋白9为阳性对照，37℃孵育1小时。
d.250μl 1×PBST洗涤5次。
e.每孔加入100μl生物素化的抗人源TGF-βRII的抗体(R&D)，37℃孵育1小时。
f.250μl 1×PBST洗涤5次。
g.每孔加入100μl TMB，室温孵育10分钟后加入100μl 1M H 2SO 4终止反应。
h.酶标仪上检测450nm测吸收值，Graphpad Prism5分析数据。
表5：融合蛋白在SD大鼠中的T1/2
Figure PCTCN2018086451-appb-000023
参见表5，PK分析结果表明，本发明融合蛋白分子融合蛋白9在大鼠体内的半衰期约为236h(9.8天)。
体内活性生物学评价
测试例8：PD-L1/TGF-βtrap对人乳腺癌MDA-MB-231小鼠皮下移植瘤的疗效
本实验应用的小鼠品系为NOD/SCID雌性小鼠(卡文斯)，实验使用的人外周血单个核细胞从新鲜采集的血液中提取获得，提取方法如下：将肝素抗凝处理的静脉血与同体积含2％FBS的PBS混合，混匀后将25ml稀释后的血液缓慢加入到含15ml淋巴细胞分离液的离心管中，室温下1200g离心10分钟，吸取淋巴细胞层转移到另一个离心管，加入PBS清洗细胞，室温下300g离心8分钟，重复一次后，用含10％FBS的RPMI-1640培养基重悬细胞，将细胞加入到事先包被好CD3抗体(OKT3，40ng/ml)的6孔板中，每孔2×10 6个细胞(2ml)，置于37℃培养箱中培养4天。
实验样品：
①空白对照：PBS；②融合蛋白9-4.8mpk；③融合蛋白9-24mpk；④PD-L1抗体-4mpk；⑤PD-L1抗体-20mpk；⑥PD-L1抗体-4mpk+对照1(20T-Fc)-2.14mpk；⑦对照1(20T-Fc)-2.14mpk。
将MDA-MB-231细胞重悬于无血清RPMI-1640培养基中，与等体积基质胶混合后100μl(2.3×10 6)接种于NOD/SCID小鼠右肋部皮下，11天后去除肿瘤体积过大过小动物后，将小鼠随机分组，每组9只。将5×10 5个刺激后的PBMC(60μl)注射到肿瘤组织中，剩余的PBMC停止刺激并继续培养，1周后将5×10 6个PBMC (100μl)腹腔注射到荷瘤小鼠体内，视为第1轮注射。整个实验周期，进行2轮半、共5次PBMC注射。首次瘤内注射当日开始腹腔给药，一周三次，共给药14次，给药方式见表6。每周2次测量瘤体积，称体重。实验结果见表7。实验结束后将荷瘤小鼠安乐死并剥瘤称重。
表6：试验分组及给药情况
组别 给药剂量
①空白对照：PBS 0
②融合蛋白9-4.8mpk 4.8mg/kg
③融合蛋白9-24mpk 24mg/kg
④PD-L1抗体-4mpk 4mg/kg
⑤PD-L1抗体-20mpk 20mg/kg
⑥PD-L1抗体-4mpk+对照1-2.14mpk 4mg/kg+2.14mg/kg
⑦对照1-2.14mpk 2.14mg/kg
表7：融合蛋白9对MDA-MB-231小鼠皮下移植瘤的疗效
Figure PCTCN2018086451-appb-000024
第0天：第一次给药时间；*p＜0.05 **p＜0.01 ***p＜0.001通过studentt检验与PBS对比。
实验结果如图8显示，抗体融合蛋白9(4.8、24mg/kg)能明显抑制人乳腺癌MDA-MB-231小鼠皮下移植瘤的生长，高低剂量间呈现剂量依赖关系，且优于各自等同摩尔剂量的参比药物PD-L1抗体(4、20mg/kg)、TGF-βRII对照分子20T-FC(2.14mg/kg)和联用组(PD-L1抗体-4mg/kg+20T-FC-2.14mg/kg)。各剂量的融合蛋白9从给药后14d开始，就一直保持理想的抑瘤效果，且其高剂量与PD-L1抗体-20mpk相比，优势非常明显(p＜0.05)；给药后25d，各抗体抑瘤效果最好，融合蛋白9和PDL-1抗体低、高剂量与联用组的抑瘤率分别为37.24％、52.38％、30.24％、28.01％、31.38％；给药后32天，融合蛋白9的抑瘤作用仍十分明显，低高剂量 组％TGI分别为36.68％和50.76％，且瘤体积与对照组相比都存在统计学差异(p＜0.05)。
测试例9：PD-L1/TGF-βtrap的物理稳定性
本测试例用于检测融合蛋白融合蛋白9和融合蛋白15的稳定性。
利用DSC(Differential scanning calorimetry，差示扫描量热法)检测不同抗体的热稳定性，比较了不同的缓冲体系下的稳定性情况，不同缓冲体系如10mM醋酸盐/135mM NaCl(pH 5.5)和10mM醋酸盐/9％海藻糖(pH 5.5)。
将样品置换到对应缓冲液中，控制样品浓度在50mg/ml左右，利用MicroCal*VP-Capillary DSC(Malvern)进行检测。检测前，将各个样品及空白缓冲液用真空脱气器脱气1～2min。样品板每个孔加入400μl样品或空白缓冲液(仪器上样量为300μl)。最后两对孔板分别加入14％Decon 90和ddH 2O，以备清洗用，样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率60℃/h。具体结果如下表8所示，在两个测试体系中融合蛋白9、融合蛋白15均表现了良好的热稳定性。
表8
Figure PCTCN2018086451-appb-000025
通过SEC-HPLC监测样品纯度考察一定浓度条件下周期性稳定性，示例性的条件比如将样品浓度控制在约50mg/ml，在10mM醋酸盐/135mM NaCl(pH 5.5)比较在比如-80℃反复冻融5次及40℃保存一个月的稳定性情况。利用Xbridge protein BEH SEC 200A(Waters)HPLC柱子检测。检测结果如下表9所示，两个融合蛋白均表现出良好的稳定性。
表9
  融合蛋白9(Δ％) 融合蛋白15(Δ％)
40℃ 3.39％ 1.8％
-80℃冻融 1.44％ 1.39％
注：Δ％表示变化率。
测试例10：融合蛋白的化学稳定性
脱酰胺修饰是抗体中可能影响后期稳定性的一种常见的化学修饰，尤其是CDR区域的部分氨基酸高度脱酰胺修饰一般选择尽量避免或者突变降低。取1600μg待测抗体溶于200μl 10mM醋酸盐/135mM NaCl(pH 5.5)中，40℃恒温箱存放；分别于0、14、28天取样，用于酶解实验。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl，8M Gμa-HCl，pH 6.0溶液中，加3μl 0.1g/mL DTT，50℃水浴1小时，后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μL 0.25mg/mL的胰蛋白酶，37℃水浴酶解过夜。Agilent 6530 Q-TOF进行LC-MS检测脱酰胺修饰情况，结果如下表10所示。
表10
Figure PCTCN2018086451-appb-000026
备注：N代表检测到修饰的天冬酰胺，数字代表所处轻链或者重链N端开始计数所处的位置。百分含量代表LC-MS检测到的脱酰胺修饰占该位点所处全部肽段信号的比例。
质谱检测结果显示两个融合蛋白都没有明显的脱酰胺修饰位点，提示其后期化学稳定性良好。

Claims (25)

  1. 一种含有TGF-β受体的融合蛋白，包含靶向部分和TGF-β受体部分，其中，所述的TGF-β受体部分为TGF-βRII胞外区的N端截短形式。
  2. 根据权利要求1所述的含有TGF-β受体的融合蛋白，其中所述的TGF-βRII胞外区的N端截短形式选自在TGF-βRII胞外区的N端上有26个以下连续氨基酸的缺失，优选14-26个连续氨基酸的缺失，更优选14-21个连续氨基酸的缺失，最优选14、19或21个连续氨基酸的缺失。
  3. 根据权利要求1或2所述的含有TGF-β受体的融合蛋白，其中所述的TGF-βRII胞外区的序列如SEQ ID NO：13所示；最优选包含SEQ ID NO：14、15或16所示的序列。
  4. 根据权利要求1-3中任一项所述的含有TGF-β受体的融合蛋白，其中所述的靶向部分为细胞特异性靶向部分；优选的，所述的靶向部分为癌细胞特异性靶向部分。
  5. 根据权利要求4所述的含有TGF-β受体的融合蛋白，其中所述的癌细胞特异性靶向部分选自抗体或其抗原结合片段、生长因子、激素、肽、受体或细胞因子。
  6. 根据权利要求5所述的含有TGF-β受体的融合蛋白，其中所述的抗体或其抗原结合片段选自全长抗体、嵌合抗体、Fab’、Fab、F(ab’) 2、单结构域抗体、Fv、scFv、小抗体、双特异性抗体、三特异性抗体或其混合物。
  7. 根据权利要求5或6所述的含有TGF-β受体的融合蛋白，其中所述的抗体或其抗原结合片段结合选自以下一种或多种多肽或蛋白：HER2、HER3、免疫检验点分子、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、CD11a、CD19、CD30、CD38、CD20、CD52、CD60、CD80、CD86、TNF-α、IL-12、IL-17、IL-23、IL-6、IL-1β、RSVF、IgE、RANK、BLyS、α4β7、PD-1、CCR4、SLAMF7、GD2、CD21、CD79b、IL20Rα、CD22、CD79a、CD72、IGF-1R和RANKL；优选所述的抗体或其抗原结合片段结合免疫检验点分子。
  8. 根据权利要求5-7中任一项所述的含有TGF-β受体的融合蛋白，其中所述的抗体为PD-L1抗体；优选的，所述的PD-L1抗体选自：MSB0010718C、MEDI4736、 BMS-936559、MPDL3280A的PD-L1抗体，或包含1个或多个选自以下的CDR区序列或CDR区序列的突变序列的抗体：
    如SEQ ID NO：1所示的HCDR1；
    如SEQ ID NO：2所示的HCDR2；
    如SEQ ID NO：3所示的HCDR3；
    如SEQ ID NO：4所示的LCDR1；
    如SEQ ID NO：5所示的LCDR2；
    如SEQ ID NO：6所示的LCDR3。
  9. 根据权利要求5-8中任一项所述的含有TGF-β受体的融合蛋白，其中所述的抗体或其抗原结合片段为嵌合抗体或其功能片段、人源化抗体或其功能片段、或人抗体或其功能片段。
  10. 根据权利要求9所述的含有TGF-β受体的融合蛋白，其中所述人源化抗体的重链可变区具有如SEQ ID NO：7所示的序列，优选具有如SEQ ID NO：9所示的序列。
  11. 根据权利要求9所述的含有TGF-β受体的融合蛋白，其中所述的人源化抗体进一步包括序列SEQ ID NO：11所示的重链。
  12. 根据权利要求9所述的含有TGF-β受体的融合蛋白，其中所述的人源化抗体包含SEQ ID NO：8或10所示的轻链可变区序列或轻链可变区的突变序列。
  13. 根据权利要求9所述的含有TGF-β受体的融合蛋白，其中所述的人源化抗体包含序列SEQ ID NO：12所示的轻链。
  14. 根据权利要求1-13中任一项所述的含有TGF-β受体的融合蛋白，其中所述的含有TGF-β受体的融合蛋白如通式(I)所示：
    Ab-L-TGF-βRII ECD(I)
    其中TGF-βRII ECD为TGF-βRII胞外区的截短形式；
    Ab为抗体；
    L为连接序列。
  15. 根据权利要求14所述的含有TGF-β受体的融合蛋白，其中所述的连接序列为(G 4S) xG，其中x为3-6，优选为4-5。
  16. 一种药物组合物，其含有治疗有效量的根据权利要求1至15中任一项所述的含有TGF-β受体的融合蛋白，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  17. 一种DNA分子，其编码根据权利要求1-15中任一项所述的含有TGF-β受体的融合蛋白。
  18. 一种表达载体，其含有根据权利要求17所述的DNA分子。
  19. 一种宿主细胞，其含有根据权利要求18所述的表达载体，所述宿主细胞选自细菌、酵母菌和哺乳动物细胞；优选哺乳动物细胞。
  20. 根据权利要求1至15中任一项所述的含有TGF-β受体的融合蛋白或根据权利要求16所述的药物组合物，在制备用于肿瘤治疗的药物中的用途；优选在制备用于治疗PD-L1介导的肿瘤的药物中的用途；更优选为表达PD-L1的癌症。
  21. 一种治疗和预防肿瘤的方法，包括给予所需患者治疗有效量的根据权利要求1至15中任一项所述的含有TGF-β受体的融合蛋白或根据权利要求16所述的药物组合物。
  22. 一种截短的TGF-βRII胞外区，其中所述的TGF-βRII胞外区的截短选自在SEQ ID NO：13的N端上有26个以下连续氨基酸的缺失，优选N端14-26个连续氨基酸的缺失，更优选N端14-21个连续氨基酸的缺失；最优选包含SEQ ID NO：14或SEQ ID NO：15所示的序列。
  23. 一种药物组合物，其含有治疗有效量的根据权利要求22所述的截短的TGF-βRII胞外区，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  24. 根据权利要求22所述的截短的TGF-βRII胞外区或根据权利要求23所述的药物组合物，在制备用于治疗、抑制癌细胞增殖或转移的疾病或病症的药物中的用途。
  25. 一种治疗和预防肿瘤的方法，包括给予所需患者治疗有效量的根据权利要求22所述的截短的TGF-βRII胞外区或根据权利要求23所述的药物组合物。
PCT/CN2018/086451 2017-05-12 2018-05-11 含有TGF-β受体的融合蛋白及其医药用途 WO2018205985A1 (zh)

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