WO2017198212A1 - 针对ctla4的单域抗体及其衍生蛋白 - Google Patents

针对ctla4的单域抗体及其衍生蛋白 Download PDF

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WO2017198212A1
WO2017198212A1 PCT/CN2017/085038 CN2017085038W WO2017198212A1 WO 2017198212 A1 WO2017198212 A1 WO 2017198212A1 CN 2017085038 W CN2017085038 W CN 2017085038W WO 2017198212 A1 WO2017198212 A1 WO 2017198212A1
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seq
ctla4
antibody
binding protein
cdr3
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PCT/CN2017/085038
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English (en)
French (fr)
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徐霆
汪皛皛
李洁
吴海燕
高丽
储倩
白玉
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苏州康宁杰瑞生物科技有限公司
张喜田
张欣
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Application filed by 苏州康宁杰瑞生物科技有限公司, 张喜田, 张欣 filed Critical 苏州康宁杰瑞生物科技有限公司
Priority to JP2019513103A priority Critical patent/JP2019522489A/ja
Priority to EP17798775.7A priority patent/EP3459597B1/en
Priority to FIEP17798775.7T priority patent/FI3459597T3/fi
Priority to US16/302,583 priority patent/US11091549B2/en
Priority to CN201780030832.XA priority patent/CN109195665B/zh
Priority to ES17798775T priority patent/ES2931223T3/es
Priority to DK17798775.7T priority patent/DK3459597T3/da
Publication of WO2017198212A1 publication Critical patent/WO2017198212A1/zh
Priority to US17/369,169 priority patent/US11912768B2/en

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Definitions

  • the present invention relates to the field of medical biology, and discloses a single domain antibody against CTLA4 and a derivative thereof.
  • the present invention discloses a CTLA4 binding protein and its use, particularly in the treatment and/or prevention of CTLA4-related diseases such as tumors.
  • Camelidae such as camels or alpacas, are capable of producing a naturally-deficient light chain heavy chain antibody containing only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but are intact. Antigen binding function, and not as easy to aggregate as artificially engineered single-chain antibody fragments (scFv). More importantly, the recombinantly expressed VHH domain has structural stability and antigen binding activity comparable to that of the original heavy chain antibody, and is the smallest known unit that can bind to the target antigen, and is called Nanoboy. Or a heavy chain single domain antibody. Due to its special structural properties, heavy chain single domain antibodies have the advantages of traditional antibodies and small molecule drugs, and overcome the defects of long development cycle, low stability and harsh storage conditions of traditional antibodies, representing a new generation of antibody therapy. The direction of development.
  • Tumor-associated antigens expressed by tumor cells are the basis for generating an effective immune response.
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • co-stimulatory signals are also required to promote activation of effector T cells.
  • Studies have shown that many tumors can escape the patient's own immune system, in part because of the lack of costimulatory signals to fully activate T cells, and most likely due to immunosuppression induced by regulatory T cells (Treg). Binding of CD80 or CD86 on antigen-presenting cells to CD28 on T cells is a key costimulatory signal.
  • CTLA4 Human cytotoxic T lymphocyte-associated antigen 4
  • CD80 and CD86 Human cytotoxic T lymphocyte-associated antigen 4
  • the immunosuppressive effect of CTLA4 plays an important role in limiting the autoimmune response.
  • CTLA4-mediated inhibition mechanism is often one of the reasons why tumor cells escape the immune system.
  • T cell mediated anti-tumor responses can be enhanced by blocking the interaction of CTLA4 with CD80 or CD86.
  • the field needs to be able to bind with high affinity to CTLA4 and be able to block CTLA4 and CD80-conjugated anti-CTLA4 antibody, particularly anti-CTLA4 heavy chain single domain antibody.
  • the inventors of the present invention obtained an anti-CTLA4 heavy chain single domain antibody (VHH) having high specificity, high affinity and high stability by screening using phage display technology.
  • VHH anti-CTLA4 heavy chain single domain antibody
  • the invention provides a CTLA4 binding protein comprising an immunoglobulin single variable domain that specifically binds to CTLA4.
  • the invention features a nucleic acid molecule encoding a CTLA4 binding protein, and an expression vector and host cell comprising the nucleic acid molecule.
  • the invention also relates to a pharmaceutical composition comprising a CTLA4 binding protein of the invention.
  • the invention also relates to methods of making the CTLA4 binding proteins described herein.
  • the invention further relates to the use of the CTLA4 binding proteins of the invention and to pharmaceutical compositions, in particular for the prevention and/or treatment of diseases associated with CTLA4.
  • Figure 1 Shows the binding curve of CTLA4 heavy chain single domain antibody to CTLA4-Fc antigen protein.
  • Figure 4 shows the results of sequence alignment of five humanized variants (A) of antibody strain C27 and four humanized variants (B) of antibody strain C1.
  • Figure 5 Shows the binding curve of CTLA4 single domain antibody Fc fusion protein to CTLA4 (ELISA method).
  • Figure 6 Shows the binding curve of CTLA4 single domain antibody Fc fusion protein to CTLA4 (ELISA method).
  • FIG. 7 Blocking curve of CTLA4 single domain antibody Fc fusion protein for CTLA4-CD80 interaction (competitive ELISA method).
  • Figure 8 Shows the binding curve of the tetravalent CTLA4 single domain antibody Fc fusion protein to CTLA4 (competitive ELISA method).
  • Figure 9 shows the bivalent and tetravalent CTLA4 single domain antibody Fc fusion protein against CTLA4/CD80 Blocking ability of interactions (cell neutralization experiments).
  • FIG. 10 Flow cytometry detects the specificity of CTLA4 single domain antibody Fc fusion protein for CTLA4 protein binding.
  • FIG. 11 Binding of the tetravalent CTLA4 single domain antibody Fc fusion protein to the monkey CTLA4 protein is shown.
  • Figure 12 Shows the activation of PBMC by the CTLA4 single domain antibody Fc fusion protein.
  • Figure 14 Shows the activation of CD4+ T cells by the CTLA4 single domain antibody Fc fusion protein.
  • Figure 16 Graph showing the plasma concentration of divalent and tetravalent CTLA4 single domain antibody Fc fusion proteins in rats versus time.
  • Figure 17 Pharmacokinetic parameters of CTLA4 single domain antibody Fc fusion protein in rat.
  • Figure 18 Shows the thermal stability of the CTLA4 single domain antibody Fc fusion protein.
  • antibody or “immunoglobulin” as used interchangeably herein, unless otherwise indicated, are used to refer to either a heavy chain antibody or a conventional 4-chain antibody, as a general term to include full length antibodies, individually A strand and all portions, domains or fragments thereof (including but not limited to antigen binding domains or fragments, eg, a VHH domain or a VH/VL domain, respectively).
  • sequence as used (eg, in terms of "immunoglobulin sequence", “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) is generally understood to include both Related amino acid sequences, in turn, include nucleic acid sequences or nucleotide sequences encoding the sequences, unless a more limited interpretation is required herein.
  • domain refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for the individual functional properties of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the rest of the protein and/or domain.
  • immunoglobulin domain refers to a spherical region of an antibody chain (eg, a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a spherical region.
  • An immunoglobulin domain is characterized in that it maintains an immunoglobulin folding characteristic of an antibody molecule consisting of two intercalations of about seven antiparallel beta-sheet strands, optionally arranged by a conserved disulfide bond, in two beta sheets. .
  • immunoglobulin variable domain refers to substantially referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3”, respectively.
  • An immunoglobulin domain consisting of "FR3”, and four “framework regions” of "framework region 4" or “FR4", wherein the framework regions are referred to in the art and hereinafter as “complementarity determining region 1", respectively.
  • the three “complementarity determining regions” or “CDRs” of "CDR1", “complementarity determining region 2" or “CDR2", and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the immunoglobulin variable domain confers specificity to the antigen for the antibody by having an antigen binding site.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding an antigenic epitope without pairing with other immunoglobulin variable domains.
  • An example of an immunoglobulin single variable domain within the meaning of the invention is a "domain antibody”, such as an immunoglobulin single variable domain VH and VL (VH domain and VL domain).
  • Another example of an immunoglobulin single variable domain is the "VHH domain” of Camelidae (or simply "VHH") as defined below.
  • VHH domain also known as heavy chain single domain antibody, VHH, VH H domain, VHH antibody fragment and VHH antibody, is an antigen called “heavy chain antibody” (ie “antibody lacking light chain”) Binding to the variable domains of immunoglobulins (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally transition antibodies devoid of light chains”; Nature 363,446 -448 (1993)).
  • the term “VHH domain” is used to bind the variable domain to a heavy chain variable domain (referred to herein as a "VH domain”) present in a conventional 4-chain antibody and to a conventional 4-chain antibody.
  • the light chain variable domain (which is referred to herein as the "VL domain") is distinguished.
  • the VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • -FR1 contains amino acid residues at positions 1-30,
  • - CDR1 contains amino acid residues at positions 31-35
  • -FR2 contains amino acids at positions 36-49,
  • - CDR2 contains amino acid residues at positions 50-65
  • -FR3 contains amino acid residues at positions 66-94
  • - CDR3 comprises amino acid residues at positions 95-102, and
  • - FR4 contains the amino acid residues at positions 103-113.
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (ie, according to One or more positions of the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more than the number of amino acid residues allowed by the Kabat number).
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • the total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domain structural and functional properties of the VHH domain and the polypeptides containing the same can be summarized as follows:
  • VHH domain which has been naturally "designed" to bind to antigen functionality in the absence of a light chain variable domain and without interaction with a light chain variable domain, can be used as a single and relatively small function
  • a sex antigen binds to a structural unit, domain or polypeptide. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies.
  • VH and VL domains are not normally suitable for practical application as single antigen-binding proteins or immunoglobulin single variable domains, but need to be
  • a functional antigen binding unit such as in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain).
  • VHH domains - either alone or as part of a larger polypeptide - provides many advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (eg Fab- or F(ab')2-fragments) Significant advantages:
  • VHH domain can be expressed from a single gene and does not require post-translational folding or modification
  • VHH domain can be easily engineered into a multivalent and multispecific format (formatted);
  • VHH domain is highly soluble and has no aggregation tendency
  • VHH domain is highly stable to heat, pH, proteases and other denaturing agents or conditions, and thus can be used without refrigeration equipment during preparation, storage or transportation, thereby achieving cost savings, time and environment;
  • VHH domain is easy to prepare and relatively inexpensive, even on the scale required for production;
  • -VHH domain is relatively small compared to conventional 4-chain antibodies and their antigen-binding fragments (large About 15kDa or about 1/10 of the size of conventional IgG, thus showing higher tissue permeability and higher doses than conventional 4-chain antibodies and antigen-binding fragments thereof;
  • the -VHH domain can display so-called cavity binding properties (especially due to its extended CDR3 loop compared to conventional VH domains), such that targets and epitopes that are unreachable to conventional 4-chain antibodies and antigen-binding fragments thereof can be reached.
  • the VHH domain derived from the camelid family can be substituted for one or more amino acid residues in the amino acid sequence of the original VHH sequence by one or more amino acid residues present at corresponding positions in the VH domain of the human conventional 4-chain antibody.
  • Humanization also referred to herein as “sequence optimization", in addition to humanization, “sequence optimization” may also encompass other modifications to the sequence by one or more mutations that provide improved properties of VHH, such as removal Potential post-translational modification sites).
  • the humanized VHH domain may contain one or more fully human framework region sequences, and in a particular embodiment, may comprise a human framework region sequence of IGHV3.
  • domain antibody As used herein, the term "domain antibody” (also referred to as “Dab” and “dAb”) is particularly used to refer to the VH or VL domain of an antibody (particularly a human 4-chain antibody) of a non-Camelid mammal.
  • dAb domain antibody
  • the domain antibody has a molecular weight of from about 13 kDa to about 16 kDa, and if derived from a fully human sequence, no humanization is required for therapeutic use, for example, in humans.
  • domain antibodies are also well expressed in prokaryotic expression systems, thereby significantly reducing overall manufacturing costs.
  • Domain antibodies are disclosed, for example, in Ward, ES, et al.: "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli"; Nature 341: 544-546 (1989); Holt, LJ Et al.: “Domain antibodies: proteins for therapy”; TRENDS in Biotechnology 21 (11): 484-490 (2003).
  • epitope or the term “antigenic determinant” as used interchangeably refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • An antigenic determinant typically comprises a chemically active surface group of a molecule, such as an amino acid or a sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "epitope” or “conformation” epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • a linear epitope can be determined, for example, by simultaneously synthesizing a large number of peptides on a solid support, wherein the peptides correspond to portions of the protein molecule and such that the peptide is still attached to the support Antibody response.
  • These techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci.
  • conformational epitopes can be identified by determining the spatial configuration of the amino acids, such as by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (ibid.).
  • Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete with antigens. High throughput methods for obtaining antibodies that bind to the same epitope based on their cross-competition are described in International Patent Application WO 03/48731. Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the invention for binding to the same epitope on CTLA4 can be obtained using conventional techniques known to those skilled in the art.
  • the term “specificity” refers to the number of different types of antigens or epitopes to which a particular antigen binding molecule or antigen binding protein (eg, an immunoglobulin single variable domain of the invention) can bind.
  • the specificity can be determined based on the affinity and/or affinity of the antigen binding protein.
  • Antigen and antibiotic The affinity expressed by the dissociation equilibrium constant (KD) of the original binding protein is a measure of the binding strength between the epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the binding between the epitope and the antigen binding protein The stronger the intensity (or the affinity can also be expressed as the association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner, depending on the particular antigen of interest.
  • Affinity is a measure of the strength of binding between an antigen binding protein (eg, an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide comprising the same) and a related antigen. Affinity is related to both the affinity between the antigen binding site on its antigen binding protein and the number of related binding sites present on the antigen binding protein.
  • CTLA4 binding protein means any protein capable of specifically binding to CTLA4.
  • the CTLA4 binding protein can include an antibody or a conjugate thereof as defined herein for CTLA4.
  • CTLA4 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR graft molecules.
  • the "CTLA4 binding protein” of the present invention may comprise at least one immunoglobulin single variable domain such as VHH that binds to CTLA4.
  • a "CTLA4 binding molecule” of the invention may comprise 2, 3, 4 or more immunoglobulin single variable domains, such as VHH, that bind to CTLA4.
  • the CTLA4 binding protein of the present invention may comprise, in addition to the immunoglobulin single variable domain of CTLA4, a linker and/or a portion having an effector function, such as a half-life extending moiety (eg, an immunoglobulin single variable structure that binds serum albumin).
  • a "CTLA4 binding protein" of the invention also encompasses a bispecific antibody comprising an immunoglobulin single variable domain that binds to a different antigen.
  • the CTLA4 binding protein of the invention will preferably be 10 -7 to 10 -10 moles per liter (M), more preferably 10 -8 to 10 -10 moles per liter, as measured in a Biacore or KinExA or Fortibio assay, or even More preferably, a dissociation constant (KD) of 10 -9 to 10 -10 or lower, and/or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , more preferably An association constant (KA) of at least 10 10 M -1 binds to the antigen to be bound (ie CTLA4). Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays described herein (eg, Radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays.
  • SPR surface plasmon resonance
  • RIA Radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competitive assays sandwich competitive assays.
  • amino acid residues will be based on standard three-letter or one-word as is well known and agreed upon in the art. The parent amino acid code is indicated.
  • amino acid difference refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence.
  • substitution will preferably be a conservative amino acid substitution, which means that the amino acid residue is replaced by another amino acid residue of similar chemical structure and its effect on the function, activity or other biological properties of the polypeptide. Smaller or substantially unaffected.
  • conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably such that one amino acid within groups (i)-(v) is substituted with another amino acid residue within the same group: (i) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) Polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) Polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is replaced by Ala Or Pro substituted; His is replaced by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Substituent; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is replaced by Trp or Phe; Val is substituted by Ile or Leu.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are the same or represent conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
  • sequence identity For the determination of sequence identity, see, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York , 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • a polypeptide or nucleic acid molecule is considered “substantially separated” when the other component (eg another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component) is separated ".
  • a polypeptide or nucleic acid molecule is considered “substantially isolated” when it has been purified at least 2 fold, in particular at least 10 fold, more particularly at least 100 fold and up to 1000 fold or more.
  • the "substantially isolated” polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by suitable techniques, such as suitable for chromatographic techniques, such as polyacrylamide gel electrophoresis.
  • An "affinity matured" anti-CTLA4 antibody particularly a VHH or domain antibody, has one or more changes in one or more CDRs that result in an affinity for CTLA4 compared to its respective parental anti-CTLA4 antibody. Increased.
  • Affinity matured anti-CTLA4 antibodies can be prepared, for example, by methods known in the art as described below: Marks et al, 1992, Biotechnology 10: 779-783 or Barbas et al, 1994, Proc. Nat. Acad. Sci , USA 91: 3809-3813.; Shier et al, 1995, Gene 169: 147-155; Yelton et al, 1995, Immunol. 155: 1994-2004; Jackson et al, 1995, J. Immunol.
  • subject means a mammal, especially a primate, especially a human.
  • the invention provides a CTLA4 binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding to CTLA4.
  • the CTLA4 binding protein comprises an immunoglobulin single variable domain that specifically binds to CTLA4.
  • the CTLA4 binding protein comprises 2, 3, 4 or more immunoglobulin single variable domains that specifically bind to CTLA4.
  • the CTLA4 binding protein comprises two or more identical immunoglobulin single variable domains that specifically bind to CTLA4.
  • the CTLA4 binding protein comprises two or more different immunoglobulin single variable domains that specifically bind to CTLA4.
  • the two or more immunoglobulin single variable domains that specifically bind to CTLA4 are directly linked to each other.
  • the two or more immunoglobulin single variable domains that specifically bind to CTLA4 They are connected to each other by a joint.
  • the linker may be a non-functional amino acid sequence having a length of 1-20 or more amino acids and no secondary structure or higher.
  • the linker is a flexible linker such as GGGGS, GS, GAP, (GGGGS) x 3, and the like.
  • the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 selected from the group consisting of:
  • CDR1 represented by SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, CDR3 of SEQ ID NO: 3 (corresponding to the CDR of antibody strain 116);
  • CDR1 represented by SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, CDR3 of SEQ ID NO: 9 (corresponding to the CDR of antibody strain 128);
  • CDR1 represented by SEQ ID NO: 13, CDR2 of SEQ ID NO: 14, CDR3 of SEQ ID NO: 15 (corresponding to the CDR of antibody strain 145);
  • CDR1 represented by SEQ ID NO: 16, CDR2 of SEQ ID NO: 17, CDR3 of SEQ ID NO: 18 (corresponding to the CDR of antibody strain 155);
  • CDR1 represented by SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, CDR3 of SEQ ID NO: 27 (corresponding to the CDR of antibody strain C1);
  • CDR1 represented by SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, CDR3 of SEQ ID NO: 33 (corresponding to the CDR of antibody strain C16);
  • CDR1 represented by SEQ ID NO: 43, CDR2 of SEQ ID NO: 44, CDR3 of SEQ ID NO: 45 (corresponding to the CDR of antibody strain C38);
  • CDR1 represented by SEQ ID NO: 46, CDR2 of SEQ ID NO: 47, CDR3 of SEQ ID NO: 48 (corresponding to the CDR of antibody strain J5);
  • CDR1 represented by SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, CDR3 of SEQ ID NO: 60 (corresponding to the CDR of antibody strain J37);
  • CDR1 represented by SEQ ID NO: 61, CDR2 of SEQ ID NO: 62, CDR3 of SEQ ID NO: 63 (corresponding to the CDR of antibody strain J38);
  • At least one immunoglobulin single variable domain of a CTLA4 binding protein of the invention is VHH.
  • the VHH comprises the amino acid sequence of any one of SEQ ID NOs: 76-100.
  • the VHH is a humanized VHH.
  • the humanized VHH comprises an amino acid having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to any of SEQ ID NOs: 76-100 sequence.
  • the amino acid sequence of the VHH comprises one or more amino acid substitutions, preferably one of SEQ ID NOs: 76-100. Conservative amino acid substitutions. For example, it contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.
  • the humanized VHH comprises the amino acid sequence of any one of SEQ ID NOs: 101-109.
  • the CTLA4 binding proteins of the invention are obtained by affinity maturation.
  • the affinity matured CTLA4 binding protein may have one or more changes in one or more CDRs that result in an increase in affinity for CTLA4 compared to the parental CTLA4 binding protein.
  • a CTLA4 binding protein of the invention comprises an immunoglobulin Fc region in addition to at least one immunoglobulin single variable domain capable of specifically binding to CTLA4.
  • the inclusion of an immunoglobulin Fc region in a CTLA4 binding protein of the invention allows the binding protein to form a dimeric molecule while extending the in vivo half-life of the binding protein.
  • the Fc regions useful in the present invention may be derived from immunoglobulins of different subtypes, for example, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM.
  • the immunoglobulin Fc region generally includes a hinge region or a portion of the hinge region, the CH2 region, and the CH3 region of the immunoglobulin constant region.
  • a mutation can be introduced on the wild type Fc sequence to alter Fc mediated related activity.
  • Such mutations include, but are not limited to, a) mutations that alter Fc-mediated CDC activity; b) mutations that alter Fc-mediated ADCC activity; or c) mutations that alter FcRn-mediated in vivo half-life.
  • Such mutations are described in Leonard G Presta, Current Opinion in Immunology 2008, 20: 460-470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al. , Nature Medicine, 2000, Volume 6, Number 4: 443-446; Paul R.
  • mutating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on the CH2 region for increasing or removing Fc-mediated ADCC or CDC activity or enhancing or attenuating FcRn Affinity.
  • protein stability can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the hinge region.
  • a mutation can be introduced on the Fc sequence such that the mutated Fc more readily forms a homodimer or a heterodimer.
  • the knob-hole model which uses the vacant interface amino acid side chain group space as mentioned in Ridgway, Presta et al. 1996 and Carter 2001, makes it easier to form heterodimers between different Fc mutations; 102558355A or CN 103388013A, by changing the Fc contact interface amino acid
  • the charge carried which in turn changes the ionic interaction between the Fc contact interfaces, makes it easier to form heterodimers between different Fc mutant pairs (CN 102558355A), or between Fcs with the same mutations. Formation of homodimer (CN 103388013A).
  • the immunoglobulin Fc region is preferably a human immunoglobulin Fc region, more preferably an Fc region of human IgG1.
  • the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO:132.
  • the N-terminal EPKSC of SEQ ID NO: 132 can be deleted or mutated to EPKSS or MDPKSS.
  • the immunoglobulin single variable domain capable of specifically binding to CTLA4 is linked to the immunoglobulin Fc region via a linker.
  • the linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length, without secondary structure or above.
  • the linker is a flexible joint such as GGGGS, GS, GAP, and the like.
  • a CTLA4 binding protein of the invention comprises an immunoglobulin single variable domain that specifically binds to CTLA4, which is linked directly or via a linker to an immunoglobulin Fc region, said immunoglobulin Fc region permitting The CTLA4 binding protein forms a dimeric molecule comprising two CTLA4 binding domains.
  • a CTLA4 binding protein is also referred to as a bivalent CTLA4 binding protein.
  • the dimer is a homodimer.
  • a CTLA4 binding protein of the invention comprises two immunoglobulin single variable domains and an immunoglobulin Fc region that specifically bind to CTLA4, directly or through a linker, said immunoglobulin Fc region
  • the CTLA4 binding protein is allowed to form a dimeric molecule comprising four CTLA4 binding domains.
  • Such a CTLA4 binding protein is also referred to as a tetravalent CTLA4 binding protein.
  • the dimer is a homodimer.
  • the CTLA4 binding protein of the invention comprising an immunoglobulin Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 114-128.
  • the CTLA4 binding protein of the invention further encompasses an anti-CTLA4 antibody molecule capable of binding to the same epitope on CTLA4 as VHH consisting of the amino acid sequence of any one of SEQ ID NOs: 76-100.
  • the CTLA4 binding protein of the invention has at least one of the following characteristics:
  • the KLA value of the CTLA4 binding protein binding CTLA4 of the present invention may be less than 1 x 10 -7 M, preferably less than 1 x 10 -8 M, more preferably less than 1 x 10 -9 M, more preferably less than 1 x 10 -10 M .
  • a CTLA4 binding protein of the invention is capable of specifically binding to human CTLA4 and blocking the interaction of CTLA4 and CD80, and/or CTLA4 and CD86.
  • the CTLA4 binding protein of the invention is capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more Preferably at least about 70%, more preferably at least about 80%.
  • CTLA4 binding protein of the present invention is resistant to heat treatment. For example, no significant aggregation or degradation was observed after treatment at 40 ° C for up to 30 days.
  • CTLA4 binding protein of the invention showed better tolerance in cynomolgus monkeys. For example, up to 30 mg/kg of the administered dose, no drug-related adverse reactions were observed.
  • the invention features a nucleic acid molecule encoding a CTLA4 binding protein of the invention.
  • the nucleic acid of the invention may be RNA, DNA or cDNA.
  • the nucleic acid of the invention is a substantially isolated nucleic acid.
  • the nucleic acids of the invention may also be in the form of a vector, which may be present in the vector and/or may be part of a vector such as a plasmid, a co-end plasmid or YAC.
  • the vector may especially be an expression vector, i.e., a vector that provides expression of the CTLA4 binding protein in vitro and/or in vivo (i.e., in a suitable host cell, host organism, and/or expression system).
  • the expression vector typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). Selection of the elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Specific examples of regulatory elements and other elements useful or essential for the expression of the CTLA4 binding protein of the invention, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.
  • the nucleic acids of the invention may be prepared or obtained in a known manner (for example by automated DNA synthesis and/or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptides of the invention presented herein, and/or may be from a suitable natural Sources are separated.
  • the invention relates to the expression or ability to express one or more of the inventions A CTLA4 binding protein and/or a recombinant host cell comprising a nucleic acid or vector of the invention.
  • Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include Gram-negative bacterial strains (eg, Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (eg, Bacillus) A cell of a genus Bacillus, a strain of Streptomyces, a strain of Staphylococcus, and a strain of Lactococcus.
  • Gram-negative bacterial strains eg, Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains eg, Bacillus
  • Bacillus A cell of a genus Bacillus, a strain of Streptomyces, a strain of Staphylococcus, and a strain of Lactococcus.
  • Suitable fungal cells include cells of the genus Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (eg, Saccharomyces cerevisiae), fission Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Han A cell of the species of the genus Hansenula.
  • Saccharomyces eg, Saccharomyces cerevisiae
  • fission Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cell in the art for expressing a heterologous protein can also be used in the present invention.
  • the invention also provides a method of producing a CTLA4 binding protein of the invention, the method generally comprising the steps of:
  • CTLA4 binding protein of the invention optionally further purification and / or modification of the CTLA4 binding protein of the invention.
  • the CTLA4 binding protein of the invention can be produced in an intracellular manner (eg, in the cytoplasm, in the periplasm, or in inclusion bodies) in a cell as described above, followed by isolation from the host cell and optionally further purification; or it can be extracellularly (for example in culture medium for culturing host cells), followed by isolation from the medium and optionally further purification.
  • CTLA4 binding proteins of the invention can also be obtained by other methods of protein production known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.
  • the invention provides a composition, such as a pharmaceutical composition, comprising a CTLA4 binding protein of the invention in one or a combination formulated with a pharmaceutically acceptable carrier.
  • a composition may comprise one or a combination (e.g., two or more different) CTLA4 binding proteins of the invention.
  • a pharmaceutical composition of the invention may contain a combination of antibody molecules that bind to different epitopes on a target antigen.
  • compositions of the invention may also be administered in combination therapy, i.e., in combination with other agents.
  • a combination therapy can include a CTLA4 binding protein of the invention in combination with at least one other anti-tumor drug.
  • a CTLA4 binding protein of the invention can be used in combination with an antibody that targets other tumor-specific antigens.
  • Such antibodies that target other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, antibodies against EGFR variants, anti-VEGFa antibodies, anti-HER2 antibodies, or anti-CMET antibodies.
  • the antibody is a monoclonal antibody.
  • the CTLA4 binding protein of the present invention can also be used in combination with other tumor immunotherapy or tumor targeting small molecule drugs.
  • the other tumor immunotherapy means include, but are not limited to, therapeutic antibodies against tumor immunomodulatory molecules such as OX40, PDL1/PD1, CD137, etc., or CAR-T treatment means and the like.
  • the pharmaceutical composition of the present invention can also be used in combination with other tumor treatment means such as radiotherapy, chemotherapy, surgery, or the like before or after radiotherapy, chemotherapy, or surgery.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., the antibody molecule, immunoconjugate
  • the active compound can be encapsulated in a material to protect the compound from acids and other natural conditions that can inactivate the compound.
  • compositions of the invention may comprise one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound without causing any undesirable toxicological effects (see, eg, Berge, SM et al. (1977) J. Pharm. Sci. 66:1). -19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those by Non-toxic inorganic acid-derived salts such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and alkanoic acids, such as aliphatic monocarboxylic and dicarboxylic acids, phenyl substituted alkanoic acids
  • Non-toxic organic acid-derived salts such as alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids.
  • Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, and such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine
  • alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like
  • Non-toxic organic amine derived salts such as choline, diethanolamine, ethylenediamine, procaine.
  • compositions of the invention may also contain a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA) ), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • the prevention of the presence of microorganisms can be ensured by a sterilization procedure or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like.
  • various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like.
  • isotonic agents for example, sugars, polyols such as mannitol, sorbitol or sodium oxide.
  • Prolonged absorption of the injectable drug can be achieved by the addition of a delay absorbent such as monostearate and gelatin to the composition.
  • the pharmaceutically acceptable carrier includes a sterile aqueous solution or dispersion and a powder for the temporary preparation of a sterile injectable solution or dispersion.
  • a sterile aqueous solution or dispersion and a powder for the temporary preparation of a sterile injectable solution or dispersion.
  • Conventional media or agents may be in the pharmaceutical compositions of the present invention, except in the range which is incompatible with the active compound.
  • Supplementary active compounds can also be incorporated into the compositions.
  • compositions must generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations.
  • the carrier can be a solvent or dispersing agent containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • a sterile injection can be prepared by mixing the active compound in the required amount in a suitable solvent, and adding one or a combination of the above-listed ingredients as needed, followed by sterile microfiltration. Cumshot.
  • dispersing agents are prepared by incorporating the active compound into a sterile vehicle which may contain a base dispersion medium and the other ingredients listed above.
  • the preferred preparation methods are vacuum drying and lyophilization (lyophilization), a solution which is pre-sterilized in such a manner as a powder of the active ingredient plus any additional desired ingredients.
  • the amount of active ingredient that can be combined with the carrier materials in a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient that can be combined with the carrier materials in a single dosage form is generally the amount of the composition that produces the therapeutic effect. Typically, this amount will range from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, by weight of the active ingredient, and pharmaceutically acceptable The carrier is combined.
  • the dosage regimen can be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be reduced or increased proportionally as needed for an emergency condition of the treatment condition. It is especially advantageous to formulate the parenteral compositions in dosage unit form for ease of administration and uniformity.
  • Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound which is calculated to produce the active compound in association with the required pharmaceutical carrier. The desired therapeutic effect.
  • the specific description of the dosage unit form of the present invention is limited to and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) inherent in the art for formulating such sensitivity for treating an individual. Limitation of active compounds.
  • the dosage range is from about 0.0001 to 100 mg/kg, more typically from 0.01 to 20 mg/kg of the recipient's body weight.
  • the dose may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight, 20 mg/kg body weight or 30 mg/kg body weight, or in the range of 1-30 mg/kg. .
  • An exemplary treatment regimen requires weekly dosing, biweekly, biweekly, biweekly, monthly, once every 3 months, every 3-6 months, or initial dosing interval Slightly shorter (eg once a week to once every three weeks) the post-dosing interval is lengthened (eg once a month to once every 3-6 months).
  • the antibody molecule can also be administered as a sustained release formulation, in which case less frequent dosing is required.
  • the dose and frequency will vary depending on the half-life of the antibody molecule in the patient.
  • human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies.
  • the dosage and frequency of administration vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over long periods of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, the patient can be administered in a prophylactic regimen.
  • the actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to achieve an amount of active ingredient which is effective to achieve the desired therapeutic response to a particular patient, composition and mode of administration without toxicity to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, and the therapeutic Duration, other drugs, compounds and/or materials used in conjunction with the particular composition of the application, age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.
  • the "therapeutically effective amount" of the CTLA4 binding protein of the present invention preferably results in a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic phase of the disease, or prevention of damage or disability caused by the pain of the disease.
  • the "therapeutically effective amount” preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably, compared to a subject who is not treated.
  • the ability to inhibit tumor growth can be evaluated in animal model systems that predict the efficacy of human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art.
  • a therapeutically effective amount of a therapeutic compound can reduce tumor size, or otherwise alleviate the symptoms of the subject, such as achieving or prolonging progression free survival of the tumor patient and prolonging the overall survival of the tumor patient.
  • One skilled in the art can determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • one skilled in the art can also determine the effective amount of a CTLA4 binding protein of the invention by examining the ability to activate T cells in vitro.
  • compositions of the invention may be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will appreciate that the route and/or manner of administration will vary depending on the desired result.
  • Preferred routes of administration of the CTLA4 binding proteins of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example Injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually injections, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.
  • CTLA4 binding proteins of the invention may also be administered by parenteral routes, such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • parenteral routes such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compound can be prepared with carriers which are not fast-released, such as a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microcapsule delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • compositions can be administered using medical devices well known in the art.
  • the therapeutic compositions of the present invention can be administered by a needleless hypodermic injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • a needleless hypodermic injection device such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • Examples of well-known implants and modules that can be used in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispersing a drug at a controlled rate; U.S. Patent No. 4,486,194, A medicinal infusion pump for delivering a drug at a precise infusion rate is disclosed in U.S.
  • Patent No. 4,447, 233 which is incorporated herein by reference.
  • a flow-injectable infusion device for the continuous delivery of a drug U.S. Patent No. 4, 439, 196, which discloses an osmotic drug delivery system having a multi-chamber compartment: and U.S. Patent No. 4,475,196, which discloses an osmotic drug Delivery system.
  • U.S. Patent No. 4,475,196 which discloses an osmotic drug Delivery system.
  • the CTLA4 binding proteins of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) blocks many highly hydrophilic compounds.
  • the therapeutic compounds of the invention are able to cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes As for the method of preparing liposomes, see, for example, US patents 4,522,811; 5,374,548 and 5,399,331.
  • Liposomes comprise one or more targeting moieties that can be selectively transported into a particular cell or organ to enhance targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685 ).
  • targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannoside (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PGBloeman et al. 1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); Surfactant Protein A Receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); P120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.
  • the invention provides the use and method of a CTLA4 binding protein, nucleic acid molecule, host cell, and pharmaceutical composition of the invention in the prevention and/or treatment of a disease associated with CTLA4.
  • CTLA4-related diseases which can be prevented and/or treated with the CTLA4 binding protein of the present invention are described in detail below.
  • Blockade of CTLA4 by the CTLA4 binding protein of the invention enhances the immune response to cancer cells in a patient.
  • the CTLA4 binding protein of the present invention can be used alone to inhibit the growth of cancerous tumors.
  • the CTLA4 binding proteins of the invention can be used in combination with other anti-tumor therapies, for example, in combination with other immunogenic agents, standard cancer therapies, or other antibody molecules.
  • the invention provides a method of preventing and/or treating cancer comprising administering to the subject a therapeutically effective amount of a CTLA4 binding protein of the invention, inhibiting tumor cell growth in a subject.
  • Preferred cancers that can be prevented and/or treated using the CTLA4 binding proteins of the invention include cancers that are generally responsive to immunotherapy.
  • Non-limiting examples of preferred cancers that can be treated include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (eg, metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant blood.
  • the party of the invention can be used Examples of other cancers treated by the law include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, Vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia , including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumor of children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system Systemic (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumor
  • the CTLA4 binding protein of the invention can be used in conjunction with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells transfected with a gene encoding an immunostimulatory cytokine.
  • immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells transfected with a gene encoding an immunostimulatory cytokine.
  • immunogenic agents include peptides of melanoma antigens, such as peptides of gp100, MAGE antigen, Trp-2, MART1 and/or tyrosinase, or cytokine GM- after transfection. Tumor cells of CSF.
  • CTLA4 blockers such as anti-CTLA4 antibodies, such as the CTLA4 binding proteins of the invention
  • CTLA4 blockers may be most effective when combined with a tumor vaccination regimen.
  • Many experimental strategies for tumor vaccination have been designed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D.2000, ASCO Educational Book Spring: 414-428; Foon, K.
  • vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a strong activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proa Natl. Acad. Sci U.S.A. 90: 3539-43).
  • tumor-specific antigens are differentiation antigens expressed in tumors and tumor-producing cells, such as gp100, MAGE antigen, and Trp-2. More importantly, many of these antigens were shown to be targets for tumor-specific T cells found in the host.
  • the CTLA4 binding proteins of the invention can be used in combination with recombinantly produced tumor-specific proteins and/or peptides to generate an immune response against these proteins. These proteins are normally considered to be autoantigens by the immune system and are therefore tolerated.
  • Tumor antigens may also include protein telomerase, which is required for telomere synthesis of chromosomes and is expressed in more than 85% of human cancers, but only in a limited number of autologous tissues (Kim, N, et al. (1994). ) Science 266: 2011-2013).
  • the tumor antigen can also be a "new antigen" expressed by cancer cells, such as a fusion protein that changes a protein sequence due to somatic mutation or that produces two unrelated sequences (eg, bcr-abl in the Philadelphia chromosome).
  • tumor vaccines may include proteins from viruses associated with human cancer, such as human papillomavirus (HPV), hepatitis virus (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV).
  • HPV human papillomavirus
  • HCV hepatitis virus
  • KHSV Kaposi's herpes sarcoma virus
  • Another form of tumor-specific antigen that can be used in conjunction with a CTLA4 blocker such as an anti-CTLA4 antibody, such as a CTLA4 binding protein of the invention
  • HSP purified heat shock protein isolated from tumor tissue itself.
  • These heat shock proteins contain fragments of proteins from tumor cells that are very effective in delivering to antigen presenting cells to elicit tumor immunity (Suot, R and Srivastava, P (1995) Science 269: 1585-1588; Tamura, Y. Et al. (1997) Science 278: 117-120).
  • DCs Dendritic cells
  • DCs are strong antigen presenting cells that can be used to elicit antigen-specific responses.
  • DCs can be produced in vitro and carry various protein and peptide antigens as well as tumor cell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332).
  • DCs can also be transduced by genetic means to express these tumor antigens as well.
  • DCs have been directly fused to tumor cells for immunization (Kugler, A. et al. (2000) Nature Medicine 6: 332-336).
  • a CTLA4 blocker such as an anti-CTLA4 antibody, such as a CTLA4 binding protein of the invention
  • CAR-T the full name of Chimeric Antigen Receptor T-Cell Immunotherapy, is another effective cell therapy for malignant tumors. law.
  • a chimeric antigen receptor T cell (CAR-T cell) is an antigen binding portion of an antibody capable of recognizing a certain tumor antigen and an intracellular portion of a CD3- ⁇ chain or Fc ⁇ RI ⁇ is coupled in vitro as a chimeric protein.
  • the transduction method transfects a patient's T cells to express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • co-stimulatory molecular signal sequences can also be introduced to increase the cytotoxic activity, proliferation and survival time of T cells, and promote the release of cytokines.
  • a CTLA4 blocker (such as an anti-CTLA4 antibody, such as the CTLA4 binding protein of the invention) can be combined with CAR-T cell therapy to activate a stronger anti-tumor response.
  • the CTLA4 binding proteins of the invention can also be combined with standard cancer therapies.
  • the CTLA4 binding protein of the invention can be effectively combined with a chemotherapy regimen.
  • the scientific principle of the combination of CTLA4 binding protein and chemotherapy in the present invention is cell death, which is the result of the cytotoxic effects of most chemotherapeutic compounds and should result in elevated levels of tumor antigens in the antigen presentation pathway.
  • Other combination therapies that can synergize with CTLA4 by cell death include radiotherapy, surgery, and hormone deprivation. These protocols all produce a source of tumor antigen in the host.
  • An angiogenesis inhibitor can also be combined with the CTLA4 binding protein of the invention. Inhibition of angiogenesis results in tumor cell death, which can provide tumor antigens to the host's antigen presentation pathway.
  • the CTLA4 binding proteins of the invention can also be used in combination with antibodies that target other tumor-specific antigens.
  • antibodies that target other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, antibodies against EGFR variants, anti-VEGFa antibodies, anti-HER2 antibodies, or anti-CMET antibodies.
  • the antibody is a monoclonal antibody.
  • the CTLA4 binding protein of the present invention can also be used in combination with a bispecific antigen that targets Fc ⁇ or Fc ⁇ receptor expression effector cells to tumor cells (see, for example, US Patent Nos. 5,922.845 and 5,837,243).
  • Bispecific antibodies can also be utilized to target two different antigens.
  • macrophages have been targeted to tumor sites using anti-Fc receptor/anti-tumor antigen (eg, Her-2/neu) bispecific antibodies. This targeting can activate tumor-specific responses more efficiently.
  • the T cell aspect of these responses can be enhanced with CTLA4 blockers.
  • antigen can be delivered directly to DC using a bispecific antibody that binds to tumor antigens and dendritic cell-specific cell surface markers.
  • Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating tumor-expressing immunosuppressive proteins. Especially including TGF- ⁇ (KehrL J. et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. and O'Garra, A. (1992) Immunology Today 13: 198-200) and Fas ligand (Hahne, M Et al. (1996) Science 274: 1363-1365). An antibody of each of these can be used in combination with the CTLA4 binding protein of the present invention to counteract the action of an immunosuppressive agent and to facilitate a tumor immune response of the host.
  • CTLA4 binding proteins of the invention Other antibodies that can be used to activate the host immune response can be used in conjunction with the CTLA4 binding proteins of the invention.
  • the anti-CD40 antibody is effective in replacing T cell helper activity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in combination with the CTLA4 binding molecule of the present invention. It is also possible to combine T cell costimulatory molecules such as OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) in order to increase the level of T cell activation. Activated antibodies of Nature Medicine 3: 682-685 (1997) and ICOS (Hutloff, A. et al.
  • CTLA-4 blocking negative costimulatory molecules
  • CTLA-4 e.g., U.S. Patent No. 5,811,097 Or an antibody that is active of BTLA (Watanabe, N. et al. (2003) Nat Immunol 4: 670-9), B7-H4 (Sica, GL et al. (2003) Immunity 18: 849-61).
  • Bone marrow transplantation is currently used to treat a variety of tumors of hematopoietic origin. Graft versus host disease is a consequence of this treatment, and the response of the graft to the tumor can provide a therapeutic benefit.
  • CTLA4 blockers can be utilized to increase the effectiveness of tumor-specific T cells.
  • There are also several experimental treatment regimens involving the ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into the receptor to combat tumors with antigen-specific T cells Greenberg, R. and Riddell, S. ( 1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV.
  • Ex vivo activation in the presence of the CTLA4 binding protein of the invention is expected to increase the frequency and activity of adoptively transferred T cells. Accordingly, the present invention also provides a method of activating immune cells (such as PBMC or T cells) ex vivo, comprising contacting the immune cells with a CTLA4 binding protein of the invention.
  • immune cells such as PBMC or T cells
  • Another aspect of the present invention provides a method for preventing and/or treating an infectious disease in a subject, comprising administering to the subject a CTLA4 binding protein of the present invention, such that the infectious disease of the subject is prevented and/or treatment.
  • the CTLA4 blocker can be used alone, Alternatively, it can be used in combination with a vaccine to stimulate an immune response to pathogens, toxins and autoantigens.
  • pathogens to which this treatment method can be particularly applied include pathogens that currently have no effective vaccine, or pathogens that are not fully effective in conventional vaccines. These include, but are not limited to, HIV, hepatitis virus (A, B, C), influenza virus, herpes virus, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.
  • CTLA4 blockers are particularly useful against infections that have been established by pathogens such as HIV, which exhibit altered antigens during infection. Upon administration of an anti-human CTLA4 antibody, these new epitopes are recognized as foreign sources, causing a strong T cell response that is unaffected by the negative signal of CTLA4.
  • Virus adenovirus, influenza virus, arbovirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, small Virus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, JC virus and arbovirus virus encephalitis virus.
  • pathogenic bacteria that cause infectious diseases that can be treated by the methods of the invention include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, Neisseria gonorrhoeae, Krebs Bacillus, Proteus, R., Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botox, Bacillus, Bacillus, Leptospira, and Lyme Bacterial bacteria.
  • pathogenic fungi that cause infectious diseases treatable by the methods of the invention include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Muscular, Absidia, Rhizopus), S. sphaericus, dermatitis bud, Brazil coccidioides, Coccidioides and collagen Cytoplasmic bacteria.
  • CTLA4 blockers can be combined with other forms of immunotherapy.
  • cytokine therapy eg, interferon, GM-CSF, G-CSF, IL-2
  • bispecific antibody treatment provides enhanced presentation of tumor antigens (see, eg, Holliger (1993) Proc .Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).
  • the CTLA4-Fc fusion protein for immunization (SEQ ID NO: 129) was expressed by HEK293 cells (pCDNA4, Invitrogen, Cat V86220) and purified by Protein A affinity chromatography.
  • a Xinjiang Bactrian camel (Camelus bactrianus) was selected for immunization.
  • 100 ml of peripheral blood lymphocytes of camel were extracted and total RNA was extracted using an RNA extraction kit supplied by QIAGEN, and the extracted RNA was reverse-transcribed into cDNA using the Super-Script III FIRST STRANDSUPERMIX kit according to the instructions.
  • a nucleic acid fragment encoding a variable region of a heavy chain antibody is amplified by nested PCR:
  • Upstream primer GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 110);
  • Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 111).
  • Upstream primer GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 112);
  • Downstream primer GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT (SEQ ID NO: 113).
  • the target heavy chain single domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pCDisplay-3 (Creative Biolabs, Cat: VPT4023) using restriction endonucleases (purchased from NEB) PstI and NotI.
  • the product was subsequently electrotransformed into E. coli electroporation competent cell TG1, and a heavy chain single domain antibody phage display library against CTLA4 was constructed and the library was assayed.
  • the size of the storage capacity is approximately 10 8 by gradient dilution plating.
  • 50 clones were randomly selected for colony PCR. The results show that the insertion rate is greater than 99%.
  • the phage specifically binding to CTLA4 was dissociated with triethylamine (100 mM), and E. coli TG1 growing in log phase was infected, and the phage was generated and purified for the next round of screening. The same screening process was repeated for 3-4 rounds. Thus, positive clones were enriched, and the purpose of screening CTLA4-specific antibodies in the antibody library by phage display technology was achieved.
  • the plate was coated with CTLA4-Fc fusion protein at 4 ° C overnight, and the obtained sample phage (control group was blank phage) was added, and reacted at room temperature for 1 hour. After washing, a anti-mouse anti-HA-tagged antibody (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added and reacted at room temperature for 1 hour.
  • a secondary anti-mouse anti-mouse alkaline phosphatase-labeled antibody purchased from Amytech Co., Ltd.
  • an alkaline phosphatase coloring solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the sample well OD value is more than 3 times the OD value of the control well, it is judged as a positive clone hole.
  • the bacteria of the positive clone wells were transferred to an LB liquid containing 100 ⁇ g per ml of ampicillin to extract a plasmid and perform sequencing.
  • the protein sequences of the individual clones were analyzed according to the sequence alignment software Vector NTI. Clones having the same CDR1, CDR2, and CDR3 sequences were regarded as the same antibody strain, and clones having different CDR sequences were regarded as different antibody strains, and the sequence terminated prematurely was excluded. A total of 34 different antibody sequences were finally obtained.
  • the coding sequence of 34 heavy chain single domain antibodies obtained by sequencing analysis was subcloned into the expression vector PET32b (Novagen, product number: 69016-3), and the correct recombinant plasmid was sequenced and transformed into the expression host strain BL1 (DE3). (Tiangen Biotechnology, product number: CB105-02), which was coated on a plate containing 100 ⁇ g of LB solid medium per ml of ampicillin at 37 ° C overnight. Single colonies were selected for inoculation and culture overnight. The next day, the overnight strains were transferred and expanded, and shaken at 37 ° C until the OD value reached 0.6-1, induced by 0.5 mM IPTG, and shaken overnight at 28 °C.
  • the bacteria were collected by centrifugation, and the cells were disrupted to obtain a crude antibody extract.
  • the antibody protein is then purified by a nickel ion affinity chromatography column. Finally, an antibody protein with a purity of more than 90% is obtained.
  • the plate was coated with CTLA4-Fc fusion protein at 4 ° C overnight, and 10 ng of the heavy chain single domain antibody obtained in Example 2.1 (the control group was a single domain antibody not bound to CTLA4-Fc protein) was added to each well, and reacted at room temperature for 1 hour.
  • a mouse anti-His tag antibody purchased from Beijing Kangwei Century Biotechnology Co., Ltd.
  • a secondary anti-goat goat anti-mouse horseradish peroxidase-labeled antibody (Shenzhou, Catalina, Catalina: SSA007200) was added and reacted at room temperature for 1 hour.
  • a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the plate was coated with Fc protein at 4 ° C overnight, and 10 ng of the heavy chain single domain antibody obtained in Example 2.1 (the control group was a single domain antibody against other unrelated targets) was added per well, and reacted at room temperature for 1 hour.
  • an anti-rabbit anti-human Fc antibody purchased from Shanghai Puxin Biotechnology Co., Ltd.
  • a secondary anti-goat anti-rabbit horseradish peroxidase-labeled antibody purchased from Shanghai Puxin Biotechnology Co., Ltd.
  • a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • Antibody strain ODa for CTLA4-Fc
  • ODb for Fc
  • ODa/ODb ODa/OD blank SEQ ID NO C1 1.201 0.053 42.9 22.7 84
  • Antibody strain ODa for CTLA4-Fc
  • ODb for Fc
  • ODa/ODb ODa/OD blank SEQ ID NO C2 1.231 0.035 44.0 35.2 85 C16 1.786 0.053 63.8 33.7 86 C22 0.848 0.06 30.3 14.1 87 C23 0.951 1.057 34.0 0.9 C24 0.231 0.114 8.3 2.0 C25 0.091 0.03 3.3 3.0 C27 1.31 0.049 46.8 26.7 88 C29 1.513 0.085 54.0 17.8 89 C34 1.022 0.945 36.5 1.1 C38 1.022 0.045 36.5 22.7 90 C46 0.621 0.241 22.2 2.6 J5 1.21 0.036 43.2 33.6 91 J17 1.4 0.046 50.0 30.4 92 J24 0.032 0.041 1.1 0.8 J29 1.439 0.036 51.4 40.0 93 J34 0.932 1.023 33.3 0.9 J35 0.823 0.036 29.4 22.9
  • CTLA4-Fc protein and the CD80-Fc protein were expressed by HEK293 cells (pCDNA4, Invitrogen, Cat V86220).
  • the biotinylated protein CD80-Fc-Biotin was obtained using a Biotinlytion kit from Thermo.
  • CTLA4-Fc fusion protein 0.5 ⁇ g/well 4 °C overnight coated plate, after which 500 ng of the heavy chain single domain antibody specifically binding to CTLA4 confirmed in Example 2.2 was added to each well (control group for He was not associated with a single domain antibody of the target, or just buffer) and 25 ng of CD80-Fc-Biotin (no additional antibody or protein was added to the blank group, only an equal volume of buffer was added) and reacted for 1 hour at room temperature. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1 hour. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm. When the sample OD value is ⁇ 0.8 compared to the control OD value, the antibody is considered to have a blocking effect.
  • the antibody strains C1, C16, C27, J37, J42, 128, 145, 155, and 165 all exhibited a blocking effect on the CD80/CTLA4 interaction.
  • the Fc fusion protein of mouse CTLA4 (SEQ ID NO: 131) was expressed by HEK293 cells (pCDNA4, Invitrogen, Cat V86220).
  • the biotinylated protein mCTLA4-Fc-Biotin was obtained using a Biotinlytion kit from Thermo.
  • Example 2.3 Using the heavy chain single domain antibody obtained in Example 2.1 (the control group was a single domain antibody against other unrelated targets), according to the results of Example 2.3, four antibodies 145, 155, C1 and C27 with better blocking effect were selected. 0.5 ⁇ g/well coated plates were incubated overnight at 4 ° C, and 100 ⁇ g of mouse CTLA4-Fc fusion protein was added to each well for 1.5 hours at room temperature. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1.5 hours. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm. The results are shown in Table 3.
  • Antibody strain OD (for mouse CTLA4) 145 0.026 155 0.032 C1 0.042 C27 0.021 Control 0.026 blank 0.027
  • the heavy chain single domain antibody of human CTLA4 of the present invention does not bind to the mouse CTLA4-Fc protein.
  • the plate was coated with the obtained CTLA4 heavy chain single domain antibody at 0.5 ⁇ g/well at 4 ° C overnight, followed by addition of a gradient dilution series of CTLA4-Fc fusion protein, and reacted at room temperature for 1 hour. After washing, goat anti-human IgG-Fc horseradish peroxidase-labeled antibody (lakepharma) was added and reacted at room temperature for 1 hour. After washing, horseradish peroxidase coloring solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis. The four-parameter fit was used to obtain the CTLA4 binding curve and EC50 value of the antibody.
  • the antibody strain 14 was about 50 ng/mL, 155 was about 13 ng/mL, and C1 was about 123 ng. /mL, C27 is about 93 ng/mL.
  • the results of 145 and 155 are shown in Figure 1A, and the results of C1 and C27 are shown in Figure 1B.
  • the plate was coated with CTLA4-Fc fusion protein 0.5 ⁇ g/well at 4 ° C overnight, and then 100 uL of the gradient dilution series of CTLA4 blocking type single domain antibody obtained in Example 2.1 was added per well (250 ng/mL CD80-Fc- in the dilution) Biotin), reacted at room temperature for 1 hour. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1 hour. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fitting was used to obtain the CD80/CTLA4 blocking curve and IC50 value of antibody strains C1, C27, 145, 155.
  • the IC50 of antibody strain C1 was 852ng. /mL
  • the antibody strain C27 was about 731 ng/ml
  • the antibody strain 145 was about 1.947 ⁇ g/ml
  • the antibody strain 155 was about 4.690 ⁇ g/ml.
  • the results of 145 and 155 are shown in Figure 2A
  • the results of C1 and C27 are shown in Figure 2B.
  • the amino acid sequence of human IgG1-Fc region was obtained from the constant region amino acid sequence of human immunoglobulin ⁇ 1 (IgG1) on the protein database Uniprot (P01857). Reverse transcription PCR, a nucleic acid fragment encoding human IgG1-Fc was obtained from human PBMC total RNA, and the nucleic acid fragment encoding the fusion protein of CTLA4 single domain antibody and Fc obtained in the above Example was obtained by overlapping PCR. This was then subcloned into the vector pCDNA4 (Invitrogen, Cat V86220).
  • Recombinantly constructed single domain antibody-Fc fusion protein particles were transfected into HEK293 cells for antibody expression.
  • the recombinant expression plasmid was diluted with Freestyle 293 medium and added to the PEI (Polyethylenimine) solution required for transformation.
  • PEI Polyethylenimine
  • Each group of plasmid/PEI mixture was separately added to the HEK293 cell suspension, and placed at 37 ° C, 10% CO 2 , 90 rpm; Add 50 ⁇ g/L IGF-1. After four hours, additional EX293 medium, 2 mM glutamine and 50 ⁇ g/L IGF-1, 135 rpm were added. Add 3.8 mM VPA after 24 hours. After 5 to 6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target CTLA4 single domain antibody-Fc fusion protein.
  • the sequences of the Fc fusion proteins of antibody strains C1, C27, 145, and 155 are shown in SEQ ID NOs: 114-117, respectively.
  • the plate was coated with CTLA4-Fc fusion protein 0.5 ⁇ g/well at 4 ° C overnight, and then 100 uL of the gradient dilution series of CTLA4 blocking type single domain antibody Fc fusion protein obtained in Example 2.7 was added per well (250 ng/mL CD80 in the dilution) -Fc-Biotin), reacted at room temperature for 1.5 hours. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1.5 hours. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the humanization method is carried out by a method of resurfacing the protein surface amino acid and a VHH CDR grafting to a universal framework.
  • the humanization procedure was as follows: the homologous modeling of the antibody strains C27 and C1 was carried out, and the modeling software was Modeller9.
  • the reference homologous sequence is the cAb-Lys3 antibody (PDB number: 1XFP), and the relative solvent accessibility of the amino acid is calculated from the three-dimensional structure of the protein. If an amino acid of antibody strains C27 and C1 is exposed to a solvent, it is replaced with an amino acid at the same position as the reference human antibody DP-47 sequence, and finally all substitutions are completed.
  • the antibody strain C27 was humanized to obtain humanized variants of five antibody strains C27; the antibody strain C1 was humanized to obtain humanized variants of four antibody strains C1.
  • Table 4 lists the sequence numbers of these humanized variants as well as the amino acid changes therein, wherein the amino acid residue numbers are numbered according to Kabat.
  • Figure 4a shows the alignment of the C27 humanized sequences and 4b shows the alignment of the C1 humanized sequences.
  • the amino acid sequence of human IgG1-Fc region was obtained from the constant region amino acid sequence of human immunoglobulin ⁇ 1 (IgG1) on the protein database Uniprot (P01857).
  • a nucleic acid fragment encoding human IgG1-Fc was obtained from human PBMC total RNA by reverse transcription PCR, and the nucleic acid fragment encoding the fusion protein of CTLA4 single domain antibody and Fc obtained in the above Examples was obtained by overlapping PCR. This was then subcloned into the vector pCDNA4 (Invitrogen, Cat V86220).
  • Recombinantly constructed single domain antibody-Fc fusion protein particles were transfected into HEK293 cells for antibody expression.
  • the recombinant expression plasmid was diluted with Freestyle 293 medium and added to the PEI (Polyethylenimine) solution required for transformation.
  • PEI Polyethylenimine
  • Each group of plasmid/PEI mixture was separately added to the HEK293 cell suspension, and placed at 37 ° C, 10% CO 2 , 90 rpm; Add 50 ⁇ g/L IGF-1. After four hours, additional EX293 medium, 2 mM glutamine and 50 ⁇ g/L IGF-1, 135 rpm were added. Add 3.8 mM VPA after 24 hours. After 5 to 6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target CTLA4 single domain antibody-Fc fusion protein.
  • CTLA4 single domain antibody-Fc fusion proteins are shown in SEQ ID NOs: 114-126, respectively, wherein SEQ ID NOs: 118-126 are Fc fusion eggs of humanized CTLA4 single domain antibodies. White.
  • These fusion proteins comprise a CTLA4 binding domain and form homodimers, which form dimeric molecules each comprising two CTLA4 binding domains, such that these fusion proteins are also referred to as bivalent CTLA4 single domain antibody-Fc fusion proteins .
  • CTLA4 antibody ipilimumab of BMS Company cloned the antibody gene by the method of antibody 10D1 in US20020086041 and cloned it into the vector pCDNA4.
  • the recombinantly constructed plasmid was transiently transfected with HEK293 cells by the same method in 4.1, and the obtained CTLA4 antibody of BMS Company was renamed to 10D1.
  • the expression level of the CTLA4 single domain antibody Fc fusion protein of the present invention is higher than 400 mg/L, and the antibody 10D1 expression level is about 150 mg/L. This result indicates that the CTLA4 single domain antibody Fc fusion protein of the present invention is more stable in structure than the known CTLA4 antibody, and can obtain a higher expression level.
  • the nucleic acid fragments encoding the fusion protein of two tandem CTLA4 single domain antibodies and Fc were obtained by overlapping PCR. This was then subcloned into the vector pCDNA4 (Invitrogen, Cat V86220). The recombinantly constructed plasmid was transiently transfected with HEK293 cells by the same method as 4.1 to obtain a fusion protein comprising two CTLA4 single domain antibodies and one Fc, which formed two of the four CTLA4 binding domains per molecule. A polymer molecule, thus the fusion protein is also referred to as a tetravalent CTLA4 single domain antibody-Fc fusion protein.
  • the transient transfection expression level of the tetravalent CTLA4 single domain antibody Fc fusion protein was about 400 mg/L, which was comparable to the bivalent CTLA4 single domain antibody Fc fusion protein.
  • CTLA4 single domain antibody Fc fusion protein obtained in Examples 2.7 and 4.1 or the 10D1 protein obtained in Example 4.2 was coated with 0.5 ⁇ g/well 4 ° C overnight, followed by addition of CTLA4-Fc-Biotin gradient dilution series, and reacted at room temperature for 1 hour. . Then joined SA-HRP (purchased from Sigma), The reaction was carried out at room temperature for 1.5 hours. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fit was used to obtain the CTLA4 binding curve and EC50 value of the antibody (the EC50 values of all test antibodies were about 60-70 ng/mL) to reflect the antibody pair. Affinity of CTLA4.
  • the abscissa is the CTLA4 single domain antibody Fc fusion protein (or 10D1 protein) concentration (in ng/mL); the antibody strain C27 is labeled with four different humanized forms of Fc fusion protein.
  • the four proteins have comparable affinities for CTLA4 and are comparable to the unhumanized C27-LdFc and the CTLA4 antibody 10D1 of known BMS.
  • CTLA4 single domain antibody Fc fusion protein obtained in Examples 2.7 and 4.1 was coated with 0.5 ⁇ g/well at 4 ° C overnight, followed by addition of a CTLA4-Fc-Biotin gradient dilution series, and reacted at room temperature for 1 hour. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1.5 hours. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fit was used to obtain the CTLA4 binding curve and EC50 value of the antibody (the EC50 values of all test antibodies were about 25-35 ng/mL) to reflect the antibody pair. Affinity of CTLA4.
  • CTLA4-Fc fusion protein 0.5 ⁇ g/well 4 °C overnight coated plate, followed by the CTLA4 single domain antibody Fc fusion protein obtained in the above Example 4.1 and the 10D1 antibody obtained in Example 4.2
  • a gradient dilution series of proteins 100 uL per well (250 ng/m LCD80-Fc-Biotin in the dilution), was reacted for 1 hour at room temperature. After washing, SA-HRP (purchased from Sigma) was added and reacted at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fitting was used to obtain the CTLA4-CD80 blocking curve and IC50 value of the antibody.
  • the results are shown in Figures 7A and 7B. It can be seen that the two different single-domain antibodies C27 and C1, whether humanized or the original sequence, have the same ability of the Fc fusion protein to block the CTLA4-CD80 interaction, and are superior to the BMS-listed antibodies. (marked as 10D1).
  • the obtained bivalent CTLA4 single domain antibody Fc fusion protein of Example 4.1 and the tetravalent CTLA4 single domain antibody Fc fusion protein obtained in Example 4.4 were coated with 0.5 ⁇ g/well overnight at 4 ° C, followed by gradient dilution of CTLA4-Fc-Biotin added. The series was reacted at room temperature for 1 hour. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1.5 hours. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fit was used to obtain the CTLA4 binding curve and EC50 value of the antibody (the EC50 values of all test antibodies were about 25-35 ng/mL) to reflect the antibody pair. Affinity of CTLA4.
  • the results are shown in Figure 8, in which the ordinate is OD405, the abscissa is the CTLA4 single domain antibody Fc fusion protein concentration (in ng/mL); the triangle represents the tetravalent CTLA4 single domain antibody Fc fusion protein huC1v4-tet-Fc (SEQ ID NO: 128), the square represents the humanized form of the antibody strain C1, the Fc fusion protein huC1v4-Fc, and the circle represents the Fc fusion protein C1-ld-Fc of the CTLA4 single domain antibody C1.
  • the EC50 of the three proteins is slightly different, but considering that the molecular weight of the tetravalent protein is about 5/4 of the divalent value, which is converted into a molar concentration, there is no difference in the affinity of the three proteins for CTLA4.
  • the binding kinetics of the tetravalent and bivalent CTLA4 single domain antibody Fc fusion proteins obtained against the recombinant human CTLA4 obtained in the above examples were measured by surface plasmon resonance (SRP) method using a BIAcore X100 instrument.
  • Recombinant camel anti-human Fc antibody was coupled to a CM5 biosensor chip to obtain approximately 1000 response units (response) Units, RU).
  • the antibody was diluted to a fixed concentration with HBS-EP+1 ⁇ buffer (GE, cat#BR-1006-69) and injected at 25 °C for 120 s to ensure a response value greater than 100 RU was obtained, after which CTLA4 and mouse Fc fusion protein CTLA4-muFc (SEQ ID NO: 133) were serially diluted three times, injected at 25 ° C for 120 s, dissociation time was 30 min, and 10 mM glycine-HCl (pH 2.0) was regenerated for 120 s.
  • Binding rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (kD) is calculated as the ratio koff/kon.
  • the binding affinities of the measured anti-CTLA4 antibodies are shown in Table 5.
  • the results show that the affinity of the tetravalent molecule is slightly lower than that of the divalent molecule, which may be due to a certain steric hindrance.
  • the 96-well plate was inoculated with 1.5 ⁇ 105 Jurkat T cells (from the Shanghai Cell Bank of Chinese Academy of Sciences), added Anti-human CD3 (50 ng/mL), and incubated at 37 ° C for 15 min, then added a gradient dilution of the bivalent or tetravalent CTLA4 single domain antibody Fc fusion protein. (30ng/ml-0.94ng/ml, 100ng/mL CTLA4-Fc fusion protein was added to the dilution) and equal-density Raji cells (from Shanghai University Library of Chinese Academy of Sciences). After 24 hours of culture, the supernatant was collected for detection of IL-2 expression. the amount. The data were processed with Soft Max to calculate the effect of inhibition of IL-2 by the CTLA4-Fc fusion protein in the bivalent or tetravalent CTLA4 single domain antibody Fc fusion protein and compared by EC50.
  • the plasmid also fused the EGFP protein to the C-terminus of the target protein, so that the expression level of the B7 family protein on the membrane can be examined by the intensity of green fluorescence.
  • Constructed transient transfected cell line package Included: 293-CTLA4-EGFP, 293-PD1-EGFP, 293-CD28-EGFP.
  • the constructed cells were resuspended in 0.5% PBS-BSA Buffer, huC1v4-tet-Fc antibody was added, and a negative control was set to 2 ⁇ g of single domain antibody against other unrelated targets, and incubated on ice for 20 min. After washing, eBioscience secondary anti-hIg-PE was added and incubated on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ l of 0.5% PBS-BSA Buffer and detected by flow cytometry.
  • the monkey CTLA4-Fc protein was purchased from Yishen Shenzhou.
  • the tetravalent CTLA4 single domain antibody Fc fusion protein obtained in Example 4.4 was obtained using the Biotinlytion kit of Thermo Scientific to obtain the biotinylated protein huC1v4-tet-Fc-Biotin.
  • the monkey CTLA4-Fc protein or human CTLA4-Fc protein 0.5 ⁇ g/well was coated overnight at 4° C., followed by addition of a gradient dilution series of huC1v4-tet-Fc-Biotin, and reacted at room temperature for 1 hour. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1.5 hours. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fit was used to obtain the binding curves and EC50 values of the antibodies against monkey CTLA4 and human CTLA4 to reflect the affinity of the antibody for CTLA4.
  • PBMC peripheral blood mononuclear cells
  • 0.3 ug/well of anti-CD3 antibody was coated overnight on a cell culture plate at 4 °C.
  • 1 ⁇ 10 5 PBMC cells were added to each well, and 10 ug/mL of CTLA4 single domain antibody Fc fusion protein huC1v4-Fc, huC27-Fc or the company's self-made BMS CTLA4 antibody (named 10D1) were added to each well. ).
  • the supernatant was taken, and the level of IFN-? in the supernatant was measured using an IFN- ⁇ ELISA test kit (ebioscience).
  • CTLA4 single domain antibody Fc fusion protein combined with anti-CD3 antibody can enhance the secretion of ⁇ -interferon by PBMC cells, that is, CTLA4 single domain antibody Fc fusion protein enhances the activation of PBMC cells.
  • CTLA4 single domain antibody Fc fusion protein enhances the activation of PBMC cells.
  • both huC1v4-Ld-Fc and huC27v3-Ld-Fc exhibited activity against the anti-CTLA4 antibody of BMS.
  • PBMC peripheral blood mononuclear cells
  • CTLA4 single domain antibody Fc fusion protein (whether bivalent or tetravalent) combined with anti-CD3 antibody can enhance the secretion of ⁇ -interferon, ie bivalent or tetravalent, in PBMC cells.
  • the CTLA4 single domain antibody Fc fusion protein significantly enhanced the activation of PBMC cells at a low dose concentration of 0.03 ug/mL.
  • the tetravalent CTLA4 single domain antibody Fc fusion protein huC1v4-tet-Fc exhibited activity superior to bivalent and BMS anti-CTLA4 antibodies.
  • Peripheral blood mononuclear cells PBMC were isolated from healthy donors' peripheral blood-enriched leukocytes using human lymphocyte separation fluid (Tianjin Minyang) density gradient centrifugation. Then, it was cultured in serum-free RPMI1640 medium for 1-2 hours to remove unattached cells, and the cells were cultured in RPMI containing 10% FBS, 10 ng/ml GM-CSF, and 20 ng/mL IL-4. After 5-6 days of culture, 10 ng/ml of TNF- ⁇ was added and incubated for 24 hours to obtain mature dendritic cells.
  • human lymphocyte separation fluid Trianjin Minyang
  • the dendritic cells obtained by this method were resuspended in RPMI complete medium, 2 x 10 5 /ml. Then 50 ⁇ l was added to each well in a 96-well U-shaped bottom plate (Costar: 3799) and cultured in an incubator.
  • CD4+ T cells were isolated from another donor PBMC using a magnetic bead separation kit (Miltenyi Biotec: 130-096-533) according to the method described.
  • the 1 ⁇ 10 4 dendritic cells obtained by the above method were mixed with 1 ⁇ 10 5 CD4+ T cells, resuspended in RPMI complete medium and added to a 96-well culture plate, and 50 ⁇ l of the cell mixture was added to each well; 100 ⁇ l of each well was diluted in each well.
  • CTLA4 single domain antibody Fc fusion protein can enhance the ⁇ -interferon secretion of CD4+ T cells in the mixed lymphocyte reaction, that is, the CTLA4 blocking type single domain antibody Fc fusion protein enhances the activation of T cells. Moreover, this biological activity was concentration-dependent, and a significant function of stimulating T cell activation was observed at a low dose concentration (0.01 ⁇ g/ml).
  • CTLA4 humanized mice i.e., the mouse expressed human CTLA4 protein
  • 5X 10 5 MC38 tumor cells 5X 10 5 MC38 tumor cells
  • test sample 100 ug of the test sample or the same amount of human immunoglobulin was intraperitoneally administered as a control group. Tumor size was measured every two days from day 5 after inoculation until day 20.
  • samples examined included a tetravalent CTLA4 single domain antibody Fc fusion protein (Tet in the figure) and a CTLA4 monoclonal antibody, ipilimumab, which has been marketed by BMS.
  • IV SD rats Single-venous (IV) SD rats were given CTLA4 single-domain antibody Fc fusion eggs, blood samples were taken at different time points, and Elisa was used to determine the concentration of the test substance in the plasma of the rats after administration of the test substances and calculate the pharmacokinetic parameters.
  • Animal selection SD rats aged 6-8 weeks were selected and weighed 200-300 g. According to the male and female, they were randomly assigned to two groups of 8 rats each.
  • IV intravenous
  • Blood collection Immediately before the drug, immediately after the drug, at various time points after the drug, about 0.5 ml of blood was collected from the jugular vein of each rat. The collected blood was quickly centrifuged to separate the serum and stored at -80 ° C until analysis.
  • CTLA4 single domain antibody Fc fusion protein The content of CTLA4 single domain antibody Fc fusion protein in serum was detected by sandwich ELISA. It was coated with recombinant human CTLA4 protein to capture CTLA4-specific antibodies, and the Fc region was detected with goat anti-human IgG (Fc specific)-HRP antibody (Sigma) to ensure detection of intact CTLA4 single domain antibody Fc fusion. protein.
  • Data processing Calculate relevant pharmacokinetic parameters using blood concentration versus time curve, including AUC (0-t), AUC (0- ⁇ ), Cmax, Tmax, T1/2, Vss, MRT, and the like.
  • the plasma concentration of divalent and tetravalent CTLA4 single domain antibody Fc fusion protein in rats is shown in Figure 16 (a is the bivalent antibody huC1v4-Ld-Fc, b is the tetravalent antibody huC1v4-tet-Fc) .
  • the pharmacokinetic parameters are shown in Figure 17.
  • the results showed that both the bivalent or tetravalent CTLA4 single domain antibody Fc fusion protein had a longer half-life in rats (more than 5 days), and the stability in vivo was better.
  • the tetravalent antibody has a half-life in vivo that is doubled (more than 11 days). It is thus concluded that the period of maintenance of the effective blood concentration of the tetravalent antibody in the body is longer, and it is expected that the clinical administration can maintain a longer administration interval.
  • the tetravalent CTLA4 single domain antibody Fc fusion protein expressed by human affinity 293HEK cells obtained by one step affinity purification was obtained by the method described in Example 4.4, followed by SE-HPLC, CE under reducing conditions, CE under non-reducing conditions, WCX, The protein was subjected to preliminary physical and chemical properties analysis by DSC and the like to further judge its drug-forming properties.
  • the specific values are as follows. From this data, it can be preliminarily judged that the tetravalent CTLA4 single domain antibody Fc fusion protein has good physical and chemical properties and is suitable for industrial scale production.
  • the CTLA4 single domain antibody Fc fusion protein was concentrated by UF/DF and exchanged into PBS buffer to prepare a 20 mg/mL solution, and the thermal destruction test was accelerated at 40 ° C for 30 days. Among them, samples of 0 days, 10 days, 20 days, and 30 days were tested for purity by SE-HPLC. Figure. It can be seen that at 40 ° C, without optimization of the preparation, although the purity of the main peak was slightly decreased, no obvious tendency of aggregation or degradation was observed. At the same time, combined with its higher Tm value, it can be judged that the protein has good thermal stability.
  • a single intravenous injection of cynomolgus monkeys at 7.5 and 30 mpk showed no significant toxicity, and NOAEL was greater than or equal to 30 mg/kg.
  • the non-clinical NOAEL reported by ipilimumab of BMS is approximately 10 mg/kg. It is speculated that the CTLA4 single domain antibody Fc fusion protein has lower toxic side effects than ipilimumab.

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Abstract

提供一种CTLA-4的抗体，所述抗体是能够特异性结合CTLA-4的单一重链单域抗体（VHH），还提供一种包含所述CTLA-4抗体的药物组合物，以及使用所述抗体制备药物的用途。

Description

针对CTLA4的单域抗体及其衍生蛋白 技术领域
本发明涉及医药生物领域，公开了针对CTLA4的单域抗体及其衍生蛋白。具体而言，本发明公开了一种CTLA4结合蛋白及其用途，特别是在治疗和/或预防CTLA4相关疾病例如肿瘤中的用途。
背景技术
骆驼或是羊驼等骆驼科动物能够产生一种天然缺失轻链的重链抗体，其分子只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，但却具有完整的抗原结合功能，而且不像人工改造的单链抗体片段(scFv)那样容易聚集。更重要的是，重组表达的单独的VHH结构域具有与原重链抗体相当的结构稳定性以及抗原结合活性，是目前已知的可结合靶抗原的最小单位，被称作纳米抗体(Nanoboy)或重链单域抗体。重链单域抗体因其特殊的结构性质，兼具了传统抗体与小分子药物的优势，克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷，代表了新一代抗体治疗开发的方向。
肿瘤细胞表达的肿瘤相关抗原是产生有效的免疫应答的基础。然而当抗原与抗原递呈细胞(APCs)表面的主要组织相容复合体(MHC)分子结合并递呈抗原后，还需要共刺激信号来促进效应T细胞的活化。研究表明，很多肿瘤能够逃逸患者本身的免疫系统，部分原因是由于缺乏共刺激信号而无法充分活化T细胞，也很可能是由于肿瘤细胞通过调节性T细胞(Treg)诱导的免疫抑制导致的。抗原递呈细胞上的CD80或CD86与T细胞上的CD28结合是一个关键的共刺激信号。人细胞毒性T淋巴细胞相关抗原4(CTLA4)是T细胞上表达的负调控因子，与CD80和CD86具有更高的亲和力，其在阻断CD28的共刺激信号同时，激活T细胞负调控通路。CTLA4的免疫抑制效果在限制自身免疫应答的过程中发挥重要作用。然而在肿瘤免疫应答中，CTLA4介导的抑制机制则往往成为肿瘤细胞逃逸免疫系统的原因之一。因此，可以通过阻断CTLA4与CD80或CD86的相互作用来增强T细胞介导的抗肿瘤反应。
本领域需要能够与CTLA4高亲和力结合，并且能够阻断CTLA4与 CD80结合的抗CTLA4抗体，特别是抗CTLA4重链单域抗体。
发明概述
本发明的发明人利用噬菌体展示技术，通过筛选得到了具有高特异性、高亲和力和高稳定性的抗CTLA4重链单域抗体(VHH)。
在第一方面，本发明提供了一种CTLA4结合蛋白，其包括特异性结合CTLA4的免疫球蛋白单一可变结构域。
在另一方面中，本发明涉及编码CTLA4结合蛋白的核酸分子以及含有所述核酸分子的表达载体和宿主细胞。
本发明还涉及包含本发明的CTLA4结合蛋白的药物组合物。
本发明还涉及制备本文所述CTLA4结合蛋白的方法。
本发明还涉及本发明所述CTLA4结合蛋白以及药物组合物的应用，尤其是预防和/或治疗与CTLA4相关的疾病的用途和方法。
附图说明
图1.示出CTLA4重链单域抗体对CTLA4-Fc抗原蛋白的结合曲线。
图2.示出CTLA4重链单域抗体对CD80与CTLA4相互作用的阻断曲线。
图3.CTLA4重链单域抗体Fc融合蛋白对CD80与CTLA4相互作用的阻断曲线。
图4.示出抗体株C27的5种人源化变体(A)以及抗体株C1的4种人源化变体(B)的序列比对结果。
图5.示出CTLA4单域抗体Fc融合蛋白对CTLA4的结合曲线(ELISA法)。
图6.示出CTLA4单域抗体Fc融合蛋白对CTLA4的结合曲线(ELISA法)。
图7.示出CTLA4单域抗体Fc融合蛋白对CTLA4-CD80相互作用的阻断曲线(竞争ELISA法)。
图8.示出四价CTLA4单域抗体Fc融合蛋白对CTLA4的结合曲线(竞争ELISA法)。
图9.示出二价以及四价CTLA4单域抗体Fc融合蛋白对CTLA4/CD80 相互作用的阻断能力(细胞中和实验)。
图10.流式细胞仪检测CTLA4单域抗体Fc融合蛋白对CTLA4蛋白结合的特异性。
图11.示出四价CTLA4单域抗体Fc融合蛋白对猴CTLA4蛋白的结合。
图12.示出CTLA4单域抗体Fc融合蛋白对PBMC的激活作用。
图13.比较二价以及四价CTLA4单域抗体Fc融合蛋白对PBMC的激活作用。
图14.示出CTLA4单域抗体Fc融合蛋白对CD4+T细胞的激活作用。
图15.示出CTLA4单域抗体Fc融合蛋白在CTLA4人源化小鼠体内对MC38肿瘤的抑制效果
图16.示出二价以及四价CTLA4单域抗体Fc融合蛋白在大鼠体内血药浓度对时间的变化曲线。
图17.示出CTLA4单域抗体Fc融合蛋白在大鼠体内的药代动力学参数。
图18.示出CTLA4单域抗体Fc融合蛋白的热稳定性。
发明详述
定义
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人，“Molecular Cloning：A Laboratory Manual”(第2版)，第1-3卷，Cold Spring Harbor Laboratory Press(1989)；Lewin，“Genes IV”，Oxford University Press，New York，(1990)；及Roitt等人，“Immunology”(第2版)，Gower Medical Publishing，London，New York(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所 用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用，术语(多肽或蛋白的)“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或结构域的功能。
如本文所用的术语“免疫球蛋白结构域”是指抗体链(例如常规4链抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征，其由排列在两个β折叠中任选由保守二硫键稳定的约7个反平行β折叠股的2层夹层组成。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
如本文所用的术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原表位的免疫球蛋白可变结构域。本发明含义中的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH及VL(VH结构域及VL结构域)。免疫球蛋白单一可变结构域的另一实例为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH结构域、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.： “Naturally occurring antibodies devoid of light chains”；Nature363，446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本发明的上下文中，术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”以及
Figure PCTCN2017085038-appb-000001
及“
Figure PCTCN2017085038-appb-000002
结构域”(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。
例如Riechmann及Muyldermans，J.Immunol.Methods 231，25-38(1999)的图2中所示，对于骆驼科的VHH结构域所应用的氨基酸残基，根据Kabat等人给出的VH结构域的一般编号法来编号(“Sequence of proteins of immunological interest”，US Public Health Services，NIH Bethesda，MD，公开案第91号)。根据该编号法，
-FR1包含在位置1-30处的氨基酸残基，
-CDR1包含在位置31-35处的氨基酸残基，
-FR2包含在位置36-49处的氨基酸，
-CDR2包含在位置50-65处的氨基酸残基，
-FR3包含在位置66-94处的氨基酸残基，
-CDR3包含在位置95-102处的氨基酸残基，且
-FR4包含在位置103-113处的氨基酸残基。
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述 替代方法还可以类似地应用于VHH结构域。然而，除非另有说明，否则在本说明书、权利要求书及附图中，将遵循如上所述的根据Kabat且适于VHH结构域的编号。
VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本文所述的目的。
VHH结构域及含有其的多肽的其他结构特性及功能性质可总结如下：
VHH结构域(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。此区分VHH结构域与常规4链抗体的VH及VL结构域，这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于这些独特性质，使用VHH结构域-单独或作为较大多肽的一部分-提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：
-仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；
-VHH结构域可自单一基因表达且不需要翻译后折叠或修饰；
-VHH结构域可容易地改造成多价及多特异性格式(格式化)；
-VHH结构域高度可溶且无聚集趋势；
-VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；
-VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；
-VHH结构域与常规4链抗体及其抗原结合片段相比相对较小(大 约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；
-VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.，Journal of Immunological Methods，240(2000)185-195；Li et al.，J Biol Chem.，287(2012)13713-13721；Deffar et al.，African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
源自骆驼科的VHH结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而经“人源化”(本文中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其他修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列，且在一具体实施方案中，可含IGHV3的人框架区序列。
如本文所用，术语“结构域抗体”(亦称为“Dab”及“dAb”)特别用于指代非骆驼科哺乳动物的抗体(特别是人4链抗体)的VH或VL结构域。为了以单一抗原结合结构域的形式(即在不与VL域或VH域分别配对的情况下)结合表位，需要例如通过使用人单一VH或VL结构域序列的文库对所述抗原结合性质进行具体选择。
与VHH一样，结构域抗体的分子量为约13kDa至约16kDa，且若源自完全人序列，则不需要进行人源化以供例如人治疗使用。正如在VHH结构域的情况下，结构域抗体在原核表达系统中也很好地得以表达，从而显著降低总制造成本。
“结构域抗体”已公开于例如以下文献中：Ward，E.S.，等人：“Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli”；Nature 341：544-546(1989)；Holt，L.J.等人：“Domain antibodies：proteins for therapy”；TRENDS in Biotechnology 21(11)：484-490(2003)。
此外，本领域技术人员还将了解，有可能将一个或多个上述CDR“移植”于其他“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。
如本文所用，术语“表位”或可互换使用的术语“抗原决定簇”指抗体的互补位所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位。参见例如Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。举例而言，线性表位可通过例如以下方法来确定：在固体支持物上同时合成大量肽，其中这些肽对应于蛋白质分子的各部分，且使这些肽在仍然与支持物连接的情况下与抗体反应。这些技术在本领域中为已知的且描述于例如美国专利第4,708,871号；Geysen等人(1984)Proc.Natl.Acad.Sci.USA 81：3998-4002；Geysen等人(1986)Molec.Immunol.23：709-715中。类似地，构象表位可通过诸如通过例如x射线结晶学及2维核磁共振确定氨基酸的空间构形加以鉴别。参见例如Epitope Mapping Protocols(同上)。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本发明的抗体分子竞争结合CTLA4上的相同表位的抗体及其抗原结合片段。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)可结合的不同类型抗原或表位的数目。可基于抗原结合蛋白的亲和力和/或亲合力确定其特异性。由抗原与抗 原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合蛋白之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合蛋白(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合蛋白上的抗原结合位点之间的亲和力，以及存在于抗原结合蛋白上的相关结合位点的数目。
如本文所用，术语“CTLA4结合蛋白”意指任何能够特异性结合CTLA4的蛋白。CTLA4结合蛋白可以包括针对CTLA4的如本文定义的抗体或其缀合物。CTLA4结合蛋白还涵盖免疫球蛋白超家族抗体(IgSF)或CDR移植分子。
本发明的“CTLA4结合蛋白”可以包含至少一个结合CTLA4的免疫球蛋白单一可变结构域如VHH。在一些实施方案中，本发明的“CTLA4结合分子”可以包含2、3、4或更多个结合CTLA4的免疫球蛋白单一可变结构域如VHH。本发明的CTLA4结合蛋白除结合CTLA4的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中，本发明的“CTLA4结合蛋白”还涵盖双特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。
通常，本发明的CTLA4结合蛋白将以如于Biacore或KinExA或Fortibio测定中测量的优选10-7至10-10摩尔/升(M)、更优选10-8至10-10摩尔/升、甚至更优选10-9至10-10或更低的解离常数(KD)，和/或以至少107M-1、优选至少108M-1、更优选至少109M-1，更优选至少1010M-1的缔合常数(KA)结合所要结合的抗原(即CTLA4)。任何大于10-4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字 母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。“序列相似性”指示相同或代表保守氨基酸取代的氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Computational Molecular Biology，Lesk，A.M.，ed.，Oxford University Press，New York，1988；Biocomputing：Informatics and Genome Projects，Smith，D.W.，ed.，Academic Press，New York，1993；Computer Analysis of Sequence Data，Part I，Griffin，A.M.，and Griffin，H.G.，eds.，Humana Press，New Jersey，1994；Sequence Analysis in Molecular Biology，von Heinje，G.，Academic Press，1987和Sequence Analysis Primer，Gribskov，M.and Devereux，J.，eds.，M Stockton Press，New York，1991。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的 其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“基本上分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“基本上分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“基本上分离的”多肽或核酸分子优选基本上为均质的。
“亲和力成熟”的抗CTLA4抗体，特别是VHH或结构域抗体，在一个或多个CDR中具有一个或多个变化，所述变化导致对CTLA4的亲和力相比于其各自的亲本抗CTLA4抗体有所增加。亲和力成熟的抗CTLA4抗体可通过例如由以下所述的本领域中已知的方法来制备：Marks等人，1992，Biotechnology 10：779-783或Barbas等人，1994，Proc.Nat.Acad.Sci，USA 91：3809-3813.；Shier等人，1995，Gene 169：147-155；Yelton等人，1995，Immunol.155：1994-2004；Jackson等人，1995，J.Immunol.154(7)：3310-9；及Hawkins等人，1992，J.MoI.Biol.226(3)：889896；KS Johnson及RE Hawkins，“Affinity maturation of antibodies using phage display”，Oxford University Press 1996。
如本文所用的术语“对象”意指哺乳动物，尤其灵长类动物，尤其是人。
本发明的CTLA4结合蛋白
在第一方面，本发明提供了一种CTLA4结合蛋白，其包含至少一个能够特异性结合CTLA4的免疫球蛋白单一可变结构域。在一些实施方案中，所述CTLA4结合蛋白包含一个特异性结合CTLA4的免疫球蛋白单一可变结构域。在一些实施方案中，所述CTLA4结合蛋白包含2、3、4或更多个特异性结合CTLA4的免疫球蛋白单一可变结构域。在一些实施方案中，所述CTLA4结合蛋白包含两个或更多个相同的特异性结合CTLA4的免疫球蛋白单一可变结构域。在另一些实施方案中，所述CTLA4结合蛋白包含两个或更多个不同的特异性结合CTLA4的免疫球蛋白单一可变结构域。在一些实施方案中，所述两个或更多个特异性结合CTLA4的免疫球蛋白单一可变结构域直接相互连接。在一些实施方案中，所述两个或更多个特异性结合CTLA4的免疫球蛋白单一可变结构域 通过接头相互连接。所述接头可以是长1-20或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如GGGGS、GS、GAP、(GGGGS)x 3等。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
(1)SEQ ID NO：1所示的CDR1，SEQ ID NO：2所示的CDR2，SEQ ID NO：3所示的CDR3(对应于抗体株116的CDR)；
(2)SEQ ID NO：4所示的CDR1，SEQ ID NO：5所示的CDR2，SEQ ID NO：6所示的CDR3(对应于抗体株119的CDR)；
(3)SEQ ID NO：7所示的CDR1，SEQ ID NO：8所示的CDR2，SEQ ID NO：9所示的CDR3(对应于抗体株128的CDR)；
(4)SEQ ID NO：10所示的CDR1，SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3(对应于抗体株138的CDR)；
(5)SEQ ID NO：13所示的CDR1，SEQ ID NO：14所示的CDR2，SEQ ID NO：15所示的CDR3(对应于抗体株145的CDR)；
(6)SEQ ID NO：16所示的CDR1，SEQ ID NO：17所示的CDR2，SEQ ID NO：18所示的CDR3(对应于抗体株155的CDR)；
(7)SEQ ID NO：19所示的CDR1，SEQ ID NO：20所示的CDR2，SEQ ID NO：21所示的CDR3(对应于抗体株165的CDR)；
(8)SEQ ID NO：22所示的CDR1，SEQ ID NO：23所示的CDR2，SEQ ID NO：24所示的CDR3(对应于抗体株188的CDR)；
(9)SEQ ID NO：25所示的CDR1，SEQ ID NO：26所示的CDR2，SEQ ID NO：27所示的CDR3(对应于抗体株C1的CDR)；
(10)SEQ ID NO：28所示的CDR1，SEQ ID NO：29所示的CDR2，SEQ ID NO：30所示的CDR3(对应于抗体株C2的CDR)；
(11)SEQ ID NO：31所示的CDR1，SEQ ID NO：32所示的CDR2，SEQ ID NO：33所示的CDR3(对应于抗体株C16的CDR)；
(12)SEQ ID NO：34所示的CDR1，SEQ ID NO：35所示的CDR2，SEQ ID NO：36所示的CDR3(对应于抗体株C22的CDR)；
(13)SEQ ID NO：37所示的CDR1，SEQ ID NO：38所示的CDR2，SEQ ID NO：39所示的CDR3(对应于抗体株C27的CDR)；
(14)SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2，SEQ ID NO：42所示的CDR3(对应于抗体株C29的CDR)；
(15)SEQ ID NO：43所示的CDR1，SEQ ID NO：44所示的CDR2，SEQ ID NO：45所示的CDR3(对应于抗体株C38的CDR)；
(16)SEQ ID NO：46所示的CDR1，SEQ ID NO：47所示的CDR2，SEQ ID NO：48所示的CDR3(对应于抗体株J5的CDR)；
(17)SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2，SEQ ID NO：51所示的CDR3(对应于抗体株J17的CDR)；
(18)SEQ ID NO：52所示的CDR1，SEQ ID NO：53所示的CDR2，SEQ ID NO：54所示的CDR3(对应于抗体株J29的CDR)；
(19)SEQ ID NO：55所示的CDR1，SEQ ID NO：56所示的CDR2，SEQ ID NO：57所示的CDR3(对应于抗体株J35的CDR)；
(20)SEQ ID NO：58所示的CDR1，SEQ ID NO：59所示的CDR2，SEQ ID NO：60所示的CDR3(对应于抗体株J37的CDR)；
(21)SEQ ID NO：61所示的CDR1，SEQ ID NO：62所示的CDR2，SEQ ID NO：63所示的CDR3(对应于抗体株J38的CDR)；
(22)SEQ ID NO：64所示的CDR1，SEQ ID NO：65所示的CDR2，SEQ ID NO：66所示的CDR3(对应于抗体株J39的CDR)；
(23)SEQ ID NO：67所示的CDR1，SEQ ID NO：68所示的CDR2，SEQ ID NO：69所示的CDR3(对应于抗体株J42的CDR)；
(24)SEQ ID NO：70所示的CDR1，SEQ ID NO：71所示的CDR2，SEQ ID NO：72所示的CDR3(对应于抗体株J69的CDR)；和
(25)SEQ ID NO：73所示的CDR1，SEQ ID NO：74所示的CDR2，SEQ ID NO：75所示的CDR3(对应于抗体株J78的CDR)。
在一些实施方案中，本发明的CTLA4结合蛋白中的至少一个免疫球蛋白单一可变结构域是VHH。在一些具体实施方案中，所述VHH包含SEQ ID NO：76-100中任一的氨基酸序列。在另一些实施方案中，所述VHH是人源化的VHH。所述人源化的VHH包含与SEQ ID NO：76-100中任一具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相同性的氨基酸序列。或者，所述VHH的氨基酸序列与SEQ ID NO：76-100中任一相比包含一或多个氨基酸取代，优选 保守氨基酸取代。例如，包含1、2、3、4、5、6、7、8、9或10个保守氨基酸取代。在一些具体实施方案中，所述人源化的VHH包含SEQ ID NO：101-109中任一的氨基酸序列。
在一些实施方案中，本发明的CTLA4结合蛋白是经过亲和力成熟获得的。经亲和力成熟的CTLA4结合蛋白可以在一个或多个CDR中具有一个或多个变化，所述变化导致对CTLA4的亲和力相比于亲本CTLA4结合蛋白有所增加。
在一些实施方案中，本发明的CTLA4结合蛋白，除了至少一个能够特异性结合CTLA4的免疫球蛋白单一可变结构域外，还包含免疫球蛋白Fc区。在本发明的CTLA4结合蛋白中包含免疫球蛋白Fc区可以使所述结合蛋白形成二聚体分子，同时延长所述结合蛋白的体内半衰期。可用于本发明的Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。免疫球蛋白Fc区一般而言包括免疫球蛋白恒定区的铰链区或部分铰链区、CH2区和CH3区。
在一些实施方案中，可以在野生型的Fc序列上引入突变用于改变Fc介导的相关活性。所述突变包括但不限于：a).改变Fc介导的CDC活性的突变；b).改变Fc介导的ADCC活性的突变；或c).改变FcRn介导的体内半衰期的突变。此类突变描述于下列文献中：Leonard G Presta，Current Opinion in Immunology 2008，20：460-470；Esohe E.Idusogie et al.，J Immunol 2000，164：4178-4184；RAPHAEL A.CLYNES et al.，Nature Medicine，2000，Volume 6，Number 4：443-446；Paul R.Hinton et al.，J Immunol，2006，176：346-356。例如，可以通过突变CH2区上的1、2、3、4、5、6、7、8、9或10个氨基酸用于增加或去除Fc介导的ADCC或CDC活性或是增强或减弱FcRn的亲和力。此外，可以通过突变铰链区的1、2、3、4、5、6、7、8、9或10个氨基酸增加蛋白的稳定性。
在一些实施方案中，Fc序列上可以引入突变，从而使得突变的Fc更容易形成同二聚体或者异二聚体。如Ridgway，Presta et al.1996以及Carter 2001中提到的利用Fc接触界面氨基酸侧链基团空间作用的knob-hole模型，使得不同Fc突变之间更容易形成异二聚体；再比如如CN 102558355A或者CN 103388013A中，通过改变Fc接触界面氨基酸 所带的电荷，进而改变Fc接触界面之间的离子相互作用力，使得不同的Fc突变对之间更容易形成异二聚体(CN 102558355A)，亦或是具有相同突变的Fc之间更容易形成同二聚体(CN 103388013A)。
所述免疫球蛋白Fc区优选是人免疫球蛋白Fc区，更优选是人IgG1的Fc区。在一些具体实施方案中，所述免疫球蛋白Fc区的氨基酸序列示于SEQ ID NO：132。在一些实施方案中，SEQ ID NO：132中N端的EPKSC可以被缺失，或被突变为EPKSS或MDPKSS。
在一些实施方案中，本发明的CTLA4结合蛋白中，能够特异性结合CTLA4的免疫球蛋白单一可变结构域与免疫球蛋白Fc区通过接头连接。所述接头可以是长1-20个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如GGGGS、GS、GAP等。
在一些实施方案中，本发明的CTLA4结合蛋白包含一个特异性结合CTLA4的免疫球蛋白单一可变结构域，其直接或通过接头与免疫球蛋白Fc区连接，所述免疫球蛋白Fc区允许所述CTLA4结合蛋白形成包含两个CTLA4结合结构域的二聚体分子。这样的CTLA4结合蛋白也称为二价CTLA4结合蛋白。在一些实施方案中，所述二聚体是同二聚体。
在一些实施方案中，本发明的CTLA4结合蛋白包含直接或通过接头相互连接的两个特异性结合CTLA4的免疫球蛋白单一可变结构域和一个免疫球蛋白Fc区，所述免疫球蛋白Fc区允许所述CTLA4结合蛋白形成包含四个CTLA4结合结构域的二聚体分子。这样的CTLA4结合蛋白也称为四价CTLA4结合蛋白。在一些实施方案中，所述二聚体是同二聚体。
在一些优选实施方案中，本发明的包含免疫球蛋白Fc区的CTLA4结合蛋白包含选自SEQ ID NO：114-128的氨基酸序列。
在另一方面，本发明的CTLA4结合蛋白还涵盖能够与由SEQ ID NO：76-100中任一的氨基酸序列组成的VHH结合CTLA4上的相同表位的抗CTLA4抗体分子。
本发明的CTLA4结合蛋白具有下述特征中的至少一个：
(a)结合人CTLA4的KD值小于1×10-7M；
(b)阻断CTLA4与CD80和/或CD86的相互作用；
(c)增强PBMC和T细胞的活化；
(d)抑制肿瘤生长。
本发明的所述CTLA4结合蛋白结合CTLA4的KD值可以小于1×10-7M，优选小于1×10-8M、更优选小于1×10-9M、更优选小于1×10-10M。
在一些实施方案中，本发明的CTLA4结合蛋白能够特异性结合人CTLA4并阻断CTLA4和CD80，和/或CTLA4和CD86的相互作用。
本发明的CTLA4结合蛋白能够抑制肿瘤生长至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。
此外，本发明的CTLA4结合蛋白对热处理具有抗性。例如，在40℃下处理多达30天未见明显聚集或降解。
最后，本发明的CTLA4结合蛋白在食蟹猴中表现出较好的耐受性。例如直至30mg/kg给药剂量，未观察到任何与药品相关的不良反应。
核酸、载体、宿主细胞
在另一方面中，本发明涉及编码本发明的CTLA4结合蛋白的核酸分子。本发明的核酸可为RNA、DNA或cDNA。根据本发明的一个实施方案，本发明的核酸是基本上分离的核酸。
本发明的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即可提供CTLA4结合蛋白体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本发明的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本发明的CTLA4结合蛋白的表达有用或必需的调控元件及其他元件的具体实例，例如启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本发明的核酸可基于关于本文给出的本发明的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
在另一方面中，本发明涉及表达或能够表达一种或多种本发明的 CTLA4结合蛋白和/或含有本发明的核酸或载体的重组宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本发明还提供产生本发明的CTLA4结合蛋白的方法，所述方法通常包含以下步骤：
-在允许表达本发明的CTLA4结合蛋白的条件下培养本发明的宿主细胞；及
-从培养物回收由所述宿主细胞表达的CTLA4结合蛋白；及
-任选进一步纯化和/或修饰本发明的CTLA4结合蛋白。
本发明的CTLA4结合蛋白可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本发明的CTLA4结合蛋白的方法中的蛋 白分离及纯化技术为本领域技术人员所公知。
然而，本发明的CTLA4结合蛋白也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
药物组合物
另一方面，本发明提供一种组合物，例如药物组合物，其含有与药学上可接受的载体配制在一起的一种或组合的本发明的CTLA4结合蛋白。这样的组合物可以包含一种或组合的(例如两种或多种不同的)本发明的CTLA4结合蛋白。例如，本发明的药物组合物可以含有结合靶抗原上的不同表位的抗体分子组合。
本发明的药物组合物也可以在联合治疗中施用，即与其他药剂联用。例如，联合治疗可包括本发明的CTLA4结合蛋白联合至少一种其他的抗肿瘤药物。例如，本发明的CTLA4结合蛋白可以与靶向其它肿瘤特异性抗原的抗体联合使用。所述靶向其它肿瘤特异性抗原的抗体包括但不限于，抗EGFR抗体、抗EGFR变体的抗体、抗VEGFa抗体、抗HER2抗体、或抗CMET抗体。优选所述抗体是单克隆抗体。本发明的CTLA4结合蛋白还可以与其它肿瘤免疫治疗手段、或肿瘤靶向性小分子类药物联用。所述其他肿瘤免疫治疗手段包括但不限于针对肿瘤免疫调节分子，如OX40、PDL1/PD1、CD137等的治疗型抗体，或CAR-T治疗手段等。
本发明的药物组合物也可以与其他肿瘤治疗手段，如放疗、化疗、手术治疗等联合使用，或是在放疗、化疗、或手术治疗之前或之后使用。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即抗体分子、免疫缀合物包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用。
本发明的药物组合物可包含一种或多种药学上可接受的盐。“药学上可接受的盐”是指保持了亲代化合物的所需生物活性而不引起任何不期望的毒理学作用的盐(参见如Berge，S.M.等(1977)J.Pharm.Sci.66：1-19)。这样的盐的例子包括酸加成盐和碱加成盐。酸加成盐包括那些由 诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等无毒性无机酸衍生的盐，以及由诸如脂族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳族酸、脂族和芳族磺酸等无毒性有机酸衍生的盐。碱加成盐包括那些由诸如钠、钾、镁、钙等碱土金属衍生的盐，以及由诸如N，N’-二苄基乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等无毒性有机胺衍生的盐。
本发明的药物组合物也可含有药学上可接受的抗氧化剂。药学上可接受的抗氧化剂的例子包括：(1)水溶性抗氧化剂，如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠，亚硫酸钠等；(2)油溶性抗氧化剂，如棕榈酸抗坏血酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚等；和(3)金属螯合剂，如柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等。
这些组合物还可含有佐剂，如防腐剂、润湿剂、乳化剂和分散剂。
可以通过灭菌程序或通过包含诸如对羟基苯甲酸酯、氯代丁醇、苯酚山梨酸等各种抗细菌剂和抗真菌剂确保防止存在微生物。在很多情况下，组合物中优选包含等渗剂，例如，糖、多元醇例如甘露糖醇、山梨糖醇或氧化钠。通过在组合物中加入延迟吸收剂，例如单硬脂酸盐和明胶，可实现注射型药物延长的吸收。
药学上可接受的载体包括无菌水溶液或分散液和用于临时制备无菌注射液或分散液的粉末剂。这些用于药学活性物质的介质和试剂的使用是本领域公知的。常规介质或试剂，除了任何与活性化合物不相容的范围外，都可能在本发明的药物组合物中。还可以向组合物中掺入补充的活性化合物。
治疗性组合物一般必须是无菌的并且在制备和贮存条件下稳定的。可以将组合物配制成溶液、微乳状液、脂质体或其他适合高药物浓度的有序结构。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液态聚乙二醇等)及其合适的混合物的溶剂或分散剂。例如，通过使用包衣，例如卵磷脂，在分散液的情况下通过保持所需的颗粒大小，以及通过使用表面活性剂，可以保持适当的流动性。
通过将活性化合物以需要的量混入合适的溶剂中，并且根据需要加入以上列举的成分中的一种或其组合，接着无菌微过滤，可制备无菌注 射液。通常，通过将活性化合物掺入到含有基本分散介质和上面所列其他所需成分的无菌载体中制备分散剂。对于用于制备无菌注射液的无菌粉末剂，优选的制备方法是真空干燥和冷冻干燥(冻干)，由其预先无菌过滤的溶液得到活性成分加任何额外所需成分的粉末。
可以与载体材料组合制备单一剂量形式的活性成分的量根据所治疗的对象和特定给药方式而不同。可以与载体材料组合制备单一剂量形式的活性成分的量一般是产生治疗效果的组合物的量。通常，以100％计，这个量的范围是大约0.01％至大约99％的活性成分，优选大约0.1％至大约70％，最优选大约1％至大约30％的活性成分，与药学上可接受的载体相组合。
可以调节剂量方案以提供最佳的期望的反应(例如，治疗反应)。例如，可以施用单一推注，可以随时间施用几次分开的剂量，或者根据治疗状况的紧急情况所需，可以按比例减小或增加剂量。特别有利的是将肠胃外组合物配制成容易给药并且剂量均匀的剂量单位形式。此处使用的剂量单位形式是指适合作为单位剂量用于所治疗的对象的物理不连续单位；每个单位含有预定量的活性化合物，经计算该预定量的活性化合物与需要的药物载体组合产生所需的治疗效果。对本发明剂量单位形式的具体说明限定于且直接依赖于(a)活性化合物的独特特性和要达到的特定治疗效果，和(b)本领域中固有的对于配制这种用于治疗个体敏感性的活性化合物的限制。
对于抗体分子的给药而言，剂量范围为约0.0001至100mg/kg，更通常为0.01至20mg/kg受者体重。例如，剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重、10mg/kg体重、20mg/kg体重或30mg/kg体重，或在1-30mg/kg范围内。示例性的治疗方案需要每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。
或者，抗体分子也可以作为持续释放制剂来给药，在此情况中需要频率较低的给药。剂量和频率根据抗体分子在患者中的半衰期而不同。通常，人抗体表现出最长的半衰期，之后是人源化抗体、嵌合抗体和非人类抗体。给药剂量和频率根据处理是预防性的还是治疗性的而不同。 在预防性应用中，在长时间内以较不频繁的间隔给予相对较低的剂量。有些患者在余生中持续接受处理。在治疗性应用中，有时需要以较短的间隔给予较高的剂量，直到疾病的进展减轻或停止，优选直到患者表现为疾病症状部分或完全改善。之后，可以以预防性方案给患者给药。
本发明药物组合物中活性成分的实际剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物或其酯、盐或酰胺的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
本发明的CTLA4结合蛋白的“治疗有效量”优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于CTLA4相关肿瘤的治疗，相对于未接受治疗的对象，“治疗有效量”优选地将细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型系统中评价。或者，也可以通过检查抑制细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。治疗有效量的治疗性化合物能够减小肿瘤大小，或者以其他方式缓解对象的症状，例如实现或延长肿瘤患者的无进展生存期，延长肿瘤患者的总生存期。本领域技术人员可以根据如下因素确定这种量，如对象的大小、对象症状的严重性和选择的特定组合物或给药途径。此外，本领域技术人员还能通过考察体外激活T细胞的能力来确定本发明的CTLA4结合蛋白的有效量。
本发明的组合物可以利用本领域公知的一种或多种方法通过一种或多种给药途径给药。本领域技术人员应当理解，给药途径和/或方式根据期望的结果而不同。本发明CTLA4结合蛋白的优选给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊柱或其他肠胃外给药途径，例如 注射或输注。本文使用的短语“肠胃外给药”是指除肠和局部给药以外的给药模式，通常是注射，包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。
或者，本发明的CTLA4结合蛋白也可以通过非肠胃外途径给药，如局部、表皮或粘膜途径给药，例如，鼻内、经口、阴道、直肠、舌下或局部。
活性化合物可以与保护化合物不被快速释放的载体一起制备，例如控释制剂，包括植入物、透皮贴剂和微胶囊递送系统。可以使用生物可降解的、生物相容的聚合物，例如乙烯乙酸乙烯酯、聚酐类、聚乙醇酸、胶原、聚原酸酯和聚乳酸。制备这样的制剂的很多方法受专利保护或者通常为本领域技术人员所公知。参见，例如，Sustainedand controlled Release Drug Delivery Systems，J.R.Robinson，ed.，Marcel Dekker，Inc.，New York，1978。
治疗性组合物可应用本领域公知的医疗装置给药。例如，在一个优选实施方案中，本发明的治疗组合物可用无针皮下注射装置给药，如在美国专利No.5,399,163；5,383,851；5,312,335；5,064,413；4,941,880；4,790,824；或4,596,556中公开的装置。可用于本发明的公知的植入物和模块的例子包括：美国专利No.4,487,603，该专利公开了用于以受控速率分散药物的可植入微量输注泵；美国专利No.4,486,194，该专利公开了用于通过皮肤给药的治疗装置；美国专利No.4,447,233，该专利公开了用于以精确的输注速率递送药物的医用输注泵；美国专利No.4,447,224，该专利公开了用于连续递送药物的变流可植入输注装置；美国专利No.4,439,196，该专利公开了具有多腔区室的渗透药物递送系统：和美国专利No.4,475,196，该专利公开了一种渗透药物递送系统。这些专利引入本文作为参考。本领域技术人员公知许多其他这样的植入物、递送系统和模块。
在某些实施方案中，本发明的CTLA4结合蛋白可配制为确保在体内的正确分布。例如，血-脑屏障(BBB)阻止了许多高度亲水性的化合物。为了确保本发明的治疗性化合物能够跨过BBB(如果需要时)，可将它们配制在如脂质体中。至于制备脂质体的方法，参见，例如，美国专利 4,522,811；5,374,548和5,399,331。脂质体包含可被选择性地转运入特定细胞或器官内的一个或多个靶向部分，从而增强靶向药物递送(参见，例如，V.V.Ranade(1989)J.Clin.Pharmacol.29：685)。靶向部分的例子包括叶酸或生物素(参见，例如，Low等的美国专利5,416,016)；甘露糖苷(Umezawa等(1988)Biochem.Biophys.Res.Commun.153：1038)；抗体(P.G.Bloeman等(1995)FEBS Lett.357：140；M.Owais等(1995)Antimicrob.Agents Chemother.39：180)；表面活性剂蛋白A受体(Briscoe等(1995)Am.J.Physiol.1233：134)；p120(Schreier等(1994)J.Biol.Chem.269：9090)；也参见K.Keinanen；M.L.Laukkanen(1994)FEBS Lett.346：123；J.J.Killion；I.J.Fidler(1994)Immunomethods 4：273。
疾病预防和治疗
在另一方面，本发明提供了本发明所述CTLA4结合蛋白、核酸分子、宿主细胞及药物组合物在预防和/或治疗与CTLA4相关的疾病中用途和方法。可用本发明的CTLA4结合蛋白预防和/或治疗的CTLA4相关的疾病如下详述。
癌症
本发明的CTLA4结合蛋白对CTLA4的阻断可以增强患者中对癌细胞的免疫应答。本发明的CTLA4结合蛋白可以单独使用，以抑制癌性肿瘤的生长。或者如以下所述，本发明的CTLA4结合蛋白可以与其它抗肿瘤治疗手段联合使用，例如与其他免疫原性剂、标准癌症疗法或其他抗体分子联合使用。
因此，在一个实施方案中，本发明提供一种预防和/或治疗癌症的方法，包括给该对象施用治疗有效量的本发明的CTLA4结合蛋白，抑制对象中的肿瘤细胞生长。
使用本发明的CTLA4结合蛋白可以预防和/或治疗的优选的癌症包括一般对免疫治疗有应答的癌症。可治疗的优选癌症的非限制性的例子包括肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤(例如转移的恶性黑色素瘤)、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本发明的方 法治疗的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病，包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。
任选地，本发明的CTLA4结合蛋白可以与免疫原性剂如癌细胞、纯化的肿瘤抗原(包括重组蛋白、肽和碳水化合物分子)、用编码免疫刺激细胞因子的基因转染的细胞联用(He等(2004)J.Immunol 173：4919-28)。可以应用的免疫原性剂的非限制性实例包括黑素瘤抗原的肽，如gp100的肽、MAGE抗原、Trp-2、MART1和/或酪氨酸酶，或转染后表达细胞因子GM-CSF的肿瘤细胞。
在人类中，已经表明一些肿瘤是具有免疫原性的，如黑素瘤。预期通过使用本发明的CTLA4结合蛋白阻断CTLA4来促进T细胞活化，可以激活宿主中的肿瘤应答。当与肿瘤接种方案联合时，CTLA4阻断剂(如抗CTLA4抗体，例如本发明的CTLA4结合蛋白)可能最有效。已经设计了针对肿瘤接种的许多实验策略(参见Rosenberg，S.，2000，Development of Cancer Vaccines，ASCO Educational Book Spring：60-62；Logothetis，C，2000，ASCO Educational Book Spring：300-302；Khayat，D.2000，ASCO Educational Book Spring：414-428；Foon，K.2000，ASCO Educational Book Spring：730-738；参见Restifo，N.和Sznol，M.，Cancer Vaccines，Ch.61，pp.3023-3043，DeVita，V.等(eds.)，1997，Cancer：Principles and Practice of Oncology.第五版)。在这些策略之一中，使用自体或异体肿瘤细胞制备疫苗。已经证明，当肿瘤细胞被转导而表达GM-CSF时，这些细胞疫苗最有效。已经表明GM-CSF是用于肿瘤接种的抗原呈递的强激活剂(Dranoff等(1993)Proa Natl.Acad.Sci U.S.A.90： 3539-43)。
各种肿瘤中基因表达和大规模基因表达模式的研究鉴定出许多所谓的肿瘤特异性抗原(Rosenberg，SA(1999)Immunity 10：281-7)。在许多情况下，这些肿瘤特异性抗原是在肿瘤和产生肿瘤的细胞中表达的分化抗原，例如gp100、MAGE抗原和Trp-2。更重要的是，证明这些抗原中的许多是在宿主中发现的肿瘤特异性T细胞的靶标。本发明的CTLA4结合蛋白可以与重组产生的肿瘤特异性蛋白和/或肽组合使用，以产生针对这些蛋白质的免疫应答。这些蛋白质在正常情况下被免疫系统看作自身抗原，因此对其耐受。肿瘤抗原也可以包括蛋白质端粒酶，该酶是染色体的端粒合成所必需的，并且在85％以上的人类癌中表达，而仅在有限数量的自身组织中表达(Kim，N等(1994)Science 266：2011-2013)。肿瘤抗原也可以是癌细胞表达的“新抗原”，例如由于体细胞突变改变蛋白质序列或产生两种无关序列的融合蛋白(例如，Philadelphia染色体中的bcr-abl)。
其他肿瘤疫苗可以包括来自与人类癌症有关的病毒的蛋白质，如人类乳头瘤病毒(HPV)、肝炎病毒(HBV和HCV)和卡波西疱疹肉瘤病毒(KHSV)。可以与CTLA4阻断剂(如抗CTLA4抗体，例如本发明的CTLA4结合蛋白)联合应用的另外一种形式的肿瘤特异性抗原是从肿瘤组织本身中分离的纯化的热休克蛋白(HSP)。这些热休克蛋白含有来自肿瘤细胞的蛋白质的片段，这些HSP在向抗原呈递细胞递送以引发肿瘤免疫方面非常有效(Suot，R和Srivastava，P(1995)Science 269：1585-1588；Tamura，Y.等(1997)Science 278：117-120)。
树突细胞(DC)是强抗原呈递细胞，可以用来引发抗原特异性应答。DC可以在体外产生，并且载有各种蛋白质和肽抗原以及肿瘤细胞提取物(Nestle，F.等(1998)Nature Medicine 4：328-332)。DC也可以通过遗传手段转导，从而也表达这些肿瘤抗原。已经为了免疫而直接将DC融合到肿瘤细胞上(Kugler，A.等(2000)Nature Medicine 6：332-336)。作为接种方法，DC免疫可以与CTLA4阻断剂(如抗CTLA4抗体，例如本发明的CTLA4结合蛋白)有效地组合，以激活更强的抗肿瘤应答。
CAR-T，全称是嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor T-Cell Immunotherapy)是另一种有效的恶性肿瘤的细胞治疗方 法。嵌合抗原受体T细胞(CAR-T细胞)是将能识别某种肿瘤抗原的抗体的抗原结合部与CD3-ζ链或FcεRIγ的胞内部分在体外偶联为一个嵌合蛋白，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。同时，还可以引入共刺激分子信号序列以提高T细胞的细胞毒活性、增殖性与存活时间，促进细胞因子的释放。患者的T细胞被“重编码”后，可在体外扩增生成大量肿瘤特异性的CAR-T细胞并回输患者体内，实现肿瘤治疗的目的。CTLA4阻断剂(如抗CTLA4抗体，例如本发明的CTLA4结合蛋白)可以与CAR-T细胞疗法联合，激活更强的抗肿瘤应答。
本发明的CTLA4结合蛋白也可以与标准癌症治疗组合。本发明的CTLA4结合蛋白可以与化疗方案有效地组合。本发明的CTLA4结合蛋白和化学疗法联用的科学原理是细胞死亡，这是大多数化疗化合物的细胞毒性作用的结果，应会导致抗原呈递途径中的肿瘤抗原水平升高。可以通过细胞死亡与CTLA4阻断协同作用的其他联合治疗有放疗、手术和激素剥夺。这些方案都在宿主中产生肿瘤抗原的来源。血管发生抑制剂也可以与本发明的CTLA4结合蛋白组合。血管发生的抑制导致肿瘤细胞死亡，这可以将肿瘤抗原提供给宿主的抗原呈递途径。
本发明的CTLA4结合蛋白还可以与靶向其它肿瘤特异性抗原的抗体联合使用。所述靶向其它肿瘤特异性抗原的抗体包括但不限于，抗EGFR抗体、抗EGFR变体的抗体、抗VEGFa抗体、抗HER2抗体、或抗CMET抗体。优选所述抗体是单克隆抗体。
本发明的CTLA4结合蛋白也可以与将Fcα或Fcγ受体表达效应细胞靶向至肿瘤细胞的双特异性抗原联合应用(参见，例如US Patent Nos.5,922.845和5,837,243)。也可以利用双特异性抗体靶向两种不同的抗原。例如，已经利用抗-Fc受体/抗肿瘤抗原(例如Her-2/neu)双特异性抗体将巨噬细胞靶向肿瘤部位。这种靶向可以更有效地激活肿瘤特异性应答。利用CTLA4阻断剂可以加强这些应答的T细胞方面。或者，可以利用结合肿瘤抗原和树突细胞特异性细胞表面标记的双特异性抗体将抗原直接递送至DC。
肿瘤通过多种机制逃避宿主的免疫监视。其中许多机制可以通过灭活肿瘤表达的免疫抑制性蛋白质来克服。尤其包括TGF-β(KehrL J.等 (1986)J.Exp.Med.163：1037-1050)、IL-10(Howard，M.和O′Garra，A.(1992)Immunology Today 13：198-200)和Fas配体(Hahne，M.等(1996)Science 274：1363-1365)。其中每种的抗体可以与本发明的CTLA4结合蛋白联用，来抵抗免疫抑制剂的作用，并且有利于宿主的肿瘤免疫应答。
可以用于激活宿主免疫应答的其他抗体可以与本发明的CTLA4结合蛋白联用。抗-CD40抗体能够有效地替代T细胞辅助活性(Ridge，J.等(1998)Nature 393：474-478)，并且可以与本发明的CTLA4结合分子联用。也可以为了提高T细胞活化的水平而联合对T细胞共刺激分子如OX-40(Weinberg，A.等(2000)Immunol 164：2160-2169)、4-1BB(Melero，I.等(1997)Nature Medicine 3：682-685(1997)和ICOS(Hutloff，A.等(1999)Nature 397：262-266)的活化抗体以及阻断阴性共刺激分子如CTLA-4(例如，美国专利No.5,811,097)或BTLA(Watanabe，N.等(2003)Nat Immunol 4：670-9)、B7-H4(Sica，GL等(2003)Immunity 18：849-61)的活性的抗体。
骨髓移植当前用来治疗造血来源的多种肿瘤。移植物抗宿主疾病是这种治疗的一种后果，移植物对抗肿瘤的应答可以获得治疗性益处。可以利用CTLA4阻断剂提高肿瘤特异性T细胞的有效性。也有几种实验治疗方案涉及抗原特异性T细胞的离体激活和扩增以及这些细胞向受体内的过继转移，以用抗原特异性T细胞对抗肿瘤(Greenberg，R.和Riddell，S.(1999)Science 285：546-51)。这些方法也可以用来激活T细胞对传染原如CMV的应答。预期在本发明的CTLA4结合蛋白存在下离体激活可以提高过继转移的T细胞的频率和活性。因此，本发明还提供了一种离体激活免疫细胞(如PBMC或T细胞)的方法，包括使所述免疫细胞与本发明的CTLA4结合蛋白接触。
感染性疾病
本发明的其他方法用于治疗暴露于特定毒素或病原体的患者。因此，本发明的另一方面提供一种预防和/或治疗对象中的感染性疾病的方法，包括给该对象施用本发明的CTLA4结合蛋白，使得所述对象的感染性疾病得到预防和/或治疗。
类似于对于如上所述的肿瘤的应用，CTLA4阻断剂可以单独使用， 或者作为佐剂与疫苗组合使用来刺激对病原体、毒素和自身抗原的免疫应答。特别可以应用该治疗方法的病原体的实例包括当前没有有效疫苗的病原体，或常规疫苗不完全有效的病原体。其中包括但不限于HIV、肝炎病毒(甲、乙、丙)、流感病毒、疱疹病毒、贾第虫、疟疾、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。CTLA4阻断剂特别可用于对抗诸如HIV等病原体已建立的感染，其在感染过程中呈现改变的抗原。在抗人CTLA4抗体给药时，这些新的表位被作为外源物识别，从而引起不受CTLA4的负信号影响的强T细胞应答。
引起可用本发明的方法治疗的感染性疾病的病原体病毒的一些实例包括HIV、肝炎(甲、乙、丙)、疱疹病毒(例如VZV、HSV-1、HAV-6，HSV-II和CMV、EB病毒)、腺病毒、流感病毒、虫媒病毒、埃可病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、流行性腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革热病毒、乳头瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病毒、JC病毒和虫媒病毒脑炎病毒。
引起可用本发明的方法治疗的感染性疾病的病原体细菌的一些实例包括衣原体、立克次氏体菌、分枝杆菌、葡萄球菌、链球菌、肺炎球菌、脑膜炎球菌和淋球菌、克雷伯氏杆菌、变形菌、雷氏菌、假单胞菌、军团杆菌、白喉杆菌、沙门氏菌、芽孢杆菌、霍乱菌、破伤风菌、肉毒杆菌、炭疽杆菌、鼠疫杆菌、钩端螺旋体、和莱姆病细菌。
引起可用本发明的方法治疗的感染性疾病的病原体真菌的一些实例包括假丝酵母(白假丝酵母、克鲁斯假丝酵母、光滑假丝酵母、热带假丝酵母等)、新型隐球菌、曲霉属(烟曲霉、黑曲霉等)、毛霉属(毛霉、犁头霉、根霉)、申克孢子丝菌、皮炎芽生菌、巴西副球孢子菌、粗球孢子菌和夹膜组织胞浆菌。
引起可用本发明的方法治疗的感染性疾病的病原体寄生虫的一些实例包括溶组织内阿米巴、结肠小袋纤毛虫、福氏耐格里阿米巴、棘阿米巴属的种、兰伯贾第虫、隐孢子虫属的种、卡氏肺囊虫、间日疟原虫、果氏巴贝虫、布氏锥虫、克氏锥虫、杜氏利什曼原虫、鼠弓形体、巴西日圆线虫。
在所有上述的方法中，CTLA4阻断剂可以与其他形式的免疫疗法如 细胞因子治疗(例如干扰素、GM-CSF、G-CSF、IL-2)或双特异性抗体治疗联合，双特异性抗体治疗提供增强的肿瘤抗原的呈递(参见，例如，Holliger(1993)Proc.Natl.Acad.Sci.USA 90：6444-6448；Poljak(1994)Structure 2：1121-1123)。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1：针对CTLA4的重链单域抗体的筛选
1.1文库的构建及筛选：
免疫用的CTLA4-Fc融合蛋白(SEQ ID NO：129)由HEK293细胞表达(pCDNA4，Invitrogen，Cat V86220)，经Protein A亲和层析纯化得到。选取一只新疆双峰驼(Camelus bactrianus)进行免疫。4次免疫结束后，提取骆驼100ml外周血的淋巴细胞并使用QIAGEN公司提供的RNA提取试剂盒提取总RNA，使用Super-Script III FIRST STRANDSUPERMIX试剂盒按照说明书将提取的RNA反转录成cDNA。用巢式PCR扩增编码重链抗体的可变区的核酸片段：
第一轮PCR：
上游引物：GTCCTGGCTGCTCTTCTACAAGGC(SEQ ID NO：110)；
下游引物：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO：111)。
第二轮PCR：
以第一轮PCR产物作模板，
上游引物：GATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO：112)；
下游引物：GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO：113)。
回收目标重链单域抗体核酸片段，并使用限制性内切酶(购自NEB)PstI及NotI将其克隆进入噬菌体展示用载体pCDisplay-3(Creative Biolabs，Cat：VPT4023)中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对CTLA4的重链单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀 释铺板，计算库容的大小为约为108。为检测文库的插入率，随机选取50个克隆做菌落PCR。结果显示插入率大于99％。
1.2针对CTLA4的重链单域抗体淘选：
用CTLA4-Fc融合蛋白以及Fc蛋白5μg/孔包被平板，4℃放置过夜。第二天用1％脱脂奶室温封闭2小时后，先在Fc包被的孔中加入100μl噬菌体(8×1011tfu，来自1.1所构建的骆驼重链单域抗体噬菌展示文库)，在室温下作用1小时。之后用PBST(PBS中含有0.05％吐温20)淘洗，将不结合Fc的噬菌体再次移入CTLA4-Fc融合蛋白包被的孔中，室温下作用1小时。最后用三乙基胺(100mM)将与CTLA4特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复3-4轮。由此，阳性的克隆被富集，达到了利用噬菌体展示技术筛取抗体库中CTLA4特异抗体的目的。
1.3用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆
3轮淘选后，获得的CTLA4结合阳性的噬菌体感染空白大肠杆菌并铺板。随后挑选96个单菌落分别培养，生产并纯化噬菌体。用CTLA4-Fc融合蛋白包被平板4℃过夜，将获得的样品噬菌体(对照组为空白噬菌体)加入，室温下反应1小时。洗涤之后加入一抗小鼠抗-HA标签抗体(购自北京康为世纪生物科技有限公司)，室温反应1小时。洗涤之后加入二抗山羊抗-小鼠碱性磷酸酶标记抗体(购自艾美捷科技有限公司)，室温反应1小时。洗涤之后加入碱性磷酸酶显色液，405nm波长读取吸收值。当样品孔OD值大于对照孔OD值3倍以上时，判为阳性克隆孔。将阳性克隆孔的菌转移至含有100微克每毫升氨苄霉素的LB液体中培养以便提取质粒并进行测序。
根据序列比对软件Vector NTI分析各个克隆的蛋白序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株，同时排除提前终止的序列。最终共获得34株不同的抗体序列。
实施例2：针对CTLA4的重链单域抗体的初步评价鉴定
2.1重链单域抗体在宿主菌大肠杆菌中表达、纯化：
将测序分析所获得34株重链单域抗体的编码序列亚克隆至表达载体PET32b(Novagen，产品号：69016-3)中，并将测序鉴定正确的重组质粒转化到表达型宿主菌BL1(DE3)(天根生化科技，产品号：CB105-02)中，其涂布在含有100微克每毫升氨苄青霉素的LB固体培养基的板上，37℃过夜。挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，37℃摇床培养至OD值达到0.6-1时，0.5mM IPTG诱导，28℃摇床培养过夜。第二天，离心收菌，并将菌体破碎以获得抗体粗提液。然后镍离子亲和层析柱纯化抗体蛋白。最终得到纯度达90％以上的抗体蛋白。
2.2检测候选CTLA4重链单域抗体对人CTLA4蛋白的特异性结合
用CTLA4-Fc融合蛋白包被平板4℃过夜，每孔加入10ng实施例2.1所得的重链单域抗体(对照组为不结合CTLA4-Fc蛋白的单域抗体)，室温下反应1小时。洗涤之后加入一抗小鼠抗-His标签抗体(购自北京康为世纪生物科技有限公司)，室温反应1小时。洗涤之后加入二抗山羊抗小鼠辣根过氧化物酶标记抗体(义翘神州，Cat：SSA007200)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。
用Fc蛋白包被平板4℃过夜，每孔加入10ng实施例2.1所得的重链单域抗体(对照组为针对其他不相关靶标的单域抗体)，室温下反应1小时。洗涤之后加入一抗兔抗人Fc抗体(购自上海普欣生物技术有限公司)，室温反应1小时。洗涤之后加入二抗山羊抗兔辣根过氧化物酶标记抗体(购自上海普欣生物技术有限公司)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。
其中当对CTLA4-Fc蛋白的OD值除以空白对照OD值的比值＞＝4时，判断候选抗体能结合CTLA4-Fc蛋白；同时上述能结合CTLA4-Fc抗原蛋白的抗体，其结合CTLA4-Fc的OD值除以其结合Fc蛋白的OD值，比值＞＝5时，则认为该候选抗体能特异性结合CTLA4部分而非Fc部分。
结果显示筛选出的34株抗体中，有25株(斜体加粗标出)能特异性结合CTLA4，而不与Fc结合。具体结果见下表1：
表1
抗体株 ODa(对CTLA4-Fc) ODb(对Fc) ODa/ODb ODa/OD空白 SEQ ID NO
C1 1.201 0.053 42.9 22.7 84
抗体株 ODa(对CTLA4-Fc) ODb(对Fc) ODa/ODb ODa/OD空白 SEQ ID NO
C2 1.231 0.035 44.0 35.2 85
C16 1.786 0.053 63.8 33.7 86
C22 0.848 0.06 30.3 14.1 87
C23 0.951 1.057 34.0 0.9  
C24 0.231 0.114 8.3 2.0  
C25 0.091 0.03 3.3 3.0  
C27 1.31 0.049 46.8 26.7 88
C29 1.513 0.085 54.0 17.8 89
C34 1.022 0.945 36.5 1.1  
C38 1.022 0.045 36.5 22.7 90
C46 0.621 0.241 22.2 2.6  
J5 1.21 0.036 43.2 33.6 91
J17 1.4 0.046 50.0 30.4 92
J24 0.032 0.041 1.1 0.8  
J29 1.439 0.036 51.4 40.0 93
J34 0.932 1.023 33.3 0.9  
J35 0.823 0.036 29.4 22.9 94
J37 1.201 0.034 42.9 35.3 95
J38 1.031 0.752 36.8 1.4 96
J39 1.747 0.042 62.4 41.6 97
J41 0.055 0.064 2.0 0.9  
J42 1.086 0.045 38.8 24.1 98
J47 0.062 0.041 2.2 1.5  
J69 0.635 0.037 22.7 17.2 99
J78 0.632 0.121 22.6 5.2 100
116 0.234 0.046 8.4 5.1 76
119 0.537 0.037 19.2 14.5 77
128 1.268 0.036 45.3 35.2 78
138 1.843 0.041 65.8 45.0 79
145 1.487 0.065 53.1 22.9 80
155 1.724 0.051 61.6 33.8 81
165 1.214 0.051 43.4 23.8 82
188 0.945 1.021 33.8 0.9 83
空白 0.028 0.035 0.8 0.8  
2.3竞争ELISA考察CTLA4重链单域抗体对CD80与CTLA4相互作用的阻断效果
CTLA4-Fc蛋白与CD80-Fc蛋白(SEQ ID NO：130)由HEK293细胞表达获得(pCDNA4，Invitrogen，Cat V86220)。利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白CD80-Fc-Biotin。
CTLA4-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入500ng实施例2.2所确认的与CTLA4特异性结合的重链单域抗体(对照组为针对其 他不相关靶标的单域抗体，或只是缓冲液)以及25ng CD80-Fc-Biotin(空白组不加入任何抗体或蛋白，只加入等体积缓冲液)，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)，室温反应1小时。之后加入显色液，405nm波长读取吸收值。当样品OD值比对照OD值＜0.8时，则认为抗体有阻断效果。
结果如表2所示，抗体株C1、C16、C27、J37、J42、128、145、155、165均表现出对CD80/CTLA4相互作用的阻断效应。
表2
样品 OD 样品 OD
对照1 2.453 J38 2.357
对照2 2.391 J39 2.133
空白 0.016 J42 0.94
C1 0.054 J5 2.504
C16 1.893 J69 2.343
C2 2.517 J78 2.413
C22 2.474 116 2.579
C27 0.04 119 2.534
C29 2.431 128 0.893
C38 2.284 138 1.918
J17 2.528 145 0.597
J29 2.502 155 0.746
J35 2.57 165 1.838
J37 1.061 188 2.633
2.4 CTLA4重链单域抗体对小鼠CTLA4蛋白的结合情况
小鼠CTLA4的Fc融合蛋白(SEQ ID NO：131)由HEK293细胞表达获得(pCDNA4，Invitrogen，Cat V86220)。利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白mCTLA4-Fc-Biotin。
用实施例2.1所得的重链单域抗体(对照组为针对其他不相关靶标的单域抗体)，根据实施例2.3的结果，选取阻断效果较好的4个抗体145、155、C1以及C27，0.5μg/孔包被平板4℃过夜，每孔加入100μg小鼠CTLA4-Fc融合蛋白室温下反应1.5小时。之后加入SA-HRP(购自Sigma公司)，室温反应1.5小时。洗涤之后加入显色液，405nm波长读取吸收值。结果见表3。
表3
抗体株 OD(对小鼠CTLA4)
145 0.026
155 0.032
C1 0.042
C27 0.021
对照 0.026
空白 0.027
可见，本发明的人CTLA4的重链单域抗体不结合小鼠CTLA4-Fc蛋白。
2.5 CTLA4重链单域抗体对CTLA4-Fc抗原蛋白的结合曲线
用得到的CTLA4重链单域抗体0.5μg/孔4℃过夜包被平板，随后加入CTLA4-Fc融合蛋白的梯度稀释系列，室温下反应1小时。洗涤之后加入山羊抗人IgG-Fc辣根过氧化物酶标记抗体(lakepharma)，室温反应1小时。洗涤之后加入辣根过氧化物酶显色液，405nm波长读取吸收值。应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对CTLA4结合曲线及EC50值，其中抗体株14约50ng/mL，155约13ng/mL，C1约为123ng/mL，C27约为93ng/mL。145和155结果见图1A，C1和C27结果见图1B。
2.6 CTLA4重链单域抗体对CD80与CTLA4相互作用的阻断曲线
用CTLA4-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入100uL实施例2.1所得的CTLA4阻断型单域抗体的梯度稀释系列(稀释液中含有250ng/mL CD80-Fc-Biotin)，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)，室温反应1小时。之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体株C1，C27，145，155对CD80/CTLA4阻断曲线及IC50值，其中抗体株C1的IC50为852ng/mL，抗体株C27约为731ng/ml，抗体株145约为1.947μg/ml，抗体株155约为4.690μg/ml。145和155结果见图2A，C1和C27结果见图2B。
2.7制备CTLA4单域抗体的Fc融合蛋白
根据蛋白数据库Uniprot上人免疫球蛋白γ1(IgG1)的恒定区氨基酸序列(P01857)，得到人IgG1-Fc区氨基酸序列(SEQ ID NO：132)。通过逆转录 PCR，从人PBMC总RNA中获得编码人IgG1-Fc的核酸片段，再通过重叠PCR得到由上述实施例获得的CTLA4单域抗体与Fc的融合蛋白的编码核酸片段。之后再亚克隆至载体pCDNA4(Invitrogen，Cat V86220)。
重组构建的单域抗体-Fc融合蛋白质粒转染HEK293细胞进行抗体表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10％CO2，90rpm中培养；同时补加50μg/L IGF-1。四小时后再补加EX293培养基，2mM谷氨酰胺和50μg/L IGF-1，135rpm培养。24小时后加3.8mM VPA。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，纯化得到目标CTLA4单域抗体-Fc融合蛋白。
抗体株C1、C27、145、155的Fc融合蛋白的序列分别示于SEQ ID NO：114-117。
2.8 CTLA4重链单域抗体Fc融合蛋白对CD80与CTLA4相互作用的阻断曲线
用CTLA4-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入100uL实施例2.7所得的CTLA4阻断型单域抗体Fc融合蛋白的梯度稀释系列(稀释液中含有250ng/mL CD80-Fc-Biotin)，室温下反应1.5小时。之后加入SA-HRP(购自Sigma公司)，室温反应1.5小时。之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体株145，155的Fc融合蛋白对CD80/CTLA4阻断曲线及IC50值，其中抗体株145的IC50值约为339ng/ml，抗体株155约为261ng/ml。结果见图3。
实施例3：CTLA4单域抗体的人源化
人源化方法采用蛋白表面氨基酸人源化(resurfacing)的方法及VHH人源化通用框架移植法(CDR grafting to a universal framework)完成。
人源化步骤如下：对抗体株C27和C1进行同源建模，建模软件为Modeller9。参考同源序列为cAb-Lys3抗体(PDB编号为：1XFP)，并根据蛋白三维结构计算氨基酸的相对溶剂可及性(relative solvent accessibility)。 如抗体株C27和C1的某个氨基酸暴露在溶剂内，则替换为参考人抗体DP-47序列相同位置的氨基酸，最终完成全部替换。
对抗体株C27进行人源化，获得5种抗体株C27的人源化变体；对抗体株C1进行人源化，获得4种抗体株C1的人源化变体。表4列出这些人源化变体的序列编号以及其中的氨基酸变化，其中氨基酸残基编号比照Kabat编号。图4a显示C27人源化序列的比对结果，4b显示C1人源化序列的比对结果。
表4
Figure PCTCN2017085038-appb-000003
实施例4：用哺乳动物细胞制备CTLA4阻断型抗体蛋白
4.1制备CTLA4单域抗体的Fc融合蛋白
根据蛋白数据库Uniprot上人免疫球蛋白γ1(IgG1)的恒定区氨基酸序列(P01857)，得到人IgG1-Fc区氨基酸序列(SEQ ID NO：132)。通过逆转录PCR，从人PBMC总RNA中获得编码人IgG1-Fc的核酸片段，再通过重叠PCR得到由上述实施例获得的CTLA4单域抗体与Fc的融合蛋白的编码核酸片段。之后再亚克隆至载体pCDNA4(Invitrogen，Cat V86220)。
重组构建的单域抗体-Fc融合蛋白质粒转染HEK293细胞进行抗体表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10％CO2，90rpm中培养；同时补加50μg/L IGF-1。四小时后再补加EX293培养基，2mM谷氨酰胺和50μg/L IGF-1，135rpm培养。24小时后加3.8mM VPA。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，纯化得到目标CTLA4单域抗体-Fc融合蛋白。
所获得的CTLA4单域抗体-Fc融合蛋白的序列分别示于SEQ ID NO：114-126，其中SEQ ID NO：118-126是人源化CTLA4单域抗体的Fc融合蛋 白。这些融合蛋白包含一个CTLA4结合结构域并形成同二聚体，其形成的二聚体分子每个包含两个CTLA4结合结构域，因此这些融合蛋白也称作二价CTLA4单域抗体-Fc融合蛋白。
4.2制备BMS公司的CTLA4抗体
BMS公司的CTLA4抗体ipilimumab，参照专利US20020086041中抗体10D1的方法克隆抗体基因，并将其克隆进载体pCDNA4中。
重组构建的质粒通过4.1相同的方法进行HEK293细胞的瞬时转染表达，得到的BMS公司的CTLA4抗体重新命名为10D1。
4.3 CTLA4单域抗体Fc融合蛋白与已知CTLA4抗体表达结果比较
利用相同的表达体系和瞬时转染条件，本发明的CTLA4单域抗体Fc融合蛋白的表达水平均高于400mg/L，而抗体10D1表达水平约为150mg/L。该结果表明，本发明的CTLA4单域抗体Fc融合蛋白相比已知CTLA4抗体，其结构更为稳定，能够获得更高表达水平。
4.4制备四价CTLA4单域抗体Fc融合蛋白
通过重叠PCR得到两个串联的CTLA4单域抗体与Fc的融合蛋白的编码核酸片段。之后再亚克隆至载体pCDNA4(Invitrogen，Cat V86220)。重组构建的质粒通过与4.1相同的方法进行HEK293细胞的瞬时转染表达，得到包含两个CTLA4单域抗体以及一个Fc的融合蛋白，其形成每个分子上包含了四个CTLA4结合结构域的二聚体分子，因此该融合蛋白也称作四价CTLA4单域抗体-Fc融合蛋白。
该四价CTLA4单域抗体Fc融合蛋白的瞬时转染表达水平均约400mg/L，与二价CTLA4单域抗体Fc融合蛋白相当。
实施例5：鉴定CTLA4单域抗体Fc融合蛋白的功能
5.1 CTLA4单域抗体Fc融合蛋白对CTLA4结合能力鉴定(ELISA法)
实施例2.7以及4.1得到的CTLA4单域抗体Fc融合蛋白或实施例4.2得到的10D1蛋白0.5μg/孔4℃过夜包被平板，之后加入的CTLA4-Fc-Biotin梯度稀释系列，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)， 室温反应1.5小时。之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对CTLA4结合曲线及EC50值(所有供试抗体EC50值均为约60～70ng/mL)以反映抗体对CTLA4的亲和能力。
结果见图5，其中纵坐标为OD405，横坐标为CTLA4单域抗体Fc融合蛋白(或10D1蛋白)浓度(单位ng/mL)；抗体株C27的四种不同人源化形式的Fc融合蛋白标注为：huC27v1-LdFc(SEQ ID NO：118)、huC27v2-LdFc(SEQ ID NO：119)、huC27v3-LdFc(SEQ ID NO：120)、huC27v4-LdFc(SEQ ID NO：121)。四种蛋白对CTLA4的亲和力相当，且与未人源化的C27-LdFc以及已知BMS的CTLA4抗体10D1相当。
5.2 CTLA4单域抗体Fc融合蛋白对CTLA4结合能力鉴定(ELISA法)
实施例2.7以及4.1得到的CTLA4单域抗体Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入的CTLA4-Fc-Biotin梯度稀释系列，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)，室温反应1.5小时。之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对CTLA4结合曲线及EC50值(所有供试抗体EC50值均为约25～35ng/mL)以反映抗体对CTLA4的亲和能力。
结果见图6，其中纵坐标为OD405，横坐标为CTLA4单域抗体Fc融合蛋白浓度(单位ng/mL)；三角代表抗体株C27的人源化形式的Fc融合蛋白huC27v3-LdFc(SEQ ID NO：120)，方形代表抗体株C1的人源化形式的Fc融合蛋白huC1v4-Ld-Fc(SEQ ID NO：126)，圆圈代表抗体株C1的Fc融合蛋白C1-ld-Fc。其中C1-ld-Fc由于反应时间过长，其显色比其他两种都高出不少，因此表现出EC50更优。但是实际上三种蛋白对CTLA4的亲和力基本没有差别。
5.2鉴定CTLA4单域抗体Fc融合蛋白对CTLA4-CD80相互作用的阻断效果(竞争ELISA法)
CTLA4-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入上述实施例4.1获得的CTLA4单域抗体Fc融合蛋白以及实施例4.2获得的10D1抗体 蛋白的梯度稀释系列，每孔100uL(稀释液中含有250ng/mLCD80-Fc-Biotin)，室温下反应1小时。洗涤之后加入SA-HRP(购自Sigma公司)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对CTLA4-CD80阻断曲线及IC50值。结果见图7A以及图7B。可以看出两株不同的单域抗体C27和C1，无论是人源化或是最初的序列，其Fc融合蛋白对CTLA4-CD80相互作用的阻断能力相当，且都优于BMS已经上市的抗体(标记为10D1)。
5.3四价CTLA4单域抗体Fc融合蛋白对CTLA4结合能力鉴定(ELISA法)
实施例4.1得到的二价CTLA4单域抗体Fc融合蛋白以及实施例4.4得到的四价CTLA4单域抗体Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入的CTLA4-Fc-Biotin梯度稀释系列，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)，室温反应1.5小时。之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对CTLA4结合曲线及EC50值(所有供试抗体EC50值均为约25～35ng/mL)以反映抗体对CTLA4的亲和能力。
结果见图8，其中纵坐标为OD405，横坐标为CTLA4单域抗体Fc融合蛋白浓度(单位ng/mL)；三角代表四价CTLA4单域抗体Fc融合蛋白huC1v4-tet-Fc(SEQ ID NO：128)，方形代表抗体株C1的人源化形式的Fc融合蛋白huC1v4-Fc，圆圈代表CTLA4单域抗体C1的Fc融合蛋白C1-ld-Fc。三种蛋白的EC50略有差异，但是考虑到四价蛋白的分子量约是二价的5/4，折算成摩尔浓度，则三种蛋白对CTLA4的亲和力并无差异。
5.4鉴定并比较四价与二价CTLA4单域抗体Fc融合蛋白对CTLA4结合能力(SPR法)
上述实施例获得的四价与二价CTLA4单域抗体Fc融合蛋白针对重组的人CTLA4的结合动力学通过表面等离振子共振(surface plasmon resonance，SRP)方法，使用BIAcore X100仪器测量。重组的骆驼抗人Fc抗体偶联于CM5生物传感器芯片上以获得大约1000应答单位(response  units，RU)。对于动力学测量，将抗体用HBS-EP+1×缓冲液(GE，cat#BR-1006-69)稀释到固定浓度，在25℃进样120s，以确保获得大于100RU的响应值，之后将CTLA4和小鼠Fc的融合蛋白CTLA4-muFc(SEQ ID NO：133)三倍连续稀释，在25℃进样120s，解离时间为30min，10mM甘氨酸-HCl(pH2.0)再生120s。使用简单一对一Languir结合模型(BIAcore评价软件3.2版(BIAcore Evaluation Software version 3.2))计算结合速率(kon)和解离速率(koff)。平衡解离常数(kD)以比率koff/kon计算。
测量的抗CTLA4抗体的结合亲和力见表5。结果表明四价分子的亲和力比二价略低，有可能是由于一定的空间位阻造成的。
表5
抗体 Ka Kd KD
HuC1V4-tet-Fc 1.318E+5 2.510E-5 1.905E-10
HuC1V4-Ld-Fc 2.335E+5 2.035E-4 8.715E-11
5.4鉴定二价以及四价CTLA4单域抗体Fc融合蛋白对CTLA4/CD80相互作用的阻断能力(细胞中和实验)
96孔板接种1.5×105Jurkat T细胞(来自中科院上海细胞库)，加入Anti-human CD3(50ng/mL)，37℃孵育15min后，加入梯度稀释的二价或四价CTLA4单域抗体Fc融合蛋白(30ng/ml-0.94ng/ml，稀释液中加入100ng/mL的CTLA4-Fc融合蛋白)及等密度的Raji细胞(来自中科院上海细胞库)，培养24h后，收取上清检测IL-2表达量。用Soft Max对数据进行处理，计算二价或四价CTLA4单域抗体Fc融合蛋白中和CTLA4-Fc融合蛋白对IL-2的抑制的效果，并通过EC50进行比较。
结果如图9所示，二价和四价CTLA4单域抗体Fc融合蛋白(分别用方框和三角表示)在对CTLA4-CD80的抑制效果上相当，且都优于BMS已经上市的CTLA4抗体(标注为10D1，用圆圈表示)。
5.5分析CTLA4单域抗体Fc融合蛋白对CTLA4蛋白结合的特异性
人HEK293细胞通过瞬时转染带有人B7家族蛋白全长基因的质粒(pCDNA4，Invitrogen，Cat V86220)，于膜上瞬时表达人CTLA4、CD28、PD1蛋白。该质粒还使得目标蛋白C端融合EGFP蛋白，从而可以通过绿色荧光强度来考察膜上B7家族蛋白的表达水平。构建好的瞬时转染细胞株包 括：293-CTLA4-EGFP，293-PD1-EGFP，293-CD28-EGFP。
取构建好的细胞，重悬于0.5％PBS-BSA Buffer中，加入huC1v4-tet-Fc抗体，同时设置阴性对照为2μg的针对其他不相关靶标的单域抗体，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μl的0.5％PBS-BSA Buffer中，流式细胞仪进行检测。
结果如图10所示。上排为对照组，下排为样品组。可以清楚的看到，huC1v4-tet-Fc只特异性结合人CTLA4蛋白，而不与其他B7家族蛋白结合。
5.6四价CTLA4单域抗体Fc融合蛋白对猴CTLA4蛋白的结合
猴CTLA4-Fc蛋白购自义翘神州。实施例4.4得到的四价CTLA4单域抗体Fc融合蛋白利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白huC1v4-tet-Fc-Biotin。
猴CTLA4-Fc蛋白或者人CTLA4-Fc蛋白0.5μg/孔4℃过夜包被平板，之后加入huC1v4-tet-Fc-Biotin的梯度稀释系列，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)，室温反应1.5小时。之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对猴CTLA4以及人CTLA4的结合曲线及EC50值以反映抗体对CTLA4的亲和能力。
结果见图11，其中纵坐标为OD405，横坐标为四价CTLA4单域抗体Fc融合蛋白浓度(单位ng/mL)；三角代表和猴CTLA4结合，方形和圆圈代表和人CTLA4结合。可以看出四价CTLA4单域抗体Fc融合蛋白可以有效的结合猴CTLA4蛋白。
5.7 CTLA4单域抗体Fc融合蛋白对PBMC的激活作用
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞(PBMC)。
0.3ug/孔的抗-CD3抗体4℃过夜包被于细胞培养板上。第二天每孔中加入1x105个PBMC细胞，同时每孔分别加入10ug/mL的CTLA4单域抗体Fc融合蛋白huC1v4-Fc、huC27-Fc或是公司自行制备的BMS的CTLA4抗体(命名为10D1)。培养5天后取上清，利用IFN-γELISA检测试剂盒 (ebioscience)检测上清中IFN-γ的水平。
结果见图12。可见在10ug/mL的浓度条件下，CTLA4单域抗体Fc融合蛋白配合anti-CD3抗体可以增强PBMC中细胞分泌γ-干扰素，即CTLA4单域抗体Fc融合蛋白增强了PBMC细胞的活化。且huC1v4-Ld-Fc、huC27v3-Ld-Fc都展示出优于BMS的anti-CTLA4抗体的活性。
5.8二价以及四价CTLA4单域抗体Fc融合蛋白对PBMC的激活作用
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞(PBMC)。
0.3ug/孔的抗-CD3抗体4℃过夜包被于细胞培养板上。第二天每孔中加入1x105个PBMC细胞，同时每孔分别加入0.03ug/mL的二价或四价CTLA4单域抗体Fc融合蛋白huC1v4-Ld-Fc(在图中标注为Ld)、huC1v4-tet-Fc(在图中标注为tet)、或是公司自行制备的BMS的CTLA4抗体(命名为10D1)。同时，以针对其他不相关靶标的单域抗体作为阴性对照组。培养5天后取上清，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ的水平。
结果见图13。可见在0.03ug/mL的低剂量浓度下，CTLA4单域抗体Fc融合蛋白(无论是二价还是四价)配合anti-CD3抗体可以增强PBMC中细胞分泌γ-干扰素，即二价或四价的CTLA4单域抗体Fc融合蛋白在0.03ug/mL的低剂量浓度下即可显著增强PBMC细胞的活化。且四价CTLA4单域抗体Fc融合蛋白huC1v4-tet-Fc展示出优于二价以及BMS的anti-CTLA4抗体的活性。
5.9在树突细胞-T细胞混合淋巴反应中测定CTLA4单域抗体Fc融合蛋白对CD4+T细胞的激活作用
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞PBMC。然后将其用无血清的RPMI1640培养基培养1-2小时，除去未贴壁的细胞，并将细胞培养于含10％FBS，10ng/ml GM-CSF以及20ng/mL IL-4的RPMI中。培养5-6天后，加入10ng/ml的TNF-α并孵育24小时，获得成熟的树突细胞。
将通过此方法获得的树突细胞重悬于RPMI完全培养基中，2x105/ml。然后在96孔U形底板(Costar：3799)中每孔加入50μl，在培养箱中培养。
利用磁珠分离试剂盒(Miltenyi Biotec：130-096-533)按照说明书方法从另一个供体PBMC中分离CD4+T细胞。
将通过上述方法获得的1x104个树突细胞和1x105个CD4+T细胞混合，于RPMI完全培养基中重悬并加入96孔培养板，每孔加入50μl细胞混液；每孔加入100μl稀释于RPMI完全培养基中的huC1v4-Ld-Fc，抗体终浓度为0.1μg/ml或0.01μg/ml。培养5-7天后取上清，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ的水平。
结果见图14。可见CTLA4单域抗体Fc融合蛋白可以增强混合淋巴细胞反应中CD4+T细胞的γ-干扰素分泌，即CTLA4阻断型单域抗体Fc融合蛋白增强了T细胞的活化。且这一生物学活性具有浓度依赖性，在低剂量浓度下(0.01μg/ml)就可观察到显著的刺激T细胞活化的功能。
5.10四价CTLA4单域抗体Fc融合蛋白在人源化小鼠中的抑瘤效果考察
取CTLA4人源化小鼠(即该小鼠表达人的CTLA4蛋白)，接种5X 105MC38肿瘤细胞。
接种后第7、10、13、16天分别腹腔给予100ug待测样品或同等量的人免疫球蛋白作为对照组。自接种后第5天起，每两天测量肿瘤大小，直至第20天。其中考察的样品包括四价CTLA4单域抗体Fc融合蛋白(图中Tet)以及BMS已经上市的CTLA4单抗药物，ipilimumab。
由下图结果可知，在约5mg/kg的剂量下，四价CTLA4单域抗体Fc融合蛋白在CTLA4人源化小鼠体内的抑瘤效果和BMS的ipilimumab相当。
实施例6：CTLA4单域抗体Fc融合蛋白在大鼠体内的药代动力学研究
单次静脉(IV)给予SD大鼠CTLA4单域抗体Fc融合蛋，于不同时间点采集血样，Elisa测定给予受试物后大鼠血浆中受试物的浓度并计算药代动力学参数。
动物选择：选取6-8周龄SD大鼠，体重200-300g。按照雌雄各半随机分配为两组，每组8只大鼠。
给药：按照10mg/kg的剂量，静脉(IV)单次给药。
采血：药前、药后即刻，药后多个时间点，每次每只大鼠颈静脉采血约0.5ml。将采集的血液迅速离心分离血清，-80℃保存待分析。
血药浓度检测：夹心ELISA法检测血清中CTLA4单域抗体Fc融合蛋白的含量。其中用重组人CTLA4蛋白包被，用以捕获CTLA4特异性抗体，用羊抗人IgG(Fc specific)-HRP抗体(Sigma)检测Fc区，这样以保证检测的是完整的CTLA4单域抗体Fc融合蛋白。
数据处理：利用血药浓度对时间曲线计算相关药代动力学参数，包括AUC(0-t)、AUC(0-∞)、Cmax、Tmax、T1/2、Vss、MRT等。
二价以及四价CTLA4单域抗体Fc融合蛋白在大鼠体内血药浓度对时间的变化曲线见图16(a为二价抗体huC1v4-Ld-Fc，b为四价抗体huC1v4-tet-Fc)。药代动力学参数见图17。结果显示，无论二价还是四价CTLA4单域抗体Fc融合蛋白在大鼠体内的半衰期都较长(超过5天)，可见其体内稳定性较好。同时，在维持最高血药浓度不变的情况下，四价的抗体其体内半衰期比二价的延长了一倍(到达11天以上)。由此推断四价抗体在体内维持有效血药浓度的周期会更长，预计到临床能维持更长的给药间隔时间。
实施例7：CTLA4单域抗体Fc融合蛋白成药性评价
7.1 CTLA4单域抗体Fc融合蛋白的理化性质分析
通过实施例4.4所述方法获得一步亲和纯化后的人293HEK细胞表达的四价CTLA4单域抗体Fc融合蛋白，随后通过SE-HPLC、还原条件下的CE、非还原条件下的CE、WCX、DSC等方法对该蛋白进行初步的理化性质分析，从而进一步判断其成药性。具体数值如下表，由此数据可以初步判断四价CTLA4单域抗体Fc融合蛋白具有较好的理化性质，适合工业放大生产。
表6
Figure PCTCN2017085038-appb-000004
7.2 CTLA4单域抗体Fc融合蛋白的热破坏试验
将CTLA4单域抗体Fc融合蛋白通过UF/DF浓缩并换液到PBS缓冲液中，制备20mg/mL的溶液，并高温40℃加速30天的热破坏试验。其中0天、10天、20天、30天的样品进行SE-HPLC纯度检测，其变化趋势见下 图。可见该蛋白在40℃时、未进行制剂优化的情况下，虽然主峰纯度略有下降，但是并未观察到明显的聚集或降解的趋势。同时结合其较高的Tm值，可以判断该蛋白具有较好的热稳定性。
实施例8：CTLA4单域抗体Fc融合蛋白初步毒性评价
试验用4只食蟹猴，随机分为2组，每组2只，雌雄各半，分别为供试品低、高剂量组(分别为7.5和30mg/kg)，后肢静脉推注单次给药(四价CTLA4单域抗体Fc融合蛋白)。于给药前(D-1)，给药后D15和D29对动物进行体重、血细胞计数、凝血功能、血液生化及免疫原性等指标的检测。
试验期间，四只动物未见死亡和濒死。单次给药食蟹猴7.5-30mpk血液学上可见lymph，Eos，Baso，Mono明显增加，其他各给药组动物临床观察、体重、凝血功能、血生化指标未见有毒理学意义的规律性改变。
由此，单次静脉注射给予食蟹猴7.5和30mpk，未见明显毒性反应，NOAEL大于等于30mg/kg。BMS的ipilimumab报道的非临床NOAEL约为10mg/kg。由此推测，该CTLA4单域抗体Fc融合蛋白的毒副作用低于ipilimumab。

Claims (27)

  1. CTLA4结合蛋白，其能够特异性结合CTLA4且包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
    (1)SEQ ID NO：1所示的CDR1，SEQ ID NO：2所示的CDR2，SEQ ID NO：3所示的CDR3；
    (2)SEQ ID NO：4所示的CDR1，SEQ ID NO：5所示的CDR2，SEQ ID NO：6所示的CDR3；
    (3)SEQ ID NO：7所示的CDR1，SEQ ID NO：8所示的CDR2，SEQ ID NO：9所示的CDR3；
    (4)SEQ ID NO：10所示的CDR1，SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
    (5)SEQ ID NO：13所示的CDR1，SEQ ID NO：14所示的CDR2，SEQ ID NO：15所示的CDR3；
    (6)SEQ ID NO：16所示的CDR1，SEQ ID NO：17所示的CDR2，SEQ ID NO：18所示的CDR3；
    (7)SEQ ID NO：19所示的CDR1，SEQ ID NO：20所示的CDR2，SEQ ID NO：21所示的CDR3；
    (8)SEQ ID NO：22所示的CDR1，SEQ ID NO：23所示的CDR2，SEQ ID NO：24所示的CDR3；
    (9)SEQ ID NO：25所示的CDR1，SEQ ID NO：26所示的CDR2，SEQ ID NO：27所示的CDR3；
    (10)SEQ ID NO：28所示的CDR1，SEQ ID NO：29所示的CDR2，SEQ ID NO：30所示的CDR3；
    (11)SEQ ID NO：31所示的CDR1，SEQ ID NO：32所示的CDR2，SEQ ID NO：33所示的CDR3；
    (12)SEQ ID NO：34所示的CDR1，SEQ ID NO：35所示的CDR2，SEQ ID NO：36所示的CDR3；
    (13)SEQ ID NO：37所示的CDR1，SEQ ID NO：38所示的CDR2，SEQ ID NO：39所示的CDR3；
    (14)SEQ ID NO：40所示的CDR1，SEQ ID NO：41所示的CDR2，SEQ ID  NO：42所示的CDR3；
    (15)SEQ ID NO：43所示的CDR1，SEQ ID NO：44所示的CDR2，SEQ ID NO：45所示的CDR3；
    (16)SEQ ID NO：46所示的CDR1，SEQ ID NO：47所示的CDR2，SEQ ID NO：48所示的CDR3；
    (17)SEQ ID NO：49所示的CDR1，SEQ ID NO：50所示的CDR2，SEQ ID NO：51所示的CDR3；
    (18)SEQ ID NO：52所示的CDR1，SEQ ID NO：53所示的CDR2，SEQ ID NO：54所示的CDR3；
    (19)SEQ ID NO：55所示的CDR1，SEQ ID NO：56所示的CDR2，SEQ ID NO：57所示的CDR3；
    (20)SEQ ID NO：58所示的CDR1，SEQ ID NO：59所示的CDR2，SEQ ID NO：60所示的CDR3；
    (21)SEQ ID NO：61所示的CDR1，SEQ ID NO：62所示的CDR2，SEQ ID NO：63所示的CDR3；
    (22)SEQ ID NO：64所示的CDR1，SEQ ID NO：65所示的CDR2，SEQ ID NO：66所示的CDR3；
    (23)SEQ ID NO：67所示的CDR1，SEQ ID NO：68所示的CDR2，SEQ ID NO：69所示的CDR3；
    (24)SEQ ID NO：70所示的CDR1，SEQ ID NO：71所示的CDR2，SEQ ID NO：72所示的CDR3；和
    (25)SEQ ID NO：73所示的CDR1，SEQ ID NO：74所示的CDR2，SEQ ID NO：75所示的CDR3。
  2. 权利要求1的CTLA4结合蛋白，其中所述免疫球蛋白单一可变结构域是VHH。
  3. 权利要求2的CTLA4结合蛋白，其中所述VHH是人源化的VHH。
  4. 权利要求2的CTLA4结合蛋白，其中所述VHH包含SEQ ID NO：76-100中任一氨基酸序列。
  5. 权利要求3的CTLA4结合蛋白，其中所述VHH包含与SEQ ID NO：76-100中任一序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。
  6. 权利要求3的CTLA4结合蛋白，其中所述人源化的VHH包含SEQ ID NO：101-109中任一氨基酸序列。
  7. CTLA4结合蛋白，其由权利要求1-6任一项的CTLA4结合蛋白经过亲和力成熟获得。
  8. 权利要求1-7任一项的CTLA4结合蛋白，其包含两个所述免疫球蛋白单一可变结构域。
  9. 权利要求1-8任一项的CTLA4结合蛋白，其还包含免疫球蛋白Fc区。
  10. 权利要求9的CTLA4结合蛋白，其中所述免疫球蛋白Fc区是人免疫球蛋白Fc区，优选是人IgG1的Fc区。
  11. 权利要求10的CTLA4结合蛋白，其中所述免疫球蛋白Fc区的氨基酸序列示于SEQ ID NO：132。
  12. 权利要求9的CTLA4结合蛋白，其包含选自SEQ ID NO：114-128的氨基酸序列。
  13. 权利要求1-12中任一项的CTLA4结合蛋白，其具有下述特征中的至少一个：
    (a)与人CTLA4结合的KD值小于1×10-7M；
    (b)阻断CTLA4与CD80和/或CD86的相互作用；
    (c)增强PBMC和/或T细胞的活化；
    (d)抑制肿瘤生长。
  14. 权利要求1-12中任一项的CTLA4结合蛋白，其结合CTLA4的KD值小于1×10-7M，优选小于1×10-8M、更优选小于1×10-9M、更优选小于1×10-10M。
  15. 核酸分子，其编码权利要求1-14中任一项的CTLA4结合蛋白。
  16. 表达载体，其包含与表达调控元件可操作地连接的权利要求15的核酸分子。
  17. 重组细胞，其包含权利要求15的核酸分子或以权利要求16的表达载体转化，并能够表达所述CTLA4结合蛋白。
  18. 产生权利要求1-14中任一项的CTLA4结合蛋白的方法，包括：
    a)在允许所述CTLA4结合蛋白表达的条件下培养权利要求17的重组细胞；
    b)从得自步骤a)的培养物回收由所述重组细胞表达的CTLA4结合蛋白；及
    c)任选进一步纯化和/或修饰得自步骤b)的CTLA4结合蛋白。
  19. 药物组合物，其包含权利要求1-14任一项的CTLA4结合蛋白以及药学上可接受的载体。
  20. 一种在对象中预防和/或治疗癌症的方法，包括给所述对象施用有效量的权利要求1-14任一项的CTLA4结合蛋白或权利要求19的药物组合物。
  21. 权利要求20的方法，其还包括给所述对象施用其它抗肿瘤治疗手段。
  22. 权利要求21的方法，其中所述其它抗肿瘤治疗手段包括化疗、放疗、靶向肿瘤特异性抗原的抗体治疗、其它肿瘤免疫治疗手段、或肿瘤靶向性小分子类药物。
  23. 权利要求21的方法，其中所述其它抗肿瘤治疗手段包括使用抗EGFR抗体、抗EGFR变体的抗体、抗VEGFa抗体、抗HER2抗体、或抗CMET抗体或其组合的抗体治疗。
  24. 权利要求20的方法，其中所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体癌、骨肉瘤。
  25. 一种在对象中预防和/或治疗感染性疾病的方法，包括给所述对象施用有效量的权利要求1-14任一项的CTLA4结合蛋白或权利要求19的药物组合物。
  26. 权利要求25的方法，其中所述感染性疾病由选自以下的病原体引起：HIV、肝炎病毒、流感病毒、疱疹病毒、贾第虫、疟原虫、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。
  27. 权利要求1-14任一项的CTLA4结合蛋白或权利要求19的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗癌症或感染性疾病。
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US11091549B2 (en) 2021-08-17
ES2931223T3 (es) 2022-12-27
EP3459597B1 (en) 2022-08-31
US20220177588A1 (en) 2022-06-09
DK3459597T3 (da) 2022-11-28
JP2021184736A (ja) 2021-12-09
EP3459597A1 (en) 2019-03-27
JP7256348B2 (ja) 2023-04-12
US11912768B2 (en) 2024-02-27
JP2019522489A (ja) 2019-08-15
EP3459597A4 (en) 2020-04-15
CN109195665A (zh) 2019-01-11
CN107400166A (zh) 2017-11-28
US20190315864A1 (en) 2019-10-17
CN109195665B (zh) 2022-09-09

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