WO2019184898A1 - 抗糖皮质激素诱导的肿瘤坏死因子受体(gitr)的小型化抗体、其聚合物及应用 - Google Patents

抗糖皮质激素诱导的肿瘤坏死因子受体(gitr)的小型化抗体、其聚合物及应用 Download PDF

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WO2019184898A1
WO2019184898A1 PCT/CN2019/079625 CN2019079625W WO2019184898A1 WO 2019184898 A1 WO2019184898 A1 WO 2019184898A1 CN 2019079625 W CN2019079625 W CN 2019079625W WO 2019184898 A1 WO2019184898 A1 WO 2019184898A1
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antibody
heavy chain
seq
variable region
sequence
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PCT/CN2019/079625
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French (fr)
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缪小牛
匡智慧
黄威峰
刘军建
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信达生物制药(苏州)有限公司
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Priority to CN201980006241.8A priority Critical patent/CN111448214B/zh
Priority to US16/771,777 priority patent/US11708417B2/en
Publication of WO2019184898A1 publication Critical patent/WO2019184898A1/zh

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Definitions

  • the invention relates to the field of antibodies.
  • the present invention relates to antibodies (including miniaturized antibodies), antibody fragments and polymers thereof that specifically bind to a glucocorticoid-induced tumor necrosis factor receptor (GITR), and conjugates and fusions containing the same or antibodies .
  • the invention also relates to nucleic acids encoding such antibodies, antibody fragments, polymers, conjugates and fusions; vectors and host cells expressing the nucleic acids.
  • the invention also relates to products comprising these antibodies and antibody fragments, polymers, immunoconjugates or fusions thereof, and to use in therapy and diagnosis.
  • GITR Glucocorticoid-induced tumor necrosis factor receptor
  • TNFRSF18 activation-inducible tumor necrosis factor receptor family members (AITR), CD357 and GITR-D
  • AITR activation-inducible tumor necrosis factor receptor
  • GITR-D The 18th member of the tumor necrosis factor receptor (TNFR) superfamily.
  • GITRL GITR ligand
  • GITR is expressed at low levels in response to resting T cells; when T cells are activated, their expression levels are significantly upregulated.
  • GITR is highly expressed constitutively in regulatory T cells (Treg, such as CD4 + CD25 + or CD8 + CD25 + cells), and when these cells are activated, expression levels are further upregulated (Nocentini and Riccardo (2005) EJImmunol. 35: 1016-1022).
  • GITR expression is not limited to T cells, and GITR has also been expressed on NK cells, macrophages, B cells, dendritic cells, mast cells, and monocytes (Nocentini and Riccardo (2005) EJImmunol. 35: 1016-1022 ).
  • GITRL GITR ligands
  • APCs antigen presenting cells
  • GITRL antigen presenting cells
  • APCs antigen presenting cells
  • GITR signaling co-stimulation in response to T cells and inhibition of Treg cell inhibitory activity.
  • GITR has several effects on effector T cells and regulatory T cells, including: co-stimulation and activation of effector T cells, making T cells more resistant to inhibition, inhibiting regulatory T cells, and reducing effector T cell regulation. Sensitivity to inhibition by sex T cells, etc. (Nocentini et al. (2007) Eur. J. Immunol. 37: 1165-1169).
  • GITR activation can lead to an increase in immune response, increased resistance to tumors and viral infections, and the like.
  • a substance capable of activating GITR can enhance a desired immune response (eg, anti-tumor, treat viral infection), induce or enhance an immune response in an individual, treat immune disorders and proliferative disorders (eg, tumors and cancer), and the like.
  • the substances capable of activating GITR in the prior art are mainly standard structure anti-GITR antibodies (see CN103951753A, CN105829343A, CN106459203A, WO2016196792A1, WO2017068186A9, etc.).
  • Such antibodies are agonists of GITR (i.e., are activating antibodies) that induce or enhance GITR signaling and are effective in treating a variety of GITR-related diseases or conditions that require an enhanced immune response. Since such an anti-GITR antibody is an activated antibody, its activation activity is a more critical indicator of its activity (for example, immunopotentiating activity) relative to its binding affinity to an antigen.
  • the above-mentioned standard structure anti-GITR antibody has some inherent defects of standard structure antibody: high production cost, long preparation cycle, complicated preparation process, large volume (poor tissue permeability), poor stability, large difference between batches, etc. , greatly limiting the scope of its application.
  • single-chain variable fragment scFv
  • single-domain antibody sdAd
  • heavy-chain antibody hcAb
  • nanobody nanobody, Nb or variable domain of Heavy chain of heavy-chain antibody (VHH)
  • small antibody-like scaffold proteins such as Affibody, DARPins, etc.
  • Single-domain antibodies and heavy-chain antibodies in such miniaturized antibodies have no light chain, and have a single heavy chain variable region that retains intact antigen-binding activity, has small molecules, high stability, good tissue permeability in vivo, and good solubility. , high melting temperature, easy to express and other characteristics.
  • Nanobodies in such miniaturized antibodies have the following advantages:
  • the longer CDR3 of the Nanobody (complementarity determining region 3), can form a stable exposed convex ring structure (disulfide bond with stable structure in the convex ring) It can penetrate deep into the antigen to better bind the antigen and improve its antigen specificity and affinity.
  • the antigen-binding surface of the conventional antibody Fab fragment and the single-chain antibody scFv often forms a concave topology, and usually only recognizes a site located on the surface of the antigen. Therefore, Nanobodies have more favorable antigen-binding properties, and even when antigenic proteins are tightly packed, hiding epitopes that are not recognized by common antibodies, Nanobodies can recognize such epitopes.
  • VH domain of a standard structural antibody When the VH domain of a standard structural antibody is expressed alone, the expressed VH domain typically forms inclusion bodies, or the exposed hydrophobic domains adhere to each other. Nano-antibodies are replaced by hydrophilic residues in the FR2 region, which makes the nano-antibody very soluble and has reduced polymerizability. It can be easily concentrated to 10 mg/mL in ordinary buffer without accumulation.
  • the antigen-binding activity is maintained under the action of high temperature (80-92 ° C) and high concentration of denaturant, even if expressed in the form of inclusion bodies, it is easy to renature, so that the Nanobody is in the chromatogram Or in the transient degeneration during aseptic processing, or in adverse environments such as pollution or organic solvents, the biological activity can be excellently maintained for a long period of time, and the utilization rate of the nano-antibody as a drug is greatly improved.
  • Nanobodies Because of its small molecular weight, simple structure, and coding by a single gene, Nanobodies can be easily synthesized in microorganisms, and can be expressed in large quantities in low-cost microorganisms such as phage and yeast for large-scale production. It has been reported that the production of Nanobodies can be increased to a yield of 1 g/L by a yeast reactor.
  • the Nanobody since the Nanobody has only one heavy chain variable region, there is no pairing problem, and thus the screening process is simpler, and often only a library is needed.
  • Nanobodies Compared with sdAb, scFv, etc., Nanobodies also have the following outstanding advantages: simpler structure, easy binding to receptors, recognition of hidden epitopes embedded in ligand grooves or sandwiched between two subunits, Moreover, the relative molecular weight is smaller, the immunogenicity is lower, the tendency of accumulation precipitation is lower, the biodispersion is better, the expression is easier (high expression in prokaryotic or eukaryotic systems), the solubility is better, the stability is stronger, and the pH is high.
  • Concentration denaturing agents or high temperature and other unfavorable physical and chemical conditions are more stable, long shelf life, oral or respiratory administration, etc.; at the same time, it is not easy to adhere to each other as single-chain antibodies (scFv), or even aggregate.
  • scFv single-chain antibodies
  • Test kits made of Nanobodies can even be stored directly at room temperature without refrigerated storage, reducing the cost of large refrigeration.
  • the standard structure of the anti-GITR antibody has disadvantages such as a complicated screening process, poor tissue permeability, and high production cost, so development of a miniaturized antibody against GITR can well compensate for these deficiencies and has many other advantages, and at the same time Can achieve the performance of anti-GITR antibodies.
  • the corresponding VHH can be obtained. Since, as described above, the amino acid sequence and length of CDR1-3 in VHH, particularly the amino acid selection and length of CDR3, are different from the corresponding HCDR1-3 (eg, HCDR3) in the standard structural antibody, the structure is also different and needs to be carefully designed and A lot of screening work can be obtained.
  • An object of the present invention is to provide an isolated antibody or antigen-binding fragment thereof (abbreviated as "an antibody of the present invention or antigen-binding fragment thereof") which specifically binds to a glucocorticoid-induced tumor necrosis factor receptor (GITR), which has One or more of the following characteristics:
  • Another object of the present invention is to provide a Nanobody (abbreviated as "the Nanobody of the present invention"), wherein the HCDR3 (heavy chain complementarity determining region 3) comprises the HCDR3 of the antibody of the present invention or an antigen-binding fragment thereof.
  • the HCDR1 (heavy chain complementarity determining region 1) and HCDR2 (heavy chain complementarity determining region 2) of the Nanobodies of the invention comprise HCDR1 and HCDR2 of an antibody or antigen-binding fragment thereof of the invention, respectively.
  • the HCDR1, HCDR2 and HCDR3 of a Nanobody of the invention comprise HCDR1, HCDR2 and HCDR3 of an antibody or antigen-binding fragment thereof of the invention, respectively.
  • the Nanobody of the invention comprises only the heavy chain variable region sequence set forth in any of SEQ ID NOs: 64-85, or variants thereof, or the Nanobody of the invention is only SEQ ID NO The heavy chain variable region sequence shown in any one of 64-85 or a variant thereof.
  • Another object of the present invention is to provide a heavy chain antibody (abbreviated as "heavy chain antibody of the present invention") comprising an Fc fragment of the Nanobody of the present invention and an IgG antibody.
  • Another object of the present invention is to provide a humanized heavy chain antibody (abbreviated as "humanized heavy chain antibody of the present invention”) which is engineered from the heavy chain antibody of the present invention.
  • humanized heavy chain antibody of the present invention a humanized heavy chain antibody which is engineered from the heavy chain antibody of the present invention.
  • the humanized heavy chain antibodies of the invention are fully human heavy chain antibodies.
  • Another object of the present invention is to provide an antibody in the form of a polymer (abbreviated as "an antibody in the form of a polymer of the present invention"), which is a heavy chain variable region of the Nanobody of the present invention, the heavy chain antibody of the present invention Or a polymeric form of the heavy chain variable region of a humanized heavy chain antibody of the invention.
  • an antibody in the form of a polymer abbreviated as "an antibody in the form of a polymer of the present invention
  • an antibody in the form of a polymer of the present invention which is a heavy chain variable region of the Nanobody of the present invention, the heavy chain antibody of the present invention Or a polymeric form of the heavy chain variable region of a humanized heavy chain antibody of the invention.
  • the antibody in the multimeric form of the invention is a heavy chain variable region of a Nanobody of the invention, a heavy chain variable region of a heavy chain antibody of the invention, or a humanized heavy chain antibody of the invention
  • the tetramerized form or the hexamed form, preferably the tetramer of the Nanobody of the present invention, the heavy chain variable region of the heavy chain antibody of the present invention or the heavy chain variable region of the humanized heavy chain antibody of the present invention Form.
  • the antibody in the multimeric form of the invention is linked by a Nanobody of the invention, a heavy chain variable region of a heavy chain antibody of the invention, or a heavy chain variable region of a humanized heavy chain antibody of the invention
  • a new structure formed by the heavy chain antibody of the present invention is polymerized.
  • the antibody in the multimeric form of the invention is linked by a Nanobody of the invention, a heavy chain variable region of a heavy chain antibody of the invention, or a heavy chain variable region of a humanized heavy chain antibody of the invention
  • the novel structure formed by the C-terminus of the heavy chain antibody of the present invention is polymerized; or the heavy chain variable region of the present invention, the heavy chain variable region of the heavy chain antibody of the present invention or the humanized heavy chain antibody of the present invention
  • the chain variable region is polymerized by a new structure formed by the N-terminus of the heavy chain antibody of the present invention (preferably linked to the humanized heavy chain antibody of the present invention).
  • the heavy chain variable region of a Nanobody of the invention a heavy chain variable region of a heavy chain antibody of the invention, or a humanized heavy chain antibody of the invention
  • the heavy chain antibody of the present invention (preferably linked to the humanized heavy chain antibody of the present invention) is linked by a linker peptide.
  • Another object of the invention is to provide a fusion protein, immunoconjugate, composition or kit comprising an antibody of the invention.
  • Another object of the invention is to provide an isolated nucleic acid encoding an antibody of the invention; a vector comprising the nucleic acid; a host cell comprising the vector; a pharmaceutical combination comprising one or more of these nucleic acids, vectors or host cells Things.
  • Another object of the invention is to provide a process for the preparation of an antibody or antigen-binding fragment thereof of the invention, a fusion protein of the invention, an immunoconjugate or composition of the invention, including a pharmaceutical composition.
  • Another object of the present invention is to provide a method of detecting the presence of GITR in a sample comprising the use of an antibody of the present invention or an antigen-binding fragment thereof.
  • Another object of the present invention is to provide a method of treating cancer, inducing or enhancing an immune response in an individual, and/or stimulating an antigen-specific T cell response, characterized by comprising administering to a desired individual an effective amount of the present invention.
  • An antibody or antigen-binding fragment thereof, a fusion protein of the invention or an immunoconjugate of the invention is an antibody or antigen-binding fragment thereof, a fusion protein of the invention or an immunoconjugate of the invention.
  • the antibody of the present invention has the advantages of simple screening process, good tissue permeability, low production cost, and the like, and is relatively simple to obtain from the standard structure anti-GITR antibody.
  • Chain antibodies, single domain antibodies, heavy chain antibodies, and the like no longer have the disadvantage of easily forming inclusion bodies or easily adhering to each other.
  • the inventors of the present invention have surprisingly found that the antibodies of the present invention are highly specific for GITR and do not interfere with the activity of other receptors, and are capable of stimulating GITR signaling at relatively low doses; Antibodies can efficiently activate the NFkB signaling pathway downstream of GITR, producing beneficial anti-immune diseases/disorders and anti-proliferative diseases/disorders.
  • Figure 1 Schematic diagram of the nanobody phage library construction process.
  • Figure 3 A graph showing the results of the binding of hcIgG to the cell surface GITR protein.
  • Figure 5 Figure showing the results of the binding of Hzhc IgG to the cell surface GITR protein.
  • FIG. 1 Schematic representation of the structure of 4xNb-IgG.
  • Figure 9 Activation of 4xNb-IgG on the NFkB signaling pathway downstream of GITR.
  • Figure 10 Inhibition of tumors in the MC38 xenograft model by anti-GITR antibody (4xNb-IgG) itself, or in combination with anti-PD-1 antibody.
  • the term "about” when used in conjunction with a numerical value is meant to encompass a numerical value within the range of a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.
  • GITR refers to "glucocorticoid-induced TNF-related genes and/or polypeptides", also known in the art as TNF receptor superfamily 18 (TNFRSF18).
  • TNF receptor superfamily 18 TNF receptor superfamily 18
  • the amino acid and nucleic acid sequences of the human and murine forms of GITR are described in WO 98/06842, which is incorporated herein by reference. See also GenBank Accession No. Q9Y5U5 (human amino acid sequence) and AF109216 (murine nucleic acid and amino acid sequence).
  • the amino acid sequence of a particular mature human GITR polypeptide is set forth in SEQ ID NO:123.
  • the term GITR as used herein also includes alleles or variants thereof that are naturally occurring.
  • the terms "antigen-binding molecule”, “antigen-binding protein” and “antibody” are used interchangeably herein to refer to a molecule comprising an antigen-binding or antigen-binding portion capable of binding to a target antigen, such as a protein or Peptide.
  • a target antigen such as a protein or Peptide.
  • the antigen-binding molecule that binds to GITR is also referred to as a GITR-binding molecule, a GITR antibody, or an anti-GITR antibody.
  • Antigen binding molecules include, for example, antibodies and antigen-binding fragments thereof, single-chain antibodies (scFv), single domain antibodies (sdAd), heavy chain antibodies (hcAb), Nanobodies (Nb or VHH); or polymeric forms of these antibodies, Various fusions and conjugates based on these antibodies, immunoconjugates, antibody drug conjugates (ADCs), poly/bispecific antibodies, chimeric antigen receptors (CAR), and the like.
  • the antigen binding portion of an antibody typically comprises amino acid residues from a "complementarity determining region" or "CDR".
  • CDR complementarity determining region
  • antibody refers to a polypeptide (immunoglobulin) comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen.
  • the term encompasses various antibody structures including, but not limited to, monoclonal antibodies; multispecific antibodies; Fab fragments, Fab' fragments, F(ab') 2 fragments or Fv fragments; diabody, single chain antibody (scFv), single Domain antibody (sdAd), heavy chain antibody (hcAb), Nanobody (Nb or VHH) or a polymeric form of these antibodies; human antibodies, humanized antibodies or chimeric antibodies; or labeled antibodies, as long as they are presented The desired antigen binding activity is sufficient.
  • full antibody As used herein, the terms “full antibody,” “full length antibody,” “complete antibody,” and “intact antibody” are used interchangeably herein and are meant to have a structure that is substantially similar in structure to the native antibody, and includes at least two Heavy chain (H) and two light chains (L).
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL).
  • the light chain constant region consists of one domain CL.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits multiple effector functions.
  • variable region As used herein, the terms "variable region (V region, Fv)" and “variable domain” are used interchangeably herein and refer to a domain of an antibody heavy or light chain that is involved in binding of an antibody to an antigen.
  • the heavy chain variable domain (VH) and light chain variable domain (VL) of a native antibody typically have a similar structure, wherein each domain comprises four conserved framework regions (FR) and three complementarity determining regions ( CDR, see, for example, Kindt et al. Kuby Immunology, Sixth Edition, WH Freeman and Co., page 91 (2007)).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • the light chain variable region and the heavy chain variable region typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems including, for example, antibody-based three-dimensional structures and Chothia of the topology of CDR loops (Chothia et al.
  • the light chain of an antibody can be divided into two types kappa ( ⁇ ) and lambda ( ⁇ ) based on the amino acid sequence of its constant domain.
  • the heavy chain of an antibody can be divided into five major different types depending on the amino acid sequence of its heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further divided into subclasses, such as , IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.
  • the heavy chain constant regions corresponding to different antibody types are referred to as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively. See, for example, Fundamental Immunology, Ch. 7 (Paul, W. Ed., Second Edition, Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety.
  • antibody fragment and "(antibody) antigen-binding fragment” are used interchangeably herein, and refer to a molecule that is not an intact antibody, which comprises an antigen in an intact antibody for binding to the intact antibody. The portion that binds to the antigen or competes with the intact antibody (ie, the intact antibody from which the antigen-binding fragment is derived) to bind to the antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab')2, Fab'-SH, Fragment of variable (Fv), single-chain Fv, diabody, Triabody, tetrabody, minibody, single domain antibody (single domain antibody, sdAb); and multispecific antibodies formed from antibody fragments.
  • a Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, for example, a Fab fragment can be obtained by digestion of a complete antibody by papain.
  • the light chain (L chain) and heavy chain (H chain) of the Fab can be fused by a linker to form a single polypeptide chain, a single chain Fab (scFab) (see for example US20070274985A1).
  • F(ab') 2 which is a dimer of Fab', is a divalent antibody fragment by digesting a complete antibody under the disulfide bond of the hinge region by pepsin.
  • F(ab') 2 can be reduced under neutral conditions by disrupting the disulfide bond in the hinge region, converting from the F(ab') 2 dimer to the Fab' monomer.
  • the Fab' monomer is essentially a Fab fragment with a hinge region.
  • the Fv fragment consists of the VL and VH domains of one arm of the antibody.
  • the term "dual-chain antibody” is an antibody fragment having two antigen-binding sites comprising VL and VH linked by a short linker in the same polypeptide chain.
  • the linker since the linker is too short, the two domains of VH and VL on the same chain cannot be paired, but are forced to pair with the complementary domain on the other chain and produce two antigen-binding sites.
  • Double-stranded antibodies can be bivalent or bispecific.
  • single-chain variable fragment also referred to as “single-chain variable region antibody fragment” refers to a region in which heavy and light chain variable regions are passed (flexible) or ( Flexible) a single polypeptide chain formed by the joining of peptides.
  • a gene encoding two domains VL and VH independently encoding an Fv fragment is ligated together by a nucleic acid sequence encoding a linker (linker) and recombinantly expressed by using a recombinant method.
  • the single polypeptide chain forms an antigen binding region.
  • Single-chain antibodies are described in detail in International Patent Application Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.
  • (flexible) linker and the term “(flexible) linker peptide” are used interchangeably herein to refer to a short peptide (peptide linker) consisting of amino acids.
  • Peptide linkers are typically enriched in glycine, which exhibits flexibility, and serine or threonine, which exhibits solubility.
  • glycine and/or serine residues can be used alone or in combination.
  • Non-limiting examples of flexible linker peptides or peptide linkers are disclosed in Shen et al., Anal. Chem.
  • the linker will facilitate pairing of VH and VL and does not interfere with the formation of functionally effective antigen binding sites for VH and VL pairs.
  • single-domain antibody refers to comprising only a single variable domain (eg, a heavy chain variable domain (VH) or a light chain variable domain (VL), An antibody that binds to an antigen, ie, it does not need to interact with another variable domain to recognize a target antigen.
  • a single domain antibody can be derived from a heavy chain variable domain of a camelid heavy chain antibody, derived from a fish IgNAR VH-like single domain (v-NAR) and the like.
  • nanobody (Nb or variable domain of heavy chain of heavy-chain antibody, VHH) refers to an antibody consisting of only one heavy chain variable region, having an activity of binding to an antigen, ie, from An antibody comprising only one strand from the C-terminus to the N-terminus: FR4-VCDR3-FR3-VCDR2-FR2-VCDR1-FR1, may be naturally produced by camelids or produced by genetic engineering techniques. Nanobodies are currently the smallest unit known to bind to a target antigen.
  • the term "heavy-chain antibody (hcAb)” refers to an antibody that does not have a light chain, and may comprise VH-CH2-CH3 from N to C, or VH-CH1-CH2-CH3 ; can form a homodimer, such as a heavy chain dimer antibody that does not have a light chain.
  • the heavy chain antibody of the present invention may comprise VH from a standard antibody or VH from a miniaturized antibody.
  • the VH in the heavy chain antibody of the present invention may be a Nanobody.
  • the term "monoclonal antibody” refers to an antibody derived from a population of substantially homogeneous antibodies, ie, in addition to a possible variant antibody that is typically present in minor amounts (eg, containing a natural mutation or in a monoclonal antibody preparation). In addition to the variant antibodies produced during the production process, the individual antibodies that make up the population are identical and/or bind to the same epitope. Monoclonal antibodies can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, yeast display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus.
  • human antibody or “full human antibody” as used herein, is used interchangeably and refers to an antibody comprising a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. . Moreover, if the antibody contains a constant region, the constant region is also derived from the human germline immunoglobulin sequence.
  • Human antibodies of the invention may include amino acid sequences that are not encoded by human germline immunoglobulin sequences (e.g., include mutations introduced by random or point-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDRs, particularly in CDR3. .
  • the term “human antibody” is not intended to include antibodies in which the CDR sequences are derived from the germline of other mammalian species (eg, mice) and transplanted into human framework sequences.
  • humanized antibody refers to an antibody that binds a CDR sequence derived from a germline of another mammalian species, such as a mouse, to a human framework sequence. Additional framework region modifications can be made within the human framework sequence.
  • chimeric antibody refers to an antibody from which a variable region sequence is derived from one species, the constant region sequence is derived from another species, eg, wherein the variable region sequence is derived from a mouse antibody, and the constant region sequence is derived.
  • An antibody to a human antibody is an antibody to a human antibody.
  • the term "isolated" antibody is an antibody that has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) HPLC) Determined purity.
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reversed phase
  • epitope refers to the region of an antigen to which an antibody binds. Epitopes may be formed from contiguous amino acids or from non-contiguous amino acids by tertiary folding of the protein.
  • the term "specifically binds” means that the antibody selectively or preferentially binds to an antigen.
  • a GITR antibody does not substantially bind to a non-GITR molecule, the antibody is considered to "specifically bind" to the GITR.
  • antibodies that specifically bind to GITR can cross-react with GITR polypeptides from different species.
  • the antibody is about 5 x 10 -7 M or less, about 1 x 10 -7 M or less, about 5 x 10 -8 M or less, about 1 x 10 -8 M Or a lower, about 5 x 10 -9 M or lower KD binds to human GITR, and the antibody is an antibody that "specifically binds to human GITR".
  • affinity or "binding affinity” refers to the inherent binding force that reflects the interaction between members of a bond.
  • the affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio Kinetic Binding Assay herein.
  • an "antibody that competes for binding" with a reference antibody that binds, for example, a GITR antigen refers to an antibody that blocks 50% or more of the binding of a reference antibody to an antigen (eg, GITR) in a competition assay.
  • An exemplary competition test is described in "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • a competitively bound antibody can bind to the same epitope region as the reference antibody, such as the same epitope, an adjacent epitope, or an overlapping epitope.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc-regions and variant Fc-regions.
  • the human IgG heavy chain Fc-region extends from Cys226 of the heavy chain or from Pro230 to the carboxy terminus.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • the numbering of amino acid residues in an Fc-region or constant region is based on the EU numbering system, also known as the EU index, such as Kabat, EA, etc., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service , National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • variant as used in relation to an antibody is meant herein to include having been substituted by at least 1, for example 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids,
  • An antibody that is deleted and/or inserted with an amino acid altered target antibody region eg, a heavy chain variable region or a light chain variable region or a heavy chain CDR region or a light chain CDR region
  • the variant substantially retains the antibody prior to alteration
  • sequence identity refers to the same degree of nucleotide-by-nucleotide or amino acid-based sequences in a comparison window.
  • the “percent sequence identity” can be calculated by comparing the two optimally aligned sequences in a comparison window to determine the presence of the same nucleic acid base in both sequences (eg, A, T, C, G, I) Or the same amino acid residues (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) The number of positions is obtained to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (ie, the window size), and the result is multiplied by 100 to generate a sequence identity percentage.
  • Optimal alignments for determining percent sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • Megalign DNASTAR
  • One of skill in the art can determine suitable parameters for aligning the sequences, including any algorithms needed to achieve maximum alignment within the full length sequence being compared or within the target sequence region.
  • the amino acid sequence identity percentage is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence in a preferred embodiment according to the Kabat numbering rule.
  • the antibody region of interest eg, the entire variable region of the heavy or light chain, or portions thereof, eg, one or more CDR regions
  • the percent sequence identity between the target antibody region and the reference antibody region is: the number of positions occupied by the same amino acid in both the target and reference antibody regions divided by the total number of aligned positions in the two regions (vacancies are not counted) and Multiply by 100 to get the percentage.
  • sequence identity may be distributed throughout the heavy chain variable region and/or the entire light chain variable region, or the percent sequence identity may be limited to only the framework region, while the corresponding CDR region The sequence remains 100% identical.
  • conservative substitution refers to an amino acid change that results in the replacement of an amino acid with a chemically similar amino acid.
  • Conservative substitution representatives that provide functionally similar amino acids are well known in the art.
  • the conservatively substituted residue is derived from the conservative substitution table A below, preferably the preferred substitution residue shown in Table A.
  • mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and large mouse.
  • rodents eg, mice and large mouse.
  • the subject is a human.
  • treatment refers to the clinical intervention intended to alter the natural course of the disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis.
  • cancer refers to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • cancer include, but are not limited to, cancer, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia.
  • cancers include: squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, nerve Glioma, cervical cancer, ovarian cancer, liver cancer such as liver cancer and hepatocellular carcinoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland cancer, Renal cancers such as renal cell carcinoma and Welman's tumor, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer.
  • squamous cell carcinoma small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, nerve
  • cancerous cells grow and multiply, they form a mass of cancerous tissue, which is a tumor that invades and destroys normal adjacent tissues.
  • a malignant tumor is a cancer. Malignant tumors can often be removed, but they may regenerate. Cells from malignant tumors can invade and destroy nearby tissues and organs.
  • cancer cells can detach from malignant tumors and enter the bloodstream or lymphatic system, which is the way cancer cells spread from primary tumors (ie, the initial cancer) to form new tumors in other organs. The spread of cancer in the body is called metastasis (What You Need to Know About Cancer-an Overview, NIH Publication No. 00-1566; published on September 26, 2000, updated September 16, 2002 (2002)).
  • the invention provides an antibody or antigen-binding fragment thereof (i.e., an antibody or antigen-binding fragment thereof of the invention) that efficiently activates the NFkB signaling pathway downstream of GITR, which specifically binds to glucocorticoid-induced tumor necrosis Factor receptor (GITR).
  • an antibody or antigen-binding fragment thereof i.e., an antibody or antigen-binding fragment thereof of the invention
  • GITR glucocorticoid-induced tumor necrosis Factor receptor
  • the antibody of the invention is a monoclonal antibody or a multispecific antibody (including a diabody); and/or is of the IgG1, IgG2, IgG3 or IgG4 type; and/or an antigen binding fragment, such as a Fab fragment a Fab' fragment, an F(ab') 2 fragment or an Fv fragment; and/or a human antibody, a humanized antibody or a chimeric antibody; and/or a labeled antibody.
  • the antibodies of the invention are miniaturized antibodies that specifically bind to GITR, such as single chain antibodies (scFv), single domain antibodies (sdAd), heavy chain antibodies (hcAb), Nanobodies that specifically bind to GITR ( Nb or VHH); or a polymeric form of these antibodies.
  • miniaturized antibodies preferably comprise HCDR3 (heavy chain complementarity determining region 3) of the heavy chain variable region of the antibody of the invention; more preferably further comprise HCDR1 (heavy chain complementarity determining region 1) and HCDR2 of the heavy chain variable region of the antibody of the invention ( Heavy chain complementarity determines region 2).
  • the invention encompasses variants of any of the antibodies described herein.
  • the antibody variant retains at least 60%, 70%, 80%, 90% or 100% of the biological activity (eg, antigen binding capacity) of the altered pre-antibody.
  • the alteration does not result in loss of binding of the antibody variant to the antigen, but may optionally impart properties such as increased antigen affinity and different effector functions.
  • the heavy chain variable region or the light chain variable region of the antibody, or each CDR region may be altered individually or in combination.
  • the amino acid change in one or more or all three heavy chain CDRs does not exceed one, two, three, four, five, six, seven, eight, nine Or 10.
  • the amino acid is changed to an amino acid substitution, preferably a conservative substitution.
  • the amino acid change in one or more or all three heavy chain CDRs does not exceed one, two, three, four, five, six, seven, eight, nine Or 10.
  • the amino acid is changed to an amino acid substitution, preferably a conservative substitution.
  • the antibody variant and the parent antibody have at least 80%, 85%, 90% or 95% or 99% or more amino acid identity over the region of the antibody sequence of interest.
  • an antibody of the invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the heavy chain variable region compared to any of the antibodies listed in Table B. Sequence identity of 95%, 96%, 97%, 98% or 99% or higher.
  • an antibody of the invention comprises at least one, two, three, four, five or six CDRs identical to the corresponding CDRs of any of the antibodies listed in Table B, or variants thereof.
  • an antibody of the invention comprises at least one, two, or three HCDRs, or variants thereof, that are identical to the corresponding heavy chain CDRs of any of the antibodies listed in Table B.
  • corresponding CDR refers to a CDR that is located at a substantially similar position in the amino acid sequence of the variable region.
  • a CDR variant is a CDR that has been modified by at least one, for example 1 or 2 or 3 amino acid substitutions, deletions and/or insertions, wherein the antibody molecule comprising the CDR variant substantially retains an antibody comprising an unmodified CDR
  • the biological properties of the molecule for example, maintain at least 60%, 70%, 80%, 90% or 100% of the biological activity (e.g., antigen binding ability).
  • each CDR may be modified individually or in combination.
  • the amino acid modification is an amino acid substitution, especially a conservative amino acid substitution, such as the preferred conservative amino acid substitutions listed in Table A.
  • an antibody or antigen-binding fragment thereof of the invention binds GITR with high specificity and high affinity.
  • an antibody or antigen-binding fragment thereof of the invention binds with high affinity to a human GITR (such as the polypeptide of SEQ ID NO: 123 or 127), for example, at less than about 50 nM, less than about 30 nM, less than about 10-25 nM. Or a low affinity of less than about 20 nM, preferably with a human GITR (such as the polypeptide of SEQ ID NO: 123 or 127) with an affinity of less than about 10 nM.
  • a human GITR such as the polypeptide of SEQ ID NO: 123 or 127
  • an antibody or antigen-binding fragment thereof of the invention has about 1-50 nM, about 5-50 nM, about 10-50 nM, about 1-30 nM, about 5-30 nM, about 10-30 nM, about 1-25 nM, about 5- Affinity of 25 nM, about 10-25 nM, about 1-20 nM, about 5-20 nM, about 10-20 nM, about 0.1-10 nM, about 1-10 nM, about 5-10 nM, about 10 nM and human GITR (eg SEQ ID NO: The polypeptide of 123 or 127) binds.
  • an antibody or antigen-binding fragment thereof of the invention binds with high affinity to a human GITR (such as the polypeptide of SEQ ID NO: 123 or 127), for example, greater than about 0.01 x 10 4 /Ms, greater than about 0.1 x. 10 4 /Ms, a binding constant greater than about 1 x 10 4 /Ms or greater than about 3 x 10 4 /Ms, preferably with a binding constant greater than about 5 x 10 4 /Ms and human GITR (eg SEQ ID NO: 123 or 127) The peptide) binds.
  • a human GITR such as the polypeptide of SEQ ID NO: 123 or 127
  • an antibody or antigen-binding fragment thereof of the invention binds to a human GITR (such as the polypeptide of SEQ ID NO: 123 or 127) with a low dissociation constant, for example, with human GITR (eg SEQ ID NO: 123 or 127)
  • the polypeptide has a dissociation constant (K d ) of less than about 2 ⁇ 10 -2 s -1 , less than about 1.5 ⁇ 10 -2 s -1 , less than about 8 ⁇ 10 -3 s -1 or less than about 5 ⁇ 10 -3 s -1 ; preferably has a dissociation constant of about 1-3 x 10 -3 s -1 for binding to human GITR (such as the polypeptide of SEQ ID NO: 123 or 127).
  • the antibody or antigen-binding fragment thereof of the present invention binds to a human GITR (such as the polypeptide of SEQ ID NO: 123 or 127) with a dissociation constant (K d ) of about 1 ⁇ 10 -4 s -1 to 2 ⁇ 10 - 2 s -1 , about 1 ⁇ 10 -3 s -1 to 2 ⁇ 10 -2 s -1 , about 3 ⁇ 10 -3 s -1 to 2 ⁇ 10 -2 s -1 , about 1 ⁇ 10 -4 s -1 to 1.5 ⁇ 10 -2 s -1 , about 1 ⁇ 10 -3 s -1 to 1.5 ⁇ 10 -2 s -1 , about 3 ⁇ 10 -3 s -1 to 1.5 ⁇ 10 -2 s -1 , about 1 ⁇ 10 -4 s -1 to 8 ⁇ 10 -2 s -1 , about 1 ⁇ 10 -3 s -1 to 8 ⁇ 10 -2 s -1 , about 3 ⁇ 10
  • an antibody or antigen-binding fragment thereof of the invention binds with high affinity to a cell surface antigen, for example with an affinity of less than about 50 nM, less than about 30 nM, less than about 25 nM, or less than about 20 nM, preferably less than about 10 nM.
  • Affinity binds to human GITR (such as the polypeptide of SEQ ID NO: 123 or 127).
  • an antibody or antigen-binding fragment thereof of the invention at about 1 nM-50 nM, about 5 nM-50 nM, about 10 nM-50 nM, about 1 nM-30 nM, about 5 nM-30 nM, about 10 nM-30 nM, about 1 nM-25 nM, about 5 nM- Affinity at 25 nM, about 10 nM-25 nM, about 1 nM-20 nM, about 5 nM-20 nM, about 10 nM-20 nM, about 0.1-10 nM, about 1-10 nM, about 5-10 nM, about 10 nM and antigen on the cell surface.
  • the antibody or antigen-binding fragment of the present invention efficiently activates NFkB signaling pathway downstream of GITR, e.g. activation of NFkB signaling pathway EC 50 value of less than about 50 nM, less than about 30 nM, less than about of 5 nM or less than about 25nM, preferably Less than about 1 nM.
  • an antibody or antigen-binding fragment of the present invention activates EC NFkB signaling pathway 50 value of about 1nM-50nM, about 5nM-50nM, about 10nM-50nM, from about 1nM-30nM, about 5nM-30nM, about 10nM-30nM, About 1 nM-25 nM, about 5 nM-25 nM, about 10 nM-25 nM, about 1 nM-20 nM, about 5 nM-20 nM, about 10 nM-20 nM, about 0.1-10 nM, about 1-10 nM, about 5-10 nM, or about 10 nM.
  • an antibody of the invention cross-competes with GITRL to bind to a GITR, preferably cross-competing with GITRL to a human GITR (such as the polypeptide of SEQ ID NO: 123 or 127).
  • an antibody of the invention can be internalized into a human CD4 cell.
  • an antibody or antigen-binding fragment thereof of the invention inhibits the inhibition of regulatory T cells.
  • an antibody or antigen-binding fragment thereof of the invention activates an effector T cell.
  • an antibody or antigen-binding fragment thereof of the invention reduces circulating regulatory T cells.
  • an antibody or antigen-binding fragment thereof of the invention is capable of binding to an Fc gamma receptor (Fc[gamma]R).
  • an antibody or antigen-binding fragment thereof of the invention has a half-life in human serum of at least about 6, about 7, about 9, or about 12 days.
  • the invention provides a Nanobody (i.e., a Nanobody of the invention) that specifically binds to GITR and efficiently activates the NFkB signaling pathway downstream of GITR, the HCDR3 of the Nanobody comprising an antibody or antigen-binding fragment of the invention HCDR3.
  • a Nanobody i.e., a Nanobody of the invention
  • the HCDR3 of the Nanobody comprising an antibody or antigen-binding fragment of the invention HCDR3.
  • the HCDR3 of the Nanobody of the invention comprises the HCDR3 contained in the heavy chain variable region sequence set forth in any one of SEQ ID NOs: 64-85, or the HCDR3 of the Nanobody of the invention is SEQ ID NO: The HCDR3 composition contained in the heavy chain variable region sequence shown in any of 64-85.
  • the HCDR3 of a Nanobody of the invention comprises at least 90%, 91%, 92%, 93% of the heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 64-85, HCDR3 contained in a 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity heavy chain variable region sequence, wherein preferred sequence differences are not present in the CDR regions, or the invention
  • the HCDR3 of the Nanobody consists of HCDR3 contained in such a heavy chain variable region sequence.
  • the HCDR3 of a Nanobody of the invention comprises one or more (preferably 1-10) of the heavy chain variable region sequences set forth in any of the sequences of SEQ ID NOs: 64-85, More preferably 1-5, most preferably 1-3) HCDR3 contained in the heavy chain variable region sequence having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein preferably the amino acid change does not occur in the CDR region
  • the HCDR3 of the Nanobody of the present invention or the Nanobody of the present invention consists of HCDR3 contained in such a heavy chain variable region sequence.
  • the HCDR3 of the Nanobody of the invention comprises the HCDR3 as set forth in any one of SEQ ID NOs: 45-63, or the HCDR3 of the Nanobody of the invention is as set forth in SEQ ID NOs: 45-63 A sequence of HCDR3 compositions is shown.
  • the HCDR3 of a Nanobody of the invention comprises one or more (preferably 1-10, more preferably 1-5) of the HCDR3 as shown in any of SEQ ID NOs: 45-63.
  • the HCDR1 (heavy chain complementarity determining region 1) and HCDR2 (heavy chain complementarity determining region 2) of the Nanobodies of the invention comprise a heavy chain as set forth in any one of SEQ ID NOs: 64-85
  • the HCDR1 and HCDR2 contained in the variable region sequence, or the HCDR1 and HCDR2 of the Nanobody of the present invention consist of HCDR1 and HCDR2 contained in the heavy chain variable region sequence as shown in any one of SEQ ID NOs: 64-85.
  • the HCDR1 and HCDR2 of a Nanobody of the invention comprise one or more of the heavy chain variable region sequences set forth in any of the sequences of SEQ ID NOs: 64-85 (preferably 1-10) , more preferably 1-5, most preferably 1-3) HCDR1 and HCDR2 contained in a heavy chain variable region sequence having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein preferably the amino acid change is not HCDR1 and HCDR2 occurring in the CDR region or the Nanobody of the present invention consist of HCDR1 and HCDR2 contained in such a heavy chain variable region sequence.
  • the HCDR1 of a Nanobody of the invention comprises HCDR1 as set forth in any one of SEQ ID NOs: 1-22, or the HCDR1 of a Nanobody of the invention is as set forth in SEQ ID NO: 1-22 A sequence of HCDR1 is shown.
  • the HCDR1 of a Nanobody of the invention comprises one or more (preferably 1-10, more preferably 1-5) of the HCDR1 as set forth in any of SEQ ID NOs: 1-22. , most preferably 1-3) HCDR1 having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein preferably the amino acid change does not occur in the CDR region, or the HCDR1 of the Nanobody of the invention consists of such HCDR1 .
  • the HCDR2 of the Nanobody of the invention comprises HCDR2 as shown in any one of SEQ ID NOs: 23-44, or the HCDR2 of the Nanobody of the invention is as set forth in SEQ ID NO: 23-44 A sequence of HCDR2 compositions.
  • the HCDR2 of a Nanobody of the invention comprises one or more (preferably 1-10, more preferably 1-5) of the HCDR2 as shown in any of SEQ ID NOs: 23-44.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 23, and HCDR3 as set forth in SEQ ID NO:45.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 2, HCDR2 as set forth in SEQ ID NO: 24, and HCDR3 as set forth in SEQ ID NO:46.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 3, HCDR2 as set forth in SEQ ID NO: 25, and HCDR3 as set forth in SEQ ID NO:47.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 4, HCDR2 as set forth in SEQ ID NO: 26, and HCDR3 as set forth in SEQ ID NO:48.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 5, HCDR2 as set forth in SEQ ID NO: 27, and HCDR3 as set forth in SEQ ID NO:49.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 6, HCDR2 as set forth in SEQ ID NO: 28, and HCDR3 as set forth in SEQ ID NO:50.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 7, HCDR2 as set forth in SEQ ID NO: 29, and HCDR3 as set forth in SEQ ID NO:51.
  • a Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 8, HCDR2 as set forth in SEQ ID NO: 30, and HCDR3 as set forth in SEQ ID NO:52.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 9, HCDR2 as set forth in SEQ ID NO: 31, and HCDR3 as set forth in SEQ ID NO:53.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 32, and HCDR3 as set forth in SEQ ID NO:51.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 33, and HCDR3 as set forth in SEQ ID NO:48.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 12, HCDR2 as set forth in SEQ ID NO: 34, and HCDR3 as set forth in SEQ ID NO:46.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 35, and HCDR3 as set forth in SEQ ID NO:54.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 14, HCDR2 as set forth in SEQ ID NO: 36, and HCDR3 as set forth in SEQ ID NO:55.
  • a Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 15, HCDR2 as set forth in SEQ ID NO: 37, and HCDR3 as set forth in SEQ ID NO:56.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 38, and HCDR3 as set forth in SEQ ID NO:57.
  • a Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 17, HCDR2 as set forth in SEQ ID NO: 39, and HCDR3 as set forth in SEQ ID NO:58.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 18, HCDR2 as set forth in SEQ ID NO: 40, and HCDR3 as set forth in SEQ ID NO:59.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 19, HCDR2 as set forth in SEQ ID NO: 41, and HCDR3 as set forth in SEQ ID NO:60.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 20, HCDR2 as set forth in SEQ ID NO: 42 and HCDR3 as set forth in SEQ ID NO:61.
  • the Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 21, HCDR2 as set forth in SEQ ID NO: 43 and HCDR3 as set forth in SEQ ID NO:62.
  • a Nanobody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 22, HCDR2 as set forth in SEQ ID NO: 44, and HCDR3 as set forth in SEQ ID NO:63.
  • a Nanobody of the invention comprises a heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 64-85, or as shown in any of SEQ ID NOs: 64-85
  • the heavy chain variable region sequence consists, preferably consisting of only one such heavy chain variable region sequence.
  • the Nanobody of the invention comprises at least 90%, 91%, 92%, 93%, 94% of the heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 64-85 a 95%, 96%, 97%, 98%, 99% or more sequence identity heavy chain variable region sequence, wherein preferably the sequence difference is not present in the CDR region, or the Nanobody of the invention is such a heavy
  • the chain variable region sequence consists, preferably consisting of only one such heavy chain variable region sequence.
  • the Nanobody of the invention comprises one or more (preferably 1-10, more preferably) of the heavy chain variable region sequences as set forth in any of SEQ ID NOs: 64-85 1-5, most preferably 1-3) heavy chain variable region sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), wherein preferably the amino acid changes do not occur in the CDR regions, or the nanoparticles of the invention
  • An antibody consists of such a heavy chain variable region sequence, preferably consisting of only one such heavy chain variable region sequence.
  • the invention provides a heavy chain antibody (ie, a heavy chain antibody of the invention) that specifically binds to GITR and efficiently activates the NFkB signaling pathway downstream of GITR, the HCDR3 of the heavy chain antibody comprising an antibody of the invention or The HCDR3 of the antigen-binding fragment, or consists of the HCDR3 of the antibody or antigen-binding fragment of the invention; the HCDR3 of the heavy chain antibody preferably comprises or consists of the HCDR3 of the above-described Nanobody of the invention; more preferably the heavy chain antibody
  • HCDR1-2 comprises HCDR1-2 of the above-described Nanobody of the present invention or HCDR1-2 of the above-described Nanobody of the present invention, respectively.
  • the heavy chain antibody of the invention is a humanized heavy chain antibody, preferably a fully humanized heavy chain antibody (human antibody).
  • the HCDR3 of a heavy chain antibody of the invention comprises a heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83 HCDR3, or HCDR3 of the heavy chain antibody of the present invention, HCDR3 contained in the heavy chain variable region sequence as shown in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79 and 83 composition.
  • the HCDR3 of a heavy chain antibody of the invention comprises a heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83 HCDR3 contained in at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity heavy chain variable region sequences, preferably The sequence difference is not present in the CDR region, or the HCDR3 of the heavy chain antibody of the present invention consists of HCDR3 contained in such a heavy chain variable region sequence.
  • the HCDR3 of a heavy chain antibody of the invention comprises a heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83
  • the HCDR3 of a heavy chain antibody of the invention comprises an HCDR3 as set forth in any one of SEQ ID NOs: 46, 47, 48, 51, 53, 57, and 61, or a heavy chain antibody of the invention HCDR3 consists of HCDR3 as shown in any of SEQ ID NOs: 46, 47, 48, 51, 53, 57 and 61.
  • the HCDR3 of a heavy chain antibody of the invention comprises one or more of the HCDR3s as set forth in any of SEQ ID NOs: 46, 47, 48, 51, 53, 57, and 61 ( Preferably, from 1 to 10, more preferably from 1 to 5, most preferably from 1 to 3, HCDR3 having an amino acid change (preferably a substitution, more preferably a conservative substitution), or an HCDR3 of a heavy chain antibody of the invention consists of such an HCDR3.
  • the HCDR1 and HCDR2 of the heavy chain antibody of the invention comprise a heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83
  • the HCDR1 and HCDR2 contained, or the HCDR1 and HCDR2 of the heavy chain antibody of the present invention are composed of HCDR1 and HCDR2 contained in such a heavy chain variable region sequence.
  • the HCDR1 and HCDR2 of the heavy chain antibody of the invention comprise a heavy chain variable region as set forth in any of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83 HCDR1 and the heavy chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity HCDR2, wherein preferred sequence differences are not present in the CDR regions, or HCDR1 and HCDR2 of the heavy chain antibodies of the invention consist of HCDR1 and HCDR2 contained in such heavy chain variable region sequences.
  • the HCDR1 and HCDR2 of the heavy chain antibody of the invention comprise a heavy chain variable region as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83
  • One or more (preferably 1-10, more preferably 1-5, most preferably 1-3) sequences in the sequence have heavy chain variable region sequences with amino acid changes (preferably substitutions, more preferably conservative substitutions)
  • HCDR1 and HCDR2 are contained, wherein preferably the amino acid change does not occur in the CDR region, or the HCDR1 and HCDR2 of the heavy chain antibody of the present invention consist of HCDR1 and HCDR2 contained in such a heavy chain variable region sequence.
  • the HCDR1 of a heavy chain antibody of the invention comprises HCDR1 as shown in any one of SEQ ID NOs: 2, 3, 4, 7, 9, 10, 16, 20, or a heavy chain of the invention
  • the HCDR1 of the antibody consists of HCDR1 as shown in any one of SEQ ID NOs: 2, 3, 4, 7, 9, 10, 16, 20.
  • the HCDR1 of a heavy chain antibody of the invention comprises one or more of HCDR1 as shown in any of SEQ ID NO: 2, 3, 4, 7, 9, 10, 16, 20 HCDR1 having an amino acid change (preferably substituted, more preferably conservative substitution), or HCDR1 of the heavy chain antibody of the present invention, is derived from such HCDR1 (preferably 1-10, more preferably 1-5, most preferably 1-3) composition.
  • the HCDR2 of a heavy chain antibody of the invention comprises HCDR2 as set forth in any one of SEQ ID NOs: 24, 25, 26, 29, 31, 32, 38, 42 or a heavy chain of the invention
  • the HCDR2 of the antibody consists of HCDR2 as shown in any of SEQ ID NOs: 24, 25, 26, 29, 31, 32, 38, 42.
  • the HCDR2 of a heavy chain antibody of the invention comprises one or more of HCDR2 as shown in any of SEQ ID NO: 24, 25, 26, 29, 31, 32, 38, 42 HCDR2 having an amino acid change (preferably substitution, more preferably conservative substitution) at a position (preferably 1-10, more preferably 1-5, most preferably 1-3), or HCDR2 of the heavy chain antibody of the present invention is derived from such HCDR2 composition.
  • the heavy chain antibodies of the invention comprise a combination of HCDR1 as set forth in SEQ ID NO: 2, HCDR2 as set forth in SEQ ID NO: 24, and HCDR3 as set forth in SEQ ID NO:46.
  • a heavy chain antibody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 3, HCDR2 as set forth in SEQ ID NO: 25, and HCDR3 as set forth in SEQ ID NO:47.
  • a heavy chain antibody of the invention comprises a combination of HCDR1 as set forth in SEQ ID NO: 4, HCDR2 as set forth in SEQ ID NO: 26, and HCDR3 as set forth in SEQ ID NO:48.
  • the heavy chain antibody of the invention comprises HCDR1 as set forth in SEQ ID NO: 7, HCDR2 as set forth in SEQ ID NO: 29, and HCDR3 as set forth in SEQ ID NO:51.
  • the heavy chain antibody of the invention comprises HCDR1 as set forth in SEQ ID NO: 9, HCDR2 as set forth in SEQ ID NO: 31, and HCDR3 as set forth in SEQ ID NO:53.
  • the heavy chain antibody of the invention comprises HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 32, and HCDR3 as set forth in SEQ ID NO:51.
  • the heavy chain antibody of the invention comprises HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 38, and HCDR3 as set forth in SEQ ID NO:57.
  • the heavy chain antibody of the invention comprises HCDR1 as set forth in SEQ ID NO: 20, HCDR2 as set forth in SEQ ID NO: 42 and HCDR3 as set forth in SEQ ID NO:61.
  • the heavy chain variable region sequence of a heavy chain antibody of the invention comprises a heavy chain as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83
  • the variable region sequence, or the heavy chain variable region sequence of the heavy chain antibody of the present invention is variable in a heavy chain as shown by any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79 and 83
  • the sequence of the region is composed.
  • the heavy chain variable region sequence of a heavy chain antibody of the invention comprises a heavy chain as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83
  • a variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity of a heavy chain variable region, wherein Preferably, the sequence difference is not present in the CDR region, or the heavy chain variable region sequence of the heavy chain antibody of the invention consists of such a heavy chain variable region sequence.
  • the heavy chain variable region sequence of a heavy chain antibody of the invention comprises a heavy chain as set forth in any one of SEQ ID NOs: 65, 66, 67, 70, 72, 73, 79, and 83
  • One or more (preferably 1-10, more preferably 1-5, most preferably 1-3) heavy chain variable regions having amino acid changes (preferably substituted, more preferably conservative substitutions) in the variable region sequence A sequence wherein preferably the amino acid change does not occur in the CDR region, or the heavy chain variable region sequence of the heavy chain antibody of the invention consists of such a heavy chain variable region sequence.
  • the heavy chain antibody of the invention comprises a heavy chain antibody sequence as set forth in any one of SEQ ID NOs: 108-115, or a heavy chain antibody of the invention consists of one, two or more such as SEQ ID NO: The composition of the heavy chain antibody sequence shown in any of 108-115.
  • the heavy chain antibody of the invention comprises at least 90%, 91%, 92%, 93%, 94% of the heavy chain antibody sequence as set forth in any one of SEQ ID NOs: 108-115, a 95%, 96%, 97%, 98%, 99% or more sequence identity heavy chain antibody sequence, wherein preferably the sequence difference is not present in the CDR region, or the heavy chain antibody of the invention consists of one, two or A plurality of such heavy chain antibody sequences are composed.
  • the heavy chain antibody of the invention comprises one or more (preferably 1-10, more preferably 1) of the heavy chain antibody sequences set forth in any of the sequences of SEQ ID NOs: 108-115 -5, most preferably 1-3) heavy chain antibody sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), wherein preferably the amino acid changes do not occur in the CDR regions, or the heavy chain antibodies of the invention are One, two or more such heavy chain antibody sequences are composed.
  • the invention provides a humanized heavy chain antibody (ie, a humanized heavy chain antibody of the invention) that specifically binds to GITR and efficiently activates the NFkB signaling pathway downstream of GITR, the humanized weight
  • a humanized heavy chain antibody ie, a humanized heavy chain antibody of the invention
  • the HCDR3 of the chain antibody comprises or consists of the HCDR3 of an antibody or antigen-binding fragment of the invention.
  • the HCDR3 of a humanized heavy chain antibody of the invention comprises or consists of the HCDR3 of a Nanobody or Heavy Chain Antibody of the invention described above, or the HCDR3 of the Nanobody or Heavy Chain Antibody of the invention described above; more preferably the inventors
  • the HCDR1-2 of the sourced heavy chain antibody preferably comprises the HCDR1-2 of the above-described Nanobody or heavy chain antibody of the present invention, or the HCDR1-2 of the above-described Nanobody or heavy chain antibody of the present invention, respectively.
  • the HCDR3 of the humanized heavy chain antibody of the invention comprises HCDR3 as contained in the heavy chain variable region sequence set forth in SEQ ID NO: 124 or 125, or the humanized heavy chain of the present invention
  • the HCDR3 in the antibody consists of the HCDR3 contained in the heavy chain variable region sequence as shown in SEQ ID NO: 124 or 125.
  • the HCDR3 of the humanized heavy chain antibody of the invention comprises at least 90%, 91%, 92%, 93% of the heavy chain variable region sequence as set forth in SEQ ID NO: 124 or 125 HCDR3 contained in a 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity heavy chain variable region sequence, wherein preferred sequence differences are not present in the CDR regions, or the invention
  • the HCDR3 in the humanized heavy chain antibody consists of HCDR3 contained in such a heavy chain variable region sequence.
  • the HCDR3 of the humanized heavy chain antibody of the invention comprises one or more (preferably 1-10) of the heavy chain variable region sequences set forth in SEQ ID NO: 124 or 125 More preferably 1-5, most preferably 1-3) HCDR3 contained in a heavy chain variable region sequence having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein preferably the amino acid change does not occur in the CDR
  • the HCDR3 in the region, or the humanized heavy chain antibody of the present invention consists of HCDR3 contained in such a heavy chain variable region sequence.
  • the HCDR3 of the humanized heavy chain antibody of the invention comprises HCDR3 selected from SEQ ID NO: 51 or 61, or the HCDR3 of the humanized heavy chain antibody of the invention is selected from the group consisting of SEQ ID NO : 51 or 61 HCDR3 composition.
  • the HCDR3 of the humanized heavy chain antibody of the invention comprises one or more (preferably 1-10, more preferably 1-5) of HCDR3 selected from SEQ ID NO: 51 or 61 , most preferably 1-3) HCDR3 having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein preferably the amino acid change does not occur in the CDR region, or the HCDR3 in the humanized heavy chain antibody of the invention It consists of such HCDR3.
  • the HCDR1 and HCDR2 of the humanized heavy chain antibody of the invention comprise HCDR1 and HCDR2, which are selected from a heavy chain variable region sequence of SEQ ID NO: 124 or 125, or a human source of the invention
  • the HCDR1 and HCDR2 in the heavy chain antibody consist of HCDR1 and HCDR2 contained in the heavy chain variable region sequence selected from SEQ ID NO: 124 or 125.
  • the HCDR1 and HCDR2 of the humanized heavy chain antibody of the invention comprise at least 90%, 91%, 92% of the heavy chain variable region sequence as set forth in SEQ ID NO: 124 or 125, HCDR1 and HCDR2 contained in the 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity heavy chain variable region sequences, wherein preferred sequence differences are not present in the CDR regions Or HCDR1 and HCDR2 in the humanized heavy chain antibody of the present invention consist of HCDR1 and HCDR2 contained in such a heavy chain variable region sequence.
  • the HCDR1 and HCDR2 in the humanized heavy chain antibody of the invention comprise one or more of the heavy chain variable region sequences set forth in SEQ ID NO: 124 or 125 (preferably 1 10, more preferably 1-5, most preferably 1-3) HCDR1 and HCDR2 contained in a heavy chain variable region sequence having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein said amino acid change is preferred HCDR1 and HCDR2 which do not occur in the CDR region or in the humanized heavy chain antibody of the present invention are composed of HCDR1 and HCDR2 contained in such a heavy chain variable region sequence.
  • the HCDR1 of the humanized heavy chain antibody of the invention comprises HCDR1 as set forth in SEQ ID NO: 7 or 20, or the HCDR1 of the humanized heavy chain antibody of the invention is represented by SEQ ID NO : HCDR1 composition shown in 7 or 20.
  • the HCDR1 of the humanized heavy chain antibody of the invention comprises one or more (preferably 1-10, more preferably 1 -) in the HCDR1 as set forth in SEQ ID NO: 7 or 20. 5, most preferably 1-3) HCDR1 having an amino acid change (preferably a substitution, more preferably a conservative substitution), or HCDR1 in a humanized heavy chain antibody of the invention consists of such HCDR1.
  • the HCDR2 of the humanized heavy chain antibody of the invention comprises HCDR2 as set forth in SEQ ID NO: 29 or 42, or the HCDR2 of the humanized heavy chain antibody of the invention is SEQ ID NO : HCDR2 composition shown in 29 or 42.
  • the HCDR2 of the humanized heavy chain antibody of the invention comprises one or more (preferably 1-10, more preferably 1-) of the HCDR2 as set forth in SEQ ID NO: 29 or 42 5, most preferably 1-3) HCDR2 having an amino acid change (preferably a substitution, more preferably a conservative substitution), or HCDR2 in a humanized heavy chain antibody of the invention consists of such HCDR2.
  • the humanized heavy chain antibody of the invention comprises a combination of HCDR1 as shown in SEQ ID NO:7, HCDR2 as set forth in SEQ ID NO: 29, and HCDR3 as set forth in SEQ ID NO: .
  • the humanized heavy chain antibody of the invention comprises a combination of HCDR1 as shown in SEQ ID NO: 20, HCDR2 as shown in SEQ ID NO: 42 and HCDR3 as set in SEQ ID NO: 61 .
  • the heavy chain variable region sequence of a humanized heavy chain antibody of the invention comprises a heavy chain variable region sequence as set forth in SEQ ID NO: 124 or 125, or a humanized heavy chain of the invention
  • the heavy chain variable region sequence of the antibody consists of the heavy chain variable region sequence set forth in SEQ ID NO: 124 or 125.
  • the heavy chain variable region sequence of a humanized heavy chain antibody of the invention comprises at least 90%, 91%, 92 of the heavy chain variable region sequence as set forth in SEQ ID NO: 124 or 125 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity heavy chain variable region sequences, wherein preferred sequence differences are not present in the CDR regions, or the invention
  • the heavy chain variable region sequence of the humanized heavy chain antibody consists of such a heavy chain variable region sequence.
  • the heavy chain variable region sequence of a humanized heavy chain antibody of the invention comprises one or more of the heavy chain variable region sequences set forth in SEQ ID NO: 124 or 125 (preferably 1-10, more preferably 1-5, most preferably 1-3) heavy chain variable region sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), wherein preferably the amino acid changes do not occur in the CDR regions
  • the heavy chain variable region sequence of the humanized heavy chain antibody of the invention, or the heavy chain variable region sequence consists of such a heavy chain variable region sequence.
  • the humanized heavy chain antibody of the invention comprises the humanized heavy chain antibody sequence set forth in SEQ ID NO: 116 or 117, or one or both of the humanized heavy chain antibodies of the invention Or consisting of a plurality of humanized heavy chain antibody sequences as set forth in SEQ ID NO: 116 or 117.
  • the humanized heavy chain antibody of the invention comprises at least 90%, 91%, 92%, 93%, 94 of the humanized heavy chain antibody sequence set forth as SEQ ID NO: 116 or 117 Humanized heavy chain antibody sequences of %, 95%, 96%, 97%, 98%, 99% or more sequence identity, wherein preferred sequence differences are not present in the CDR regions, or the humanized weight of the invention
  • a chain antibody consists of one, two or more such humanized heavy chain antibody sequences.
  • the humanized heavy chain antibody of the invention comprises one or more (preferably 1-10, more) of the humanized heavy chain antibody sequences set forth in SEQ ID NO: 116 or 117 Preferred are 1-5, most preferably 1-3) humanized heavy chain antibody sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), wherein preferably the amino acid changes do not occur in the CDR regions, or the invention
  • a humanized heavy chain antibody consists of one, two or more such humanized heavy chain antibody sequences.
  • the heavy chain antibody or humanized heavy chain antibody of the invention comprises any of the above heavy chain variable regions and Fc fragments, preferably the Fc fragment comprises one of the following sequences (a)-(d) Or consist of one of the following sequences (a)-(d):
  • the heavy chain antibody or humanized heavy chain antibody of the invention comprises two Fc sequences selected from the above sequences (a) to (d).
  • an interchain disulfide bond is present between the two Fc sequences (a)-(d) of the same heavy chain antibody or humanized heavy chain antibody of the invention.
  • the interchain disulfide The bond is a two-chain disulfide bond formed between amino acid residues C in a CPPC sequence contained in the heavy chain antibody or the humanized heavy chain antibody of the present invention, and the CPPC sequence is a heavy chain antibody or human of the present invention.
  • the invention provides an antibody (i.e., an antibody of the multimeric form of the invention) that specifically binds to GITR and efficiently activates the NFkB signaling pathway downstream of GITR, the polymeric form
  • the HCDR3 of the antibody comprises, or consists of, the HCDR3 of an antibody or antigen-binding fragment of the invention.
  • the HCDR3 of the antibody of the multimeric form of the invention comprises the HCDR3 of a Nanobody, a Heavy Chain Antibody or a Humanized Heavy Chain Antibody of the invention described above, or a Nanobody, a heavy chain antibody or a human of the invention described above
  • the HCDR3 composition of the antibody-derived heavy chain antibody; more preferably, the HCDRs 1-2 of the antibody of the multimeric form of the invention preferably comprise the HCDRs 1-2 of the above-described Nanobody, heavy chain antibody or humanized heavy chain antibody of the invention, respectively, or The HCDR1-2 composition of the above-described Nanobody, heavy chain antibody or humanized heavy chain antibody of the present invention.
  • the antibody in the multimeric form of the invention is a nanobody of the invention, a heavy chain variable region of a heavy chain antibody, a polymeric form of a heavy chain variable region of a humanized heavy chain antibody
  • the Nanobody of the invention, the heavy chain variable region of the heavy chain antibody, the tetrameric form or the hexamed form of the heavy chain variable region of the humanized heavy chain antibody most preferably the Nanobody of the invention
  • the NFkB signaling pathway downstream of the GITR can be more efficiently activated by forming an antibody that forms a multi-polymer form of the nanobody of the present invention, the heavy chain variable region of the heavy chain antibody, and the heavy chain variable region of the humanized heavy chain antibody.
  • the half-life of the Nanobody, heavy chain antibody, and humanized heavy chain antibody of the present invention in vivo can be appropriately extended.
  • the HCDR3 in the heavy chain variable region of the antibody of the present invention comprises the HCDR3 sequence contained in the heavy chain variable region sequence set forth in SEQ ID NO: 125, or
  • the HCDR3 in the heavy chain variable region of the antibody in the form of a polymer consists of the HCDR3 contained in the heavy chain variable region sequence set forth in SEQ ID NO: 125.
  • the HCDR3 in the heavy chain variable region of the antibody of the multimeric form of the invention comprises at least 90%, 91%, 92 of the heavy chain variable region sequence as set forth in SEQ ID NO: HCDR3 sequences contained in the heavy chain variable region sequences of %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity, wherein preferred sequence differences are not present in the CDR regions
  • the HCDR3 in the heavy chain variable region of an antibody, or a multimeric form of the invention consists of such an HCDR3 sequence.
  • the HCDR3 in the heavy chain variable region of the antibody of the present invention is contained in one or more of the heavy chain variable region sequences set forth in SEQ ID NO: 125 (preferably 1-10, more preferably 1-5, most preferably 1-3) HCDR3 contained in a heavy chain variable region sequence having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein said amino acid change is preferred HCDR3 that does not occur in the CDR regions, or in the heavy chain variable region of the antibody of the present invention, consists of such HCDR3 sequences.
  • the HCDR3 in the heavy chain variable region of the antibody of the present invention comprises the HCDR3 sequence set forth in SEQ ID NO: 61, or the antibody of the multimeric form of the invention.
  • the HCDR3 in the chain variable region consists of the HCDR3 sequence shown as SEQ ID NO:61.
  • the HCDR3 in the heavy chain variable region of the antibody of the present invention is contained in one or more (preferably 1-10, more preferably 1) of the HCDR3 of SEQ ID NO:61 -5, most preferably 1-3) HCDR3 sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), or HCDR3 in the heavy chain variable region of an antibody of the multimeric form of the invention are derived from such HCDR3 Sequence composition.
  • the HCDR1 and HCDR2 in the heavy chain variable region of the antibody of the present invention comprise the HCDR1 and HCDR2 sequences contained in the heavy chain variable region sequence set forth in SEQ ID NO: 125,
  • HCDR1 and HCDR2 in the heavy chain variable region of the antibody of the multimeric form of the invention consists of the HCDR1 and HCDR2 sequences contained in the heavy chain variable region sequence set forth in SEQ ID NO: 125.
  • the HCDR1 and HCDR2 in the heavy chain variable region of the antibody of the present invention comprise at least 90%, 91% of the heavy chain variable region sequence as set forth in SEQ ID NO: 125 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the heavy chain variable region sequences of sequence identity contained in HCDR1 and HCDR2, wherein preferred sequence differences are not present HCDR1 and HCDR2 in the CDR region, or in the heavy chain variable region of an antibody of the present invention, consist of such HCDR1 and HCDR2 sequences.
  • the HCDR1 and HCDR2 in the heavy chain variable region of the antibody of the present invention comprise one or more of the heavy chain variable region sequences set forth in SEQ ID NO: 125 (preferably 1 to 10, more preferably 1 to 5, most preferably 1 to 3) HCDR1 and HCDR2 contained in the heavy chain variable region sequence having an amino acid change (preferably substitution, more preferably conservative substitution), wherein preference is preferred
  • the amino acid changes do not occur in the CDR regions, or HCDR1 and HCDR2 in the heavy chain variable region of the antibody of the multimeric form of the invention consist of such HCDR1 and HCDR2 sequences.
  • the HCDR1 of the heavy chain variable region of the antibody of the present invention comprises the HCDR1 sequence set forth in SEQ ID NO: 20, or the heavy chain of the antibody of the multimeric form of the invention
  • the HCDR1 in the variable region consists of the HCDR1 sequence set forth in SEQ ID NO:20.
  • the HCDR1 in the heavy chain variable region of the antibody of the present invention is contained in one or more (preferably 1-10, more preferably 1) of the HCDR1 of SEQ ID NO:20 -5, most preferably 1-3) HCDR1 sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), or HCDR1 in the heavy chain variable region of an antibody of the multimeric form of the invention are derived from such HCDR1 Sequence composition.
  • the HCDR2 in the heavy chain variable region of the antibody of the present invention comprises the HCDR2 sequence set forth in SEQ ID NO: 42 or the heavy chain of the antibody in the multimeric form of the invention HCDR2 in the variable region consists of the HCDR2 sequence as set forth in SEQ ID NO:42.
  • the HCDR2 in the heavy chain variable region of the antibody of the present invention is contained in one or more (preferably 1-10, more preferably 1) of the HCDR2 of SEQ ID NO:42 -5, most preferably 1-3) HCDR2 sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), or HCDR2 in the heavy chain variable region of an antibody of the multimeric form of the invention are derived from such HCDR2 Sequence composition.
  • the heavy chain variable region of the antibody of the multimeric form of the invention comprises HCDR1 as set forth in SEQ ID NO: 20, HCDR2 as set forth in SEQ ID NO: 42 and SEQ ID NO: 61 The combination of HCDR3 shown.
  • the heavy chain variable region of the antibody of the present invention comprises a heavy chain variable region sequence as set forth in SEQ ID NO: 125, or the antibody of the multimeric form of the invention.
  • the chain variable region consists of the heavy chain variable region sequence set forth in SEQ ID NO:125.
  • the heavy chain variable region of the antibody of the multimeric form of the invention comprises at least 90%, 91%, 92%, 93 of the heavy chain variable region sequence as set forth in SEQ ID NO: %, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity heavy chain variable region sequences, wherein preferred sequence differences are not present in the CDR regions, or the multimerization of the invention
  • the heavy chain variable region of the antibody in the form of a composition consists of such a sequence of heavy chain variable regions.
  • the heavy chain variable region of the antibody of the multimeric form of the invention comprises one or more of the heavy chain variable region sequences set forth in SEQ ID NO: 125 (preferably 1-10) , more preferably from 1 to 5, and most preferably from 1 to 3, heavy chain variable region sequences having amino acid changes (preferably substitutions, more preferably conservative substitutions), wherein preferably the amino acid changes do not occur in the CDR regions, or
  • the heavy chain variable region of an antibody of the present invention is composed of such a heavy chain variable region sequence.
  • the antibody of the multimeric form of the invention comprises a sequence selected from any one of SEQ ID NOs: 118-120, or the antibody of the multimeric form of the invention consists of the same two, three or more A sequence consisting of SEQ ID NOs: 118-120.
  • the antibody of the multimeric form of the invention comprises at least 90%, 91%, 92%, 93%, 94% of the sequence as set forth in any one of SEQ ID NOs: 118-120, a sequence of 95%, 96%, 97%, 98%, 99% or more sequence identity, wherein preferably the sequence difference is not present in the CDR region, or the antibody of the multimeric form of the invention consists of the same two, Three or more such sequences are composed.
  • the antibody of the multimeric form of the invention comprises one or more (preferably 1-10, more preferably 1) of the sequences set forth in any of the sequences of SEQ ID NOs: 118-120 -5, most preferably 1-3) a sequence having an amino acid change (preferably a substitution, more preferably a conservative substitution), wherein preferably the amino acid change does not occur in the CDR region, or the antibody in the polymer form of the invention consists The same two, three or more such sequences are composed.
  • the Nanobody of the antibody of the present invention is linked to a humanized heavy chain antibody of the invention.
  • the Nanobody of the invention, the heavy chain variable region of the heavy chain antibody, the heavy chain variable region of the humanized heavy chain antibody in the antibody of the multimeric form of the invention are linked to the invention The C-terminus of the humanized heavy chain antibody; or the nanobody of the invention, the heavy chain variable region of the heavy chain antibody, the heavy chain variable region of the humanized heavy chain antibody, linked to the humanized heavy chain of the invention The N-terminus of the antibody.
  • the antibody of the multimeric form of the invention comprises two identical Nanobodies of the invention, a heavy chain variable region of a heavy chain antibody, a heavy chain variable region of a humanized heavy chain antibody
  • the two identical Nanobodies of the invention, the heavy chain variable region of the heavy chain antibody, and the heavy chain variable region of the humanized heavy chain antibody are ligated to two identical humanized heavy chain antibodies of the invention, respectively C-terminus; or the two identical Nanobodies of the invention, the heavy chain variable region of a heavy chain antibody, the heavy chain variable region of a humanized heavy chain antibody are linked to two identical human sources of the invention The N-terminus of the heavy chain antibody.
  • the antibody in the multimeric form of the invention is a tetramer comprising four identical Nanobodies of the invention, a heavy chain variable region of a heavy chain antibody, a humanized heavy chain antibody a heavy chain variable region) comprising two identical strands, each strand comprising a Nanobody, a heavy chain variable region of a heavy chain antibody, a heavy chain variable region of a humanized heavy chain antibody, and a present invention Humanized heavy chain antibodies. That is, the antibody in the multimeric form of the present invention comprises two identical strands (identical first strand and second strand), each strand comprising only one additional Nanobody, heavy chain antibody of the invention.
  • a heavy chain variable region, a heavy chain variable region of a humanized heavy chain antibody, and a humanized heavy chain antibody of the invention wherein the heavy chain of the Nanobody, heavy chain antibody of the present invention contained in the first strand is variable
  • the humanized heavy chain antibody contained in the first strand is identical to the humanized heavy chain antibody contained in the second strand.
  • interchain disulfide bond between the two identical strands, which is the sequence of the heavy chain variable region and Fc to which the humanized heavy chain antibody of each strand is ligated (for example, In the CPPC sequence), two interchain disulfide bonds formed between amino acid residues C. That is, the amino acid residue C in the sequence between the heavy chain variable region and the Fc in the humanized heavy chain antibody of the first strand and the heavy chain variable region in the humanized heavy chain antibody of the second strand are An interchain disulfide bond is formed between amino acid residues C in the sequence between Fc.
  • the Nanobody of the invention, the heavy chain variable region of a heavy chain antibody, the heavy chain variable region of a humanized heavy chain antibody in a multimeric form of the antibody of the invention are linked by a linker peptide
  • the humanized heavy chain antibody of the present invention is preferably a flexible linker peptide, more preferably a linker peptide having an amino acid sequence of (G4S)n (n is an integer of 0-7).
  • the present invention provides Nanobodies, heavy chain antibodies, humanized heavy chain antibodies, and tetrameric forms of antibodies that specifically bind to GITR (eg, human GITR), isolated and characterized in the Examples.
  • GITR eg, human GITR
  • the amino acid sequences and nucleotide sequences of these exemplary antibodies of the invention are set forth in Table B below.
  • the sequences shown in bold italics from the N-terminus to the C-terminus are exemplary CDR sequences, respectively, by any of various methods known in the art for determining the precise amino acid sequence boundaries of the CDRs. Or a combination thereof, for example, the following exemplary CDR sequences can be determined according to the methods described herein above and with reference to other factors, such as conservation.
  • Table B Amino acid sequence and nucleotide sequence of an exemplary Nanobody of the invention, amino acid sequence of a heavy chain antibody, amino acid sequence of a humanized heavy chain antibody, and amino acid sequence of a tetrameric form of the antibody, and SEQ ID NO Numbering.
  • the invention provides a fusion protein or immunoconjugate (abbreviated as a fusion protein or immunoconjugate of the invention), wherein the antibody of the invention is fused to one or more heterologous molecules (second molecule) Or conjugation, wherein the heterologous molecule includes, but is not limited to, a protein/polypeptide, a label, a drug, or a cytotoxic agent.
  • a fusion protein or immunoconjugate of the invention wherein the antibody of the invention is fused to one or more heterologous molecules (second molecule) Or conjugation, wherein the heterologous molecule includes, but is not limited to, a protein/polypeptide, a label, a drug, or a cytotoxic agent.
  • the drug of the fusion protein or immunoconjugate of the invention is an anti-tumor drug.
  • the invention provides a composition or kit (abbreviated as a composition or kit of the invention) comprising an antibody of the invention.
  • compositions or kits of the invention include vectors and/or instructions.
  • an antibody in a composition or kit of the invention is conjugated to a diagnostic or detectable agent.
  • the invention provides an isolated nucleic acid (abbreviated as an isolated nucleic acid of the invention or a nucleic acid (molecule) of the invention) encoding an antibody of the invention or a fragment thereof (including an antigen-binding fragment), the invention A fusion protein or an immunoconjugate of the invention.
  • a nucleic acid molecule of the invention is a substantially purified nucleic acid molecule.
  • a nucleic acid molecule of the invention comprises a nucleotide sequence that encodes a heavy chain variable region of any of the antibodies set forth in Table B, or a variant thereof.
  • the nucleic acid molecule is the nucleotide sequence of the Nanobody listed in Table B.
  • a nucleic acid molecule of the invention comprises a nucleotide sequence encoding at least one CDR region and generally all three CDR regions of a heavy chain of any of the antibodies set forth in Table B, or variants thereof.
  • nucleic acid molecules of the invention are substantially identical (e.g., at least 65%, 80%, 95%, or 99% identical) to the nucleotide sequence encoding the nucleic acid molecule of the amino acid sequence set forth in Table B.
  • Polypeptides encoded by these polynucleotides are capable of exhibiting GITR antigen binding ability when expressed in a suitable expression vector.
  • each antibody or polypeptide amino acid sequence can be encoded by a plurality of nucleic acid sequences because of codon degeneracy.
  • nucleic acid sequences of the invention encode any of the above-described Nanobodies of the invention.
  • a nucleic acid sequence of the invention encoding a Nanobody comprises the nucleotide sequence of SEQ ID NO: 86-107 or a sequence substantially identical thereto.
  • a nucleic acid sequence of the invention encoding a Nanobody consists of the nucleotide sequence of SEQ ID NO: 86-107 or a sequence substantially identical thereto.
  • a "substantially identical" nucleotide sequence means having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 in sequence with a reference nucleotide sequence. Sequence of %, 98% or 99% or higher identity.
  • the identity of the nucleotide sequences can be determined using various sequence alignment methods well known in the art. For example, the BLAST sequence alignment search tool can be obtained from the website of NCBI (National Center for Biotechnology Information, Bethesda, MD). In general, percent identity is performed using the default parameters of NCBI Blast.
  • a polynucleotide sequence encoding a GITR antibody or a binding fragment thereof can be produced by de novo solid phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding a GITR antibody or a binding fragment thereof (for example, the sequences shown in Tables B and 2).
  • Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester of Brown et al., Meth. Enzymol. 68:109, 1979. Method; Beaucage et al, Tetra.
  • the invention provides a vector comprising the nucleic acid of the invention (abbreviated as a vector of the invention).
  • the vector of the invention is an expression vector, ie, a multinucleus that can be used to express an antibody chain (eg, any of the antibodies of the invention) or a polypeptide (eg, any of the fusions and conjugates of the invention) that binds to a GITR. Glycosylate.
  • Non-viral vectors and systems comprise plasmids, episomal vectors and artificial chromosomes, typically containing expression cassettes for expression of proteins or RNA (see, for example, Harrington et al, Nat Genet 15:345, 1997).
  • Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papillomavirus, HBP EB virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See, Smith, Annu. Rev. Microbiol. 49: 807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.
  • the invention provides a host cell (abbreviated as a host cell of the invention) comprising a vector of the invention.
  • a host cell of the invention is any host cell, including prokaryotic or eukaryotic cells, suitable for cloning or expression of a vector of the invention.
  • the host cell of the invention is a bacterium, a yeast cell, a mammalian cell, or an immune effector cell (such as a T cell).
  • antibodies can be produced in bacteria when glycosylation and Fc effector functions are not required.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized", which results in partial or Production of antibodies in a fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al, Nat. Biotech (2006) 24: 210-215.
  • Suitable host cells for expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates).
  • invertebrate cells include plant and insect cells.
  • a number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417, 429 (described PLANTIBODIESTM technology for the production of antibodies in transgenic plants).
  • Vertebrate cells that can be used as hosts include, for example, suspension growth-adapted mammalian cell lines can be useful.
  • SV40 transformed monkey kidney CV1 line COS-7
  • human embryonic kidney line HEK293 or as described, for example, in Graham, FL et al, J. Gen Virol. 36 (1997) 59. HEK293 cells
  • BHK baby hamster kidney cells
  • mouse Sertoli cells eg TM4 cells described in Mather, JP, Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2) Mouse breast tumor (MMT 060562); TRI cells, such as those described in Mather, JP et al, Annals NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells; FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al, Proc. Natl. Acad. Sci.
  • E. coli cells TG1 cells
  • human embryonic kidney (293 cells) or human cervical cancer cells HELA
  • HELA human cervical cancer cells
  • the present invention provides a method comprising the preparation of the antibody of the present invention or an antigen-binding fragment thereof, the fusion protein of the present invention or the immunoconjugate of the present invention (abbreviated as the preparation method of the present invention or the method of the present invention), Including: culturing a host cell of the invention under conditions suitable for expression of an antibody of the invention, or an antigen binding fragment thereof, a fusion protein or an immunoconjugate.
  • the methods of the invention further comprise any expression means that facilitate expression and purification of an antibody or antigen-binding fragment thereof, fusion protein or immunoconjugate of the invention, for example, using a secretion signal sequence, an expression enhancing element A highly efficient promoter, such as any expression or regulatory element known to those skilled in the art.
  • the present invention provides a pharmaceutical composition (abbreviated as a pharmaceutical composition of the present invention) comprising the antibody of the present invention or an antigen-binding fragment thereof, the fusion protein of the present invention or the immunoconjugate of the present invention, and A pharmaceutically acceptable carrier is selected.
  • a pharmaceutical composition of the present invention comprising the antibody of the present invention or an antigen-binding fragment thereof, the fusion protein of the present invention or the immunoconjugate of the present invention, and A pharmaceutically acceptable carrier is selected.
  • the pharmaceutical compositions of the present invention further comprise other ingredients, such as immunotherapeutic agents, anti-angiogenic agents, and chemotherapeutic agents.
  • an immunotherapeutic agent in a pharmaceutical composition of the invention can induce or enhance an immune response, including, for example: 1) a dendritic cell activator; 2) a vaccine adjuvant; 3) a T cell stimulator; 4) Immunological checkpoint inhibitors, and 5) inhibitors of inhibitory cells, cytokines and/or enzymes.
  • the pharmaceutical compositions of the invention may include other compounds, drugs, and/or agents for treating cancer, including but not limited to chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response against a given cancer.
  • the therapeutic composition can include: an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody, an anti-CD137 antibody Or anti-LAG-3 antibodies.
  • the invention provides the use of a GITR binding molecule (antibody) of the invention in the diagnosis and/or detection (abbreviated as the use of the invention, or the diagnostic and/or detection use of the invention) and compositions for use therein.
  • a GITR binding molecule antibody
  • Any of the anti-GITR antibodies provided herein can be used to detect (quantitatively or qualitatively detect) the presence of GITR in a biological sample.
  • the presence of GITR in a biological sample is detected, for example, by immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic binding of antibody molecules, ELISA assay, PCR-technology (e.g., RT-PCR), and the like.
  • the biological sample comprises a bodily fluid, a cell, or a tissue.
  • the biological sample is a blood, serum or other liquid sample of biological origin.
  • an anti-GITR antibody for use in a diagnostic or detection method.
  • a method of detecting the presence of GITR in a biological sample comprises contacting a biological sample with an anti-GITR antibody described herein under conditions that allow binding of the anti-GITR antibody to GITR, and detecting whether a complex is formed between the anti-GITR antibody and the GITR.
  • Such methods can be in vitro or in vivo methods.
  • an anti-GITR antibody is used to select a subject for treatment with an anti-GITR antibody, for example, when the GITR is a biomarker for patient selection.
  • Exemplary conditions that can be diagnosed using the antibodies of the invention include immune disorders or regenerative disorders.
  • a method of stratifying a patient with an immune disorder or a regenerative disorder with an antibody of the invention is provided.
  • the anti-GITR antibody is any of the foregoing immunoconjugates of the invention conjugated to a diagnostic or detectable agent.
  • the invention provides a kit for use in diagnosis or detection comprising a GITR binding molecule of the invention, such as any of the anti-GITR antibodies of the invention.
  • the invention provides a method of detecting the presence of a GITR in a sample, comprising:
  • the invention provides methods of screening, identifying and characterizing antibodies of the invention.
  • the anti-GITR antibodies provided herein can be screened, identified, or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.
  • Phage/phagemids that bind with high affinity to the antigen of interest can be selected from the phage/phagemid display nanobody library.
  • a variety of methods have been presented for displaying or displaying antibodies or antibody fragments on the surface of phage/phagemids and for screening libraries.
  • the antibodies of the invention may be identified or characterized for their antigen binding activity, for example by known methods, such as ELISA, aLISA, Western blot, antibodies or reverse phase arrays, and the methods described in the Examples.
  • antibodies can be detected using the ForteBio assay.
  • the ForteBio affinity assay can be carried out according to the existing method (Estep, P, et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8).
  • the AHQ sensor can be lined down in the assay buffer for 30 minutes and then detected on line for 60 seconds to establish a baseline. Thereafter, the AHQ sensor loaded with the purified antibody was exposed to 100 nM antigen for 5 minutes, and the sensor was transferred to assay buffer for 5 minute offline measurement. Kinetic analysis was performed using a 1:1 binding model.
  • the invention provides a method of treating a cancerous condition, inducing or enhancing an immune response in an individual, and/or stimulating an antigen-specific T cell response, characterized by comprising administering to the individual an effective amount of the invention An isolated antibody or antigen-binding fragment thereof, a fusion protein of the invention or an immunoconjugate of the invention.
  • the immune response is produced against a tumor antigen or against an infectious agent.
  • the above methods of the invention may treat or prevent an "immune disorder", including, for example, pathological inflammation, inflammatory conditions, and autoimmune disorders or diseases, as well as infections, persistent infections, and proliferative disorders, such as cancer. , tumors and angiogenesis, including infections, tumors and cancers that are immune to the immune system.
  • an "immune disorder” including, for example, pathological inflammation, inflammatory conditions, and autoimmune disorders or diseases, as well as infections, persistent infections, and proliferative disorders, such as cancer.
  • tumors and angiogenesis including infections, tumors and cancers that are immune to the immune system.
  • the above methods of the invention may treat or prevent an "immune disorder", wherein the immune disorder refers to a disease in a mammal that is caused, mediated, or otherwise contributes to the onset of disease by a mammalian immune system component; A disease that interferes with the development of the disease by interfering with the immune response.
  • immune disorders include autoimmune diseases, immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, and immunodeficiency diseases.
  • immune-related diseases and inflammatory diseases that can be treated using the antibodies of the invention (some of which are immune or T cell mediated) include: systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondylolisthesis Disease, systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immunization) Pneumocytopenia, paroxysmal nocturnal hemoglobinuria, autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves' disease, Hashimoto's thyroid Inflammation, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated nephropathy (glomerulonephritis,
  • the above methods of the invention may treat or prevent a "cancerous condition" including, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions, such as dysplasia.
  • a "cancerous condition” including, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions, such as dysplasia.
  • the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer, more preferably a solid cancer or a blood cancer; most preferably melanoma, lung cancer, head and neck cancer, colorectal cancer, non-small cell lung cancer, Squamous cell carcinoma of the head and neck, bladder cancer, breast cancer, uterus/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colon cancer, kidney cancer, stomach cancer, germ cells Cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, new organisms of the central nervous system, lymphoma, leukemia, myeloma, sarcoma or virus-related cancer.
  • melanoma lung cancer, head and neck cancer, colorectal cancer, non-small cell lung cancer, Squamous cell carcinoma of the head and neck, bladder cancer, breast cancer, uterus/cervical cancer, ovarian cancer, prostate cancer, testicular cancer,
  • an effective amount of an antibody of the invention is an amount that achieves one or more of the following in an individual: a) a regulatory T cell inhibition that reduces effector T cell activity; b) a decrease in circulating regulatory T cell levels c) activating effector T cells; d) inducing or enhancing effector T cell proliferation; e) inhibiting tumor growth; f) inducing tumor regression; and g) increasing IL-2 and/or IFN- ⁇ in T cells expressing GITR Produce and increase T cell proliferation.
  • the above methods of the invention further comprise: a) administering chemotherapy; b) administering radiation therapy; and/or c) administering one or more additional therapeutic agents, preferably immunostimulating agents.
  • the immunostimulatory agent in the above methods of the invention is selected from the group consisting of T-VEC, a PD1 antagonist, a PDL1 antagonist, a CTLA-4 antagonist, and BiTE.
  • the chemotherapy, radiation therapy, or therapeutic agent in the above methods of the invention is administered prior to, concurrently with, or subsequent to the antibody or antigen-binding fragment thereof.
  • the 1 mg antigen GITR-His (Acro biosystems) was mixed with Freund's adjuvant (Sigma) in equal volume and divided into two tubes to immunize two Xinjiang Bactrian camels. So, immunize once a week, a total of 7 times, stimulate B lymphocytes to express antigen-specific Nanobodies. After seven immunizations, the serum titer of the GITR-His Nanobody produced in Xinjiang Bactrian camel can reach 1:1000 or more, thereby confirming that the desired Nanobody is produced in the camel.
  • RNA obtained in the step (1) is reverse transcribed into cDNA by RT-PCR, and then amplified by nested PCR (two PCR) to obtain VHH, and the amplification principle thereof is shown in FIG. 1;
  • phage specifically binding to the antigenic protein GITR-His was dissociated with a 100 mM TEA (Sigma) eluate, followed by infection of E. coli TG1 cells in log phase growth. Incubate at 37 ° C for 1 hour, expand the cultured phage for the next round of screening;
  • PE-ELISA Post-Enrichment Enzyme-linked Immunosorbent Assay
  • HEK293 cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1 ⁇ 10 6 /ml one day before transfection. The cell density on the day of transfection was about 2 ⁇ 10 6 /ml;
  • PEI polyethyleneimine, Polysciences, 23966
  • the desired hcIgG protein was purified by HiTrapTM MabSelect SuReTM 5 ml (GE Healthcare). The specific process is as follows:
  • the AKTA protein purification system was detoxified (overnight) with 0.1 M NaOH. On the day of collection, the above cell supernatant was centrifuged at 7,500 rpm for 30 minutes, and filtered using SARTOPORE (Sartorius, 5441307H4). The system was washed with 5 column volumes of binding buffer (Tris 20 mM, NaCl 150 mM, pH 7.2) and the column was equilibrated before purification. The supernatant obtained by centrifugation described above was passed through a column. Re-equilibrate with 5-10 column volumes of binding buffer to baseline equilibration.
  • the antibody was eluted with an elution buffer (citric acid + sodium citrate 100 mM, pH 3.5), and samples were collected according to the ultraviolet absorption value. Each 1 ml of the collection solution was neutralized by adding 80 ⁇ l of a neutralization buffer (Tris-HCl 2M).
  • a neutralization buffer Tris-HCl 2M
  • the heavy chain antibodies hcIgG-02, hcIgG-03, hcIgG-04, hcIgG-07, hcIgG-09, hcIgG-10, hcIgG-16 and hcIgG-20 were obtained, the specific sequences of which are shown in Table B.
  • This experiment uses the Fortebio method (Estep, P, et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-278) to determine antigen-antibody binding kinetics and affinity, GITR Inc
  • the anti-GITR antibody is used as a positive control (referred to as TRX518 in the present application, cloned according to the sequence provided in US20130183321A1, the sequences of the light and heavy chains are SEQ ID NO: 44, and SEQ ID NO: 54, respectively, in US20130183321A1, Transiently transfected HEK293 cells for expression).
  • TRX518 cloned according to the sequence provided in US20130183321A1, the sequences of the light and heavy chains are SEQ ID NO: 44, and SEQ ID NO: 54, respectively, in US20130183321A1, Transiently transfected HEK293 cells for expression.
  • the specific measurement method is
  • Detection step centrifugation at 400 g for 5 minutes to remove the cell culture medium.
  • the gradient-diluted sample was added to the U-shaped plate and the cells were resuspended, 100 ⁇ l per well, and allowed to stand on ice for 30 minutes.
  • the supernatant was removed by 400 g for 5 minutes, and the cells were washed 1 time with PBS.
  • PBS was removed, and 100 ⁇ l of 1:200 diluted PE-anti-human Fc antibody (Jackson Immuno Research) was added to each well. Incubate on ice for 30 minutes in the dark.
  • the supernatant was removed by centrifugation at 400 g for 5 minutes, and the cells were washed 1 time with PBS.
  • the cells were resuspended in 100 ⁇ l of PBS.
  • DSF Differential Scanning Fluorescence
  • SYPRO Orange protein gel stain (GIBCO) was diluted 50-fold with PBS, i.e., 4 ⁇ l of SYPRO Orange protein gel stain mother liquor plus 196 ⁇ l of PBS. The sample was loaded in a 96-well PCR plate, 50 ⁇ l of the diluted antibody sample + 10 ⁇ l of SYPRO Orange protein gel stain dilution + 40 ⁇ l of water. The test was carried out in a 7500 real time PCR system, and the results are shown in Table 3.
  • This experiment used promega's ADCC Report Bioassay kit to detect hcIgG-mediated antibody-dependent (cell-mediated) cytotoxicity (ADCC).
  • the effector cells used were Jurkat cells stably transfected with the NFAT-Luciferase reporter gene, and the target cells were CHO cells (CHO-GITR) overexpressing GITR.
  • the specific experimental process is as follows:
  • the target cells were diluted to 2.5 ⁇ 10 5 /ml with ADCC Assay Buffer, and 50 ⁇ l of target cells were added to each well;
  • the effector cells were diluted to 1.5 ⁇ 10 6 /ml with ADCC Assay Buffer, and 25 ⁇ l per well;
  • the reaction was carried out at 37 ° C for 6 hours; then an equal volume of room temperature Bio-Glo Luciferase Reagent (Promega) was added to each well and incubated for 10 minutes at room temperature.
  • the hcIgG (hcIgG-02, hcIgG-03, hcIgG-04, hcIgG-07, hcIgG-09, hcIgG-10, hcIgG-16, and hcIgG-20) of the present invention are capable of causing ADCC. effect.
  • Humanization was performed by selecting hcIgG-07 and hcIgG-20, and the humanization process was according to the existing method (Ce'cile Vincke et al., General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold. JOURNAL OF OF BIOLOGICAL CHEMISTRY, 2009.5 (284): p. 3273-3284) was carried out to obtain humanized antibodies HzhcIgG-07 and HzhcIgG-20 (see Table B for specific sequences).
  • This experiment used the Fortebio method to determine the binding kinetics and affinity of the antibody hcIgG-20 before humanization and the antibody HzhcIgG-20 after humanization, in the same manner as in Example 3.
  • the results of ForteBio are shown in Table 4.
  • hcIgG-20 and HzhcIgG-20 maintained binding ability to GITR with higher affinity than control antibody TRX518.
  • the detection method was the same as in Example 4.
  • the cell binding results by flow cytometry are shown in Figure 5.
  • Both hcIgG-20 and HzhcIgG-20 maintained cell binding activity (EC50 from 1.631 nM to 1.873 nM).
  • Wild-type Hela cells were engineered to overexpress GITR protein on the surface.
  • the plasmid expressing the NFkB binding site and the fluorescent reporter gene (Luciferase) was transferred into Hela-GITR cells to obtain a stable cell line Hela-GITR-NFkB which simultaneously expressed GITR and NFkB fluorescent reporter enzyme gene.
  • -Luc-Rep This experiment used this cell to detect the activation of NFkB signaling pathway downstream of GITR by hcIgG-20 and HzhcIgG-20.
  • the specific experimental process is as follows:
  • HzhcIgG-20 has an activation effect on NFkB signaling pathway.
  • the activation EC50 of hcIgG-20, HzhcIgG-20 and control antibody TRX518 are 0.6894nM, 0.6632nM and 2.097nM, respectively.
  • hcIgG-20 and HzhcIgG-20 are better than control antibody. Activation of the NFkB pathway.
  • a naturally occurring monoclonal antibody can exist as a multimeric structure such as IgM, which increases the affinity for its target antigen on the immobilized surface. Because agonistic effects of TNFR require receptor aggregation, tetravalent or hexavalent monoclonal antibodies can provide increased aggregation and receptor agonism.
  • the antibody in the form of a multimer of the present invention comprises four identical Nanobody regions, the specific construction methods of which are as follows:
  • VHH portion of 4xNb-IgG-I:HzhcIgG-20 (ie, the heavy chain variable region VH in HzhcIgG-20) is linked to the C-terminus of HzhcIgG-20 via a flexible linker of GGGGS (Fig. 7A);
  • 4xNb-IgG-II The VHH portion of HzhcIgG-20 is linked to the N-terminus of HzhcIgG-20 via a flexible linker of GGGGS (Fig. 7B);
  • 4xNb-IgG-III The VHH portion of Hzhc IgG-20 was directly ligated to the N-terminus of Hzhc IgG-20 (Fig. 7C).
  • the construct was cloned into the XhoI/NotI multiple cloning site of the expression vector pTT5.1 of HEK293 cells by conventional methods.
  • the constructed plasmid was transfected into HEK293 cells, and the expressed and secreted 4xNb-IgG molecules were screened and the correct molecule was selected for further experiments.
  • the expression and purification method of 4xNb-IgG was the same as in Example 2.
  • the purified protein was purified by SEC.
  • the purity of 4xNb-IgG-I, 4xNb-IgG-II and 4xNb-IgG-III was 97.5%, 96.86% and 97.21%, respectively, after one-step purification. .
  • the detection method was basically similar to that of Example 4, but was optimized to reduce the antibody concentration and increase the sensitivity of the detection.
  • the anti-tumor activity of the anti-human GITR antibody 4xNb-IgG-III alone or in combination with the anti-mouse PD-1 antibody "Antibody C” was investigated using a mouse model of MC38 xenograft tumor.
  • mice Male human GITR transgenic mice (approximately 8 weeks old) were purchased from Biostech Biotech Co., Ltd. The mice were domesticated for 7 days after arrival and the study was started.
  • Mouse colon cancer cells MC38 (ATCC) were purchased from ATCC and routinely subcultured for subsequent in vivo experiments in strict accordance with ATCC requirements. The cells were collected by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 5 x 10 6 /ml. A tumor-bearing mouse model was established by inoculating 0.2 ml of the cell suspension on day 0 into the right abdomen region of human GITR transgenic mice.
  • mice were divided into four groups (8 mice per group), and each group was injected subcutaneously with the following doses of antibody:
  • Mouse IgG control (equitech-Bio), 10 mg/kg;
  • mice meeting the experimental requirements were randomly divided into groups of 6 each. Each group of mice was administered as above with the above four groups of reagents on days 8, 11, 14, and 17, respectively.
  • Tumors and body weight were measured twice weekly throughout the study, and mice were euthanized when the tumor reached the endpoint or when the mice had >20% weight loss.
  • the anti-GITR antibody 4xNb-IgG-III of the present invention significantly inhibits tumor growth compared to the IgG control (equitech-Bio);
  • the combination of the anti-GITR antibody 4xNb-IgG-III of the present invention and the anti-PD-1 monoclonal antibody "Antibody C” can significantly inhibit tumor growth as compared with the use of IgG and the two antibodies, respectively.

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Abstract

本发明公开了特异性结合糖皮质激素诱导的肿瘤坏死因子受体(GITR)的抗体、抗体片段及其聚合物、以及含有该抗体或抗体片段的缀合物和融合物。本发明还公开了编码上述抗体、抗体片段、聚合物、缀合物和融合物的核酸、表达所述核酸的载体和宿主细胞。本发明还公开了包含上述抗体、抗体片段、聚合物、缀合物或融合物的组合物，以及其在治疗和诊断中的用途。

Description

抗糖皮质激素诱导的肿瘤坏死因子受体(GITR)的小型化抗体、其聚合物及应用
相关申请的交叉引用
本申请要求于2018年3月26日递交的中国专利申请第201810255332.2号优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
技术领域
本发明涉及抗体领域。本发明尤其涉及特异性结合糖皮质激素诱导的肿瘤坏死因子受体(GITR)的抗体(包括小型化抗体)、抗体片段及其聚合物，以及含有该抗体或抗体片段的缀合物和融合物。本发明还涉及编码这些抗体、抗体片段、聚合物、缀合物和融合物的核酸；载体和表达该核酸的宿主细胞。此外，本发明也涉及包含这些抗体及其抗体片段、聚合物、免疫缀合物或融合物的产品，及在治疗和诊断中的应用。
背景技术
糖皮质激素诱导的肿瘤坏死因子受体(glucocorticoid induced tumor necrosis factor receptor，GITR，亦称作TNFRSF18、活化诱导型肿瘤坏死因子受体(TNFR)家族成员(AITR)、CD357及GITR-D)，是肿瘤坏死因子受体(tumor necrosis factor receptor，TNFR)超家族的第18个成员。GITR由其同源配体，GITR配体(GITRL)活化，可结合NFkB二聚体，激活下游的NFkB信号通路。
GITR在应答静息T细胞中低水平表达；当T细胞活化后，其表达水平显著上调。GITR在调节性T细胞(Treg，如CD4 +CD25 +或CD8 +CD25 +细胞)中高水平组成型表达，并且当这些细胞被活化时，表达水平进一步上调(Nocentini和Riccardi(2005)E.J.Immunol.35:1016-1022)。GITR表达不限于T细胞，已报道，GITR在NK细胞、巨噬细胞、B细胞、树突状细胞、肥大细胞及单核细胞上也表达(Nocentini和Riccardi(2005)E.J.Immunol.35:1016-1022)。
GITR配体(GITRL)主要在抗原递呈细胞(APC，包括巨噬细胞、B细胞、树突状细胞及内皮细胞)上表达。APC上的GITRL与应答T细胞上GITR的结合触发GITR信号传导，共刺激应答T细胞并抑制Treg细胞的抑制活性。由此，GITR对效应T细胞和调节性T细胞具有若干作用，包括：共刺激和活化效应T细胞，使T细胞对抑制更具抗性、抑制调节性T细胞、降低效应T细胞对由调节性T细胞引起的抑制的敏感性等(Nocentini等(2007)Eur.J.Immunol.37:1165-1169)。
这些作用意味着GITR活化可以导致免疫应答的增强，增加对肿瘤和病毒感染等的抗性。因此，能够活化GITR的物质可以增强需要的免疫反应(例如，抗肿瘤、治疗病毒感染)，诱导或增强个体中的免疫应答，治疗免疫障碍和增生性障碍(例如肿瘤和癌症)等。
目前，现有技术中能够活化GITR的物质主要是标准结构的抗GITR抗体(参见CN103951753A、CN105829343A、CN106459203A、WO2016196792A1、WO2017068186A9等)。这样的抗体是GITR的激动剂(即，是激活型抗体)，可诱导或增强GITR信号传导，在治疗需要增强免疫应答的多种GITR相关疾病或病症中是有效的。由于这样的抗GITR抗体是激活型抗体，故相对于其与抗原的结合亲和力，其激活活性的高低是其发挥活性(例如。免疫增强活性)更为关键的指标。
但是，上述标准结构的抗GITR抗体具有标准结构抗体的一些固有缺陷：生产成本高、制备周期长、制备工序复杂、体积大(组织渗透性差)、稳定性差、不同批次之间差异较大等，大大限制了其应用范围。
同时，包括单链抗体(single-chain variable fragment,scFv)、单域抗体(single-domain antibody,sdAd)、重链抗体(heavy-chain antibody,hcAb)、纳米抗体(nanobody,Nb或variable domain of heavy chain of heavy-chain antibody,VHH)、小型的类抗体支架蛋白(如Affibody、DARPins 等)等小型化抗体由于克服了标准结构抗体的一些固有缺陷，其研发已引起生物技术应用领域的广泛关注，近年得以快速发展。
这类小型化抗体中的单域抗体和重链抗体没有轻链，具有的单一重链可变区保留了完整的抗原结合活性，具有分子小、稳定性高、体内组织渗透性好、可溶性好、熔解温度高、易表达等的特性。
例如，这类小型化抗体中的纳米抗体具有如下优点：
1.由于具有独特的结构，由此具有有益的抗原结合特性：纳米抗体较长的CDR3(互补决定区3)，可形成稳定暴露的凸环结构(凸环中具有稳定结构的二硫键)，能够深入抗原内部，从而更好地结合抗原，提高其抗原特异性和亲和力。而传统抗体Fab片段及单链抗体scFv的抗原结合表面常形成凹形拓扑结构,通常只能识别位于抗原表面的位点。因此，纳米抗体具有更有益的抗原结合性能，甚至当抗原蛋白紧密包裹，隐藏了普通抗体无法识别的表位时，纳米抗体也可以识别这样的表位。
2.当单独表达标准结构抗体的VH结构域时，表达出的VH结构域通常形成包涵体，或者暴露的疏水域相互黏附。而纳米抗体由于其FR2区域中的疏水残基被亲水残基所取代，使得纳米抗体的可溶性非常好，聚合性减少，在普通缓冲液中可轻易被浓缩到10mg/mL而不出现积聚。并且由于纳米抗体具有可逆重折叠性质，在高温(80～92℃)和高浓度变性剂作用下仍保持抗原结合活性，即使以包涵体形式表达，也很容易复性，使纳米抗体在层析或无菌处理时的短暂变性中，或在污染或有机溶剂等不良环境中，仍能出色地长期维持生物学活性，大大提高纳米抗体作为药物的利用率。
3.因为分子量小、结构简单，且由单一的基因编码，纳米抗体很容易在微生物中合成，能在价格低廉的噬菌体、酵母等微生物中大量表达，进行大规模生产。据报道，可通过酵母反应器将纳米抗体的产量提高至1克/升的产量。
同时，由于纳米抗体仅仅具有一个重链可变区，不存在配对问题，由此其筛选过程更为简便，常常仅仅需要文库即可。
4.纳米抗体由于分子很小，组织渗透性强，可以穿透或转移进入血脑屏障，也可以快速经肾脏滤过(肾脏截留值约60kDa)，血液中半寿期约2小时，表位靶向唯一，是优异的治疗载体。
5.与sdAb、scFv等相比，纳米抗体也具有如下突出优点：结构更简单，易与受体结合、可以识别嵌入配体沟槽或夹在两个亚基之间的隐藏抗原表位，而且相对分子量更小，免疫原性更低、积聚沉淀趋势更低、生物分散性更好，更易表达(原核或真核系统中高表达)、可溶性更好、稳定性更强，在极端pH、高浓度变性剂或高温等不利理化环境条件下更稳定，保质期长，可以口服或呼吸道给药等；同时不易如单链抗体(scFv)那样容易相互粘连，甚至聚集成块。
由纳米抗体制成的检测试剂盒甚至可以直接储存在室温下，不用冷藏保存，减少巨大的冷藏费用。
结合上述能够活化GITR的物质的诸多作用和优点，本领域存在制备能够活化GITR的小型化抗体的需要，例如能够活化GITR的纳米抗体的需要，以便更好地诱导或增强个体中的免疫应答，治疗免疫障碍和增生性障碍(例如肿瘤和癌症)。
发明内容
如上所述，标准结构的抗GITR抗体具有如筛选流程复杂、组织渗透性差、生产成本高等不足之处，故开发抗GITR的小型化抗体可以很好地弥补这些不足，具有诸多其他优点，同时还能实现抗GITR抗体的性能。
然而，并不是简单地将已知标准结构抗体的重链可变区分离，就可以获得相应的VHH。因为如上所述，VHH中CDR1-3的氨基酸序列和长度，特别是CDR3的氨基酸选择和长度与 标准结构抗体中对应的HCDR1-3(例如HCDR3)不同，结构也不同，需要进行精心地设计和大量的筛选工作才能获得。
本发明的目的是提供一种分离的特异性结合糖皮质激素诱导的肿瘤坏死因子受体(GITR)的抗体或其抗原结合片段(简称“本发明的抗体或其抗原结合片段”)，其具有以下一种或多种特性：
(i)以高亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合；
(ii)以低解离常数与人GITR(如SEQ ID NO:123或127的多肽)结合；
(iii)与细胞表面的抗原高亲和力结合；
(iv)高效激活GITR下游的NFkB信号通路；
(v)与GITRL交叉竞争结合至GITR；
(vi)可以内化到人CD4细胞中；
(vii)抑制调节性T细胞的抑制作用；
(viii)活化效应T细胞；
(ix)减少循环调节性T细胞；
(x)能够结合Fcγ受体(FcγR)；和
(xi)在人血清中具有至少6、7、9或12天的半衰期。
本发明的另一目的是提供一种纳米抗体(简称“本发明的纳米抗体”)，其中的HCDR3(重链互补决定区3)包含本发明的抗体或其抗原结合片段的HCDR3。
在一些实施方案中，本发明的纳米抗体的HCDR1(重链互补决定区1)和HCDR2(重链互补决定区2)分别包含本发明的抗体或其抗原结合片段的HCDR1和HCDR2。
在一些实施方案中，本发明的纳米抗体的HCDR1、HCDR2和HCDR3分别包含本发明的抗体或其抗原结合片段的HCDR1、HCDR2和HCDR3。
在一些实施方案中，本发明的纳米抗体仅包含如SEQ ID NO:64-85中任一序列所示的重链可变区序列或其变体，或本发明的纳米抗体仅由SEQ ID NO:64-85中任一序列所示的重链可变区序列或其变体组成。
本发明的另一目的是提供一种重链抗体(简称“本发明的重链抗体”)，包含本发明的纳米抗体和IgG抗体的Fc片段。
本发明的另一目的是提供一种人源化重链抗体(简称“本发明的人源化重链抗体”)，其改造自本发明的重链抗体。
在一些实施方案中，本发明的人源化重链抗体是全人源重链抗体。
本发明的另一目的是提供一种多聚物形式的抗体(简称“本发明的多聚物形式的抗体”)，是本发明的纳米抗体、本发明的重链抗体的重链可变区或本发明的人源化重链抗体的重链可变区的多聚物形式。
在一些实施方案中，本发明的多聚物形式的抗体是本发明的纳米抗体、本发明的重链抗体的重链可变区或本发明的人源化重链抗体的重链可变区的四聚化形式或六聚化形式，优选是本发明的纳米抗体、本发明的重链抗体的重链可变区或本发明的人源化重链抗体的重链可变区的四聚化形式。
在一些实施方案中，本发明的多聚物形式的抗体由本发明的纳米抗体、本发明的重链抗体的重链可变区或本发明的人源化重链抗体的重链可变区连接到本发明的重链抗体(优选连接到本发明的人源化重链抗体)形成的新的结构聚合而成。
在一些实施方案中，本发明的多聚物形式的抗体由本发明的纳米抗体、本发明的重链抗体的重链可变区或本发明的人源化重链抗体的重链可变区连接到本发明的重链抗体的C末端形成的新的结构聚合而成；或者由本发明的纳米抗体、本发明的重链抗体的重链可变区或本发明的人源化重链抗体的重链可变区连接到本发明的重链抗体(优选连接到本发明的人源化重链抗体)的N末端形成的新的结构聚合而成。
在一些实施方案中，本发明的多聚物形式的抗体中，本发明纳米抗体、本发明的重链抗体的重链可变区或本发明的人源化重链抗体的重链可变区通过连接肽连接到本发明的重链抗体(优选连接到本发明的人源化重链抗体)。
本发明的另一目的是提供一种包含本发明的抗体的融合蛋白、免疫缀合物、组合物或试剂盒。
本发明的另一目的是提供一种编码本发明的抗体的分离的核酸；包含该核酸的载体；包含该载体的宿主细胞；包含这些核酸、载体或宿主细胞中的一个或多个的药物组合物。
本发明的另一目的是提供一种制备本发明的抗体或其抗原结合片段、本发明的融合蛋白、本发明的免疫缀合物或组合物(包括药物组合物)的方法。
本发明的另一目的是提供一种检测样品中GITR的存在的方法，包括使用本发明的抗体或其抗原结合片段。
本发明的另一目的是提供一种治疗癌症，诱导或增强个体中的免疫应答，和/或刺激抗原特异性T细胞应答的方法，其特征在于包括向所需个体施用有效量的本发明的抗体或其抗原结合片段、本发明的融合蛋白或本发明的免疫缀合物。
与现有技术中已有的标准结构抗GITR抗体相比，本发明的抗体不但具有筛选流程简便、组织渗透性好、生产成本低等优点；且相对于直接获自标准结构抗GITR抗体的单链抗体、单域抗体、重链抗体等，不再具有容易形成包涵体或者容易相互黏附的缺点。进一步，本发明的发明人令人惊讶地发现，本发明的抗体对GITR是高度特异性的，且不会干扰其他受体的活性，能够以相对低剂量刺激GITR信号传递；同时，本发明的抗体能够高效激活GITR下游的NFkB信号通路，产生有益的抗免疫疾病/障碍和抗增生性疾病/障碍的效果。
在下面的附图和具体实施方案中进一步说明本发明。然而，这些附图和具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。
附图说明
图1.纳米抗体噬菌体文库构建流程示意图。
图2.SEC检测纯化后的hcIgG的纯度结果图。
图3.hcIgG和细胞表面GITR蛋白的结合情况检测结果图。
图4.hcIgG引起的ADCC作用图。
图5.HzhcIgG和细胞表面GITR蛋白的结合情况检测结果图。
图6.人源化后的hcIgG对GITR下游的NFkB信号通路的激活作用图。
图7. 4xNb-IgG的结构示意图。
图8.SEC检测纯化后的4xNb-IgG的纯度结果图。
图9. 4xNb-IgG对GITR下游的NFkB信号通路的激活作用图。
图10.抗GITR抗体(4xNb-IgG)本身，或与抗PD-1抗体联合使用在MC38移植瘤模型中对肿瘤的抑制作用图。
具体实施方式
以下结合实施例对本发明进行详细描述。所用的所有实验试剂和仪器设备，如无特别说明均为普通市售试剂和设备。
1.定义
除非本文中另外定义，否则本文中所用的所有术语均具有本领域普通技术人员通常理解的含义。此外，除非上下文另外要求，否则单数术语将包括复数术语且复数术语将包括单数术语。为了更好地解释和理解本发明，下面具体定义一些本文中使用的术语。
如本文所用，术语“约”在与数字数值联合使用时，意为涵盖比指定数字数值小5％的下 限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项。
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
如本文所用，术语“GITR”是指“糖皮质激素诱导的TNF相关基因和/或多肽”，在本领域中也称为TNF受体超家族18(TNFRSF18)。人和鼠形式的GITR的氨基酸和核酸序列描述于WO98/06842中，其以引用的方式并入本文。也参见GenBank登记号Q9Y5U5(人氨基酸序列)和AF109216(鼠核酸和氨基酸序列)。一个特定成熟人GITR多肽的氨基酸序列陈述于SEQ ID NO:123中。如本文所用的术语GITR也包括其天然存在的等位基因或变体。
如本文所用，术语“抗原结合分子”、“抗原结合蛋白”与“抗体”在本文中可以互换使用，均指包含能够与靶抗原结合的抗原结合区或抗原结合部分的分子，例如蛋白质或多肽。在本发明中，当靶抗原是糖皮质激素诱导的肿瘤坏死因子受体(GITR)时，结合GITR的抗原结合分子也称作GITR结合分子、GITR抗体或抗GITR抗体。抗原结合分子包括例如抗体及其抗原结合片段、单链抗体(scFv)、单域抗体(sdAd)、重链抗体(hcAb)、纳米抗体(Nb或VHH)；或这些抗体的多聚物形式、基于这些抗体的各种融合物和缀合物、免疫缀合物、抗体药物偶联物(ADC)、多/双特异性抗体、嵌合抗原受体(CAR)等。如本领域技术人员所知，抗体的抗原结合部分通常包含来自“互补决定区”或“CDR”的氨基酸残基。因此，在本发明中术语“GITR结合分子”与“本发明的抗体”、“GITR抗体”或“抗GITR抗体”中的任一个可以互换使用。
如本文所用，术语“抗体”是指至少包含轻链或重链免疫球蛋白可变区的多肽(免疫球蛋白)，其特异性识别并结合抗原。该术语涵盖各种抗体结构，包括但不限于单克隆抗体；多特异性抗体；Fab片段、Fab’片段、F(ab’) 2片段或Fv片段；双抗体、单链抗体(scFv)、单域抗体(sdAd)、重链抗体(hcAb)、纳米抗体(Nb或VHH)或这些抗体的多聚物形式；人抗体、人源化抗体或嵌合抗体；或经标记的抗体，只要它们呈现期望的抗原结合活性即可。
如本文所用，术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换使用，均指具有基本上与天然抗体结构相似的结构，包含至少两条重链(H)和两条轻链(L)。每条重链由重链可变区(本文中缩写为VH)和重链恒定区(本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区(本文中缩写为CL)组成。轻链恒定区由一个结构域CL组成。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
如本文所用，术语“可变区(V区，Fv)”和“可变结构域”在本文中可以互换使用，均指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链可变结构域(VH)和轻链可变结构域(VL)通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和三个互补决定区(CDR，参见，例如，Kindt等Kuby Immunology，第六版，W.H.Freeman and Co.，91页(2007))。单个VH或VL结构域可足以给予抗原结合特异性。轻链可变区和重链可变区从N-末端到C-末端通常包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。
在一个给定的VH或VL氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国 际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以根据本领域的任何方案或其组合及人为评估确定边界。
抗体的轻链可以基于其恒定结构域的氨基酸序列分为两种类型kappa(κ)和lambda(λ)。抗体的重链可以取决于其重链恒定区的氨基酸序列而分为主要5种不同的类型：IgA、IgD、IgE、IgG和IgM，并且这些类型中的几种可以进一步划分成亚类，如，IgG1、IgG2、IgG3和IgG4、IgA1以及IgA2。对应于不同抗体类型的重链恒定区分别称作α、δ、ε、γ和μ。参见例如Fundamental Immunology，Ch.7(Paul，W.编辑，第二版，Raven Press，N.Y.(1989))(其为所有目的以其整体在此引作参考)。
如本文所用，术语“抗体片段”和“(抗体的)抗原结合片段”在本文中可互换使用，均指并非完整抗体的分子，其包含完整抗体中用于结合该完整抗体所结合的抗原的部分，能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体，来制备抗原结合片段。抗原结合片段包括但不限于Fab、scFab、Fab’、F(ab’)2、Fab’-SH、可变区抗体片段(Fragment of variable,Fv)、单链Fv、双链抗体(diabody)、三链抗体(triabody)、四链抗体(tetrabody)、微抗体(minibody)、单结构域抗体(单域抗体，sdAb)；以及从抗体片段形成的多特异性抗体。Fab片段是一种由VL、VH、CL和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。可以借助接头将Fab的轻链(L链)和重链(H链)融合构建成单一多肽链，即单链Fab(scFab)(参见例如US20070274985A1)。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体可以产生F(ab’) 2，其为Fab’的二聚体，是二价的抗体片段。F(ab’) 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，从F(ab’) 2二聚体转化为Fab’单体。Fab’单体基本上是具有铰链区的Fab片段。Fv片段由抗体单臂的VL和VH结构域组成。
如本文所用，术语“双链抗体”是具有两个抗原结合位点的抗体片段，该片段在同一多肽链中包含通过短接头连接的VL和VH。在双链抗体中，由于接头过短，同一链上的VH和VL两个结构域之间无法配对，而被迫与另一链上的互补结构域配对并且产生两个抗原结合位点。双链抗体可以是二价的或双特异性的。双链抗体的更详细描可以参见例如，EP 404,097；WO 1993/01161；Hudson等,Nat.Med.9:129-134(2003)；以及Hollinger等，PNAS USA 90:6444-6448(1993)。
三链抗体和四链抗体和微抗体也描述于Hudson等，Nat.Med.9:129-134(2003)，和邵荣光等(编辑)，抗体药物研究与应用，人民卫生出版社(2013)。
如本文所用，术语“单链抗体(single-chain variable fragment,scFv)”又称为“单链可变区抗体片段”，是指其中重链和轻链可变区通过(柔性)接头或(柔性)连接肽连接形成的单多肽链。例如，通过使用重组方法，将独立编码Fv片段的两个结构域VL和VH的基因，通过编码连接肽(接头)的核酸序列连接在一起，重组表达而形成。该单多肽链形成抗原结合区。单链抗体详述于国际专利申请公布号WO88/01649及美国专利号4,946,778和5,260,203中。
术语“(柔性)接头”和术语“(柔性)连接肽”在本文中可以互换使用，是指由氨基酸组成的短肽(肽接头)。通过这样的肽接头可以连接本发明的抗体中的各个可变结构域，例如VH和VL区。肽接头通常富含表现柔性的甘氨酸以及表现溶解性的丝氨酸或苏氨酸。例如可以单独或组合使用甘氨酸和/或丝氨酸残基。柔性连接肽或肽接头的非限定性例子公开于Shen等，Anal.Chem.80(6):1910-1917(2008)、WO2012/138475和WO2014/087010，将其内容全文并入作为参考。如本领域已知的，在scFv的构建中，接头将利于促使VH和VL配对，且不干扰VH和VL对形成功能有效的抗原结合位点。
如本文所用，术语“单域抗体(single-domain antibody,sdAb)”是指仅包含单个可变结构域(例如，重链可变结构域(VH)或轻链可变结构域(VL)，就可以结合抗原的抗体，即其不需要与另一可变结构域相互作用即以识别靶抗原。单域抗体可以衍生自骆驼科重链抗体的 重链可变结构域、衍生自鱼类IgNAR的VH样单结构域(v-NAR)等。
如本文所用，术语“纳米抗体(nanobody,Nb或variable domain of heavy chain of heavy-chain antibody,VHH)”是指仅由一个重链可变区组成，具有与抗原结合的活性的抗体，即从C端到N端仅包含一条链：FR4-VCDR3-FR3-VCDR2-FR2-VCDR1-FR1的抗体，可由骆驼天然产生或由基因工程技术产生。纳米抗体是目前已知的可结合目标抗原的最小单位。
如本文所用，术语“重链抗体(heavy-chain antibody,hcAb)”是指不具有轻链的抗体，从N段到C段可以包含VH-CH2-CH3，或包含VH-CH1-CH2-CH3；可以构成同型二聚体，例如不具有轻链的重链二聚体抗体。本发明的重链抗体中可以包含来自标准抗体的VH或者来自小型化抗体的VH。例如，本发明的重链抗体中的VH可以就是纳米抗体。
如本文所用，术语“单克隆抗体”表示得自基本上同质的抗体群体的抗体，即，除了通常以很少量存在的可能变体抗体(例如，含有天然突变或在单克隆抗体制品的生产过程中产生的变体抗体)以外，构成所述群体的各个抗体是相同的和/或结合相同表位。单克隆抗体可以通过多种技术来制备，该技术包括、但不限于杂交瘤方法、重组DNA方法、酵母展示方法，和使用包含人免疫球蛋白基因座的全部或部分的转基因动物的方法。
如本文所用，术语“人抗体”或“全人源抗体”在本文中可以互换使用，指包括其中构架区和CDR区二者均源自人种系免疫球蛋白序列的可变区的抗体。而且，如果抗体含有恒定区，恒定区也源自人种系免疫球蛋白序列。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸序列(例如，包含通过体外随机或点特异诱变或体内体细胞突变引入的突变)，例如在CDR──尤其在CDR3中。然而，术语“人抗体”不意欲包括其中的CDR序列衍生自其他哺乳动物物种(如，小鼠)的种系而移植入人构架序列的抗体。
如本文所用，术语“人源化抗体”是指将源自其他哺乳动物物种例如小鼠种系的CDR序列接到人构架序列上的抗体。可以在人构架序列内进行额外的构架区修饰。
如本文所用，术语“嵌合抗体”是指可变区序列源自一物种、恒定区序列源自另一物种的抗体，例如，其中可变区序列源自小鼠抗体、恒定区序列源自人抗体的抗体。
如本文所用，术语“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的纯度。对于用于评估抗体纯度的方法的综述参见，例如，Flatman等，J.Chromatogr.B848:79-87(2007)。
如本文所用，术语“表位”是指抗体所结合的抗原区域。表位可以由连续的氨基酸形成或者通过蛋白的三级折叠而由非连续氨基酸形成。
如本文所用，术语“特异性结合”表示抗体选择性地或优先结合抗原。当GITR抗体基本不结合非GITR分子时，认为该抗体“特异性地结合”GITR。然而，特异性结合GITR的抗体可与来自不同物种的GITR多肽交叉反应。如果在生物光干涉测量中，抗体以约5×10 -7M或更低、约1×10 -7M或更低、约5×10 -8M或更低、约1×10 -8M或更低、约5×10 -9M或更低的KD与人GITR结合，则该抗体是“与人GITR特异性结合”的抗体。
如本文所用，“亲和力”或“结合亲和力”指反映结合的成员之间相互作用的固有结合力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(K D)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
与结合例如GITR抗原的参考抗体“竞争结合的抗体”是指在竞争检验中阻断参考抗体与抗原(例如GITR)结合50％或更多的抗体。示例性竞争检验描述于：“Antibodies”,Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harbor,NY)。竞争结合的抗体可以与参考抗体结合相同的表位区，例如相同表位、相邻表位或重叠表位。
如本文所用，术语“Fc区”用于定义含有至少一部分的恒定区的免疫球蛋白重链的C-末端区域。该术语包括天然序列Fc-区和变体Fc-区。在一个实施方案中，人IgG重链Fc-区从 重链的Cys226或从Pro230延伸至羧基端。然而，Fc-区的C-端赖氨酸(Lys447)可以存在或可以不存在。除非本文中另外指出，Fc-区或恒定区中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。
与抗体相关的术语“变体”在本文中是指包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的目标抗体区域(例如重链可变区或轻链可变区或重链CDR区或轻链CDR区)的抗体，其中变体基本上保持改变之前的抗体分子的生物学特性。
在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在本发明中，就抗体序列而言，氨基酸序列同一性百分数通过将候选抗体序列与参考抗体序列最佳比对后，在一个优选方案中按照Kabat编号规则进行最佳比对后，予以确定。比对后，将目标抗体区域(例如，重链或轻链的整个可变区、或其部分例如一个或多个CDR区)与参考抗体的相同区域进行比较。目标抗体区域和参考抗体区域之间的序列同一性百分数为：在目标和参考抗体区域两者中被相同氨基酸占据的位置的数目除以两个区域的比对位置总数(空位不计入)并乘以100得到的百分数。在本文中，在不指定目标抗体区域的情况下，将适用于在参考抗体序列的全长上进行比对。在一些实施方案中，就抗体而言，序列同一性可以分布在整个重链可变区和/或整个轻链可变区上，或序列百分数同一性可以仅限定于构架区，而对应CDR区的序列保持100％相同。
在本文中，“保守性取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。提供功能上相似氨基酸的保守取代表是本领域熟知的。在本发明任一实施方案中，在一个优选的方面，保守取代残基来自以下的保守替代表A，优选地为表A中所示的优选取代残基。
表A
原始残基 示例性取代 优选的保守氨基酸取代
Ala(A) Val、Leu、Ile Val
Arg(R) Lys、Gln、Asn Lys
Asn(N) Gln、His、Asp、Lys、Arg Gln
Asp(D) Glu、Asn Glu
Cys(C) Ser、Ala Ser
Gln(Q) Asn、Glu Asn
Glu(E) Asp、Gln Asp
Gly(G) Ala Ala
His(H) Asn、Gln、Lys、Arg Arg
Ile(I) Leu、Val、Met、Ala、Phe、正亮氨酸 Leu
Leu(L) 正亮氨酸、Ile、Val、Met、Ala、Phe Ile
Lys(K) Arg、Gln、Asn Arg
Met(M) Leu、Phe、Ile Leu
Phe(F) Trp、Leu、Val、Ile、Ala、Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val、Ser Ser
Trp(W) Tyr、Phe Tyr
Tyr(Y) Trp、Phe、Thr、Ser Phe
Val(V) Ile、Leu、Met、Phe、Ala、正亮氨酸 Leu
在本文中，术语“个体”或“受试者”可互换使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，受试者是人。
在本文中，术语“治疗”指意欲改变正在接受治疗的个体中疾病的天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。
在本文中，术语“癌症”、“肿瘤”、“癌性的”和“恶性的”是指或描述哺乳动物中特征通常为细胞生长不受调节的生理状况。癌症的实例包括但不限于：癌，包括腺癌、淋巴瘤、母细胞瘤、黑素瘤、肉瘤和白血病。此类癌症的更具体实例包括：鳞状细胞癌、小细胞肺癌、非小细胞肺癌、胃肠癌、何杰金氏和非何杰金氏淋巴瘤、胰腺癌、胶质母细胞瘤、神经胶质瘤、宫颈癌、卵巢癌、肝癌诸如肝的癌和肝细胞瘤、膀胱癌、乳癌、结肠癌、结肠直肠癌、子宫内膜癌、骨髓瘤(诸如多发性骨髓瘤)、唾液腺癌、肾癌诸如肾细胞癌和威尔曼瘤、基底细胞癌、黑素瘤、前列腺癌、外阴癌、甲状腺癌、睾丸癌、食管癌、及各种类型的头颈癌。
随着癌性细胞生长和倍增，它们形成癌性组织块，这就是肿瘤，其侵入和破坏正常的邻近组织。恶性肿瘤是癌症。恶性肿瘤经常可以切除，但是它们可能再生。来自恶性肿瘤的细胞可以侵入和破坏附近的组织和器官。另外，癌细胞可以脱离恶性肿瘤，并进入血流或淋巴系统，这是癌细胞从原发肿瘤(即，最初的癌)扩散以在其他器官中形成新肿瘤的途径。癌症在体内的扩散被称作转移(What You Need to Know About Cancer-an Overview,NIH公开号00-1566；2000年9月26日公布，2002年9月16日更新(2002))。
2.详细描述
除非明确指明相反，否则本发明的实施将采用本领域内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。这些方法的描述可以参见，例如，Sambrook等，Molecular Cloning:A Laboratory Manual(第3版,2001)；Sambrook等，Molecular Cloning:A Laboratory Manual(第2版,1989)；Maniatis等，Molecular Cloning:A Laboratory Manual(1982)；Ausubel等，Current Protocols in Molecular Biology(John Wiley和Sons,2008年7月更新)；Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates和Wiley-Interscience；Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985)；Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)；Transcription and Translation(B.Hames&S.Higgins编著,1984)；Perbal,A Practical Guide to Molecular Cloning(1984)；Harlow和Lane，Antibodies,(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，,N.Y.，1998)Current Protocols in Immunology Q.E.Coligan，A.M.Kruisbeek，D.H.Margulies，E.M.Shevach和W.Strober编著,1991)；Annual Review of Immunology；以及期刊专著如Advances in Immunology。
一方面，本发明提供了一种可以高效激活GITR下游的NFkB信号通路的抗体或其抗原结合片段(即，本发明的抗体或其抗原结合片段)，其特异性结合糖皮质激素诱导的肿瘤坏死因子受体(GITR)。
在一些实施方案中，本发明的抗体是单克隆抗体或多特异性抗体(包括双抗体)；和/或是IgG1、IgG2、IgG3或IgG4型；和/或是抗原结合片段，例如是Fab片段、Fab’片段、F(ab’) 2 片段或Fv片段；和/或是人抗体、人源化抗体或嵌合抗体；和/或是经标记的抗体。
在一些实施方案中，本发明的抗体是特异性结合GITR的小型化抗体，例如特异性结合GITR的单链抗体(scFv)、单域抗体(sdAd)、重链抗体(hcAb)、纳米抗体(Nb或VHH)；或这些抗体的多聚物形式。这些小型化抗体优选包含本发明抗体重链可变区的HCDR3(重链互补决定区3)；更优选还包含本发明抗体重链可变区的HCDR1(重链互补决定区1)和HCDR2(重链互补决定区2)。
本发明涵盖在本文中所述及的任何抗体的变体。在一个实施方案中，抗体变体保持改变前抗体的至少60％、70％、80％、90％或100％的生物学活性(例如抗原结合能力)。在一些实施方案中，该改变不导致抗体变体丧失对抗原的结合，但任选地可以赋予诸如提高的抗原亲和力和不同的效应子功能等性质。可以理解的，抗体的重链可变区或轻链可变区，或各CDR区可以单独改变或组合改变。在一些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。优选地，所述氨基酸改变为氨基酸取代，优选保守取代。在一些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。优选地，所述氨基酸改变为氨基酸取代，优选保守取代。在一些实施方案中，抗体变体与亲本抗体在目的抗体序列区域上具有至少80％、85％、90％或95％或99％或更高的氨基酸同一性。在一个实施方案中，本发明抗体与表B所列任一抗体相比，在重链可变区上具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性。
例如，本发明的抗体包含与表B所列任一抗体的对应CDR相同的至少一个、两个、三个、四个、五个或六个CDR，或其变体。在一些实施方案中，本发明的抗体包含与表B所列任一抗体的对应重链CDR相同的至少一个、两个、或三个HCDR，或其变体。在本文中，“对应CDR”是指在可变区氨基酸序列中位于基本相似位置上的CDR。在本文中，CDR变体是已经通过至少一个，例如1或2或3个氨基酸取代、缺失和/或插入而修饰的CDR，其中包含CDR变体的抗体分子基本上保持包含未修饰CDR的抗体分子的生物学特性，例如，保持至少60％、70％、80％、90％或100％的生物学活性(例如抗原结合能力)。可以理解，各CDR可以单独修饰或组合修饰。优选地，氨基酸修饰为氨基酸取代，尤其是保守氨基酸取代，例如表A中列出的优选保守氨基酸置换。
在一些实施方案中，本发明的抗体或其抗原结合片段以高特异性和高亲合性结合GITR。
在一些实施方案中，本发明的抗体或其抗原结合片段以高亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合，例如以小于约50nM、小于约30nM、小于约10-25nM或小于约20nM的高亲和力，优选以小于约10nM的亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合。
例如，本发明的抗体或其抗原结合片段以约1-50nM、约5-50nM、约10-50nM、约1-30nM、约5-30nM、约10-30nM、约1-25nM、约5-25nM、约10-25nM、约1-20nM、约5-20nM、约10-20nM、约0.1-10nM、约1-10nM、约5-10nM、约10nM的亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合。
在一些实施方案中，本发明的抗体或其抗原结合片段以高亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合，例如以大于约0.01×10 4/Ms、大于约0.1×10 4/Ms、大于约1×10 4/Ms或大于约3×10 4/Ms的结合常数，优选以大于约5x 10 4/Ms的结合常数与人GITR(如SEQ ID NO:123或127的多肽)结合。
在一些实施方案中，本发明的抗体或其抗原结合片段以低解离常数与人GITR(如SEQ ID NO:123或127的多肽)结合，例如与人GITR(如SEQ ID NO:123或127的多肽)结合的解离常数(K d)小于约2×10 -2s -1、小于约1.5×10 -2s -1、小于约8×10 -3s -1或小于约5×10 -3s -1；优选与人GITR(如SEQ ID NO:123或127的多肽)结合的解离常数为约1-3×10 -3s -1。
例如，本发明的抗体或其抗原结合片段与人GITR(如SEQ ID NO:123或127的多肽)结合的解离常数(K d)为约1×10 -4s -1至2×10 -2s -1、约1×10 -3s -1至2×10 -2s -1、约3×10 -3s -1至2×10 -2s -1、约1×10 -4s -1至1.5×10 -2s -1、约1×10 -3s -1至1.5×10 -2s -1、约3×10 -3s -1至1.5×10 -2s -1、约1×10 -4s -1至8×10 -2s -1、约1×10 -3s -1至8×10 -2s -1、约3×10 -3s -1至1.5×10 -2s -1、约1×10 -4s -1至5×10 -2s -1、约1×10 -3s -1至5×10 -2s -1、约3×10 -3s -1至1.5×10 -2s -1、1×10 -3s -1至2×10 -250nM、约3×10 -3s -1至1.5×10 -2s -1。
在一些实施方案中，本发明的抗体或其抗原结合片段与细胞表面的抗原高亲和力结合，例如以小于约50nM、小于约30nM、小于约25nM或小于约20nM的亲和力，优选以小于约10nM的亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合。
例如，本发明的抗体或其抗原结合片段以约1nM-50nM、约5nM-50nM、约10nM-50nM、约1nM-30nM、约5nM-30nM、约10nM-30nM、约1nM-25nM、约5nM-25nM、约10nM-25nM、约1nM-20nM、约5nM-20nM、约10nM-20nM、约0.1-10nM、约1-10nM、约5-10nM、约10nM的亲和力与细胞表面的抗原。
在一些实施方案中，本发明的抗体或其抗原结合片段高效激活GITR下游的NFkB信号通路，例如激活NFkB信号通路的EC 50值小于约50nM、小于约30nM、小于约25nM或小于约5nM，优选小于约1nM。
例如，本发明的抗体或其抗原结合片段激活NFkB信号通路的EC 50值为约1nM-50nM、约5nM-50nM、约10nM-50nM、约1nM-30nM、约5nM-30nM、约10nM-30nM、约1nM-25nM、约5nM-25nM、约10nM-25nM、约1nM-20nM、约5nM-20nM、约10nM-20nM、约0.1-10nM、约1-10nM、约5-10nM或约10nM。
在一些实施方案中，本发明的抗体或其抗原结合片段与GITRL交叉竞争结合至GITR，优选与GITRL交叉竞争结合至人GITR(如SEQ ID NO:123或127的多肽)。
在一些实施方案中，本发明的抗体或其抗原结合片段可以内化到人CD4细胞中。
在一些实施方案中，本发明的抗体或其抗原结合片段抑制调节性T细胞的抑制作用。
在一些实施方案中，本发明的抗体或其抗原结合片段活化效应T细胞。
在一些实施方案中，本发明的抗体或其抗原结合片段减少循环调节性T细胞。
在一些实施方案中，本发明的抗体或其抗原结合片段能够结合Fcγ受体(FcγR)。
在一些实施方案中，本发明的抗体或其抗原结合片段在人血清中具有至少约6、约7、约9或约12天的半衰期。
另一方面，本发明提供了一种特异性结合GITR，并高效激活GITR下游的NFkB信号通路的纳米抗体(即，本发明的纳米抗体)，该纳米抗体的HCDR3包含本发明抗体或抗原结合片段的HCDR3。
在一些实施方案中，本发明的纳米抗体的HCDR3包含SEQ ID NO:64-85中任一序列所示的重链可变区序列所含的HCDR3，或本发明的纳米抗体的HCDR3由SEQ ID NO:64-85中任一序列所示的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的纳米抗体的HCDR3包含与如SEQ ID NO:64-85中任一序列所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列所含的HCDR3，其中优选序列差异不存在于CDR区域中，或本发明的纳米抗体的HCDR3由这样的重链可变区序列中所含的HCDR3组成。
在一些实施方案中，本发明的纳米抗体的HCDR3包含在如SEQ ID NO:64-85中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR3，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的纳米抗体的HCDR3由这样的重链可变区序列中所含的HCDR3组成。
在一些实施方案中，本发明的纳米抗体的HCDR3包含如SEQ ID NO:45-63中任一序列 所示的HCDR3，或本发明的纳米抗体的HCDR3由如SEQ ID NO:45-63中任一序列所示的HCDR3组成。
在一些实施方案中，本发明的纳米抗体的HCDR3包含在如SEQ ID NO:45-63中任一序列所示的HCDR3中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR3，或本发明的纳米抗体的HCDR3由这样的HCDR3组成。
在一些实施方案中，本发明的纳米抗体的HCDR1(重链互补决定区1)和HCDR2(重链互补决定区2)包含如SEQ ID NO:64-85中任一序列所示的重链可变区序列所含的HCDR1和HCDR2，或本发明的纳米抗体的HCDR1和HCDR2由如SEQ ID NO:64-85中任一序列所示的重链可变区序列所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的纳米抗体的HCDR1和HCDR2包含在如SEQ ID NO:64-85中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR1和HCDR2，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的纳米抗体的HCDR1和HCDR2由这样的重链可变区序列中所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的纳米抗体的HCDR1包含如SEQ ID NO:1-22中任一序列所示的HCDR1，或本发明的纳米抗体的HCDR1由如SEQ ID NO:1-22中任一序列所示的HCDR1组成。
在一些实施方案中，本发明的纳米抗体的HCDR1包含在如SEQ ID NO:1-22中任一序列所示的HCDR1中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR1，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的纳米抗体的HCDR1由这样的HCDR1组成。
在一些实施方案中，本发明的纳米抗体的HCDR2包含如SEQ ID NO:23-44中任一序列所示的HCDR2，或本发明的纳米抗体的HCDR2由如SEQ ID NO:23-44中任一序列所示的HCDR2组成。
在一些实施方案中，本发明的纳米抗体的HCDR2包含在如SEQ ID NO:23-44中任一序列所示的HCDR2中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR2，或本发明的纳米抗体的HCDR2由这样的HCDR2组成。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:23所示的HCDR2和如SEQ ID NO:45所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:24所示的HCDR2和如SEQ ID NO:46所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:3所示的HCDR1、如SEQ ID NO:25所示的HCDR2和如SEQ ID NO:47所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:4所示的HCDR1、如SEQ ID NO:26所示的HCDR2和如SEQ ID NO:48所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:5所示的HCDR1、如SEQ ID NO:27所示的HCDR2和如SEQ ID NO:49所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:6所示的HCDR1、如SEQ ID NO:28所示的HCDR2和如SEQ ID NO:50所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:7所示的HCDR1、如SEQ ID NO:29所示的HCDR2和如SEQ ID NO:51所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:8所示的HCDR1、如SEQ ID NO:30所示的HCDR2和如SEQ ID NO:52所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:9所示的HCDR1、如SEQ ID NO:31所示的HCDR2和如SEQ ID NO:53所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:10所示的HCDR1、如SEQ ID NO:32所示的HCDR2和如SEQ ID NO:51所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:33所示的HCDR2和如SEQ ID NO:48所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:12所示的HCDR1、如SEQ ID NO:34所示的HCDR2和如SEQ ID NO:46所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:13所示的HCDR1、如SEQ ID NO:35所示的HCDR2和如SEQ ID NO:54所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:14所示的HCDR1、如SEQ ID NO:36所示的HCDR2和如SEQ ID NO:55所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:15所示的HCDR1、如SEQ ID NO:37所示的HCDR2和如SEQ ID NO:56所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:16所示的HCDR1、如SEQ ID NO:38所示的HCDR2和如SEQ ID NO:57所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:17所示的HCDR1、如SEQ ID NO:39所示的HCDR2和如SEQ ID NO:58所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:18所示的HCDR1、如SEQ ID NO:40所示的HCDR2和如SEQ ID NO:59所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:19所示的HCDR1、如SEQ ID NO:41所示的HCDR2和如SEQ ID NO:60所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:20所示的HCDR1、如SEQ ID NO:42所示的HCDR2和如SEQ ID NO:61所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:21所示的HCDR1、如SEQ ID NO:43所示的HCDR2和如SEQ ID NO:62所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:22所示的HCDR1、如SEQ ID NO:44所示的HCDR2和如SEQ ID NO:63所示的HCDR3的组合。
在一些实施方案中，本发明的纳米抗体包含如SEQ ID NO:64-85中任一序列所示的重链可变区序列，或由如SEQ ID NO:64-85中任一序列所示的重链可变区序列组成，优选仅由一条这样的重链可变区序列组成。
在一些实施方案中，本发明的纳米抗体包含与如SEQ ID NO:64-85中任一序列所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列，其中优选序列差异不存在于CDR区域中，或本发明的纳米抗体由这样的重链可变区序列组成，优选仅由一条这样的重链可变区序列组成。
在一些实施方案中，本发明的纳米抗体包含在如SEQ ID NO:64-85中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的纳米抗体由这样的重链可变区序列组成，优选仅由一条这样的重链可变区序列组成。
另一方面，本发明提供了一种特异性结合GITR，并高效激活GITR下游的NFkB信号通路的重链抗体(即，本发明的重链抗体)，该重链抗体的HCDR3包含本发明抗体或抗原结合片段的HCDR3，或由本发明抗体或抗原结合片段的HCDR3组成；该重链抗体的HCDR3优选包含上述本发明纳米抗体的HCDR3或由上述本发明纳米抗体的HCDR3组成；更优选该重链抗体的HCDR1-2优选分别包含上述本发明纳米抗体的HCDR1-2或分别由上述本发明纳米 抗体的HCDR1-2组成。
在一些实施方案中，本发明的重链抗体是人源化重链抗体，优选是全人源重链抗体(人抗体)。
在一些实施方案中，本发明的重链抗体的HCDR3包含如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列所含的HCDR3，或本发明的重链抗体的HCDR3由如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的重链抗体的HCDR3包含与如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列所含的HCDR3，其中优选序列差异不存在于CDR区域中，或本发明的重链抗体的HCDR3由这样的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的重链抗体的HCDR3包含在如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR3，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的重链抗体的HCDR3由这样的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的重链抗体的HCDR3包含如SEQ ID NO:46、47、48、51、53、57和61中任一序列所示的HCDR3，或本发明的重链抗体的HCDR3由如SEQ ID NO:46、47、48、51、53、57和61中任一序列所示的HCDR3组成。
在一些实施方案中，本发明的重链抗体的HCDR3包含在如SEQ ID NO:46、47、48、51、53、57和61中任一序列所示的HCDR3中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR3，或本发明的重链抗体的HCDR3由这样的HCDR3组成。
在一些实施方案中，本发明的重链抗体的HCDR1和HCDR2包含如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列所含的HCDR1和HCDR2，或本发明的重链抗体的HCDR1和HCDR2由这样的重链可变区序列所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的重链抗体的HCDR1和HCDR2包含与如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列所含的HCDR1和HCDR2，其中优选序列差异不存在于CDR区域中，或本发明的重链抗体的HCDR1和HCDR2由这样的重链可变区序列所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的重链抗体的HCDR1和HCDR2包含在如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR1和HCDR2，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的重链抗体的HCDR1和HCDR2由这样的重链可变区序列所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的重链抗体的HCDR1包含如SEQ ID NO:2、3、4、7、9、10、16、20中任一序列所示的HCDR1，或本发明的重链抗体的HCDR1由如SEQ ID NO:2、3、4、7、9、10、16、20中任一序列所示的HCDR1组成。
在一些实施方案中，本发明的重链抗体的HCDR1包含在如SEQ ID NO:2、3、4、7、9、10、16、20中任一序列所示的HCDR1中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR1，或本发明的重链抗体的 HCDR1由这样的HCDR1组成。
在一些实施方案中，本发明的重链抗体的HCDR2包含如SEQ ID NO:24、25、26、29、31、32、38、42中任一序列所示的HCDR2，或本发明的重链抗体的HCDR2由如SEQ ID NO:24、25、26、29、31、32、38、42中任一序列所示的HCDR2组成。
在一些实施方案中，本发明的重链抗体的HCDR2包含在如SEQ ID NO:24、25、26、29、31、32、38、42中任一序列所示的HCDR2中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR2，或本发明的重链抗体的HCDR2由这样的HCDR2组成。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:24所示的HCDR2和如SEQ ID NO:46所示的HCDR3的组合。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:3所示的HCDR1、如SEQ ID NO:25所示的HCDR2和如SEQ ID NO:47所示的HCDR3的组合。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:4所示的HCDR1、如SEQ ID NO:26所示的HCDR2和如SEQ ID NO:48所示的HCDR3的组合。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:7所示的HCDR1、如SEQ ID NO:29所示的HCDR2和如SEQ ID NO:51所示的HCDR3。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:9所示的HCDR1、如SEQ ID NO:31所示的HCDR2和如SEQ ID NO:53所示的HCDR3。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:10所示的HCDR1、如SEQ ID NO:32所示的HCDR2和如SEQ ID NO:51所示的HCDR3。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:16所示的HCDR1、如SEQ ID NO:38所示的HCDR2和如SEQ ID NO:57所示的HCDR3。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:20所示的HCDR1、如SEQ ID NO:42所示的HCDR2和如SEQ ID NO:61所示的HCDR3。
在一些实施方案中，本发明的重链抗体的重链可变区序列包含如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列，或本发明的重链抗体的重链可变区序列由如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列组成。
在一些实施方案中，本发明的重链抗体的重链可变区序列包含与如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列，其中优选序列差异不存在于CDR区域中，或本发明的重链抗体的重链可变区序列由这样的重链可变区序列组成。
在一些实施方案中，本发明的重链抗体的重链可变区序列包含在如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的重链抗体的重链可变区序列由这样的重链可变区序列组成。
在一些实施方案中，本发明的重链抗体包含如SEQ ID NO:108-115中任一序列所示的重链抗体序列，或本发明的重链抗体由一条、两条或多条如SEQ ID NO:108-115中任一序列所示的重链抗体序列组成。
在一些实施方案中，本发明的重链抗体包含与如SEQ ID NO:108-115中任一序列所示的重链抗体序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链抗体序列，其中优选序列差异不存在于CDR区域中，或本发明的重链抗体由一条、两条或多条这样的重链抗体序列组成。
在一些实施方案中，本发明的重链抗体包含在如SEQ ID NO:108-115中任一序列所示的重链抗体序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链抗体序列，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的重链抗体由一条、两条或多条这样的重链抗体序列组成。
另一方面，本发明提供了一种特异性结合GITR，并高效激活GITR下游的NFkB信号通路的人源化重链抗体(即，本发明的人源化重链抗体)，该人源化重链抗体的HCDR3包含本发明抗体或抗原结合片段的HCDR3，或由本发明抗体或抗原结合片段的HCDR3组成。
在一些实施方案中，本发明的人源化重链抗体的HCDR3包含上述本发明纳米抗体或重链抗体的HCDR3，或由上述本发明纳米抗体或重链抗体的HCDR3组成；更优选本发明人源化重链抗体的HCDR1-2优选分别包含上述本发明纳米抗体或重链抗体的HCDR1-2，或分别由上述本发明纳米抗体或重链抗体的HCDR1-2组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR3包含如SEQ ID NO:124或125所示的重链可变区序列所含的HCDR3，或本发明的人源化重链抗体中的HCDR3由如SEQ ID NO:124或125所示的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR3包含与如SEQ ID NO:124或125所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列所含的HCDR3，其中优选序列差异不存在于CDR区域中，或本发明的人源化重链抗体中的HCDR3由这样的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR3包含在如SEQ ID NO:124或125所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR3，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的人源化重链抗体中的HCDR3由这样的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR3包含选自SEQ ID NO:51或61的HCDR3，或本发明的人源化重链抗体中的HCDR3由选自SEQ ID NO:51或61的HCDR3组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR3包含在选自SEQ ID NO:51或61的HCDR3中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR3，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的人源化重链抗体中的HCDR3由这样的HCDR3组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR1和HCDR2包含选自SEQ ID NO:124或125的重链可变区序列所含的HCDR1和HCDR2，或本发明的人源化重链抗体中的HCDR1和HCDR2由选自SEQ ID NO:124或125的重链可变区序列所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR1和HCDR2包含与如SEQ ID NO:124或125所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列所含的HCDR1和HCDR2，其中优选序列差异不存在于CDR区域中，或本发明的人源化重链抗体中的HCDR1和HCDR2由这样的重链可变区序列所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR1和HCDR2包含在如SEQ ID NO:124或125所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR1和HCDR2，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的人源化重链抗体中的HCDR1和HCDR2由这样的重链可变区序列所含的HCDR1和HCDR2组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR1包含如SEQ ID NO:7或20所示的HCDR1，或本发明的人源化重链抗体中的HCDR1由如SEQ ID NO:7或20所示的HCDR1组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR1包含在如SEQ ID NO:7或20所示的HCDR1中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR1，或本发明的人源化重链抗体中的HCDR1由这样的HCDR1组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR2包含如SEQ ID NO:29或42所示的HCDR2，或本发明的人源化重链抗体中的HCDR2由如SEQ ID NO:29或42所示的HCDR2组成。
在一些实施方案中，本发明的人源化重链抗体中的HCDR2包含在如SEQ ID NO:29或42所示的HCDR2中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR2，或本发明的人源化重链抗体中的HCDR2由这样的HCDR2组成。
在一些实施方案中，本发明的人源化重链抗体包含如SEQ ID NO:7所示的HCDR1、如SEQ ID NO:29所示的HCDR2和如SEQ ID NO:51所示的HCDR3的组合。
在一些实施方案中，本发明的人源化重链抗体包含如SEQ ID NO:20所示的HCDR1、如SEQ ID NO:42所示的HCDR2和如SEQ ID NO:61所示的HCDR3的组合。
在一些实施方案中，本发明的人源化重链抗体的重链可变区序列包含如SEQ ID NO:124或125所示的重链可变区序列，或本发明的人源化重链抗体的重链可变区序列由如SEQ ID NO:124或125所示的重链可变区序列组成。
在一些实施方案中，本发明的人源化重链抗体的重链可变区序列包含与如SEQ ID NO:124或125所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列，其中优选序列差异不存在于CDR区域中，或本发明的人源化重链抗体的重链可变区序列由这样的重链可变区序列组成。
在一些实施方案中，本发明的人源化重链抗体的重链可变区序列包含在如SEQ ID NO:124或125所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的人源化重链抗体的重链可变区序列由这样的重链可变区序列组成。
在一些实施方案中，本发明的人源化重链抗体包含如SEQ ID NO:116或117所示的人源化重链抗体序列，或本发明的人源化重链抗体由一条、两条或多条如SEQ ID NO:116或117所示的人源化重链抗体序列组成。
在一些实施方案中，本发明的人源化重链抗体包含与如SEQ ID NO:116或117所示的人源化重链抗体序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的人源化重链抗体序列，其中优选序列差异不存在于CDR区域中，或本发明的人源化重链抗体由一条、两条或多条这样的人源化重链抗体序列组成。
在一些实施方案中，本发明的人源化重链抗体包含在如SEQ ID NO:116或117所示的人源化重链抗体序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的人源化重链抗体序列，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的人源化重链抗体由一条、两条或多条这样的人源化重链抗体序列组成。
在一些实施方案中，本发明的重链抗体或人源化重链抗体包含上述任一种重链可变区和Fc片段，优选该Fc片段包含下述序列(a)-(d)之一或由下述序列(a)-(d)之一组成:
(a)人IgG1的Fc片段；
(b)如SEQ ID NO:121所示的Fc片段；或
(c)与如SEQ ID NO:121所示的Fc片段具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的Fc片段；或
(d)在如SEQ ID NO:121所示的Fc片段中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的Fc片段。
在优选的实施方案中，本发明的重链抗体或人源化重链抗体包含两条选自上述序列(a)-(d)中的Fc序列。
在更优选的实施方案中，同一个本发明的重链抗体或人源化重链抗体的两条Fc序列(a)-(d)之间存在链间二硫键。
最优选的实施方案中，同一个本发明的重链抗体或人源化重链抗体的两条Fc序列(a)-(d)之间存在两个链间二硫键，该链间二硫键是本发明的重链抗体或人源化重链抗体中包含的CPPC序列中，氨基酸残基C之间形成的两个链间二硫键，该CPPC序列是本发明的重链抗体或人源化重链抗体中的纳米抗体与Fc之间连接的序列。
另一方面，本发明提供了一种特异性结合GITR，并高效激活GITR下游的NFkB信号通路的多聚物形式的抗体(即，本发明的多聚物形式的抗体)，该多聚物形式的抗体的HCDR3包含本发明抗体或抗原结合片段的HCDR3，或由本发明抗体或抗原结合片段的HCDR3组成。
在一些实施方案中，本发明的多聚物形式的抗体的HCDR3包含上述本发明纳米抗体、重链抗体或人源化重链抗体的HCDR3，或由上述本发明纳米抗体、重链抗体或人源化重链抗体的HCDR3组成；更优选本发明多聚物形式的抗体的HCDR1-2优选分别包含上述本发明纳米抗体、重链抗体或人源化重链抗体的HCDR1-2，或分别由上述本发明纳米抗体、重链抗体或人源化重链抗体的HCDR1-2组成。
在优选的实施方案中，本发明的多聚物形式的抗体是本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区的多聚物形式，优选是本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区的四聚化形式或六聚化形式，最优选是本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区的四聚化形式。
通过形成将本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区形成多聚物形式的抗体，可以更有效地激活GITR下游的NFkB信号通路，同时可以适当延长本发明的纳米抗体、重链抗体、人源化重链抗体在活体内的半衰期。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR3包含如SEQ ID NO:125所示的重链可变区序列所含的HCDR3序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR3由如SEQ ID NO:125所示的重链可变区序列所含的HCDR3组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR3包含与如SEQ ID NO:125所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列所含的HCDR3序列，其中优选序列差异不存在于CDR区域中，或本发明的多聚物形式的抗体的重链可变区中的HCDR3由这样的HCDR3序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR3包含在如SEQ ID NO:125所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR3，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的多聚物形式的抗体的重链可变区中的HCDR3由这样的HCDR3序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR3包含所示SEQ ID NO:61所示的HCDR3序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR3由所示SEQ ID NO:61所示的HCDR3序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR3包含在SEQ  ID NO:61的HCDR3中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR3序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR3由这样的HCDR3序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR1和HCDR2包含如SEQ ID NO:125所示的重链可变区序列所含的HCDR1和HCDR2序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR1和HCDR2由如SEQ ID NO:125所示的重链可变区序列所含的HCDR1和HCDR2序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR1和HCDR2包含与如SEQ ID NO:125所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列所含的HCDR1和HCDR2，其中优选序列差异不存在于CDR区域中，或本发明的多聚物形式的抗体的重链可变区中的HCDR1和HCDR2由这样的HCDR1和HCDR2序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR1和HCDR2包含在如SEQ ID NO:125所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR1和HCDR2，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的多聚物形式的抗体的重链可变区中的HCDR1和HCDR2由这样的HCDR1和HCDR2序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR1包含如SEQ ID NO:20所示的HCDR1序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR1由如SEQ ID NO:20所示的HCDR1序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR1包含在SEQ ID NO:20的HCDR1中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR1序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR1由这样的HCDR1序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR2包含如SEQ ID NO:42所示的HCDR2序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR2由如SEQ ID NO:42所示的HCDR2序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区中的HCDR2包含在SEQ ID NO:42的HCDR2中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR2序列，或本发明的多聚物形式的抗体的重链可变区中的HCDR2由这样的HCDR2序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区包含如SEQ ID NO:20所示的HCDR1、如SEQ ID NO:42所示的HCDR2和如SEQ ID NO:61所示的HCDR3的组合。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区包含如SEQ ID NO:125所示的重链可变区序列，或本发明的多聚物形式的抗体的重链可变区由如SEQ ID NO:125所示的重链可变区序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区包含与如SEQ ID NO:125所示的重链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的重链可变区序列，其中优选序列差异不存在于CDR区域中，或本发明的多聚物形式的抗体的重链可变区由这样的重链可变区序列组成。
在一些实施方案中，本发明的多聚物形式的抗体的重链可变区包含在如SEQ ID NO:125所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的多聚物形式的抗体的重链可变区由这样的重链可变区序列组成。
在一些实施方案中，本发明的多聚物形式的抗体包含选自SEQ ID NO:118-120的任一序 列，或本发明的多聚物形式的抗体由相同的两条、三条或多条选自SEQ ID NO:118-120的序列组成。
在一些实施方案中，本发明的多聚物形式的抗体包含与如SEQ ID NO:118-120中任一序列所示的序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，其中优选序列差异不存在于CDR区域中，或本发明的多聚物形式的抗体由相同的两条、三条或多条这样的序列组成。
在一些实施方案中，本发明的多聚物形式的抗体包含在如SEQ ID NO:118-120中任一序列所示的序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的序列，其中优选所述氨基酸改变不发生在CDR区域中，或本发明的多聚物形式的抗体由相同的两条、三条或多条这样的序列组成。
在一些实施方案中，本发明的多聚物形式的抗体中的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区(简称“本发明的抗体的重链可变区”)连接到本发明的人源化重链抗体。
在优选的实施方案中，本发明的多聚物形式的抗体中的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区连接到本发明的人源化重链抗体的C末端；或者本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区连接到本发明的人源化重链抗体的N末端。
在更优选的实施方案中，本发明的多聚物形式的抗体包含两条相同的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区，该两条相同的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区分别连接到两条相同的本发明的人源化重链抗体的C末端；或者该两条相同的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区分别连接到两条相同的本发明的人源化重链抗体的N末端。
在最优选的实施方案中，本发明的多聚物形式的抗体是四聚体(包含四个相同的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区)，其包含两条相同的链，每条链包含一个纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区，和一个本发明的人源化重链抗体。即，本发明的多聚物形式的抗体包含两条完全相同的链(完全相同的第一链和第二链)，每条链均只包含一个额外的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区和一个本发明的人源化重链抗体，其中第一链包含的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区和第二链包含的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区相同；第一链包含的人源化重链抗体和第二链包含的人源化重链抗体相同。这两条相同的链之间还可以存在链间二硫键，该链间二硫键是每条链的人源化重链抗体中连接其中的重链可变区与Fc的序列(例如，CPPC序列)中，氨基酸残基C之间形成的两个链间二硫键。即，第一链的人源化重链抗体中的重链可变区与Fc之间的序列中的氨基酸残基C和第二链的人源化重链抗体中的重链可变区与Fc之间的序列中的氨基酸残基C之间形成链间二硫键。
在一些实施方案中，本发明的多聚物形式的抗体中的本发明的纳米抗体、重链抗体的重链可变区、人源化重链抗体的重链可变区通过连接肽连接到本发明的人源化重链抗体，该连接肽优选是柔性连接肽，更优选是氨基酸序列为(G4S)n(n为0-7整数)的连接肽。
本发明提供了如实施例中分离并表征的特异性结合GITR(例如人GITR)的纳米抗体、重链抗体、人源化重链抗体和四聚体形式的抗体。下表B中列出了本发明这些示例性抗体的氨基酸序列和核苷酸序列。其中，抗体的氨基酸序列中，从N端至C端以加粗斜体示出的序列分别是示例性CDR序列(通过本领域已知的各种确定CDR的精确氨基酸序列边界的方法中的任一种或其组合确定，例如可以按照本文上述介绍的方法，并参考其他因素，例如保守性，确定如下示例性CDR序列)。
表B.本发明示例性纳米抗体的氨基酸序列和核苷酸序列、重链抗体的氨基酸序列、人源化 重链抗体的氨基酸序列和四聚体形式的抗体的氨基酸序列，及其SEQ ID NO编号。
Figure PCTCN2019079625-appb-000001
Figure PCTCN2019079625-appb-000002
Figure PCTCN2019079625-appb-000003
Figure PCTCN2019079625-appb-000004
Figure PCTCN2019079625-appb-000005
Figure PCTCN2019079625-appb-000006
Figure PCTCN2019079625-appb-000007
Figure PCTCN2019079625-appb-000008
下表C中给出本发明示例的序列对应的SEQ ID NO序号。
表C本发明示例的序列的SEQ ID NO序号
Figure PCTCN2019079625-appb-000009
Figure PCTCN2019079625-appb-000010
另一方面，本发明提供了一种融合蛋白或免疫缀合物(简称本发明的融合蛋白或免疫缀合物)，其中本发明的抗体与一个或多个异源分子(第二分子)融合或缀合，其中该异源分子包括但不限于蛋白/多肽、标记物、药物或细胞毒性剂。
蛋白质、多肽或肽与抗体融合或缀合的方法是本领域已知的。参见，例如，美国专利号5,336,603、5,622,929和EP 367,166。
在一些实施方案中，本发明的融合蛋白或免疫缀合物中药物是抗肿瘤药物。
另一方面，本发明提供了一种组合物或试剂盒(简称本发明的组合物或试剂盒)，其包含本发明的抗体。
在一些实施方案中，本发明的组合物或试剂盒包括载体和/或说明书。
在一些实施方案中，本发明的组合物或试剂盒中的抗体与诊断剂或可检测试剂缀合。
另一方面，本发明提供了一种分离的核酸(简称本发明的分离的核酸或本发明的核酸(分子))，其编码本发明的抗体或其片段(包括抗原结合片段)、本发明的融合蛋白或本发明的免疫缀合物。
在一些实施方案中，本发明的核酸分子是基本上纯化的核酸分子。
在一些实施方案中，本发明的核酸分子包含编码表B所示任一抗体的重链可变区或其变体的核苷酸序列。在一个具体实施方案中，核酸分子是表B中列出的纳米抗体的核苷酸序列。本发明的核酸分子包含编码表B所示任一抗体的重链至少一个CDR区和通常全部三个CDR区，或其变体的核苷酸序列。
本发明的一些其他核酸分子与表B中所示的氨基酸序列的编码核酸分子的核苷酸序列基本上相同(例如，至少65％、80％、95％、或99％同一性)。在适宜的表达载体表达时，由这些多核苷酸编码的多肽能够显示GITR抗原结合能力。
如本领域技术人员所知的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。
在一些实施方案中，本发明的核酸序列编码本发明的上述任何纳米抗体。在一些实施方案中，编码纳米抗体的本发明核酸序列包含SEQ ID NO:86-107的核苷酸序列或与其基本相同的序列。在一些实施方案中，编码纳米抗体的本发明核酸序列由SEQ ID NO:86-107的核苷酸序列或与其基本相同的序列组成。
“基本相同”的核苷酸序列是指与参考核苷酸序列在序列上具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％,、98％或99％或更高同一性的序列。核苷酸序列的同一性可以使用本领域公知的各种序列比对方法确定。例如可以从NCBI(National Center for Biotechnology Information,Bethesda,MD)的网站上获得BLAST序列比对检索工具。一般而言，百分比同一性采用NCBI Blast的默认参数进行。
可以通过从头固相DNA合成或通过PCR诱变编码GITR抗体或其结合片段的现有序列(例如，表B和2中所示的序列)，产生GITR抗体或其结合片段的多核苷酸序列。核酸的直接化学合成可以通过本领域已知的方法完成，如Narang等,1979,Meth.Enzymol.68:90的磷酸三酯法；Brown等,Meth.Enzymol.68:109,1979的磷酸二酯法；Beaucage等,Tetra.Lett.,22:1859,1981的二乙基磷酰亚胺法；和美国专利号4,458,066的固相支持法。通过PCR向多核苷酸序列引入突变可以如同例如PCR Technology:Principles and Applications for DNA Amplification,H.A.Erlich(编著),Freeman Press,NY,NY,1992；PCR Protocols:A Guide to  Methods and Applications,Innis等(编著),Academic Press,San Diego,CA,1990；Mattila等,Nucleic Acids Res.19:967,1991；and Eckert等,PCR Methods and Applications 1:17,1991中所述那样进行。
另一方面，本发明提供包含本发明的核酸的载体(简称本发明的载体)。
在一些实施方案中，本发明的载体是表达载体，即可以用来表达编码结合GITR的抗体链(例如本发明的任何抗体)或多肽(例如本发明的任何融合物和缀合物)的多核苷酸。
基于病毒的表达载体和非病毒表达载体都可用于在哺乳动物宿主细胞中产生抗体。非病毒载体和系统包含质粒、游离型载体和人工染色体，一般含有用于表达蛋白质或RNA的表达盒(参见，例如，Harrington等,Nat Genet 15:345,1997)。有用的病毒载体包括基于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒的载体，基于SV40、乳头瘤病毒、HBP EB病毒、痘苗病毒载体和Semliki Forest病毒(SFV)的载体。参见，Smith,Annu.Rev.Microbiol.49:807,1995；和Rosenfeld等,Cell 68:143,1992。
另一方面，本发明提供包含本发明的载体的宿主细胞(简称本发明的宿主细胞)。
在一些实施方案中，本发明的宿主细胞是适合本发明载体的克隆或表达的任何宿主细胞，包括原核或真核细胞。
在一些实施方案中，本发明的宿主细胞是细菌、酵母细胞、哺乳动物细胞或免疫效应细胞(如T细胞)。
例如，当不需要糖基化和Fc效应子功能时，抗体可以在细菌中生产。对于抗体片段和多肽在细菌中的表达，参见例如US 5,648,237、US 5,789,199和US 5,840,523。除了原核生物外，真核微生物例如丝状真菌或酵母是用于抗体编码载体的合适克隆或表达宿主，包括糖基化途径已被“人源化”的真菌和酵母菌株，这导致具有部分或完全人糖基化模式的抗体的生产。参见Gerngross，Nat.Biotech.22(2004)1409-1414；和Li,H.等，Nat.Biotech(2006)24:210-215。用于糖基化抗体表达的合适宿主细胞也可以源自多细胞生物(无脊椎动物和脊椎动物)。无脊椎动物细胞的实例包括植物和昆虫细胞。已经鉴定了众多杆状病毒株，其可以与昆虫细胞结合使用，特别用于草地贪夜蛾(Spodoptera frugiperda)细胞的转染。植物细胞培养物也可以用作宿主。参见，例如，US 5,959,177、US 6,040,498、US 6,420,548、US 7,125,978和US 6,417,429(描述用于在转基因植物中生产抗体的PLANTIBODIESTM技术)。可以用作宿主的脊椎动物细胞包括，例如，悬浮生长适应化的哺乳动物细胞系可以是有用的。有用的哺乳动物宿主细胞系的其他实例是SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293或例如Graham,F.L.等，J.Gen Virol.36(1997)59中描述的HEK293细胞)；幼仓鼠肾细胞(BHK)；小鼠塞尔托利细胞(例如Mather,J.P.，Biol.Reprod.23(1980)243-251中描述的TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人宫颈癌细胞(HELA)；犬肾细胞(MDCK)；Buffalo大鼠肝细胞(BRL 3A)；人肺细胞(W138)；人肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞，例如Mather,J.P.等，Annals N.Y.Acad.Sci.383(1982)44-68中描述的；MRC 5细胞；FS4细胞。其他有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞(Urlaub,G.等，Proc.Natl.Acad.Sci.USA 77(1980)4216-4220)；和骨髓瘤细胞系，如Y0、NS0和Sp2/0。对于适合于抗体生产的一些哺乳动物宿主细胞系的综述，参见，例如，Yazaki,P.和Wu,A.M.，Methods in Molecular Biology，Vol.248，Lo.B.K.C.(编辑)，Humana Press，Totowa，NJ(2004)pp.255-268。
在一些优选的实施方案中，大肠杆菌细胞(TG1细胞)、人胚肾系(293细胞)或人宫颈癌细胞(HELA)可以用来表达并产生结合GITR的本发明抗体。
另一方面，本发明提供一种包含制备本发明的抗体或其抗原结合片段、本发明的融合蛋白或本发明的免疫缀合物的方法(简称本发明的制备方法或本发明的方法)，包括：在适于表达本发明的抗体或其抗原结合片段、融合蛋白或免疫缀合物的条件下，培养本发明的宿主细胞。
在一些实施方案中，本发明的方法中还包括采用有利于本发明的抗体或其抗原结合片段、融合蛋白或免疫缀合物表达和纯化的任何表达手段，例如采用分泌信号序列、表达增强元件、高效的启动子等本领域技术人员已知的任何表达、调节元件。
另一方面，本发明提供一种药物组合物(简称本发明的药物组合物)，其包含本发明的抗体或其抗原结合片段、本发明的融合蛋白或本发明的免疫缀合物，以及任选地药用载体。
在一些实施方案中，本发明的药物组合物进一步包含其他成分，例如免疫治疗剂、抗血管生成剂及化疗剂。
在一些实施方案中，本发明的药物组合物中的免疫治疗剂可以诱导或增强免疫应答，包括例如：1)树突细胞活化剂；2)疫苗佐剂；3)T细胞刺激物；4)免疫检查点抑制剂，以及5)抑制性细胞、细胞因子和/或酶的抑制剂。
在一些实施方案中，本发明的药物组合物可包括用于治疗癌症的其他化合物、药物及/或药剂，包括但不限于刺激针对给定癌症的免疫应答的化学疗法药物、小分子药物或抗体。在一些情况下，治疗组合物可包括：抗CTLA-4抗体、抗PD-1抗体、抗PDL-1抗体、抗OX40(亦称作CD134、TNFRSF4、ACT35及/或TXGP1L)抗体、抗CD137抗体或抗LAG-3抗体。
另一方面，本发明提供本发明GITR结合分子(抗体)在诊断和/或检测中的用途(简称本发明的用途，或本发明的诊断和/或检测用途)及其中使用的组合物。本文中提供的任何抗GITR抗体均可以用于检测(定量或定性检测)生物样品中GITR的存在。例如通过免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)等检测生物样品中GITR的存在。在一些实施方案中，生物样品包括体液、细胞或组织。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。
在一个实施方案中，提供了抗GITR抗体用于诊断或检测方法。在进一步的方面中，提供了检测生物样品中GITR存在的方法。在一些实施方案中，该方法包括将生物样品在允许抗GITR抗体与GITR结合的条件下接触本文中所述的抗GITR抗体，并且检测抗GITR抗体和GITR之间是否形成了复合物。这样的方法可以是体外或体内方法。
在一个实施方案中，将抗GITR抗体用于选择适宜使用抗GITR抗体治疗的受试者，例如，当GITR是用于患者选择的生物标志物时。可以使用本发明的抗体诊断的示例性病症包括免疫障碍或再生性障碍。在一些实施方案中，提供了用本发明的抗体对免疫障碍或再生性障碍患者进行分层的方法。在一些实施方案中，抗GITR抗体是与诊断剂或可检测剂缀合的前述本发明任何免疫缀合物。在一些实施方案中，本发明提供用于诊断或检测的试剂盒，其包含本发明的GITR结合分子，例如本发明的任何抗GITR抗体。
在一个实施方案中，本发明提供一种检测样品中GITR的存在的方法，包括：
(a)将所述样品与本发明的分离的抗体或其抗原结合片段、本发明的融合蛋白或本发明的免疫缀合物接触；和
(b)检测所述抗体或其抗原结合片段、或融合蛋白或免疫缀合物和GITR蛋白之间复合物的形成。
另一方面，本发明提供本发明抗体的筛选、鉴定和表征方法。可以通过本领域已知的各种试验，针对其物理/化学特性和/或生物活性，筛选、鉴定、或表征本文中提供的抗GITR抗体。
可以从噬菌体/噬菌粒展示纳米抗体文库中选择与所关注的目标抗原以高亲合力结合的噬菌体/噬菌粒。已有多种在噬菌体/噬菌粒表面呈递或展示抗体或抗体片段以及筛选文库的方法。
对于抗体的鉴定，可以就其抗原结合活性，例如通过已知方法，如ELISA、αLISA、蛋白质印迹、抗体或反相阵列等、以及实施例中所述的方法，鉴定或表征本发明的抗体。
例如，可以采用ForteBio测定法检测抗体。ForteBio亲和力测定可以按照现有的方法(Estep,P等，High throughput solution Based measurement of antibody-antigen affinity and  epitope binning.MAbs,2013.5(2):第270-8页)进行。例如，可以将AHQ传感器在分析缓冲液中线下平衡30分钟，然后线上检测60秒建立基线。之后，将在线加载了经纯化的抗体的AHQ传感器，暴露于100nM抗原作用5分钟，将传感器转移至分析缓冲液进行5分钟的线下测量。使用1:1结合模型进行动力学分析。
另一方面，本发明提供一种治疗癌性病症、诱导或增强个体中的免疫应答，和/或刺激抗原特异性T细胞应答的方法，其特征在于包括向所述个体施用有效量的本发明的分离的抗体或其抗原结合片段、本发明的融合蛋白或本发明的免疫缀合物。
在一些实施方案中，免疫应答针对肿瘤抗原产生或针对感染性因子产生。
在一些实施方案中，本发明的上述方法可以治疗或预防“免疫病症”，例如包括病理性炎症、炎性病症和自身免疫性病症或疾病，也包括感染、持续感染和增生性病症，例如癌症、肿瘤和血管发生，包括抗免疫系统根除的感染、肿瘤和癌症。
在一些实施方案中，本发明的上述方法可以治疗或预防“免疫障碍”，其中免疫障碍指哺乳动物中由哺乳动物免疫系统成分引起、介导、或以其他方式促成发病的疾病；或刺激或干预免疫应答对疾病发展具有改善作用的疾病。“免疫障碍”包括自身免疫病、免疫介导的炎性疾病、非免疫介导的炎性疾病、传染病和免疫缺陷病。
可使用本发明抗体治疗的这些免疫相关疾病和炎性疾病的实例(其中有些是免疫或T细胞介导的)包括：系统性红斑狼疮、类风湿性关节炎、幼年型慢性关节炎、脊椎关节病、系统性硬化病(硬皮病)、特发性炎性肌病(皮肌炎、多肌炎)、干燥综合征、系统性血管炎、结节病、自身免疫性溶血性贫血(免疫性全血细胞减少症、阵发性夜间血红蛋白尿)、自身免疫性血小板减少症(特发性血小板减少性紫癜、免疫介导的血小板减少症)、甲状腺炎(格雷夫斯氏病、桥本甲状腺炎、幼年型淋巴细胞性甲状腺炎、萎缩性甲状腺炎)、糖尿病、免疫介导的肾病(肾小球肾炎、肾小管间质性肾炎)、中枢和周围神经系统的脱髓鞘病诸如多发性硬化病、特发性脱髓鞘多神经病或格-巴二氏(Guillain-Barre)综合征、和慢性炎性脱髓鞘多神经病、肝胆病诸如传染性肝炎(甲型、乙型、丙型、丁型、戊型和其他非嗜肝病毒肝炎)、自身免疫性慢性活动性肝炎、原发性胆汁性肝硬化、肉芽肿性肝炎、和硬化性胆管炎、炎性和纤维化肺病诸如炎性肠病(溃疡性结肠炎：克罗恩病)、麸胶敏感性肠病、和惠普尔氏(Whipple)病、自身免疫性或免疫介导的皮肤病包括大疱性皮肤病、多形红斑和接触性皮炎、银屑病、变应性疾病诸如哮喘、变应性鼻炎、特应性皮炎、食物超敏反应和荨麻疹、肺的免疫学疾病诸如嗜曙红细胞性肺炎、特发性肺纤维化和超敏感性肺炎、移植相关疾病包括移植物排斥和移植物抗宿主疾病。传染病包括AIDS(HIV感染)、甲型、乙型、丙型、丁型和戊型肝炎、细菌感染、真菌感染、原生动物感染和寄生虫感染。
在一些实施方案中，本发明的上述方法可以治疗或预防“癌性病症”，包括例如癌症、癌细胞、肿瘤、血管发生和癌变前病症，例如发育异常。
在一些实施方案中，癌症是转移性癌症、难治性癌症或复发性癌症，更优选是实体癌或血液癌；最优选是黑色素瘤、肺癌、头颈癌、结肠直肠癌、非小细胞肺癌、头颈部的鳞状细胞癌、膀胱癌、乳腺癌、子宫/子宫颈癌、卵巢癌、前列腺癌、睾丸癌、食道癌、胃肠癌、胰腺癌、结肠癌、肾癌、胃癌、生殖细胞癌、骨癌、肝癌、甲状腺癌、皮肤癌、中枢神经系统的新生物、淋巴瘤、白血病、骨髓瘤、肉瘤或病毒相关癌症。
在一些实施方案中，本发明抗体的有效量是在个体中实现以下一项或多项的量：a)减轻效应T细胞活性的调节性T细胞抑制；b)降低循环调节性T细胞的水平；c)活化效应T细胞；d)诱导或增强效应T细胞增殖；e)抑制肿瘤生长；f)诱导肿瘤消退；以及g)增加表达GITR的T细胞中的IL-2及/或IFN-γ产生、增加T细胞增殖。
在一些实施方案中，本发明的上述方法进一步包括以：a)施用化疗；b)施用放射疗法；和/或c)施用一种或多种另外的治疗剂，优选是免疫刺激剂。
在一些实施方案中，本发明的上述方法中的免疫刺激剂选自T-VEC、PD1拮抗剂、PDL1 拮抗剂、CTLA-4拮抗剂及BiTE。
在一些实施方案中，本发明的上述方法中的化疗、放射疗法或治疗剂是在所述抗体或其抗原结合片段之前、同时或之后施用。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1.抗GITR纳米抗体的筛选
1.骆驼免疫
将1mg抗原GITR-His(Acro biosystems)与弗氏佐剂(Sigma)等体积混合后平分于两个管子中，免疫两只新疆双峰驼。如此每周免疫一次，共免疫7次，刺激B淋巴细胞表达抗原特异性的纳米抗体。免疫七次之后，新疆双峰驼中产生的GITR-His纳米抗体的血清滴度可达到1:1000以上，由此确定骆驼中产生期望的纳米抗体。
2.噬菌体展示纳米抗体文库的构建
(1)7次免疫结束后，提取100ml骆驼血液，分离外周血淋巴细胞，提取总RNA；
(2)利用RT-PCR将步骤(1)中得到的总RNA反转录为cDNA，然后利用巢式PCR(两次PCR)扩增得到VHH，其扩增原理如图1所示；
(3)按照制造商的使用说明书，利用限制性内切酶PstI(NEB)及NotI(NEB)酶切20μg噬菌体展示载体及10μg VHH片段，然后利用T4连接酶(NEB)连接双酶切得到的两个片段；
(4)将连接产物电转化至感受态大肠杆菌细胞TG1中，构建纳米抗体文库。
3.抗GITR纳米抗体的筛选
(1)将溶解在100mM NaHCO 3(pH8.2)中的抗原蛋白GITR-His(Acro biosystems)偶联在酶标板(Nunc)上，4℃放置过夜；
(2)第二天加入100μl封闭液(5％BSA(Jackson))，室温封闭2小时；
(3)2小时后，加入100μl噬菌体(5×10 11PFU(plaque forming unit，噬菌体形成单位)上述构建的噬菌体展示纳米抗体文库)，室温下作用1小时；
(4)用PBST(PBS+0.05％Tween-20(Bio Basic Inc.,BBI))洗涤5次，以洗掉非结合的噬菌体；
(5)用100mM TEA(Sigma)洗脱液将与抗原蛋白GITR-His特异性结合的噬菌体解离下来，之后感染处于对数期生长的大肠杆菌TG1细胞。37℃培养1小时，扩大培养噬菌体用于下一轮的筛选；
(6)步骤(1)至(5)的筛选过程重复3轮，得到富集的与抗原蛋白GITR-His特异性结合的噬菌体。
4.PE-ELISA鉴定阳性克隆
(1)从含有上述3轮筛选后得到的噬菌体的细胞培养皿中，随机挑选1000个单菌落并接种于含有氨苄青霉属(Amresco)的TB培养基(2％胰蛋白胨、2.4％酵母提取物、72mM K 2HPO 4、17mM KH 2PO 4、0.4％甘油)中。菌落生长至对数期后，加入IPTG(Sigma)至终浓度为1mM，28℃培养过夜；
(2)利用常规渗透冲击法获得粗提抗体，并将粗提抗体转移到已过夜包被抗原GITR-His的酶标板(Nunc)中，在室温下孵育1小时；
(3)用PBST洗去未结合的抗体，加入小鼠抗-HA tag抗体(SinoBiological)，在室温下放置1小时；
(4)用PBST洗去未结合的抗体，加入山羊抗小鼠碱性磷酸酶标记抗体(Millipore)，在室温下放置1小时；
(5)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液(新赛美M30100)，于ELISA仪(Thermo(MULTISCAN FC))上，在405nm波长读取吸收值；当样品孔OD值大于对照孔OD值(Ratio:+/-)3倍以上时，判为阳性克隆孔；
(6)PE-ELISA(Post-Enrichment Enzyme-linked Immunosorbent Assay，富集后)结果共出现486颗阳性克隆，其比值(Ratio:+/-)在3-30之间，随后，将所有阳性克隆转至LB培养基中培养过夜，提取质粒并进行测序。
5.测序结果分析
根据序列比对软件Vector NTI分析各个克隆株的基因序列，将CDR1、CDR2、CDR3序列相同的株视为同一克隆株，而其序列不同的株视为不同克隆株。最终获得22株序列不同的纳米抗体，其ELISA结果如表1所示。
表1.纳米抗体Nb-01至Nb-22的ELISA结果
抗体编号 氨基酸SEQ ID NO 样品OD值(+) 对照OD值(-) 比率(+/-)
Nb-01 64 0.467 0.075 6.227
Nb-02 65 1.425 0.069 20.652
Nb-03 66 0.786 0.067 11.728
Nb-04 67 1.628 0.074 22.000
Nb-05 68 1.412 0.066 21.518
Nb-06 69 0.314 0.095 3.305
Nb-07 70 2.346 0.072 32.583
Nb-08 71 1.705 0.074 23.041
Nb-09 72 1.449 0.063 23.107
Nb-10 73 2.269 0.076 29.855
Nb-11 74 0.552 0.071 7.775
Nb-12 75 0.444 0.098 4.531
Nb-13 76 0.263 0.080 3.288
Nb-14 77 0.460 0.115 4.000
Nb-15 78 1.254 0.249 5.036
Nb-16 79 1.674 0.081 20.667
Nb-17 80 1.037 0.069 15.112
Nb-18 81 1.010 0.103 9.800
Nb-19 82 1.324 0.072 18.469
Nb-20 83 1.414 0.116 12.212
Nb-21 84 0.522 0.069 7.548
Nb-22 85 0.606 0.060 10.031
实施例2.重链抗体的构建及表达、纯化
1.构建重链抗体(hcIgG)：
(1)PCR扩增纳米抗体Nb-02、Nb-03、Nb-04、Nb-07、Nb-09、Nb-10、Nb-16、Nb-20的cDNA。
(2)胶回收获得各条PCR片段，通过同源重组的方法(Vazyme，C112-01/02)构建入HEK293表达载体pTT5(Biotechnology Research Institute；Montreal,Canada)中，其中已包含DNA序列如下的IgG1Fc片段：
Figure PCTCN2019079625-appb-000011
Figure PCTCN2019079625-appb-000012
(3)通过测序(金唯智公司)验证载体构建的正确性。
2.hcIgG蛋白的表达
(1)根据所需转染体积传代HEK293细胞(Invitrogen)，转染前一天将细胞密度调整至1×10 6/ml。转染当天细胞密度约为2×10 6/ml；
(2)取终体积1/10的F17(Gibco，A13835-01)培养基作为转染缓冲液，每毫升转染缓冲液加入10ug构建的HEK293表达载体pTT5，混匀；
(3)每毫升转染缓冲液加入30ug PEI(聚乙烯亚胺，Polysciences，23966)到质粒中，混匀后室温孵育10分钟。将混合物轻柔倒入HEK293细胞，36.5℃，8％CO 2培养；
(4)过夜后各补加转染体积1/50的200g/L的FEED(Sigma，H6784-100G)和200g/L的葡萄糖母溶液，20小时后加VPA(丙戊酸Gibco，11140-050)至2mM/L；
(5)连续培养至第7天时，离心收集细胞上清用以纯化。
3.hcIgG蛋白纯化
通过HiTrapTM MabSelect SuReTM 5ml(GE Healthcare)纯化目的hcIgG蛋白。具体过程如下：
(1)用0.1M NaOH将AKTA蛋白纯化系统除去内毒(过夜)。收样当天将上述细胞上清7500转/分钟离心30分钟，使用SARTOPORE(Sartorius，5441307H4)过滤。纯化前用5倍柱体积的结合缓冲液(Tris 20mM，NaCl 150mM，pH 7.2)清洗系统以及平衡柱子。将上述离心后得到的细胞上清通过柱子。用5～10倍柱体积结合缓冲液再平衡，至基线平衡。用洗脱缓冲液(柠檬酸+柠檬酸钠100mM，pH 3.5)洗脱抗体，根据紫外吸收值来收集样品。每1ml的收集液加80μl的中和缓冲液(Tris-HCl 2M)中和备用。
(2)纯化后的蛋白利用SECC-HPLC(分子排阻高效液相色谱法)检测纯度，结果如图2所示，所有hcIgG经一步亲和纯化后纯度均>97％。
得到重链抗体hcIgG-02、hcIgG-03、hcIgG-04、hcIgG-07、hcIgG-09、hcIgG-10、hcIgG-16和hcIgG-20，其具体序列示于表B中。
实施例3.重链抗体的性质鉴定
1.FortiBio KD测定
本实验使用Fortebio方法(Estep,P等，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):第270-278页)测定抗原-抗体结合动力学及亲和力，GITR Inc.的抗GITR抗体作为阳性对照(在本申请中称为TRX518，根据US20130183321A1提供的序列进行克隆，轻链和重链的序列分别为US20130183321A1中的SEQ ID NO:44，和SEQ ID NO:54，瞬时转染HEK293细胞进行表达获得)。具体测定方法如下：
(1)准备传感器：实验开始前半个小时，将AHQ传感器(Pall，1506091)浸泡于SD缓冲液(PBS 1×，BSA 0.1％，Tween-20 0.05％)中；
(2)实验过程：取100μl的SD buffer、抗体(hcIgG-02、hcIgG-03、hcIgG-04、hcIgG-07、hcIgG-09、hcIgG-10、hcIgG-16和hcIgG-20，对照抗体)、GITR-His(Acro biosystems)分别加入到96孔黑色聚苯乙烯半量微孔板中。根据样品位置布板，选择传感器位置，设置运行步骤及时间：Baseline、Loading～1nm、Baseline、Association和Dissociation时间取决于样品结合、解离速度，转速为400转/分钟，温度为30℃。
(3)使用1:1结合模型进行动力学分析，发现本发明抗体(hcIgG-02、hcIgG-03、hcIgG-04、hcIgG-07、hcIgG-09、hcIgG-10、hcIgG-16和hcIgG-20)均具有比对照抗体TRX518更高的亲和力，结果如表2所示。
表2.hcIgG亲和力测定结果
Figure PCTCN2019079625-appb-000013
2.检测hcIgG和细胞表面的抗原结合情况
(1)细胞准备：将携带克隆至多克隆位点MCS的人GITR cDNA的pCHO1.0载体(Invitrogen)转染至中国仓鼠卵巢癌细胞(CHO)(Invitrogen)，产生过量表达人GITR的CHO细胞(CHO-GITR)，将由此构建的表面过表达人GITR的CHO细胞(CHO-GITR)计数，并稀释至1×10 6个细胞/ml，向U型底96孔板中加入100μl/孔；
(1)检测步骤：400g离心5分钟，去除细胞培养基。将梯度稀释的样品加入U型板并重悬细胞，每孔加100μl，冰上静置30分钟。400g 5分钟去除上清，PBS洗细胞1遍。400g离心5分钟，去除PBS，每孔加入100μl 1:200稀释的PE-抗人Fc抗体(Jackson Immuno Research)。冰上避光孵育30分钟。400g离心5分钟去除上清，PBS洗细胞1遍。用100μl PBS重悬细胞。
(3)FACS检测。检测结果如图3所示，所有测定的hcIgG(hcIgG-02、hcIgG-03、hcIgG-04、hcIgG-07、hcIgG-09、hcIgG-10、hcIgG-16和hcIgG-20)均可以和细胞表面表达的抗原相结合。
3.差示扫描荧光检测抗体T m
差示扫描荧光法(DSF)可根据图谱中的荧光变化过程提供有关结构稳定性的信息，检测蛋白的构型变化。荧光曲线绝对值最大时对应的温度为该蛋白的T m。本研究利用DSF法，测定了抗GITR hcIgG的T m值，用PBS稀释抗体(hcIgG-02、hcIgG-03、hcIgG-04、hcIgG-07、hcIgG-09、hcIgG-10、hcIgG-16和hcIgG-20)样品至1mg/ml。用PBS将SYPRO Orange protein gel stain(GIBCO)稀释50倍，即4μl SYPRO Orange protein gel stain母液加196μl PBS。在96孔PCR板中加样，50μl稀释后抗体样品+10μl SYPRO Orange protein gel stain稀释液+40μl水。置于7500real time PCR system进行检测，结果如表3所示。
表3.hcIgG Tm值测定结果
Figure PCTCN2019079625-appb-000014
Figure PCTCN2019079625-appb-000015
检测hcIgG介导的ADCC作用
本实验利用Promega公司的ADCC Report Bioassay试剂盒对hcIgG介导的抗体依赖的(细胞介导的)细胞毒性作用(ADCC)作用进行了检测。所用的效应细胞为稳定转染NFAT-Luciferase报告基因的Jurkat细胞，靶细胞为过表达GITR的CHO细胞(CHO-GITR)。具体实验过程如下：
用ADCC Assay Buffer将靶细胞稀释至2.5×10 5个/ml，每孔加入50μl靶细胞；
加入25μl梯度稀释的抗体hcIgG-02、hcIgG-03、hcIgG-04、hcIgG-07、hcIgG-09、hcIgG-10、hcIgG-16和hcIgG-20(每种抗体的终浓度为3.33μg/ml、1.11μg/ml、0.37μg/ml、0.12μg/ml、0.041μg/ml、0.013μg/ml、0.0046μg/ml、0.0015μg/ml)；
用ADCC Assay Buffer将效应细胞稀释至1.5×10 6个/ml，每孔加入25μl；
37℃反应6小时；之后每孔加入等体积的室温Bio-Glo Luciferase Reagent(Promega)，室温孵育10分钟。
GloMax Multi+Plate Reader读数。结果如图4所示。从图中曲线趋势可以看出，本发明的hcIgG(hcIgG-02、hcIgG-03、hcIgG-04、hcIgG-07、hcIgG-09、hcIgG-10、hcIgG-16和hcIgG-20)均能够引起ADCC作用。
实施例4.抗GITR的重链抗体的人源化
1.hcIgG人源化改造
选择hcIgG-07和hcIgG-20进行人源化改造，人源化过程按照已有方法(Ce′cile Vincke等，General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold.JOURNAL OF BIOLOGICAL CHEMISTRY,2009.5(284):第3273-3284页)进行，得到人源化抗体HzhcIgG-07和HzhcIgG-20(具体序列参见表B)。
2.FortiBio KD测定
本实验使用Fortebio方法测定人源化之前的抗体hcIgG-20和人源化之后的抗体HzhcIgG-20的结合动力学及亲和力，方法同实施例3。ForteBio结果如表4。hcIgG-20和HzhcIgG-20维持了和GITR的结合能力，且亲和力高于对照抗体TRX518。
表4.hcIgG-20和HzhcIgG亲和力测定结果
Figure PCTCN2019079625-appb-000016
3.检测hcIgG和细胞表面的抗原结合情况
检测方法同实施例4。流式细胞仪检测细胞结合结果如图5。hcIgG-20和HzhcIgG-20均保持了细胞结合活性(EC50从1.631nM到1.873nM)。
4.检测hcIgG对GITR介导的信号通路的激活作用
野生型Hela细胞通过工程改造后，表面过表达GITR蛋白。同时将表达NFkB结合位点 和荧光报告素酶(Luciferase)表达基因的质粒转入Hela-GITR细胞中获得稳定的、同时表达GITR及NFkB荧光报告素酶基因的检测用细胞株Hela-GITR-NFkB-Luc-Rep。本实验利用该细胞检测了hcIgG-20和HzhcIgG-20对GITR下游的NFkB信号通路的激活作用。具体实验过程如下：
将表面过表达Hela-GITR-NFkB-Luc-Rep细胞调节细胞密度至1×10 6个/ml，第一孔加入150μl细胞悬液，其他孔100μl，第一孔加入抗体，终浓度500nM，吸取50μl细胞悬液入下一孔，混匀，以此类推(抗体稀释终浓度为500nM、166.67nM、55.56nM、18.52nM、6.17nM、2.06nM、0.69nM、0.23nM、0.08nM、0.025nM)，置于二氧化碳培养箱，过夜孵育。
室温放置10分钟后，每孔加入80μl的室温Bio-Glo Luciferase Reagent(Promega)，室温孵育5分钟。
GloMax Multi+Plate Reader读数。流式细胞仪检测细胞结合结果如图6。HzhcIgG-20对NFkB信号通路有激活作用，hcIgG-20、HzhcIgG-20及对照抗体TRX518的激活EC50分别为0.6894nM、0.6632nM和2.097nM，hcIgG-20、HzhcIgG-20均具有比对照抗体更优的NFkB通路激活能力。
实施例5.抗GITR 4xNb-IgG的构建及活性检测
1.构建4xNb-IgG分子
天然存在的单克隆抗体可以作为多聚体结构如IgM存在，其增加对固定表面上的其靶抗原的亲合性。因为TNFR的激动作用需要受体聚集，所以四价或六价单克隆抗体可以提供增加的聚集和受体激动作用。本发明的多聚物形式的抗体包含四个相同的纳米抗体区域，其具体构建方法如下：
4xNb-IgG-I：HzhcIgG-20的VHH部分(即HzhcIgG-20中的重链可变区VH)通过GGGGS的柔性连接肽连接到HzhcIgG-20的C末端(如图7A)；
4xNb-IgG-II：HzhcIgG-20的VHH部分通过GGGGS的柔性连接肽连接到HzhcIgG-20的N末端(如图7B)；
4xNb-IgG-III：HzhcIgG-20的VHH部分直接连接到HzhcIgG-20的N末端(如图7C)。
其中4xNb-IgG-I、4xNb-IgG-II和4xNb-IgG-III的具体序列参见表B。
将构建体通过常规方法克隆到HEK293细胞的表达载体pTT5.1的XhoI/NotI多克隆位点中。将构建好的质粒转染HEK293细胞，筛选表达和分泌的4xNb-IgG分子，并选择正确的分子用于进一步的实验。
2. 4xNb-IgG蛋白的表达及纯化
4xNb-IgG的表达及纯化方法同实施例2。纯化后的蛋白利用SEC检测纯度，结果如图8所示，4xNb-IgG-I、4xNb-IgG-II和4xNb-IgG-III经一步亲和纯化后纯度分别为97.5％、96.86％和97.21％。
3.FortiBio KD测定
本实验使用Fortebio方法测定抗原-抗体结合动力学及亲和力，方法同实施例4。ForteBio结果如表5所示。
表5. 4xNb-IgG的亲和力检测
Figure PCTCN2019079625-appb-000017
4.检测4xNb-IgG对GITR介导的信号通路的激活作用
检测方法和实施例4基本类似，但是进行了优化，将抗体浓度降低，增加检测的灵敏度。
实验过程如下：
将表面过表达Hela-GITR-NFkB-Luc-Rep细胞调节细胞密度至5×10 5个/ml，第一孔加入200μl细胞悬液，其他孔100μl，第一孔加入抗体，终浓度10nM，吸取100μl细胞悬液入下一孔，以此类推(抗体稀释终浓度为10nM、5.00nM、2.50nM、1.25nM、0.63nM、0.31nM、0.16nM、0.08nM、0.04nM、0.02nM、0.01nM)，置于二氧化碳培养箱，孵育7h。
室温放置10分钟，每孔加入80μl的室温Bio-Glo Luciferase Reagent(Promega)，室温孵育5分钟。
GloMax Multi+Plate Reader读数。结果如图9所示，4xNb-IgG-I、4xNb-IgG-II和4xNb-IgG-III均显示出比阳性对照更强的激活活性。
5.本发明抗GITR抗体的抗肿瘤活性
利用MC38移植瘤小鼠模型研究了抗人GITR抗体4xNb-IgG-III单独使用，或与抗鼠PD-1抗体“Antibody C”(WO2017/133540)联合使用的抗肿瘤活性。
小鼠：雄性人GITR转基因小鼠(约8周大)购自百奥赛图基因生物技术有限公司。小鼠在到达后驯化7天，随后开始研究。
细胞：小鼠结肠癌细胞MC38(ATCC)购自ATCC，并严格按照ATCC要求进行常规传代培养用于后续体内实验。离心收集细胞，在无菌PBS中重悬细胞并调整细胞密度为5×10 6个/ml。在第0天取0.2ml细胞悬液皮下接种至人GITR转基因小鼠右侧腹部区域中来建立荷瘤小鼠模型。
给药：将小鼠分为四组(每组8只小鼠)，每组皮下注射如下剂量的抗体：
(1)小鼠IgG对照(equitech-Bio)，10mg/kg；
(2)PD-1(Antibody C),1mg/kg；
(3)4xNb-IgG-III,10mg/kg；
(4)4xNb-IgG-III，10mg/kg PBS+PD-1(Antibody C),1mg/kg。
试剂注射：接种后第8天，将符合实验要求的小鼠随机分组，每组6只。分别用如上四组试剂在第8天、第11天、第14天和第17天为每组小鼠按如上剂量给药。
分析：在整个研究期间每周测量两次肿瘤和体重，当肿瘤达到端点时或当小鼠具有>20％体重减轻时，使小鼠安乐死。采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。将来自每组的小鼠的肿瘤尺寸与时间作图。使用方差分析(ANOVA)来确定统计显著性。P＜0.05的值被视为在所有分析中具有统计显著性。
实验结果见图10，可见:
-本发明的抗GITR抗体4xNb-IgG-III与IgG对照(equitech-Bio)相比，能显著抑制肿瘤的生长；
-本发明的抗GITR抗体4xNb-IgG-III和抗PD-1单克隆抗体“Antibody C”联合使用与IgG及这两个抗体分别使用相比，能显著抑制肿瘤的生长。

Claims (16)

  1. 一种特异性结合糖皮质激素诱导的肿瘤坏死因子受体(GITR)的纳米抗体，其特征在于其中的HCDR3包含下述序列(a)-(c)之一或由下述序列(a)-(c)之一组成:
    (a)如SEQ ID NO:64-85中任一序列所示的重链可变区序列所含的HCDR3序列；或
    (b)与如SEQ ID NO:64-85中任一序列所示的重链可变区序列具有至少90％序列同一性的重链可变区序列所含的HCDR3序列，其中优选序列差异不存在于CDR区域中；或
    (c)在如SEQ ID NO:64-85中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR3序列，其中优选所述氨基酸改变不发生在CDR区域中。
  2. 权利要求1的纳米抗体，其特征在于所述HCDR3包含下述序列(a)-(c)之一或由下述序列(a)-(c)之一组成:
    (a)如SEQ ID NO:45-63中任一序列所示的HCDR3序列；或
    (b)在如SEQ ID NO:45-63中任一序列所示的HCDR3中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的HCDR3序列。
  3. 一种特异性结合糖皮质激素诱导的肿瘤坏死因子受体(GITR)的重链抗体，其特征在于所述重链抗体中的HCDR3包含下述序列(a)-(c)之一或由下述序列(a)-(c)之一组成:
    (a)如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列所含的HCDR3；或
    (b)与如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列具有至少90％序列同一性的重链可变区序列所含的HCDR3，其中优选序列差异不存在于CDR区域中；或
    (c)在如SEQ ID NO:65、66、67、70、72、73、79和83中任一序列所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR3，其中优选所述氨基酸改变不发生在CDR区域中，
    优选所述抗体是人源化重链抗体，更优选是全人源重链抗体。
  4. 权利要求3的重链抗体，其特征在于所述重链抗体是人源化重链抗体，其中所述人源化重链抗体中的HCDR3包含下述序列(a)-(c)之一或由下述序列(a)-(c)之一组成:
    (a)如SEQ ID NO:124或125所示的重链可变区序列所含的HCDR3序列；或
    (b)与如SEQ ID NO:124或125所示的重链可变区序列具有至少90％序列同一性的重链可变区序列所含的HCDR3序列，其中优选序列差异不存在于CDR区域中；或
    (c)在如SEQ ID NO:124或125所示的重链可变区序列中的一处或多处(优选1-10个，更优选1-5个，最优选1-3个)具有氨基酸改变(优选取代、更优选保守取代)的重链可变区序列中所含的HCDR3序列，其中优选所述氨基酸改变不发生在CDR区域中。
  5. 权利要求1的抗体或其抗原结合片段，其特征在于所述抗体是多聚物形式的抗体，优选是权利要求4的人源化重链抗体的重链可变区的多聚物形式，更优选是权利要求4的人源化重链抗体的重链可变区的四聚化形式或六聚化形式，最优选是权利要求4的人源化重链抗体的重链可变区的四聚化形式。
  6. 一种融合蛋白，其包含权利要求1-5任一项的抗体或其抗原结合片段，和第二分子。
  7. 一种免疫缀合物、组合物或试剂盒，其包含权利要求1-5中任一项的抗体或其抗原结合片段，任选包括载体和/或说明书。
  8. 分离的核酸，其编码权利要求1-5中任一项的分离的抗体或其抗原结合片段、权利要求6的融合蛋白或权利要求7的免疫缀合物。
  9. 包含权利要求8的核酸的载体，优选所述载体是表达载体。
  10. 包含权利要求9的载体的宿主细胞，优选地，所述宿主细胞选自酵母细胞、哺乳动物细胞、免疫效应细胞(如T细胞)。
  11. 制备权利要求1至5中任一项的分离的抗体或其抗原结合片段、权利要求6的融合蛋白或权利要求7的免疫缀合物的方法，包括在适于表达所述抗体或其抗原结合片段、融合蛋白或免疫缀合物的条件下，培养权利要求10的宿主细胞。
  12. 药物组合物，其包含权利要求1至5中任一项的分离的抗体或其抗原结合片段、权利要求6的融合蛋白或权利要求7的免疫缀合物，以及任选的药用载体。
  13. 一种检测样品中GITR的存在的方法，包括：
    (a)将所述样品与权利要求1至5中任一项的分离的抗体或其抗原结合片段、权利要求6的融合蛋白或权利要求7的免疫缀合物接触；和
    (b)检测所述抗体或其抗原结合片段、或融合蛋白或免疫缀合物和GITR蛋白之间复合物的形成。
  14. 一种治疗癌症、诱导或增强个体中的免疫应答，和/或刺激抗原特异性T细胞应答的方法，其特征在于包括向所述个体施用有效量的权利要求1-5中任一项的分离的抗体或其抗原结合片段、权利要求6的融合蛋白或权利要求7的免疫缀合物。
  15. 一种分离的特异性结合糖皮质激素诱导的肿瘤坏死因子受体(GITR)的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段具有以下一种或多种特性：
    (i)以高亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合，例如以小于50nM、小于30nM、小于25nM或小于20nM的亲和力，优选以小于10nM的亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合；
    (ii)以高亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合，例如以大于0.01 x 10 4/Ms、大于0.1 x 10 4/Ms、大于1 x 10 4/Ms或大于3 x 10 4/Ms的结合常数，优选以大于5 x10 4/Ms的结合常数与人GITR(如SEQ ID NO:123或127的多肽)结合；
    (iii)以低解离常数与人GITR(如SEQ ID NO:123或127的多肽)结合，例如与人GITR(如SEQ ID NO:123或127的多肽)结合的解离常数(K d)小于2×10 -2s -1、小于1.5×10 -2s -1、小于8×10 -3s -1或小于5×10 -3 s -1；优选与人GITR(如SEQ ID NO:123或127的多肽)结合的解离常数为约1-3×10 -3 s -1；
    (iv)与细胞表面的抗原高亲和力结合，例如以小于50nM、小于30nM、小于25nM或小于20nM的亲和力，优选以小于10nM的高亲和力与人GITR(如SEQ ID NO:123或127的多肽)结合；
    (v)高效激活GITR下游的NFkB信号通路，例如激活NFkB信号通路的EC 50值小于50nM、小于30nM、小于10-25nM或小于5nM，优选小于1nM。
  16. 权利要求15的抗体或其抗原结合片段，其特征在于所述抗体是
    (a)双抗体、单链抗体(scFv)、单域抗体(sdAd)、重链抗体(hcAb)、纳米抗体(Nb或VHH)；或这些抗体的多聚物形式；和/或
    (b)人抗体、人源化抗体或嵌合抗体，其中所述Fab、单链抗体、单域抗体、重链抗体、纳米抗体；或这些抗体的人源化形式、全人源形式或多聚物形式包含权利要求1-5或15中任一项的抗体的HCDR3，优选还包含权利要求1-5或15中任一项的抗体的HCDR1和HCDR2。
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