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WO2020020281A1 - 抗tigit抗体及其用途 - Google Patents

抗tigit抗体及其用途 Download PDF

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Publication number
WO2020020281A1
WO2020020281A1 PCT/CN2019/097665 CN2019097665W WO2020020281A1 WO 2020020281 A1 WO2020020281 A1 WO 2020020281A1 CN 2019097665 W CN2019097665 W CN 2019097665W WO 2020020281 A1 WO2020020281 A1 WO 2020020281A1
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amino acid
seq
acid sequence
antibody
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PCT/CN2019/097665
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English (en)
French (fr)
Inventor
史新震
张攀
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 US16/965,687 priority Critical patent/US11370837B2/en
Priority to AU2019310803A priority patent/AU2019310803B2/en
Priority to BR112021001201-0A priority patent/BR112021001201A2/pt
Priority to CA3092456A priority patent/CA3092456C/en
Priority to CN201980022679.5A priority patent/CN112135626B/zh
Priority to EP19842191.9A priority patent/EP3838289A4/en
Application filed by 信达生物制药(苏州)有限公司 filed Critical 信达生物制药(苏州)有限公司
Priority to KR1020207028253A priority patent/KR20210040827A/ko
Priority to SG11202100746WA priority patent/SG11202100746WA/en
Priority to JP2020552889A priority patent/JP7524065B2/ja
Publication of WO2020020281A1 publication Critical patent/WO2020020281A1/zh
Priority to ZA2021/00676A priority patent/ZA202100676B/en
Priority to JP2024027487A priority patent/JP2024063083A/ja

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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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Definitions

  • the present invention relates to novel antibodies and antibody fragments that specifically bind TIGIT and compositions containing said antibodies or antibody fragments.
  • the present invention relates to a nucleic acid encoding the antibody or an antibody fragment thereof, a host cell comprising the same, and related uses.
  • the invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments.
  • TIGIT Ig and ITIM domain-containing T cell immune receptors, also known as WUCAM, Vstm3, or Vsig9 was originally discovered as a member of the CD28 family using bioinformatics alignment methods.
  • TIGIT is a cosuppressive receptor expressed on the surface of a variety of immune cells (natural killer cells / activated T cells / memory T cells / regulatory T cells / follicular T helper cells, etc.). A member of the protein superfamily.
  • TIGIT may play a regulatory role on the immune system through the following three mechanisms: 1) TIGIT competitively binds to a co-stimulatory receptor CD226 and is expressed on the surface of dendritic cells or cancer cells, the shared ligand CD155 / CD112, Passing inhibitory signals to TIGIT-expressing cells to inhibit the activation of such cells; 2) TIGIT directly interacts with the costimulatory receptor CD226, destroying homodimerization of CD226 and blocking its downstream activation signal; 3) TIGIT After binding to CD155 expressed in dendritic cells, the intracellular ITIM sequence conducts inhibitory signals and then expresses immunosuppressive cytokines to suppress the activation of the immune system.
  • TIGIT may play a role in a variety of cells in the tumor microenvironment. These cells may be tumor infiltrating CD8 positive T cells, they may be regulatory T cells, or they may be NK cells. Overall, numerous studies have shown that the first mechanism is probably the most critical mechanism by which TIGIT exerts its immunosuppressive effects.
  • Antibodies that bind human TIGIT have been shown to be useful in the treatment of cancer. See, for example, WO 2006/124667.
  • antibody blocking of both PD-L1 and TIGIT can lead to a synergistic increase in CD8 + T cell-mediated tumor rejection.
  • TIGIT is considered an attractive target for cancer immunotherapy. Therefore, there is a need in the art to develop new TIGIT antibodies, especially CD155 / CD112 blocking antibodies that target TIGIT, especially human anti-TIGIT antibodies, and combinations thereof for the treatment of diseases, especially cancer treatment.
  • the invention provides an anti-TIGIT antibody and a coding gene and application thereof.
  • the present inventors screened fully human antibodies against human TIGIT from the human antibody library displayed on the surface of yeast, and further obtained high-affinity anti-human TIGIT antibodies matured on this basis.
  • the fully human antibody molecule of the present invention can effectively block the binding of TIGIT with its ligand CD155, reduce or eliminate inhibitory signals transmitted to cells, increase IL-2 production, and inhibit tumor growth when administered in vivo, especially in When combined with anti-PD-1 antibodies, the tumor suppressive effect is particularly significant. Therefore, the antibodies of the present invention can be used for a variety of purposes, including but not limited to enhancing immune response, inhibiting tumor growth, anti-infection, and detecting TIGIT protein.
  • the present invention therefore provides a novel fully human antibody that binds human TIGIT, and an antigen-binding fragment thereof.
  • an anti-TIGIT antibody of the invention has one or more of the following characteristics:
  • the present invention provides an antibody or antigen-binding fragment thereof that binds TIGIT, comprising: HCDR1, 2 and 3 sequences of one of the heavy chain variable regions shown in SEQ ID NO: 84-103, and / or SEQ ID NO: 104-110 LCDR1, 2 and 3 sequences of the light chain variable region, or a variant of the CDR sequence combination.
  • the invention also provides an anti-TIGIT antibody or antigen-binding fragment thereof that binds to an exemplary antibody of the invention (such as an antibody having a combination of the VH and VL sequences of the antibodies listed in Table B) to the same or overlapping tables Position and / or competition binds TIGIT, and / or inhibits (eg, competitively inhibits) an exemplary antibody of the invention.
  • an exemplary antibody of the invention such as an antibody having a combination of the VH and VL sequences of the antibodies listed in Table B
  • Position and / or competition binds TIGIT, and / or inhibits (eg, competitively inhibits) an exemplary antibody of the invention.
  • the invention provides a nucleic acid encoding an antibody of the invention or an antigen-binding fragment thereof, a vector comprising the nucleic acid, and a host cell comprising the vector.
  • the invention provides methods for making an antibody or antigen-binding fragment thereof of the invention.
  • the invention provides immunoconjugates, pharmaceutical compositions and combination products comprising an antibody of the invention.
  • the invention also provides methods of using the antibodies of the invention to block the binding of TIGIT to CD155 (eg, CD155 expressed on the surface of dendritic cells or cancer cells) in a subject.
  • TIGIT-expressing cells especially T cells, natural killer cells
  • NK cell activation In some embodiments, the production of IL-2 in T cells is increased by the method.
  • CD155-expressing cells e.g., dendritic cells
  • activation of the immune system is promoted by the method. Therefore, the present invention also provides a method for preventing or treating cancer or infection using the antibody of the present invention.
  • the invention also relates to a method for detecting TIGIT in a sample.
  • Figures 1A-F show the binding of yeast-expressed antibodies to human TIGIT expressed on the surface of CHO cells before and after affinity maturation.
  • Figure 2 shows the binding of seven candidate antibodies expressed by CHO cells to human TIGIT expressed on the surface of CHO cells.
  • Figures 3A-F show the ability of yeast-expressed antibodies to block CD155 before and after affinity maturation.
  • Figure 4 shows the blocking ability of CD155 by 7 candidate antibodies expressed by CHO cells.
  • FIGS 5A-E show the detection of MOA biological activity of antibodies expressed by yeast before and after affinity maturation
  • Figure 6 shows the detection of MOA biological activity of 7 candidate antibodies expressed by CHO cells
  • Figure 7 shows the pharmacodynamic study of candidate molecules ADI-30293 and ADI-30278 administered alone (10 mg / kg) in a human TIGIT knock-in-MC38 mouse model.
  • Figures 8A-B show the pharmacodynamics of the candidate molecules ADI-30293 and ADI-30278 in combination with the anti-PD1 antibody antigen C (WO2017133540A1) (10 + 1 mg / kg) in a human TIGIT knock-in-MC38 mouse model .
  • FIG. 9 shows an exemplary human TIGIT sequence used in an embodiment of the present invention.
  • the term “comprising” or “including” means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps.
  • the terms “comprising” or “including” are used, unless otherwise indicated, the case of consisting of the mentioned elements, integers, or steps is also covered.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region consisting of that particular sequence.
  • antibody refers to a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an antigen.
  • the term encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, or Chimeric antibodies or humanized antibodies, full-length antibodies, and antibody fragments are sufficient as long as they exhibit the desired antigen-binding activity.
  • “whole antibodies” (which are used interchangeably herein with “full-length antibodies”, “full antibodies” and “full antibodies”) comprise at least two heavy chains (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.
  • 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.
  • the light chain constant region consists of a domain CL.
  • a variable region is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to its antigen.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but displays a variety of effector functions.
  • the light chain of an antibody can be classified into one of two types (called kappa ( ⁇ ) and lambda ( ⁇ )) based on the amino acid sequence of its constant domain.
  • the heavy chain of an antibody can be divided into five main types depending on the amino acid sequence of the constant region of its heavy chain: 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 the five different antibody types are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the term "isotype" refers to the type of antibody determined by the constant region of the antibody heavy chain. See, for example, Fundamental Immunology, Ch. 7 (Editor Paul, W., Second Edition, Raven Press, N.Y. (1989)) (which is incorporated herein by reference in its entirety for all purposes).
  • antigen-binding portion of an antibody refers to a molecule that is not an intact antibody, which contains the intact antibody used to bind the intact antibody Part of the antigen.
  • the antigen-binding portion of an antibody typically contains amino acid residues from a "complementarity determining region" or "CDR".
  • Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of whole antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab ', F (ab') 2 , Fab'-SH, Fv, single-chain Fv, double-chain antibody (diabody), triple-chain antibody (triabody), four-chain antibody ( tetrabody), minibody, single domain antibody (sdAb).
  • human antibody or “fully human antibody” are used interchangeably herein and refer to antibodies that include variable regions 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 a human germline immunoglobulin sequence.
  • the human antibodies of the invention may include amino acid sequences that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or point-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDRs-particularly CDR3.
  • the term "human antibody” does not include antibodies in which the CDR sequences are derived from the germline of other mammalian species (eg, mice) and transplanted into human framework sequences.
  • recombinant human antibody includes all human antibodies that have been produced, expressed, produced or isolated by recombinant means, for example, (a) an animal (e.g., a mouse) that has been transgenic or transchromosomic with a human immunoglobulin gene or Antibodies isolated from hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed into human antibody expression such as transfected tumors, (c) antibodies isolated from recombinant, combined human antibody libraries such as yeast display libraries, and ( d) An antibody prepared, expressed, produced or isolated by any other means, including splicing of human immunoglobulin genes to other DNA sequences.
  • recombinant human antibodies have framework regions and CDR regions that are derived from variable regions of human germline immunoglobulin sequences.
  • the recombinant human antibody can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using human Ig sequence transgenic animals), and the amino acid sequences of the VH and VL regions of the recombinant antibody thus obtained, although derived from and related to human germline VH and VL sequences, they are not naturally found in human antibody germline libraries in vivo.
  • chimeric antibody refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species. .
  • humanized antibody refers to an antibody that attaches CDR sequences derived from other mammalian species such as mouse germline to human framework sequences. Additional framework region modifications can be made within the human framework sequence.
  • an “isolated” antibody is one that has been separated from components in its natural environment.
  • the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase Phase HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reversed-phase Phase HPLC
  • epitope refers to the region of an antigen to which an antibody binds.
  • An epitope can be formed from consecutive amino acids or discontinuous amino acids juxtaposed by the tertiary folding of a protein.
  • TIGIT refers to "T-cell immune receptors containing Ig and ITIM domains.” The expression also includes variants, isotypes, homologs, and species homologs of TIGIT.
  • human TIGIT refers to the human sequence TIGIT. A specific human TIGIT sequence is shown in SEQ ID NO: 208. In some embodiments, the human TIGIT sequence has at least 95%, even at least 96%, 97%, 98%, or 99% amino acid sequence identity with the human TIGIT amino acid sequence of SEQ ID NO: 208.
  • the TIGIT protein may also include fragments of TIGIT, such as fragments containing an extracellular domain, for example, fragments that retain the ability to bind to any antibody of the invention.
  • CD155 also known as PVR (Poliovirus Receptor); PVS; HVED; CD155; NECL5; TAGE4; Necl-5, which interacts with TIGIT to induce immunosuppressive signals.
  • the term "specifically binds” means that an antibody selectively or preferentially binds an antigen. If in the biological light interferometry, the antibody is about 5x10 -7 M or lower, about 1x10 -7 M or lower, about 5x10 -8 M or lower, about 1x10 -8 M or lower, about 5x10 -9 M or lower K D , which binds to human TIGIT, the antibody is an antibody that "specifically binds to human TIGIT".
  • antibodies that specifically bind human TIGIT can be cross-reactive with TIGIT proteins from other species. For example, antibodies specific for human TIGIT, in some embodiments, can cross-react with TIGIT proteins of non-human species. In other embodiments, human TIGIT-specific antibodies may be completely specific to human TIGIT without showing species or other types of cross-reactivity, or showing only cross-reactivity to TIGIT of certain species.
  • cross-reactivity refers to the ability of an antibody to bind TIGIT from different species.
  • the human TIGIT-binding antibodies described herein can also bind TIGIT from other species (eg, monkey and / mouse TIGIT).
  • Methods for determining cross-reactivity include the methods described in the examples as well as standard assays known in the art, such as by using biological light interference, or flow cytometry techniques.
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on, respectively ).
  • K D equilibrium dissociation constant
  • k dis and k on, respectively association rate constant
  • Affinity can be measured by common methods known in the art.
  • One specific method for measuring affinity is the ForteBio kinetic binding assay herein.
  • high affinity for an IgG antibody refers to the antibody 1x10 -7 M or less, preferably 5x10 -8 M or less, more preferably about 1x10 -8 M or less, even more preferably from about 5x10 - 9 M or lower K D , which binds to the target antigen.
  • “high affinity” binding can vary with antibody isotype.
  • the IgM isotype “high affinity” refers to an antibody having 1x10 -6 M or lower, preferably 1x10 -7 M or less, more preferably about 1x10 -8 M or less K D.
  • a “competitively bound antibody” to a reference antibody that binds an antigen such as TIGIT refers to an antibody that blocks 50% or more of the binding of the reference antibody to an antigen (such as TIGIT) in a competition test, and in turn, A reference antibody blocks 50% or more of the antibody's binding to an antigen (eg, TIGIT) in a competition test.
  • Exemplary competition tests are described in: "Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • Competitively bound antibodies may bind the same epitope region as the reference antibody, such as the same epitope, adjacent epitopes, or overlapping epitopes.
  • An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90% or 95% .
  • the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more.
  • the binding of an antibody to its antigen can be measured by affinity (eg, equilibrium dissociation constant). Methods for determining affinity are known in the art.
  • An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the reference antibody's binding Affinity and / or specificity. This can be determined by any method known in the art to determine binding affinity and / or specificity.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region.
  • the term includes natural 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 the 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 Institute of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • variant in relation to an antibody means herein that, as compared to a reference antibody, it has passed at least one, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or An antibody having a target antibody region of amino acid change with 5 amino acid substitutions, deletions and / or insertions, wherein the variant substantially retains at least one biological property (eg, antigen-binding ability) of the antibody molecule before the change.
  • the target antibody region may be the full length of the antibody, or a heavy chain light region or a light chain variable region or a combination thereof, or a heavy chain CDR region (s) or a light chain CDR region (s) or a combination thereof .
  • An antibody region having an amino acid change relative to a reference antibody region is also referred to herein as a "variant" of the antibody region.
  • sequence identity refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-based basis in a comparison window.
  • the “percent sequence identity” can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of the same nucleic acid base in both sequences (for example, A, T, C, G, I ) Or the same amino acid residue (e.g.
  • the number of positions to get the number of matching positions divides the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiplies the result by 100 to produce a percent sequence identity.
  • the optimal alignment for determining the percent sequence identity can be achieved 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. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment within the full-length sequence range being compared or within the region of the target sequence.
  • the percent amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence and performing optimal alignment in accordance with the Kabat numbering rule in a preferred scheme.
  • sequence identity may be distributed over the entire heavy chain variable region and / or the entire light chain variable region, or the percent sequence identity may be limited to the framework region only, and the corresponding CDR regions The sequence remains 100% identical.
  • candidate antibodies with amino acid changes in the target antibody region relative to the reference antibody can be determined.
  • “conservative substitution” refers to an amino acid change that results in the replacement of a certain amino acid with a chemically similar amino acid.
  • Amino acid modifications, such as substitutions can be introduced into the antibodies of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative substitution residues are from the conservative substitution Table A below, preferably the preferred conservative substitution residues shown in Table A.
  • an antibody or antigen-binding fragment thereof that specifically binds TIGIT, preferably a human TIGIT protein (such as the human TIGIT sequence of SEQ ID NO: 208), especially a fully human antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment of an antibody of the invention is an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, a single chain antibody such as scFv, a (Fab') 2 fragment, a single domain antibody , Diabody (dAb) or linear antibody.
  • the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention binds to human TIGIT with high affinity, for example, the dissociation equilibrium constant (K D ) is less than 100x10 -9 M, less than or equal to about 50x10 -9 M, It is more preferably about 1-30x10 -9 M, more preferably less than or equal to about 1x10 -9 M, and still more preferably about 1-10x10 -10 M.
  • K D is determined by using a bio-optic interferometry (for example, Fortebio affinity measurement). In some embodiments, by measuring the K D value of antibody Fab form (e.g.
  • the monovalent affinity to human TIGIT determined K D value is preferably a monovalent 1-100x10 -10 M, more preferably 1-50x10 - 10 M, more preferably 1-10x10 -10 M or 2-5x10 -10 M.
  • the K D value is determined by measuring the monovalent affinity of an intact antibody (eg, an intact antibody expressed by CHO cells) to human TIGIT, preferably a monovalent K D value of 1-50x10 -10 M, more preferably 1- 30x10 -10 M or 1-10x10 -10 M.
  • an anti-TIGIT antibody or antigen-binding fragment thereof of the invention cross-reacts with monkey TIGIT.
  • the antibody binds to monkey TIGIT with high affinity, where the K D value (as determined by measuring the monovalent affinity of the intact antibody to monkey TIGIT) is about 0.1-100x10 -9 M, more preferably 0.1-50x10 -9 M or 1-30x10 -10 M.
  • the antibody cross-reacts with murine TIGIT, wherein the K D value (for example, the monovalent affinity of murine TIGIT determined by measuring the intact antibody) is about 1-100x10 -9 M, for example, 1-10x10 -7 M or 1 -10x10 -8 M, or 1-10x10 -9 M.
  • the anti-TIGIT antibodies of the invention do not cross-react with murine TIGIT.
  • the antibodies or antigen-binding fragments thereof of the invention bind to TIGIT expressed on the cell surface with high affinity.
  • the cell that expresses human TIGIT on the surface is a CHO cell.
  • the EC50 value of the antibody binding to human TIGIT expressing cells is determined by flow cytometry (e.g. FACS).
  • the antibody is a yeast-expressed intact antibody with an EC50 value of less than about 10 nM, such as 0.1-1 nM, preferably, less than or equal to about 1 nM, more preferably about 0.2-0.9 nM, such as 0.9 nM, 0.6M, or 0.4nM.
  • the antibody is in the form of a whole antibody expressed by CHO cells with an EC50 value of less than about 10 nM, such as 0.1-1 nM, preferably, about 0.1-0.3 nM, such as about 0.3 nM, 0.2M, or 0.1 nM.
  • an antibody of the invention or an antigen-binding fragment thereof inhibits TIGIT-related activity.
  • an antibody of the invention or an antigen-binding fragment thereof blocks the binding of TIGIT to its ligand, CD155.
  • the ability of an antibody to block human TIGIT (TIGIT expressed on cells) from binding to human CD155 is determined by flow cytometry (e.g., FACS).
  • the antibody is a yeast expressed intact antibody with an IC50 value of less than about 10 nM, such as 0.1-2 nM, preferably, about 0.1-1.0 nM, such as 0.8 nM, 0.6M, 0.4 nM, or 0.2 nM.
  • the antibody is in the form of a whole antibody expressed by CHO cells with an EC50 value of less than about 1 nM, such as 0.1-0.5 nM, preferably, about 0.3 nM, 0.2M, or 0.1 nM.
  • the antibodies or antigen-binding fragments thereof of the invention reduce or eliminate inhibitory signaling caused by TIGIT binding to CD155.
  • an antibody or fragment of the invention reduces or eliminates TIGIT-mediated inhibitory signaling in TIGIT-expressing cells, especially T cells.
  • the antibodies or antigen-binding fragments thereof of the invention induce expression of genes downstream of the IL2 promoter in T cells, and in some embodiments increase IL-2 production by T cells.
  • the ability of the antibody to reduce or eliminate inhibitory signaling caused by TIGIT binding to CD155 is detected by a fluorescent reporter test (such as the MOA test of Example 5).
  • the antibody is a yeast-expressed intact antibody, and the EC50 value is preferably less than about 10 nM, such as 0.1-5 nM, preferably, about 0.1-3.0 nM, such as about 2.nM, 1.5nM, 1.0nM, or 0.5nM.
  • the antibody is a whole antibody expressed by CHO cells, and the EC50 value is preferably less than 5 nM, such as about 0.1-3.0 nM, such as about 1.6 nM, 1.2 nM, 1.0 nM, or about 0.18-0.5 nM.
  • an antibody or antigen-binding fragment thereof of the invention inhibits the growth of a tumor containing an infiltrating lymphocyte expressing human TIGIT.
  • the tumor cell is a gastrointestinal tumor, preferably a colorectal cancer.
  • in vivo tumor transplantation models such as in MC38 mice, inhibit the growth of colon cancer cells.
  • an antibody of the invention is used in combination with an anti-PD1 antibody to obtain a significantly better antitumor effect than when an antibody is administered alone.
  • an antibody of the invention or an antigen-binding fragment thereof exhibits at least one, more preferably at least two, more preferably at least three, four, or five, even more preferably all of the properties described above.
  • a “complementarity determining region” or “CDR region” or “CDR” (which is used interchangeably with the hypervariable region “HVR” herein) is an amino acid region in an antibody variable region that is mainly responsible for binding to an epitope.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • VH and VL sequence combinations of some exemplary antibodies of the invention are given in Table B below:
  • CDR complementarity determining region
  • Kabat complementarity determining region is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interface, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. ( 1991)). Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)).
  • AbM HVR is a compromise between Kabat HVR and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software.
  • a "Contact" HVR is based on an analysis of the complex crystal structures available. According to different CDR determination schemes, the residues of each HVR / CDR in these HVRs are described below.
  • the HVR can also be an HVR sequence located at the following Kabat residue positions according to the Kabat numbering system:
  • Positions 24-36 or 24-34 (LCDR1) in VL, positions 46-56 or 50-56 (LCDR2), and positions 89-97 or 89-96 (LCDR3); and positions 26-35 or in VH 27-35B (HCDR1), positions 50-65 or 49-65 (HCDR2), and positions 93-102, 94-102, or 95-102 (HCDR3).
  • the HVR of an antibody of the invention is an HVR sequence located at the following Kabat residue position according to the Kabat numbering system:
  • the HVR of an antibody of the invention is an HVR sequence located at the following Kabat residue position according to the Kabat numbering system:
  • HVRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (such as any of the exemplary CDRs of the invention).
  • CDR or “CDR sequence” or “HVR” or “HVR sequence” encompasses HVR or CDR sequences determined in any of the ways described above.
  • the CDR sequences of the present invention are shown in Table 2, wherein HCDR1 is a CDR sequence determined according to the AbM scheme; the remaining CDRs are CDR sequences determined according to the Kabat scheme.
  • CDR sequences of the present invention are shown in Table 1.
  • Antibodies with different specificities have different CDRs.
  • CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding.
  • the minimum binding unit may be a sub-portion of the CDR.
  • the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Accordingly, the invention also contemplates any variant of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • an antibody of the invention has at least one, two, three, four, five, or six CDRs that are the same as the corresponding CDRs in the variable region sequence of any of the antibodies listed in Table B, or Its a variant.
  • an antibody of the invention has at least one, two, or three HCDRs that are the same as the corresponding heavy chain CDRs in the variable region sequence of any of the antibodies listed in Table B, or are variants thereof.
  • the antibody of the invention has at least one, two, or three LCDRs that are the same as the corresponding light chain CDRs in the variable region sequence of any of the antibodies listed in Table B, or are variants thereof.
  • corresponding CDR refers to a CDR that is located at the most similar position to the CDR of the reference antibody in the variable region amino acid sequence of the candidate antibody after optimal alignment.
  • a CDR variant is a CDR that has been modified by at least one, such as 1 or 2 or 3 amino acid substitutions, deletions, and / or insertions, wherein the antigen-binding molecule comprising the CDR variant substantially remains unmodified
  • the biological characteristics of the antigen-binding molecule for example, maintain at least 60%, 70%, 80%, 90%, or 100% biological activity (eg, antigen-binding capacity). It is understood that each CDR can be modified individually or in combination.
  • 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.
  • amino acid substitutions preferably occur with a consensus CDR sequence provided herein (e.g., SEQ ID NO: 5, 10, 15, 21, 24, 30, 33, 37, 40, 47, 52, 56, 62 , 65, 68, 74, 77, 80) at the amino acid position corresponding to the X residue.
  • the CDR3 region independent of the CDR1 and / or CDR2 regions, alone can determine the binding specificity of an antibody to an associated antigen. And, based on a common CDR3 sequence, a variety of other antibodies can be generated with the same binding specificity. See, for example, US Patents Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. All these references are incorporated herein by reference.
  • an antibody of the invention comprises a CDR3 sequence from the heavy and / or light chain variable region sequences of any of the antibodies shown in Table B, wherein the antibody is capable of specifically binding human TIGIT.
  • the antibodies may further comprise CDR2 from the heavy and / or light chain variable regions of the same antibody, or CDR2 from the heavy and / or light chain variable regions of different TIGIT antibodies.
  • the antibodies may further comprise CDR1 from the heavy and / or light chain variable regions of the same antibody, or CDR1 from the heavy and / or light chain variable regions of different TIGIT antibodies.
  • the activity of these antibodies can be characterized by the assays described herein, including the binding activity to human TIGIT, the activity to block the binding of TIGIT to the CD155 molecule, and / or the activity to inhibit tumor growth.
  • the VHCDR1, 2 and 3 sequences and the VLCDR1, 2 and 3 sequences can be "mixed and matched" (ie CDRs from different antibodies that bind to the same TIGIT antigen can be mixed and matched, although each antibody preferably contains VH, CDR1, 2 and 3 and VL, CDR1, 2 and 3) to produce other molecules of the invention that bind TIGIT.
  • the binding of such "mixed and matched" antibodies to TIGIT can be tested using binding assays known in the art (eg, ELISA, SET, Biacore) and those described in the examples.
  • the CDR1, CDR2 and / or CDR3 sequences from a particular VH sequence are preferably replaced with structurally similar CDR sequences.
  • the CDR1, CDR2 and / or CDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences.
  • “Mixing and matching" of CDRs can be performed between the antibodies shown in Table 3 of the present invention.
  • the structurally similar CDR sequences of the antibodies shown herein can also be replaced by one or more VH and / or VL CDR region sequences from other different antibodies to generate other antibody.
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region comprising a heavy chain complementarity determining region 3 (HCDR3), said HCDR3:
  • HCDR3 (i) or (ii) contains at least 1 (preferably 1-2 or more) 1 amino acid change (preferably a substitution, more preferably a conservative substitution) relative to the HCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, and the antibody has a heavy chain complementarity determining region 3 (HCDR3) and a light chain complementarity determining region 3 ( LCDR3):
  • HCDR3 heavy chain complementarity determining region 3
  • LCDR3 light chain complementarity determining region 3
  • an antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises:
  • an antibody of the invention or an antigen-binding fragment thereof comprises a light chain variable region (VL), wherein the VL comprises:
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein said antibody comprises:
  • the total of 6 CDR regions contains at least one and no more than 10 or 5, 4, 3, 2, 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of.
  • an antibody or antigen-binding fragment thereof of the invention comprises:
  • HCDR1 2 and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 84, 85, 86, or 87, and LCDR1, of the light chain variable region as shown in SEQ ID NO: 104. 2 and 3 sequences, or
  • the antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions HCDRs of the variable region of the heavy chain, and three complementarity determining regions LCDR of the variable region of the light chain, wherein
  • HCDR1 contains or consists of an amino acid sequence selected from SEQ ID NO: 1-5 and 178-181
  • HCDR2 contains or consists of an amino acid sequence selected from SEQ ID NO: 6-10
  • HCDR3 contains IDR NO: 11-15 and 182-185 amino acid sequence or consisting of it
  • LCDR1 contains or consists of SEQ ID ID NO: 16 amino acid sequence
  • LCDR2 contains or consists of SEQ ID NO: 17 amino acid sequence
  • LCDR3 Contains or consists of the amino acid sequence of SEQ ID NO: 18; or
  • HCDR1 contains or consists of an amino acid sequence selected from SEQ ID NO: 19-21 and 186-187
  • HCDR2 contains or consists of an amino acid sequence selected from SEQ ID NO: 22-24
  • HCDR3 contains SEQ ID ID NO : 25 and 188 amino acid sequences or consisting of
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 26
  • LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 27
  • LCDR3 contains the amino acid sequence selected from SEQ ID ID NO : The amino acid sequence of 28-30 or consisting of it; or
  • HCDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 31-33 and 189-190
  • HCDR2 comprises or consists of an amino acid sequence selected from SEQ ID NO: 34-37
  • HCDR3 comprises IDR NO: 38-40 and 191-192 amino acid sequence or consisting of it
  • LCDR1 contains the amino acid sequence of SEQ ID NO: 41 or consists of it
  • LCDR2 contains the amino acid sequence of ID NO: 42 or consists of it
  • LCDR3 Comprising or consisting of the amino acid sequence of SEQ ID NO: 43; or
  • HCDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 44-47 and 193-195
  • HCDR2 comprises or consists of an amino acid sequence selected from SEQ ID NO: 48-52
  • HCDR3 comprises The amino acid sequences of IDNO: 53-56 and 196-198 or consist of them
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 57
  • LCDR2 contains or consists of the amino acid sequence of SEQ ID: NO: 58
  • LCDR3 Contains or consists of the amino acid sequence of SEQ ID NO: 59; or
  • HCDR1 contains or consists of an amino acid sequence selected from SEQ ID NO: 60-62 and 199-200
  • HCDR2 contains or consists of an amino acid sequence selected from SEQ ID NO: 6 and 63-65
  • HCDR3 contains From or consisting of the amino acid sequences shown in SEQ ID NO: 66-68 and 201-202
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 69
  • LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 17
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 70; or
  • HCDR1 contains or consists of an amino acid sequence selected from SEQ ID NO: 71-74 and 203-205
  • HCDR2 contains or consists of an amino acid sequence selected from SEQ ID NO: 22 and 75-77
  • HCDR3 contains an alternative From or consisting of the amino acid sequences shown in SEQ ID NO: 78-80 and 206-207
  • LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 81
  • LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 83.
  • an antibody of the invention or an antigen-binding fragment thereof comprises 6 CDR sequences in one of the combinations listed in Table C.
  • an antibody of the invention or an antigen-binding fragment thereof comprises 6 CDR sequences in one of the combinations listed in Table D.
  • a “variable region” or “variable domain” is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to its antigen.
  • the heavy chain variable region (VH) and light chain variable region (VL) can be further divided into hypervariable regions (HVR, also known as complementarity determining regions (CDR)), with more conservative regions (i.e., frameworks) interposed between them. (FR)).
  • HVR hypervariable regions
  • FR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some cases, a single VH or VL domain is sufficient to confer antigen-binding specificity.
  • antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains (see, for example, Portolano, S. et al., J. Immunol. 150 (1993 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).
  • one or more residues in one or both of the two variable regions can be modified, for example, one or more CDR regions and / or one Or more than one framework region is subject to residue modification, especially conservative residue substitution, while the modified antibody still substantially retains at least one biological property (eg, antigen-binding ability) of the previous antibody molecule.
  • residues in the CDR regions can be mutated to improve one or more binding properties (e.g., affinity) of the antibody.
  • the antibody-binding or other functional properties of the mutated antibody can be assessed in in vitro or in vivo assays.
  • conservative substitutions are introduced.
  • framework region residues can be mutated, for example, to improve the properties of the antibody.
  • one or more framework residues can be "backmutated" to corresponding germline sequence residues.
  • CDR grafting is another modification of antibody variable region known in the art. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants can be constructed that mimic the properties of known antibodies. In this antibody variant, CDR sequences from known antibodies are grafted onto the framework regions of different antibodies with different properties. Therefore, in one embodiment, the present invention relates to an anti-TIGIT antibody, or an antigen-binding fragment thereof, which comprises the CDR sequences of the heavy and light chain variable regions from one of the antibodies of Table B, but has a different architecture Zone sequence. Framework region sequences for replacement can be obtained from public DNA databases, including germline antibody gene sequences, or from TIGIT antibody sequences reported in published literature.
  • germline DNA encoding human heavy and light chain variable region genes can be obtained from the GenBank database.
  • the antibody protein sequence can be compared with the protein sequence in the database using a sequence similarity search tool such as Gapped BLAST.
  • the framework sequence used for substitution has structural similarity to the framework sequence of the antibody of the present invention selected for change, for example, having sequence identity of at least 80%, 85%, 90%, or 95%, 96%, 97 %, 98%, 99% or more framework sequences.
  • VH and VL from an exemplary antibody of the invention can be "mixed and matched" Sequence to generate other antibodies of the invention that bind TIGIT.
  • the VH sequences from a particular VH / VL pairing are preferably replaced with structurally similar VH sequences.
  • VL sequences from a particular VH / VL pair are preferably replaced with structurally similar VL sequences.
  • Such "mixed and matched" antibodies can be tested for binding to TIGIT using binding assays known in the art (eg, ELISA, and other assays described in the Examples section).
  • the antibody of the invention comprises or consists of the heavy chain variable region VH sequence of any of the antibodies listed in Table B.
  • an antibody of the invention comprises a variant of said VH sequence.
  • an antibody of the invention comprises or consists of the light chain variable region VL sequence of any of the antibodies listed in Table B. In yet another embodiment, an antibody of the invention comprises a variant of said VL sequence.
  • an antibody of the invention comprises:
  • VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO: 84 or 85 or 86 or 87, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO: 104, or
  • VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO: 94 or 95 or 96 or 97, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO: 108, or
  • VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO: 101 or 102 or 103, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO: 110;
  • the variant of the VH sequence is at least 80%, 85% in amino acid sequence compared to the reference VH sequence (preferably over the full length or over the three regions of CDR1, 2 and 3). %, 90%, 92%, 95%, 97%, 98%, 99% or higher identity. In one embodiment, the variant of the VH sequence is at least one and no more than the reference VH sequence (preferably over the full length or over the three regions of CDR1, 2 and 3) in the amino acid sequence. 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions). Preferably sequence differences do not occur in the CDR regions.
  • the variant of the VL sequence is at least 80% in amino acid sequence compared to the reference VL sequence (preferably over the full length or in the three regions of CDR1, 2 and 3) , 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity.
  • the variant of the VL sequence is in amino acid sequence, compared to the reference VL sequence, (preferably over the full length or over the three regions of CDR1, 2 and 3), comprising at least one and No more than 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • sequence differences do not occur in the CDR regions.
  • the antibody of the invention comprises or consists of the heavy chain variable region and light chain variable region VH / VL sequence pairs of any of the antibodies listed in Table B.
  • the invention also provides variants of the antibody, such as variants that have at least 95-99% identity on VH, VL or VH and VL or contain no more than 10 amino acid changes.
  • the heavy chain variable region of the antibody variant contains no more than 10, preferably no more than 1 CDR regions on one or more CDRs (preferably all 3 CDRs) relative to the reference antibody.
  • 5 eg, 3, 2, 1 or 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the light chain variable region VL of the antibody variant contains no more than 10, preferably not more than 1 CDR regions relative to the reference antibody, preferably on one or more CDRs (preferably all 3 CDRs). More than 5 (e.g., 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • an antibody of the invention comprises a heavy chain Fc region, such as an Fc region of the IgG1, IgG2, or IgG4 isotype.
  • an antibody of the invention contains an IgG4-Fc region with a serine to proline mutation (S228P) at amino acid residue position 228 (EU numbering).
  • an antibody of the invention comprises an IgG4-PAA Fc portion.
  • the IgG4-PAA Fc portion has a serine to proline mutation (S228P) at position 228, a phenylalanine to alanine mutation at position 234 (EU number), and a leucine at position 235 (EU number) To alanine mutation.
  • the S228P mutation is a mutation in the hinge region of the constant region of tumors, which can reduce or eliminate heterosulfide bridge heterogeneity.
  • the F234A and L235A mutations can further reduce the effector function of the human IgG4 isotype (which already has low effector function).
  • the antibodies of the invention contain an IgG4-PAA Fc portion with the heavy chain C-terminal lysine (des-Lys) removed.
  • an antibody of the invention comprises a kappa light chain constant region, such as a human kappa light chain constant region.
  • the Fc region comprises the amino acid sequence of SEQ ID NO: 177, or the amino acid sequence of SEQ ID NO: 177 contains at least one, two or three, but not more than 20, 10 Or an amino acid sequence of 5 amino acid changes, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 177.
  • an antibody of the invention comprises a light chain constant region.
  • the light chain constant region is a human kappa light chain constant region.
  • the light chain constant region comprises the amino acid sequence of SEQ ID NO: 209, or the amino acid sequence of SEQ ID NO: 209 contains at least one, two or three, but not more than 20, 10 Amino acid sequence with one or five amino acids changed, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 209.
  • the antibody of the invention comprises a heavy chain
  • the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 111-130, or at least one, two, or three relative to it, but not Over 20, 10 or 5 amino acid sequence changes, or an amino acid sequence with at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity .
  • the amino acid changes do not occur in the CDR regions, and more preferably, they do not occur in the variable regions.
  • the antibody of the invention comprises a light chain
  • the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 137-143, or at least one, two, or three relative to it, but not Over 20, 10 or 5 amino acid sequence changes, or an amino acid sequence with at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity .
  • the amino acid changes do not occur in the CDR regions, and more preferably, they do not occur in the variable regions.
  • an antibody of the invention comprises a heavy chain sequence and / or a light chain sequence selected from:
  • the variant contains at least one, two or three, but no more than 20, 10 or 5 amino acid altered amino acid sequences compared to the corresponding reference sequence, or has at least 80%, 85%, 90% , 92%, 95%, 97%, 98%, 99% or higher amino acid sequence identity.
  • the amino acid changes do not occur in the CDR regions, and more preferably do not occur in the variable regions.
  • residue modifications are made in the constant region of the antibody to, for example, alter the properties of the antibody, such as effector function.
  • the heavy and / or light chain of an anti-TIGIT antibody or fragment thereof of the invention further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG.
  • the invention provides fully human antibodies that specifically bind to TIGIT (eg, human TIGIT) as isolated and characterized in the examples.
  • TIGIT eg, human TIGIT
  • the VH and VL sequences of the antibody variable regions of these exemplary antibodies of the invention are listed in Table 3 below.
  • Exemplary CDR sequences of the antibodies are listed in Tables 1 and 2 below.
  • the sequence listing shows the heavy and light chain amino acid sequences of the exemplary antibodies of the present invention and the nucleotide sequences encoding the variable regions VH and VL of the exemplary antibodies of the present invention.
  • the invention provides any antibody described herein, particularly variants of the exemplary antibodies listed in Table B.
  • the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen-binding capacity) of the pre-modified antibody.
  • the alteration does not cause the antibody variant to lose binding to the antigen, but optionally can confer properties such as increased antigen affinity and different effector functions.
  • variable region of the heavy chain or the variable region of the light chain of the antibody, or each CDR region can be changed individually or in combination.
  • the amino acid is changed to an amino acid substitution, preferably a conservative substitution.
  • the amino acid is changed to an amino acid substitution, preferably a conservative substitution.
  • the antibody variant and the reference antibody have at least 80%, 85%, 90%, or 95% or 99% or higher amino acid identity on the region of the target antibody sequence.
  • an antibody of the invention has at least 90%, 91%, 92%, 93% on 3 heavy chain CDR regions compared to a reference antibody (such as one of the antibodies listed in Table 3), 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.
  • the antibody of the invention has at least 90%, 91%, 92%, 93%, 94% of the three light chain CDR regions compared to a reference antibody (eg, one of the antibodies listed in Table 3). , 95%, 96%, 97%, 98%, or 99% or higher identity.
  • an antibody of the invention has at least 90%, 91%, 92%, 93%, 94% of the 6 CDR regions compared to a reference antibody (eg, one of the antibodies listed in Table 3), 95%, 96%, 97%, 98%, or 99% or higher identity.
  • an antibody of the invention has at least 80%, 85%, 90%, 91%, 92% of the heavy chain variable region compared to a reference antibody (such as one of the antibodies listed in Table 3). , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In yet another embodiment, an antibody of the invention has at least 80%, 85%, 90%, 91%, 92% of the light chain variable region compared to a reference antibody (such as one of the antibodies listed in Table 3). , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In yet another embodiment, an antibody of the invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.
  • changes can be made to the Fc region of the antibody. Changes in the Fc region can be made alone or in combination with changes to the framework and / or CDR regions described above.
  • the Fc region can be altered, for example, to alter one or more functions of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity.
  • the antibodies of the invention can also be chemically modified (e.g., linked to PEG) or their glycosylation pattern can be changed.
  • the Fc region may comprise an Fc-region having one or more amino acid substitutions that increase ADCC activity, for example, substitutions at positions 298, 333, and / or 334 of the Fc-region (EU numbering of residues) .
  • changes can also be made to the Fc-region to cause altered (ie, increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC) (see, for example, US 6,194,551, WO99 / 51642 and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184).
  • the Fc can be altered to increase or decrease its degree of glycosylation and / or change its glycosylation pattern.
  • Addition or deletion of a glycosylation site of an Fc can be conveniently achieved by altering the amino acid sequence in order to create or remove one or more glycosylation sites.
  • one or more amino acid substitutions can be performed to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site.
  • Antibodies with altered types of glycosylation can be made, such as low or no fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisected GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies.
  • the invention also contemplates antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. These antibody variants may have improved CDC function.
  • the invention also contemplates antibody variants that have some but not all effector functions, which makes them ideal candidates for certain applications in which the in vivo half-life of the antibody is important, but Certain effector functions (such as complement and ADCC) are unnecessary or harmful.
  • the Fc region may contain mutations that eliminate or reduce effector functions, such as a human IgG1 Fc region with mutations P329G and / or L234A and L235A, or a human IgG4 Fc region with mutations P329G and / or S228P and L235E.
  • cysteine engineered antibody such as a "thio MAb"
  • cysteine residues in the hinge region of an antibody can be changed to, for example, facilitate assembly of the light and heavy chains or increase or decrease the stability of the antibody. See, for example, U.S. Patent No. 5,677,425.
  • the antibodies provided herein can be further modified to contain a non-proteinaceous moiety.
  • Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG) to, for example, increase the half-life of an antibody (eg, serum).
  • PEG polyethylene glycol
  • Methods for PEGylation of proteins are known in the art and can be applied to antibodies of the invention. See for example EP 0154 316 and EP 0401384.
  • the invention provides a nucleic acid encoding any of the above anti-TIGIT antibodies or fragments thereof.
  • a vector comprising the nucleic acid is also provided.
  • the vector is an expression vector.
  • a host cell comprising the nucleic acid or the vector is also provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells).
  • the host cell is prokaryotic.
  • the invention provides a nucleic acid encoding any of the above anti-TIGIT antibodies or fragments thereof.
  • the nucleic acid may comprise a nucleic acid encoding an amino acid sequence of a light chain and / or heavy chain variable region of an antibody, or a nucleic acid comprising an amino acid sequence of a light and / or heavy chain of an antibody.
  • Exemplary nucleic acid sequences encoding the variable region of an antibody heavy chain comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, and nucleic acid sequences selected from SEQ ID NOs: 150-169, A nucleic acid sequence that is 95%, 96%, 97%, 98%, or 99% identical, or contains a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 150-169.
  • Exemplary nucleic acid sequences encoding the variable region of an antibody light chain include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, and nucleic acid sequences selected from the group consisting of SEQ ID NOs: 170-176, A nucleic acid sequence that is 95%, 96%, 97%, 98%, or 99% identical, or comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 170-176.
  • the polypeptide encoded by these polynucleotides can show TIGIT antigen-binding ability.
  • polynucleotides that encode at least one CDR region and generally all three CDR regions from the heavy chain VH or light chain VL sequences of the TIGIT-binding antibody described above.
  • the polynucleotide encodes a complete or substantially complete variable region sequence of the heavy and / or light chain of a TIGIT-binding antibody described above.
  • each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences.
  • a nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a heavy chain Fc region, such as the Fc region sequence shown in SEQ ID NO: 177 or a sequence substantially the same.
  • the nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a light chain constant region sequence, such as the sequence shown in SEQ ID NO: 209 or a sequence substantially identical thereto.
  • one or more vectors comprising a nucleic acid of the invention are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • the expression vector of the invention is a pTT5 expression vector.
  • a host cell comprising the vector.
  • Suitable host cells for cloning or expressing a vector encoding an antibody include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • For the expression of antibody fragments and polypeptides in bacteria see, for example, U.S. Pat. , Pages 245-254, which describe the expression of antibody fragments in E. coli. After expression, the antibodies in the soluble fraction can be separated from the bacterial cell paste and can be further purified.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for use in making antibodies or antigen-binding fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies.
  • fungal and yeast strains whose glycosylation pathways have been "humanized” result in the production of antibodies with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • Host cells suitable for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines that have been engineered to be suitable for suspension growth can be used.
  • useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293HEK or 293 cells, such as, for example, Graham et al., J. Gen Virol. 36:59 (1977 )) And so on.
  • Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al., Proc. Natl.
  • a method of making an anti-TIGIT antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for antibody expression, as provided above, and optionally The antibody is recovered from the host cell (or host cell culture medium).
  • a nucleic acid encoding an antibody (such as the antibody described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell.
  • nucleic acids are easy to isolate and sequence using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains).
  • anti-TIGIT antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.
  • the antibodies of the invention are tested for their antigen-binding activity.
  • binding to human TIGIT can be determined by methods known in the art, such as ELISA, Western blot, etc., or exemplary methods disclosed in the examples herein.
  • the assay can be performed using flow cytometry in which the antibody reacts with a cell line expressing human TIGIT, such as CHO cells expressing human TIGIT on the cell surface after transfection. Other cells are also suitable for flow cytometry, including T cells expressing native TIGIT.
  • the antibody binding including binding kinetics (e.g., K D value), using recombinant TIGIT protein, in a biological assay of optical interference. In some embodiments, for example, Fortebio affinity measurement is used.
  • competition assays can be used to identify antibodies that compete with any of the anti-TIGIT antibodies disclosed herein for binding to TIGIT.
  • such competitive antibodies bind to an epitope (e.g., a linear or conformational epitope) that is the same as or overlaps with the epitope bound by any of the anti-TIGIT antibodies disclosed herein.
  • an epitope e.g., a linear or conformational epitope
  • an epitope e.g., a linear or conformational epitope
  • For detailed exemplary methods for locating the epitope bound by an antibody see Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
  • the invention also provides an assay for identifying a biologically active anti-TIGIT antibody.
  • Biological activities may include, for example, binding to TIGIT (e.g., human TIGIT), blocking the binding of TIGIT (e.g., human TIGIT) to the CD155 molecule, blocking TIGIT-mediated inhibitory signaling, increasing IL2 production by T cells, and / or inhibiting Tumor growth.
  • TIGIT e.g., human TIGIT
  • TIGIT e.g., human TIGIT
  • any of the above assays can be performed using the immunoconjugate or multispecific antibody of the invention in place of or in addition to an anti-TIGIT antibody.
  • the invention provides a multispecific (including bispecific) antibody molecule that specifically binds TIGIT, preferably human TIGIT.
  • a multispecific antibody an antibody of the invention (or an antigen-binding fragment thereof) forms a first binding specificity for TIGIT.
  • the multispecific antibody further has a second specificity, or in a further embodiment further comprises a second and a third binding specificity.
  • the multispecific antibody is a bispecific antibody.
  • the binding specificity is provided by the "binding site” or "antigen binding site” of the antibody (the region of the antibody molecule that actually binds to the antigen).
  • the antigen binding site consists of a VH / VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • VL antibody light chain variable domain
  • VH antibody heavy chain variable domain
  • a "multispecific" antibody is an antibody having at least two antigen-binding sites, each of said at least two antigen-binding sites may be a different list of the same antigen Or binding to different epitopes of different antigens.
  • the invention provides immunoconjugates produced by conjugating an antibody of the invention to a heterologous molecule.
  • an antibody (or antigen-binding fragment thereof) of the invention is conjugated to a therapeutic or diagnostic agent.
  • an antibody of the invention may be conjugated to a heterologous molecule in the form of a full-length antibody or antibody fragment. For example, conjugation is performed in the form of Fab fragments, Fab 'fragments, F (ab)' 2 fragments, single-chain scFab antibodies, and single-chain scFv.
  • an antibody of the invention is conjugated to a therapeutic molecule.
  • Linkers can be used to covalently link antibodies to therapeutic assays. Suitable linkers include chemical linkers or peptide linkers.
  • antibodies of the invention can be conjugated to a diagnostic or detectable agent.
  • conjugates can be used as part of a clinical test (such as determining the efficacy of a particular therapy) for monitoring or predicting the onset, formation, progression, and / or severity of a disease or disorder.
  • diagnosis and detection can be achieved by coupling antibodies to a detectable agent.
  • the present invention also includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising an anti-TIGIT antibody or an immunoconjugate or a multispecific antibody thereof, and a polynuclear core comprising an anti-TIGIT antibody or an immunoconjugate or a multispecific antibody thereof.
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.
  • the composition further comprises a second therapeutic agent.
  • the second therapeutic agent may be selected from the group consisting of, but not limited to, for example, anti-PD-1 antibodies and anti-PD-L1 antibodies.
  • the second therapeutic agent is a PD-1 antagonist, especially an anti-PD-1 antibody.
  • Pharmaceutically acceptable carriers suitable for the present invention can be sterile liquids, such as water and oils, including those from petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • water is the preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , Glycerin, propylene, glycol, water, ethanol, etc.
  • compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents.
  • these compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, and saccharin.
  • An anti-TIGIT antibody, immunoconjugate or multispecific antibody of the invention having the required purity can be obtained by combining one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A (1980)) to prepare a pharmaceutical formulation comprising the present invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • a pharmaceutical formulation comprising the present invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffers.
  • the pharmaceutical composition or formulation of the present invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other .
  • active ingredients such as a PD-1 binding antagonist or a PD-L1 binding antagonist, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the active ingredients are suitably present in combination in an amount effective for the intended use.
  • sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.
  • the invention also provides a combination product comprising an antibody of the invention or an antigen-binding fragment thereof, a bispecific antibody or an immunoconjugate, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies , Cytotoxic agents, vaccines, anti-infective agents, etc.).
  • chemotherapeutic agents e.g., chemotherapeutic agents, other antibodies , Cytotoxic agents, vaccines, anti-infective agents, etc.
  • the combination product of the present invention can be used in the treatment method of the present invention.
  • the present invention provides a combination product, wherein the other therapeutic agent is, for example, a therapeutic agent, such as an antibody, effective to stimulate an immune response to further enhance, stimulate or up-regulate the immune response in a subject.
  • the other antibody is, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the combination product is used to prevent or treat a tumor.
  • the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, etc .; or a skin cancer such as malignant melanoma.
  • the combination product is used to prevent or treat infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats mouse).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., human and non-human primates such as monkeys
  • rabbits and rodents e.g., mice and rats mouse.
  • the subject is a human.
  • treatment refers to a clinical intervention intended to alter the natural process of a disease in an individual being treated.
  • the desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the state of the disease, and alleviating or improving the prognosis.
  • the invention relates to a method of enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of any of the anti-TIGIT antibodies or fragments thereof described herein, or comprising An immunoconjugate, a multispecific antibody, or a pharmaceutical composition of the antibody or fragment.
  • an anti-TIGIT antibody or antigen-binding portion thereof of the invention is administered to a tumor-bearing subject to stimulate an anti-tumor immune response.
  • an antibody of the invention or an antigen-binding portion thereof is administered to a subject carrying an infection to stimulate an anti-infective immune response.
  • the invention in another aspect, relates to a method of treating a tumor, such as cancer, in a subject, said method comprising administering to said subject an effective amount of any of the anti-TIGIT antibodies or fragments thereof described herein, or comprising said An antibody or fragment is an immunoconjugate, a multispecific antibody, or a pharmaceutical composition.
  • Cancer can be early, intermediate, or advanced or metastatic.
  • TIGIT is highly expressed on human tumor infiltrating CD8 + T cells.
  • the methods of the invention are used to treat cancer, especially solid tumors that are tumor infiltrating lymphocytes infiltrating TIGIT.
  • the cancer is a gastrointestinal cancer such as a colon cancer.
  • the tumor or tumor cell may be selected from colorectal tumor, ovarian tumor, pancreatic tumor, lung tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical Tumors, bladder tumors, glioblastomas, and head and neck tumors.
  • the cancer can be selected from colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glia Cell tumor and head and neck cancer.
  • a tumor such as a cancer
  • methods of treating a tumor comprising administering to a subject an anti-TIGIT antibody and an antagonist anti-PD-1 antibody of the invention.
  • it is used to treat tumors that co-express TIGTI and PD-1, for example, melanoma that co-expresses TIGIT and PD-1 (Chauvin et al. (2015) J. Clin. Invest. 125: 2046) , And non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC).
  • NSCLC non-small cell lung cancer
  • RRCC renal cell carcinoma
  • the invention in another aspect, relates to a method of treating an infectious disease, such as a chronic infection, in a subject, said method comprising administering to said subject an effective amount of any of the anti-TIGIT antibodies or fragments thereof described herein, or An immunoconjugate, a multispecific antibody, or a pharmaceutical composition comprising the antibody or fragment.
  • the infection is a viral infection.
  • the infectious disease is caused by a viral infection.
  • pathogenic viruses include (A, B, and C) hepatitis viruses, (A, B, and C) influenza viruses, HIV, herpes viruses (e.g., VZV, HSV-1, HAV-6 , HSV-II, CMV, Epstein Barr virus +), adenovirus, flavivirus, acovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, Rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma, molluscum virus, polio virus, rabies virus, JC virus and arbovirus.
  • the methods described herein further comprise administering to the subject one or more therapies (eg, a treatment modality and / or other therapeutic agent).
  • the treatment modality includes surgical treatment and / or radiation therapy.
  • a T cell response can be stimulated by a combination of an anti-TIGIT antibody of the invention and one or more therapeutic agents.
  • the method of the invention in addition to administering an antibody of the invention, the method of the invention also includes administering at least one other immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, and these antibodies may be, for example, fully human , Chimeric, or humanized antibodies.
  • other therapeutic agents that can be used in combination with the antibodies of the invention are selected from PD-1 binding antagonists and PD-L1 binding antagonists.
  • Alternative names for "PD-1” include CD279 and SLEB2.
  • Alternative names for "PD-L1” include B7-H1, B7-4, CD274, and B7-H.
  • PD-1, PD-L1 are human PD-1, PD-L1.
  • a PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand-binding partner.
  • the PD-1 ligand binding partner is PD-L1.
  • the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner.
  • the PD-L1 binding partner is PD-1.
  • Antagonists can be antibodies, antigen-binding fragments, immunoadhesins, fusion proteins or oligopeptides.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD-1 antibody is selected from the group consisting of IBI308 (sintilizumab, WO2017 / 025016A1), MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), and CT-011 (Pidilizumab).
  • the anti-PD-1 antibody is MDX-1106.
  • the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4).
  • the anti-PD-1 antibody is an "Antibody C" described herein.
  • an anti-TIGIT antibody or fragment thereof alone or in combination with a PD-1 binding antagonist or in combination with a PD-L1 binding antagonist can also be combined with one or more other therapies such as a treatment modality and / or Other therapeutic agents are administered in combination.
  • the treatment modalities include surgery (e.g., tumor resection); radiation therapy (e.g., exoparticle beam therapy, which involves three-dimensional conformal radiation therapy in which the illuminated area is designed), local irradiation (e.g., pointing to a preselected target Or organ irradiation) or focused irradiation.
  • an anti-TIGIT antibody or antigen-binding fragment thereof of the invention can be administered in combination with chemotherapy or a chemotherapeutic agent. In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof of the invention may be administered in combination with radiotherapy or a radiotherapy agent. In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof of the invention can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof of the invention may be administered in combination with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody.
  • the antibodies of the invention can be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, And, if local treatment is needed, it is administered intralesionally.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection.
  • Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • an antibody of the invention when used alone or in combination with one or more other therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease , Whether the antibody is administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to a patient in one treatment or after a series of treatments.
  • compositions, multispecific antibody, or immunoconjugate of the present invention may be administered instead of the antibody or antigen-binding portion of the present invention.
  • a composition, a multispecific antibody, or an immunoconjugate of the present invention may be further administered.
  • the present invention provides the use of the anti-TIGIT antibody, composition, immunoconjugate, multispecific antibody of the present invention in the manufacture of a medicament for the aforementioned method (for example, for treatment).
  • the invention relates to a method and kit for detecting TIGIT in a sample, wherein the method comprises: (a) contacting the sample with an antibody of the invention or an antigen-binding fragment or immunoconjugate thereof; and (b) The formation of a complex between the antibody or antigen-binding fragment or immunoconjugate and a TIGIT protein is detected.
  • the sample is from a cancer patient, such as a skin cancer patient. The detection may be in vitro or in vivo.
  • the term "detection" as used herein includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays Method, PCR-technology (eg, RT-PCR).
  • the biological sample is blood, serum, or other liquid samples of biological origin.
  • the biological sample comprises cells or tissue.
  • the biological sample is from a hyperproliferative or cancerous lesion.
  • the TIGIT to be detected is human TIGIT.
  • an anti-TIGIT antibody is used to select a subject suitable for treatment with an anti-TIGIT antibody, for example where TIGIT is a biomarker used to select the subject.
  • a cancer or tumor can be diagnosed using an antibody of the invention, such as to evaluate (e.g., monitor) a subject for the treatment or progression of a disease described herein (e.g., a hyperproliferative or cancerous disease), its diagnosis, and / or Staging.
  • Labels include, but are not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels), and portions that are detected indirectly, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.
  • Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and their derivatives, rhodamine and its derivatives, dansyl, umbrella Umbelliferone, luciferase, for example, firefly luciferase and bacterial luciferase (US Patent No.
  • fluorescein 2,3-dihydrophthalazine dione, horseradish peroxidase (HR), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lyase, sugar oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as urase and xanthine oxidase, and enzymes using peroxidase dyes such as HR, lactoperoxidase, or microperoxidase, biotin / avidin , Spin labeling, phage labeling, stable free radicals, and more.
  • HR horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lyase sugar oxidase
  • Yeast-based antibody display libraries were amplified according to existing methods (WO2009036379; WO 2010105256; W02012009568), where the diversity of each library reached 1 ⁇ 10 9 .
  • the first two rounds of screening used the Miltenyi company's MACS system for magnetic bead cell sorting.
  • FACS washing buffer phosphate buffer, containing 0.1% bovine serum protein
  • the buffer contains 100 nM biotin Human TIGIT antigen (Acro Biosystems, TIT-H52H3).
  • LC-FITC FITC-labeled anti-human immunoglobulin kappa light chain antibody, Southern Biotech
  • SA -633 Streptavidin-633, Molecular Probes
  • SA-PE Streptavidin-PE, Sigma
  • the yeast cells expressing the anti-TIGIT antibody obtained by screening were induced by shaking at 30 ° C for 48 hours to express the anti-TIGIT antibody. After induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 min, and the supernatant was harvested. The anti-TIGIT antibody in the supernatant was purified using Protein A and eluted with a pH 2.0 acetic acid solution. The anti-TIGIT antibody was harvested, and the antibody purity was> 95%. The corresponding Fab fragments can be obtained by digestion with papain and purification with KappaSelect (GE Life Medical Group). The anti-TIGIT antibodies ADI-27301, ADI-27238, ADI-27278, ADI-27243, ADI-27297, and ADI-27291 were obtained in this screening.
  • This method uses conventional mismatch PCR to introduce mutations into the antibody heavy chain region.
  • the base mismatch probability was increased to about 0.01 bp by using 1uM highly mutated base analogs dPTP and 8-oxo-dGTP.
  • mismatched PCR products were constructed into a vector containing a heavy chain constant region by a method of homologous recombination.
  • a method of homologous recombination under the screening pressure including TIGIT antigen titer, unlabeled antigen competition, and competition using parent antibody, we obtained a secondary library with a library capacity of 1 ⁇ 10 7 .
  • Three successful screenings were performed by the FACS method.
  • the CDRH3 gene of the progeny antibody obtained by the VHmut method was constructed into a 1 ⁇ 10 8 diversity CDRH1 / CDRH2 gene library, and 3 rounds of screening were performed. In the first round, the MACS method was used, while in the second and third rounds, the FACS method was used to pressurize the antibody-antigen conjugates to screen the antibodies with the highest affinity.
  • the yeast cells expressing the anti-TIGIT antibody obtained by screening were induced by shaking at 30 ° C for 48 hours to express the anti-TIGIT antibody. After induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 min, and the supernatant was harvested. The anti-TIGIT antibody in the supernatant was purified using Protein A and eluted with a pH 2.0 acetic acid solution. The anti-TIGIT antibody was harvested, and the antibody purity was> 95%.
  • the corresponding Fab fragments can be obtained by digestion with papain and purification with KappaSelect (GE Life Medical Group).
  • 22G2 is an anti-human TIGIT antibody from Bristol-Myers Squibb Company transiently expressed in HEK293 cells, with light and heavy chain variable region sequences and patent application WO2016 /
  • the sequence of the antibody "22G2" in 106302A1 is the same.
  • 31C6 is an anti-human TIGIT antibody from Merck, transiently expressed in HEK293 cells.
  • the sequence of the light and heavy chain variable regions is the same as that of the antibody "31C6" in patent application WO2016 / 028656 A1.
  • Both 10A7 and 1F4 are anti-human TIGIT antibodies from Genentech, transiently expressed in HEK293 cells.
  • sequences of the light and heavy chain variable regions are the same as the sequences of the antibodies "10A7" and “1F4" in the patent application WO2015009856A2, respectively.
  • the constant regions of the four control antibodies used wild-type IgG4 sequences.
  • the vector pTT5 was used for transient expression of antibodies in HEK293 cells.
  • the heavy and light chains of the antibody were cloned into separate pTT5 vectors.
  • Chemical transfection was used to transfer the pTT5 vector carrying the heavy and light chains of the antibody molecule into HEK293 cells.
  • the chemical transfection reagent used was PEI (purchased from Polysciences), and the cultured HEK293 cells were transiently transfected according to the protocol provided by the manufacturer. First prepare plasmid DNA and transfection reagents in a clean bench.
  • ADI-30268, ADI-30278, ADI-30286, ADI-30288, ADI-30293, ADI-30306, ADI-30336 were expressed and purified in CHO cells.
  • Horizon's HD-BIOP1 (GS Null CHO-K1) was used to generate antibody-expressing CHO cell lines according to the manufacturer's instructions.
  • the DNA sequences of the heavy and light chains of the antibody molecule were inserted into the pD2531 plasmid from ATUM Company.
  • the transfected plasmid was then transfected into a CHO cell line by electrotransfection. After 24 hours of transfection, ForteBio was used to detect antibody production to determine transfection efficiency.
  • the transfected cells were subjected to pressure screening to obtain a pool of cells with high expression of antibodies. Afterwards, the cell pool was expanded to express a large amount of the antibody, and the cell supernatant was collected and purified by Protein A and gel filtration, so that the purity of the antibody was> 95%.
  • the bio-optic interferometry (ForteBio) assay was used to determine the equilibrium dissociation constant (K D ) of the 20 exemplary antibodies of the present invention that bind to human TIGIT.
  • TIGIT-FC the sensor is equilibrated for 20 minutes below the midline in the analysis buffer, and then detected online for 120 seconds to establish a baseline.
  • the antigen-loaded sensor was placed in a solution containing 100 nM Fab until the plateau phase, after which the sensor was transferred to the analysis buffer for dissociation for at least 2 minutes for dissociation rate measurement.
  • Kinetic analysis was performed using a 1: 1 binding model.
  • the binding ability of the above 20 exemplary antibodies of the present invention to human TIGIT expressed on the surface of CHO cells was measured based on a flow cytometry assay.
  • the binding ability of different antibodies to human TIGIT expressed on the surface of CHO cells was compared by comparing their binding curves.
  • yeast is used to express parental and progeny antibodies. After the antibodies were obtained (about 70% purity), experiments were performed to bind these antibodies to human TIGIT expressed on the surface of CHO cells.
  • the specific experimental procedure is as follows: (1) CHOS cells were transfected with the human TIGIT plasmid constructed on pCHO1.0, and the transfected cells were screened with methotrexate and puromycin to obtain a stable cell pool. (2) Incubate the stable cell pool at 4 ° C for 30 minutes with candidate antibodies of different concentrations. After washing three times with PBS, it was incubated with PE-labeled mouse anti-human secondary antibody at 4 ° C for 30 minutes.
  • the 7 molecules selected were expressed and purified using the CHO system to obtain high-purity antibodies, and binding experiments with human TIGIT expressed on the surface of CHO cells were performed.
  • ADI-30268, ADI-30336, ADI-30293, ADI-30286, ADI-30288, ADI-30278 and ADI-30306 combined with human TIGIT, EC50 overexpressed on CHO cells
  • the values are 0.1392nM, 0.1300nM, 0.2288nM, 0.1758nM, 0.1860nM, 0.1321nM, and 0.1999nM, respectively, which are higher than the binding capacity of the control antibodies 22G2, 1F4 and human TIGIT overexpressed on CHO cells (the values of control antibodies 22G2 and 1F4 EC50 values were 0.7249 nM and 0.6073 nM, respectively, similar to the control antibody 31C6 (EC50 value was 0.1846 nM).
  • control antibody 10A7 has similar EC50 values (0.1949 nM) to the seven candidate antibodies, the binding curve of the antibody shows that the upper platform of the curve is lower than that of the other seven candidate antibodies. This is believed to be caused by its faster dissociation characteristics (see Figure 2). Therefore, it is also believed that the binding ability of seven candidate antibodies to human TIGIT expressed on the surface of CHO cells is stronger than 10A7.
  • Flow cytometry was used to determine the ligand blocking ability of candidate antibodies.
  • the specific method is as follows: construct a pool of CHOS cells stably overexpressing human TIGIT, and incubate 50 nM human CD155 protein with mouse IgG2aFC segment (ACRO, TIT-H5253-1MG) and antibodies at different concentrations for 30 minutes at 4 degrees Celsius . After washing with PBS three times, the residual ligand CD155 on the cells was stained with 1% concentration of goat anti-mouse FC and a secondary antibody (Biolegend, 405308) labeled with APC fluorescein. After washing with PBS three times, the intermediate fluorescence values of the corresponding channels (C6, 4 channels) were detected by flow cytometry.
  • IgG1 was used as the negative control antibody, and 10A7, 22G2, and 31C6 were used as the positive control antibodies.
  • Figure 3 and the table below show the ability of yeast-expressed antibodies to block CD155 before and after affinity maturation (IC50: nM).
  • CD155 and TIGIT blocking experiments were performed using high-purity antibodies expressed by 7 CHO strains.
  • IgG4 was used as a negative control
  • 22G2, 31C6, and 10A7 were used as positive controls.
  • the IC50s of ADI-30268, ADI-30336, and ADI-30278 are 0.1402nM, 0.1219nM, and 0.1164nM, which are lower than 22G2 (0.6069nM), 31C6 (0.1659nM), and 10A7 (0.3252nM).
  • Anti-TIGIT antibody can release the inhibitory effect of CD155 on downstream IL2 signaling pathway by blocking the binding of TIGIT and CD155.
  • the MOA detection cell line (J2201) provided by Promega is used to detect the expression of the fluorescent reporter gene and reflect the activation of the IL2 signal, thereby detecting the inhibitory effect of the antibody on the binding of TIGIT and CD155.
  • the test was performed according to the standard method provided by Promega, the process is as follows: Jurkat cells stably expressing the luciferase reporter gene under the control of the human TIGIT and IL2 promoter, and CHO-K1 cells stably expressing human CD155 and T cell activating elements, and Anti-human TIGIT antibodies with different concentration gradients were co-incubated in a 37 ° C carbon dioxide cell incubator, and the fluorescence signal was detected after 6 hours.
  • MOA biological activity experiments were performed using high-purity antibodies expressed by 7 CHO strains.
  • IgG4 was used as a negative control and 31C6 was used as a positive control.
  • ADI- The EC50 of 30268, ADI-30286, ADI-30288, and ADI-30306 are 0.5065nM, 0.3963nM, 0.3844nM, and 0.3984nM, respectively, showing similar biological activity as the control antibody 31C6; EC50 of ADI-30336 and ADI-30293 1.274nM and 1.624nM, respectively, showing weak biological activity.
  • the results are shown in the following table and Figure 6 (EC50: nM).
  • human TIGIT knock-in-MC38 mouse tumor model was used to study the antitumor activity of anti-TIGIT antibodies.
  • mice in each group were injected subcutaneously with antibodies, and on the 6th, 10th, 13th, 17th and 17th days after transplantation. Tumor volume was measured on days 21 and 25, and then mice were euthanized. The results are shown in Figure 7.
  • the anti-TIGIT monoclonal antibodies ADI-30293 and ADI-30278 of the present application have a certain inhibitory effect on tumor growth when used alone compared with the Ig4 negative control antibody.
  • the tumor suppressive effect of the two candidate antibodies when used alone was not significantly different from that of 31C6.
  • Mouse colon cancer cell MC38 (Shanghai Heyuan Biotechnology, HYC0116) was cultured in DMEM medium. 1 ⁇ 10 6 MC38 cells in a 0.2 ml DMEM basal medium suspension were transplanted to the right of female TIGIT transgenic mice (Shanghai Ncapturing Model Biotechnology Co., Ltd.). Tumor size and weight were measured twice a week throughout the study period.
  • mice with a size of 71 mm3 were randomly divided into 6 groups, and the mice were euthanized when the tumor volume reached the end of detection or the weight of the mice was reduced by more than 20%.
  • mice in each group were injected subcutaneously with h-IgG (10mg / kg), antibody C (1mg / kg), and 10mg / kg. anti-TIGIT antibody or its isotype.
  • the endpoint tumor average volume was measured on day 29 after cell transplantation, after which experimental mice were euthanized. The results are shown in Figure 8.
  • mice in this study had no significant change in body weight after 29 days of vaccination.

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Abstract

特异性结合TIGIT的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物。编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途，包括抗体和抗体片段的治疗和诊断用途。

Description

抗TIGIT抗体及其用途 技术领域
本发明涉及特异性结合TIGIT的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物。此外，本发明涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途。此外，本发明涉及这些抗体和抗体片段的治疗和诊断用途。
背景技术
TIGIT(含Ig和ITIM结构域的T细胞免疫受体，也称为WUCAM、Vstm3或Vsig9)最初是利用生物信息学比对的方法作为CD28家族成员之一被发现。TIGIT是一种表达于多种免疫细胞(自然杀伤细胞/激活的T细胞/记忆T细胞/调节性T细胞/滤泡性T辅助细胞等)膜表面的一种共抑制受体，为免疫球蛋白超家族成员之一。据认为，TIGIT分子可能通过以下三种机制对免疫系统发挥调节作用：1)TIGIT与一种共刺激受体CD226竞争性结合表达于树突状细胞或癌细胞表面的共享配体CD155/CD112，向表达TIGIT的细胞传递抑制信号，抑制这类细胞的激活；2)TIGIT与共刺激受体CD226直接相互作用，破坏CD226的同源二聚化进而阻断其向下游传递的激活信号；3)TIGIT与表达于树突状细胞的CD155结合后，胞内段ITIM序列传导抑制信号进而表达免疫抑制细胞因子抑制免疫系统的激活。TIGIT可能在肿瘤微环境中的多种细胞中发挥作用。这些细胞可能是肿瘤浸润的CD8阳性T细胞，可能是调节性T细胞，也可能是NK细胞。总体来说，众多研究都表明第一种机制可能是TIGIT发挥其免疫抑制性作用的最关键机制。
已经证实，结合人TIGIT的抗体可用于治疗癌症。参见例如WO 2006/124667。在小鼠模型中，PD-L1和TIGIT二者的抗体阻断可以导致协同提高CD8+T细胞介导的肿瘤排斥。Grogan et al.(2014)J.Immunol.192(1)Suppl.203.15；Johnston et al.(2014)Cancer Cell 26：1-15。在黑色素瘤的动物模型中得到类似结果。Inozume et al.(2014)J.Invest.Dermatol.134：S121-Abstract 693。
鉴于TIGIT在免疫反应中的作用，TIGIT被认为是一个有吸引力的癌症免疫治疗靶点。因此，本领域存在着开发新的TIGIT抗体的需求，尤其是靶向TIGIT的CD155/CD112阻断型抗体，特别是人抗TIGIT抗体，及其组合疗法，用于疾病治疗，尤其是癌症治疗。
发明概述
本发明提供了抗TIGIT抗体及其编码基因与应用。通过基因工程手段和酵母表面展示技术，本发明人从展示在酵母表面的人抗体库中筛选出抗人TIGIT的全人源抗体，在此基础上进一步获得经亲和力成熟的高亲和力抗人TIGIT抗体。本发明的全人源抗体分子能够有效地阻断TIGIT与其配体CD155的结合，减少或消除传递至细胞的抑制性信号，增加IL-2产生，并且在体内施用时抑制肿瘤生长，尤其是在与抗PD-1抗体联用时，抑瘤效果尤为显著。因此，本发明的抗体可以用于多种用途，包括但不限于增强免疫反应、抑制肿瘤生长、抗感染和检测TIGIT蛋白。
本发明因此提供了一种新的结合人TIGIT的全人源抗体，以及其抗原结合片段。
在一些实施方案中，本发明的抗TIGIT抗体具有以下一个或多个特性：
(i)以高亲和力与人TIGIT结合；
(ii)具有与猴和/或鼠TIGIT的交叉免疫反应性；
(iii)与细胞表面的TIGIT有效结合；
(iv)阻断TIGIT与其配体CD155的结合；
(v)解除CD155与TIGIT的结合对TIGIT下游IL-2信号通路的抑制作用；
(vi)增加T细胞中的IL-2产生；
(vii)具有抗肿瘤活性，例如抑制肿瘤的生长；
(viii)与抗PD-1抗体组合具有更好的肿瘤抑制作用，例如能够更好地抑制肿瘤生长。
在一些实施方案中，本发明提供了结合TIGIT的抗体或其抗原结合片段，包含：SEQ ID NO：84-103所示的重链可变区之一的HCDR1、2和3序列，和/或SEQ ID NO：104-110所示的轻链可变区的LCDR1、2和3序列，或所述CDR序列组合的变体。
在一些实施方案中，本发明也提供抗TIGIT抗体或其抗原结合片段，所述抗体与本发明示例性抗体(如具有表B所列抗体VH和VL序列组合的抗体)结合相同或重叠的表位和/或竞争结合TIGIT、和/或抑制(例如，竞争性抑制)本发明示例性抗体。
在一些实施方案中，本发明提供了编码本发明抗体或其抗原结合片段的核酸，包含所述核酸的载体，包含所述载体的宿主细胞。
在一些实施方案中，本发明提供了制备本发明抗体或其抗原结合片段的方法。
在一些实施方案中，本发明提供了包含本发明抗体的免疫缀合物、药物组合物和组合产品。
本发明还提供了利用本发明抗体在受试者中阻断TIGIT与CD155的结合(例如，树突细胞或癌细胞表面表达的CD155)的方法。在一些实施方案中，通过所述方法，在表达TIGIT的细胞(尤其是T细胞、自然杀伤细胞)中降低或消除TIGIT介导的抑制信号传导，刺激T细胞和NK细胞的激活。在一些实施方案中，通过所述方法，增加T细胞中IL-2的产生。在一些实施方案中，通过所述方法，在表达CD155的细胞(例如树突细胞)中减少由CD155介导的抑制信号传导，减少免疫抑制细胞因子的表达。在一些实施方案中，通过所述方法，促进免疫系统的激活。因此，本发明也提供了利用本发明抗体预防或治疗癌症或感染的方法。
本发明还涉及在样品中检测TIGIT的方法。
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1A-F显示了亲和力成熟前后酵母表达的抗体对表达于CHO细胞表面的人TIGIT的结合。
图2显示了CHO细胞表达的7个候选抗体对表达于CHO细胞表面的人TIGIT的结合。
图3A-F显示了亲和力成熟前后酵母表达的抗体对CD155的阻断能力。
图4显示了CHO细胞表达的7个候选抗体对CD155的阻断能力。
图5A-E显示了亲和力成熟前后酵母表达的抗体的MOA生物学活性检测
图6显示了CHO细胞表达的7个候选抗体的MOA生物学活性检测
图7显示了候选分子ADI-30293和ADI-30278单独给药(10mg/kg)在人TIGIT敲入-MC38小鼠模型中的药效学研究。
图8A-B显示了候选分子ADI-30293和ADI-30278与抗PD1抗体antibody C(WO2017133540A1)联合给药(10+1mg/kg)在人TIGIT敲入-MC38小鼠模型中的药效学研究。
图9显示了用于本发明实施例中的示例性人TIGIT序列。
发明详述
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小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。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或多项的组合。
如本文中所用，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、整数或步骤组成的情形。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。
在本文中，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，所述免疫球蛋白可变区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、多克隆抗体、单链抗体或多链抗体、单特异性或多特异性抗体(例如双特异性抗体)、全人源抗体或嵌合抗体或人源化抗体、全长抗体和抗体片段，只要它们呈现期望的抗原结合活性即可。
本领域技术人员明了，“全抗体”(在本文中可与“全长抗体”、“完全抗体”和“完整抗体”互换使用)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)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。可变区是抗体的重链或轻链中参与抗体与其抗原结合的结构域。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。抗体的轻链可以基于其恒定结构域的氨基酸序列归入两种类型(称为kappa(κ)和lambda(λ))中的一种。抗体的重链可以取决于其重链恒定区的氨基酸序列而划分为主要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。对应于该5种不同抗体类型的重链恒定区分别称作α、δ、ε、γ和μ。术语“同种型”是指由抗体重链恒定区确 定的抗体类型。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)。
术语抗体的“抗原结合部分”(在本文中可与“抗体片段”和“抗原结合片段”互换使用)，是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。如本领域技术人员理解的，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。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、scFab、Fab’、F(ab’) 2、Fab’-SH、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(sdAb)。关于抗体片段的更详细的描述，可以参见：基础免疫学(Fundamental Immunology)，W.E.Paul编辑，Raven Press，N.Y.(1993)；邵荣光等人(编辑)，抗体药物研究与应用，人民卫生出版社(2013)；Hollinger等人，PNAS USA 90：6444-6448(1993)；Hudson等人，Nat.Med.9：129-134(2003)。
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，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，如本文所使用的，术语“人抗体”不包括其中的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“重组人抗体”包括所有通过重组方式制备、表达、产生或分离的人抗体，例如，(a)自用人免疫球蛋白基因进行转基因或转染色体的动物(例如小鼠)或由其制备的杂交瘤分离的抗体，(b)自转化成表达人抗体的宿主细胞例如转染瘤分离的抗体，(c)自重组、组合人抗体文库例如酵母展示文库分离的抗体，和(d)通过包括剪接人免疫球蛋白基因至其他DNA序列的任意其他方式制备、表达、产生或分离的抗体。这些重组人抗体具有构架区和CDR区源自人种系免疫球蛋白序列的可变区。然而，在某些实施方案中，可以对重组人抗体进行体外诱变(或使用人Ig序列转基因动物时为体内体细胞诱变)，由此得到的重组抗体的VH和VL区的氨基酸序列，尽管源自人种系VH和VL序列并与之相关、但是并不天然存在于体内的人抗体种系库中。
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。
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。
“分离的”抗体是已经与它的天然环境中的组分分离的抗体。在一些实施方案中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE、等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)确定。关于评价抗体纯度的方法的综述，参见，例如，Flatman，S.等，J.Chrom.B 848(2007)79-87。
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并置的非连续氨基酸形成。
在本文中，TIGIT是指″含Ig和ITIM结构域的T细胞免疫受体″。该表述也包括TIGIT 的变体、同种型、同源物、和物种同源物。术语“人TIGIT”指的是人序列TIGIT。一个具体的人TIGIT序列示于SEQ ID NO：208中。在一些实施方案中，人TIGIT序列与SEQ ID NO：208的人TIGIT氨基酸序列具有至少95％，甚至至少96％，97％，98％，或99％氨基酸序列同一性。TIGIT蛋白也可包括TIGIT的片段，诸如包含胞外结构域的片段，例如保持与本发明任何抗体结合能力的片段。在本文中，CD155，也称作PVR(脊髓灰质炎病毒受体)；PVS；HVED；CD155；NECL5；TAGE4；Necl-5，其与TIGIT相互作用诱导免疫抑制信号。
术语“特异性结合”表示抗体选择性地或优先地结合抗原。如果在生物光干涉测量中，抗体以大约5x10 -7M或更低、大约1x10 -7M或更低、大约5x10 -8M或更低、大约1x10 -8M或更低、大约5x10 -9M或更低的K D，与人TIGIT结合，则该抗体是“与人TIGIT特异性结合”的抗体。然而，特异性结合人TIGIT的抗体可以与来自其它物种的TIGIT蛋白具有交叉反应性。例如，特异于人TIGIT的抗体，在一些实施方案中，可以与非人物种的TIGIT蛋白交叉反应。在另一些实施方案中，特异于人TIGIT的抗体可以完全特异于人TIGIT而不表现出物种或其它类型的交叉反应性，或仅表现出对某些物种的TIGIT的交叉反应性。
如本文所用的，术语“交叉反应”是指抗体结合来自不同物种的TIGIT的能力。例如，本文所述的结合人TIGIT的抗体还可结合来自其它物种的TIGIT(例如，猴和/鼠TIGIT)。测定交叉反应性的方法包括实施例中所述的方法以及本领域已知的标准测定法，例如通过使用生物光干涉，或流式细胞术技术。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。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k dis和k on)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。
术语“高亲和力”对于IgG抗体指，该抗体以1x10 -7M或更低、优选地5x10 -8M或更低、更优选地大约1x10 -8M或更低、甚至更优选地大约5x10 -9M或更低的K D，与靶抗原结合。然而，“高亲和力”结合可以随抗体同种型而变。例如对于IgM同种型，“高亲和力”指抗体具有1x10 -6M或更低、优选地1x10 -7M或更低、更优选地大约1x10 -8M或更低的K D.
与结合例如TIGIT的抗原的参考抗体“竞争结合的抗体”是指这样的抗体，所述抗体在竞争检验中阻断参考抗体与抗原(例如TIGIT)结合的50％或更多，并且反过来，参考抗体在竞争检验中阻断该抗体与抗原(例如TIGIT)结合的50％或更多。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。
抑制(例如竞争性抑制)参照抗体与其抗原的结合的抗体是指这样的抗体，其抑制50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体抑制50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。抗体与其抗原的结合可以亲和力(例如平衡解离常数)衡量。测定亲和力的方法是本领域已知的。
与参照抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有参照抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。
本文中的术语“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-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”在本文中指，与参考抗体相比，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的抗体，其中变体基本上保持改变之前的抗体分子的至少一个生物学特性(例如，抗原结合能力)。目标抗体区域可以是抗体全长、或重链可变区或轻链可变区或其组合、或(一个或多个)重链CDR区或(一个或多个)轻链CDR区或其组合。在本文中，相对于参考抗体区域具有氨基酸改变的抗体区域，也称作该抗体区域的“变体”。
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。在本文中，在不指定比较窗(即待比较的目标抗体区域)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。
类似地，就抗体序列而言，基于比对，可以确定相对于参考抗体在目标抗体区域具有氨基酸改变的候选抗体。
在本发明中，“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。可以通过本领域已知的标准方法，例如定点诱变和PCR介导的诱变，将氨基酸修饰如取代引入本发明的抗体中。
提供功能上相似氨基酸的保守性置换表是本领域熟知的。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中所示的优选保守取代残基。
表A
原始残基 示例性取代 优选的保守取代
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
本发明的各方面将在下面各小节中进一步详述。
I.本发明的抗TIGIT抗体
本发明一方面提供特异性地结合TIGIT，优选人TIGIT蛋白质(例如SEQ ID NO：208的人TIGIT序列)的抗体或其抗原结合片段，尤其是全人源抗体或其抗原结合片段。在一些实施方案中，本发明抗体的抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体例如scFv、(Fab’) 2片段、单结构域抗体、双抗体(dAb)或线性抗体。
抗体的有利生物性质
在一些实施方案中，本发明的抗TIGIT抗体或其抗原结合片段以高亲和力与人TIGIT结合，例如，解离平衡常数(K D)小于100x10 -9M，小于或等于大约50x10 -9M，更优选地大约1-30x10 -9M，更优选地小于或等于大约1x10 -9M，更优选地大约1-10x10 -10M。优选地，K D通过使用生物光干涉测定法(例如Fortebio亲和测量法)测定。在一些实施方案中，K D值通过测量抗体Fab形式(例如酵母表达的Fab)与人TIGIT的单价亲和力来确定，优选地单价K D值为1-100x10 -10M，更优选1-50x10 -10M，更优选1-10x10 -10M或2-5x10 -10M。在另一些实施方案中，K D值通过测量完整抗体(例如CHO细胞表达的完整抗体)与人TIGIT的单价亲和力来确定，优选地单价K D值为1-50x10 -10M，更优选1-30x10 -10M或1-10x10 -10M。
在一些实施方案中，本发明的抗TIGIT抗体或其抗原结合片段与猴TIGIT交叉反应。在一些实施方案中，抗体以高亲和力与猴TIGIT结合，其中K D值(例如通过测量完整抗体与猴TIGIT的单价亲和力来确定)为大约0.1-100x10 -9M，更优选0.1-50x10 -9M或1-30x10 -10M。 在一些实施方案中，抗体与鼠TIGIT交叉反应，其中K D值(例如通过测量完整抗体与鼠TIGIT的单价亲和力来确定)为大约1-100x10 -9M，例如1-10x10 -7M或1-10x10 -8M，或1-10x10 -9M。在另一些实施方案中，本发明抗TIGIT抗体不与鼠TIGIT交叉反应。
在一些实施方案中，本发明抗体或其抗原结合片段以高亲和力结合细胞表面表达的TIGIT。在一个实施方案中，表面表达人TIGIT的细胞是CHO细胞。优选地，以流式细胞术(例如FACS)测定，抗体与表达人TIGIT的细胞结合的EC50值。在一些实施方案中，抗体为酵母表达的完整抗体，EC50值小于大约10nM，例如0.1-1nM，优选地，小于或等于大约1nM，更优选地大约0.2-0.9nM，如0.9nM、0.6M或0.4nM。在另一些实施方案中，抗体为CHO细胞表达的完整抗体形式，EC50值小于大约10nM，例如0.1-1nM，优选地，大约0.1-0.3nM，如约0.3nM、0.2M或0.1nM。
在一些实施方案中，本发明抗体或其抗原结合片段抑制TIGIT的相关活性。在一些实施方案中，本发明抗体或其抗原结合片段阻断TIGIT与其配体CD155的结合。优选地，以流式细胞术(例如FACS)测定抗体阻断人TIGIT(细胞上表达的TIGIT)与人CD155结合的能力(如IC50值)。在一些实施方案中，抗体为酵母表达的完整抗体，IC50值小于大约10nM，例如0.1-2nM，优选地，大约0.1-1.0nM，如0.8nM、0.6M、0.4nM或0.2nM。在另一些实施方案中，抗体为CHO细胞表达的完整抗体形式，EC50值小于大约1nM，例如0.1-0.5nM，优选地，约0.3nM、0.2M或0.1nM。
在一些实施方案中，本发明抗体或其抗原结合片段减少或消除由TIGIT结合CD155引起的抑制性信号传导。在一些实施方案中，本发明抗体或片段在表达TIGIT的细胞(尤其是T细胞)中降低或消除TIGIT介导的抑制性信号传导。在一些实施方案中，本发明抗体或其抗原结合片段诱导T细胞中IL2启动子下游基因的表达，在一些实施方案中增加T细胞的IL-2产生。在一些实施方案中，通过荧光报告基因试验(如实施例5的MOA试验)，检测抗体减少或消除由TIGIT结合CD155引起的抑制性信号传导的能力(如EC50值)。在一些实施方案中，抗体为酵母表达的完整抗体，EC50值优选小于大约10nM，例如0.1-5nM，优选地，大约0.1-3.0nM，如约2.nM、1.5nM、1.0nM、或0.5nM。在另一些实施方案中，抗体为CHO细胞表达的完整抗体，EC50值优选地小于5nM，例如大约0.1-3.0nM，如约1.6nM、1.2nM、1.0nM、或约0.18-0.5nM。
在一些实施方案中，本发明的抗体或其抗原结合片段抑制含有表达人TIGIT的浸润淋巴细胞的肿瘤的生长。在一个实施方案中，肿瘤细胞是胃肠道肿瘤，优选结肠直肠癌。例如，在体内肿瘤移植模型中，例如MC38小鼠中，抑制结肠癌细胞的生长。在一些实施方案中，本发明抗体与抗PD1抗体联用，获得显著好于单独一种抗体施用时的抗肿瘤效果。
优选地，本发明抗体或其抗原结合片段表现出上述性质中的至少一个、更优选地至少二个、更优选地至少三个、四个、或五个，甚至更优选地上述所有性质。
抗体CDR区
“互补决定区”或“CDR区”或“CDR”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。
下表B中给出了本发明的一些示例性抗体的VH和VL序列组合：
Figure PCTCN2019097665-appb-000001
本领域公知多种用于在一个给定的VH或VL氨基酸序列中确定其CDR序列的方案。例如，Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196：901-917(1987))。AbM HVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。根据不同的CDR确定方案，这些HVR中的每一个HVR/CDR的残基如下所述。
Figure PCTCN2019097665-appb-000002
Figure PCTCN2019097665-appb-000003
HVR也可以是根据Kabat编号系统位于如下Kabat残基位置的HVR序列：
VL中的位置24-36或24-34(LCDR1)，位置46-56或50-56(LCDR2)，和位置89-97或89-96位置(LCDR3)；和VH中的位置26-35或27-35B(HCDR1)，位置50-65或49-65(HCDR2)，和位置93-102、94-102或95-102(HCDR3)。
在一个实施方案中，本发明抗体的HVR是根据Kabat编号系统位于如下Kabat残基位置的HVR序列：
VL中的位置24-34(LCDR1)、位置50-56(LCDR2)、和位置89-97(LCDR3)，以及VH中的位置27-35B(HCDR1)、位置50-65(HCDR2)、和位置93-102(HCDR3)。
在一个实施方案中，本发明抗体的HVR是根据Kabat编号系统位于如下Kabat残基位置的HVR序列：
VL中的位置24-34(LCDR1)、位置50-56(LCDR2)、和位置89-97(LCDR3)，以及VH中的位置26-35B(HCDR1)、位置50-65(HCDR2)、和位置95-102(HCDR3)。
HVR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”或“HVR”或“HVR序列”涵盖以上述任一种方式确定的HVR或CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.(1991))的编号位置。
在一个优选的实施方案中，本发明CDR序列如表2所示，其中HCDR1为根据AbM方案确定的CDR序列；其余CDR为根据Kabat方案确定的CDR序列。
在另一个优选的实施方案中，本发明CDR序列如表1所示。
下表C给出了本发明的一些示例性CDR序列组合：
Figure PCTCN2019097665-appb-000004
Figure PCTCN2019097665-appb-000005
下表D给出了本发明的另一些示例性CDR序列组合：
Figure PCTCN2019097665-appb-000006
Figure PCTCN2019097665-appb-000007
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat，Chothia，AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
在一些实施方案中，本发明的抗体有至少一个、两个、三个、四个、五个或六个CDR与表B所列任一抗体的可变区序列中的对应CDR相同，或是其变体。在一些实施方案中，本发明的抗体有至少一个、两个、或三个HCDR与表B所列任一抗体的可变区序列中的对应重链CDR相同，或是其变体。在一些实施方案中，本发明的抗体有至少一个、两个、或三个LCDR与表B所列任一抗体的可变区序列中的对应轻链CDR相同，或是其变体。在本文中，“对应CDR”是指，在最佳比对后，在候选抗体的可变区氨基酸序列中与参考抗体的CDR位于最相似位置上的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变体的抗原结合分子基本上保持包含未修饰CDR的抗原结合分子的生物学特性，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，氨基酸取代优选发生在与本文提供的共有CDR序列(例如，SEQ ID NO：5，10，15，21，24，30，33，37，40，47，52，56，62，65，68，74，77，80)的X残基相对应的氨基酸位置上。
此外，本领域已知，CDR3区，独立于CDR1和/或CDR2区，单独可以确定抗体对关联抗原的结合特异性。并且，可以基于共同CDR3序列，产生具有相同结合特异性的多种其它抗体。参见例如，US Patents Nos.6,951,646；6,914,128；6,090,382；6,818,216；6,156,313； 6,827,925；5,833,943；5,762,905和5,760,185。所有这些参考文献并入本文作为参考。
因此，在一个实施方案中，本发明抗体包含来自表B所示任一抗体的重链和/或轻链可变区序列的CDR3序列，其中所述抗体能够特异地结合人TIGIT。在再一实施方案中，所述抗体还可以包含来自同一抗体的重链和/或轻链可变区的CDR2，或来自不同的TIGIT抗体的重链和/或轻链可变区的CDR2。在再一实施方案中，所述抗体还可以包含来自同一抗体的重链和/或轻链可变区的CDR1，或来自不同的TIGIT抗体的重链和/或轻链可变区的CDR1。可以通过本文描述的测定方法，表征这些抗体的活性，包括与人TIGIT的结合活性、阻断TIGIT与CD155分子结合的活性、和/或抑制肿瘤生长的活性。
再一方面，考虑到抗原结合特异性主要由CDR1、2和3区提供，在一些实施方案中，可以将VH CDR1、2和3序列和VL CDR1、2和3序列“混合并匹配”(即，可以混合并匹配来自结合同一TIGIT抗原的不同抗体的CDR，不过每种抗体优选地含有VH CDR1、2和3和VL CDR1、2和3)，以产生结合TIGIT的本发明其他分子。可以使用本领域已知的结合测定法(例如，ELISA、SET、Biacore)和实施例中描述的那些测定法，测试这类“混合和匹配的”抗体与TIGIT的结合。当混合并匹配VH CDR序列时，来自特定VH序列的CDR1、CDR2和/或CDR3序列优选地替换为结构上相似的CDR序列。同样，当混合并匹配VL CDR序列时，来自特定VL序列的CDR1、CDR2和/或CDR3序列优选地替换为结构上相似的CDR序列。可以在本发明表3所示抗体之间进行CDR的“混合和匹配”。此外，本领域技术人员明了，也可以通过用来自其它不同抗体的一个或多个VH和/或VL CDR区序列，置换本文中所示抗体的结构上相似的CDR序列，以产生本发明的其它抗体。
因此，在一些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区，所述重链可变区包含重链互补决定区3(HCDR3)，所述HCDR3：
(i)与表B列出的任一抗体的重链可变区的HCDR3相同；或
(ii)与表C或D所列任一HCDR3序列相同；或
(iii)相对于(i)或(ii)的HCDR3，包含至少1个(优选1-2个或更优选1个)氨基酸改变(优选取代、更优选保守取代)。
在一些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，且所述抗体的重链互补决定区3(HCDR3)和轻链互补决定区3(LCDR3)：
(i)与表B列出的任一抗体的重链和轻链可变区序列的HCDR3和LCDR3相同；或
(ii)与表C或D所列任一组合中的HCDR3和LCDR3序列相同；或
(iii)相对于(i)或(ii)的HCDR3和LCDR3，共包含至少1个(优选1-2个或更优选1个)氨基酸改变(优选取代、更优选保守取代)。
在一个实施方案中，本发明抗体或其抗原结合片段包含重链可变区(VH)，其中所述VH包含：
(i)表B所列任一抗体的VH序列中所含的HCDR1、HCDR2和HCDR3序列；或
(ii)表C或D所列任一组合中的HCDR1、HCDR2和HCDR3序列；或
(iii)相对于(i)或(ii)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2、或1个氨基酸改变(优选氨基酸取代，优选保守取代)的序列。
在另一个实施方案中，本发明抗体或其抗原结合片段包含轻链可变区(VL)，其中所述VL 包含：
(i)表B所列任一抗体的VL序列中所含的LCDR1、LCDR2和LCDR3序列；或
(ii)表C或D所列任一组合中的LCDR1、LCDR2和LCDR3序列；或
(iii)相对于(i)或(ii)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2、或1个氨基酸改变(优选氨基酸取代，优选保守取代)的序列。
在另一个实施方案中，本发明抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述抗体包含：
(i)表B所列任一抗体的VH和VL序列中所含的6个CDR序列；或
(ii)表C或D所列任一组合中的6个CDR序列；或
(iii)相对于(i)或(ii)的序列，在6个CDR区上共包含至少一个且不超过10或5、4、3、2、1个氨基酸改变(优选氨基酸取代，优选保守取代)的序列。
在一个实施方案中，本发明抗体或其抗原结合片段包含：
(i)如SEQ ID NO：84、85、86、或87所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：104所示的轻链可变区的LCDR1、2和3序列，或者
(ii)如SEQ ID NO：88、89或90所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：105或106所示的轻链可变区的LCDR1、2和3序列，或者
(iii)如SEQ ID NO：91、92或93所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：107所示的轻链可变区的LCDR1、2和3序列，或者
(iv)如SEQ ID NO：94、95、96或97所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：108所示的轻链可变区的LCDR1、2和3序列，或者
(v)如SEQ ID NO：98、99或100所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：109所示的轻链可变区的LCDR1、2和3序列，或者
(vi)如SEQ ID NO：101、102或103所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：110所示的轻链可变区的LCDR1、2和3序列。
在一个优选实施方案中，本发明抗体或其抗原结合片段包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中
(i)HCDR1包含选自SEQ ID NO：1-5和178-181的氨基酸序列或由其组成，HCDR2包含选自SEQ ID NO：6-10的氨基酸序列或由其组成，HCDR3包含选自SEQ ID NO：11-15和182-185的氨基酸序列或由其组成，LCDR1包含SEQ ID NO：16的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：17的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO：18的氨基酸序列或由其组成；或者
(ii)HCDR1包含选自SEQ ID NO：19-21和186-187的氨基酸序列或由其组成，HCDR2包含选自SEQ ID NO：22-24的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：25和188的氨基酸序列或由其组成，LCDR1包含SEQ ID NO：26的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：27的氨基酸序列或由其组成，且LCDR3包含选自SEQ ID NO：28-30的氨基酸序列或由其组成；或者
(iii)HCDR1包含选自SEQ ID NO：31-33和189-190的氨基酸序列或由其组成，HCDR2包含选自SEQ ID NO：34-37的氨基酸序列或由其组成，HCDR3包含选自SEQ ID NO：38-40和 191-192的氨基酸序列或由其组成，LCDR1包含SEQ ID NO：41的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：42的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO：43的氨基酸序列或由其组成；或者；
(iv)HCDR1包含选自SEQ ID NO：44-47和193-195的氨基酸序列或由其组成，HCDR2包含选自SEQ ID NO：48-52的氨基酸序列或由其组成，HCDR3包含选自SEQ ID NO：53-56和196-198的氨基酸序列或由其组成，LCDR1包含SEQ ID NO：57的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：58的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO：59的氨基酸序列或由其组成；或者
(v)HCDR1包含选自SEQ ID NO：60-62和199-200的氨基酸序列或由其组成，HCDR2包含选自SEQ ID NO：6和63-65的氨基酸序列或由其组成，HCDR3包含选自SEQ ID NO：66-68和201-202所示的氨基酸序列或由其组成，LCDR1包含SEQ ID NO：69的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：17的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO：70的氨基酸序列或由其组成；或者
(vi)HCDR1包含选自SEQ ID NO：71-74和203-205的氨基酸序列或由其组成，HCDR2包含选自SEQ ID NO：22和75-77的氨基酸序列或由其组成，HCDR3包含选自SEQ ID NO：78-80和206-207所示的氨基酸序列或由其组成，LCDR1包含SEQ ID NO：81的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：82的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO：83的氨基酸序列或由其组成。
在一个优选实施方案中，本发明抗体或其抗原结合片段包含表C所列组合之一的6个CDR序列。
在另一个优选实施方案中，本发明抗体或其抗原结合片段包含表D所列组合之一的6个CDR序列。
抗体可变区
“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。重链可变区(VH)和轻链可变区(VL)可以进一步再划分为高变区(HVR，又称作互补决定区(CDR))，其间插有较保守的区域(即，构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。在一些情况下，单个VH或VL结构域足以赋予抗原-结合特异性。此外，结合特定抗原的抗体可以使用来自结合所述抗原的抗体的VH或VL结构域筛选互补VL或VH结构域文库而分离(参见，例如，Portolano，S.等，J.Immunol.150(1993)880-887；Clackson，T.等，Nature 352(1991)624-628)。
本领域已知，可以对两个可变区之一或两者(即VH和/或VL)中的一个或多个残基进行修饰，例如，对一个或多个CDR区和/或对一个或多个构架区进行残基修改，尤其是保守残基取代，而修饰后的抗体仍基本上保持改变之前的抗体分子的至少一个生物学特性(例如，抗原结合能力)。例如，可以突变CDR区的残基，改善抗体的一种或多种结合性质(例如亲和性)。可以在体外或体内测定试验中，评估突变后的抗体的抗体结合性质或其它功能性质。优选地，引入保守替代。优选地，在CDR区中引入的残基改变不超过1个、2个、3个、4个或5个。此外，可以突变构架区残基，例如以改善抗体的性质。例如，可以将一个或多个构架残基“回复突变”为对应的种系序列残基。
CDR移植是本领域已知的另一种抗体可变区修饰方式。由于CDR序列负责大多数抗体- 抗原相互作用，故可以构建模拟已知抗体的性质的重组抗体变体。在该抗体变体中，来自已知抗体的CDR序列被移植到具有不同性质的不同抗体的构架区上。因此，在一个实施方案中，本发明涉及这样的抗TIGIT抗体或其抗原结合片段，所述抗体包含来自表B抗体之一的重链和轻链可变区的CDR序列，但具有不同的构架区序列。可以从公共DNA数据库，包括种系抗体基因序列，或从公开文献报道的TIGIT抗体序列，获得用于替换的构架区序列。例如，可以从GenBank数据库获得编码人重链和轻链可变区基因的种系DNA。可以将抗体蛋白序列与数据库中的蛋白序列，使用序列相似性检索工具，例如Gapped BLAST，进行比较。优选，用于替代的构架序列，与选择进行改变的本发明抗体的构架序列，具有结构相似性，例如，具有序列同一性至少80％，85％，90％，或95％、96％、97％、98％、99％以上的构架序列。
在再一实施方案中，可以“混合并匹配”来自本发明示例性抗体(表B所示抗体之一)与其它不同抗TIGIT抗体(优选地，表B所示另一抗体)的VH和VL序列，以产生结合TIGIT的本发明其他抗体。在混合和匹配这些链时，优选地，将来自具体VH/VL配对的VH序列替换为结构相似的VH序列。同样，来自特定VH/VL配对的VL序列优选地替换为结构上相似的VL序列。可以使用本领域已知的结合测定法(例如，ELISA，和实施例部分中描述的其他测定法)测试这类“混合和匹配的”抗体与TIGIT的结合。
因此，在一个实施方案中，本发明的抗体包含表B所列任一抗体的重链可变区VH序列，或由所述氨基酸序列组成。在再一实施方案中，本发明的抗体包含所述VH序列的变体。
在另一个实施方案中，本发明的抗体包含表B所列任一抗体的轻链可变区VL序列，或由所述氨基酸序列组成。在再一实施方案中，本发明的抗体包含所述VL序列的变体。
在再一实施方案中，本发明的抗体包含：
(i)包含SEQ ID NO：84或85或86或87所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO：104所示的氨基酸序列的VL序列或其变体，或者
(ii)包含SEQ ID NO：88或89或90所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO：105或106所示的氨基酸序列的VL序列或其变体，或者
(iii)包含SEQ ID NO：91或92或93所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO：107所示的氨基酸序列的VL序列或其变体，或者
(iv)包含SEQ ID NO：94或95或96或97所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO：108所示的氨基酸序列的VL序列或其变体，或者
(v)包含SEQ ID NO：98或99或100所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO：109所示的氨基酸序列的VL序列或其变体；
(vi)包含SEQ ID NO：101或102或103所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO：110所示的氨基酸序列的VL序列或其变体。
在一个实施方案中，VH序列的变体在氨基酸序列上，与参考VH序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，VH序列的变体在氨基酸序列上，与参考VH 序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，包含至少一个且不超过30个、10个、或5、4、3、2、1、0氨基酸改变(优选氨基酸取代，优选保守取代)。优选地序列差异不发生在CDR区中。
在一个优选的实施方案中，VL序列的变体在氨基酸序列上，与参考VL序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性。在一个优选的实施方案中，VL序列的变体在氨基酸序列上，与参考VL序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，包含至少一个且不超过30个、10个、或5、4、3、2、1、0氨基酸改变(优选氨基酸取代，优选保守取代)。优选地序列差异不发生在CDR区中。
在一个优选的实施方案中，本发明的抗体包含表B所列任一抗体的重链可变区和轻链可变区VH/VL序列对，或由所述氨基酸序列对组成。本发明也提供该抗体的变体，例如在VH、VL或VH和VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在上述任一实施方案中，优选地，相对于参考抗体，抗体变体的重链可变区在1个或多个CDR(优选全部3个CDR)区域上包含不超过10个，优选不超过5个(例如，3、2、1或0个)氨基酸改变(优选氨基酸取代，优选保守取代)。
在上述任一实施方案中，优选地，相对于参考抗体，抗体变体的轻链可变区VL在1个或多个CDR(优选全部3个CDR)区域上包含不超过10个，优选不超过5个(例如，3、2、1或0个)氨基酸改变(优选氨基酸取代，优选保守取代)。
抗体重链和轻链
在一些实施方案中，本发明抗体包含重链Fc区，例如IgG1，IgG2或IgG4同种型的Fc区。在一个实施方案中，本发明抗体含有IgG4-Fc区，其中在氨基酸残基228位置(EU编号)具有丝氨酸至脯氨酸突变(S228P)。在再一优选实施方案中，本发明抗体包含IgG4-PAA Fc部分。该IgG4-PAA Fc部分在位置228具有丝氨酸至脯氨酸突变(S228P)，并且在位置234(EU编号)具有苯丙氨酸至丙氨酸突变，在位置235(EU编号)具有亮氨酸至丙氨酸突变。S228P突变是肿瘤恒定区的铰链区的突变，可以减少或消除重链间二硫桥异质性。F234A和L235A突变可以进一步降低(已经具有低效应子功能的)人IgG4同种型的效应子功能。在一些实施方案中，本发明抗体含有去除了重链C末端赖氨酸(des-Lys)的IgG4-PAA Fc部分。在一些实施方案中，本发明抗体包含κ轻链恒定区，例如人κ轻链恒定区。
在再一优选的实施方案中，Fc区包含SEQ ID NO：177的氨基酸序列，或相对于SEQ ID NO：177的氨基酸序列包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与SEQ ID NO：177的氨基酸序列具有至少95-99％同一性的序列。
在一个优选的实施方案中，本发明抗体包含轻链恒定区。在一个优选实施方案中，轻链恒定区为人κ轻链恒定区。在再一优选实施方案中，轻链恒定区包含SEQ ID NO：209的氨基酸序列，或相对于SEQ ID NO：209的氨基酸序列包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与SEQ ID NO：209的氨基酸序列具有至少95-99％同一性的序列。
在一些优选的实施方案中，本发明抗体包含重链，并且所述重链包含选自SEQ ID  NO：111-130的氨基酸序列、或相对于其包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。优选地，氨基酸改变不发生在CDR区中，更优选地，不发生在可变区中。
在一些优选的实施方案中，本发明抗体包含轻链，并且所述轻链包含选自SEQ ID NO：137-143的氨基酸序列、或相对于其包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。优选地，氨基酸改变不发生在CDR区中，更优选地，不发生在可变区中。
在一个优选实施方案中，本发明的抗体包含选自以下的重链序列和/或轻链序列：
(a)包含选自SEQ ID NO：111-114的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO：137的氨基酸序列的轻链序列或其变体；
(b)包含选自SEQ ID NO：115-117的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO：138或139的氨基酸序列的轻链序列或其变体；
(c)包含选自SEQ ID NO：118-120的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO：140的氨基酸序列的轻链序列或其变体；
(d)包含选自SEQ ID NO：121-124的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO：141的氨基酸序列的轻链序列或其变体；
(e)包含选自SEQ ID NO：125-127的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO：142的氨基酸序列的轻链序列或其变体；
(d)包含选自SEQ ID NO：128-130的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO：143的氨基酸序列的轻链序列或其变体，
其中所述变体与对应的参考序列相比包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。优选地，氨基酸改变不发生在CDR区，更优选地不发生在可变区。
在一个实施方案中，在抗体的恒定区中进行残基修饰，以例如改变抗体的性质，例如效应子功能。
在一些实施方案中，本发明抗TIGIT抗体或其片段的重链和/或轻链还包含信号肽序列，例如METDTLLLWVLLLWVPGSTG。
示例性抗体序列
本发明提供如实施例中分离并表征的特异性结合TIGIT(例如人TIGIT)的全人源抗体。下表3中列出了本发明这些示例性抗体的抗体可变区VH和VL序列。下表1和2中列出了抗体的示例性CDR序列。序列表显示了本发明示例性抗体的重链和轻链氨基酸序列和本发明示 例性抗体可变区VH和VL的编码核苷酸序列。
抗体变体
在一方面，本发明提供在本文中所述及的任何抗体，尤其是表B所列示例性抗体的变体。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，所述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轻链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。在一些实施方案中，在一个或多个或全部6个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，抗体变体与参考抗体在目标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。例如，在一个实施方案中，本发明抗体，与参考抗体(例如表3所列抗体之一)相比，在3个重链CDR区域上具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％或更高的同一性。在一个实施方案中，本发明抗体，与参考抗体(例如表3所列抗体之一相比)，在3个轻链CDR区域上具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％或更高的同一性。在另一实施方案中，本发明抗体，与参考抗体(例如表3所列抗体之一相比)，在6个CDR区域上具有至少90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％或更高的同一性。在再一实施方案中，本发明抗体，与参考抗体(例如表3所列抗体之一)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。在再一实施方案中，本发明抗体，与参考抗体(例如表3所列抗体之一)相比，在轻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。在再一实施方案中，本发明抗体，与参考抗体(例如表3所列抗体之一)相比，在重链可变区和/或轻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。
此外，可以对抗体的Fc区进行改变。Fc区的改变可以单独进行，或与上述对构架和/或CDR区的改变相组合。可以改变Fc区，例如，以改变抗体的一种或多种功能，例如血清半衰期，补体固定、Fc受体结合、和/或抗原依赖性细胞毒性。此外，还可以对本发明抗体进行化学修饰(例如，与PEG连接)或改变其糖基化模式。
在某些实施方案中，Fc区可以包含具有一个或多个提高ADCC活性的氨基酸置换的Fc-区，例如，Fc-区的位置298、333和/或334的置换(残基的EU编号)。在一些实施方案中，也可以对Fc-区进行改变，以导致改变的(即，提高的或降低的)C1q结合和/或补体依赖性细胞毒性(CDC)(参见，例如，US6,194,551、WO99/51642和Idusogie，E.E.等，J.Immunol.164(2000)4178-4184)。
在另一些实施方案中，可以对Fc进行改变以增加或降低其糖基化程度和/或改变其糖基 化模式。对Fc的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。举例而言，可实施一或多种氨基酸取代以消除一或多个糖基化位点，由此消除该位点处的糖基化。可制备具有改变类型的糖基化的抗体，例如具有减小量的岩藻糖基残基的低或无岩藻糖化抗体或具有增加的等分GlcNac结构的抗体。这类改变的糖基化模式已显示可增加抗体的ADCC能力。本发明也考虑在与Fc区连接的寡糖中具有至少一个半乳糖残基的抗体变体。这些抗体变体可具有提高的CDC功能。
在某些实施方案中，本发明也考虑具有一些但非所有效应子功能的抗体变体，这使其成为某些应用的理想候选物，在所述应用中抗体的体内半衰期是重要的，但某些效应子功能(如补体和ADCC)是不必要或有害的。例如，Fc区可以包含消除或减弱效应子功能的突变，例如具有突变P329G和/或L234A和L235A的人IgG1Fc区，或具有突变P329G和/或S228P和L235E的人IgG4Fc区。
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。例如，可以改变抗体铰链区中的半胱氨酸残基数目，以例如利于轻链和重链的装配或增加或降低抗体的稳定性。参见例如美国专利号5,677,425。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有非蛋白质部分。适合抗体衍生的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)，以例如增加抗体的(例如血清)半衰期。用于蛋白质PEG化的方法是本领域已知的，可以将其应用于本发明的抗体。参见例如EP 0154 316和EP 0401384。
II.多核苷酸、载体和宿主
本发明提供编码以上任何抗TIGIT抗体或其片段的核酸。还提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。还提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)。在另一个实施方案中，宿主细胞是原核的。
在一方面，本发明提供编码以上任何抗TIGIT抗体或其片段的核酸。所述核酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的核酸，或包含编码抗体的轻链和/或重链的氨基酸序列的核酸。示例性的编码抗体重链可变区的核酸序列包含与选自SEQ ID NO：150-169的核酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的核酸序列，或者包含选自SEQ ID NO：150-169的核酸序列。示例性的编码抗体轻链可变区的核酸序列包括与选自SEQ ID NO：170-176的核酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的核酸序列，或者包含选自SEQ ID NO：170-176的核酸序列。从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TIGIT抗原结合能力。
本发明中还提供多核苷酸，所述多核苷酸编码来自上文所述的结合TIGIT的抗体的重链VH或轻链VL序列的至少一个CDR区和通常全部三个CDR区。一些进一步的实施方案中，多核苷酸编码上文所述的结合TIGIT的抗体的重链和/或轻链的完整或基本上完整可变区序列。
如本领域技术人员明了的，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。
在一个优选实施方案中，编码抗体的本发明核酸还包含编码重链Fc区的核苷酸序列，例如SEQ ID NO：177中所示的Fc区序列或与其基本相同的序列。
在一个优选实施方案中，编码抗体的本发明核酸还包含编码轻链恒定区序列的核苷酸序列，例如SEQ ID NO：209中所示的序列或与其基本相同的序列。
可以采用本领域熟知的方法，通过从头固相DNA合成或通过PCR诱变编码结合TIGIT的抗体或其抗原结合片段的现有序列(例如，SEQ ID NO：150-169中所示的VH DNA序列，SEQ ID NO：170-176中所示的VL DNA序列)，产生这些多核苷酸序列。
在一个实施方案中，提供包含本发明核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在优选的实施方案中，本发明的表达载体是pTT5表达载体。
在一个实施方案中，提供包含所述载体的宿主细胞。用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，抗体可在细菌中产生，特别当不需要糖基化和Fc效应子功能时。对于抗体片段和多肽在细菌中的表达，见，例如，美国专利号5,648,237，5,789,199和5,840,523，还见Charlton，Methods in Molecular Biology，卷248(B.K.C.Lo，编辑，Humana Press，Totowa，NJ，2003)，第245-254页，其描述抗体片段在大肠杆菌中的表达。在表达后，在可溶级分中的抗体可以与细菌细胞糊状物分离，并且可以进一步纯化。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是用于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖基化模式的抗体。参见Gerngross，Nat.Biotech.22：1409-1414(2004)，和Li等，Nat.Biotech.24：210-215(2006)。适于表达糖基化抗体的宿主细胞也可以衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的一些实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(293HEK或293细胞，如例如Graham等，J.Gen Virol.36：59(1977)中所描述的)等。其它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等，Proc.Natl.Acad.Sci.USA 77：216(1980))；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。关于适合产生抗体的某些哺乳动物宿主细胞系的综述见例如Yazaki和Wu，Methods in Molecular Biology，卷248(B.K.C.Lo，ed.，Humana Press，Totowa，NJ)，第255-268页(2003)。
III.抗体的制备
在一个实施方案中，提供了制备抗TIGIT抗体的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体的核酸的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。为了重组产生抗TIGIT抗体，分离编码抗体(例如上文所描述的抗体)的核酸，并将其插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序(例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针进行)。
IV.测定法
可以通过本领域中已知的多种测定法对本文中提供的抗TIGIT抗体鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
一方面，对本发明的抗体测试其抗原结合活性。例如，可以通过本领域已知的方法，诸如ELISA，Western印迹等，或本文实施例公开的例示性方法，来测定与人TIGIT的结合。例如，可以使用流式细胞术进行测定，其中抗体与表达人TIGIT的细胞系，例如经转染在细胞表面上表达人TIGIT的CHO细胞，进行反应。其它的细胞也适用于流式细胞术，包括表达天然TIGIT的T细胞。备选地，抗体的结合，包括结合动力学(例如K D值)，可以使用重组TIGIT蛋白，在生物光干涉测定法中测定。在一些实施方案中，使用例如Fortebio亲和测量法。
另一方面，可使用竞争测定法来鉴定与本文中公开的任何抗TIGIT抗体竞争结合TIGIT的抗体。在某些实施方案中，此类竞争性抗体结合与本文中公开的任何抗TIGIT抗体所结合表位相同或重叠的表位(例如线性或构象表位)。用于定位抗体所结合表位的详细例示性方法见Morris(1996)“Epitope Mapping Protocols”，Methods in Molecular Biology vol.66(Humana Press，Totowa，NJ)。
本发明还提供了用于鉴定具有生物学活性的抗TIGIT抗体的测定法。生物学活性可以包括例如结合TIGIT(例如人TIGIT)，阻断TIGIT(例如人TIGIT)与CD155分子的结合、阻断TIGIT介导的抑制性信号传导、增加T细胞的IL2产生、和/或抑制肿瘤生长。例如，在体内肿瘤抑制模型中(参见例如实施例6)，测试抗体抑制肿瘤生长的能力。在本发明也提供在体内和/或在体外具有此类生物学活性的抗体。
可以理解的是，能够使用本发明的免疫缀合物或多特异性抗体替换或补充抗TIGIT抗体来进行任何上述测定。
V.多特异性抗体
在再一方面，本发明提供特异性地结合TIGIT(优选人TIGIT)的多特异性(包括双特异性)抗体分子。在一个实施方案中，在多特异性抗体中，本发明的抗体(或其抗原结合片段)形成针对TIGIT的第一结合特异性。在再一实施方案中，所述多特异性抗体还具有第二特异性、或在再一实施方案中还包含第二和第三结合特异性。在再一实施方案中，所述多特异性抗体是双特异性抗体。
在一个实施方案中，结合特异性由抗体的“结合位点”或“抗原结合位点”(抗体分子中与抗原实际结合的区域)提供。在一个优选的实施方案中，抗原结合位点由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)组成的VH/VL对构成。因此，在一个实施方案中，“多特异性”抗体是具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点可以与相同抗原的不同表位或与不同抗原的不同表位结合。
有关多特异性抗体及其制备，可以参见例如WO 2009/080251、WO 2009/080252、WO 2009/080253、WO 2009/080254、WO 2010/112193、WO 2010/115589、WO 2010/136172、WO 2010/145792和WO 2010/145793中的描述。
VI.免疫缀合物
再一方面，本发明提供通过将本发明抗体缀合于异源分子而产生的免疫缀合物。在一个实施方案中，在免疫缀合物中，本发明的抗体(或其抗原结合片段)与治疗剂或诊断剂缀合。在一些实施方案中，本发明抗体可以以全长抗体或抗体片段的形式与异源分子缀合。例如，以Fab片段、Fab’片段、F(ab)’2片段、单链scFab抗体、单链scFv等片段形式进行缀合。
在一些实施方案中，本发明的抗体与治疗性分子缀合。可以使用接头来共价连接抗体与治疗性分析。适宜的接头包括化学接头或肽接头。
在另一些实施方案中，本发明的抗体可以与诊断剂或可检测剂缀合。这类缀合物可以作为临床检验方法的部分(如确定特定疗法的效力)，用于监测或预测疾病或病症的发作、形成、进展和/或严重性。可以通过将抗体与可检测剂偶联实现这类诊断和检测。
VII.药物组合物和药物制剂
本发明还包括包含抗TIGIT抗体或其免疫缀合物或多特异性抗体的组合物(包括药物组合物或药物制剂)和包含编码抗TIGIT抗体或其免疫缀合物或多特异性抗体的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
在一个实施方案中，组合物还包含第二治疗剂。所述第二治疗剂可以选自包括但不限于：例如抗-PD-1抗体和抗-PD-L1抗体。优选地，第二治疗剂是PD-1拮抗剂，尤其是抗PD-1抗体。
适用于本发明的药用载体可以是无菌液体，如水和油，包括来自石油、动物、植物或合成起源的那些，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的药用赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”，第五版，R.C.Rowe，P.J.Seskey和S.C.Owen，PharmaceuticalPress，London，Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服制剂可以包含标准载体，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
可以通过将具有所需纯度的本发明抗TIGIT抗体、免疫缀合物或多特异性抗体，与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合，来制备包含本发明的药物制剂，优选地以冻干制剂或水溶液的形式。
示例性的冻干抗体制剂描述于美国专利号6,267,958。水性抗体制剂包括美国专利号6,171,586和WO2006/044908中所述的那些，后一种制剂包括组氨酸-乙酸盐缓冲剂。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。例如，理想的是还提供其它抗癌活性成分，例如PD-1结合拮抗剂或PD-L1结合拮抗剂，例如抗PD-1 抗体或抗PD-L1抗体。所述活性成分以对于目的用途有效的量合适地组合存在。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
关于药物制剂的其它组分，还可以参见WO2015/153513中公开的那些。
VIII.组合产品
在一方面，本发明还提供了组合产品，其包含本发明的抗体或其抗原结合片段，双特异性抗体或免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、其他抗体、细胞毒性剂、疫苗、抗感染活性剂等)。本发明的组合产品可用于本发明的治疗方法中。
在一些实施方案中，本发明提供组合产品，其中所述其它治疗剂为例如有效刺激免疫反应从而进一步增强、刺激或上调受试者的免疫反应的治疗剂如抗体。在一些实施方案中，其它抗体为例如抗PD-1抗体或抗PD-L1抗体。
在一些实施方案中，所述组合产品用于预防或治疗肿瘤。在一些实施方案中，肿瘤为癌症，例如胃肠道癌症，例如胃癌、直肠癌、结肠癌、结肠直肠癌等；或皮肤癌，例如恶性黑素瘤。在一些实施方案中，所述组合产品用于预防或治疗感染，例如细菌感染、病毒感染、真菌感染、原生动物感染等。
IX.治疗方法和用途
在本文中，术语“个体”或“受试者”可互换地使用，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之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。
在一方面中，本发明涉及在受试者中增强机体的免疫应答的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗TIGIT抗体或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。在一些实施方案，将本发明的抗TIGIT抗体或其抗原结合部分施用于携带肿瘤的受试者，刺激抗肿瘤免疫应答。在另一些实施方案中，将本发明的抗体或其抗原结合部分施用于携带感染的受试者，刺激抗感染免疫应答。
在另一方面中，本发明涉及治疗受试者肿瘤，例如癌症的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗TIGIT抗体或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。癌症可以处于早期、中期或晚期或是转移性癌。
已经证实，TIGIT在人肿瘤浸润性CD8+T细胞上高表达。在一些实施方案中，本发明方法用于治疗癌症，尤其是表达TIGIT的肿瘤浸润性淋巴细胞浸润的实体肿瘤。
在一个实施方案中，癌症是胃肠道癌症如结肠癌。
在一些实施方案中，肿瘤或肿瘤细胞可以选自结直肠肿瘤、卵巢肿瘤、胰腺肿瘤、肺肿瘤、肺肿瘤，肝肿瘤，乳房肿瘤，肾肿瘤，前列腺肿瘤，胃肠肿瘤，黑素瘤，宫颈肿瘤，膀 胱肿瘤，成胶质细胞瘤和头颈部肿瘤。在一些实施方案中，癌症可以选自结直肠癌，卵巢癌，胰腺癌，肺癌，肝癌，乳腺癌，肾癌，前列腺癌，胃肠癌，黑素瘤，宫颈癌，膀胱癌，成胶质细胞瘤和头颈癌。
在一些实施方案中，本文提供了治疗肿瘤(如癌症)的方法，包括给予受试者本发明的抗-TIGIT抗体和拮抗性抗PD-1抗体。在一些实施方案中，用于治疗共表达TIGTI和PD-1的肿瘤，例如，共表达TIGIT和PD-1的黑素瘤(Chauvin et al.(2015)J.Clin.Invest.125：2046)，和非小细胞肺癌(NSCLC)和肾细胞癌(RCC)。
在另一方面中，本发明涉及治疗受试者感染性疾病，例如慢性感染的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗TIGIT抗体或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。在一个实施方案中，所述感染是病毒感染。
在一些实施方案中，所述感染性疾病是由于病毒感染引起的。致病性病毒的一些例子包括(甲型、乙型和丙型)肝炎病毒、(甲型、乙型和丙型)流感病毒、HIV、疱疹病毒(例如，VZV、HSV-1、HAV-6、HSV-II、CMV、Epstein Barr病毒+)、腺病毒、黄病毒、艾柯病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞体病毒、腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革病毒、乳头状瘤、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、JC病毒和虫媒脑炎病毒。
一些实施方案中，本文所述的方法还包括向所述受试者联合施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方案中，治疗方式包括手术治疗和/或放射疗法。
在一些实施方案中，可通过本发明的抗-TIGIT抗体和一种或多种治疗剂的组合，刺激T细胞反应。在一些实施方案中，除了施用本发明抗体外，本发明方法还包括施用至少一种其它的免疫刺激性抗体，例如抗PD-1抗体、抗PD-L1抗体，这些抗体可以是例如全人源的、嵌合的、或人源化的抗体。
在另一些实施方案中，可以与本发明抗体联合使用的其它治疗剂选自PD-1结合拮抗剂和PD-L1结合拮抗剂。″PD-1″的备选名称包括CD279和SLEB2。″PD-L1″的备选名称包括B7-H1、B7-4、CD274和B7-H。在一些实施方案中，PD-1、PD-L1是人PD-1、PD-L1。在一些实施方案中，PD-1结合拮抗剂是抑制PD-1结合其配体结合配偶的分子。在一个具体方面，PD-1配体结合配偶是PD-L1。在另一个实施方案中，PD-L1结合拮抗剂是抑制PD-L1结合其结合配偶的分子。在一个具体方面，PD-L1结合配偶是PD-1。拮抗剂可以是抗体，抗原结合片段、免疫粘附素、融合蛋白或寡肽。在一些实施方案中，PD-1结合拮抗剂是抗PD-1抗体(例如人抗体，人源化抗体，或嵌合抗体)。在一些实施方案中，抗PD-1抗体选自下组：IBI308(信迪利单抗，WO2017/025016A1)，MDX-1106(nivolumab，OPDIVO)，Merck 3475(MK-3475，pembrolizumab，KEYTRUDA)和CT-011(Pidilizumab)。在一些实施方案中，抗PD-1抗体是MDX-1106。在一些实施方案中，抗PD-1抗体是nivolumab(CAS注册号：946414-94-4)。在优选的实施方案中，抗PD-1抗体是本文所述的“Antibody C”。
在进一步的一些实施方案中，单独或与PD-1结合拮抗剂或与PD-L1结合拮抗剂组合的抗TIGIT抗体或其片段，还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用。在一些实施方案中，治疗方式包括外科手术(例如肿瘤切除术)；放射疗法(例如，外粒子束疗法，它涉及其中设计照射区域的三维适形放射疗法)、局部照射(例如，指向预选靶或器官的 照射)或聚焦照射等。
在一些实施方案中，本发明的抗TIGIT抗体或其抗原结合片段可以与化疗或化疗剂联合施用。在一些实施方案中，本发明的抗TIGIT抗体或其抗原结合片段可以与放疗或放疗剂联合施用。在一些实施方案中，本发明的抗TIGIT抗体或其抗原结合片段可以与靶向疗法或靶向治疗剂联合施用。在一些实施方案中，本发明的抗TIGIT抗体或其抗原结合片段可以与免疫疗法或免疫治疗剂，例如单克隆抗体联合施用。
本发明的抗体(以及包含其的药物组合物或免疫缀合物，以及任何另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的抗体的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、所述抗体是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
在上述本发明方法中，可以替代本发明抗体或抗原结合部分，施用本发明的组合物、多特异性抗体或免疫缀合物。或者，在这些方法中，除了施用本发明抗体或抗原结合部分，还可以进一步施用本发明的组合物、多特异性抗体或免疫缀合物。
再一方面，本发明提供本发明抗TIGIT抗体、组合物、免疫缀合物、多特异性抗体在制备用于前述方法(例如用于治疗)的药物中的用途。
X.用于诊断和检测的方法和组合物
再一方面，本发明涉及检测样品中TIGIT的方法和试剂盒，其中所述方法包括：(a)将所述样品与本发明抗体或其抗原结合片段或免疫缀合物接触；和(b)检测所述抗体或其抗原结合片段或免疫缀合物和TIGIT蛋白之间复合物的形成。在一些实施方案中，样品来自癌症患者，例如皮肤癌患者。所述检测可以是体外的或体内的。
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、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)。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。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自过度增生性或癌性病灶。在某些实施方案中，待检测的TIGIT是人TIGIT。
在一个实施方案中，抗TIGIT抗体被用于选择适合利用抗TIGIT抗体的治疗的受试者，例如其中TIGIT是用于选择所述受试者的生物标记物。在一个实施方案中，可以使用本发明抗体诊断癌症或肿瘤，例如评价(例如，监测)对象中本文所述疾病(例如，过度增生性或癌性疾病)的治疗或进展、其诊断和/或分期。
在某些实施方案中，提供标记的抗TIGIT抗体。标记包括但不限于，被直接检测的标记 或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。示例性标记包括但不限于，放射性同位素32P、14C、125I、3H和131I，荧光团如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰(dansyl)，伞形酮(umbelliferone)，荧光素酶(luceriferase)，例如，萤火虫荧光素酶和细菌荧光素酶(美国专利号4,737,456)，荧光素，2，3-二氢酞嗪二酮，辣根过氧化物酶(HR)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶解酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶如尿酸酶和黄嘌呤氧化酶，以及利用过氧化氢氧化染料前体的酶如HR，乳过氧化物酶，或微过氧化物酶(microperoxidase)，生物素/亲和素，自旋标记，噬菌体标记，稳定的自由基，等等。
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。
Figure PCTCN2019097665-appb-000008
Figure PCTCN2019097665-appb-000009
Figure PCTCN2019097665-appb-000010
Figure PCTCN2019097665-appb-000011
Figure PCTCN2019097665-appb-000012
Figure PCTCN2019097665-appb-000013
Figure PCTCN2019097665-appb-000014
本发明下面实施例涉及的20个示例性抗体(ADI-27238，ADI-30263，ADI-30267，ADI-30268，ADI-27243，ADI-30302，ADI-30336，ADI-27278，ADI-30293，ADI-30296，ADI-27291，ADI-30283，ADI-30286，ADI-30288，ADI-27297，ADI-30272，ADI-30278，ADI-27301，ADI-30306，ADI-30311)、以及这些抗体的CDR区、轻链可变区和重链可变区、轻链和重链的氨基酸序列，以及对应的核苷酸序列在本申请的表1-3和序列表部分列出，并且其对应序列编号汇总在表4中。
实施例
实施例1：抗体的制备
酵母展示技术筛选抗TIGIT全人源抗体
基于酵母的抗体展示文库(Adimab)，按照现有的方法(WO2009036379；WO 2010105256；W02012009568)进行扩增，其中每个库的多样性达到1×10 9。简言之，前两轮的筛选使用Miltenyi公司的MACS系统进行磁珠细胞分选。首先，将文库的酵母细胞(～1×10 10细胞/文库)分别在FACS洗涤缓冲液中(磷酸盐缓冲液，含有0.1％牛血清蛋白)室温孵化15分钟，缓冲液中含有100nM生物素标记的人TIGIT抗原(Acro Biosystems，TIT-H52H3)。使用50ml预冷的FACS洗涤缓冲液洗一次，再用40ml相同洗涤缓冲液重悬细胞，并加入500μl链霉亲和素微珠(Miltenyi LS)于4℃孵化15分钟。1000rpm离心5min弃去上清后用5ml FACS洗涤缓冲液重悬细胞，将细胞溶液加到Miltenyi LS柱中。加样完成后，用FACS洗涤缓冲液洗柱3次，每次3ml。从磁性区域取下Miltenyi LS柱，用5ml生长培养基洗脱，收集洗脱的酵母细胞并在37℃过夜生长。
使用流式细胞仪进行下一轮的分选：将经过MACS系统筛选获得的大约1×10 8的酵母细胞用FACS缓冲液洗三次，于含有低浓度生物素(100-1nM)标记的TIGIT抗原中室温下培养。弃去培养液，细胞用FACS洗涤缓冲液洗两次之后，将细胞与LC-FITC(FITC标记的抗人免疫球蛋白kappa轻链抗体，Southern Biotech)(1∶100稀释)混合，并与SA-633(链霉亲和素-633，Molec ular Probes)(1∶500稀释)或SA-PE(链霉亲和素-PE，Sigma)(1∶50稀释)试剂混合，4℃下培养15分钟。用预冷的FACS洗涤缓冲液洗脱两次，并重悬于0.4ml缓冲液中，将细胞转移到带滤器的分离管中。使用FACS ARIA(BD Biosciences)分选细胞。
将通过筛选获得的表达抗TIGIT抗体的酵母细胞在30℃下震荡诱导48小时以表达抗TIGIT的抗体。诱导结束之后，1300rpm离心10min去除酵母细胞，收获上清液。使用Protein A对上清液中的抗TIGIT抗体进行纯化，pH2.0醋酸溶液洗脱，收获抗TIGIT抗体，抗体纯度＞95％。使用木瓜蛋白酶消化并用KappaSelect(GE生命医疗集团)进行纯化可获得相应的Fab片段。本次筛选获得抗TIGIT抗体ADI-27301，ADI-27238，ADI-27278，ADI-27243，ADI-27297，ADI-27291。
抗人TIGIT抗体的亲和力优化
为了获得更高亲和力的抗人TIGIT抗体，我们通过以下方法对抗体ADI-27301，ADI-27238，ADI-27278，ADI-27243，ADI-27297，ADI-27291进行了优化。
VHmut筛选
该方法是通过常规的错配PCR的方法向抗体重链区域引入突变。PCR过程中，通过使用1uM高突变的碱基类似物dPTP和8-oxo-dGTP，从而将碱基错配概率提高至约0.01bp。
获得的错配PCR的产物通过同源重组的方法构建入含有重链恒定区的载体中。通过这种方法，在包括TIGIT抗原滴度、未标记抗原竞争以及使用母抗体竞争的筛选压力下，我们获得了库容量为1×10 7的次级库。通过FACS方法进行了3轮成功筛选。
CDRH1/CDRH2筛选
把VHmut方法获得的子代抗体的CDRH3基因构建入1×10 8多样性的CDRH1/CDRH2基因库中，并对其进行了3轮筛选。第一轮使用MACS方法，而第二、三轮使用FACS方法，对抗体抗原结合物进行亲和力加压，筛选出最高亲和力的抗体。
经过以上对6株亲本抗体的亲和力成熟过程，我们获得了14株亲和力提高的抗人TIGIT单克隆抗体ADI-30263/ADI-30267/ADI-30268(ADI-27238子代)、ADI-30302/ADI-30336(ADI-27243子代)、ADI-30293/ADI-30296(ADI-27278子代)、ADI-30283/ADI-30286/ADI-30288(ADI-27291子代)、ADI-30272/ADI-30278(ADI-27297子代)、ADI-30306/ADI-30311(ADI-27301子代)。
将通过筛选获得的表达抗TIGIT抗体的酵母细胞在30℃下震荡诱导48小时以表达抗TIGIT的抗体。诱导结束之后，1300rpm离心10min去除酵母细胞，收获上清液。使用Protein A对上清液中的抗TIGIT抗体进行纯化，pH2.0醋酸溶液洗脱，收获抗TIGIT抗体，抗体纯度＞95％。使用木瓜蛋白酶消化并用KappaSelect(GE生命医疗集团)进行纯化可获得相应的Fab片段。
抗体的表达及纯化
本申请涉及的各个抗人TIGIT抗体的序列信息及编号如表1-9所示，其中亲本以及亲和力成熟后的子代抗体均以上述方法由酵母表达纯化。
在HEK293细胞中表达并且纯化实施例中使用的下述对照抗体：22G2是在HEK293细胞中瞬时表达的来自百时美施贵宝公司的抗人TIGIT抗体，其轻重链可变区序列与专利申请WO2016/106302A1中的抗体“22G2”的序列相同。31C6是在HEK293细胞中瞬时表达的来自默沙东的抗人TIGIT抗体，其轻重链可变区序列与专利申请WO2016/028656 A1中的抗体“31C6”的序列相同。10A7和1F4均是在HEK293细胞中瞬时表达的来自基因泰克公司的抗人TIGIT抗体，其轻重链可变区序列与专利申请WO2015009856A2中的抗体“10A7”、“1F4”的序列分别相同。4个对照抗体的恒定区均采用野生型IgG4序列。
对于HEK293细胞中抗体的瞬时表达，使用载体pTT5。首先将抗体的重链和轻链分别克隆到单独的pTT5载体中。使用化学转染的方法将带有抗体分子重链和轻链的pTT5载体转入HEK293细胞中。采用的化学转染试剂为PEI(购自Polysciences)，按照生产产商提供的方案瞬时转染培养的HEK293细胞。首先在超净工作台中准备质粒DNA和转染试剂，将F17培养基(Gibco)(体积为转染体积的1/5)各一半加入50ml离心管中，一份加入已过滤的质粒(130μg/100ml)，另一半加入已过滤的PEI(1g/L，Polysciences)(质量比(质粒∶PEI)＝1∶3)，混匀5min，将二者轻柔混匀20次，静置15-30min，不要超过30min。将DNA/PEI混合物轻柔倒入HEK293细胞并混匀，在37℃，8％CO2的条件下培养细胞7天，每48小时流加新鲜培养基。7天后或者连续培养至细胞活力≤60％时，13000rpm离心20min。取上清液，用Protein A纯化上清液，使抗体的纯度＞95％。
此外，在CHO细胞中表达并且纯化完整抗体ADI-30268，ADI-30278，ADI-30286，ADI-30288，ADI-30293，ADI-30306，ADI-30336。
CHO细胞中表达和纯化：根据制造商的说明书，使用Horizon的HD-BIOP1(GS Null  CHO-K1)产生表达抗体的CHO细胞系。首先将抗体分子重链和轻链的DNA序列分别插入到来自ATUM公司的pD2531质粒中。之后采用电转染法将构建的质粒转入CHO细胞系，转染24小时之后利用ForteBio检测抗体产量以判断转染效率。转染后的细胞经过加压筛选得到高表达抗体的细胞池(pool)。之后扩增细胞池，大量表达抗体，并收集细胞上清用Protein A以及凝胶过滤纯化上清液，使抗体的纯度＞95％。
实施例2 抗体亲和力测定
采用生物光干涉测量(ForteBio)测定法测定本发明上述20个示例抗体结合人TIGIT的平衡解离常数(K D)。
ForteBio亲和力测定按照现有的方法(Estep，P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs，2013.5(2)：p.270-8)进行。
测量候选抗体Fab与TIGIT-FC的单价亲和力：传感器在分析缓冲液中线下平衡20分钟，然后线上检测120秒建立基线。加载人TIGIT-FC至AHQ传感器(ForteBio)上进行ForteBio亲和测量。将已加载抗原的传感器置于含100nM Fab的溶液中直至平台期，之后将传感器转移至分析缓冲液解离至少2分钟用于解离速率测量。使用1∶1结合模型进行动力学的分析。
测量完整候选抗体与人(ACRO，TIT-H52H3)、鼠(ACRO，TIT-M52E6)、猴TIGIT-his(ACRO，TIT-C5223)的单价亲和力：传感器在分析缓冲液中线下平衡20分钟，然后线上检测120秒建立基线，加载经纯化的抗体至AHQ传感器(ForteBio)上至厚度为1纳米进行ForteBio亲和测量。将已加载抗体的传感器于100nM的TIGIT-his抗原中作用至平台期，之后将传感器转移至分析缓冲液解离至少2分钟用于解离速率测量。使用1∶1结合模型进行动力学的分析。
在如以上测定法所述进行的实验中，20个酵母表达的候选抗体(Fab)的K D值如表5所示。
表5. 20个酵母表达的候选抗体(Fab)与人TIGIT-FC的单价KD
Figure PCTCN2019097665-appb-000015
在如以上测定法所述进行的实验中，7个CHO表达的候选完整抗体的人、鼠、猴TIGIT单价KD如表6所示。
表6. 7个CHO表达候选抗体及对照抗体与人、鼠、猴TIGIT的单价KD
序号 KD(人) KD(猴) KD(鼠)
ADI-30268 3.29E-09 6.23E-08 4.64E-08
ADI-30278 1.75E-09 4.53E-09 N.B.
ADI-30286 1.76E-09 1.06E-08 1.34E-07
ADI-30288 1.31E-09 7.19E-09 5.70E-09
ADI-30293 7.89E-10 3.49E-08 1.30E-08
ADI-30306 2.70E-09 3.44E-09 N.B.
ADI-30336 1.46E-09 5.56E-10 N.B.
22G2(BMS) 3.50E-09 4.20E-09 N.B.
31C6(MSD) 2.34E-09 4.72E-10 N.B.
由以上图表结果可见：(1)亲和力成熟后，各子代抗体与其亲代抗体相比，与人TIGIT的亲和力均有显著提高；(2)亲和力成熟后选取的7株CHO表达的抗体与人TIGIT的亲和力与对照抗体相比类似或更高；7株候选抗体均能与猴TIGIT交叉反应；7株候选抗体中有4株(ADI-30268/ADI-30286/ADI-30288/ADI-30293)能与小鼠交叉反应。
实施例3 抗TIGIT抗体与表达于细胞上的人TIGIT的结合
基于流式细胞术测定法测量本发明的上述20个示例抗体与表达于CHO细胞表面的人TIGIT的结合能力。通过比较不同抗体与表达于CHO细胞表面的人TIGIT的结合曲线来测定其结合能力。
具体地，在亲和力成熟后，使用酵母表达亲代及子代抗体。得到抗体后(纯度约70％)，进行这些抗体与表达于CHO细胞表面的人TIGIT结合实验。具体实验过程为：(1)使用构建于pCHO1.0的人TIGIT质粒转染CHOS细胞，使用氨甲喋呤以及嘌呤霉素筛选转染后的细胞得到稳定细胞pool。(2)使用不同浓度的候选抗体与稳定细胞pool于4℃孵育30分钟。PBS清洗三次后，使用PE标记的鼠抗人二抗与之4℃孵育30分钟。PBS再次清洗三次后，使用100ul PBS重悬细胞。(3)使用流式细胞仪测定其二通道中位荧光度值。以抗体浓度以10为底的对数为横坐标，以二通道中位荧光度值为纵坐标作图，比较其EC50及曲线峰值。结果如下表和图1所示(EC50：nM)。
抗体 IgG1 10A7 1F4 22G2 31C6  
EC50 ～1.388e+007 0.2690 0.7096 0.5106 0.4358  
抗体 ADI-27238 ADI-30267 ADI-30268 ADI-27243 ADI-30302 ADI-30336
EC50 0.7512 0.7402 0.3293 0.5873 0.3262 0.6291
抗体 ADI-27278 ADI-30293 ADI-30296 ADI-27291 ADI-30286 ADI-30288
EC50 0.9682 0.9003 0.3714 0.8522 0.4459 0.4755
抗体 ADI-27297 ADI-30272 ADI-30278 ADI-27301 ADI-30306 ADI-30311
EC50 0.4958 0.6098 0.4086 0.8990 0.7634 0.7566
由上面的实验结果可以得出如下结论：(1)亲和力成熟后，各子代抗体与表达于CHO细胞表面的人TIGIT的结合能力较其亲代抗体有增强(表现为结合曲线上平台更高)。(2)亲和力成熟后的子代抗体与表达于CHO细胞表面的人TIGIT的结合能力均强于来自基因泰克公司的两个分子10A7和1F4，与来自百时美施贵宝公司的分子22G2以及来自默沙东的分子31C6的结合能力相当。
此外，在亲和力成熟后，对筛选出来的7个分子使用CHO系统表达纯化得到高纯度的抗体，进行与表达于CHO细胞表面的人TIGIT的结合实验。在如以上测试法所述进行的实验中，ADI-30268、ADI-30336、ADI-30293、ADI-30286、ADI-30288、ADI-30278和ADI-30306结合CHO细胞上过表达的人TIGIT，EC50值分别为0.1392nM、0.1300nM、0.2288nM、0.1758nM、0.1860nM、0.1321nM和0.1999nM，高于对照抗体22G2、1F4与CHO细胞上过表达的人TIGIT的结合能力(对照抗体22G2及1F4的EC50值分别为0.7249nM和0.6073nM)，与对照抗体31C6类似(EC50值为0.1846nM)。虽然对照抗体10A7与7个候选抗体有着类似的EC50值(0.1949nM)，但是观察该抗体的结合曲线可知：与其他7个候选抗体相比，其曲线的上平台较低。一般认为这是由其较快的解离特性导致(参见图2)。因此也认为7个候选抗体与表达于CHO细胞表面的人TIGIT结合能力强于10A7。
实施例4 抗TIGIT抗体对其配体CD155的阻断
采用流式细胞术的方法测定候选抗体的配体阻断能力。具体方法为：构建稳定过表达人TIGIT的CHOS细胞pool，将50nM的带小鼠IgG2aFC段的人CD155蛋白(ACRO，TIT-H5253-1MG)以及不同浓度的抗体与细胞于4摄氏度共孵育30分钟。PBS清洗三遍后，使用1％浓度的羊抗鼠FC、带APC荧光素标记的二抗(Biolegend，405308)染色细胞上残留的配体CD155。PBS再次清洗3遍后，上流式细胞仪检测相应通道(C6，4通道)的中间荧光度值。以抗体浓度(nM)的以10为底的对数为横坐标，以各抗体浓度点对应的中间荧光度值为纵坐标作图。通过分析曲线的IC50区分不同抗体对CD155的阻断能力。
以IgG1为阴性对照抗体，以10A7、22G2、31C6为阳性对照抗体，做亲和力成熟前后的候选分子对CD155的阻断能力检测。图3和下表显示了亲和力成熟前后酵母表达的抗体对CD155的阻断能力(IC50：nM)。
抗体 IgG1 10A7 22G2 31C6    
IC50 ～0.03904 0.7408 0.1786 0.1536    
抗体 ADI-27238 ADI-30267 ADI-30268 ADI-27243 ADI-30302 ADI-30336
IC50 0.4181 0.2775 0.2209 0.8188 0.2808 0.4093
抗体 ADI-27278 ADI-30293 ADI-30296 ADI-27291 ADI-30286 ADI-30288
IC50 0.5859 0.8586 0.2781 1.614 0.2965 0.6677
抗体 ADI-27297 ADI-30272 ADI-30278 ADI-27301 ADI-30306 ADI-30311
IC50 0.6029 0.4036 0.2175 0.6411 0.5281 0.5313
从实验结果可得出：大部分亲和力成熟后的子代抗体较其亲本对CD155的阻断能力有提高，与对照抗体22G2和31C6类似且强于10A7。
使用7株CHO表达的高纯度抗体做CD155与TIGIT的阻断实验。在如以上测试法所述进行的实验中，以IgG4为阴性对照，22G2、31C6以及10A7为阳性对照。由实验结果可得出：ADI-30268、ADI-30336、ADI-30278的IC50分别为0.1402nM、0.1219nM、0.1164nM，低于22G2(0.6069nM)、31C6(0.1659nM)、10A7(0.3252nM)三个对照抗体，表明这三株抗体有较对照抗体更强的配体(CD155)阻断能力；其他4个候选抗体ADI-30293、ADI-30286、ADI-30288、ADI-30306的IC50分别为0.2510nM、0.1893nM、0.2238nM、0.2420nM，分别都小于对照抗体22G2和10A7但大于31C6，表明这四株抗体有较22G2和10A7更强但较31C6弱的配体(CD155)阻断能力。结果如下表和图4所示(IC50：nM)。
抗体 IgG1 22G2 31C6 10A7
IC50 ～1.152 0.6069 0.1659 0.3252
抗体 ADI-30268 ADI-30336 ADI-30293 ADI-30286
IC50 0.1402 0.1219 0.2510 0.1893
抗体 ADI-30288 ADI-30278 ADI-30306  
IC50 0.2238 0.1164 0.2420  
实施例5 MOA方法检测候选抗体的生物学活性
抗TIGIT抗体能够通过阻断TIGIT和CD155的结合，从而解除CD155对下游IL2信号通路的抑制作用。本实施例使用Promega公司提供的MOA检测细胞株(J2201)，通过检测荧光报告基因的表达，反映IL2信号的激活情况，从而检测抗体对TIGIT与CD155结合的抑制作用。按照Promega提供的标准方法进行该检测，过程如下：稳定表达人TIGIT以及IL2启动子控制下的荧光素酶报告基因的Jurkat细胞，与稳定表达人CD155以及T细胞激活元件的CHO-K1细胞、以及不同浓度梯度的抗人TIGIT抗体，于37℃二氧化碳细胞培养箱共同孵育，6小时后检测荧光信号。
实验结果表明：亲和力成熟后的子代分子(由酵母直接表达，纯度约70％)较亲代分子在体外生物学活性上有显著提升，大都达到与22G2和31C6两个对照抗体类似的水平。结果如下表和图5所示(EC50：nM)。
抗体 IgG1 10A7 22G2 31C6
EC50 ～272.8 ～77.84 2.113 0.8383
抗体 ADI-27238 ADI-30267 ADI-30268  
EC50 68.70 2.064 1.224  
抗体 ADI-27243 ADI-30302 ADI-30336  
EC50 297.1 0.9831 3.334  
抗体 ADI-27278 ADI-30293 ADI-30296  
EC50 ～1.405e+008 1.559 1.088  
抗体 ADI-27297 ADI-30272 ADI-30278  
EC50 ～25059 1.489 0.5228  
抗体 ADI-27301 ADI-30306 ADI-30311  
EC50 ～1.085e+012 1.645 1.824  
使用7株CHO表达的高纯度抗体做MOA生物学活性实验。在如以上测试法所述进行的实验中，以IgG4为阴性对照，31C6为阳性对照。由实验结果可得出：候选抗体ADI-30278在该实验中的EC50为0.1856nM，较其他候选抗体以及对照抗体31C6(0.3424nM)更小，显示出其更高的体外生物学活性；ADI-30268、ADI-30286、ADI-30288、ADI-30306的EC50分别为0.5065nM、0.3963nM、0.3844nM、0.3984nM，显示出与对照抗体31C6类似的生物学活性；ADI-30336以及ADI-30293的EC50分别为1.274nM和1.624nM，显示出其较弱的生物学活性。结果如下表和图6所示(EC50：nM)。
抗体 IgG4 31C6 ADI-30268
EC50 0.0 0.3424 0.5065
抗体 ADI-30336 ADI-30293 ADI-30286
EC50 1.274 1.624 0.3963
抗体 ADI-30288 ADI-30278 ADI-30306
EC50 0.3844 0.1856 0.3984
实施例6 动物体内药效试验
本实验利用人TIGIT敲入-MC38小鼠肿瘤模型研究抗TIGIT抗体的抗肿瘤活性。
研究了候选分子ADI-30293和ADI-30278单独给药(10mg/kg)在人TIGIT敲入-MC38小鼠模型中的药效学。小鼠结肠癌MC38细胞(上海和元生物，HYC0116)被移植到雌性TIGIT转基因小鼠(北京百奥赛图基因生物技术有限公司)。细胞移植后的第6天，第10天，第13天，第17天，给各组小鼠分别经皮下注射抗体，移植后第6天、第10天、第13天、第17天、第21天、第25天测量肿瘤体积，之后对小鼠实施安乐死。结果如图7所示。
观察图7实验结果，可见本申请的抗TIGIT单克隆抗体ADI-30293和ADI-30278与Ig4阴性对照抗体相比，在单独使用时对肿瘤的生长有一定的抑制作用。与31C6阳性对照相比，两个候选抗体单独使用时其肿瘤抑制作用均与31C6无明显区别。
进一步，研究了候选分子ADI-30293和ADI-30278与抗PD1抗体antibody C(WO2017133540A1)联合给药(10+1mg/kg)在人TIGIT敲入-MC38小鼠模型中的药效学。
小鼠结肠癌细胞MC38(上海和元生物，HYC0116)用DMEM培养基培养。在0.2ml DMEM基础培养基悬液中的1×10 6MC38细胞被移植到雌性TIGIT转基因小鼠(上海南方模式生物科技股份有限公司)的右侧。肿瘤大小和体重在整个研究周期中每周测定2次，肿瘤细胞移植8天后，使用游标卡尺测量肿瘤长度和宽度，根据如下公式计算肿瘤体积：宽2×长/2(mm3)；肿瘤体积平均约为71mm3的小鼠按每组6只随机分组，当肿瘤体积达到检测终点或小鼠体重降低20％以上时即对小鼠实施安乐死。细胞移植后的第8天，第12天，第15天，第19天，给各组小鼠分别经皮下注射h-IgG(10mg/kg)，antibody C(1mg/kg)以及10mg/kg的anti-TIGIT抗体或其同型抗体。终点肿瘤平均体积在细胞移植后的第29天测定，之后对实验小鼠实施安乐死。结果如图8所示。
观察图8实验结果可知：当使用抗TIGIT抗体ADI-30293与抗PD1抗体antibody C联合用药时，表现出了较抗TIGIT抗体或抗PD1抗体单独给药时更强的肿瘤抑制效果，且ADI-30293+anti-PD1抗体的联用效果好于31C6+anti-PD1抗体的联用效果。同样的，ADI-30278与anti-PD1抗体联合用药的效果好于31C6+anti-PD1抗体antibody C的联用效果。
本研究中所有组小鼠在接种后29天，小鼠体重均无明显变化。

Claims (19)

  1. 结合TIGIT的抗体或其抗原结合片段，其包含
    (i)如SEQ ID NO：84、85、86、或87所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：104所示的轻链可变区的LCDR1、2和3序列，或者
    (ii)如SEQ ID NO：88、89或90所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：105或106所示的轻链可变区的LCDR1、2和3序列，或者
    (iii)如SEQ ID NO：91、92或93所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：107所示的轻链可变区的LCDR1、2和3序列，或者
    (iv)如SEQ ID NO：94、95、96或97所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：108所示的轻链可变区的LCDR1、2和3序列，或者
    (v)如SEQ ID NO：98、99或100所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：109所示的轻链可变区的LCDR1、2和3序列，或者
    (vi)如SEQ ID NO：101、102或103所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO：110所示的轻链可变区的LCDR1、2和3序列。
  2. 结合TIGIT的抗体或其抗原结合片段，其包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
    (a)HCDR1包含SEQ ID NO：1或2或3或4或5所示的氨基酸序列，HCDR2包含SEQ ID NO：6或7或8或9或10所示的氨基酸序列，HCDR3包含SEQ ID NO：11或12或13或14或15所示的氨基酸序列，LCDR1包含SEQ ID NO：16所示的氨基酸序列，LCDR2包含SEQ ID NO：17所示的氨基酸序列，且LCDR3包含SEQ ID NO：18所示的氨基酸序列；或
    (b)HCDR1包含SEQ ID NO：19或20或21所示的氨基酸序列，HCDR2包含SEQ ID NO：22或23或24所示的氨基酸序列，HCDR3包含SEQ ID NO：25所示的氨基酸序列，LCDR1包含SEQ ID NO：26所示的氨基酸序列，LCDR2包含SEQ ID NO：27所示的氨基酸序列，且LCDR3包含SEQ ID NO：28或29或30所示的氨基酸序列；或
    (c)HCDR1包含SEQ ID NO：31或32或33所示的氨基酸序列，HCDR2包含SEQ ID NO：34或35或36或37所示的氨基酸序列，HCDR3包含SEQ ID NO：38或39或40所示的氨基酸序列，LCDR1包含SEQ ID NO：41所示的氨基酸序列，LCDR2包含SEQ ID NO：42所示的氨基酸序列，且LCDR3包含SEQ ID NO：43所示的氨基酸序列；或
    (d)HCDR1包含SEQ ID NO：44或45或46或47所示的氨基酸序列，HCDR2包含SEQ ID NO：48或49或50或51或52所示的氨基酸序列，HCDR3包含SEQ ID NO：53或54或55或56所示的氨基酸序列，LCDR1包含SEQ ID NO：57所示的氨基酸序列，LCDR2包含SEQ ID NO：58所示的氨基酸序列，且LCDR3包含SEQ ID NO：59所示的氨基酸序列；或
    (e)HCDR1包含SEQ ID NO：60或61或62所示的氨基酸序列，HCDR2包含SEQ ID NO：6或63或64或65所示的氨基酸序列，HCDR3包含SEQ ID NO：66或67或68所示的氨基酸序列，LCDR1包含SEQ ID NO：69所示的氨基酸序列，LCDR2包含SEQ ID NO：17所示的氨 基酸序列，且LCDR3包含SEQ ID NO：70所示的氨基酸序列；或
    (f)HCDR1包含SEQ ID NO：71或72或73或74所示的氨基酸序列，HCDR2包含SEQ ID NO：22或75或76或77所示的氨基酸序列，HCDR3包含SEQ ID NO：78或79或80所示的氨基酸序列，LCDR1包含SEQ ID NO：81所示的氨基酸序列，LCDR2包含SEQ ID NO：82所示的氨基酸序列，且LCDR3包含SEQ ID NO：83所示的氨基酸序列；或
    (g)HCDR1包含SEQ ID NO：178或179或180或181所示的氨基酸序列，HCDR2包含SEQ ID NO：6或7或8或9所示的氨基酸序列，HCDR3包含SEQ ID NO：182或183或184或185所示的氨基酸序列，LCDR1包含SEQ ID NO：16所示的氨基酸序列，LCDR2包含SEQ ID NO：17所示的氨基酸序列，且LCDR3包含SEQ ID NO：18所示的氨基酸序列；或
    (h)HCDR1包含SEQ ID NO：186或187所示的氨基酸序列，HCDR2包含SEQ ID NO：22或23所示的氨基酸序列，HCDR3包含SEQ ID NO：188所示的氨基酸序列，LCDR1包含SEQ ID NO：26所示的氨基酸序列，LCDR2包含SEQ ID NO：27所示的氨基酸序列，且LCDR3包含SEQ ID NO：28或29所示的氨基酸序列；或
    (i)HCDR1包含SEQ ID NO：189或190所示的氨基酸序列，HCDR2包含SEQ ID NO：34或35或36所示的氨基酸序列，HCDR3包含SEQ ID NO：191或192所示的氨基酸序列，LCDR1包含SEQ ID NO：41所示的氨基酸序列，LCDR2包含SEQ ID NO：42所示的氨基酸序列，且LCDR3包含SEQ ID NO：43所示的氨基酸序列；或
    (j)HCDR1包含SEQ ID NO：193或194或195所示的氨基酸序列，HCDR2包含SEQ ID NO：48或49或50或51所示的氨基酸序列，HCDR3包含SEQ ID NO：196或197或198所示的氨基酸序列，LCDR1包含SEQ ID NO：57所示的氨基酸序列，LCDR2包含SEQ ID NO：58所示的氨基酸序列，且LCDR3包含SEQ ID NO：59所示的氨基酸序列；或
    (k)HCDR1包含SEQ ID NO：199或200所示的氨基酸序列，HCDR2包含SEQ ID NO：6或63或64所示的氨基酸序列，HCDR3包含SEQ ID NO：201或202所示的氨基酸序列，LCDR1包含SEQ ID NO：69所示的氨基酸序列，LCDR2包含SEQ ID NO：17所示的氨基酸序列，且LCDR3包含SEQ ID NO：70所示的氨基酸序列；或
    (1)HCDR1包含SEQ ID NO：203或204或205所示的氨基酸序列，HCDR2包含SEQ ID NO：22或75或76所示的氨基酸序列，HCDR3包含SEQ ID NO：206或207所示的氨基酸序列，LCDR1包含SEQ ID NO：81所示的氨基酸序列，LCDR2包含SEQ ID NO：82所示的氨基酸序列，且LCDR3包含SEQ ID NO：83所示的氨基酸序列。
  3. 权利要求1至2中任一项的抗体或其抗原结合片段，其包含
    (i)包含与SEQ ID NO：84或85或86或87所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的重链可变区，和/或包含与SEQ ID NO：104所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的轻链可变区，或者
    (ii)包含与SEQ ID NO：88或89或90所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的重链可变区，和/或包含与SEQ ID NO：105或106所示的氨基酸序 列具有至少80％、85％或90％序列同一性的氨基酸序列的轻链可变区，或者
    (iii)包含与SEQ ID NO：91或92或93所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的重链可变区，和/或包含与SEQ ID NO：107所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的轻链可变区，或者
    (iv)包含与SEQ ID NO：94或95或96或97所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的重链可变区，和/或包含与SEQ ID NO：108所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的轻链可变区；
    (v)包含与SEQ ID NO：98或99或100所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的重链可变区，和/或包含与SEQ ID NO：109所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的轻链可变区；
    (vi)包含与SEQ ID NO：101或102或103所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的重链可变区，和/或包含与SEQ ID NO：110所示的氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列的轻链可变区。
  4. 权利要求1至3中任一项的抗体或其抗原结合片段，其包含选自以下的重链可变区和轻链可变区
    (i)包含SEQ ID NO：84或85或86或87所示的氨基酸序列的重链可变区，和包含SEQ ID NO：104所示的氨基酸序列的轻链可变区，或者
    (ii)包含SEQ ID NO：88或89或90所示的氨基酸序列的重链可变区，和包含SEQ ID NO：105或106所示的氨基酸序列的轻链可变区，或者
    (iii)包含SEQ ID NO：91或92或93所示的氨基酸序列的重链可变区，和包含SEQ ID NO：107所示的氨基酸序列的轻链可变区，或者
    (iv)包含SEQ ID NO：94或95或96或97所示的氨基酸序列的重链可变区，和包含SEQ ID NO：108所示的氨基酸序列的轻链可变区；
    (v)包含SEQ ID NO：98或99或100所示的氨基酸序列的重链可变区，和包含SEQ ID NO：109所示的氨基酸序列的轻链可变区；
    (vi)包含SEQ ID NO：101或102或103所示的氨基酸序列的重链可变区，和包含SEQ ID NO：110所示的氨基酸序列的轻链可变区。
  5. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体是IgG1，IgG2，或IgG4形式的抗体或其抗原结合片段，优选具有S228P，F234A和L235A突变的IgG4Fc区。
  6. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体是全人源抗体、或人源化抗体、或嵌合抗体。
  7. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab'-SH、Fv、单链抗体例如scFv、(Fab')2片段、单结构域抗体、双抗体(dAb)或线性抗体。
  8. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体具有以下一个或多个特 性：
    (i)以高亲和力与人TIGIT结合；
    (ii)具有与猴和/或鼠TIGIT的交叉免疫反应性；
    (iii)与细胞表面的TIGIT有效结合；
    (iv)阻断TIGIT与其配体CD155的结合；
    (v)解除TIGIT与CD155结合对下游IL-2信号通路的抑制作用；
    (vi)增加T细胞中的IL-2产生；
    (vii)具有抗肿瘤活性，例如抑制肿瘤的生长；
    (viii)与抗PD-1抗体组合具有更好的肿瘤抑制作用，例如能够更好地抑制肿瘤生长。
  9. 一种分离的核酸，其编码前述权利要求任一项的抗TIGIT抗体或其抗原结合片段。
  10. 一种载体，其包含权利要求9的核酸，优选地所述载体是表达载体。
  11. 一种宿主细胞，其包含权利要求9的核酸或权利要求10的载体，优选地，所述宿主细胞是原核的或真核的，更优选的酵母细胞、哺乳动物细胞(例如293细胞或CHO细胞)。
  12. 制备抗TIGIT抗体或其抗原结合片段的方法，所述方法包括在适于表达编码前述权利要求1-8中任一项的抗体或其抗原结合片段的核酸的条件下培养权利要求11的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述抗TIGIT抗体或其抗原结合片段。
  13. 免疫缀合物，其包含与治疗剂或诊断剂缀合的前述权利要求1-8中任一项的抗体或其抗原结合片段。
  14. 包含前述权利要求1-8任一项的抗体或其抗原结合片段的多特异性抗体，优选地，所述多特异性抗体是双特异性抗体。
  15. 药物组合物，其包含前述权利要求1-8任一项的抗体或其抗原结合片段，以及任选地药用辅料。
  16. 权利要求15的药物组合物，其包含第二治疗剂；优选地，所述第二治疗剂选自抗PD-1抗体或抗PD-L1抗体。
  17. 在受试者中预防或治疗肿瘤或感染性疾病的方法，所述方法包括向所述受试者施用有效量的前述权利要求1-8任一项的抗TIGIT抗体或其抗原结合片段、或权利要求15或16的药物组合物，优选地，所述肿瘤是胃肠道癌症，例如结肠癌。
  18. 在受试者中阻断TIGIT与CD155的结合以降低或消除TIGIT的免疫抑制作用的方法，所述方法包括向所述受试者施用有效量的前述权利要求1-8任一项的抗TIGIT抗体或其抗原结合片段、或权利要求15或16的药物组合物。
  19. 检测样品中TIGIT的方法，所述方法包括
    (a)将样品与前述权利要求1-8中任一项的抗体或其抗原结合片段接触；和
    (b)检测所述抗体或其抗原结合片段和TIGIT间的复合物的形成；任选地，抗体是被可检测地标记的。
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