CN118165107A - Antibody molecules that bind TIGIT - Google Patents

Abstract

The present disclosure relates to antibodies against TIGIT or antigen-binding fragments thereof, derivatives comprising said antibodies or antigen-binding fragments thereof, related pharmaceutical compositions, and the use of the same for the preparation of a medicament for the treatment of tumors.

Description

Antibody molecules that bind TIGIT

Technical Field

The present disclosure relates to antibody molecules or antigen-binding fragments thereof that specifically bind TIGIT, pharmaceutical compositions comprising the same, and uses thereof.

Background

TIGIT (T CELL IG AND ITIM domain, also known as WUCAM, vstm3, VSIG 9) is a member of the poliovirus receptor (PVR)/Nectin family. TIGIT consists of an extracellular immunoglobulin variable region (IgV) domain, a type I transmembrane domain, and an intracellular domain with a classical Immunoreceptor Tyrosine Inhibitory Motif (ITIM) and an Immunoglobulin Tyrosine Tail (ITT) motif. TIGIT is highly expressed in lymphocytes, particularly in effector and regulatory cd4+ T cells, follicular helper cd4+ T cells and effector cd8+ T cells, and Natural Killer (NK) cells.

TIGIT has become an important immune checkpoint capable of inhibiting each step of the cancer immune cycle. TIGIT may prevent NK cells from releasing tumor antigens, impair dendritic cell activation of T cells, or inhibit killing of cancer cells by cd8+ T cells. Numerous preclinical studies indicate that TIGIT will be a suitable target for cancer patients. TIGIT blocking not only reduces the inhibition of tumor infiltration-modulating T cells, but also activates anti-tumor T cell responses and enhances anti-tumor NK cell responses. Targeting TIGIT is probably the most effective when combined with other immunotherapies. In preclinical studies, targeting TIGIT and PD-1/PD-LI pathway showed better tumor suppression compared to either monotherapy. TIGIT blocking and PD-1/PD-L1 blocking act synergistically to enhance the anti-tumor effect of cd8+ T cells.

Many clinical trials of anti-TIGIT monoclonal antibodies are currently underway, which would help to determine the effect of TIGIT blocking. Of course, designing an optimal combination strategy for TIGIT blocking in cancer patients would also help to develop therapies for TIGIT to treat other chronic diseases.

Summary of The Invention

The present disclosure provides an antibody or antigen binding fragment thereof against TIGIT, which is capable of specifically binding to human TIGIT and cynomolgus TIGIT, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 being respectively:

(1) SEQ ID NO:9, 10, 11, 12, 13, 14; or (b)

(2) SEQ ID NO:15, 16, 17, 18, 19, 20; or (b)

(3) SEQ ID NO:21, 22, 23, 24, 25, 26; or (b)

(4)SEQ ID NO：27，28，29，30，31，32。

According to one aspect of the disclosure, the antibody or antigen-binding fragment thereof against TIGIT comprises a heavy chain variable region and a light chain variable region, which have at least 80% to 100% sequence identity to any one of the following sets of heavy chain variable region and light chain variable region, respectively:

(1) SEQ ID NOs 1 and 2; or (b)

(2) SEQ ID NOs 3 and 4; or (b)

(3) SEQ ID NOs 5 and 6; or (b)

(4) SEQ ID NOS 7 and 8; or (b)

(5) SEQ ID NOS.33 and 34.

In some preferred embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof described in the present disclosure are murine, chimeric, or humanized antibodies.

In some preferred embodiments, the anti-TIGIT antibody is a monoclonal antibody.

In some preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and/or a light chain constant region, preferably, the heavy chain constant region comprises a native Fc or a modified variant Fc, the Fc being of murine or human origin.

In some preferred embodiments, the anti-TIGIT antibody is a full length antibody.

In some preferred embodiments, the anti-TIGIT antibodies of the present disclosure, or antigen-binding fragments thereof, are in the form of IgG1, igG2, igG3, or IgG 4.

In some preferred embodiments, the antigen binding fragments described in the present disclosure are Fab, fv, scFv, F (ab') ₂, linear antibodies, single domain antibodies.

In some embodiments, the present disclosure provides a conjugate formed by coupling the aforementioned antibody or antigen-binding fragment thereof to a capture or detection label. Such detection labels include, but are not limited to, radionuclides, luminescent substances (e.g., luciferin), colored substances, or enzymes.

In some embodiments, the present disclosure provides a fusion protein wherein one fused moiety comprises an antibody of the disclosure against TIGIT or antigen-binding fragment thereof.

In some embodiments, the disclosure provides a bispecific antibody or a multispecific antibody, one antigen-binding domain of which comprises an antibody of the disclosure that is anti-TIGIT or antigen-binding fragment thereof.

In some embodiments, the disclosure provides nucleic acids encoding any one of the foregoing antibodies or antigen binding fragments thereof. And, the present disclosure also provides recombinant vectors comprising the nucleic acids.

In some embodiments, the present disclosure provides a host cell comprising an expression vector as described in the present disclosure or a nucleic acid encoding the antibody or antigen binding fragment thereof. In some preferred embodiments, the host cell may be a prokaryotic cell, such as E.coli; eukaryotic cells, such as yeast or mammalian cells, mammalian cells such as CHO cells or HEK293 cells, are also possible.

In some embodiments, the disclosure provides methods of making the antibodies or antigen binding fragments thereof, comprising: the host cells of the present disclosure are cultured under suitable conditions and purified from the cells to obtain an expression product.

In some embodiments, the disclosure provides for the use of the antibodies, or antigen binding fragments thereof, for the treatment of tumors.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, or comprising an effective amount of a nucleic acid encoding the antibody or antigen-binding fragment thereof, or comprising an effective amount of a recombinant vector comprising the encoding nucleic acid, or comprising an effective amount of a host cell comprising the encoding nucleic acid, or comprising an effective amount of a fusion protein of the present disclosure, or comprising an effective amount of a bispecific antibody or multispecific antibody of the present disclosure. In some more preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In some specific embodiments, the pharmaceutical composition further comprises one or more additional other therapeutic agents. The additional therapeutic agent is a pharmaceutical formulation that can be administered to a subject in combination with the pharmaceutical composition of the present disclosure.

In some embodiments, the present disclosure provides for the use of the pharmaceutical composition in the manufacture of a medicament for treating a disease, wherein the disease may be a tumor, further wherein the tumor may be a cancer.

Drawings

The accompanying drawings further illustrate novel features disclosed in this specification. The features and advantages disclosed in this specification will be better understood by reference to the drawings, which are included merely for purposes of illustration of specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims.

FIG. 1 shows the binding of humanized antibody TAb07 to human TIGIT and cynomolgus TIGIT. Among them, FIG. 1A shows the binding activity of TAb07 to human TIGIT, and FIG. 1B shows the binding activity of TAb07 to cynomolgus TIGIT.

FIG. 2 shows the binding of humanized antibody TAb07 to both overexpressing human TIGIT cells and overexpressing cynomolgus monkey TIGIT cells. Wherein, FIG. 2A shows the binding activity of TAb07 to cells overexpressing human TIGIT, and FIG. 2B shows the binding activity of TAb07 to cells overexpressing cynomolgus TIGIT.

Detailed Description

Terminology

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Before the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Certain embodiments disclosed herein encompass a range of values, and certain aspects of the disclosure may be described by way of the range. Unless otherwise indicated, it should be understood that the numerical ranges or the manner in which the ranges are described are for the purpose of brevity and convenience only and should not be construed as a strict limitation on the scope of the present disclosure. Accordingly, the description of a range format should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within the range as if such sub-ranges and numerical points had been explicitly written herein. For example, a description of a range from 1 to 6 should be considered to specifically disclose sub-ranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as specific numerical points within these ranges, such as 1,2, 3, 4, 5, 6. The above principle applies equally regardless of the breadth of the values. When a range description is employed, the range includes the endpoints of the range.

The term "about" when referring to a measurable value such as an amount, temporal duration, or the like, is meant to include a change of + -20%, or in some cases + -10%, or in some cases + -5%, or in some cases + -1%, or in some cases + -0.1% of the specified value.

The amino acid three-letter codes and one-letter codes used herein are as described in J.biol. Chem,243, p3558 (1968).

The term "TIGIT" as used herein refers to any form of TIGIT and variants thereof that retain at least part of TIGIT activity.

The term "anti-TIGIT antibody" or "TIGIT-binding antibody" as used herein refers to an antibody that is capable of binding to human TIGIT, cynomolgus TIGIT with sufficient affinity, i.e., that the antibody is capable of recognizing and binding to both human TIGIT and cynomolgus TIGIT.

TIGIT of human origin is expressed as: hTIGIT thus, the expressions "antibody against human TIGIT", "antibody against hTIGIT" etc. are specific for being able to bind hTIGIT with sufficient affinity that the antibody can be used as a diagnostic and/or therapeutic agent targeting hTIGIT.

Cynomolgus monkey-derived TIGIT is denoted cyno TIGIT.

The term "antibody" as used herein, typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have such a structure. Each light chain consists of one light chain variable domain (VL) and one light chain constant domain (CL). Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH), alternatively referred to as a heavy chain constant region (CH).

Five main classes of antibodies are known in the art: igA, igD, igE, igG and IgM, the corresponding heavy chain constant domains are referred to as α, δ, epsilon, γ and μ, respectively, igG and IgA can be further divided into different subclasses, e.g., igG can be divided into IgG1, igG2, igG3, igG4, igA can be divided into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be identified as one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains.

In the case of IgG, igA and IgD antibodies, the heavy chain constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have the fourth domain CH 4). In the IgG, igA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable length proline and cysteine rich segment. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.

The term "variable region" or "variable domain" shows a significant change in amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light/heavy chain pair forms an antigen binding site such that the complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each comprise three regions of extreme variability, known as hypervariable regions (HVRs), or more generally as Complementarity Determining Regions (CDRs), each of VH and VL having 4 framework regions FR, denoted FR1, FR2, FR3, FR4, respectively. Thus, CDR and FR sequences are typically found in the following sequences of the heavy chain variable domain (VH) (or light chain variable domain (VL): FR1-HCDR1 (LCDR 1) -FR2-HCDR2 (LCDR 2) -FR3-HCDR3 (LCDR 3) -FR4.

The term "Fc" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as described in Kabat et al ,Sequences of Proteins of Immunological Interest,5thEd.Public Health Service,National Institutes of Health,Bethesda,MD,1991.

As used herein, the broad class of "antibodies" can include, for example, polyclonal (polyclonal antibodies), monoclonal, chimeric, humanized and primatized antibodies, CDR-grafted antibodies (CDR-grafted antibodies), human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

The terms "full length antibody," "intact antibody," and "intact antibody" may be used interchangeably herein to refer to an antibody that is substantially similar in structure to a native antibody structure or has an Fc region.

The term "monoclonal antibody" (or "mab") refers to an antibody that is produced by a single cell clone that is substantially homogeneous and directed against only a particular epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, synthetic techniques, combinations thereof, or the like.

The term "chimeric antibody" is a construct in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies. In a narrow sense, the chimeric antibody comprises all or most of the selected murine heavy and light chain variable regions operably linked to human light and heavy chain constant regions. The constant region sequences, or variants or derivatives thereof, may be operably associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide full length antibodies which may be used per se or which may be incorporated into the anti-TIGIT of the present disclosure.

The term "humanized antibody" is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof, that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) having the desired specificity, affinity and properties, such as mouse, rat, rabbit or primate. In some cases, the framework region residues of the human immunoglobulin are replaced with corresponding non-human residues. In some cases, a "back mutation" may be introduced into a humanized antibody in which residues in one or more of the FR of the variable region of the recipient human antibody are replaced with corresponding residues from a non-human species donor antibody. Such back mutations may help to maintain the appropriate three-dimensional configuration of one or more grafted CDRs and thus improve affinity and antibody stability. Antibodies from a variety of donor species may be used, including but not limited to mice, rats, rabbits, or non-human primates. In addition, humanized antibodies may contain novel residues not found in the recipient antibody or in the donor antibody in order to further improve antibody performance.

It is to be noted that the division of the CDRs and FRs of the variable regions of the monoclonal antibodies of the present disclosure is determined according to the Kabat definition. While other naming and numbering systems, such as Chothia, IMGT or AHo, etc., are also known to those skilled in the art. Thus, humanized antibodies comprising one or more CDRs derived from any naming system based on the mab sequences of the present disclosure are expressly maintained within the scope of the present disclosure.

The term "sequence identity" or "sequence similarity" or "sequence homology" refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence after aligning the sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.

The term "antibody fragment" encompasses at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or epitope thereof, or a fusion protein product further derived from such fragment, e.g., a single chain antibody, an extracellular binding region in a chimeric antigen receptor, and the like. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), fab fragments, F (ab') ₂ fragments, fd fragments, fv fragments, single domain antibodies, linear antibodies, single chain antibodies (scFv), bispecific or multispecific antibodies formed from antibody fragments, and the like.

The term "Fab fragment" includes the heavy chain variable region and the light chain variable region, and also includes the constant region of the light chain and the first constant region CH1 of the heavy chain, which is a monovalent antibody fragment. The term "F (ab') ₂ fragment" comprises 2 Fab fragments as well as hinge regions, which are bivalent antibody fragments.

The term "Fd fragment" generally comprises the heavy chain variable region and the constant region CH1; the term "Fv fragment" encompasses a minimum antibody fragment that contains the antibody heavy and light chain variable regions, but no constant region, and has all antigen binding sites.

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

The term "fusion protein" refers to a polypeptide/protein that is joined together by genetic recombination or chemical means to form a larger molecule, either with or without the use of a linker.

The term "multispecific antibody" refers to an antibody that is functionally linked (e.g., chemically coupled, genetically fused, non-covalently bound, or otherwise) to one or more other binding molecules, thereby forming a novel antibody construct that binds to two or more different sites and/or targets. Among these, more "bispecific antibodies" are used, which specifically refer to antibody constructs specific for two different antigens. Typically, a bispecific antibody or multispecific antibody comprises at least 2 antigen-binding domains.

The term "antigen" refers to a substance recognized and specifically bound by an antibody or antigen binding fragment thereof, and in a broad sense, an antigen may include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, or an intact extracellular domain (ECD) or protein.

The term "epitope," also known as an "antigenic determinant," refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes generally consist of 3-15 amino acid residues.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine as well as D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by the indicated nucleic acid sequences.

The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and/or deletions in the polypeptide sequence. "amino acid substitution" or "substitution" means the replacement of an amino acid at a particular position in the parent polypeptide sequence with another amino acid. For example, substitution S32A refers to replacement of serine at position 32 with alanine.

The sequence identity or homology of the humanized antibody variable region to the human acceptor variable region may be determined as discussed herein and when so measured will preferably share at least 60% or 65% sequence identity, more preferably at least 70%, 75%, 80%, 85% or 90% sequence identity, even more preferably at least 93%, 95%, 98% or 99% sequence identity. Preferably, the different residue positions differ by conservative amino acid substitutions. A "conservative substitution" is an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids containing basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions or in the framework regions of the antibodies of the present disclosure may be replaced with other similarly pendant amino acid residues. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct the conservative nature of the substitution.

In the monoclonal antibody production process, various post-translational modification (PTM) variants, such as glycosylation, oxidation, saccharification, deamidation, isomerization, end group cyclization and the like, are easily generated by different physical and chemical factors, and the PTM can cause physical and chemical property changes of the antibody, change interaction with an Fc receptor of the antibody and influence the binding activity with a target antigen; some PTM occurrences may even decrease antibody stability, cause immunogenicity, etc. (JARASCH et al, JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). The negative effects of amino acid modifications, e.g. conservative substitutions, to the PTM site may be eliminated. Amino acid substitutions of antibody CDRs for the purpose of engineering PTMs are also expressly within the scope of the present disclosure.

Antibodies of the present disclosure may also include substitutions or modifications of the constant region (e.g., fc), including but not limited to amino acid residue substitutions, mutations, and/or modifications, that result in compounds having the following preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased yield, altered binding to Fc receptors (FcR), enhanced or reduced ADCC or CDC, altered glycosylation and/or disulfide bonds, and modified binding specificity.

The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a particular antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, flow cytometry, and the like.

The term "pharmaceutical composition" refers to a formulation or combination of formulations comprising one, two or more active ingredients, which allows the active ingredients contained therein to be present in a biologically active effective form, and which does not comprise additional ingredients having unacceptable toxicity to the subject to whom the formulation is administered. When the "pharmaceutical composition" is in the form of a combination of separate preparations containing different, more than two active ingredients, it can be administered simultaneously, sequentially, separately or at intervals, with the aim of exerting the biological activity of the various active ingredients, together for the treatment of diseases.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

The term "effective amount" refers to the dose of a pharmaceutical formulation of an antibody or antigen-binding fragment thereof of the present disclosure that produces a desired effect in a treated patient after administration to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician as a person skilled in the art by considering a number of factors: such as race differences; body weight, age and health; specific diseases involved; severity of disease; response of individual patients; specific antibodies administered; mode of administration; the bioavailability characteristics of the administration formulation; a selected dosing regimen; and the use of any concomitant therapy.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny having the same function or biological activity as screened or selected in the initially transformed cell.

The term "transfection" as used herein refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection may be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics (biolistics).

The term "stable transfection" or "stable transfection" refers to the introduction and integration of an exogenous nucleic acid, DNA or RNA into the genome of a transfected cell. The term "stable transfectant" (stable transfectant) refers to a cell that stably integrates foreign DNA into genomic DNA.

The term "isolated polynucleotide" or "isolated nucleic acid" refers to a nucleic acid molecule, DNA, or RNA that has been removed from its natural environment. For example, for the purposes of this disclosure, recombinant polynucleotides encoding polypeptides contained in a vector are considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or (partially or substantially) purified polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell that normally contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present disclosure, as well as positive and negative strand forms and double stranded forms.

The terms "nucleic acid molecule encoding", "encoding DNA sequence" and "encoding DNA" refer to the sequence of deoxyribonucleotides along the strand of a deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, the nucleic acid sequence encodes an amino acid sequence.

Methods for producing and purifying antibodies and antigen binding fragments are well known and can be found in the art, e.g., in the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. The antibodies or antigen binding fragments thereof described herein are genetically engineered to incorporate one or more human FR regions in CDR regions of non-human origin. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http:// imgt. Cines. FR, or from the journal of immunoglobulins, (2001) ISBN: 012441351.

The engineered antibodies of the present disclosure, or antigen binding fragments thereof, can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bound to human antigens. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.

The term "individual" or "subject" as used herein refers to any animal, such as a mammal or a pouched animal. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus or other types of macaque), mice, pigs, horses, donkeys, cattle, sheep, rats, and any variety of poultry.

The term "disease" or "condition" or "disorder" or the like as used herein refers to any change or disorder that impairs or interferes with the normal function of a cell, tissue or organ. For example, the "disease" includes but is not limited to: tumors, pathogen infection, autoimmune diseases, T cell dysfunctional diseases, or defects in immunological tolerance (e.g., transplant rejection), and the like.

The term "tumor" as used herein refers to a disease characterized by pathological proliferation of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is generally uncontrolled and progressive, not inducing or inhibiting normal cell proliferation. Tumors include "cancers," which generally refer to all malignant tumors.

The term "treatment" as used herein refers to a clinical intervention in an attempt to alter the course of a disease caused by an individual or a treated cell, either prophylactically or during a clinical pathology. Therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of progression of a disease, improving or alleviating a condition, alleviating or improving prognosis, and the like.

The term "combination" as used herein relates to a therapeutic regimen that provides at least two or more different therapies, either physically, such as radiation therapy, or chemically, such as administration of a drug to a subject, including combinations of drugs, to achieve a given therapeutic effect.

Examples

The disclosure is further illustrated below in conjunction with specific embodiments. It should be understood that these examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, which are not specified in the following examples, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning Experimental guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

EXAMPLE 1 animal immunization

BALB/c mice (Vetolihua, animal use license number SYXK (Lu) 2021-0014) 6-8 weeks old were selected for animal immunization. Each mouse was given 25 μg of recombinant human TIGIT protein, the first time TIGIT protein dose was 50 μg/mouse, every other week. Immune serum is subjected to titer detection by ELISA, the immune response condition of animals is determined, after 4 rounds of immunization, mice with higher serum titers are selected for euthanasia, and spleens are collected for preparing hybridoma cells.

EXAMPLE 2 preparation of murine monoclonal antibody against TIGIT

Lymph node and spleen cells of immunized mice were collected and treated with Sp2/0 myeloma cells at 1:1, and then using a PEG solution to perform fusion, re-suspending the fused cells in HAT medium (dmem+20% fbs+1×hat), mixing to prepare a cell suspension, and transferring the cell suspension into a 96-well plate. The fused cells were cultured in a 5% CO ₂ incubator at 37℃for 12 days. Preliminary screening of hybridoma cells was performed by ELISA, screening positive clones for binding to human TIGIT antigen, and then transferring them to 24-well plate for culture. After 3 days, performing FACS detection on hybridoma supernatant again, selecting positive clones combined with the over-expressed human TIGIT cells and the over-expressed cynomolgus monkey TIGIT cells, and subcloning the selected positive clones to obtain stable single hybridoma cells; the obtained monoclonal is screened again through ELISA detection, FACS measurement and other experiments, and 4 hybridoma cell lines which are combined with the over-expressed human TIGIT cells and the over-expressed cynomolgus monkey TIGIT cells are selected.

Total RNA was extracted from cells by a conventional biological method, and cDNA was synthesized by reverse transcription using HISCRIPT III.sup.1 st Strand cDNASynthesis Kit (Vazyme) with the total RNA as a template. Amplification was performed using cDNA as a template and the constant region primers of the antibody. The PCR products were separated by agarose gel electrophoresis, and the DNA fragments were purified and recovered, and the amino acid sequences of the variable regions of the 4 murine monoclonal antibodies of the present disclosure were obtained after nucleic acid sequencing and translation, and the results are shown in Table 1.

TABLE 14 amino acid sequences of variable regions of murine monoclonal antibodies

Clone ID	Heavy chain variable region (VH)	Light chain variable region (VL)
			18D7E11	SEQ ID NO：1	SEQ ID NO：2
19C6E8	SEQ ID NO：3	SEQ ID NO：4
			19D4F4	SEQ ID NO：5	SEQ ID NO：6
22G9E9	SEQ ID NO：7	SEQ ID NO：8

Based on the amino acid sequences of the variable regions disclosed in Table 1, the CDR and FR of the variable regions of the antibodies are divided by the Kabat numbering convention, and the 6 CDR sequences of each antibody are organized as shown in Table 2 below, with the numerals in parentheses in Table 2 representing the sequence numbers, e.g., (9) representing SEQ ID NO:9.

TABLE 24 amino acid sequences of CDRs of murine monoclonal antibody variable regions

EXAMPLE 3 humanization of murine anti-TIGIT antibodies

Humanized modification is carried out on the selected murine monoclonal antibody 18D7E 11: chimeric antibodies were prepared using human constant domains instead of the constant domains of the parent (murine) antibodies, and humanized by selecting the humanized antibody sequences based on the homology of the parent and human antibodies.

(1) Design of humanized antibodies

According to the sequence of the human IgG germ line (germline) reported in literature (Antibody humanization by framework shuffling, doi: 10.1016/j.ymeth.2005.01.005), two FW1, two FW2 and two FW3 were selected as the framework regions, respectively, for the heavy chain and the light chain. The heavy chain CDR regions of the murine antibody 18D7E11 sequence, namely HCDR1 (SEQ ID NO: 9), HCDR2 (SEQ ID NO: 10) and HCDR3 (SEQ ID NO: 11), were grafted into the framework regions of the heavy chain selected as described above; the light chain CDR regions of 18D7E11, namely LCDR1 (SEQ ID NO: 12), LCDR2 (SEQ ID NO: 13) and LCDR3 (SEQ ID NO: 14), were grafted into the framework regions of the light chain selected as described above.

(2) Expression of humanized antibodies

After the heavy chain and light chain genes of various humanized variants are synthesized and cloned into expression plasmids, respectively, the heavy chain expression plasmid and the light chain expression plasmid which are properly sequenced are co-transfected into CHO-S cells for expression. After screening various expressed humanized variants, obtaining a molecule with better activity, namely TAb07, wherein the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO:33, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 34.

EXAMPLE 4 binding Activity of humanized antibody TAb07

ELISA method was selected to determine the binding activity of the humanized antibody TAb07 obtained in example 3 to human TIGIT and cynomolgus monkey TIGIT.

The experimental procedure is approximately as follows: 100. Mu.L/well of antigen (hTIGIT-his: 1. Mu.g/mL; cyno TIGIT: 2. Mu.g/mL) was coated overnight at 4 ℃. Blocking was performed with 200. Mu.L of blocking solution (1% BSA in PBS) for 1 hour at room temperature. 100. Mu.L of diluted antibody was added per well at an initial concentration of 3. Mu.g/mL or 20nM, according to 1: dilution was done at 3-fold ratio. Incubate at room temperature for 2 hours. PBST was washed 3 times. mu.L of diluted HRP anti-human secondary antibody was added to each well and incubated for 1 hour at room temperature. PBST was washed 3 times. The reaction was stopped by adding 50. Mu.L of LTMB substrate per well, incubating for 5 minutes at room temperature, and then adding 50. Mu.L of stop solution per well. The microplate reader (TECAN SPARK) reads the OD at a wavelength of 450 nm. The results are shown in FIGS. 1A and 1B, respectively.

Half maximal binding concentrations (EC 50) were calculated using GRAPHPAD PRISM 8.0.0 software, with EC50 of TAb07 and human TIGIT, cynomolgus TIGIT being 0.0237nM, 1.28nM, respectively. The result shows that the humanized antibodies TAb07 and hTIGIT and the cyno TIGIT have strong binding activity.

EXAMPLE 5 get the information in person force detection of humanized antibody TAb07

To determine if TAb07 binds to CHO-K1 cells overexpressing human TIGIT or to CHO-K1 cells overexpressing cynomolgus monkey TIGIT, FACS cell binding assays were performed using CHO-K1 cells overexpressing human TIGIT or CHO-K1 cells overexpressing cynomolgus monkey TIGIT, respectively. Briefly, cells with good growth status were seeded at a density of 2×10 ⁵ cells/well in 96-well plates, centrifuged to remove supernatant, and blocked with 200 μl of blocking solution (5% bsa in PBS) at 4 ℃ for 30 min. mu.L of diluted antibody was added per well at a concentration of 3. Mu.g/mL or 20nM, 2. Mu.g/mL or 13.33nM, 1. Mu.g/mL or 6.67nM. After incubation at 4℃for 1 hour, the 96-well plates were washed 3 times with wash solution (1% BSA in PBS). After that, diluted PE anti-human IgG Fc was added. After incubation at 4 ℃ for 40 min, the 96-well plates were washed 3 times with wash solution and then examined for cellular fluorescence using FACS detector (BD), the results are shown in fig. 2A and 2B, respectively. The result shows that the humanized antibody TAb07 has strong binding activity with CHO-K1 cells which over-express human TIGIT and CHO-K1 cells which over-express cynomolgus monkey TIGIT.

The embodiments of the present disclosure described above are exemplary only, and any person skilled in the art may recognize or be able to ascertain numerous specific compounds, materials, and operational equivalents without departing from the general purpose. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims (16)

1. An antibody or antigen-binding fragment thereof against TIGIT, the antibody or antigen-binding fragment thereof being capable of specifically binding to human TIGIT, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 being respectively:

(1) SEQ ID NO 9, 10, 11, 12, 13, 14; or (b)

(2) 15, 16, 17, 18, 19, 20; Or (b)

(3) 21, 22, 23, 24, 25, 26; Or (b)

(4)SEQ ID NO:27，28，29，30，31，32。

2. An antibody against TIGIT, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, which have at least 80% to 100% sequence identity to any one of the following sets of heavy chain variable region and light chain variable region, respectively:

(1) SEQ ID NOs 1 and 2; or (b)

(2) SEQ ID NOs 3 and 4; or (b)

(3) SEQ ID NOs 5 and 6; or (b)

(4) SEQ ID NOS 7 and 8; or (b)

(5) SEQ ID NOS.33 and 34.

3. The anti-TIGIT antibody or antigen-binding fragment thereof of claim 1 or 2, further having one or more of the following characteristics:

(1) It also includes a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region comprises Fc; more preferably, the Fc is derived from murine or human; more preferably, the sequence of the Fc is a native or modified variant;

(2) It is a murine, chimeric, humanized or fully human antibody;

(3) It is a monoclonal antibody; or a full-length antibody, or an antigen-binding fragment thereof of Fab, fv, scFv, F (ab') ₂, a linear antibody, a single domain antibody;

(4) It is in the form of IgG1, igG2, igG3 or IgG 4.

4. A conjugate formed by coupling an antibody or antigen binding fragment thereof against TIGIT of any of the foregoing with a capture label or a detection label comprising a radionuclide, luminescent material, colored material, or enzyme.

5. A fusion protein wherein one fused moiety comprises an antibody or antigen-binding fragment thereof against TIGIT of any of the preceding.

6. A bispecific or multispecific antibody wherein one antigen-binding domain comprises an antibody or antigen-binding fragment thereof of any one of the foregoing anti-TIGIT.

7. Nucleic acid encoding an antibody or antigen binding fragment thereof against TIGIT of any of the preceding.

8. A recombinant vector comprising the nucleic acid of claim 7.

9. A host cell comprising the recombinant vector of claim 8 or the nucleic acid of claim 7.

10. The host cell of claim 9, which is a prokaryotic cell, such as e.coli; or it is a eukaryotic cell, such as a yeast or a mammalian cell, such as CHO cells or HEK293 cells.

11. A method of making any of the foregoing antibodies against TIGIT, or antigen-binding fragments thereof, comprising: culturing the host cell of claim 9 or 10 under suitable conditions and purifying the expression product from said cell.

12. A pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof against TIGIT of any one of claims 1-3, or comprising an effective amount of the fusion protein of claim 5, or comprising an effective amount of the bispecific or multispecific antibody of claim 6, or comprising an effective amount of the nucleic acid of claim 7, or comprising an effective amount of the recombinant vector of claim 8, or comprising an effective amount of the host cell of claim 9 or 10.

13. The pharmaceutical composition of claim 12, further comprising a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, further comprising an additional other therapeutic agent.

15. Use of a pharmaceutical composition according to any one of claims 12-14 for the preparation of a medicament for the treatment of a tumor.

16. The use of claim 15, wherein the tumor is cancer.