KR102769148B1 - Fusion protein comprising anti-TIGIT antibody, interleukin-15, and interleukin-15 receptor alpha sushi domain, and uses thereof - Google Patents

Abstract

Provided are an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, a fusion protein comprising interleukin-15 (IL-15), and an interleukin-15 receptor alpha (IL-15Rα) sushi domain, and pharmaceutical uses of the fusion protein.

Description

Fusion protein comprising anti-TIGIT antibody, interleukin-15, and interleukin-15 receptor alpha sushi domain, and uses thereof

The present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, a fusion protein comprising interleukin-15 (IL-15), and an interleukin-15 receptor alpha (IL-15Rα) sushi domain, and a pharmaceutical use of the fusion protein.

Cancer immunotherapy is a method of treating cancer by using the body's immune system, and is called the fourth treatment for cancer after chemotherapy, surgery, and radiotherapy. Cancer immunotherapy requires a mechanism that activates the immune system to increase recognition and response to tumor cells. Immune system activation is a complex mechanism that involves the functions of various cells, such as antigen-presenting cells, which are important in initiating antigen-specific responses, and effector cells, which are responsible for destroying tumor cells.

A representative example of the above effector cells is cytotoxic T cells.

Meanwhile, TIGIT (T cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibition motif) domain), also called WUCAM, VSIG9 or Vstm3, is a co-inhibitory receptor preferentially expressed on NK, CD8+ and CD4+ T cells as well as regulatory T cells ("Treg"). TIGIT is a transmembrane protein containing an ITIM domain, a transmembrane domain, and an immunoglobulin variable domain within the cell.

TIGIT expression is increased in tumor infiltrating lymphocytes (TILs) and in disease settings such as infection. TIGIT expression may be a marker of exhausted T cells with reduced effector function compared to TIGIT-negative cells. Furthermore, Treg cells expressing TIGIT exhibit enhanced immunosuppressive activity compared to TIGIT-negative Treg populations.

Based on these points, drugs that have an antagonistic effect on TIGIT (e.g., antagonistic anti-TIGIT antibodies) are expected to have a beneficial effect on disease treatment by inducing immune system activation and enhancing immune responses in disease states such as cancer.

Meanwhile, based on the immune activation and anticancer effects of cytokines, proteins that mimic them are being developed as immune activators and immuno-anticancer agents, but the risk of their action as immunogens in vivo has not been verified.

In this situation, the development of drugs that are safe, non-immunogenic, and have excellent immune-activating effects is required.

One example provides a fusion protein comprising (1) an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically recognizes TIGIT, and (2) a cytokine-like polypeptide.

In the above fusion protein, the cytokine-like polypeptide may be linked to the C-terminus and/or N-terminus of the heavy chain (heavy chain constant region or heavy chain variable region) or the light chain (light chain constant region or light chain variable region) of the anti-TIGIT antibody or antigen-binding fragment thereof via a linker (first linker) or directly linked without a linker.

The above cytokine-like polypeptide may be a fusion polypeptide comprising (a) an interleukin-15 receptor alpha (IL-15Rα) sushi domain and (b) interleukin-15 (IL-15).

The cytokine-like polypeptide may be one in which interleukin-15 is linked to the C-terminus and/or the N-terminus of an interleukin-15 receptor alpha sushi domain via a linker (second linker) or is directly linked without a linker. For example, the cytokine-like polypeptide may include, in order from the N-terminus to the C-terminus, an interleukin-15 receptor alpha sushi domain and interleukin-15, or an interleukin-15 receptor alpha sushi domain, a linker (second linker), and interleukin-15.

The first linker (linking the anti-TIGIT antibody or antigen-binding fragment thereof and the cytokine-like polypeptide) and/or the second linker (linking interleukin-15 and the interleukin-15 receptor alpha sushi domain) may be a peptide linker, and the linkage between the anti-TIGIT antibody or antigen-binding fragment thereof and the cytokine-like polypeptide and/or between interleukin-15 and the interleukin-15 receptor alpha sushi domain may be via a peptide bond.

Another example provides a nucleic acid molecule encoding the fusion protein.

Another example provides a recombinant vector comprising a nucleic acid molecule encoding the fusion protein.

Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector.

Another example provides an immunostimulant or an immunostimulant pharmaceutical composition comprising as an active ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a pharmaceutical composition for preventing and/or treating an immune-related disease, comprising as an active ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides an anticancer agent or a pharmaceutical composition for preventing and/or treating cancer, comprising as an active ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a method for enhancing immunity, comprising administering to a subject in need of enhancing immunity a pharmaceutically effective amount of at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a method for preventing and/or treating an immune-related disease, comprising administering to a subject in need of prevention and/or treatment of an immune-related disease a pharmaceutically effective amount of at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a method for preventing and/or treating cancer, comprising administering to a subject in need of prevention and/or treatment of cancer a pharmaceutically effective amount of at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides an immunostimulant use or use for producing an immunostimulant selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a use for preventing and/or treating one or more immune-related diseases selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector, or a use for preparing a drug for preventing and/or treating an immune-related disease.

Another example provides a use for preventing and/or treating one or more cancers selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector, or a use for preparing a drug for preventing and/or treating cancer.

In the present specification, a fusion protein comprising an anti-TIGIT antibody or an antigen-binding fragment thereof that binds to TIGIT, and a cytokine-like polypeptide, and a pharmaceutical use thereof are provided. The fusion protein has the activity of (1) blocking the action of TIGIT by the anti-TIGIT antibody or the antigen-binding fragment thereof to activate immunity (e.g., enhancing effector T cell function, controlling Treg activity, increasing cytokine secretion by immune cells (T cells, NK cells, etc.)), and (2) having an advantage of excellent binding affinity to a wild-type interleukin-15/interleukin-2 receptor while having low immunogenicity by the cytokine-like polypeptide, and thus can be applied as various immune activators and/or immunotherapeutic agents.

Below, it is explained in more detail.

Definition of terms

In this specification, the expression that a nucleic acid molecule (which may be used interchangeably with a “polynucleotide” or a “gene”) or a polypeptide (which may be used interchangeably with a “protein”) “comprises a particular nucleic acid sequence or amino acid sequence or consists of or is represented by a particular nucleic acid sequence or amino acid sequence” may mean that the nucleic acid molecule or the polypeptide essentially includes the particular nucleic acid sequence or amino acid sequence, and may be interpreted to include (or not exclude) a “substantially equivalent sequence” in which a mutation (deletion, substitution, modification, and/or addition) is added to the particular nucleic acid sequence or amino acid sequence to the extent that the original function and/or the desired function of the nucleic acid molecule or the polypeptide is maintained.

In one example, the nucleic acid sequence or amino acid sequence provided herein may include a modification by conventional mutagenesis, such as directed evolution and/or site-directed mutagenesis, to the extent that it maintains its original function or desired function. In one example, a nucleic acid molecule or a polypeptide "comprises, consists of, or is represented by a particular nucleic acid sequence or amino acid sequence" can mean that the nucleic acid molecule or polypeptide (i) consists essentially of the particular nucleic acid sequence or amino acid sequence, or (ii) consists of or essentially consists of an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the particular nucleic acid sequence or amino acid sequence and retains its original function and/or desired function.

The nucleic acid sequence described herein may be modified in various ways in the coding region within a range that does not change the amino acid sequence and/or function of the protein expressed from the coding region, taking into account the codon preferred in the microorganism that is intended to express the protein due to the degeneracy of the codon.

As used herein, the term "homology" means the degree of identity with a given nucleic acid sequence or amino acid sequence, and may be expressed as a percentage (%). In the case of homology for nucleic acid sequences, for example, it can be determined using well-known algorithms such as BLAST or FASTA, and based on the algorithm BLAST, programs called BLASTN or BLASTX have been developed (reference: http://www.ncbi.nlm.nih.gov).

fusion protein

In the present specification, an example provides a fusion protein comprising (1) an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically recognizes TIGIT and (2) a cytokine-like polypeptide.

In the above fusion protein, the anti-TIGIT antibody or the antigen-binding fragment thereof may have an activity that blocks the action of TIGIT to activate immunity (e.g., enhancing effector T cell function, controlling Treg activity, increasing cytokine secretion of immune cells (T cells, NK cells, etc.)). In addition, the cytokine-like polypeptide may have an activity similar to interleukin-15 and/or interleukin-2, while having excellent binding affinity to receptors and low immunogenicity compared to wild-type interleukin-15/interleukin-2, and may be a safe polypeptide that has an excellent immune-enhancing effect when administered to a living body while having a low risk of inducing an immune response recognizing it as an immunogen. Therefore, the fusion protein can be applied as various immune activators and/or immunotherapeutic agents.

In one example, in the fusion protein, the cytokine-like polypeptide may be linked to the C-terminus and/or N-terminus of the heavy chain (heavy chain constant region or heavy chain variable region) or the light chain (light chain constant region or light chain variable region) of the anti-TIGIT antibody or antigen-binding fragment thereof via a linker (first linker) or directly linked without a linker.

More specifically, the fusion protein comprises (1) an anti-TIGIT antibody (e.g., an IgG type antibody comprising two heavy chains and two light chains) and (2) a cytokine-like polypeptide, wherein the cytokine-like polypeptide may be linked to the C-terminus (the C-terminus of the constant region) or the N-terminus of either one of the two heavy chains or one of the two light chains of the anti-TIGIT antibody, with or without a peptide linker (a first linker).

In another specific embodiment, the fusion protein comprises (1) an anti-TIGIT antibody (e.g., an IgG type antibody comprising two heavy chains and two light chains) and (2) two cytokine-like polypeptides, wherein the two cytokine-like polypeptides may be linked to the C-terminus or the N-terminus of the two heavy chains or the two light chains of the anti-TIGIT antibody, respectively, with or without a peptide linker (first linker).

For example, in the above fusion protein,

(i) one of the two cytokine-like polypeptides is linked to the C-terminus (i.e., the C-terminus of the heavy chain constant region (e.g., Fc)) or N-terminus (i.e., the N-terminus of the heavy chain variable region) of one of the two heavy chains of the anti-TIGIT antibody, and the other is linked to the C-terminus (i.e., the C-terminus of the heavy chain constant region (e.g., Fc)) or N-terminus (i.e., the N-terminus of the heavy chain variable region) of the other heavy chain of the anti-TIGIT antibody, with or without a linker (first linker), or

(ii) one of the two cytokine-like polypeptides may be linked to the C-terminus (i.e., the C-terminus of the light chain constant region) or the N-terminus (i.e., the N-terminus of the light chain variable region) of one of the two light chains of the anti-TIGIT antibody, and the other may be linked to the C-terminus (i.e., the C-terminus of the light chain constant region) or the N-terminus (i.e., the N-terminus of the light chain variable region) of the other light chain of the anti-TIGIT antibody, with or without a linker (first linker).

More specifically, in the fusion protein,

(i) one of the two cytokine-like polypeptides is linked to the C-terminus of the Fc of one of the two heavy chains of the anti-TIGIT antibody, and the other is linked to the C-terminus of the Fc of the other of the anti-TIGIT antibody, with or without a linker (first linker), or

(ii) One of the two cytokine-like polypeptides may be linked to the C-terminus of the constant region of one of the two light chains of the anti-TIGIT antibody, and the other may be linked to the C-terminus of the constant region of the other light chain of the anti-TIGIT antibody, with or without a linker (first linker).

The first linker may be a peptide linker, and in one example, 1 to 50, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 10, 2 to 50, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 18, 2 to 15, 2 to 10, 5 to 50, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 18, 5 to 15, 5 to 10, 10 to It may comprise 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 18, 10 to 15, 20 to 50, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 30 to 50, 30 to 40, 30 to 35, 32 to 50, 32 to 40, 32 to 35, 35 to 50, or 35 to 40 amino acids. For example, the first linker is [(G)mS]l (wherein m is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) representing the number of amino acids G(Gly), and l is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) representing the number of [(G)mS]; for example, GGGGS (SEQ ID NO: 96), GGGGSGGGGS (SEQ ID NO: 97), GGGGSGGGGSGGGGS (SEQ ID NO: 85), GS, GSGS (SEQ ID NO: 98), GSGSGS (SEQ ID NO: 99), GSGSGSGS (SEQ ID NO: 100), GSGSGSGSGS (SEQ ID NO: 101), etc.), [(S)o(G)pS]q[XQ]r (wherein o is the number of amino acids S (Ser) and is 0 (absent) or 1, p is the number of amino acids G (Gly) and is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), q is the number of the units [(S)o(G)pS] and is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), and when there are two or more of the units (when q is 2, 3, 4, or 5), each unit may be the same or different, X is leucine (Leu; I) or isoleucine (Ile; I), and r is the number of the dipeptide [XQ] and is 0 (absent) or 1; for example, GGGGSGGGGSLQ (SEQ ID NO: 102), GSGGGGSGGGGSLQ (SEQ ID NO: 103), GGGGSGGGGSIQ (SEQ ID NO: 104), GSGGGGSGGGGSIQ (SEQ ID NO: 105), SGGSGGGGSGGGSGGGGSIQ (SEQ ID NO: 106), SGGGSGGGGSGGGGSGGGGSGGGSIQ (SEQ ID NO: 107), SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108), SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 109)), SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110), SGGGGSGGGSGGGGGSGGGSGSG (SEQ ID NO: 111) etc.) [EAAAK]n (n is the number of [EAAAK] (SEQ ID NO: 112) and is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); For example, EAAAKEAAAKEAAAK (SEQ ID NO: 44), etc.), A EAAAK A (SEQ ID NO: 113), A EAAAK EAAAK A (SEQ ID NO: 114), A EAAAK EAAAK EAAAK A (SEQ ID NO: 115), A EAAAK EAAAK EAAAK EAAAK A (SEQ ID NO: 116), A EAAAK EAAAK EAAAK EAAAK EAAAK A (SEQ ID NO: 117), AEAAAKEAAAKAG (SEQ ID NO: 118), AEAAAKEAAAKAGS (SEQ ID NO: 119), GGGGG (SEQ ID NO: 120), GGAGG (SEQ ID NO: 121), GGGGGGGG (SEQ ID NO: 122), GAGAGAGAGA (SEQ ID NO: 123), RPLSYRPPFPFGFPSVRP (SEQ ID NO: 124), YPRSIYIRRRHPSPSLTT (SEQ ID NO: 125), TPSHLSHILPSFGLPTFN (SEQ ID NO: 126), RPVSPFTFPRLSNSWLPA (SEQ ID NO: 127), SPAAHFPRSIPRPGPIRT (SEQ ID NO: 128), APGPSAPSHRSLPSRAFG (SEQ ID NO: 129), PRNSIHFLHPLLVAPLGA (SEQ ID NO: 130), MPSLSGVLQVRYLSPPDL (SEQ ID NO: 131), SPQYPSPLTLTLPPHPSL (SEQ ID NO: 132), NPSLNPPSYLHRAPSRIS (SEQ ID NO: 133), LPWRTSLLPSLPLRRRP (SEQ ID NO: 134), PPLFAKGPVGLLSRSFPP (SEQ ID NO: 135), VPPAPVVSLRSAHARPPY (SEQ ID NO: 136), LRPTPPRVRSYTCCPTP (SEQ ID NO: 137), PNVAHVLPLL TVPWDNLR (SEQ ID NO: 138), CNPLLPLCARSPAVRTFP (SEQ ID NO: 139), LGTPTPTPTPTGEF (SEQ ID NO: 140), EDFTRGKL (SEQ ID NO: 141), L EAAAR EAAAR EAAAR EAAAR (SEQ ID NO: 142), L EAAAR EAAAR EAAAR (SEQ ID NO: 143), L EAAAR (SEQ ID NO: 144), L EAAAR EAAAR (SEQ ID NO: 145), EAAAR EAAAR EAAAR EAAAR (SEQ ID NO: 146), EAAAR EAAAR EAAAR (SEQ ID NO: 147), EAAAR EAAAR (SEQ ID NO: 148), EAAAR (SEQ ID NO: 149), LTEEQQEGGG (SEQ ID NO: 150), TEEQQEGGG LTEEQQEGGG (SEQ ID NO: 151), The present invention may be directed to, but is not limited to, one or more selected from the group consisting of LAKLKQKTEQLQDRIAGGG (SEQ ID NO: 152), LELKTPLGDT (SEQ ID NO: 153), THTCPRCPEP (SEQ ID NO: 154), KSCDTPPPCP (SEQ ID NO: 155), RCPEPKSCDT (SEQ ID NO: 156), PPPCPRCPEP (SEQ ID NO: 157), KSCDTPPPCP (SEQ ID NO: 158), RCPGG (SEQ ID NO: 159), and LEPKSSDKTHTSPPSPGG (SEQ ID NO: 160). In one embodiment, the first linker is a rigid peptide linker (e.g., [EAAAK]n (where n is the number of [EAAAK] and is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); e.g., EAAAKEAAAKEAAAK (SEQ ID NO: 44)) or a flexible peptide linker (e.g., [(G)mS]l (wherein m is the number of amino acids G(Gly) and is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) and l is the number of [(G)mS] and is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); e.g., GGGGS (SEQ ID NO: 96), GGGGSGGGGS (SEQ ID NO: 97, GGGGSGGGGSGGGGS (SEQ ID NO: 85), GS, GSGS (SEQ ID NO: 98), GSGSGS (SEQ ID NO: 99), GSGSGSGS (SEQ ID NO: 100), GSGSGSGSGS (SEQ ID NO: 101), etc.)) but are not limited thereto.

Anti-TIGIT antibody or antigen-binding fragment thereof

The above anti-TIGIT antibody or antigen-binding fragment thereof,

A polypeptide comprising an amino acid sequence of sequence number 1, 2, or 3 (CDR-H1),

A polypeptide comprising an amino acid sequence of sequence number 4, 5, or 6 (CDR-H2),

A polypeptide comprising an amino acid sequence of sequence number 7, 8, or 9 (CDR-H3),

A polypeptide comprising an amino acid sequence of sequence number 10, 13, or 14 (CDR-L1),

A polypeptide comprising an amino acid sequence of SEQ ID NO: 15, 16, or 17 (CDR-L2), and

A polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 19, or 20 (CDR-L3)

may include.

The polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 10 may comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

In this specification, the complementarity determining region (CDR) is determined based on the CDR definition according to the Kabat system.

In one specific example, the six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that can be included in the anti-TIGIT antibody or antigen-binding fragment thereof provided herein are summarized in Table 1 below:

order Sequence number CDR-H1 SDYAWN 1 CDR-H1 GSTFTEYTMH 2 CDR-H1 GYSFTDYIMN 3 CDR-H2 YISYSGSARYNPSLKS 4 CDR-H2 GLNPNNGGTSYNQRFKD 5 CDR-H2 LSIPYNGGTSYNQKFEG 6 CDR-H3 KGYPAYFAY 7 CDR-H3 GTYYDYSFAY 8 CDR-H3 GIKGYFAMDY 9 CDR-L1 XASQDVSTAVA (X=K or R) 10 CDR-L1 KASQDVSTAVA 11 CDR-L1 RASQDVSTAVA 12 CDR-L1 KASHYVSTAVA 13 CDR-L1 RSSQSLVNSFGKTYLS 14 CDR-L2 SASYRYT 15 CDR-L2 SPSYRYT 16 CDR-L2 GVSNRFS 17 CDR-L3 QHHYSTPYT 18 CDR-L3 HQHYSTPWT 19 CDR-L3 LQGTHQPWT 20

(In Table 1 above, CDR-H1, CDR-H2, and CDR-H3 represent the heavy chain complementarity determining region, and CDR-L1, CDR-L2, and CDR-L3 represent the light chain complementarity determining region)

In a specific embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof is

A heavy chain variable region comprising CDR-H1 of SEQ ID NO: 1, 2, or 3, CDR-H2 of SEQ ID NO: 4, 5, or 6, and CDR-H3 of SEQ ID NO: 7, 8, or 9, and

A light chain variable region comprising a CDR-L1 of SEQ ID NO: 10 (e.g., SEQ ID NO: 11 or SEQ ID NO: 12), 13, or 14, a CDR-L2 of SEQ ID NO: 15, 16, or 17, and a CDR-L3 of SEQ ID NO: 18, 19, or 20.

may include.

More specifically, the anti-TIGIT antibody or antigen-binding fragment thereof,

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, or 28, and

A light chain variable region comprising the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, or 36

may include.

In some cases (e.g., when produced recombinantly), the heavy chain variable region and/or the light chain variable region may additionally comprise an appropriate signal sequence at the N terminus.

The amino acid sequences of the heavy chain variable region and light chain variable region that can be included in the anti-TIGIT antibody or antigen-binding fragment thereof provided herein are exemplified in Table 2 below:

Variable area Amino acid sequence (N→C) Sequence number Heavy chain variable region (VH0) DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YISYSGSARYNPSLKS RISITRDTSMNQFFLQLNSVTAEDTATYYCAR KGYPAYFAY WGQGTLVTVSS 21 Heavy chain variable region (VH1) DVQLQESGPGLVKPSQTLSLTCTVTGYSIT SDYAWN WIRQPPGKGLEWMG YISYSGSARYNPSLKS RITISRDTSMNQFSLKLNSVTAEDTATYYCAR KGYPAYFAY WGQGTLVTVSS 22 Heavy chain variable region (VH2) DVQLQESGPGLVKPSQTLSLTCTVTGYSIT SDYAWN WIRQPPGKGLEWMG YISYSGSARYNPSLKS RITISRDTSKNQFSLKLSSVTAEDTATYYCAR KGYPAYFAY WGQGTLVTVSS 23 Heavy chain variable region (VH3) QVQLQESGPGLVKPSQTLSLTCTVTGYSIT SDYAWN WIRQPPGKGLEWMG YISYSGSARYNPSLKS RVTISRDTSKNQFSLKLSSVTAEDTATYYCAR KGYPAYFAY WGQGTLVTVSS 24 Heavy chain variable region (VH4) QVQLQESGPGLVKPSQTLSLTCTVTGYSIT SDYAWN WIRQPPGKGLEWMG YISYSGSARYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR KGYPAYFAY WGQGTLVTVSS 25 Heavy chain variable region (VH5) QVQLQESGPGLVKPSQTLSLTCTVTGYSIT SDYAWN WIRQPPGKGLEWMG YISYSGSARYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR KGYPAYFAY WGQGTLVTVSS 26 Heavy chain variable region EVQLQQSGPELVKPGASVKISCKTS GSTFTEYTMH WVKQSHGKSLEWIG GLNPNNGGTSYNQRFKD RATLTVDKSSSTAYMELRSLTSEDSAVYYCTR GTYYDYSFAY WGQGTLVTVSA 27 Heavy chain variable region EVQLQQSGPELVKPGASMKISCKAS GYSFTDYIMN WVKQSHGKNLEWIG LSIPYNGGTSYNQKFEG KATLTVDKSSSTAYMELLSLTSEDSAVYYCAR GIKGYFAMDY WGQGTSVTVSS 28 Light chain variable region (Vk0) DIVMTQSHKFMSTSVGDRVSISC KASQDVSTAVA WYQQKPGQSPELLIY SASYRYT GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC QHHYSTPYT FGGGTKLEMK 29 Light chain variable region (Vk1) DIVMTQSHSFLSASVGDRVSITC KASQDVSTAVA WYQQKPGQAPELLIY SASYRYT GVPDRFTGSGSGTDFTLTISSLQAEDVAVYYC QHHYSTPYT FGQGTKLEMK 30 Light chain variable region (Vk2) DIVMTQSPSSLSASVGDRVSITC KASQDVSTAVA WYQQKPGQAPRLLIY SASYRYT GVPDRFTGSGSGTDFTLTISSLQAEDVAVYYC QHHYSTPYT FGQGTKLEIK 31 Light chain variable region (Vk3) DIQMTQSPSSLSASVGDRVSITC KASQDVSTAVA WYQQKPGQAPRLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QHHYSTPYT FGQGTKLEIK 32 Light chain variable region (Vk4) DIQMTQSPSSLSASVGDRVTITC RASQDVSTAVA WYQQKPGQAPRLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QHHYSTPYT FGQGTKLEIK 33 Light chain variable region (Vk5) DIQMTQSPSSLSASVGDRVSITC KASQDVSTAVA WYQQKPGQAPRLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFATYYC QHHYSTPYT FGQGTKLEIK 34 Light chain variable region DIVMTQSHKFMSTSVGDRVSITC KASHYVSTAVA WYQQKPGQSPKLLIY SPSYRYT GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC HQHYSTPWT FGGGTKLEIK 35 Light chain variable region DVVVTQTPLSLPVSFGDQVSISC RSSQSLVNSFGKTYLS WFLHKPGQSPQLIMY GVSNRFS GVPDRFSGSGSGTDFTLKISTIKPEDLGMYYC LQGTHQPWT FGGGTKLEIK 36

(In Table 2, the underlined regions represent CDR1, CDR2, and CDR3 of the heavy and light chains, respectively.)

In one example, the anti-TIGIT antibody or antigen-binding fragment thereof provided herein can bind to a TIGIT protein, e.g., one or more, or two or more (e.g., consecutively positioned) amino acids selected from the region of amino acid residues 51-70 (TAQVTQVNWEQQDQLLAICN; SEQ ID NO: 38) of human TIGIT protein (NCBI Reference Sequence NP_776160.2; UniProtKB/SwissProt Q495A1-1), but is not limited thereto.

[Human TIGIT protein (SEQ ID NO: 37)]

1 MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST TAQVTQVNWE

61 QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV NDTGEYFCIY HTYPDGTYTG

121 RIFLEVLESS VAEHGARFQI PLLGAMAATL VVICTAVIVV VALTRKKKAL RIHSVEGDLR

181 RKSAGQEEWS PSAPSPPGSC VQAEAAPAGL CGEQRGEDCA ELHDYFNVLS YRSLGNCSFF

241 TETG

In this specification, the term "antibody" refers to a general term for a protein that specifically binds to a specific antigen, and may be a protein produced by stimulation of an antigen in the immune system, or a protein produced chemically synthesized or recombinantly, and its type is not particularly limited. The antibody may be non-naturally produced, for example, recombinantly or synthetically produced. The antibody may be an animal antibody (e.g., mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.

The heavy chain CDR and light chain CDR regions, or the heavy chain variable region and the light chain variable region, as defined above in the anti-TIGIT antibodies or antigen-binding fragments thereof provided herein may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), for example, may be derived from the framework region, and/or the light chain constant region and/or the heavy chain constant region of any of the above subtypes of immunoglobulins. In one example, the anti-TIGIT antibodies provided herein may be, but are not limited to, human IgG type antibodies, such as, but not limited to, antibodies of the IgG1, IgG2, IgG3, or IgG4 type.

A complete antibody (e.g., IgG type) has two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) as subclasses. The constant region of the light chain has kappa (κ) and lambda (λ) types.

In one example, the anti-TIGIT antibody provided herein may comprise, but is not limited to, an IgG constant region as the heavy chain constant region and a kappa constant region as the light chain constant region.

In one specific embodiment, the constant region of the IgG (e.g., human IgG1) may be wild-type. In another specific embodiment, the constant region of the IgG may be a mutant comprising one or more mutations selected from the group consisting of S240D (S at position 240 is replaced with D, hereinafter amino acid mutations are expressed in the same manner), A331L, I333E, N298A, S299A, E334A, K335A, L235A, L236A, and P330G, based on human IgG1, and for example, may be a mutant comprising the following mutations:

(1) S240D, A331L, and I333E;

(2) N298A;

(3) S299A, E334A, and K335A; or

(4) L235A, L236A and P330G.

The term "heavy chain" is interpreted to mean both a full-length heavy chain and fragments thereof, comprising a variable region domain V _H comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains C _H1 , C _H2 and C _H3 and a hinge. Furthermore, the term "light chain" is interpreted to mean both a full-length light chain and fragments thereof, comprising a variable region domain V _L comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a constant region domain C _L .

The term "CDR (complementarity determining region)" refers to a region among the variable regions of an antibody that confers binding specificity to an antigen, and refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulins. The heavy and light chains may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide major contact residues for binding of the antibody to an antigen or epitope. Meanwhile, in the present specification, the terms "specifically bind" or "specifically recognize" have the same meaning as that commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to cause an immunological reaction.

The complementarity determining region (CDR) described in this specification is determined based on the CDR definition according to the Kabat system.

In this specification, an antibody is understood to include an antigen-binding fragment of an antibody possessing antigen-binding ability, unless otherwise specified.

The term "antigen-binding fragment" refers to any form of a polypeptide that comprises a portion capable of binding an antigen (e.g., six CDRs as defined herein). For example, but not limited to, scFv, scFv-Fc, (scFv) ₂ , Fab, Fab' or F(ab') ₂ of an antibody.

Among the above antigen-binding fragments, Fab has one antigen-binding site in a structure having variable regions of the light and heavy chains, constant regions of the light chain, and first constant regions (C _H1 ) of the heavy chain.

Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain.

F(ab') ₂ antibodies are produced by disulfide bonding between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art.

In a two-chain Fv, the heavy chain variable region and the light chain variable region are non-covalently linked, and in a single-chain Fv, the heavy chain variable region and the single chain variable region are covalently linked, usually via a peptide linker, or are directly linked at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv.

The above antigen-binding fragment can be obtained using a protein hydrolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab') ₂ fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology.

The term "hinge region" refers to a region contained in the heavy chain of an antibody, which exists between the CH1 and CH2 regions and functions to provide flexibility to the antigen binding site within the antibody.

The antibody provided herein may be a monoclonal antibody. The monoclonal antibody may be produced by a method widely known in the art. For example, it may be produced using a phage display technique. Alternatively, the antibody may be produced as a monoclonal antibody derived from an animal (e.g., a mouse) by a conventional method.

Meanwhile, individual monoclonal antibodies can be screened based on their binding ability to the receptor binding domain of TIGIT using a typical Enzyme-Linked ImmunoSorbent Assay (ELISA) format. The inhibitory activity can be tested using functional assays such as Competitive ELISA to test molecular interactions for the binders or cell-based assays. Then, the affinities (Kd values) of the monoclonal antibody members selected based on strong inhibitory activity to the receptor binding domain of TIGIT can be tested.

The final selected antibodies can be manufactured and used as humanized antibodies as well as antibodies in which the remainder, excluding the antigen-binding portion, is converted to human immunoglobulin antibodies. Methods for manufacturing humanized antibodies are well known in the art.

The antigen-binding fragment of the anti-TIGIT antibody provided herein means a fragment derived from the anti-TIGIT antibody and having binding ability to an antigen (TIGIT), and may be any polypeptide including six CDRs of the anti-TIGIT antibody, for example, scFv, scFv-Fc, scFv-Ck (kappa constant region), scFv-Cλ (lambda constant region), (scFv) ₂ , Fab, Fab' or F(ab') ₂ , but is not limited thereto. In one example, the antigen-binding fragment can be, but is not limited to, a scFv, or a fusion polypeptide (scFv-Fc) in which the scFv is fused to the Fc portion of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, etc.) or a fusion polypeptide (scFv-Ck or scFv-Cλ) in which the scFv is fused to the constant region of a light chain (e.g., kappa or lambda).

In the present specification, the phrase “an antibody (e.g., a CDR, a variable region, or a heavy/light chain, an antigen-binding fragment, etc.) comprises, consists of, or includes a particular amino acid sequence” may mean both cases where the antibody essentially includes the amino acid sequence, and cases where a meaningless mutation (e.g., substitution, deletion, and/or addition of an amino acid residue) that does not significantly affect antibody activity (e.g., antigen affinity, pharmacological activity, etc.) is introduced into the amino acid sequence.

The anti-TIGIT antibody or antigen-binding fragment thereof provided herein can have a binding affinity (K _D ) to TIGIT (eg, human TIGIT) of, for example, as measured by surface plasmon resonance (SPR) of 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM or less, 0.1 mM or less, 0.05 mM or less, 0.01 mM or less, 0.005 mM or less, or 0.001 mM or less, for example, 0.0001 nM to 10 mM, 0.0005 nM to 10 mM, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM. 10 mM, 0.1 nM to 10 mM, 0.5 nM to 10 mM, 1 nM to 10 mM, 0.0001 nM to 5 mM, 0.0005 nM to 5 mM, 0.001 nM to 5 mM, 0.005 nM to 5 mM, 0.01 nM to 5 mM, 0.05 nM to 5 mM, 0.1 nM to 5 mM, 0.5 nM to 5 mM, 1 nM to 5 mM, 0.0001 nM to 1 mM, 0.0005 nM to 1 mM, 0.001 nM to 1 mM, 0.005 nM to 1 mM, 0.01 nM to 1 mM, 0.05 nM to 1 mM, 0.1 nM to 1 mM, 0.5 nM to 1 mM, 1 nM to 1 mM, 0.0001 nM to 0.5 mM, 0.0005 nM to 0.5 mM, 0.001 nM to 0.5 mM, 0.005 nM to 0.5 mM, 0.01 nM to 0.5 mM, 0.05 nM to 0.5 mM, 0.1 nM to 0.5 mM, 0.5 nM to 0.5 mM, 1 nM to 0.5 mM, 0.0001 nM to 0.2 mM, 0.0005 nM to 0.2 mM, 0.001 nM to 0.2 mM, 0.005 nM to 0.2 mM, 0.01 nM to 0.2 mM, 0.05 nM to 0.2 mM, 0.1 nM to 0.2 mM, 0.5 nM to 0.2 mM, 1 nM to 0.2 mM, 0.0001 nM to 0.1 mM, 0.0005 nM to 0.1 mM, 0.001 nM to 0.1 mM, 0.005 nM to 0.1 mM, 0.01 nM to 0.1 mM, 0.05 nM to 0.1 mM, 0.1 nM to 0.1 mM, 0.5 nM to 0.1 mM, 1 nM to 0.1 mM, 0.0001 nM to 0.05 mM, 0.0005 nM to 0.05 mM, 0.001 nM to 0.05 mM, 0.005 nM to 0.05 mM, 0.01 nM to 0.05 mM, 0.05 nM to 0.05 mM, 0.1 nM to 0.05 mM, 0.5 nM to 0.05 mM, 1 nM to 0.05 mM, 0.0001 nM to 0.01 mM, 0.0005 nM to 0.01 mM, 0.001 nM to 0.01 mM, 0.005 nM to 0.01 mM, 0.01 nM to 0.01 mM, 0.05 nM to 0.01 mM, 0.1 nM to 0.01 mM, 0.5 nM to 0.01 mM, or 1 nM to 0.01 mM However, it is not limited to these.

Cytokine-like polypeptide

The above cytokine-like polypeptides are,

(a) interleukin-15 receptor alpha (IL-15Rα) sushi domain, and

(b) Interleukin-15 (IL-15)

A fusion polypeptide comprising:

In addition to the cytokine-like polypeptide, (a) an interleukin-15 receptor alpha (IL-15Rα) sushi domain and (b) interleukin-15, a peptide linker (a second linker) may be further included. The second linker may be positioned between interleukin-15 and the interleukin-15 receptor alpha sushi domain to connect them.

In one example, the cytokine-like polypeptides are, in order from the N-terminus to the C-terminus,

Containing interleukin-15 receptor alpha sushi domain and interleukin-15, or

It may comprise an interleukin-15 receptor alpha sushi domain, a peptide linker (a second linker), and interleukin-15.

In one specific embodiment, the interleukin-15 receptor alpha sushi domain may comprise an amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 88, or an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[Sequence number 39]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP

[Sequence number 88]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPPS

The above interleukin-15 may be human interleukin-15 (wild type) or a variant of human interleukin-15, and the variant of human interleukin-15 may include an amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[Sequence number 40]

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Interleukin-15 (IL-15) and interleukin-2 (IL-2) have structural similarity to each other and bind to the IL-2/IL-15 receptor beta chain (CD122) and common gamma chain (gamma-C, CD132) complex, thereby participating in signal transduction. In one example, interleukin-15 may be of human origin, such as, but not limited to, NCBI Accession No. NP_000576.1 (amino acid residues 49 to 162). Interleukin-2 may be of human origin, such as, but not limited to, NCBI Accession No. NP_000577.2.

The above cytokine-like polypeptide may include additional oligopeptides for additional purposes, such as, but not limited to, a Tag for labeling at the N-terminus for convenience of protein purification (e.g., MAPRRARGCRTLGLPALLLLLLLRPPATRGDYKDDDDKIEGR (SEQ ID NO: 89), etc.).

The above cytokine-like polypeptide is a synthetic protein (engineered protein) using interleukin-15/interleukin-2 protein as a basic framework, and is characterized by having the activity of interleukin-15 and lower immunogenicity when administered in the body compared to other synthetic proteins having the activity of interleukin-15.

The second linker may be a peptide linker, and in one example, 1 to 50, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 10, 2 to 50, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 18, 2 to 15, 2 to 10, 5 to 50, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 18, 5 to 15, 5 to 10, 10 to It may comprise 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 18, 10 to 15, 20 to 50, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 30 to 50, 30 to 40, 30 to 35, 32 to 50, 32 to 40, 32 to 35, 35 to 50, or 35 to 40 amino acids. For example, the second linker is [(G)mS]l (wherein m is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) representing the number of amino acids G(Gly), and l is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) representing the number of [(G)mS]; for example, GGGGS (SEQ ID NO: 96), GGGGSGGGGS (SEQ ID NO: 97), GGGGSGGGGSGGGGS (SEQ ID NO: 85), GS, GSGS (SEQ ID NO: 98), GSGSGS (SEQ ID NO: 99), GSGSGSGS (SEQ ID NO: 100), GSGSGSGSGS (SEQ ID NO: 101), etc.), [(S)o(G)pS]q[XQ]r (wherein o is the number of amino acids S (Ser) and is 0 (absent) or 1, p is the number of amino acids G (Gly) and is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), q is the number of the units [(S)o(G)pS] and is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), and when there are two or more of the units (when q is 2, 3, 4, or 5), each unit may be the same or different, X is leucine (Leu; I) or isoleucine (Ile; I), and r is the number of the dipeptide [XQ] and is 0 (absent) or 1; for example, GGGGSGGGGSLQ (SEQ ID NO: 102), GSGGGGSGGGGSLQ (SEQ ID NO: 103), GGGGSGGGGSIQ (SEQ ID NO: 104), GSGGGGSGGGGSIQ (SEQ ID NO: 105), SGGSGGGGSGGGSGGGGSIQ (SEQ ID NO: 106), SGGGSGGGGSGGGGSGGGGSGGGSIQ (SEQ ID NO: 107), SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108), SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 109)), SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110), SGGGGSGGGSGGGGGSGGGSGSG (SEQ ID NO: 111) etc.) [EAAAK]n (n is the number of [EAAAK] (SEQ ID NO: 112) and is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); For example, EAAAKEAAAKEAAAK (SEQ ID NO: 44), etc.), A EAAAK A (SEQ ID NO: 113), A EAAAK EAAAK A (SEQ ID NO: 114), A EAAAK EAAAK EAAAK A (SEQ ID NO: 115), A EAAAK EAAAK EAAAK EAAAK A (SEQ ID NO: 116), A EAAAK EAAAK EAAAK EAAAK EAAAK A (SEQ ID NO: 117), AEAAAKEAAAKAG (SEQ ID NO: 118), AEAAAKEAAAKAGS (SEQ ID NO: 119), GGGGG (SEQ ID NO: 120), GGAGG (SEQ ID NO: 121), GGGGGGGG (SEQ ID NO: 122), GAGAGAGAGA (SEQ ID NO: 123), RPLSYRPPFPFGFPSVRP (SEQ ID NO: 124), YPRSIYIRRRHPSPSLTT (SEQ ID NO: 125), TPSHLSHILPSFGLPTFN (SEQ ID NO: 126), RPVSPFTFPRLSNSWLPA (SEQ ID NO: 127), SPAAHFPRSIPRPGPIRT (SEQ ID NO: 128), APGPSAPSHRSLPSRAFG (SEQ ID NO: 129), PRNSIHFLHPLLVAPLGA (SEQ ID NO: 130), MPSLSGVLQVRYLSPPDL (SEQ ID NO: 131), SPQYPSPLTLTLPPHPSL (SEQ ID NO: 132), NPSLNPPSYLHRAPSRIS (SEQ ID NO: 133), LPWRTSLLPSLPLRRRP (SEQ ID NO: 134), PPLFAKGPVGLLSRSFPP (SEQ ID NO: 135), VPPAPVVSLRSAHARPPY (SEQ ID NO: 136), LRPTPPRVRSYTCCPTP (SEQ ID NO: 137), PNVAHVLPLL TVPWDNLR (SEQ ID NO: 138), CNPLLPLCARSPAVRTFP (SEQ ID NO: 139), LGTPTPTPTPTGEF (SEQ ID NO: 140), EDFTRGKL (SEQ ID NO: 141), L EAAAR EAAAR EAAAR EAAAR (SEQ ID NO: 142), L EAAAR EAAAR EAAAR (SEQ ID NO: 143), L EAAAR (SEQ ID NO: 144), L EAAAR EAAAR (SEQ ID NO: 145), EAAAR EAAAR EAAAR EAAAR (SEQ ID NO: 146), EAAAR EAAAR EAAAR (SEQ ID NO: 147), EAAAR EAAAR (SEQ ID NO: 148), EAAAR (SEQ ID NO: 149), LTEEQQEGGG (SEQ ID NO: 150), TEEQQEGGG LTEEQQEGGG (SEQ ID NO: 151), The present invention may be directed to, but is not limited to, one or more selected from the group consisting of LAKLKQKTEQLQDRIAGGG (SEQ ID NO: 152), LELKTPLGDT (SEQ ID NO: 153), THTCPRCPEP (SEQ ID NO: 154), KSCDTPPPCP (SEQ ID NO: 155), RCPEPKSCDT (SEQ ID NO: 156), PPPCPRCPEP (SEQ ID NO: 157), KSCDTPPPCP (SEQ ID NO: 158), RCPGG (SEQ ID NO: 159), and LEPKSSDKTHTSPPSPGG (SEQ ID NO: 160). In one example, the second linker may be at least one selected from the group consisting of SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108), GGGGSGGGGSGGGGS (SEQ ID NO: 85), GSGGGGSGGGGSLQ (SEQ ID NO: 103), GSGGGGSGGGGSIQ (SEQ ID NO: 105), SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110), SGGGGSGGGSGGGGGSGGGSG (SEQ ID NO: 111), and the like.

In one specific embodiment, the cytokine-like polypeptide may comprise an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54, or an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[Sequence number 41] (RS15.0)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[SEQ ID NO: 52] (RS15.1)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[SEQ ID NO: 53] (RS15.2)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[Sequence number 54] (RS15.3)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[Sequence number 64] (RS15.4)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[Sequence number 65] (RS15.5)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

(IL-15R alpha sushi domain: bold; second linker: italics; IL-15: underlined)

Nucleic acid molecules, recombinant vectors, and recombinant cells

Another example provides a nucleic acid molecule encoding the fusion protein.

Another example provides a recombinant vector comprising the nucleic acid molecule. The vector can be used as an expression vector for the nucleic acid molecule.

Another example provides a recombinant cell comprising the nucleic acid molecule or a recombinant vector comprising the same. The cell can be used for production of the fusion protein.

Another example provides a method for producing the fusion protein, comprising the step of expressing the nucleic acid molecule in a suitable host cell. The step of expressing the nucleic acid molecule in a suitable host cell may comprise the step of culturing the recombinant cell described above under conventional conditions.

The term "vector" refers to a means for expressing a target gene in a host cell. For example, it includes a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The vector that can be used as the above recombinant vector can be produced by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.) or a virus (e.g., SV40, etc.) that is often used in the art.

In the above recombinant vector, the nucleic acid molecule encoding the fusion protein may be operably linked to a promoter. The term "operatively linked" refers to a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. The regulatory sequence can regulate transcription and/or translation of the other nucleotide sequence by being "operatively linked".

The above recombinant vector can be constructed as a vector for cloning or a vector for expression. The expression vector can be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The above recombinant vector can be constructed by various methods known in the art.

The above recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, if the vector used is an expression vector and a prokaryotic cell is used as a host, it generally includes a strong promoter capable of initiating transcription (e.g., pL ^λ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription/translation terminator sequence. If a eukaryotic cell is used as a host, the replication origin that operates in a eukaryotic cell included in the vector includes, but is not limited to, the f1 replication origin, the SV40 replication origin, the pMB1 replication origin, the adeno-replication origin, the AAV replication origin, and the BBV replication origin. Additionally, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be utilized, which generally have a polyadenylation sequence as a transcription termination sequence.

The above recombinant cell may be obtained by introducing the above recombinant vector into an appropriate host cell. The host cell may be any host cell known in the art that can stably and continuously clone or express the recombinant vector. Prokaryotic cells include, for example, strains of the genus Bacillus such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. In the case of transforming eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, plant cells, and animal cells, for example, Sp2/0, CHO (Chinese hamster ovary) K1, CHO, DG44, PER.C6, Cell lines that can be used include, but are not limited to, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK.

The delivery (introduction) of the above nucleic acid molecule or the recombinant vector containing it into the host cell can be carried out using a delivery method widely known in the art. For example, if the host cell is a prokaryotic cell, the CaCl ₂ method or the electroporation method can be used, and if the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome-mediated transfection method, the gene bombardment method, etc. can be used, but are not limited thereto.

The method for selecting the host cell transformed (introduced with a recombinant vector) can be easily performed according to a method widely known in the art by utilizing a phenotype expressed by a selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the recombinant cell into which the recombinant vector has been introduced can be easily selected by culturing in a medium containing the antibiotic.

Medicinal Uses

The anti-TIGIT antibody or antigen-binding fragment thereof included in the fusion protein provided herein has the function of blocking the action of TIGIT (e.g., interaction between TIGIT and its ligand CD155 (PVR), etc.) to activate immunity (e.g., enhancing effector T cell function, controlling Treg activity, increasing cytokine secretion of immune cells (T cells, NK cells, etc.)), and also has excellent anticancer effects (e.g., killing cancer cells, inhibiting cancer cell proliferation, etc.). In addition, the cytokine-like polypeptide has an activity similar to interleukin-15, but has excellent binding affinity to receptors (IL-2/IL-15 receptors) and low immunogenicity compared to anaplastic interleukin-15, so that when administered to a living body, it has an excellent immune-enhancing effect such as increasing cytokine secretion, but does not act as an immunogen, so there is a low risk of inducing an immune response recognizing it as an immunogen, and thus safety can be secured, and it also has an excellent anticancer effect (e.g., cancer cell death, inhibition of cancer cell proliferation, etc.). Therefore, the fusion peptide has the advantages of both the anti-TIGIT antibody or antigen-binding fragment thereof and the cytokine-like polypeptide, and can be usefully applied to immune enhancement, prevention and/or treatment of immune-related diseases, and particularly, prevention and/or treatment of cancer.

One example provides an immunostimulant or an immunostimulant pharmaceutical composition comprising as an active ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a pharmaceutical composition for preventing and/or treating an immune-related disease, comprising as an active ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides an anticancer agent or a pharmaceutical composition for preventing and/or treating cancer, comprising as an active ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a method for enhancing immunity, comprising administering to a subject in need of enhancing immunity a pharmaceutically effective amount of at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector. The method for enhancing immunity may further comprise, prior to the administering step, a step of identifying a subject in need of enhancing immunity.

Another example provides a method for preventing and/or treating an immune-related disease, comprising administering to a subject in need of prevention and/or treatment of an immune-related disease at least one pharmaceutically effective amount selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector. The method for preventing and/or treating an immune-related disease may further comprise, prior to the administering step, a step of identifying a subject in need of prevention and/or treatment of the immune-related disease.

Another example provides a method for preventing and/or treating cancer, comprising administering to a subject in need of prevention and/or treatment of cancer at least one pharmaceutically effective amount selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector. The method for preventing and/or treating cancer may further comprise, prior to the administering step, a step of identifying a subject in need of prevention and/or treatment of cancer.

Other examples provide uses for enhancing immunity, preventing and/or treating immune-related diseases, and/or preventing and/or treating cancer, or uses for preparing a pharmaceutical composition for enhancing immunity, preventing and/or treating immune-related diseases, and/or preventing and/or treating cancer, selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

As used herein, the term "immune enhancement" may mean inducing an initial immune response to an antigen or increasing an existing immune response, and may be interchangeable with terms such as immune enhancement, immune activation, etc. as equivalent meanings. More specifically, the "immune enhancement effect" of the fusion protein provided herein may be, but is not limited to, enhancement of function (activation) and/or proliferation of immune cells (NK cells, NKT cells, CD3+ T cells, effector T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), etc.), inhibition of activity and/or depletion of regulatory T (Treg) cells, increased production and/or secretion of immune proteins (e.g., cytokines (IL-2, IFN-gamma, etc.), etc.).

As used herein, the term “immune-related disease” encompasses all diseases caused by disorders and/or insufficient activity of the immune system, and may be, for example, one or more selected from the group consisting of cancer, infectious diseases, autoimmune diseases, inflammatory diseases, etc., but is not limited thereto.

The cancer may be a solid cancer or a hematological cancer, and is not limited thereto, but may be at least one selected from the group consisting of squamous cell carcinoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, etc.), peritoneal cancer, skin cancer, melanoma (e.g., cutaneous or intraocular melanoma, etc.), rectal cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, liver cancer, stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, head and neck cancer, brain cancer, osteosarcoma, etc.

The above cancer prevention and/or treatment effect (anticancer effect or antitumor effect) includes not only the effect of inhibiting the occurrence and/or growth of cancer cells, but also the effect of inhibiting the worsening of cancer due to migration, invasion, metastasis, etc.

The above infectious diseases, autoimmune diseases, and inflammatory diseases may be selected from all infectious diseases, autoimmune diseases, and inflammatory diseases that can be treated, alleviated, and/or prevented by the aforementioned immune enhancement (e.g., enhancing the function (activation) and/or proliferation of immune cells (NK cells, CD3+ T cells, effector T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), etc.)), suppressing the activity of regulatory T (Treg) cells and/or eliminating them (depleting them), increasing the production and/or secretion of immune proteins (e.g., cytokines (IL-2, IFN-gamma, etc.)), etc.). For example, the infectious disease is a collective term for diseases caused by the transmission or invasion of a pathogen causing a disease, such as a virus (e.g., influenza virus, coronavirus (e.g., Severe Acute Respiratory Syndrome (SARS) virus (SARS-CoV, SARS-CoV-2, etc.), Middle East Respiratory Syndrome (MERS) virus (MERS-CoV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV)), bacteria, fungi, or parasites, into a living organism (e.g., animals including humans), and may be an infection by one or more pathogens selected from the group consisting of viruses, bacteria, fungi, parasites, etc., or a disease (infection) caused by the infection. The autoimmune disease may be rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, lupus, Sjogren's syndrome, myasthenia gravis, scleroderma, hypothyroidism, hyperthyroidism, psoriasis, It may be selected from the group consisting of vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, cytokine storm, etc., but is not limited thereto. The above inflammatory disease is a general term for inflammation (e.g., chronic inflammation or acute inflammation) or diseases caused by inflammation, for example, cardiac inflammation (e.g., coronary artery disease, angina pectoris, myocardial infarction, pericarditis, myocarditis, etc.), vascular inflammation (e.g., atherosclerosis, vasculitis, disseminated intravascular coagulation (DIC), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), anemia, etc.), upper respiratory tract inflammation (e.g., acute nasopharyngitis, allergic rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, etc.), lower respiratory tract and/or lung inflammation (e.g., bronchitis, bronchiectasis, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, interstitial lung disease, tuberculosis, etc.), inflammation of the upper gastrointestinal tract (e.g., gastritis, esophagitis, etc.), inflammation of the lower gastrointestinal tract (e.g., enteritis, ulcerative colitis, Crohn's disease, celiac disease, diverticulitis, irritable bowel syndrome, appendicitis, fistulas, etc.), inflammation of the liver, biliary tract and/or pancreas (e.g., hepatitis, fatty liver disease, cholangitis, cholecystitis, pancreatitis, type 1 diabetes, etc.), inflammation of the kidney (upper urinary tract) (e.g., pyelonephritis, glomerulonephritis, urinary tract infections, etc.), inflammation of the lower urinary tract (e.g., urinary tract infections, ureteritis, urethritis, cystitis, prostatitis/chronic pelvic pain syndrome, etc.), inflammation of the thyroid and/or parathyroid glands (e.g., thyroiditis, parathyroiditis, etc.), inflammation of the adrenal glands (e.g., adrenalitis, etc.), inflammation of the reproductive organs (e.g., pelvic inflammatory disease, oophoritis, orchitis, epididymitis, etc.), inflammation of the bones and/or joints (e.g., osteoarthritis, The present invention relates to, but is not limited to, one or more of the following: inflammatory bowel disease (inflammatory bowel disease), autoimmune disease (AAD), autoimmune disease (AAD), autoimmune disease (OA), autoimmune disease (AAD), autoimmune disease (AVD ...

The subject of administration of the fusion protein, and/or pharmaceutical composition provided herein may be any animal or cell, and may be, for example, an animal selected from mammals including humans, primates such as monkeys, rodents such as rats and mice, or cells, tissues, body fluids (e.g., serum) derived (isolated) from said animals, or a culture thereof, and may be, for example, a human or cells, tissues, body fluids (e.g., serum) isolated from a human.

The pharmaceutical composition may further comprise, in addition to the fusion protein, nucleic acid molecule, vector and/or cell as an active ingredient, a pharmaceutically acceptable carrier, and the carrier may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. The pharmaceutical composition may further comprise at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, which are commonly used in the preparation of pharmaceutical compositions.

The content of the fusion protein, nucleic acid molecule, vector and/or cell as an active ingredient in the pharmaceutical composition may be, but is not limited to, 0.00001 to 99 wt%, 0.0001 to 99 wt%, 0.001 to 99 wt%, 0.01 to 99 wt%, 0.1 to 99 wt%, 1 to 99 wt%, 10 to 99 wt%, 15 to 99 wt%, 20 to 99 wt%, 25 to 99 wt%, 30 to 99 wt%, 35 to 99 wt%, 40 to 99 wt%, 45 to 99 wt%, or 50 to 99 wt%, based on the total weight of the composition.

Administration of the above fusion proteins, nucleic acid molecules, vectors, cells, and/or pharmaceutical compositions may be via oral or parenteral routes. In the case of parenteral administration, administration may be via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. Since proteins or peptides are digested when administered orally, oral compositions should be formulated to coat the active agent or protect it from degradation in the stomach.

In addition, the fusion proteins, nucleic acid molecules, vectors, cells, and/or pharmaceutical compositions may be formulated in the form of solutions, suspensions, syrups or emulsions in oily or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, injections, etc., and may additionally contain dispersants or stabilizers for the formulation.

The content of the active ingredients, such as fusion proteins, nucleic acid molecules, vectors, and/or cells, in the pharmaceutical composition can be varied depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate, and response sensitivity. The daily dosage of the pharmaceutical composition is 0.00001 to 1000 mg/kg, 0.00001 to 500 mg/kg, 0.00001 to 100 mg/kg, 0.00001 to 50 mg/kg, 0.0001 to 1000 mg/kg, 0.0001 to 500 mg/kg, 0.0001 to 100 mg/kg, 0.0001 to 50 mg/kg, 0.001 to 1000 mg/kg, 0.001 to 500 mg/kg, 0.001 to 100 mg/kg, 0.001 to 50 mg/kg, 0.01 to 1000 mg/kg, 0.01 to The range may be, but is not limited to, 500 mg/kg, 0.01 to 100 mg/kg, 0.01 to 50 mg/kg, 0.1 to 1000 mg/kg, 0.1 to 500 mg/kg, 0.1 to 100 mg/kg, or 0.1 to 50 mg/kg. The above daily dosage may be formulated as a single preparation in the form of a unit dose, formulated in an appropriate amount, or manufactured by placing it in a multi-dose container. In addition, the pharmaceutically effective amount in this specification means the amount of the effective ingredient that can exhibit the desired pharmaceutical activity, and may be within the dosage range mentioned above.

Another example provides an immune-enhancing or metabolism-enhancing health functional food comprising as an effective ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a health functional food for preventing and/or improving an immune-related disease, comprising as an active ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

Another example provides a health functional food for preventing and/or improving cancer, comprising as an effective ingredient at least one selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

'Health functional food' refers to a food manufactured (processed) using nutrients that are easily deficient in daily meals or raw materials or ingredients (hereinafter, 'functional raw materials') that have functions useful to the human body, and all foods that help maintain health or prevent and/or improve certain diseases or symptoms, and there are no special restrictions on the final product form. For example, the above health functional food may be selected from the group consisting of various foods, beverages, food additives, etc., but is not limited thereto.

The above food composition may include acceptable food additives and may further include suitable carriers, excipients and diluents commonly used in the manufacture of health functional foods.

When the above food composition is used as a food additive, the composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

The content of the effective ingredient contained in the food composition can be appropriately determined without special limitation according to the form of the food, the desired effect, the purpose, etc., and may be, for example, 0.00001 to 99 wt%, 0.0001 to 99 wt%, 0.001 to 99 wt%, 0.01 to 99 wt%, 0.1 to 99 wt%, 1 to 99 wt%, or 10 to 99 wt% of the total food weight, but is not limited thereto.

The fusion protein provided herein has the advantages of both an anti-TIGIT antibody or an antigen-binding fragment thereof and a cytokine-like polypeptide, and has effects of enhancing immunity, preventing and/or treating immune-related diseases, and anticancer effects (e.g., killing cancer cells, inhibiting cancer cell proliferation, etc.), and thus can be usefully applied as an immunostimulant and an anticancer agent.

Figures 1a and 1b are graphs showing the binding affinity of a fusion protein to human TIGIT according to one embodiment.
Figure 2 is a graph showing the ability of a fusion protein to migrate to the surface of an immune cell according to one embodiment.
Figure 3 is a graph showing dimerization of IL2RB/IL2RG of a fusion protein according to one embodiment.
Figures 4a to 4d are graphs showing the phosphorylation effect of STAT5 in immune cells of a fusion protein according to one embodiment (4a: CD8+ T cells, 4b: CD4+ T cells, 4c: CD56+ NK cells, 4d: NK cells).
Figures 5a and 5b are graphs showing the cytokine production effect of a fusion protein according to one embodiment in PBMC compared to a comparative antibody (5a: IL-2, 5b: IFN-γ).
Figures 6a to 6c are graphs showing the cytokine production effect of a fusion protein in immune cells according to one embodiment compared to a comparative fusion protein (6a: CD4+ T cells, 6b: CD8+ T cells, 6c: CD56+ NK cells).
Figures 7a to 7h are graphs showing the cytokine production effect in immune cells of fusion proteins having various linkers according to one embodiment.
Figures 8a to 8c are graphs showing the cytokine production effect of a fusion protein according to one embodiment in immune cells compared to an anti-TIGIT antibody (8a: CD4+ T cells, 8b: CD8+ T cells, 8c: CD56+ NK cells).
Figures 9a to 9c are graphs showing the cytokine production effect of a fusion protein in immune cells according to one embodiment compared to combined treatment (9a: CD4+ T cells, 9b: CD8+ T cells, 9c: CD56+ NK cells).
Figure 10 is a graph showing the cytokine production effect of a fusion protein in cancer patient-derived immune cells according to one embodiment.
Figure 11 shows the results showing the cytotoxicity of a fusion protein against cancer cells through NK cells according to one embodiment.
Figures 12a and 12b are graphs showing changes in tumor size (12a) and body weight (12b) in mice administered a fusion protein according to one embodiment.
Figures 13a to 13c are graphs showing changes in body weight (13a), AST and ALT levels (13b), and complete blood counts (CBC) (13c) in mice administered a fusion protein according to one embodiment.

Hereinafter, the present invention will be described in more detail by way of examples, but these are merely exemplary and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that the examples described below can be modified without departing from the essential gist of the invention.

Example 1. Preparation of anti-TIGIT antibody (TIGIT mAb) and cytokine (IL-15)-like polypeptide (RS15) fusion protein

1.1. Expression and purification of fusion proteins

Fusion proteins were designed to have the structures shown in Table 3 below, in which anti-TIGIT antibodies (7A6 (7A6_VH3/Vk5), 2B7, or 3F8) and cytokine (IL-15)-like polypeptides (IL-15Rα sushi domain and fusion polypeptide of IL-15 (RS15); RS15.0, RS15.1, RS15.2, RS15.3, RS15.4, or RS15.5) were fused with or without a linker (flexible linker or rigid linker):

Structure of the TIGIT mAb-RS15 fusion protein Fusion protein name Structure Linker 7A6-RS15
(Fusion protein of TIGIT mAb7A6 with RS15 at LC linking) TIGIT mAb-(LC-(EAAAK)3- RS15)2 RS15 fused to LC C-terminus (LC variant) with rigid linker TIGIT mAb-(LC-(GGGGS)3-RS15)2 RS15 fused to LC C-terminus (LC variant) with flexible linker 7A6-RS15-Fc
(Fusion protein of TIGIT mAb7A6 with RS15 at Fc linking) TIGIT mAb-Fc-(RS15)2 RS15fused to Fc C-terminus
(Fc variant) without linker TIGIT mAb-Fc-((EAAAK)3- RS15)2 RS15 fused to Fc C-terminus (Fc variant) with rigid linker TIGIT mAb-Fc-((GGGGS)3-RS15)2 RS15 fused to Fc C-terminus (Fc variant) with flexible linker 2B7-RS15
(Fusion protein of TIGIT mAb2B7 with RS15 at LC linking) TIGIT mAb-(LC-(EAAAK)3-RS15)2 RS15fused to LC C-terminus (LC variant) with rigid linker TIGIT mAb-(LC-(GGGGS)3-RS15)2 RS15 fused to LC C-terminus (LC variant) with flexible linker 2B7-RS15-Fc
(Fusion protein of TIGIT mAb2B7 with RS15 at Fc linking) TIGIT mAb-Fc-(RS15)2 RS15fused to Fc C-terminus
(Fc variant) without linker TIGIT mAb-Fc-((EAAAK)3-RS15)2 RS15 fused to Fc C-terminus (Fc variant) with rigid linker TIGIT mAb-Fc-((GGGGS)3-RS15)2 RS15 fused to Fc C-terminus (Fc variant) with flexible linker 3F8-RS15
(Fusion protein of TIGIT mAb3F8 with RS15 at LC linking) TIGIT mAb-(LC-(EAAAK)3- RS15)2 RS15fused to LC C-terminus (LC variant) with rigid linker TIGIT mAb-(LC-(GGGGS)3-RS15)2 RS15 fused to LC C-terminus (LC variant) with flexible linker 3F8-RS15-Fc
(Fusion protein of TIGIT mAb3F8 with RS15 at Fc linking) TIGIT mAb-Fc-(RS15)2 RS15fused to Fc C-terminus
(Fc variant) without linker TIGIT mAb-Fc-((EAAAK)3-RS15)2 RS15 fused to Fc C-terminus (Fc variant) with rigid linker TIGIT mAb-Fc-((GGGGS)3-RS15)2 RS15 fused to Fc C-terminus (Fc variant) with flexible linker

(In Table 3 above, anti-TIGIT monoclonal antibodies (TIGIT mAb): 7A6 (7A6_VH3/Vk5), 2B7, 3F8;

RS15 (fusion polypeptide of IL-15Rα sushi domain and IL-15); RS15.0, RS15.1, RS15.2, RS15.3, RS15.4, or RS15.5;

LC variant: RS15 is fused to the C-terminus of the light chain constant region (LC) of the antibody;

Fc variants: RS15 is fused to the C-terminus of the antibody's heavy chain constant region (Fc; including the ΔK478 mutation)

For the production of fusion proteins, the genes encoding the heavy chain strand including the heavy chain of TIGIT mAb and the light chain strand including the light chain variable region (RS15 is included in the heavy chain or light chain strand; see Table 4-27) were synthesized and cloned into the pcDNA3.1 (Invitrogen) vector. CHO-K1 cells (ATCC) were seeded on plates and cultured to 70-90% confluent, and then 1 μg/μl of the gene DNA and Lipofectamine reagent (Thermo) were each diluted in Opti-MEM culture medium and mixed 1:1. After incubation for 5 minutes, the DNA-lipid complex was dropped onto CHO-K1 cells. The cells were then cultured at 37°C for transient expression. The culture supernatant was purified with Protein A and preparative SEC. The produced protein was buffer-exchanged with PBS, quantified by measuring OD280 nm, and analyzed by SDS-PAGE.

The IL-15-like polypeptides (RS15) used in the production of fusion proteins are summarized below:

[RS15.0 (SEQ ID NO: 41)]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence (SEQ ID NO: 90): )

[RS15.1 (SEQ ID NO: 52)]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence (SEQ ID NO: 91): )

[RS15.2 (SEQ ID NO: 53)]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence (SEQ ID NO: 92): )

[RS15.3 (SEQ ID NO: 54)]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence (SEQ ID NO: 93): )

[RS15.4 (SEQ ID NO: 64)]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence (SEQ ID NO: 94): )

[RS15.5 (SEQ ID NO: 65)]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence (SEQ ID NO: 95): )

(IL-15R alpha sushi domain: bold font;

Linker2: Italic font;

IL-15: underlined)

Design of anti-TIGIT antibody-RS15 fusion protein

A fusion protein having the following structure was designed and manufactured:

(N-terminus)-[anti-TIGIT antibody]-[1st linker (connected to the light chain C-terminus of the antibody)]-[IL-15 receptor alpha sushi domain]-[2nd linker]-[hIL-15]-(C-terminus)

The first linker (linking the antibody and RS15) included in the anti-TIGIT antibody-RS15 fusion protein prepared in this example was named as follows:

TL-1: Flexible linker connected to the light chain C-terminus of the antibody (GGGGSGGGGSGGGGS (SEQ ID NO: 85); GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC (SEQ ID NO: 86))

TL-2: Rigid linker connected to the light chain C-terminus of the antibody (EAAAKEAAAKEAAAK (SEQ ID NO: 44); GAGGCCGCCGCCAAGGAGGCCGCCGCCAAGGAGGCCGCCGCCAAG (SEQ ID NO: 87))

The anti-TIGIT antibody-RS15 fusion proteins prepared in this example were named as follows according to the types of the first linker and the second linker (RS15 internal linker):

7A6RS15TL-2 (7A6-RS15.0) (first linker: EAAAKEAAAKEAAAK (SEQ ID NO: 44); second linker: SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108));

7A6RS15TL-2DL-2 (7A6-RS15.1) (first linker: EAAAKEAAAKEAAAK (SEQ ID NO: 44); second linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85));

7A6RS15TL-2DL-1 (7A6-RS15.2) (first linker: EAAAKEAAAKEAAAK (SEQ ID NO: 44); second linker: GSGGGGSGGGGSLQ (SEQ ID NO: 103));

7A6RS15TL-2DL-0 (7A6-RS15.3) (first linker: EAAAKEAAAKEAAAK (SEQ ID NO: 44); second linker: GSGGGGSGGGGSIQ (SEQ ID NO: 105));

7A6RS15TL-2DL-3 (7A6-RS15.4) (first linker: EAAAKEAAAKEAAAK (SEQ ID NO: 44); second linker: SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110));

7A6RS15TL-2DL-4 (7A6-RS15.5) (first linker: EAAAKEAAAKEAAAK (SEQ ID NO: 44); second linker: SGGGGSGGGSGGGGGSGGGSG (SEQ ID NO: 111));

7A6RS15TL-1 (7A6-RS15.0) (first linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85); second linker: SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108));

7A6RS15TL-1DL-2 (7A6-RS15.1) (first linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85); second linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85));

7A6RS15TL-1DL-1 (7A6-RS15.2) (first linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85); second linker: GSGGGGSGGGGSLQ (SEQ ID NO: 103));

7A6RS15TL-1DL-0 (7A6-RS15.3) (first linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85); second linker: GSGGGGSGGGGSIQ (SEQ ID NO: 105));

7A6RS15TL-1DL-3 (7A6-RS15.4) (first linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85); second linker: SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110));

7A6RS15TL-1DL-4 (7A6-RS15.5) (first linker: GGGGSGGGGSGGGGS (SEQ ID NO: 85); second linker: SGGGGSGGGSGGGGGSGGGSG (SEQ ID NO: 111)).

The fusion proteins manufactured in this example are summarized in Tables 4 to 27 below:

7A6-RS15.0(7A6 VH3/Vk5-RS15.0; 7A6RS15TL-2) 7A6-RS15 or 7A6RS15TL-2 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGACCGGCTACAGCATCACCAGCGACTACGCCTGGAACTGGATCAGGCAGCCCCCCGGCAAGGGCCTGGAGGTGGATGGGCTACATCAGCTACAGCGGCAGCGCCAGG TACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCAGGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGAGGACACCGCCACCTACTACTGCGCCAGGAAGGGCTACCCCGCCTACTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC 77 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 GCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGG AACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCT GCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTT CCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGAGGAGATGACCAAGAACCAGGTGAGCC TGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCT GTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG 79 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGAGCATCACCTGCAAGGCCAGCCAGGACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACAGCGCCAGCTACA GGTACACCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCACCACTACAGCACCCCCTACACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG 78 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 AGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGG AGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 80 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 41 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 46

7A6-RS15.0(7A6 VH3/Vk5-RS15.0; 7A6RS15TL-1) 7A6-RS15 or 7A6RS15TL-1 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker GGGGSGGGGSGGGGS 85 Cytokine-like polypeptide (RS15) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 41 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 47

2B7-RS15.0 2B7-RS15 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSA 27 GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGACCAGCGGCAGCACCTTCACCGAGTACACCATGCACTGGGTGAAGCAGAGCCACGGCAAGAGCCTGGAGTGGATCGGCGGCCTGAACCCCAACAACGGCGGCACCAGCT ACAACCAGAGGTTCAAGGACAGGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCACCAGGGGCACCTACTACGACTACAGCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC 81 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 48 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIK 35 GACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGACAGGGGTGAGCATCACCTGCAAGGCCAGCCACTACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAGCCCCAGCTACA GGTACACCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCGTGCAGGCCGAGGACCTGGCCGTGTACTACTGCCACCAGCACTACAGCACCCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG 82 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 41 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 49

3F8-RS15.0 3F8-RS15 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSS 28 GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCATGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGACTACATCATGAACTGGGTGAAGCAGAGCCACGGCAAGAACCTGGAGTGGATCGGCCTGAGCATCCCCTACAACGGCGGCACCAGCT ACAACCAGAAGTTCGAGGGCAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGCTGAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGGCATCAAGGGCTACTTCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC 83 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 50 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 36 GACGTGGTGGTGACCCAGACCCCCCTGAGCCTGCCCGTGAGCTTCGGCGACCAGGTGAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGAAACAGCTTCGGCAAGACCTACCTGAGCTGGTTCCTGCACAAGCCCGGCCAGAGCCCCCAGCTGATCATGTACGGCGTG AGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCACCATCAAGCCCGAGGACCTGGCATGTACTACTGCCTGCAGGGCACCCACCAGCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG 84 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 41 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 51

7A6-RS15.1(7A6 VH3/Vk5-RS15.1; 7A6RS15TL-2DL-2) 7A6-RS15.1 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.1) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 55

7A6-RS15.1(7A6 VH3/Vk5-RS15.1; 7A6RS15TL-1DL-2) 7A6-RS15.1 or 7A6RS15TL-1DL-2 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker GGGGSGGGGSGGGGS 85 Cytokine-like polypeptide (RS15.1) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 72

2B7-RS15.1 2B7-RS15.1 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSA 27 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 47 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIK 35 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.1) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 56

3F8-RS15.1 3F8-RS15.1 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSS 28 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 36 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.1) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 57

7A6-RS15.2(7A6 VH3/Vk5-RS15.2; 7A6RS15TL-2DL-1) 7A6-RS15.2 or 7A6RS15TL-2DL-1 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.2) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 53 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 58

7A6-RS15.2(7A6 VH3/Vk5-RS15.2; 7A6RS15TL-1DL-1) 7A6-RS15.2 or 7A6RS15TL-1DL-1 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker GGGGSGGGGSGGGGS 85 Cytokine-like polypeptide (RS15.2) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 53 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 73

2B7-RS15.2 2B7-RS15.2 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSA 27 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 47 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIK 35 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.2) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 53 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 59

3F8-RS15.2 3F8-RS15.2 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSS 28 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 36 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.2) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 53 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 60

7A6-RS15.3(7A6 VH3/Vk5-RS15.3; 7A6RS15TL-2DL-0) 7A6-RS15.3 or 7A6RS15TL-2DL-0 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.3) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 54 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GSGGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 61

7A6-RS15.3(7A6 VH3/Vk5-RS15.3; 7A6RS15TL-1DL-0) 7A6-RS15.3 or 7A6RS15TL-1DL-0 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker GGGGSGGGGSGGGGS 85 Cytokine-like polypeptide (RS15.3) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 54 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 74

2B7-RS15.3 2B7-RS15.3 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSA 27 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 47 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIK 35 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.3) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 54 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GSGGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 62

3F8-RS15.3 3F8-RS15.3 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSS 28 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 36 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.3) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 54 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP GSGGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 63

7A6-RS15.4(7A6 VH3/Vk5-RS15.4; 7A6RS15TL-2DL-3) 7A6-RS15.4 or 7A6RS15TL-2DL-3 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.4) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 64 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 66

7A6-RS15.4(7A6 VH3/Vk5-RS15.4; 7A6RS15TL-1DL-3) 7A6-RS15.4 or 7A6RS15TL-1DL-3 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker GGGGSGGGGSGGGGS 85 Cytokine-like polypeptide (RS15.4) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 64 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 75

2B7-RS15.4 2B7-RS15.4 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSA 27 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 47 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIK 35 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.4) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 64 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 67

3F8-RS15.4 3F8-RS15.4 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSS 28 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 36 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.4) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 64 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 68

7A6-RS15.5(7A6 VH3/Vk5-RS15.5; 7A6RS15TL-2DL-4) 7A6-RS15.5 or 7A6RS15TL-2DL-4 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.5) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 65 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 69

7A6-RS15.5(7A6 VH3/Vk5-RS15.5; 7A6RS15TL-1DL-4) 7A6-RS15.5 or 7A6RS15TL-1DL-4 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSS 24 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker GGGGSGGGGSGGGGS 85 Cytokine-like polypeptide (RS15.5) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 65 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 76

2B7-RS15.5 2B7-RS15.5 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSA 27 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 47 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIK 35 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.5) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 65 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 70

3F8-RS15.5 3F8-RS15.3 Amino acid sequence (N→C) or nucleic acid sequence (5'→3') Sequence number Heavy chain strand (anti-TIGIT heavy chain (IgG1)) anti-TIGIT heavy chain variable region EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSS 28 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT Double Chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 36 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 Linker EAAAKEAAAKEAAAK 44 Cytokine-like polypeptide (RS15.5) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 65 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 71

Reference examples: Preparation of reference antibodies and reference fusion proteins

The comparative fusion proteins used for comparison in the examples below were prepared by fusing the following anti-TIGIT antibodies with the cytokine-like polypeptide RS15.0 (313M32-RS15 and 4.1D3-RS15): The antibodies were prepared based on the sequence information described in the corresponding patents or obtained from the manufacturers. The corresponding patents or manufacturers of each antibody are summarized below:

4.1D3 (Genentech): WO2017/053748 A2,

313M32 (Mereo): US2016/0376365 A1.

In addition, the reference antibodies used for comparison in the examples below were also prepared based on the sequence information described in the corresponding patents or obtained from the manufacturers. The corresponding patents or manufacturers of each antibody are summarized below:

22G2(BMS): US2016/0176963 A1;

31C6 (Merck): WO2016028656 A1,

TIG1(Arcus): WO2017/152088 A1,

4.1D3 (Genentech): WO2017/053748 A2,

10A7 (Genentech): Obtained from Creative Biolabs.

Example 2. Evaluation of the characteristics of fusion proteins

2.1. Measuring Binding Affinity for Human TIGIT

2.1.1. Affinity measurement of fusion proteins using multi-cycle kinetic analysis

To measure the binding affinity of the fusion proteins prepared in Example 1 to human TIGIT antigen, multi-cycle kinetic analysis was performed on the purified proteins (fusion proteins) 7A6-RS15 (fusion of 7A6 and RS15.0), 2B7-RS15 (fusion of 2B7 and RS15.0), and 3F8-RS15 (fusion of 3F8 and RS15.0). Kinetic experiments were performed using a Biacore T200 (GE Healthcare, Uppsala, Sweden) running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 at 25°C.

HBS-P+ (GE Healthcare, Uppsala, Sweden) supplemented with 1% BSA w/v (Sigma, Dorset, UK) was used as the running buffer, and was also used for ligand and analyte dilutions. Purified lead fusion proteins were diluted to 1 μg/mL in running buffer and loaded onto Fc2, Fc3, and Fc4 on an anti-human IgG CM5 sensor chip (GE Healthcare, Little Chalfont, UK) at the beginning of each cycle. Antibodies were captured at a flow rate of 10 μl/min, resulting in an immobilization level (RL) of ∼150 RU. The surface was then allowed to stabilize. Multi-cycle kinetic data were obtained using recombinant human TIGIT (acrobiosystems, China) as the analyte, which was injected at a flow rate of 50 μl/min to minimize potential mass transfer effects. Antigen (TIGIT) was diluted 2-fold (seven points) in the concentration range of 0.406 nM to 30 nM in running buffer. No regeneration was allowed between each concentration. At each concentration, the association phases were monitored for 240 s and the dissociation phase was measured for 900 s. The sensor chip surface was regenerated between cycles by injecting 3 M MgCl ₂ twice. To check the stability of both the surface and the analyte over the kinetic cycles, multiple repetitions of blank and repetitions of a single concentration of analyte were programmed as kinetic runs. Representative results for 7A6-RS15, 2B7-RS15, and 3F8-RS15 fusion proteins are shown in Table 28:

Binding affinity of fusion proteins Fusion antibody K _a (1/Ms) K _d (1/s) K _D (M) R _MAX Chi ² (RU ² ) Parental Ab ( 7A6) 1.78E+06 1.58E-03 8.84E-10 24.2 0.12 7A6-RS15 2.37E+06 1.79E-03 7.56E-10 18.1 0.13 2B7-RS15 2.61E+05 3.86E-04 1.48E-09 30.9 0.0925 3F8-RS15 1.89E+05 2.16E-04 1.14E-09 26 0.048

Table 28 above shows the kinetic constants determined from the 1:1 model fitted curves. As shown in Table 28, the binding affinities (KD) for human TIGIT protein were 756 pM for 7A6-RS15, 1.48 nM for 2B7-RS15, and 1.14 nM for 3F8-RS15, and all three fusion proteins showed binding affinities comparable to those of the parent antibody to which RS15 was not fused.

2.1.2. Measurement of binding affinity of fusion proteins with various RS15 internal linkers to TIGIT (ELISA)

ELISA PVC plates were coated with recombinant human TIGIT protein (Acro Biosystems; in PBS) at a concentration of 1 μg/mL and incubated overnight at 4°C. The coating solution was removed, and the plates were washed three times by filling the wells with 200 μL of wash buffer containing 0.5% Tween-20 (Sigma) diluted in PBS (Gibco). The wells were rapidly inverted, and the supernatant was removed. Nonspecific binding was blocked for 1 h using 200 μL per well of blocking buffer containing 3% skim milk (Daeil Bio) diluted in PBS. The plates were washed three times by filling the wells with 200 μL of wash buffer. Anti-TIGIT antibody-RS15 fusion proteins with different types of RS15 internal linkers (7A6RS15TL-2, 7A6RS15TL-2DL-0, 7A6RS15TL-2DL-1, 7A6RS15TL-2DL-2, 7A6RS15TL-2DL-3, 7A6RS15TL-2DL-4) or parental antibody (7A6 VH3/Vκ5 (7A6)) were prepared by serial dilution 1:3 from a starting concentration of 61.097 nM in PBS.

100 μL of the diluted fusion protein or antibody was added to the prepared plate and incubated at room temperature for 1 hour at 0.65 x g. 100 μL of anti-mouse/human IgG-HRP antibody (1:10000, Promega) was added to each well. The plate was covered with a black microplate lid and shaken at 0.65 x g for 1 hour at room temperature. The HRP-conjugated antibody was removed and the plate was washed three times with wash buffer. The plate was developed with tetramethylbenzidine (TMB) and stopped with 1 N HCl. The OD value (absorbance) at 450 nM was measured using GloMax® Explorer Fully Loaded.

The results obtained above are shown in Figs. 1a and 1b (Fig. 1b shows the results of repeating the same process for 7A6RS15TL-2 and 7A6). As shown in Figs. 1a and 1b, all of the anti-TIGIT-RS15 fusion proteins having various RS15 internal linkers tested showed high binding affinity to human TIGIT, similar to that of the parent antibody (7A6 VH3/Vκ5). These results show that in the fusion protein in which the anti-TIGIT antibody is fused with the IL-15 superagonist, the addition of IL-15 does not interfere with the antigen (TIGIT) binding domain of the fusion protein, so that the fusion protein can maintain all the functional properties of the parent antibody.

2.2. Binding Affinity Measurement for Various Human Fc Receptors (FcγR) (SPR analysis)

The fusion protein (7A6RS15: 7A6RS15TL-2) was purified from CHO cell culture supernatant using MabSelect SuRe (Cytiva, Little Chalfont, UK). The column was washed with Dulbecco's phosphate-buffered saline (DPBS) and the protein was eluted with 0.1 M sodium citrate at pH 3.0. After elution, the buffer of the relevant fractions was exchanged with PBS containing 100 mM L-Arginine. The protein was filtered and prepared in two ways: i) direct dilution of the fusion protein (7A6RS15TL-2) into PBS buffer, pH 6.0; and ii) buffer exchange of the fusion protein into PBS buffer, pH 6.0 prior to experimental setup. The binding capacity of the fusion protein (7A6RS15TL-2) to different FcγRs was determined using purified proteins and single-cycle Biacore analysis. Trastuzumab (IgG1) was used as a positive control. Human Fc receptors, FcγRI, FcγRIIa (167R and 167H polymorphisms), FcγRIIb, FcγRIIIa (176F and 176V polymorphisms), and FcγRIIIb used in the test were all obtained from Sino Biological (Beijing, China).

The results obtained are shown in Table 29:

CD16A
(176 Phe) CD16A
(176 Val) CD16B CD32A
(167 Arg) CD32A
(167 His) CD32B CD64 FcgRIIIA176F FcgRIIIA176V FcgRIIIB FcgRIIA167R FcgRIIA167H FcgRIIB FcgRI Trastuzumab 1.79E-06 7.02E-07 5.35E-06 1.38E-05 1.14E-05 2.21E-05 3.93E-09 7A6RS15 3.01E-06 1.10E-06 4.46E-06 4.89E-06 7.60E-06 8.52E-06 5.19E-09

As shown in Table 29, RS15 included in the fusion protein (7A6RS15TL-2) bound to all tested Fc gamma receptors with a high affinity comparable to that of trastuzumab without inhibiting the binding affinity to human Fc receptors. This suggests that the anti-TIGIT-RS15 fusion protein can maintain the Fc receptor-mediated activity of the parental anti-TIGIT antibody.

2.3. Measurement of movement of fusion proteins to the immune cell surface

In the fusion protein provided in the present application, it was confirmed that the anti-TIGIT antibody was fused to RS15, thereby translocating RS15 to the surface of immune cells (human CD4+ T cells, CD8+ T cells, CD56+ NK cells).

To measure the RS15 migration ability as described above, human peripheral blood mononuclear cells (PBMC; Stemcell technologies) were plated in 24 wells at a density of approximately 0.5 x 10 ⁶ cells/well, spun down, the supernatant was removed, and the pellet was disrupted by vortexing. The obtained cells were resuspended in 50 μl of RPMI medium supplemented with 10% (v/v) fetal calf serum (FCS), penicillin, and streptomycin.

Serial dilutions of 7A6RS15 fusion protein (containing RS15.0 and the first linker EAAAKEAAAKEAAAK (SEQ ID NO: 44)) were prepared. Fifty μl of each diluted fusion protein was added to the wells containing the prepared PBMCs. The final concentration of the fusion protein was 10 μg/ml, 1 μg/ml, 0.5 μg/ml, 50 ng/ml, 5 ng/ml, or 0 μg/ml. The cells (PBMCs) to which the fusion protein was added were incubated at room temperature for 10 minutes. Unbound antibody was removed by adding 200 μl of PBS, spinning the plate at 500 × g for 5 minutes, and removing the supernatant. Anti-IL-15-PE antibody that can bind to RS15 (Thermo fisher; clone 34559, cat# MA5-23561) (1.25 ml PBS + 50 μl anti-IL-15 (1/25)) was added to all wells and the cells were stained by incubating for 10 min at room temperature. 200 μl of PBS was added and the supernatant was removed by spinning. The cells were resuspended in staining buffer containing anti-CD3 antibody (Biolegend, at 1:50), anti-CD4 antibody (Biolegend, at 1:50), anti-CD8 antibody (Biolegend, at 1:50), and anti-CD56-BV421 (Biolegend, at 1:50). For three of the above fusion proteins (7A6RS15TL-3, 7A6RS15TL-4, 7A6RS15TL-5), anti-TIGIT-PE-Cy7 (Biolegend, 1/50) was also treated together to stain TIGIT. The cells were incubated with the above antibodies at room temperature for 10 minutes and then washed in 200 μl of PBS. The cells were resuspended in 100 μl of PBS, and the surface marker labeling of each cell and the fluorescent labeling for TIGIT were measured using a flow cytometer (CytoFLEX, Beckman Coulter). The obtained data were analyzed with Flowjo software (BD Biosciences) and graphed with GraphPad Prism 9 software (Dotmatics).

The results are shown in Fig. 2. As shown in Fig. 2, all the tested fusion proteins dose-dependently transported RS15 to the surface of CD4+ and CD8+ T cells and CD56+ NK cells. When the fusion proteins were treated together with anti-TIGIT-PE-Cy7 antibody, it was confirmed that the amount of free TIGIT on the cell surface decreased. Therefore, it was confirmed that the binding of the fusion protein to TIGIT on the cell surface of the antibody was related to the transport of RS15 to immune cells.

The above obtained results suggest that the fusion protein provided in the present application has the effect of transporting RS15 into immune cells (effector T cells and NK cells) through TIGIT binding.

2.4. By fusion proteins IL12RB1/IL12RB2 dimerization

Cells (PathHunter eXpress Dimerization Cells, PathHunter® eXpress Dimerization Assay Kit (Discoverx)) were plated in 96-well plates and cultured at 37°C, 5% CO ₂ for cell seeding. The cells were then stimulated with 7A6RS15TL-2 fusion proteins at the concentrations indicated in Fig. 1c. After stimulation, signals were measured using PathHunter® eXpress IL2RB/IL2RG/IL2RA and IL2RB/IL2RG Dimerization Assay (Eurofins Discoverx, USA) according to the manufacturer's protocol. The PathHunter® eXpress functional assay detects ligand-induced dimerization of two subunits of the IL-15 receptor family. When a ligand binds to one receptor subunit, it induces interaction with its heterodimer partner, resulting in the complementary pair of enzyme fragments linked to each receptor subunit, resulting in a functional enzyme complex that hydrolyzes the substrate and generates a chemiluminescent signal.

The results of the above assay are shown in Fig. 3 (Buffer: PBS (untreated fusion protein group). As shown in the results, when the 7A6RS15TL-2 fusion protein was added as a ligand, luminescence was induced in a dose-dependent manner, indicating dimerization of IL2RB/IL2RG. The EC50 value of dimerization was 0.248 nM. The results show that the fusion protein maintains the functional activity of IL-15.

2.5. Induction of STAT5 phosphorylation by fusion proteins

2.5.1. Induction of STAT5 phosphorylation in immune cells by fusion proteins (1)

Cells (human PBMC) were thawed and 500,000 cells per well were seeded in 100 μl R10 medium (RPMI1640 medium (Gibco) containing 10% (v/v) FBS (Gibco), Penicillin-Streptomycin (Sigma), L-glutamine (Sigma)). The fusion protein 7A6RS15TL-2 was diluted to 68.7 nM, and this concentration was used as a starting concentration and serially diluted 10-fold to 69 fM. The cells and fusion protein were mixed well by pipetting and incubated at 37°C for 15 minutes. After incubation, the cells were pelleted by centrifugation at 400 × g for 3 minutes at 4°C. The supernatant was removed, the pellet was vortexed to disrupt, and the cells were resuspended in PBS containing viability marker (eBioScience, 1:500), anti-CD3 antibody (Biolegend, 1:50), anti-CD4 antibody (Biolegend, 1:50), anti-CD56 antibody (Biolegend, 1:50), and anti-CD8 antibody (Biolegend, 1:50). The cells were stained at 4°C for 10 minutes and then centrifuged at 400 × g for 3 minutes to pellet the cells. The supernatant was removed, the pellet was vortexed to disrupt, and the cells were resuspended in 200 μL of Fixation Buffer (Biolegend) prewarmed at 37°C for 15 minutes at room temperature. The cells were then pelleted again by centrifugation at 400 × g for 3 minutes. The supernatant was removed, the pellet was disrupted by vortexing, and the cells were resuspended in 200 μL ice-cold True-Phos 1X Perm Buffer (Biolegend). The cells were then stored in the freezer for 1 h and centrifuged at 400 × g for 3 min to pellet the cells. The supernatant was removed, the pellet was disrupted by vortexing, and the cells were resuspended in staining buffer containing anti-pSTAT5 antibody (Biolegend, 1:50) at room temperature for 15 min. The cells were pelleted by centrifugation at 400 × g for 3 min. The supernatant was removed, the pellet was disrupted by vortexing, the cells were resuspended in PBS, and pelleted again as described above. Finally, the cells were resuspended in 100 μL of PBS, and data were acquired using a flow cytometer (CytoFlex, BeckmanCoulter). Data were analyzed with FlowJo (BD, USA).

The pSTAT5 signal at 68.7 nM was set to 100%, and the obtained data were normalized to the 68.7 nM condition. Non-linear regression curves were calculated using Graphpad Prism 9 and displayed on the graph.

The obtained results are shown in Fig. 4a (CD8+ T cells), 4b (CD4+ T cells), and 4c (CD56+ NK cells). In primary cells, receptor heterodimerization should induce signal transduction across the cell membrane. Signal transduction of IL-15 can be assessed by phosphorylation of STAT5. As shown in Fig. 4a to 4c, a fusion protein (7A6RS15TL-2) containing an IL-15-like polypeptide (RS15) linked to an anti-TIGIT monoclonal antibody induced STAT5 phosphorylation in both T cells (CD8+ T cells, CD4+ T cells) and NK cells. This shows that the ability of the IL-15-like polypeptide to effectively transmit signals is not inhibited even when linked to an anti-TIGIT antibody.

2.5.2. Induction of STAT5 phosphorylation in immune cells by fusion proteins (2)

Human PBMCs were plated at approximately 500,000 cells per well. The cells were pelleted, the supernatant was removed, and the pellet was vortexed to disrupt the pellet as described in Example 2.5.1 above. The cells were resuspended in 50 μl of RPMI medium supplemented with 10% (v/v) FCS, penicillin, and streptomycin. The fusion protein 7A6RS15TL-2 and IL-15 (control) were diluted to 1938 pmol and 3876 pmol, respectively, and added to the prepared PBMCs, respectively. The PBMCs were cultured at 37°C for 1 h in the presence or absence of the fusion protein or IL-15, and then washed five times. In addition, IL-15 was added to the IL-15 continuous control wells at a final concentration of 3876 pmol for 24 h.

All cells were resuspended in 200 μl of RPMI medium supplemented with 10% (v/v) FCS, penicillin, and streptomycin and incubated at 37°C for an additional 24 h. The cells were then pelleted, the supernatant removed, the pellet disrupted by vortexing, the cells resuspended in PBS, and pelleted by centrifugation at 500 × g. This process was repeated twice more. Finally, the cells were resuspended in staining buffer (50 μl/well) containing viability marker (eBioScience, 1:500), anti-CD3 antibody (Biolegend, 1:50), anti-CD4 antibody (Biolegend, 1:50), anti-CD8 antibody (Biolegend, 1:50), and anti-CD56-BV421 antibody (Biolegend, 1:50) and incubated for an additional 30 min at room temperature in the dark. Cells were then washed with 100 μL of PBS and resuspended in pre-warmed Fixation Buffer (Biolegend, UK) at room temperature in the dark for 30 min. 200 μL of True-Nuclear 1X Perm Buffer (Biolegend) was added to each well and incubated at -20°C for at least 1 h. Cells were then pelleted and resuspended in staining buffer (50 μL/well) containing anti-pSTAT5 antibody (Biolegend, at 1:25). Cells were further incubated at room temperature in the dark for 15 min and then washed with 200 μL of PBS. Finally, cells were resuspended in 100 μL of PBS and data were acquired using a flow cytometer (CytoFlex, Beckman Coulter). Data were analyzed using FlowJo (BD, USA).

The obtained results are shown in Fig. 4d. As shown in the results, when stimulated with the fusion protein 7A6RS15TL-2 for 1 hour, STAT5 phosphorylation (pSTAT5) was detected in T cells and NK cells even after 24 hours. However, when the initial stimulation was performed with IL-15, pSTAT5 was not significantly detected. In addition, it was shown that stimulation with the fusion protein for only 1 hour could induce pSTAT5 levels equivalent to those continuously stimulated with IL-15 for 24 hours. These results show that the IL-15-induced pSTAT5 level significantly decreased after 24 hours, indicating that the signal disappeared, whereas the fusion protein provides a sustained signal through the IL-15 receptor complex for a long period of time (for 24 hours after treatment).

Example 3. Immune-enhancing effect of fusion protein

3.1. Superior cytokine secretion increase effect of fusion protein compared to comparative antibodies (Multiplex Array)

To test cytokine secretion in immune cells by fusion protein treatment, human PBMC (Stemcell Technologies) from healthy donors were resuspended in PRMI1640 medium (Gibco) containing 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), L-glutamine (Sigma), and HEPES (Sigma) and seeded in 96-well plates. For T cell activation, PBMC were stimulated with 3 μg/ml of anti-CD3 monoclonal antibody (BD Biosciences, clone HIT3). Upon stimulation with anti-CD3 antibody, PBMC were also treated with the fusion protein 7A6RS15TL-2 or five comparator anti-TIGIT monoclonal antibodies (see Examples). PBMCs were incubated at 37°C and 5% _CO2 for 44 hours (antibody concentrations; 13.76 nM, 34.39 nM, and 68.79 nM). The cultured cells were harvested after incubation by centrifugation at 400 ×g for 10 min at 4°C. Each supernatant was collected into a 1.5 ml microcentrifuge tube to secure all samples for each individual and stored in a -80°C deep freezer. For the PBMC supernatants obtained as described above, cytokine levels were measured by performing multiplex array using BioPlex200 (Luminex™ instrument) according to the manufacturer's protocol.

The results obtained above are shown in Fig. 5a (IL-2) and 5b (IFN-γ) (Benchmark mAb1: 22G2, Benchmark mAb2: 31C6 (Merck), Benchmark mAb3: TIG1, Benchmark mAb4: 4.1D3, Benchmark mAb5: 10A7 clones). As shown in the results, the fusion protein (7A6RS15TL-2) induced higher levels of cytokine (IL-2 and IFN-γ) secretion in human PBMCs compared to the comparative antibodies.

3.2. Comparison of fusion proteins Superior cytokine secretion increase effect compared to fusion proteins

Human PBMCs (Stemcell technologies) obtained from healthy donors were cultured with anti-CD3 monoclonal antibody (BD Biosciences, 0.2 ug/ml) and anti-CD28 monoclonal antibody (BD Biosciences, 1 ug/ml) in the presence of fusion protein 7A6RS15TL-2 (6.65 ug/ml). Anti-TIGIT 7A6 (5 ug/ml; heavy chain: SEQ ID NO: 45; light chain: SEQ ID NO: 34+43) antibody, 313M32 (5 ug/ml) antibody, IL-15 alone (775 pM) (Peprotech), RS15 alone (RS15.0) protein (775 pM), 313M32RS15TL-2 fusion protein (313M32-RS15.0 fusion; 6.65 ug/ml; Reference Example) were used as comparators. The group that was not treated with anti-TIGIT antibody, cytokine (IL-15), or fusion proteins (treated only with anti-CD3 antibody and anti-CD28 antibody) was used as the control group. Cytokine production in human CD4+ T and CD8+ T cells and CD56+ NK cells was measured. After culture, the cells were spun down at 400 × g for 10 min at 4°C, and the cytokine concentrations in T cells and NK cells were measured. For this, intracellular cytokines were stained as follows: Cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) and labeled with fluorophore-conjugated antibodies to CD4+ T and CD8+ T cell surface markers on ice for 30 min. Anti-CD3, anti-CD4, anti-CD8, and anti-CD56 antibodies used here were all obtained from Biolegend.

Cells were fixed and permeabilized using the eBioscience™ Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent kit (eBiosciences) according to the manufacturer’s instructions. The cells were stained with a fluorophore-conjugated antibody to intracellular IFNγ (Biolegend). The cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter), and data were analyzed with Flowjo software (BD Biosciences).

The results are shown in Fig. 6a (CD4+ T cells), 6b (CD8+ T cells), and 6c (CD56+ NK cells), respectively. As shown in the results, the fusion protein of the present application (7A6RS15TL-2) was confirmed to increase the production of Th1/Tc1 cytokines (IFN-gamma) in CD4+ T cells, CD8+ T cells, and CD56+ NK cells, thereby enhancing the immune response. The obtained results suggest that the fusion protein of the present application has a more potent effector T cell function enhancing effect than anti-TIGIT antibodies. In addition, the fusion protein of the present application shows a more potent effector T cell function enhancing effect than IL-15 alone or RS15. In addition, the fusion protein of the present application showed a higher IFN-gamma level than the control group (313M32RS15TL-2), which suggests that the fusion protein of the present application has a superior immune-enhancing effect than the fusion protein in which RS15 is conjugated to other reference TIGIT antibodies.

3.3. Cytokine production effects of fusion proteins with various RS15 internal linkers

Cytokine production in human PBMCs treated with 7A6-RS15 fusion proteins having various RS15 internal linkers was investigated. For this, human PBMCs (STEMCELL) obtained from healthy donors were resuspended in PRMI1640 medium (Gibco) containing 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), L-glutamine (Sigma), and HEPES (Sigma) and seeded in 96-well plates. For T cell activation, PBMCs were stimulated with 0.2 μg/mL anti-CD3 monoclonal antibody (BD Biosciences, clone HIT3) and 1 μg/mL anti-CD28 monoclonal antibody (BD Biosciences, clone CD28.2). Upon stimulation with anti-CD3/CD28, PBMCs were also treated with anti-TIGIT antibody-RS15 fusion proteins (7A6RS15TL-2, 7A6RS15TL-2DL-0, 7A6RS15TL-2DL-1, 7A6RS15TL-2DL-2, 7A6RS15TL-2DL-3, 7A6RS15TL-2DL-4) (fusion protein treatment concentrations: 13.76 nM, 34.39 nM, and 68.79 nM).

After PBMCs were cultured for 4 hours at 37°C and 5% _CO2 , Brefeldin A (Sigma) was added, and then cultured for an additional 15 hours. The cultured cells were spun down by centrifugation at 400 × g for 6 minutes at 4°C, harvested, and cultured. The supernatant was removed, and the cells were washed twice with phosphate-buffered saline (PBS) (Thermo) under the same centrifugation conditions. The cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) diluted 1:400 in PBS for 20 minutes at room temperature. The cells were washed with FACS buffer (PBS containing 1% FBS) and labeled with fluorophore-conjugated antibodies including anti-CD3 antibody (Biolegend, 1:100), anti-CD4 antibody (Biolegend, 1:100), CD8 antibody (Biolegend, at 1:100), and anti-CD56 antibody (Biolegend, at 1:100) (for 30 min in the dark on ice). The cells were washed with FACS buffer and resuspended in Fixation/perm buffer (Thermo) for 45 min at room temperature in the dark. The cells were then washed twice with permeabilization wash buffer (Thermo) and pelleted by centrifugation at 400×g for 10 min. For cytokine staining, cells were resuspended in permeabilization wash buffer (Thermo) containing anti-IL-2 antibody (Biolegend, 1:50), anti-IFN-γ antibody (Biolegned, 1:50), and anti-TNF-α antibody (Biolegned, 1:50). Cells were incubated in the dark at room temperature for 30 min. After washing cells with FACS buffer, fluorescence intensity was measured and analyzed by flow cytometry (CytoFLEX, Beckman Coulter) and data were analyzed using Flowjo software (BD). Cytokine production was measured in CD4+ T cells (CD3 ⁺ CD4 ⁺ CD8 ^- ), CD8+ T cells (CD3 ⁺ CD8 ⁺ CD4 ^- ), and CD56+ NK cells (CD3 ^- CD56 ⁺ ).

The results obtained above are shown in Figs. 7a to 7h (fusion protein treatment concentration - Bright bar: 34.39 nM, Dark bar: 68.79 nM). As shown in Figs. 7a to 7h, all the tested fusion proteins increased cytokine (e.g., IL-2, IFN-γ, TNF-α) production in both CD4+ T cells and CD8+ T cells compared to the control group (untreated with fusion proteins), regardless of the type of RS15 internal linker, and also showed a superior effect of increasing cytokine (e.g., IL-2, IFN-γ, TNF-α) production in CD3-CD56+ NK cells compared to the control group.

3.4. Cytokine secretion of fusion proteins in response to anti-TIGIT antibodies Increased effect

To measure cytokine production in human CD3+ T cells and CD56+ NK cells, 500,000 human PBMCs (Stemcell technologies) from healthy donors were cultured in the presence of anti-TIGIT (7A6) antibody (10 μg/ml) or fusion protein (7A6RS15TL-2, 68.79 nM), with anti-CD3 monoclonal antibody (BD Biosciences, 0.2 μg/ml) and anti-CD28 monoclonal antibody (BD Biosciences, 1 μg/ml). The group not treated with anti-TIGIT antibody and fusion protein served as the control. After 4 h of culture in an incubator with 5% CO ₂ at 37°C, Brefeldin A (10 μg/ml) was treated to block cytokine secretion and incubated again in the incubator for 15 h, for a total of 19 h of culture.

To analyze the proportion of cells producing cytokines, cells were washed twice by spinning down at 400 × g for 10 min at 4°C, and then stained as follows: Cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) for 20 min at room temperature, washed by spinning down at 400 × g for 10 min, and labeled with fluorophore-conjugated antibodies to CD3+ T cell or CD56+ NK cell surface markers for 30 min on ice. Both anti-CD3 and anti-CD56 antibodies used here were obtained from Biolegend.

Cells were fixed and permeabilized using the eBioscience™ Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent kit (eBiosciences) according to the manufacturer's instructions. The cells were stained with fluorophore-conjugated antibodies to intracellular IFN-γ and TNF-α (Biolegend) for 30 min at room temperature. The cells were washed, and surface marker labeling of each cell and fluorophore fluorescence of cytokines were detected by flow cytometry (CytoFLEX, Beckman Coulter). Data were analyzed using Flowjo software (BD Biosciences) and plotted using GraphPad Prism 9 software (Dotmatics) (statistical analysis: One-Way ANOVA corrected for multiple comparisons with a Dunnett’s test (n=7), * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001)).

The results are shown in Figs. 8a (CD4+ T cells), 8b (CD8+ T cells), and 8c (CD56+ NK cells). As shown in the results, the fusion protein of the present invention was confirmed to increase the production of Th1/Tc1 cytokines (IFN-gamma) and TNF-alpha in T cells and NK cells, thereby enhancing the immune response. The obtained results suggest that 7A6RS15TL-2 has a more potent and dose-dependent effector T cell and NK cell function enhancement effect than anti-TIGIT (7A6) antibody.

3.5. Effect of fusion protein on cytokine secretion compared to combined treatment

Human PBMCs (Stemcell technologies) obtained from healthy donors were cultured with anti-CD3 monoclonal antibody (BD Biosciences, 0.2 μg/ml) and anti-CD28 monoclonal antibody (BD Biosciences, 1 μg/ml) in the presence of fusion protein 7A6RS15TL-2 (2 μg/ml). For comparison, IL-15 alone (775 pM) (Peprotech), RS15 protein alone (RS15.0; 775 pM), and anti-TIGIT antibody (7A6; heavy chain: SEQ ID NO: 45; light chain: SEQ ID NO: 34+43; 2 μg/ml) were used. The group that was not treated with anti-TIGIT antibody, IL-15 cytokine, or fusion proteins served as a control. Cytokine production in human CD4+ T cells, CD8+ T cells, and CD56+ NK cells was measured. After incubation, cells were spun down at 400 × g for 10 min at 4°C, and cytokine concentrations within T cells and NK cells were measured. For this, intracellular cytokines were stained as follows: Cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) and labeled with fluorophore-conjugated antibodies to CD4+ T and CD8+ T cell surface markers on ice for 30 min. Anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, and anti-CD56 antibody used here were all obtained from Biolegend.

Cells were fixed and permeabilized using the eBioscience™ Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent kit (eBiosciences) according to the manufacturer’s instructions. The cells were stained with a fluorophore-conjugated antibody to intracellular IFNγ (Biolegend). The cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter), and data were analyzed with Flowjo software (BD Biosciences).

The results are shown in Figs. 9a (CD4+ T cells), 9b (CD8+ T cells), and 9c (CD56+ NK cells), respectively. As shown in the results, the fusion protein of the present invention can increase the production of Th1/Tc1 cytokines (IFN-gamma) in CD4+ T, CD8+ T cells, and CD56+ NK to a higher level compared to the case of combined treatment with the anti-TIGIT antibody and RS15 protein that compose it, thereby enhancing the immune responses of T cells and NK cells.

3.6. Immune-enhancing effect of fusion proteins in PBMCs derived from cancer patients

In this example, the cytokine production ability of the fusion protein in PBMCs derived from cancer patients was compared with that of the parent antibody.

For this, human PBMCs (Cureline) derived from hepatocellular carcinoma (HCC) patients were resuspended in PRMI1640 medium (Gibco) containing 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), L-glutamine (Sigma), and HEPES (Sigma) and seeded in 96-well plates. For T cell activation, PBMCs were stimulated with 0.2 μg/mL anti-CD3 monoclonal antibody (BD Biosciences, clone HIT3) and 1 μg/mL anti-CD28 monoclonal antibody (BD Biosciences, clone CD28.2). Upon stimulation with anti-CD3/CD28, PBMCs were also treated with the fusion protein (7A6RS15TL-2, 10 μg/ml) or parental antibody (7A6, 10 μg/ml).

After PBMCs were cultured for 4 hours at 37°C and 5% _CO2 , Brefeldin A (Sigma) was added, and then cultured for an additional 15 hours. The cultured cells were spun down by centrifugation at 400 × g for 6 minutes at 4°C, harvested, and cultured. The supernatant was removed, and the cells were washed twice with phosphate-buffered saline (PBS) (Thermo) under the same centrifugation conditions. The cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) diluted 1:400 in PBS for 20 minutes at room temperature. The cells were washed with FACS buffer (PBS containing 1% FBS) and labeled with fluorophore-conjugated antibodies including anti-CD3 antibody (Biolegend, 1:100), anti-CD4 antibody (Biolegend, 1:100), CD8 antibody (Biolegend, at 1:100), and anti-CD56 antibody (Biolegend, at 1:100) (for 30 min in the dark on ice). The cells were washed with FACS buffer and resuspended in Fixation/perm buffer (Thermo) for 45 min at room temperature in the dark. The cells were then washed twice with permeabilization wash buffer (Thermo) and pelleted by centrifugation at 400×g for 10 min. For cytokine staining, cells were resuspended in permeabilization wash buffer (Thermo) containing anti-IL-2 antibody (Biolegend, 1:50), anti-IFN-γ antibody (Biolegned, 1:50), and anti-TNF-α antibody (Biolegned, 1:50). Cells were incubated in the dark at room temperature for 30 min. After washing cells with FACS buffer, fluorescence intensity was measured and analyzed by flow cytometry (CytoFLEX, Beckman Coulter) and data were analyzed using Flowjo software (BD). Cytokine production was measured in CD4+ T cells (CD3 ⁺ CD4 ⁺ CD8 ^- ), CD8+ T cells (CD3 ⁺ CD8 ⁺ CD4 ^- ), and CD56+ NK cells (CD3 ^- CD56 ⁺ ).

The results obtained above are shown in Fig. 10. As shown in the results above, the fusion protein of the present application showed an excellent effect of increasing cytokine (e.g., IL-2, IFN-γ, TNF-α) production compared to the parent antibody in both CD4+ T cells and CD8+ T cells derived from cancer patients.

Example 4. Anticancer effect of fusion protein

4.1. Cancer cell killing effect by NK cells ( in vitro )

NK cells were isolated from healthy human PBMCs using an NK cell isolation kit (Miltenyi) according to the manufacturer's instructions. NK cells were seeded at 200,000 cells/well in 96-well U-bottom plates in R10 (PRMI1640 medium (Gibco) containing 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), L-glutamine (Sigma), and HEPES (Sigma)). The cells were stimulated with 3.876 nM fusion protein 7A6RS15TL-2 or IL-15 for 1 h at 37°C and 5% CO ₂ , then centrifuged at 400 × g for 3 min and washed five times with R10. The cells were allowed to rest for 24 h at 37°C and 5% CO ₂ .

GFP-expressing MCF7 breast cancer cell line (MacroPhox Ltd) was seeded at 10,000 cells/well (in 50 μl R10). Cells were allowed to attach to the plate for 2 hours, and then stimulated PBMCs were added to each well line (100,000 PBMCs per well in 50 μl R10). Cells were then cultured in IncuCyte for 120 hours at 37°C and 5% CO ₂ . Four images were captured at 10x magnification every 4 hours from each well. Cytotoxicity was evaluated by calculating the Log2 Fold value of the target cell area per well normalized to 0 hour time point. Raw data were processed using Incucyte 2021A software, and graphs and statistics were generated using GraphPad Prism 9.

The results are shown in Fig. 11. As shown in Fig. 11, the fusion protein 7A6RS15TL-2 exhibited a superior tumor cell killing effect that lasted for a long time, compared to the case of combined treatment with soluble IL-15 and the parent antibody (7A6). This result can be said to be because the fusion protein of the present application provides a long-lasting signal to NK cells that control tumor cell growth, compared to the combined treatment of its components.

4.2. Anticancer effect in colon cancer model ( in vivo )

CT-26 tumor cells (CrownBio), a colon cancer cell line, were maintained in RPMI1640 medium supplemented with 10% (v/v) FBS at 37°C and 5% CO ₂ atmosphere. Cells in the exponential growth phase were harvested and quantified by cell counter before tumor cell inoculation.

The prepared tumor cell solution (5x10 ⁵ tumor cells in 0.1 mL of PBS solution) was subcutaneously inoculated into the right posterior flank of BALB/c hTIGIT knock-in mice (GemPharmatech Co., Ltd), and tumors were allowed to grow. The day of tumor inoculation was defined as “Day 0”. Randomization was performed when the average tumor size (volume) reached 70-100 mm ³ . A total of 30 mice were used in this study, and they were randomly divided into 5 groups (5 mice/group). After tumor cell inoculation, the body weights of the animals were measured.

PBS solution was used as a vehicle (control group; group 1), and the fusion protein 7A6RS15TL-1 was treated at doses of 2 mg/kg and 3 mg/kg, respectively (groups 2 and 3, respectively).

The vehicle-treated group (control group) ended the trial on day 17 when the mean tumor burden reached 3000 mm ³ , and the other experimental groups ended the trial on day 24 (see Table 30).

Group Treatment Dose level
(mg/kg) ROA Dosing Frequency & Duration 1 Vehicle - i.v. 1QW x 3 weeks 2 7A6RS15TL-1 2 i.v. 1QW x 3 weeks 3 7A6RS15TL-1 3 i.v. 1 administration at first dosing

QW: weekly(once a week)TGI% was calculated using the following formula:

TGI(%) = 100 x(1-T/C)

(T and C are the mean tumor volume (or weight) of the test group (T) and the control group (C) on a specific day, respectively).

The tumor sizes (mm ³ ) measured during the test period are shown in Fig. 12a. As shown in the results above, on the 24th day, tumor growth reductions of TGI 90.06% and 95.26% were observed in both the 2 mg/kg and 3 mg/kg treatment groups of the fusion protein 7A6RS15TL-1, respectively. Complete tumor removal of less than 100 mm ³ was observed in the 2 mg/kg 7A6RS15TL-1 treatment group (2 mice) and the 3 mg/kg 7A6RS15TL-1 treatment group (4 mice) on the 24th day.

In addition, the weight change of the mice measured during the test period is shown in Fig. 12b. As shown in Fig. 12b, there was no significant difference in weight among the test groups treated with the fusion protein (7A6RS15TL-1). Considering that the side effect of general cytokine therapy is significant weight loss, the above results show that the fusion protein of the present application has a favorable effect compared to existing cytokine therapy.

Example 5. Tolerability of fusion proteins

The body weight (A), AST/ALT levels (B), and total blood count (C) of mice administered the fusion protein of the present invention were analyzed to evaluate the biotolerance of the fusion protein.

To this end, healthy wild type BALB/c mice (8-10 week old female; Beijing Vital River Laboratory Animal Technology Co., Ltd) were injected with the fusion protein (7A6RS15TL-1) or vehicle (PBS) as described in Table 31 below.

Group No Treatment Dose level
(mg/kg) concentration
(mg/mL) Dosing Volume
(μL/g) ROA Dosing Frequency & Duration 1 3 Vehicle (PBS) - - 10 i.v. QW x 1 week 2 3 7A6RS15TL-1 fusion antibody 3 0.3 10 i.v. QW x 1 week 3 3 7A6RS15TL-1 fusion antibody 1 0.1 10 i.p. QW x 2 weeks

Body weight, AST (aspartate transaminase) level, ALT (alanine transferase) level, and complete blood cell count of Groups 1 to 3 prepared as above were measured daily. AST, ALT, and blood cell count were measured by collecting blood and performing a blood test. Body weight measurement was performed in a Laminar Flow Cabinet using StudyDirector ^TM software (version 3.1.399.19).

The obtained results are shown in Figs. 13a (body weight), 13b (AST and ALT levels), and 13c (complete blood count). A slight decrease in body weight was observed in all groups during the test period (group 1 max. loss or gain = -2.3%, +1.9%, group 2 max. loss or gain = -3.2%, +1.7%, group 3 max. loss or gain = -0.6%, +9.6%). In addition, compared to the control group (Group 1), no significant difference in AST and ALT levels was observed in all test groups (Groups 2 & 3). AST and ALT levels were well maintained within the normal range expected for 8- to 10-week-old female mice (ALT low 40, high 170; AST low 67, high 381). Additionally, complete blood counts (CBC) were measured within the normal expected range for 8- to 10-week-old female mice (White blood cells (WBC) low 5.69 K/μL, high 14.84 K/μL; Lymphocytes (LYM) low 3.6 K/μL, 11.56 K/μL; Monocytes (MON) low 0.34 K/μL, high 1.37 K/μL; Neutrophils (NEU) low 0.74 K/μL, high 3.01 K/μL). These results suggest that the fusion protein of the present application is tolerable and non-toxic to the body.

Attach electronic file of sequence list

Claims (22)

(1) Anti-TIGIT antibody or antigen-binding fragment thereof and
(2) Interleukin-15 (IL-15)-like polypeptide
As a fusion protein comprising:
The above interleukin-15-like polypeptide comprises (a) an interleukin-15 receptor alpha (IL-15Rα) sushi domain, and (b) interleukin-15.
The above interleukin-15-like polypeptide is linked to the N-terminus or C-terminus of the heavy chain or light chain of the anti-TIGIT antibody or antigen-binding fragment thereof,
The above interleukin-15 is linked to the N-terminus or C-terminus of the interleukin-15 receptor alpha sushi domain,
The above anti-TIGIT antibody or antigen-binding fragment thereof,
CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising an amino acid sequence of SEQ ID NO: 4, CDR-H3 comprising an amino acid sequence of SEQ ID NO: 7, CDR-L1 comprising an amino acid sequence of SEQ ID NO: 10, CDR-L2 comprising an amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising an amino acid sequence of SEQ ID NO: 18; or
CDR-H1 comprising an amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising an amino acid sequence of SEQ ID NO: 6, CDR-H3 comprising an amino acid sequence of SEQ ID NO: 9, CDR-L1 comprising an amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising an amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising an amino acid sequence of SEQ ID NO: 20
which includes,
Fusion protein. delete In the first paragraph, the anti-TIGIT antibody or antigen-binding fragment thereof,
CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising an amino acid sequence of SEQ ID NO: 4, CDR-H3 comprising an amino acid sequence of SEQ ID NO: 7, CDR-L1 comprising an amino acid sequence of SEQ ID NO: 11, CDR-L2 comprising an amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising an amino acid sequence of SEQ ID NO: 18
A fusion protein comprising: In the first paragraph, the anti-TIGIT antibody or antigen-binding fragment thereof,
a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, or 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, or 34; or
A heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28, and a light chain variable region of SEQ ID NO: 36
A fusion protein comprising: In claim 1, the anti-TIGIT antibody is a fusion protein which is an animal antibody, a chimeric antibody, or a humanized antibody. In the first aspect, the antigen-binding fragment is a fusion protein which is scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2 of the anti-TIGIT antibody. A fusion protein in claim 1, wherein the interleukin-15 receptor alpha sushi domain comprises an amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 88. In claim 1, the interleukin-15 is a fusion protein comprising an amino acid sequence of SEQ ID NO: 40. A fusion protein in claim 1, which does not contain a peptide linker between the anti-TIGIT antibody or antigen-binding fragment thereof and the interleukin-15-like polypeptide. A fusion protein comprising a peptide linker (first linker) between an anti-TIGIT antibody or an antigen-binding fragment thereof and an interleukin-15-like polypeptide in claim 1. In the 10th paragraph, the first linker is expressed as follows: a fusion protein:
[EAAAK]n, where n is the number of [EAAAK], an integer from 1 to 5; or
[(G)mS]l, where m is the number of G and is an integer from 1 to 5, and l is the number of [(G)mS] and is an integer from 1 to 5. A fusion protein comprising a peptide linker (second linker) between the interleukin-15 receptor alpha (IL-15Rα) sushi domain and interleukin-15 in claim 1. In claim 12, the second linker is a fusion protein expressed as follows:
[(G)mS]l, where m is the number of G, an integer from 1 to 5, and l is the number of [(G)mS], an integer from 1 to 5; or
[(S)o(G)pS]q[XQ]r, where o is the number of S, which is 0 or 1, p is the number of G, which is an integer from 1 to 5, q is the number of the monomer [(S)o(G)pS], which is an integer from 1 to 5, and when there are two or more of the monomers, the monomers can be the same or different, X is leucine (Leu; I) or isoleucine (Ile; I), and r is the number of the dipeptide [XQ], which is 0 or 1. A fusion protein in claim 12, wherein the second linker is SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108), GGGGSGGGGSGGGGS (SEQ ID NO: 85), GSGGGGSGGGGSLQ (SEQ ID NO: 103), GSGGGGSGGGGSIQ (SEQ ID NO: 105), SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110), or SGGGGSGGGSGGGGGSGGGSG (SEQ ID NO: 111). A nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 14. A recombinant vector comprising the nucleic acid molecule of claim 15. A recombinant cell comprising the nucleic acid molecule of claim 15 or a recombinant vector comprising the same. A pharmaceutical composition for preventing or treating cancer, comprising a fusion protein according to any one of claims 1 and 3 to 14. A pharmaceutical composition for preventing or treating cancer, wherein the cancer in claim 18 is a solid cancer or a blood cancer. delete A pharmaceutical composition for preventing or treating an infectious disease, comprising a fusion protein according to any one of claims 1 and 3 to 14. A pharmaceutical composition for preventing or treating an infectious disease, wherein the infectious disease in claim 21 is a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or an infection caused by the above infection.