WO2023239228A1 - Fusion protein comprising anti-tigit antibody, interleukin-15, and interleukin-15 receptor alpha sushi domain, and use thereof - Google Patents

Abstract

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

Description

Anti-TIGIT antibody, interleukin-15, and fusion protein comprising interleukin-15 receptor alpha sushi domain and uses thereof

Cross-citation with related applications

This application is based on Korean Patent Application No. 10-2022-0070914 dated June 10, 2022, Korean Patent Application No. 10-2022-0167014 dated December 2, 2022, and Korean Patent Application No. 10 dated January 18, 2023. The benefit of priority based on -2023-0007618 is claimed, and all content disclosed in the document of the relevant Korean patent application is incorporated as part of this specification.

An anti-TIGIT antibody or 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 the fusion protein. It concerns medicinal uses.

Cancer immunotherapy is a method of treating cancer using the body's immune system, and is called the fourth treatment for cancer following chemotherapy, surgery, and radiation therapy. Cancer immunotherapy requires mechanisms to activate the immune system to increase recognition and response to tumor cells. Immune system activation is a complex mechanism involving the functions of a variety of cells, such as antigen-presenting cells, which are important for the initiation of antigen-specific responses, and effector cells, which are responsible for tumor cell destruction.

Representative examples of the effector cells include cytotoxic T cells.

Meanwhile, TIGIT (T cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibition motif) domain), also referred to as WUCAM, VSIG9, or Vstm3, is used to regulate NK, CD8+, and CD4+ T cells as well as regulatory T cells (“Treg”). It is also a co-inhibitory receptor preferentially expressed. TIGIT is a transmembrane protein containing an intracellular ITIM domain, a transmembrane domain, and an immunoglobulin variable domain.

TIGIT expression is increased in disease settings such as tumor infiltrating lymphocytes (TIL) and infections. TIGIT expression may be a marker of exhausted T cells with lower effector function compared to TIGIT negative cells. Additionally, Treg cells expressing TIGIT exhibit enhanced immunosuppressive activity compared to the TIGIT negative Treg population.

Based on this, drugs that have an antagonistic effect on TIGIT (e.g., antagonistic anti-TIGIT antibodies, etc.) induce immune system activation and enhance immune responses in disease states such as cancer, thereby providing a beneficial effect in disease treatment. It is expected to show.

Meanwhile, based on the immune activity and anti-cancer effects of cytokines, proteins mimicking these are being developed as immune activators and anti-cancer agents, but the risk of their action as immunogens in vivo has not been verified.

In this situation, there is a need for the development of drugs that are safe and have excellent immune activating effects without being immunogenic in vivo.

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

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

The cytokine-like polypeptide may be a fusion polypeptide comprising (a) 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 N-terminus of the interleukin-15 receptor alpha sushi domain through a linker (second linker) or directly linked without a linker. For example, the cytokine-like polypeptide comprises, in order from N-terminus to C-terminus, an interleukin-15 receptor alpha sushi domain and an interleukin-15, or an interleukin-15 receptor alpha sushi domain, a linker ( 2 linker), and may include interleukin-15.

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

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

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

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

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provides an immune-enhancing agent or pharmaceutical composition for immune-enhancing.

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provided is a pharmaceutical composition for preventing and/or treating immune-related diseases.

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provided is an anticancer agent or a pharmaceutical composition for preventing and/or treating cancer.

Another example is immunization with 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provided is a method of enhancing immunity comprising administering to a subject in need of enhancement.

Another example is immunization with 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provided is a method for preventing and/or treating an immune-related disease, comprising administering to a subject in need of prevention and/or treatment of the related disease.

Another example is 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector to treat cancer. Provided is a method for preventing and/or treating cancer, comprising administering to a subject in need of such prevention and/or treatment.

Other examples include at least one type selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector, for use in enhancing immunity or immunization. Provided is use for the manufacture of enhancers.

Another example is the prevention of 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. and/or use for treatment or for the manufacture of drugs for the prevention and/or treatment of immune-related diseases.

Another example is the prevention of one or more types of cancer selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Or it provides a use for treatment or for the production of a drug for the prevention and/or treatment of cancer.

Herein, a fusion protein comprising an anti-TIGIT antibody or antigen-binding fragment thereof that binds to TIGIT, and a cytokine-like polypeptide, and a medicinal use thereof are provided. The fusion protein activates immunity by (1) blocking the action of TIGIT by anti-TIGIT antibody or antigen-binding fragment thereof (e.g., strengthening effector T cell function, controlling Treg activity, immune cells (T cells, NK cells, etc.) (2) It has the advantage of having excellent binding affinity to the receptor and low immunogenicity compared to wild-type interleukin-15/interleukin-2 due to the cytokine-like polypeptide, thereby improving various immune systems. It can be applied as an activator and/or immunotherapeutic agent.

Hereinafter, it will be described in more detail.

Definition of Terms

As used herein, a nucleic acid molecule (may be used interchangeably with “polynucleotide” or “gene”) or a polypeptide (may be used interchangeably with “protein”) “comprises a particular nucleic acid sequence or amino acid sequence or a particular nucleic acid sequence or “Consisting of or expressed as an amino acid sequence” may mean that the nucleic acid molecule or polypeptide essentially includes the specific nucleic acid sequence or amino acid sequence, and may be used to determine the original function and/or desired function of the nucleic acid molecule or polypeptide. Includes (or does not exclude said mutations) a "substantially equivalent sequence" in which mutations (deletions, substitutions, modifications, and/or additions) are made to the specific nucleic acid sequence or amino acid sequence to the extent of maintaining function. It can be interpreted.

In one example, the nucleic acid sequence or amino acid sequence provided herein can be modified by conventional mutagenesis methods, such as directed evolution and/or site-specific methods, to the extent of maintaining their original or desired function. It may include modifications made by site-directed mutagenesis, etc. In one example, a nucleic acid molecule or polypeptide "comprises, consists of, or is represented by a particular nucleic acid sequence or amino acid sequence" means that the nucleic acid molecule or polypeptide (i) comprises the particular nucleic acid sequence or amino acid sequence. Essentially comprising, or (ii) 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% of the specific nucleic acid sequence or amino acid sequence It consists of or essentially contains an amino acid sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology, and has the original function and/ Or, it may mean maintaining the intended function.

The nucleic acid sequence described herein is 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 preferred codon in microorganisms seeking to express the protein due to codon degeneracy. Various modifications can be made to the coding area within.

As used herein, the term “homology” refers to the degree of matching with a given nucleic acid sequence or amino acid sequence and can be expressed as a percentage (%). In the case of homology to nucleic acid sequences, for example, it can be determined using the well-known algorithms BLAST or FASTA, and based on this algorithm BLAST, programs called BLASTN or BLASTX have been developed (see: 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 antigen-binding fragment thereof that specifically recognizes TIGIT, and (2) a cytokine-like polypeptide.

In the fusion protein, the anti-TIGIT antibody or antigen-binding fragment thereof blocks the action of TIGIT to activate immunity (e.g., strengthening effector T cell function, controlling Treg activity, activating immune cells (T cells, NK cells, etc.) It may have an activity that increases cytokine secretion, etc. In addition, the cytokine-like polypeptide has similar activity to interleukin-15 and/or interleukin-2, has excellent binding affinity to the receptor and low immunogenicity compared to wild-type interleukin-15/interleukin-2, and thus has immunity when administered to a living body. It may be a safe polypeptide that has excellent enhancing effects and has a low risk of inducing an immune response that recognizes it as an immunogen. Accordingly, 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 is C of the heavy chain (heavy chain constant region or heavy chain variable region) or light chain (light chain constant region or light chain variable region) of the anti-TIGIT antibody or antigen-binding fragment thereof. It may be connected to the -terminus and/or N-terminus through a linker (first linker) or directly 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, and the cytokine The similar polypeptide is connected to the C-terminus (C-terminus of the constant region) or N-terminus of one of the two heavy chains or one of the two light chains of the anti-TIGIT antibody through a peptide linker (first linker). It may be connected without being connected.

In another 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, The two cytokine-like polypeptides may be linked to the C-terminus or N-terminus of the two heavy chains or two light chains of the anti-TIGIT antibody, respectively, with or without a peptide linker (first linker).

For example, in the fusion protein,

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

(ii) either of the two cytokine-like polypeptides is bound to the C-terminus (i.e., the C-terminus of the light chain constant region) or the N-terminus (i.e., the light chain) of either of the two light chains of the anti-TIGIT antibody. one at 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. It may be connected to the terminal) with or without a linker (first linker).

More specifically, in the fusion protein,

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

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

The first linker may be a peptide linker, 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 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 may contain 35 to 40 amino acids. For example, the first linker is [(G)mS]l (m is the number of amino acids G (Gly) and is an integer of 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.), [(S)o(G)pS]q[XQ]r (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 1 to 5 is an integer (e.g., 1, 2, 3, 4, or 5), and q is the number of units [(S)o(G)pS] and is an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5), when there are two or more units (q is 2, 3, 4, or 5), each unit may be the same or different from each other, and X is leucine (Leu; I) or isoleucine (Ile; I) ), and r is the number of dipeptides [ , SGGSGGGGSGGGSGGGGSIQ (SEQ ID NO: 106), SGGGSGGGGSGGGGSGGGGSGGGSIQ (SEQ ID NO: 107), SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108), SGGGSGGGGSGGGGSGGGGSGGGSSLQ (SEQ ID NO: 109)), SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110), SGGGG SGGGSGGGGGSGGGSG (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, EAAAAKEAAAAKEAAAK (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), APGPSAP SHRSLPSRAFG (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), PPLFAKGPVGLLSRSSFPP (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 EAAAAR 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), LAKLKQKTEQLQDRIAGGG (SEQ ID NO: 152), LELKTPLGDT (SEQ ID NO: 153), THTCPRCPEP (SEQ ID NO: 154), KSCDTPPPCP (SEQ ID NO: 155) ), It may be one or more selected from the group consisting of 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), but is limited thereto. That is not the case. 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); For example, EAAAAKEAAAAKEAAAK (SEQ ID NO: 44)) or a flexible peptide linker (for example, [(G)mS]l (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 is not limited thereto.

Anti-TIGIT antibody or antigen-binding fragment thereof

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

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, or 3 (CDR-H1),

A polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 5, or 6 (CDR-H2),

A polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8, or 9 (CDR-H3),

A polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, or 14 (CDR-L1),

A polypeptide comprising the 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)

It may include.

The polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 10 may include 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 embodiment, 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 One 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 refer to the heavy chain complementarity determining region, and CDR-L1, CDR-L2, and CDR-L3 refer to the light chain complementarity determining region)

In an 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

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

It 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

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

It may include.

In some cases (eg, when produced recombinantly), the heavy chain variable region and/or light chain variable region may additionally include 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 shown 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 WYQQKPGQAPRLIY 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, in that order)

In one example, the anti-TIGIT antibody or antigen-binding fragment thereof provided herein is a TIGIT protein, e.g., a region of amino acid residues 51-70 of human TIGIT protein (NCBI Reference Sequence NP_776160.2; UniProtKB/SwissProt Q495A1-1) ( TAQVTQVNWEQQDQLLAICN; SEQ ID NO: 38) may bind to one or more or two or more (e.g., consecutively located) amino acids, 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

As used herein, “antibody” refers to a general term for proteins that specifically bind to a specific antigen, and may be a protein produced by stimulation of an antigen within the immune system or a protein produced chemically or recombinantly. The 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 polyclonal antibody.

In the anti-TIGIT antibody or antigen-binding fragment thereof provided herein, the regions other than the heavy chain CDR and light chain CDR regions, or the heavy chain variable region and light chain variable region, as defined above, are used to control immunoglobulins of all subtypes (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), such as the framework region of immunoglobulins of all of the above subtypes, and/or the light chain constant region and/or the heavy chain constant region. It may be derived from the area. In one example, the anti-TIGIT antibody provided herein may be a human IgG type antibody, such as IgG1, IgG2, IgG3, or IgG4, but is not limited thereto.

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

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

In one embodiment, the constant region of the IgG (eg, human IgG1) may be wild type. In another embodiment, the constant region of the IgG is, based on human IgG1, S240D (S, the 240th amino acid, is replaced with D; hereinafter, amino acid changes are expressed in the same way), A331L, I333E, N298A, S299A, E334A , K335A, L235A, L236A and P330G, and may be a variant containing one or more mutations selected from the group consisting of, for example, 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" refers to a variable region domain V _H containing an amino acid sequence with 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 ( It is interpreted to include both the full-length heavy chain including the hinge and fragments thereof. In addition, the term "light chain" refers to both a full-length light chain and fragments thereof comprising a variable region domain V _L and a constant region domain C _L containing an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. It is interpreted to mean inclusive.

The term "CDR (complementarity determining region)" refers to a region among the variable regions of an antibody that confers antigen-binding specificity, 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 contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDR may provide key contact residues for the antibody to bind to the antigen or epitope. Meanwhile, in the present specification, the term "specifically binds" or "specifically recognizes" has the same meaning commonly known to those skilled in the art, and refers to a condition in which an antigen and an antibody specifically interact to produce an immunological reaction. means that

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

As used herein, unless otherwise specified, an antibody may be understood to include an antigen-binding fragment of an antibody that possesses antigen-binding ability.

The term “antigen-binding fragment” refers to any form of polypeptide containing a portion to which an antigen can bind (e.g., the six CDRs defined herein). For example, the antibody may be scFv, scFv-Fc, (scFv) ₂ , Fab, Fab', or F(ab') ₂ , but is not limited thereto.

Among the above antigen-binding fragments, Fab has one antigen-binding site with a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (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.

The F(ab') ₂ antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment containing 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.

Double-chain Fv (two-chain Fv) is a non-covalent bond in which the heavy chain variable region and light chain variable region are connected, and single-chain Fv (single-chain Fv) is generally shared between the heavy chain variable region and the short chain variable region through a peptide linker. They can be connected by a bond or directly connected at the C-terminus to form a dimer-like structure, such as double-chain Fv.

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

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

Antibodies provided herein may be monoclonal antibodies. Monoclonal antibodies can be prepared according to methods well known in the art. For example, it can be manufactured using phage display techniques. Alternatively, the antibody may be prepared as a monoclonal antibody derived from an animal (eg, 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 ELISA (Enzyme-Linked ImmunoSorbent Assay) format. Conjugates can be tested for inhibitory activity through functional assays such as competitive ELISA or cell-based assays to test molecular interactions. The respective affinities (Kd values) for the receptor binding domain of TIGIT can then be assayed for monoclonal antibody members selected based on their strong inhibitory activity.

The final selected antibodies can be prepared and used as humanized antibodies as well as antibodies in which the remaining portion, excluding the antigen-binding site, is a human immunoglobulin antibody. Methods for producing humanized antibodies are well known in the art.

The antigen-binding fragment of the anti-TIGIT antibody provided herein refers to a fragment derived from the anti-TIGIT antibody and retaining binding ability to the antigen (TIGIT), and includes the six CDRs of the anti-TIGIT antibody described above. Any polypeptide, for example scFv, scFv-Fc, scFv-Ck (kappa constant region), scFv-Cλ (lambda constant region), (scFv) ₂ , Fab, Fab' or F(ab') ₂ days. However, it is not limited to this. In one example, the antigen-binding fragment is an scFv, or a fusion polypeptide in which an scFv is fused to the Fc region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, etc.) (scFv-Fc), or a constant region of a light chain (e.g., It may be a fusion polypeptide (scFv-Ck or scFv-Cλ) fused with (kappa or lambda), but is not limited thereto.

In the present specification, an antibody (e.g., CDR, variable region, or heavy/light chain, antigen-binding fragment, etc.) "comprises a specific amino acid sequence, or consists of or is comprised of a specific amino acid sequence" means that the amino acid sequence is essentially the same. Cases where meaningless mutations (e.g., substitution, deletion, and/or addition of amino acid residues) that do not have a significant effect on antibody activity (e.g., antigen affinity, pharmacological activity, etc.) are introduced into the amino acid sequence. It could mean anything.

The anti-TIGIT antibody or antigen-binding fragment thereof provided herein has a binding affinity (K _D ) for TIGIT (eg, human TIGIT) measured by, for example, surface plasmon resonance (SPR). Based on the case, it may be 10mM or less, 5mM or less, 1mM or less, 0.5mM or less, 0.2mM or less, 0.1mM or less, 0.05mM or less, 0.01mM or less, 0.005mM or less, or 0.001mM or less, for example. , 0.0001nM to 10mM, 0.0005nM to 10mM, 0.001nM to 10mM, 0.005nM to 10mM, 0.01nM to 10mM, 0.05nM to 10mM, 0.1nM to 10mM, 0.5nM to 10mM, 1nM to 10mM, 0.0001nM to 5mM, 0.0005nM to 5mM, 0.001nM to 5mM, 0.005nM to 5mM, 0.01nM to 5mM, 0.05nM to 5mM, 0.1nM to 5mM, 0.5nM to 5mM, 1nM to 5mM, 0.0001nM to 1mM, 0.0 005nM to 1mM, 0.001 nM to 1mM, 0.005nM to 1mM, 0.01nM to 1mM, 0.05nM to 1mM, 0.1nM to 1mM, 0.5nM to 1mM, 1nM to 1mM, 0.0001nM to 0.5mM, 0.0005nM to 0.5mM, 0.001 nM to 0.5mM , 0.005nM to 0.5mM, 0.01nM to 0.5mM, 0.05nM to 0.5mM, 0.1nM to 0.5mM, 0.5nM to 0.5mM, 1nM to 0.5mM, 0.0001nM to 0.2mM, 0.0005nM to 0.2mM, 0.001nM to 0.2mM, 0.005nM to 0.2mM, 0.01nM to 0.2mM, 0.05nM to 0.2mM, 0.1nM to 0.2mM, 0.5nM to 0.2mM, 1nM to 0.2mM, 0.0001nM to 0.1mM, 0.0005nM to 0.1mM , 0.001nM to 0.1mM, 0.005nM to 0.1mM, 0.01nM to 0.1mM, 0.05nM to 0.1mM, 0.1nM to 0.1mM, 0.5nM to 0.1mM, 1nM to 0.1mM, 0.0001nM to 0.05mM, 0.0005nM to 0.05mM, 0.001nM to 0.05mM, 0.005nM to 0.05mM, 0.01nM to 0.05mM, 0.05nM to 0.05mM, 0.1nM to 0.05mM, 0.5nM to 0.05mM, 1nM to 0.05mM, 0.0 001nM to 0.01mM , 0.0005nM to 0.01mM, 0.001nM to 0.01mM, 0.005nM to 0.01mM, 0.01nM to 0.01mM, 0.05nM to 0.01mM, 0.1nM to 0.01mM, 0.5nM to 0.01mM, or 1nM to Could be 0.01mM However, it is not limited to this.

Cytokine-like polypeptide

The cytokine-like polypeptide,

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

(b) Interleukin-15 (IL-15)

It is a fusion polypeptide containing.

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

In one example, the cytokine-like polypeptide is, in order from N-terminus to C-terminus:

Contains interleukin-15 receptor alpha sushi domain and interleukin-15,

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

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

[SEQ ID NO: 39]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP

[SEQ ID NO: 88]

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPPS

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

[SEQ ID NO: 40]

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Interleukin-15 (IL-15) and interleukin-2 (IL-2) have structural similarities to each other and are involved in the IL-2/IL-15 receptor beta chain (CD122) and common gamma chain (gamma-C, CD132) complex. It binds and participates in signal transmission through this. In one example, interleukin-15 may be of human origin, e.g., NCBI Accession No. It may be NP_000576.1 (amino acid residues 49 to 162), but is not limited thereto. Interleukin-2 may be of human origin, e.g., NCBI Accession No. It may be NP_000577.2, etc., but is not limited thereto.

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

The cytokine-like polypeptide is a synthetic protein (engineered protein) with the interleukin-15/interleukin-2 protein as the basic skeleton, and has the activity of interleukin-15, compared to other synthetic proteins that have the activity of interleukin-15. It is characterized by low immunogenicity when administered in the body.

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 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 may contain 35 to 40 amino acids. For example, the second linker is [(G)mS]l (m is the number of amino acids G (Gly) and is an integer of 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.), [(S)o(G)pS]q[XQ]r (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 1 to 5 is an integer (e.g., 1, 2, 3, 4, or 5), and q is the number of units [(S)o(G)pS] and is an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5), when there are two or more units (q is 2, 3, 4, or 5), each unit may be the same or different from each other, and X is leucine (Leu; I) or isoleucine (Ile; I) ), and r is the number of dipeptides [ , SGGSGGGGSGGGSGGGGSIQ (SEQ ID NO: 106), SGGGSGGGGSGGGGSGGGGSGGGSIQ (SEQ ID NO: 107), SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108), SGGGSGGGGSGGGGSGGGGSGGGSSLQ (SEQ ID NO: 109)), SGGGGSGGGSGGGGGSGG (SEQ ID NO: 110), SGGGG SGGGSGGGGGSGGGSG (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, EAAAAKEAAAAKEAAAK (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), APGPSAP SHRSLPSRAFG (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), PPLFAKGPVGLLSRSSFPP (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 EAAAAR 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), LAKLKQKTEQLQDRIAGGG (SEQ ID NO: 152), LELKTPLGDT (SEQ ID NO: 153), THTCPRCPEP (SEQ ID NO: 154), KSCDTPPPCP (SEQ ID NO: 155) ), It may be one or more selected from the group consisting of 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), but is limited thereto. That is not the case. In one example, 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), SGGGGSGGGSGGGGGSGGGSG (SEQ ID NO: 110) Column number 111) It may be one or more types selected from the group consisting of etc.

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

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[SEQ ID NO: 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 containing the above nucleic acid molecule. The vector can be used as an expression vector for the nucleic acid molecule.

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

Another example provides a method of making the fusion protein, comprising expressing the nucleic acid molecule in a suitable host cell. The step of expressing the nucleic acid molecule in an appropriate host cell may include culturing the previously described recombinant cell under normal conditions.

The term “vector” refers to a means for expressing a gene of interest in a host cell. Examples include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated viral vectors. Vectors that can be used as the recombinant vector include plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.).

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

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

The recombinant vector can be constructed using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of advancing transcription (e.g., pL ^λ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and transcription/translation termination sequences. In the case of using a eukaryotic cell as a host, the origin of replication operating in the eukaryotic cell included in the vector includes the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, and the BBV origin of replication. It is not limited. Additionally, promoters derived from the genome of mammalian cells (e.g., metallothioneine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV) can be used and typically have a polyadenylation sequence as the transcription termination sequence.

The recombinant cell may be obtained by introducing the recombinant vector into an appropriate host cell. The host cell is a cell capable of stably and continuously cloning or expressing the recombinant vector, and any host cell known in the art can be used. Prokaryotic cells include, for example, E. coli JM109, E. coli Bacillus genus strains such as BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli Enterobacteriaceae strains such as Tia marcescens and various Pseudomonas species are used as host cells when transformed into eukaryotic cells, such as yeast ( Saccharomyce cerevisiae ), insect cells, plant cells, and animal cells, such as Sp2/0. , CHO (Chinese hamster ovary) K1, CHO, DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. can be used, but are not limited thereto.

The nucleic acid molecule or the recombinant vector containing it can be transported (introduced) into a host cell using a transport method widely known in the art. For example, if the host cell is a prokaryotic cell, the transport method may be used, such as the CaCl ₂ method or electroporation method, and if the host cell is a eukaryotic cell, microinjection method, calcium phosphate precipitation method, electroporation method, etc. Liposome-mediated transfection and gene bombardment may be used, but are not limited thereto.

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

medicinal use

The anti-TIGIT antibody or antigen-binding fragment thereof included in the fusion protein provided herein blocks the action of TIGIT (e.g., the interaction between TIGIT and its ligand, CD155 (PVR), etc.) to activate immunity (e.g., effector). It not only has the function of strengthening T cell function, controlling Treg activity, increasing cytokine secretion of immune cells (T cells, NK cells, etc.), but also has excellent anticancer effects (e.g., killing cancer cells, inhibiting cancer cell proliferation, etc.). In addition, cytokine-like polypeptides have similar activity to interleukin-15, and have excellent binding ability to the receptor (IL-2/IL-15 receptor) and low immunogenicity compared to the subtype interleukin-15, so they can be administered to the living body. Not only does it have the advantage of ensuring safety as it has excellent immune-enhancing effects, such as increased cytokine secretion, but does not act as an immunogen, so the risk of inducing an immune response that recognizes it as an immunogen is low, as well as excellent anti-cancer effects (e.g., cancer cell killing, inhibits 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 is useful for enhancing immunity, preventing and/or treating immune-related diseases, and especially preventing and/or treating cancer. It can be usefully applied.

One example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provides an immune-enhancing agent or pharmaceutical composition for immune-enhancing.

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provided is a pharmaceutical composition for preventing and/or treating immune-related diseases.

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provides an anticancer agent or a pharmaceutical composition for preventing and/or treating cancer.

Another example is immunization with 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. A method for enhancing immunity is provided, including the step of administering to a subject in need of enhancement. The immune enhancement method may further include the step of identifying a subject in need of immune enhancement prior to the administering step.

Another example is immunization with 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provided is a method for preventing and/or treating an immune-related disease, comprising administering to a subject in need of prevention and/or treatment of the related disease. The method of preventing and/or treating an immune-related disease may further include the step of identifying a subject in need of prevention and/or treatment of an immune-related disease before the administering step.

Another example is 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector to treat cancer. Provided is a method for preventing and/or treating cancer, comprising administering to a subject in need of such prevention and/or treatment. The cancer prevention and/or treatment method may further include the step of identifying a subject in need of cancer prevention and/or treatment before the administering step.

Another example is one or more types of immunopotentiating, immune-related methods selected from the group consisting of the fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Pharmaceutical compositions for use in the prevention and/or treatment of diseases, and/or in the prevention and/or treatment of cancer, or in the enhancement of immunity, in the prevention and/or treatment of immune-related diseases, and/or in the prevention and/or treatment of cancer It provides use for the manufacture of.

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 and immune activation. More specifically, the “immune enhancing effect” of the fusion protein provided herein refers to immune cells (NK cells, NKT cells, CD3+ T cells, effector T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), etc. )) function enhancement (activation) and/or proliferation, inhibition of activity and/or depletion of regulatory T (Treg) cells, production of immune proteins (e.g., cytokines (IL-2, IFN-gamma, etc.)) and/or increased secretion, etc., but is not limited thereto.

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

The cancer may be a solid cancer or a hematological cancer, but is not limited to, squamous cell cancer, 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., skin 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, and liver cancer. It may be one or more types selected from the group consisting of subspecies, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrium or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulva cancer, head and neck cancer, brain cancer, osteosarcoma, etc. .

The cancer prevention and/or treatment effect (anticancer effect or antitumor effect) is not only the effect of suppressing the occurrence and/or growth of cancer cells, but also the effect of cancer caused by migration, invasion, metastasis, etc. It has the effect of suppressing the deterioration of.

The infectious diseases, autoimmune diseases, and inflammatory diseases are caused by strengthening immunity as described above (e.g., immune cells (NK cells, CD3+ T cells, effector T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), etc.)). Enhancement (activation) and/or proliferation of functions, inhibition of activity and/or depletion of regulatory T (Treg) cells, production of immune proteins (e.g., cytokines (IL-2, IFN-gamma, etc.)) and/ or increased secretion, etc.) may be selected from among all infectious diseases, autoimmune diseases, and inflammatory diseases that can be treated, alleviated, and/or prevented. For example, the infectious diseases include viruses (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 (e.g., A disease that occurs when disease-causing pathogens such as bacteria, fungi, and parasites (MERS-CoV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), etc.) spread and invade living organisms (e.g., animals, including humans). is a general term for, and may be an infection or a disease (infectious disease) caused by one or more pathogens selected from the group consisting of viruses, bacteria, fungi, parasites, etc. The autoimmune diseases include rheumatoid arthritis, type 1 diabetes, and Crohn's disease. , ulcerative colitis, Behçet syndrome, lupus, Sjögren syndrome, myasthenia gravis, scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, It may be selected from the group consisting of cytokine storm, etc., but is not limited thereto. The 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 (e.g. 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 lung disease) (COPD), pneumonia, interstitial lung disease, tuberculosis, etc.), upper gastrointestinal tract inflammation (e.g., gastritis, esophagitis, etc.), lower gastrointestinal tract inflammation (e.g., enterocolitis, ulcerative colitis, Crohn's disease, celiac disease, diverticulitis, irritable bowel) syndrome, appendicitis, fistula, etc.), inflammation of the liver, biliary tract and/or pancreas (e.g. hepatitis, fatty liver, cholangitis, cholecystitis, pancreatitis, type 1 diabetes, etc.), kidney (upper urinary tract) inflammation (e.g. pyelonephritis, glomerulonephritis) , urinary tract infection, etc.), lower urinary tract inflammation (e.g., urinary tract infection, ureteritis, urethritis, cystitis, prostatitis/chronic pelvic pain syndrome, etc.), thyroid and/or parathyroid inflammation (e.g., thyroiditis, parathyroiditis, etc.), adrenal inflammation (e.g. Reproductive tract inflammation (e.g. pelvic inflammatory disease, oophoritis, orchitis, epididymitis, etc.), bone and/or joint inflammation (e.g. osteoarthritis, rheumatoid arthritis, osteomyelitis, synovitis, etc.), skin inflammation (e.g. , Skin: cellulitis, erysipelas, rash, athlete's foot, acne, etc.), muscle inflammation (e.g., myositis, etc.), brain inflammation (e.g., encephalitis, major depressive disorder, etc.), nerve inflammation (e.g., eyes, ears, etc.) Neuritis of various areas, complex regional pain syndrome, Guillain-Barre syndrome, etc.), eye inflammation (e.g. stye, uveitis, conjunctivitis, etc.), ear inflammation (e.g. otitis media, mastoiditis, etc.), oral inflammation (e.g. stomatitis, It may be one or more selected from the group consisting of periodontitis, gingivitis, etc.), systemic inflammation (e.g., systemic inflammatory response syndrome (sepsis, etc.), metabolic syndrome-related disease, etc.), peritonitis, reperfusion injury, transplant rejection reaction, hypersensitivity reaction, etc. It is not limited.

The subject of administration of the fusion protein and/or pharmaceutical composition provided herein may be any animal or cell, for example, an animal selected from mammals including humans, primates such as monkeys, rodents such as rats, mice, etc., Alternatively, it may be a cell, tissue, body fluid (such as serum) derived from (isolated) the animal, or a culture thereof, for example, a human or a cell, tissue, or body fluid (such as serum) isolated from a human.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, in addition to fusion proteins, nucleic acid molecules, vectors and/or cells as active ingredients, and the carrier is commonly used in the formulation of protein drugs. , lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl It may be one or more selected from the group consisting of hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. commonly used in the preparation of pharmaceutical compositions.

The content of fusion proteins, nucleic acid molecules, vectors and/or cells as active ingredients in the pharmaceutical composition is 0.00001 to 99% by weight, 0.0001 to 99% by weight, 0.001 to 99% by weight, 0.01 to 99% by weight, based on the total weight of the composition. Weight %, 0.1 to 99 weight %, 1 to 99 weight %, 10 to 99 weight %, 15 to 99 weight %, 20 to 99 weight %, 25 to 99 weight %, 30 to 99 weight %, 35 to 99 weight % , 40 to 99% by weight, 45 to 99% by weight, or 50 to 99% by weight, but is not limited thereto.

Administration of the fusion protein, nucleic acid molecule, vector, cell, and/or pharmaceutical composition may be via oral or parenteral routes. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. When administered orally, proteins or peptides are digested, so oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach.

Additionally, the fusion protein, nucleic acid molecule, vector, cell, and/or pharmaceutical composition may be in the form of a solution, suspension, syrup, or emulsion in oil or aqueous medium, or in the form of extract, powder, powder, granule, tablet or capsule, injection, etc. It may be formulated in a form, and a dispersant or stabilizer may be additionally included for formulation.

The content of the active ingredient, fusion protein, nucleic acid molecule, vector, and/or cell, in the pharmaceutical composition is determined by the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration interval, administration route, It can be prescribed in various ways depending on factors such as excretion rate and reaction 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 based on the active ingredient (fusion protein, nucleic acid molecule, vector, cell). /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 ㎎/ kg, 0.001 to 50 mg/kg, 0.01 to 1000 mg/kg, 0.01 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, but is not limited thereto. The daily dosage may be formulated as a single preparation in unit dosage form, may be formulated in appropriate portions, or may be prepared by placing it in a multi-dose container. In addition, as used herein, the pharmaceutically effective amount refers to the amount of the active ingredient that can exhibit the desired pharmaceutical activity, and may be within the dosage range described above.

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. We provide health functional foods for strengthening immunity or improving metabolism.

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provides health functional foods for preventing and/or improving immune-related diseases.

Another example includes 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 containing the nucleic acid molecule, and a recombinant cell containing the nucleic acid molecule or the recombinant vector. Provides health functional foods for preventing and/or improving cancer.

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

The food composition may contain acceptable food supplements and may further include appropriate carriers, excipients, and diluents commonly used in the production of health functional foods.

When using the food composition 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 conventional methods.

The content of the active ingredient contained in the food composition may be appropriately determined without particular limitation depending on the form of the food, desired effect, purpose, etc., for example, 0.00001 to 99% by weight, 0.0001 to 99% by weight of the total food weight, It may be 0.001 to 99% by weight, 0.01 to 99% by weight, 0.1 to 99% by weight, 1 to 99% by weight, or 10 to 99% by weight, but is not limited thereto.

The fusion protein provided herein has the advantages of both an anti-TIGIT antibody or antigen-binding fragment thereof and a cytokine-like polypeptide, and has immune enhancement, prevention and/or treatment of immune-related diseases, and anticancer effects (e.g., cancer cell killing). , inhibition of cancer cell proliferation, etc.), so it can be usefully applied as an immune enhancer and anticancer agent.

1A and 1B are graphs showing the binding affinity of a fusion protein to human TIGIT according to an example.

Figure 2 is a graph showing the ability of a fusion protein to move to the surface of an immune cell according to one embodiment.

Figure 3 is a graph showing dimerization of IL2RB/IL2RG fusion protein according to one embodiment.

Figures 4a to 4d are graphs showing the phosphorylation effect of STAT5 on immune cells of a fusion protein according to an example (4a: CD8+ T cells, 4b: CD4+ T cells, 4c: CD56+ NK cells, 4d: NK cells).

Figures 5a and 5b are graphs showing the effect of a fusion protein according to an example on cytokine production in PBMC compared with a comparative antibody (5a: IL-2, 5b: IFN-γ).

Figures 6a to 6c are graphs showing the cytokine production effect in immune cells of a fusion protein according to an example 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 effect of fusion proteins with various linkers on cytokine production in immune cells according to one embodiment.

Figures 8a to 8c are graphs showing the cytokine production effect of the fusion protein in immune cells according to one embodiment compared to the 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 the fusion protein in immune cells according to one embodiment compared to the combined treatment (9a: CD4+ T cells, 9b: CD8+ T cells, 9c: CD56+ NK cells).

Figure 10 is a graph showing the effect of a fusion protein according to an example on cytokine production in immune cells derived from cancer patients.

Figure 11 is a result showing the cytotoxicity of a fusion protein against cancer cells through NK cells according to an example.

Figures 12a and 12b are graphs showing the change in tumor size (12a) and change in body weight (12b) in mice administered a fusion protein according to an embodiment.

Figures 13a to 13c are graphs showing body weight change (13a), AST and ALT levels (13b), and complete blood counts (CBC) (13c) in mice administered a fusion protein according to an embodiment. .

Hereinafter, the present invention will be described in more detail through examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the embodiments 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

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

Structure of 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-15Ra 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 fused to the C-terminus of the heavy chain constant region of the antibody (Fc; including ΔK478 mutation)

To produce the fusion protein, genes encoding the heavy chain strand containing the heavy chain of TIGIT mAb and the light chain strand containing the light chain variable region (RS15 is included in the heavy chain strand or light chain strand; see Table 4-27) were synthesized, and pcDNA3 It was cloned into the .1 (Invitrogen) vector. CHO-K1 cells (ATCC) were seeded on a plate and cultured to reach 70-90% cell density, and then 1 μg/μl of the genetic DNA and Lipofectamine reagent (Thermo) were 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. Afterwards, the cells were cultured at 37°C and transiently expressed. Culture supernatants were purified by Protein A and preparative SEC. The produced protein was buffer exchanged with PBS, quantified by OD280nm measurement, and analyzed by SDS-PAGE.

The IL-15-like polypeptide (RS15) used to prepare the fusion protein is summarized below:

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA Sequence (SEQ ID NO: 90): )

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence Column number 91): )

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence Column number 92): )

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (DNA sequence (from Column number 93): )

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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP 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 with the following structure was designed and prepared:

(N-terminus)-[anti-TIGIT antibody]-[first linker (connected to light chain C-terminus of antibody)]-[IL-15 receptor alpha sushi domain]-[second linker]-[hIL-15] -(C-terminal)

The first linker (connecting antibody to 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 (EAAAKEAAAAKEAAAK (SEQ ID NO: 44); GAGGCCGCCGCCAAGGAGGCCGCCGCCAAGGAGGCCGCCGCCAAG (SEQ ID NO: 87))

The anti-TIGIT antibody-RS15 fusion protein prepared in this example was 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: EAAAAKEAAAAKEAAAK (SEQ ID NO: 44); second linker: SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 108));

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

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

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

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

7A6RS15TL-2DL-4 (7A6-RS15.5) (first linker: EAAAAKEAAAAKEAAAK (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 prepared 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 CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGACCGGCTACAGCATCACCAGCGACTACGCCTGGAACTGGATCAGGCAGCCCCCCGGCAAGGGCCTGGAGTGGATGGGCTACATCAGCTACAGCGGCAGCGCCAGGTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCAGGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGA GCAGCGTGACCGCCGAGGACACCGCCACCTACTACTGCGCCAGGAAGGGCTACCCCGCCTACTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC 77 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 GCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACG TGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCTGTGGAGGTGCACAACAA CGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTC TACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG 79 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK 34 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGAGCATCACCTGCAAGGCCAGCCAGGACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACAGCGCCAGCTACAGGTACACCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCAC CTACTACTGCCAGCACCACTACAGCACCCCCTACACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG 78 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 AGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGG TGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGGCGAGTGC 80 linker EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 41 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKE CEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES GCKECEELEEKNIKEFLQSFVHIVQMFINTS 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 GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGACCAGCGGCAGCACCTTCACCGAGTACACCATGCACTGGGTGAAGCAGAGCCACGGCAAGAGCCTGGAGTGGATCGGCGGCCTGAACCCCAACAACGGCGGCACCAGCTACAACCAGAGGTTCAAGGACAGGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGGAGC CTGACCAGCGAGGACAGCGCCGTGTACTACTGCACCAGGGGCACCTACTACGACTACAGCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC 81 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 48 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIK 35 GACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGACAGGGTGAGCATCACCTGCAAGGCCAGCCACTACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAGCCCCAGCTACAGGTACACCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCGTGCAGGCCGAGGACCTGGCC GTGTACTACTGCCACCAGCACTACAGCACCCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG 82 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 linker EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 41 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS 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 GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCATGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGACTACATCATGAACTGGGTGAAGCAGAGCCACGGCAAGAACCTGGAGTGGATCGGCCTGAGCATCCCCTACAACGGCGGCACCAGCTACAACCAGAAGTTCGAGGGCAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGCTGAGCCT GACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGGCATCAAGGGCTACTTCGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC 83 anti-TIGIT heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 50 Light chain strand ([Anti-TIGIT light chain (kappk)]-[linker]-[cytokine-like polypeptide]) Anti-TIGIT light chain variable region DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIK 36 GACGTGGTGGTGACCCAGACCCCCCTGAGCCTGCCCGTGAGCTTCGGCGACCAGGTGAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGGTGAACAGCTTCGGCAAGACCTACCTGAGCTGGTTCCTGCACAAGCCCGGCCAGAGCCCCCAGCTGATCATGTACGGCGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCACCATCAAGC CCGAGGACCTGGCATGTACTACTGCCTGCAGGGCACCCACCAGCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG 84 Anti-TIGIT light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 linker EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 41 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSS NGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.1) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.1) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.1) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GGGGSGGGGSGGGGS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES GCKECEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.2) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 53 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.2) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 53 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.2) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 53 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES GCKECEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.3) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 54 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC GGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKE CEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.3) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 54 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.3) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 54 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP GSGGGGSGGGGSIQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES GCKECEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.4) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 64 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEEL EEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKE CEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.4) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 64 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEE KNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.4) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 64 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNV TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.5) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 65 light chain strand DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKE CEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES GCKECEELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASVKISCKTSGSTFTEYTMHWVKQSHGKSLEWIGGLNPNNGGTSYNQRFKDRATLTVDKSSSTAYMELRSLTSEDSAVYYCTRGTYYDYSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.5) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 65 light chain strand DIVMTQSHKFMSTSVGDRVSITCKASHYVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCHQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Anti-TIGIT heavy chain EVQLQQSGPELVKPGASMKISCKASGYSFTDYIMNWVKQSHGKNLEWIGLSIPYNGGTSYNQKFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARGIKGYFAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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 EAAAAKEAAAAKEAAAK 44 Cytokine-like polypeptide (RS15.5) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 65 light chain strand DVVVTQTPLSLPVSFGDQVSISCRSSQSLVNSFGKTYLSWFLHKPGQSPQLIMYGVSNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECEAAAAKEAAAAKEAAAKITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SGGGGSGGGSGGGGGSGGGSG NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSS NGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 71

Reference example: Preparation of reference antibodies and reference fusion proteins

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

4.1D3 (Genentech): WO2017/053748 A2,

313M32 (Mereo): US2016/0376365 A1.

In addition, reference antibodies used for comparison in the following examples were made based on sequence information described in the corresponding patent or obtained from the manufacturer. 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 fusion protein properties

2.1. Binding Affinity Measurement 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 the human TIGIT antigen, purified 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) were performed. 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 a running buffer and also used for dilution of ligands and analytes. 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 start of each cycle. Antibodies were captured at a flow rate of 10 μl/min, resulting in an immobilization level (RL) of ~150 RU. Afterwards, the surface was allowed to stabilize. To minimize potential mass transfer effects, recombinant human TIGIT (acrobiosystems, China) injected at a flow rate of 50 μl/min was used as an analyte to obtain multi-cycle kinetic data. Antigen (TIGIT) was diluted two-fold (7 points) in the running buffer in a concentration range of 0.406 nM to 30 nM. It was used so that there was no regeneration between each concentration. At each concentration, association phases were monitored for 240 seconds and dissociation phases were measured for 900 seconds. 3 M MgCl ₂ was injected twice to regenerate the sensor chip surface between cycles. To confirm the stability of both the surface and the analyte over kinetic cycles, multiple repetitions of blank and 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 kinetic constants determined from 1:1 model fitted curves. As shown in Table 28, the binding affinity (KD) for human TIGIT protein was 756 pM for 7A6-RS15, 1.48 nM for 2B7-RS15, and 1.14 nM for 3F8-RS15, and the three types of fusions All proteins showed binding affinity equivalent to that of the parent antibody without RS15 fusion.

2.1.2. Measurement of binding affinity to TIGIT of fusion proteins with various RS15 internal linkers (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 well was quickly inverted to remove the supernatant. Non-specific binding was blocked for 1 hour using blocking buffer containing 3% skim milk (Daeil Bio) diluted in PBS in an amount of 200 μL per well. Plates were washed three times by filling the wells with 200 μL of wash buffer. Anti-TIGIT antibody-RS15 fusion proteins with various types of RS15 internal linkers (7A6RS15TL-2, 7A6RS15TL-2DL-0, 7A6RS15TL-2DL-1, 7A6RS15TL-2DL-2, 7A6RS15TL-2DL-3, 7A6RS15TL-2DL-4 ) or parent antibody (7A6 VH3/Vκ5 (7A6)) was prepared by serial dilution of 1:3 in PBS from a starting concentration of 61.097 nM.

100 μL of the diluted fusion protein or antibody was added to the prepared plate and incubated with shaking at 0.65 x g for 1 hour at room temperature. 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 0.65xg for 1 hour at room temperature. HRP-conjugated antibodies were removed and plates were washed three times with wash buffer. Plates were developed with tetramethylbenzidine (TMB) and stopped with 1N HCl. The OD value (absorbance) at 450nM was measured with GloMax® Explorer Fully Loaded.

The obtained results are shown in Figures 1a and 1b (Figure 1b shows the results of repeated tests using the same process for 7A6RS15TL-2 and 7A6). As shown in Figures 1A and 1B, all tested anti-TIGIT-RS15 fusion proteins with various RS15 internal linkers 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 an anti-TIGIT antibody is fused with an IL-15 superagonist, the addition of IL-15 does not interfere with the antigen (TIGIT) binding region of the fusion protein, so the fusion protein has all the functional properties of the parent antibody. It shows that it can be maintained.

2.2. Binding affinity measurement (SPR analysis) for various human Fc receptors (FcγR)

The fusion protein (7A6RS15: 7A6RS15TL-2) was purified from CHO cell culture supernatants using MabSelect SuRe (Cytiva, Little Chalfont, UK). The column was washed with DPBS (Dulbecco's phosphate-buffered saline), and the protein was eluted using 0.1 M sodium citrate at pH 3.0. After elution, the buffer of the relevant fractions was exchanged with PBS containing 100mM L-Arginine. Proteins were filtered and prepared in two ways: i) directly diluting the fusion protein (7A6RS15TL-2) in pH 6.0 PBS buffer; and ii) buffer exchange of the fusion protein into pH 6.0 PBS buffer prior to experimental setup. The binding capacity of the fusion protein (7A6RS15TL-2) to different FcγRs was measured using purified proteins and single-cycle Biacore analysis. Trastuzumab (IgG1) was used as a positive control. The human Fc receptors used in the test, FcγRI, FcγRIIa (167R and 167H polymorphisms), FcγRIIb, FcγRIIIa (176F and 176V polymorphisms), and FcγRIIIb, 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 contained in the fusion protein (7A6RS15TL-2) does not inhibit the binding affinity to human Fc receptors and binds to all tested Fc gamma receptors with high affinity, comparable to that of trastuzumab. did. This suggests that the anti-TIGIT-RS15 fusion protein can maintain the Fc receptor mediating ability of the parent anti-TIGIT antibody.

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

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

To measure RS15 migration ability as described above, human-derived peripheral blood mononuclear cells (PBMC; Stemcell technologies) were plated in 24 wells at an amount of approximately 0.5 x 10 ⁶ cells/well, spun, and the supernatant was After removal, the pellet was crushed 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.

The 7A6RS15 fusion protein (including RS15.0 and the first linker EAAAAKEAAAAKEAAAK (SEQ ID NO: 44)) was prepared by serial dilution. 50 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. Cells (PBMC) to which the fusion protein was added were cultured at room temperature for 10 minutes. Unbound antibody was removed by adding 200 μl of PBS, spinning the plate at 500 x g for 5 minutes, and removing the supernatant. Add anti-IL-15-PE antibody (Thermo fisher; clone 34559, cat# MA5-23561) (1.25 ml PBS + 50 μl anti-IL-15 (1/25)), which can bind to RS15, to all wells. After staining the cells by incubating at room temperature for 10 minutes, 200 μl of PBS was added and the supernatant was removed by rotation. The cells were incubated with 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) and resuspended in staining buffer containing In the case of three of the above fusion proteins (7A6RS15TL-3, 7A6RS15TL-4, 7A6RS15TL-5), anti-TIGIT-PE-Cy7 (Biolegend, 1/50) was also treated to stain TIGIT. The cells were incubated with the above-mentioned 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 label and fluorescent label for TIGIT of each cell were measured using a flow cytometer (CytoFLEX, Beckman Coulter). The obtained data were analyzed with Flowjo software (BD Biosciences) and plotted with GraphPad Prism 9 software (Dotmatics).

The results are shown in Figure 2. As shown in Figure 2, all tested fusion proteins transported RS15 to the surface of CD4+ and CD8+ T cells and CD56+ NK cells in a dose-dependent manner. When treated with anti-TIGIT-PE-Cy7 antibody, it was confirmed that the amount of free-TIGIT on the cell surface was reduced when the fusion protein was treated. Therefore, it was confirmed that there is a correlation between the binding of the fusion protein to TIGIT on the cell surface of the antibody and the transport of RS15 to immune cells.

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

2.4. by fusion protein 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 and 5% CO2 for cell seeding. Cells were then stimulated with 7A6RS15TL-2 fusion protein at the concentrations indicated in Figure 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, causing the two enzyme fragments linked to each receptor subunit to complement each other, resulting in hydrolysis of the substrate and generating a chemiluminescent signal. A complex of functional enzymes is produced.

The results of the assay are shown in Figure 3 (Buffer: PBS (fusion protein untreated group). As shown in the results, addition of the 7A6RS15TL-2 fusion protein as a ligand induced luminescence in a dose-dependent manner, which is consistent with IL2RB /Indicates dimerization of 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 protein

2.5.1. Induction of STAT5 phosphorylation in immune cells by fusion protein (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), and L-glutamine (Sigma). ) was seeded. The fusion protein 7A6RS15TL-2 was diluted to 68.7 nM, and using this concentration as the starting concentration, it was serially diluted 10 times to 69 fM. The cells and the fusion protein were mixed well by pipetting and incubated at 37°C for 15 minutes. After incubation, cells were pelleted by centrifugation at 400 x g for 3 minutes at 4°C. The supernatant was removed, the pellet was disrupted by vortexing, and the cells were incubated with a viability marker (eBioScience, 1:500), anti-CD3 antibody (Biolegend, 1:50), and anti-CD4 antibody (Biolegend, 1:50). ), anti-CD56 antibody (Biolegend, 1:50), and anti-CD8 antibody (Biolegend, 1:50). After staining the cells for 10 minutes at 4°C, the cells were pelleted by centrifugation at 400 x g for 3 minutes. The supernatant was removed, the pellet was disrupted by vortexing, and the cells were resuspended in 200 μL of Fixation Buffer (Biolegend) preheated at 37°C for 15 minutes at room temperature. Then, the cells were pelleted again by centrifugation at 400 x g for 3 minutes. The supernatant was removed, the pellet was disrupted by vortexing, and the cells were resuspended in 200 μL of ice-cold True-Phos 1X Perm Buffer (Biolegend). Then, the cells were stored in the freezer for 1 hour and then centrifuged at 400 x g for 3 minutes 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) for 15 minutes at room temperature. Cells were pelleted by centrifugation at 400 x g for 3 minutes. The supernatant was removed, the pellet was disrupted by vortexing, and the cells were resuspended in PBS and pelleted again as previously described. Finally, cells were resuspended in 100 μl of PBS, and data were acquired by flow cytometry (CytoFlex, BeckmanCoulter). Data were analyzed with FlowJo (BD, USA).

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

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

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

Human PBMCs were plated at approximately 500,000 per well. Referring to Example 2.5.1 above, cells were pelleted, the supernatant was removed, and the pellet was disrupted by vortexing. Cells were resuspended in 50 μl of RPMI medium supplemented with 10% (v/v) FCS, penicillin, and streptomycin. The fusion proteins 7A6RS15TL-2 and IL-15 (control) were diluted to concentrations of 1938 pmol and 3876 pmol, respectively, and added to the PBMC prepared above. The PBMCs were cultured for 1 hour at 37°C in the presence or absence of the fusion protein or IL-15 and then washed 5 times. Additionally, IL-15 was added to the IL-15 continuous control well at a final concentration of 3876 pmol for 24 hours.

All cells were resuspended in 200 μl of RPMI medium supplemented with 10% (v/v) FCS, penicillin, and streptomycin and cultured at 37°C for another 24 hours. Afterwards, the cells were pelleted, the supernatant was removed, the pellet was disrupted by vortexing, and the cells were resuspended in PBS and pelleted by centrifugation at 500 x g. The above process was repeated two more times. Finally, cells were stained with viability marker (eBioScience, 1:500), anti-CD3 antibody (Biolegend, 1:50), anti-CD4 antibody (Biolegend, 1:50), and anti-CD8 antibody (Biolegend, 1:50). , and anti-CD56-BV421 antibody (Biolegend, 1:50) was resuspended in staining buffer (50 μl/well) and incubated for another 30 minutes in the dark and at room temperature. Then, the cells were washed with 100 μL of PBS and resuspended in preheated Fixation Buffer (Biolegend, UK) at room temperature in dark conditions for 30 minutes. 200 μL of True-Nuclear 1X Perm Buffer (Biolegend) was added to each well, and then incubated at -20°C for at least 1 hour. Afterwards, cells were pelleted and resuspended in staining buffer (50 μL/well) containing anti-pSTAT5 antibody (Biolegned, at 1:25). The cells were incubated for another 15 minutes at room temperature in dark conditions and then washed with 200 μL of PBS. Finally, cells were resuspended in 100 μL of PBS, and data were acquired by flow cytometry (CytoFlex, Beckman Coulter). Data were analyzed with FlowJo (BD, USA).

The obtained results are shown in Figure 4d. As shown in the above 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 initial stimulation was performed with IL-15, pSTAT5 was not significantly detected. In addition, it was shown that stimulation with the fusion protein for just 1 hour could induce pSTAT5 levels equivalent to 24-hour continuous stimulation with IL-15. These results show that while IL-15-induced pSTAT5 levels decrease significantly after 24 hours and the signal disappears, the fusion protein provides continuous signaling through the IL-15 receptor complex for a long period of time (24 hours after treatment).

Example 3. Immune strengthening effect of fusion protein

3.1. Excellent increased cytokine secretion effect of fusion protein compared to comparative antibody (Multiplex Array)

To test cytokine secretion from immune cells by fusion protein treatment, human PBMCs (Stemcell Technologies) obtained from healthy donors were incubated with 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), and L-glutamine. (Sigma) and HEPES (Sigma) were resuspended in PRMI1640 medium (Gibco) and seeded in 96-well plates. For activation of T cells, PBMCs were stimulated with 3 μg/ml of anti-CD3 monoclonal antibody (BD Biosciences, clone HIT3). Upon stimulation with anti-CD3 antibody, PBMCs were also treated with fusion protein 7A6RS15TL-2 or five comparative anti-TIGIT monoclonal antibodies (see Reference Example). 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 incubated at 4°C and centrifuged at 400 x g for 10 minutes and then harvested. Each supernatant was collected in a 1.5 ml microcentrifuge tube to obtain all samples for each individual, and stored in a deep freezer at -80°C. For the PBMC supernatant obtained as above, multiplex array was performed using BioPlex200 (Luminex™ instrument) according to the manufacturer's protocol to measure cytokine levels.

The obtained results are shown in Figures 5a (IL-2) and 5b (IFN-γ) (Benchmark mAb1: 22G2, Benchmark mAb2: 31C6 (Merck), Benchmark mAb3: TIG1, Benchmark mAb4: 4.1D3, Benchmark mAb5: 10A7 clone). As shown in the above results, the fusion protein (7A6RS15TL-2) induced higher levels of cytokine (IL-2 and IFN-γ) secretion in human PBMC 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 incubated 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). ml). Comparative groups include anti-TIGIT 7A6 (5ug/ml; heavy chain: SEQ ID NO: 45; light chain: SEQ ID NO: 34+43) antibody, 313M32 (5ug/ml) antibody, IL-15 alone (775pM) (Peprotech), and RS15 alone. (RS15.0) protein (775pM) and 313M32RS15TL-2 fusion protein (313M32-RS15.0 fusion; 6.65ug/ml; reference example) were used. A group not treated with anti-TIGIT antibody, cytokine (IL-15), or fusion proteins (treated with only 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 incubation, cells were spun down at 400 x g for 10 minutes at 4°C, and cytokine concentrations in T cells and NK cells were measured. For this purpose, intracellular cytokines were stained using the following method: cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) and incubated with fluorophore for CD4+ T and CD8+ T cell surface markers on ice for 30 minutes. )-labeled with a conjugated antibody. The 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 (Biolegend) against intracellular IFNγ. The cells were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter), and data were analyzed using Flowjo software (BD Biosciences).

The results are shown in Figures 6a (CD4+ T cells), 6b (CD8+ T cells), and 6c (CD56+ NK cells), respectively. As shown in the above results, the fusion protein (7A6RS15TL-2) of the present application increases Th1/Tc1 cytokine (IFN-gamma) production in CD4+ T cells, CD8+ T cells, and CD56+ NK cells, thereby increasing immunity. It was confirmed that the response was strengthened. The results obtained above suggest that the fusion protein of the present application has a more powerful effect of enhancing the function of effector T cells than the anti-TIGIT antibody. In addition, the fusion protein of the present application shows that it has a more powerful effect on enhancing the function of effector T cells than IL-15 alone or RS15. In addition, the fusion protein of the present application showed a higher IFN-gamma level than the comparison group (313M32RS15TL-2), which means that the fusion protein of the present application has a superior immune-enhancing effect than the fusion protein in which RS15 is bound to another reference TIGIT antibody. It suggests having.

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

Cytokine production was confirmed in human PBMC treated with 7A6-RS15 fusion proteins with various RS15 internal linkers. For this purpose, human PBMCs (STEMCELL) obtained from healthy donors were cultured in PRMI1640 medium containing 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), L-glutamine (Sigma), and HEPES (Sigma). Gibco) and seeded in a 96-well plate. 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, anti-TIGIT antibody-RS15 fusion proteins (7A6RS15TL-2, 7A6RS15TL-2DL-0, 7A6RS15TL-2DL-1, 7A6RS15TL-2DL-2, 7A6RS15TL-2DL-3, 7A6RS15TL-2DL) -4) was also treated with PBMC (fusion protein treatment concentration: 13.76 nM, 34.39 nM, and 68.79 nM).

PBMCs were cultured at 37°C and 5% CO2 for 4 hours, then Brefeldin A (Sigma) was added, and cultured for an additional 15 hours. The cultured cells were spun down by centrifugation at 400xg for 6 minutes at 4°C, cultured, and then harvested. The supernatant was removed, and the cells were washed twice with PBS (phosphate-buffered saline) (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 incubated with anti-CD3 antibody (Biolegend, 1:100), anti-CD4 antibody (Biolegend, 1:100), and CD8 antibody (Biolegend, at 1:100). 100), and were labeled with fluorophore-conjugated antibodies, including anti-CD56 antibody (Biolegend, at 1:100) (for 30 min in the dark on ice). After washing the cells with FACS buffer, they were resuspended in Fixation/perm buffer (Thermo) for 45 minutes in the dark at room temperature. Then, cells were washed twice with permeabilization wash buffer (Thermo) and pelleted by centrifugation at 400xg for 10 minutes. For cytokine staining, cells were incubated with anti-IL-2 antibody (Biolegned, 1:50), anti-IFN-γ antibody (Biolegned, 1:50), and anti-TNF-α antibody (Biolegned, 1:50). It was resuspended in permeabilization wash buffer (Thermo) containing it. Cells were incubated for 30 minutes in the dark at room temperature. After washing the cells with FACS buffer, the fluorescence intensity was measured and analyzed using a flow cytometer (CytoFLEX, Beckman Coulter), and the 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 obtained results are shown in Figures 7a to 7h (fusion protein treatment concentration - Bright bar: 34.39 nM, Dark bar: 68.79 nM). As shown in Figures 7a-7h, all fusion proteins tested, largely regardless of the type of RS15 internal linker, significantly increased cytokines (e.g., IL-2, It increased the production of cytokines (e.g., IL-2, IFN-γ, and TNF-α) in CD3-CD56+ NK cells compared to the control group.

3.4. Cytokine secretion from fusion protein compared to anti-TIGIT antibody increase effect

To measure cytokine production in human CD3+ T cells and CD56+ NK cells, 500,000 human PBMCs (Stemcell technologies) from healthy donors were incubated with anti-TIGIT (7A6) antibody (10 μg/ml) or fusion protein (7A6RS15TL-2). , 68.79 nM) and incubated 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 or fusion protein was used as the control group. After culturing for 4 hours in an incubator at 37°C and 5% CO2, the cells were treated with Brefeldin A (10 μg/ml) to prevent secretion of cytokines and cultured again in the incubator for 15 hours, for a total of 19 hours.

To analyze the proportion of cells producing cytokines, the washing method was repeated twice by spinning down the cells at 400 After staining with cell dye solution (Biolegend) at room temperature for 20 minutes, spin down at 400 x g for 10 minutes and wash. Then, they were labeled with fluorophore-conjugated antibodies against CD3+ T cells or CD56+ NK cell surface markers on ice for 30 minutes. The anti-CD3 antibody and anti-CD56 antibody used at this time 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 fluorophore-conjugated antibodies (Biolegend) against intracellular IFN-γ and TNF-α at room temperature for 30 minutes. After washing the cells, surface marker labeling of each cell and fluorophore fluorescence of cytokines were detected using a flow cytometer (CytoFLEX, Beckman Coulter). Data were analyzed with Flowjo software (BD Biosciences) and plotted with 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 Figures 8a (CD4+ T cells), 8b (CD8+ T cells), and 8c (CD56+ NK cells). As shown in the above results, it was confirmed that the fusion protein of the present application increases the production of Th1/Tc1 cytokine (IFN-gamma) and TNF-alpha in T cells and NK cells, thereby strengthening the immune response. . The obtained results suggest that 7A6RS15TL-2 has a more potent and dose-dependent effect on effector T cell and NK cell function enhancement than the anti-TIGIT (7A6) antibody.

3.5. Increased cytokine secretion effect of fusion protein compared to combined treatment

Human PBMCs (Stemcell technologies) obtained from healthy donors were incubated 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). ) was cultured with. The comparison group included sole IL-15 (775pM) (Peprotech), sole RS15 protein (RS15.0; 775pM), anti-TIGIT antibody (7A6; heavy chain: SEQ ID NO: 45; light chain: SEQ ID NO: 34+43; 2μg/ml ) was used. A group not treated with anti-TIGIT antibody, IL-15 cytokine, or fusion proteins was used as the control group. Cytokine production was measured in human CD4+ T cells, CD8+ T cells, and CD56+ NK cells. After incubation, cells were spun down at 400 x g for 10 minutes at 4°C, and cytokine concentrations in T cells and NK cells were measured. For this purpose, intracellular cytokines were stained using the following method: cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) and incubated with fluorophore for CD4+ T and CD8+ T cell surface markers on ice for 30 minutes. )-labeled with conjugated antibody. The 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 (Biolegend) against intracellular IFNγ. The cells were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter), and data were analyzed using Flowjo software (BD Biosciences).

The results are shown in Figures 9a (CD4+ T cells), 9b (CD8+ T cells), and 9c (CD56+ NK cells), respectively. As shown in the above results, the fusion protein of the present application reduces Th1/Tc1 within CD4+ T cells, CD8+ T cells, and CD56+ NK compared to the case of combined treatment of the anti-TIGIT antibody and RS15 protein that constitutes it. It was confirmed that cytokine (IFN-gamma) production could be increased to a higher level, thereby further strengthening the immune response of T cells and NK cells.

3.6. Immune enhancing effect of fusion protein in PBMC derived from cancer patients

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

For this purpose, human PBMC (Cureline) derived from hepatocellular carcinoma (HCC) patients were mixed with 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), L-glutamine (Sigma), and HEPES (Sigma). It was resuspended in PRMI1640 medium (Gibco) and seeded in a 96-well plate. 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, fusion protein (7A6RS15TL-2, 10 μg/ml) or parent antibody (7A6, 10 μg/ml) was also treated with PBMC.

PBMCs were cultured at 37°C and 5% CO2 for 4 hours, then Brefeldin A (Sigma) was added, and cultured for an additional 15 hours. The cultured cells were spun down by centrifugation at 400xg for 6 minutes at 4°C, cultured, and then harvested. The supernatant was removed, and the cells were washed twice with PBS (phosphate-buffered saline) (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 incubated with anti-CD3 antibody (Biolegend, 1:100), anti-CD4 antibody (Biolegend, 1:100), and CD8 antibody (Biolegend, at 1:100). 100), and were labeled with fluorophore-conjugated antibodies, including anti-CD56 antibody (Biolegend, at 1:100) (for 30 min in the dark on ice). After washing the cells with FACS buffer, they were resuspended in Fixation/perm buffer (Thermo) for 45 minutes in the dark at room temperature. Then, the cells were washed twice with permeabilization wash buffer (Thermo) and pelleted by centrifugation at 400xg for 10 minutes. For cytokine staining, cells were incubated with anti-IL-2 antibody (Biolegned, 1:50), anti-IFN-γ antibody (Biolegned, 1:50), and anti-TNF-α antibody (Biolegned, 1:50). It was resuspended in permeabilization wash buffer (Thermo) containing it. Cells were incubated for 30 minutes in the dark at room temperature. After washing the cells with FACS buffer, the fluorescence intensity was measured and analyzed using a flow cytometer (CytoFLEX, Beckman Coulter), and the 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 obtained results are shown in Figure 10. As shown in the above results, the fusion protein of the present application produces superior cytokines (e.g., IL-2, IFN-γ, TNF-α) compared to the parent antibody in both CD4+ T cells and CD8+ T cells derived from cancer patients. It showed an effect of increasing production.

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 cultured in R10 (PRMI1640 medium (Gibco) containing 10% (v/v) FBS (Gibco), Penicilin-Streptomycin (Sigma), L-glutamine (Sigma), and HEPES (Sigma)) in 96-well U. -The bottom plate was seeded at an amount of 200,000 cells/well. Cells were stimulated with fusion protein 7A6RS15TL-2 or IL-15 at a concentration of 3.876 nM for 1 hour at 37°C and 5% CO2, then centrifuged at 400 x g for 3 minutes and washed five times with R10. The cells were rested at 37°C and 5% CO2 for 24 hours.

MCF7 breast cancer cell line expressing GFP (MacroPhox Ltd) was seeded at an amount of 10,000 cells/well (in 50 μl R10). The 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% CO2. Four images were taken at 10x magnification in each well every 4 hours. Cytotoxicity was evaluated by calculating the Log2 Fold value of the target cell area per well normalized to the 0 hour time point. Raw data was processed using Incucyte 2021A software, and graph creation and statistical processing were performed using GraphPad Prism 9.

The results are shown in Figure 11. As shown in Figure 11, the fusion protein 7A6RS15TL-2 showed a long-lasting and excellent tumor cell killing effect compared to the case of combined treatment with soluble IL-15 and the parent antibody (7A6). This result can be attributed to the fact that the fusion protein of the present application provides a long-lasting signal to NK cells that controls 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 stored in RPMI1640 medium supplemented with 10% (v/v) FBS at 37°C and 5% CO2 atmosphere. Cells in exponential growth phase were harvested and quantified using a 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 rear flank of a BALB/c hTIGIT knock-in mouse (GemPharmatech Co., Ltd.) and tumors were grown. The day of tumor inoculation was designated 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 test, and they were randomly divided into 5 groups (5 mice/group). After tumor cell inoculation, the animals were weighed.

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

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

Group Treatment Dose level
(mg/kg) ROA Dosing Frequency & Duration One 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 average tumor volume (or weight) of the test group (T) and control group (C) on a particular day, respectively).

The tumor size (mm ³ ) measured during the test period is shown in Figure 12a. As shown in the above results, on day 24, a reduction in tumor growth of 90.06% and 95.26% in TGI was observed in both the 2 mg/kg and 3 mg/kg treatment groups of fusion protein 7A6RS15TL-1, respectively. On day 24, complete removal of tumors less than 100 mm ³ was observed in the 2 mg/kg 7A6RS15TL-1 treatment group (2 animals) and the 3 mg/kg 7A6RS15TL-1 treatment group (4 animals).

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

Example 5. Tolerability of fusion proteins

The body weight (A), AST/ALT level (B), and complete blood count (C) of mice administered the fusion protein of the present application were analyzed to evaluate the biotolerability of the fusion protein.

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

Group No Treatment Dose level
(mg/kg) concentration
(mg/mL) Dosing Volume
(μL/g) ROA Dosing Frequency & Duration One 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 One 0.1 10 i.p. QW x 2 weeks

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

The results obtained above are shown in Figures 13a (body weight), 13b (AST and ALT levels), and 13c (complete blood count). A slight weight loss 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%). Additionally, compared to the control group (Group 1), no significant differences in AST and ALT levels were observed in all test groups (Group 2 & 3). AST and ALT levels remained well within the normal range expected for female mice 8 to 10 weeks of age (ALT low 40, high 170; AST low 67, high 381). In addition, complete blood counts (CBC) were also 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). The above results suggest that the fusion protein of the present application is acceptable and does not cause harm to living organisms.

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 interleukin-15-like polypeptide includes (a) an interleukin-15 receptor alpha (IL-15Rα) sushi domain, and (b) interleukin-15, Fusion protein. The method of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 2, or 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, 5, or 6, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7, 8, or 9, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, 13, or 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, 16, or 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, 19, or 20 which includes, fusion protein The method of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is: CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, CDR-H3 containing the amino acid sequence of SEQ ID NO: 7, CDR-H3 containing the amino acid sequence of SEQ ID NO: 11 or 12 CDR-L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; CDR-H1 containing the amino acid sequence of SEQ ID NO: 2, CDR-H2 containing the amino acid sequence of SEQ ID NO: 5, CDR-H3 containing the amino acid sequence of SEQ ID NO: 8, CDR- containing the amino acid sequence of SEQ ID NO: 13 L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19; or CDR-H1 containing the amino acid sequence of SEQ ID NO: 3, CDR-H2 containing the amino acid sequence of SEQ ID NO: 6, CDR-H3 containing the amino acid sequence of SEQ ID NO: 9, CDR- containing the amino acid sequence of SEQ ID NO: 14 L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20 A fusion protein containing a. The method of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is: A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, or 28, and Light chain variable region comprising the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, or 36 A fusion protein containing a. The fusion protein of claim 1, wherein the anti-TIGIT antibody is an animal antibody, a chimeric antibody, or a humanized antibody. The fusion protein of claim 1, wherein the antigen-binding fragment is scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2 of the anti-TIGIT antibody. The fusion protein of claim 1, wherein the interleukin-15 receptor alpha sushi domain comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 88. The fusion protein of claim 1, wherein the interleukin-15 comprises the amino acid sequence of SEQ ID NO: 40. The fusion protein of claim 1, which does not include a peptide linker between the anti-TIGIT antibody or antigen-binding fragment thereof and the interleukin-15-like polypeptide. The fusion protein according to claim 1, comprising a peptide linker (first linker) between the anti-TIGIT antibody or antigen-binding fragment thereof and the interleukin-15-like polypeptide. The method of claim 10, wherein the first linker is [EAAAK]n (n is the number of [EAAAK] and is an integer of 1 to 5) or [(G)mS]l (m is the number of G and is an integer of 1 to 5) , l is the number of [(G)mS] and is an integer from 1 to 5). The fusion protein of claim 1, comprising a peptide linker (second linker) between the interleukin-15 receptor alpha (IL-15Rα) sushi domain and interleukin-15. The method of claim 12, wherein the second linker is [(G)mS]l (m is the number of G and is an integer of 1 to 5, l is the number of [(G)mS] and is an integer of 1 to 5) or [ (S)o(G)pS]q[XQ]r (o is the number of S and is 0 or 1, p is the number of G and is an integer from 1 to 5, and q is the unit [(S)o(G) pS] is an integer of 1 to 5, and when there are two or more units, each unit may be the same or different from each other, X is leucine (Leu; I) or isoleucine (Ile; I), and r is a dipeptide [XQ] is 0 or 1). The method of 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 SGGGGSGGGSGGGGGSGG GSG(sequence Number 111), a fusion protein. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 14. A recombinant vector containing the nucleic acid molecule of claim 15. A recombinant cell containing the nucleic acid molecule of claim 15 or a recombinant vector containing the same. A pharmaceutical composition for preventing or treating cancer comprising the fusion protein of any one of claims 1 to 14. The pharmaceutical composition for preventing or treating cancer according to claim 18, wherein the cancer is solid cancer or blood cancer. A pharmaceutical composition for enhancing immunity, comprising the fusion protein of any one of claims 1 to 14. A pharmaceutical composition for preventing or treating immune-related diseases, comprising the fusion protein of any one of claims 1 to 14. The pharmaceutical composition for preventing or treating an immune-related disease according to claim 21, wherein the immune-related disease is an infectious disease, an inflammatory disease, or an autoimmune disease.

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