WO2023234743A1 - Bispecific antibody including anti-tigit antibody, and use thereof - Google Patents

Abstract

The present invention relates to a bispecific antibody including: an anti-TIGIT antibody or a fragment thereof; and an IL-2 protein. The bispecific antibody including an anti-TIGIT antibody and an IL-2 variant according to the present invention can modulate mechanisms related to TIGIT and has the same or similar functions as IL-2. That is, the bispecific antibody can not only regulate the binding of TIGIT and CD155, but also activate immune cells. In addition, the bispecific antibody significantly inhibited tumor growth in xenograft mouse tumor models and allograft mouse tumor models. Thus, the bispecific antibody can be used as an anticancer agent.

Description

Bispecific antibodies, including anti-TIGIT antibodies, and uses thereof

The present invention relates to an anti-TIGIT antibody or fragment thereof; and a bispecific antibody containing the IL-2 protein and a pharmaceutical composition for treating or preventing cancer using the same.

Cancer immunotherapy is a method of treating cancer using the body's immune system. Cancer immunotherapy can trigger the immune system to attack cancer cells by targeting antigens, such as cancer cell surface proteins. In particular, it has been reported that anti-cancer immunity can be activated through blocking the immune checkpoint pathway. Immune checkpoints are one of the main mechanisms by which tumor cells result in immune evasion. Therefore, inhibition or blockade of immune checkpoints can increase T cell activation, thereby enhancing anti-tumor immunity.

Meanwhile, IL-2 is mainly synthesized by activated T cells, especially CD4+ helper T cells. IL-2 stimulates the proliferation and differentiation of T cells and promotes the production of cytotoxic T lymphocytes (CTL). However, IL-2 has a dual function in the immune response in that it not only mediates the increase and activity of immune cells, but is also important in maintaining immune tolerance. Additionally, it has been reported that IL-2 may be suboptimal for inhibiting tumor growth. This is because, in the presence of IL-2, activation-induced cell death (AICD) may occur in the generated cytotoxic T lymphocytes, and the immune response may be suppressed by IL-2-dependent regulatory T cells (Treg). (Imai et al., Cancer Sci 98, 416-423, 2007).

Meanwhile, TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an immune checkpoint protein (receptor) that binds to CD155 present in dendritic cells, macrophages, etc. to activate T cells and natural killer cells in vivo. is known to inhibit.

Accordingly, the present inventors conducted research to develop a new combination of dual-specific antibodies that enhance the activity of immune cells, and as a result confirmed that dual-specific antibodies including anti-TIGIT antibodies and IL-2 variants effectively regulate immune cells. did. Based on this, the present invention was completed by confirming that the bispecific antibody has an anticancer effect.

To achieve the above object, one aspect of the present invention is an antibody or fragment thereof that specifically binds to TIGIT; and IL-2 protein.

Another aspect of the present invention provides a polynucleotide encoding the bispecific antibody, an expression vector containing the polynucleotide, and a transformed cell into which the expression vector has been introduced.

Another aspect of the present invention provides a method for producing a bispecific antibody, comprising culturing the transformed cells and obtaining a bispecific antibody.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the bispecific antibody as an active ingredient.

Another aspect of the present invention provides the use of the bispecific antibody for preparing a medicament for preventing or treating cancer.

Another aspect of the invention provides the use of the bispecific antibody for preventing or treating cancer.

Another aspect of the present invention provides a method for preventing or treating cancer comprising administering the bispecific antibody to a subject.

Bispecific antibodies, including anti-TIGIT antibodies and IL-2 variants according to the present invention, can modulate TIGIT-related mechanisms and have the same or similar functions as IL-2. In other words, the bispecific antibody can not only regulate the binding of TIGIT and CD155, but also activate immune cells. Additionally, tumor growth was significantly inhibited in mouse tumor models. Therefore, the bispecific antibody can be used as an anticancer agent.

Figure 1 is a diagram schematically illustrating the structure of the GI-106 bispecific antibody, which is an example.

Figure 2 is a diagram showing the results of confirming the purified bispecific antibody (GI-106) by SDS-PAGE.

Figure 3 is a diagram schematically illustrating the structure of the GI-106B7NH06Kv3 bispecific antibody, which is an example.

Figure 4 is a diagram showing the results of confirming the purified bispecific antibody (GI-106B7NH06Kv3) by SDS-PAGE.

Figure 5 is a diagram schematically illustrating the mechanism of action of the TIGIT/CD155 inhibition assay (Blockade Bioassay) for measuring the anti-hTIGIT antibody activity of GI-106.

Figure 6 is a graph showing the degree of inhibition of TIGIT/CD155 binding by concentration of GI-106 and anti-hTIGIT antibody in the TIGIT/CD155 inhibition assay.

Figure 7 is a graph showing the results of treating HEK-Blue ^TM IL-2 reporter cells with GI-106 or Proleukin® and measuring JAK-STAT signaling pathway activity.

Figure 8 is a graph showing the results of measuring the tumor volume of each group after administering PBS or GI-106 to mice implanted with human-derived breast cancer cells (MDA-MB-231).

Figure 9 is a graph showing the tumor volume of each individual in each group after administering PBS or GI-106 to mice implanted with human-derived breast cancer cells (MDA-MB-231).

Figure 10 is a graph showing the tumor volume for each individual after administration of PBS to mice implanted with human-derived breast cancer cells (MDA-MB-231).

Figure 11 is a graph showing the tumor volume for each individual after administering GI-106 to mice implanted with human-derived breast cancer cells (MDA-MB-231).

Figure 12 is a graph showing the tumor growth inhibition (TGI) of each group of mice after administering PBS or GI-106 to mice implanted with human-derived breast cancer cells (MDA-MB-231).

Figure 13 is a graph showing the results of measuring the tumor volume of each group after administering hIgG4 or GI-106 B7NH06Kv3 to mice implanted with mouse-derived breast cancer cells (EMT-6).

Figure 14 is a graph showing the tumor growth inhibition rate of each group of mice after administering hIgG4 or GI-106 B7NH06Kv3 to mice implanted with mouse-derived breast cancer cells (EMT-6).

Figure 15 shows the results of each group after administering hIgG4, GI-106B7NH06Kv3, or GI-106B7NH06K-CN alone or in combination with GI-106B7NH06K-CN and Fc-IL-2v3 to mice implanted with mouse-derived colon cancer cells (MC38). This is a graph showing the results of measuring tumor volume.

Figure 16 shows the results of each group after single administration of hIgG4, GI-106B7NH06Kv3, or GI-106B7NH06K-CN or combined administration of GI-106B7NH06K-CN and Fc-IL-2v3 in mice implanted with mouse-derived colon cancer cells (MC38). This is a graph showing the tumor growth inhibition rate.

Figure 17 shows the results of each group after single administration of hIgG4, GI-106B7NH06Kv3, or GI-106B7NH06K-CN or combined administration of GI-106B7NH06K-CN and Fc-IL-2v3 in mice implanted with mouse-derived colon cancer cells (MC38). This is a graph showing the survival rate until the 25th day.

anti-TIGIT antibody or fragment thereof; and a bispecific antibody containing the IL-2 protein.

One aspect of the present invention is an antibody or fragment thereof that specifically binds to TIGIT; and IL-2 protein.

As used herein, the term “antibody” refers to an immunoglobulin molecule that reacts immunologically with a specific antigen, and refers to a protein molecule that specifically recognizes the antigen. The heavy and light chains of immunoglobulins may each include a constant region and a variable region. The light and heavy chain variable regions of immunoglobulins contain three variable regions called complementarity determining regions (CDRs) and four framework regions (FRs). The CDR is an antigen binding site that mainly binds to the epitope of the antigen.

As used herein, the term “dual specific antibody” refers to a substance that binds to at least one target or antigen. In the present invention, the bispecific antibody may include an antigen binding site that specifically binds to TIGIT and an IL-2 protein or a variant thereof.

The term “TIGIT” as used herein is also known as WUCAM and Vstm2. TIGIT is an immune receptor present on some T cells and natural killer cells (NK cells), and binds to CD155 present on dendritic cells and macrophages to inhibit the activity of T cells and natural killer cells in vivo. Inhibits and reduces immune response.

The antibody or fragment thereof that specifically binds to TIGIT may collectively refer to molecules capable of antigen-antibody binding specifically to TIGIT. The antibody or fragment thereof that specifically binds to TIGIT may have any form as long as it contains an antigen-binding site capable of specifically binding to TIGIT. The antibody or fragment thereof may contain other amino acids that are not directly involved in binding or amino acids whose effect is blocked by amino acid residues in the antigen binding site.

The antibody or fragment thereof that specifically binds to TIGIT is a heavy chain variable region consisting of HCDR1 containing the amino acid sequence of SEQ ID NO: 9, HCDR2 containing the amino acid sequence of SEQ ID NO: 10, and HCDR3 containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable region consisting of LCDR1 including the amino acid sequence of SEQ ID NO: 12, LCDR2 including the amino acid sequence (GVK) of SEQ ID NO: 13, and LCDR3 including the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the antibody or fragment thereof that specifically binds to TIGIT may be composed of a heavy chain variable region including the amino acid sequence of SEQ ID NO: 2 and a light chain variable region including the amino acid sequence of SEQ ID NO: 7. In addition, in one embodiment of the present invention, the anti-TIGIT antibody or fragment thereof includes a heavy chain constant region 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 3 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 24. (CL) may be included.

As used herein, the terms “IL-2” or “interleukin-2” include mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise noted. refers to any wild type IL-2 obtained from any vertebrate source. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. Additionally, the IL-2 may be wild-type IL-2 or a variant thereof.

In the present specification, IL-2 or its variants may be collectively used interchangeably with the terms “IL-2 protein,” “IL-2 polypeptide,” or “IL-2.” IL-2, IL-2 protein, IL-2 polypeptide and IL-2 variants, for example, bind specifically to the IL-2 receptor. The specific binding can be confirmed through methods known to those skilled in the art.

The IL-2 may be in a mature form. Specifically, the mature IL-2 may not contain a signal sequence and may contain a truncated fragment of the N-terminus or C-terminus of wild-type IL-2. At this time, the IL-2 may have the amino acid sequence of SEQ ID NO: 17.

As used herein, the term “IL-2 variant” refers to a form in which some amino acids of the full-length IL-2 or fragment of IL-2 are substituted. That is, the IL-2 variant may have an amino acid sequence that differs from wild-type IL-2 or a fragment thereof. However, the IL-2 variant may have equivalent or similar activity to wild-type IL-2. Here, “IL-2 activity” may mean, for example, specific binding to the IL-2 receptor, and this specific binding can be measured through methods known to those skilled in the art.

Specifically, the IL-2 variant may be one in which some amino acids of wild-type IL-2 have been substituted. One specific example of an IL-2 variant resulting from amino acid substitution may be one in which at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 17 is substituted. According to one embodiment, three amino acids may be substituted as long as IL-2 activity is maintained. The IL-2 variant may be one in which amino acids at the 38th, 42nd, and 61st positions in the amino acid sequence of SEQ ID NO: 17 are substituted.

Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 17. The IL-2 variant may have R38A, F42A, and E61R substitutions in the amino acid sequence of SEQ ID NO: 17. In one embodiment, the IL-2 variant may have the amino acid sequence of SEQ ID NO:6.

An antibody or fragment thereof that specifically binds to the TIGIT; And the IL-2 protein or its variant may be bound by a linker or carrier. In this specification, linker and carrier are also used interchangeably. One specific example of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, Fc (region), etc. The Fc may be the Fc domain of an immunoglobulin.

At this time, the Fc region of the immunoglobulin includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of the immunoglobulin, but does not include the variable regions of the heavy and light chains and the light chain constant region (CL) of the immunoglobulin. refers to proteins that do not The immunoglobulin Fc region may be derived from IgG, IgA, IgE, IgD or IgM. Specifically, the immunoglobulin Fc region may be IgG1, IgG2, IgG3, or IgG4, which are subclasses of IgG, and preferably may be derived from IgG4.

Additionally, the Fc domain of the immunoglobulin may be not only a wild-type Fc domain, but also an Fc domain variant. In addition, the term "Fc domain variant" as used herein refers to a glycosylation pattern that is different from that of the wild-type Fc domain, has an increased sugar chain compared to the wild-type Fc domain, has a decreased sugar chain compared to the wild-type Fc domain, or has a sugar chain removed ( It may be in a deglycosylated form. Additionally, an aglycosylated Fc domain is also included. The Fc domain or variant may have an adjusted number of sialic acids, fucosylation, and glycosylation through culture conditions or genetic manipulation of the host.

Additionally, the sugar chain of the Fc domain of an immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Additionally, the Fc domain variant may be a mixture of the Fc regions of immunoglobulins IgG, IgA, IgE, IgD, or IgM. Additionally, the Fc domain variant may be a form in which some amino acids of the Fc domain are replaced with other amino acids.

In one embodiment, the Fc region may have the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 23.

The bispecific antibody of the present invention is a fusion protein comprising the light chain variable region (VL) and light chain constant region (CL) of an anti-TIGIT antibody, and the heavy chain variable region (VH) and heavy chain constant region 1 (CH1) of an anti-TIGIT antibody. , may include a fusion protein containing an Fc domain and an IL-2 protein. In addition, the bispecific antibody of the present invention is a fusion protein comprising the light chain variable region, light chain constant region, and IL-2 protein of an anti-TIGIT antibody, and the heavy chain variable region, heavy chain constant region 1 (CH1), and Fc of the anti-TIGIT antibody. It may contain a fusion protein containing a domain.

Specifically, the bispecific antibody comprising the anti-TIGIT antibody and IL-2 protein or a variant thereof has the following structural formulas (I) and (II); Or it may include (III) and (IV).

N'-X-[Linker (1)]o-Fc region fragment or variant thereof-[Linker (2)]p-Y-C' (I) and

N'-X'-C' (II);

or

N'-X-[Linker (1)]o-Fc region fragment or variant thereof-C' (III) and

N'-X'-[Linker (3)]q-Y-C' (IV)

At this time, in the structural formulas (I), (II), (III) and (IV),

Wherein N' is the N terminus,

Wherein C' is the C terminus,

The X is the antigen binding site of the heavy chain of the anti-TIGIT antibody and includes a variable region (VH) and a CH1 region,

The X' is the light chain antigen binding site of the anti-TIGIT antibody and includes a variable region (VL) and a constant region (CL),

Y is IL-2 protein or a variant thereof,

The linker (1), linker (2), and linker (3) are each independently peptide linkers,

The o, p and q are each independently O or 1.

At this time, anti-TIGIT antibody, antigen binding site, variable region, constant region; IL-2 protein, IL-2 variant, and Fc region (or Fc domain) are as described above. Additionally, in the bispecific antibody, the IL-2 protein or a variant thereof may be bound to the C terminus of the Fc region or the C terminus of the light chain constant region of the anti-TIGIT antibody. At this time, the IL-2 protein or its variant and the Fc region or light chain constant region may be combined through a peptide linker.

The peptide linker (1) may be composed of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one embodiment, the peptide linker (1) may be composed of 12 amino acids. Additionally, the peptide linker 1 may include at least one cysteine. Specifically, it may contain one, two or three cysteines. Additionally, the peptide linker (1) may be derived from the hinge of immunoglobulin. For example, the hinge can be selected from the hinge regions of various IgG subtype antibodies. Additionally, the hinge may be a form in which some amino acids in the hinge region derived from immunoglobulin are replaced with other amino acids, or may be a sequence in which some amino acid sequences are added. In one embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 18.

The peptide linker (2) or linker (3) may be composed of 1 to 30 consecutive amino acids, or 2 to 20 consecutive amino acids, or 2 to 10 amino acids. In one embodiment, the peptide linker (2) or linker (3) may be (G4S)n (where n is an integer of 1 to 10). At this time, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) or linker (3) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 5.

The amino acid sequences of each region constituting the bispecific antibody are shown in Tables 1 and 2 below. Specifically, Table 1 describes the amino acid sequences of anti-hTIGIT HC-hIgG4 Fc-hIL2v3 and anti-hTIGIT LC (lambda). Additionally, Table 2 describes the amino acid sequences of anti-hTIGIT HC-hIgG4FcM1 and anti-hTIGIT LC(kappa)-hIL2v3.

division		amino acid sequence	sequence number
Anti-hTIGIT HC-hIgG4Fc- hIL2v3	signal peptide (mIgG)	MEWSWVFLFFLSVTTGVHS	One
	Anti-hTIGIT VH	QVQLQESGPGLVKPSGTLSLTCAVS GVSIRQGHW WSWVRQPPGKGLEWIGE IYQTGRT NYNPSLKSRVTISVGKSRNHISLKLSSVTAADTAVYYC TTGSGWYPIDY WGQGTLVTVSS	2
	Anti-hTIGIT CH1	ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV	3
	first linker		ESKYGPPCPPCP	18
	F02(hIgG4 Fc)	APEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVLHEALHNHYTQKSLLSLSLG	4
	2nd linker		GGGGS	5
	hIL2v3		APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT	6
Anti-hTIGIT LC(lambda)	signal peptide (mIgG)	MEWSWVFLFFLSVTTGVHS	One
	Anti-hTIGIT VL	SYELTQDPAVSVALGQTVRITCQGD SLRGFY ASWYQQKPGQAPLLVLY GVK DRPSGIPDRFSGSRSGTTASLIITGAQAEDEADYYC NSRDSNGAMM FGGGTKLTVL	7
	anti-hTIGIT lambda CL		QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS	8

division		amino acid sequence	sequence number
Anti-hTIGIT HC-hIgG4FcM1	signal peptide (mIgG)	MEWSWVFLFFLSVTTGVHS	One
	Anti-hTIGIT VH	QVQLQESGPGLVKPSGTLSLTCAVS GVSIRQGH WWSWVRQPPGKGLEWIGE IYQTGRT NYNPSLKSRVTISVGKSRNHISLKLSSVTAADTAVYYC TTGSGWYPIDY WGQGTLVTVSS	2
	Anti-hTIGIT CH1	ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV	3
	first linker		ESKYGPPCPPCP	18
	F02K(hIgG4 FcM1)	APEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVLHEALHNHYTQKSLLSLSLGK	23
Anti-hTIGIT LC(kappa)-hIL2v3	signal peptide (mIgG)	MEWSWVFLFFLSVTTGVHS	One
	Anti-hTIGIT VL	SYELTQDPAVSVALGQTVRITCQGD SLRGFY ASWYQQKPGQAPLLVLY GVK DRPSGIPDRFSGSRSGTTASLIITGAQAEDEADYYC NSRDSNGAMM FGGGTKLTVL	7
	Anti-hTIGIT kappa CL		RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	24
	2nd linker		GGGGS	5
	hIL2v3		APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT	6

Polynucleotides encoding bispecific antibodies

Another aspect of the invention is an anti-TIGIT antibody or fragment thereof; and a polynucleotide encoding a bispecific antibody comprising the IL-2 protein. The anti-TIGIT antibody, fragment of anti-TIGIT antibody, IL-2 protein and bispecific antibody are the same as described above.

The polynucleotide may include the base sequence of SEQ ID NO: 32, which encodes the heavy chain variable region of an anti-TIGIT antibody. Additionally, the polynucleotide may include the base sequence of SEQ ID NO: 33, which encodes the light chain variable region of an anti-TIGIT antibody. In one embodiment of the present invention, the polynucleotide encoding the dual-specific antibody may include the base sequences of SEQ ID NO: 15 and SEQ ID NO: 16. In one embodiment of the present invention, the polynucleotide encoding the dual-specific antibody may include the base sequences of SEQ ID NO: 27 and SEQ ID NO: 28.

Additionally, if the polynucleotide encodes the same polypeptide, one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. When preparing a polynucleotide sequence by chemical synthesis, synthesis methods well known in the art, for example, methods described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), can be used. and triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on solid supports.

According to one embodiment, the polynucleotide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88% of the base sequence of SEQ ID NO: 15. %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98 %, at least about 99%, or at least about 100% identity.

According to one embodiment, the polynucleotide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88% of the base sequence of SEQ ID NO: 16. %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98 %, at least about 99%, or at least about 100% identity.

According to one embodiment, the polynucleotide has the base sequence of SEQ ID NO: 27 and at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, and at least about 88%. %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98 %, at least about 99%, or at least about 100% identity.

According to one embodiment, the polynucleotide has the base sequence of SEQ ID NO: 28 and at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, and at least about 88%. %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98 %, at least about 99%, or at least about 100% identity.

The polynucleotide may additionally include a signal sequence or leader sequence. The term “signal sequence” used herein refers to a nucleic acid encoding a signal peptide that directs secretion of a target protein. The signal peptide is cleaved after translation in the host cell. Specifically, the signal sequence of the present invention is a polynucleotide encoding an amino acid sequence that initiates the movement of a protein across the ER (endoplasmic reticulum) membrane.

Signal sequences are well known in the art, and typically contain 16 to 30 amino acid residues, but may contain more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence throughout the membrane lipid bilayer while the immature polypeptide moves.

After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. At this time, the signal sequence may be tPa (tissue Plasminogen Activation), HSV gDs (signal sequence of Herpes simplex virus glycoprotein D), IgG signal sequence, or growth hormone secretion signal sequence. Preferably, a secretion signal sequence used in higher eukaryotic cells, including mammals, can be used. Signal sequences useful in the present invention include antibody light chain signal sequences, such as antibody 14.18 (Gillies et al., J. Immunol. Meth 1989. 125:191-202), and antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal. sequence (Sakano et al., Nature, 1980. 286: 676-683) and other signal sequences known in the art (e.g., see Watson et al., Nucleic Acid Research, 1984. 12:5145-5164). . In one specific example, the signal sequence may include the amino acid sequence of SEQ ID NO: 1.

Vector loaded with polynucleotide

Another aspect of the invention is the anti-TIGIT antibody or fragment thereof; and a vector loaded with a polynucleotide encoding a bispecific antibody containing the IL-2 protein. At this time, the polynucleotide may include the base sequence of SEQ ID NO: 32, which encodes the heavy chain variable region of the anti-TIGIT antibody, and SEQ ID NO: 33, which encodes the light chain variable region of the anti-TIGIT antibody. Specifically, the bispecific antibody may be encoded by the base sequences of SEQ ID NO: 15 and SEQ ID NO: 16, or may be encoded by the base sequences of SEQ ID NO: 27 and SEQ ID NO: 28.

As used herein, the term “vector” can be introduced into a host cell and recombined and inserted into the host cell genome. Alternatively, the vector is understood as a nucleic acid vehicle containing a nucleotide sequence capable of spontaneous replication as an episome. The vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, mini-chromosomes and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

Specifically, the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis. plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retroviruses) ), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.), etc. Since the expression level and modification of the protein of the vector varies depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.

Additionally, the plasmid may contain a selection marker, such as an antibiotic resistance gene, and host cells maintaining the plasmid may be cultured under selective conditions.

As used herein, the term “gene expression” or “expression” of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a bispecific antibody product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or variants thereof. The expression vector may include a human CMV (cytomegalovirus) promoter to promote continuous transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence to increase the steady-state level of RNA after transcription. You can.

transformed cells

Another aspect of the invention is the anti-TIGIT antibody or fragment thereof; and an expression vector containing a polynucleotide encoding a bispecific antibody containing the IL-2 protein is provided. At this time, the anti-TIGIT antibody, anti-TIGIT antibody fragment, IL-2 protein, and bispecific antibody are the same as described above.

As used herein, the term “transformed cell” refers to prokaryotic cells and eukaryotic cells into which a recombinant expression vector can be introduced. The transformed cells can be produced by introducing a vector into a host cell and transforming it. Additionally, the bispecific antibody of the present invention can be produced by expressing the polynucleotide contained in the vector.

The transformation can be performed by various methods. There is no particular limitation thereto, as long as the bispecific antibody of the present invention can be produced. Specifically, the transformation method is a CaCl ₂ precipitation method, which increases efficiency by using a reducing substance called DMSO (dimethyl sulfoxide) in the CaCl ₂ precipitation method. Hanahan method, electroporation, calcium phosphate precipitation, protoplast fusion method, stirring method using silicon carbide fiber, Agrobacteria-mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and Drying/inhibition-mediated transformation methods, etc. may be used. Additionally, the target can be delivered into cells using virus particles through infection. Additionally, vectors can be introduced into host cells by gene bombardment or the like.

Additionally, the host cell used to produce the transformed cell is not particularly limited as long as it is capable of producing the antibody of the present invention. Specifically, the host cells may include, but are not limited to, cells of prokaryotic, eukaryotic, mammalian, plant, insect, fungal or cellular origin. An example of the prokaryotic cell may be Escherichia coli. Additionally, yeast can be used as an example of a eukaryotic cell. In addition, the mammalian cells include CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2/0 cells, and human lymphoblastoids. ), NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, or HEK293T cells can be used, but are not limited to these, and any cell that can be used as a mammalian host cell known to those skilled in the art can be used.

In addition, in order to optimize the characteristics of the antibody as a therapeutic agent or for other purposes, the glycosylation-related genes of the host cell are manipulated through methods known to those skilled in the art to manipulate the antibody's sugar chain pattern (e.g., sialic acid, fucose). misfire, saccharification) can be adjusted.

Method for producing bispecific antibodies

Another aspect of the invention is the anti-TIGIT antibody or fragment thereof; and a method for producing a bispecific antibody comprising the IL-2 protein.

The method for producing the bispecific antibody includes i) culturing the transformed cells; and ii) obtaining a bispecific antibody. At this time, the anti-TIGIT antibody, anti-TIGIT antibody fragment, IL-2 protein, and bispecific antibody are the same as described above.

The term “culture” used in this specification refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions.

The method of culturing the transformed cells can be performed using methods widely known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the antibody of the present invention. Specifically, the culture may be continuously cultured in a batch process or fed batch or repeated fed batch process.

Additionally, the step of obtaining the antibody from the culture may be performed by methods known in the art. Specifically, the obtaining method is not particularly limited as long as the produced bispecific antibody of the present invention can be obtained. Preferably, the obtaining method includes centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, It may be a method such as affinity, hydrophobicity, and size exclusion).

Uses of Bispecific Antibodies

In another aspect of the invention, the anti-TIGIT antibody or fragment thereof; and a bispecific antibody containing IL-2 protein as an active ingredient. It provides a pharmaceutical composition for preventing or treating cancer. Here, the anti-TIGIT antibody, fragment of anti-TIGIT antibody, IL-2 protein and bispecific antibody are the same as described above.

As used herein, the term "cancer" is classified as a disease in which normal tissue cells proliferate indefinitely for some reason and continue to grow rapidly regardless of the living phenomenon of the living body or the condition of surrounding tissues, etc. Cancer in the present invention is Various cancers of the human body, such as stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreas cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and It may be any cancer selected from the group consisting of lymphoma, but is not limited to the above types. Additionally, for the purposes of the present invention, cancer may be resistant to radiation, but is not limited thereto.

In the pharmaceutical composition for preventing or treating cancer of the present invention, the bispecific antibody may be included in an arbitrary amount (effective amount) depending on the use, formulation, formulation purpose, etc., as long as it exhibits anti-cancer activity or, in particular, can exhibit a cancer treatment effect. A typical effective amount will be determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, “effective amount” refers to the amount of an active ingredient that can improve the condition of a disease or induce a treatment effect, especially an improvement in the condition of cancer or a treatment effect. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

As used herein, the term “treatment” can be used to include both therapeutic treatment and preventive treatment, and includes both application and any form of medication to treat diseases in mammals, including humans. The term also includes inhibiting or slowing the progression of a disease; Restoring or repairing damaged or missing function, thereby partially or completely relieving a disease; or stimulating inefficient processes; It includes the meaning of alleviating serious diseases. The term “prevention” may be used to mean alleviating or reducing the pathological condition or disease of an individual.

Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate may also affect efficacy. Accordingly, “enhanced efficacy” (e.g., improvement in efficacy) may be due to improved pharmacokinetic parameters and improved efficacy, measured by comparing parameters such as clearance rate and treatment or amelioration of cancer disease in test animals or human subjects. It can be.

Meanwhile, the pharmaceutical composition of the present invention is administered in a “therapeutically effective amount.”

The term “administration” used herein means introducing a predetermined substance into an individual by an appropriate method, and the composition may be administered through any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonaryly, or rectally, but is not limited thereto.

As used herein, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of a compound or composition effective in preventing or treating a target disease, which means treating the disease at a reasonable benefit/risk ratio applicable to medical treatment. It refers to an amount that is sufficient for treatment and does not cause side effects. The level of the effective amount is determined by factors including the patient's health status, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and It may be determined based on other factors well known in the medical field. In one embodiment, a therapeutically effective amount refers to an amount of drug that is effective in treating cancer.

At this time, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier that is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. Pharmacologically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition may be prepared into a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier. Here, “pharmaceutically acceptable” means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what is acceptable for the subject of application (prescription).

When the pharmaceutical composition is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine, or sterile for injection. Isotonic solutions such as water or 5% dextrose can be used. Regarding the formulation of pharmaceutical compositions, it is known in the art, and specifically, references can be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)]. The above documents are considered part of this specification.

The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. The preferred dosage of the pharmaceutical composition may range from 0.0001 μg/kg to 100 g/kg per day depending on the patient's condition, weight, gender, age, patient's severity, and administration route. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

The subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, and humans are particularly preferred. The pharmaceutical composition of the present invention may further include any compounds or natural extracts known to have cancer treatment effects.

Another aspect of the present invention is the anti-TIGIT antibody or fragment thereof for preparing a medicament for preventing or treating cancer; and the use of a bispecific antibody comprising the IL-2 protein. Here, the cancer, prevention, treatment, anti-TIGIT antibody, fragment of anti-TIGIT antibody, IL-2 protein and bispecific antibody are the same as described above.

Another aspect of the present invention is the anti-TIGIT antibody or fragment thereof for preventing or treating cancer; and the use of a bispecific antibody comprising the IL-2 protein. Here, the cancer, prevention, treatment, anti-TIGIT antibody, fragment of anti-TIGIT antibody, IL-2 protein and bispecific antibody are the same as described above.

Another aspect of the invention is the anti-TIGIT antibody or fragment thereof; and administering to a subject a bispecific antibody containing the IL-2 protein. Here, cancer, prevention, treatment, anti-TIGIT antibody, fragment of anti-TIGIT antibody, IL-2 protein and bispecific antibody and administration are the same as described above. The subject may be a mammal, preferably a human. Additionally, the individual may be a patient suffering from cancer or an individual with a high risk of suffering from cancer.

Additionally, the bispecific antibody may be administered in combination with any compound or natural extract known to have a cancer treatment effect, or may be formulated in the form of a combination preparation with other drugs.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Preparation of bispecific antibodies

Example 1.1. Preparation of anti-TIGIT-IgG4 Fc-IL-2 variant: GI-106

To produce a bispecific antibody comprising an anti-TIGIT antibody and an IL-2 variant that specifically binds to TIGIT, the signal peptide (SEQ ID NO: 1), the variable region (SEQ ID NO: 2) of the anti-TIGIT antibody heavy chain and the constant IL-2 variant in which region (SEQ ID NO: 3), linker (1) (SEQ ID NO: 18), IgG4 Fc domain (SEQ ID NO: 4), linker (2) (SEQ ID NO: 5) and three amino acids are substituted (SEQ ID NO: 6) ), a polynucleotide (SEQ ID NO: 34) encoding a signal peptide (SEQ ID NO: 1), and a polynucleotide (SEQ ID NO: 35) encoding the variable region (SEQ ID NO: 7) and constant region (SEQ ID NO: 8) of the anti-TIGIT antibody light chain. ) was loaded into the pCGS3 vector (Sigma-Aldrich®) using BioXp ^TM 3250 SYSTEM (Codex DNA).

Additionally, the vector was introduced into CHO cells (Expi-CHO ^™ , Thermo Fisher Scientific) to express bispecific antibodies. After introducing the vector, it was cultured at 37°C, 125 rpm, and 8% CO ₂ conditions. Afterwards, when the cell survival rate was 50%, the culture medium was collected and the bispecific antibody was purified. The purified bispecific antibody was named “GI-106” (Figure 1).

Specifically, it was purified using chromatography containing Protein A resin. After filtering the collected culture, the filtered culture was flowed through a column and combined. Afterwards, the bispecific antibody was collected with 50 mM glycine, pH 3.4. The buffer containing the collected bispecific antibodies was changed to PBS buffer using dialysis, and the concentration was measured. When detected using NanoDrop equipment (Thermo Fisher Scientific), it was confirmed that a bispecific antibody was included at a concentration of 1.52 mg/mL.

The molecular weight of the separated and purified bispecific antibody (GI-106) was confirmed through SDS-PAGE under reduced (R, reduced) or non-reduced (NR, non-reduced) conditions (Figure 2), and size exclusion chromatography (SEC). ) Purity was confirmed through analysis. As a result, the purity of the separated and purified bispecific antibody was confirmed to be 87.42%.

Example 1.2. Preparation of anti-TIGIT-IgG4 Fc-IL-2 variant: GI-106B7NH06kv3

To produce a bispecific antibody comprising an anti-TIGIT antibody that specifically binds to TIGIT and an IL-2 variant, the signal peptide (SEQ ID NO: 1), the variable region (SEQ ID NO: 2) of the anti-TIGIT antibody heavy chain and the constant region (SEQ ID NO: 3), a polynucleotide (SEQ ID NO: 36) encoding the linker (1) (SEQ ID NO: 18) and an IgG4 FcM1 domain (SEQ ID NO: 23) and a signal peptide (SEQ ID NO: 1) of the anti-TIGIT antibody light chain. A polynucleotide encoding an IL-2 variant (SEQ ID NO: 6) with a variable region (SEQ ID NO: 7), a constant region (SEQ ID NO: 24), a linker (3) (SEQ ID NO: 5), and three amino acids substituted (SEQ ID NO: 37) ) was loaded into the pCGS3 vector (Sigma-Aldrich®) using BioXp ^TM 3250 SYSTEM.

Additionally, the vector was introduced into CHO cells (Expi-CHO ^™ , Thermo Fisher Scientific) to express bispecific antibodies. After introducing the vector, it was cultured for 7 days at 37°C, 127 rpm, 5% CO ₂ , and 80% humidity. Afterwards, the culture medium was collected and the bispecific antibody was purified. The purified bispecific antibody was named “GI-106B7NH06Kv3” (Figure 3).

As a specific purification method for the bispecific antibody, it was purified using affinity chromatography containing Protein A resin. After removing impurities including cell debris generated during the cell culture process through filtration, the filtered culture medium was flowed through the column and combined. Afterwards, a buffer (pH 3.0) containing 100mM glycine and 100mM arginine was sequentially flowed to collect the bispecific antibody. The buffer containing the collected bispecific antibodies was changed to a buffer containing 4% sucrose, 50 mM histidine, and 50 mM arginine (pH 7.4) using dialysis, and the concentration was measured. When detected using Nanodrop equipment, it was confirmed that a bispecific antibody was included at a concentration of 1.87 mg/mL.

The molecular weight of the separated and purified bispecific antibody (GI-106B7NH06Kv3) was confirmed by SDS-PAGE under reducing (R, reduced) or non-reducing (NR, non-reduced) conditions (Figure 4), and size exclusion chromatography (SEC). Purity was confirmed through analysis. As a result, the purity of the separated and purified bispecific antibody was confirmed to be 88.24%.

Example 1.3. Preparation of anti-TIGIT antibodies

To produce a control anti-TIGIT antibody, signal peptide (SEQ ID NO: 1), variable region (SEQ ID NO: 2) and constant region (SEQ ID NO: 3) of the anti-TIGIT antibody heavy chain, and linker (1) (SEQ ID NO: 18) , and a polynucleotide (SEQ ID NO: 22) and signal peptide (SEQ ID NO: 1) encoding the IgG4 Fc domain (SEQ ID NO: 4), the variable region (SEQ ID NO: 7) and the constant region (SEQ ID NO: 8) of the anti-TIGIT antibody light chain. The polynucleotide (SEQ ID NO: 35) encoding was synthesized through GenScript's Gene Synthesis service and loaded into the pcDNA3.4 vector.

Additionally, the vector was introduced into CHO cells (Expi-CHO ^™ , Thermo Fisher Scientific) to express the control anti-TIGIT antibody. After introducing the vector, the culture medium was cultured at 37°C, 125 rpm, 8% CO ₂ , and when the cell survival rate was 50%, the culture medium was collected and the control anti-TIGIT antibody was purified. Purification was performed in the same manner as Example 1.1.

Example 1.4. Manufacturing of GI-106B7NH06-CN

Signal peptide (SEQ ID NO: 1), encoding the variable region (SEQ ID NO: 2) and constant region (SEQ ID NO: 3) of the anti-TIGIT antibody heavy chain, linker (1) (SEQ ID NO: 18), and IgG4 FcM1 domain (SEQ ID NO: 23) A polynucleotide (SEQ ID NO: 36), a signal peptide (SEQ ID NO: 1), and a polynucleotide (SEQ ID NO: 38) encoding the variable region (SEQ ID NO: 7) and constant region (SEQ ID NO: 24) of the anti-TIGIT antibody light chain were used as BioXp. It was loaded into pCGS3 vector (Sigma-Aldrich®) using ^TM 3250 SYSTEM.

Additionally, the vector was introduced into CHO cells (Expi-CHO ^™ , Thermo Fisher Scientific) to express the fusion protein. After introducing the vector, it was cultured for 7 days at 37°C, 127 rpm, 5% CO ₂ , and 80% humidity. Afterwards, the culture medium was collected and the fusion protein was purified. The purified fusion protein was named “GI-106B7NH06K-CN”.

Example 1.5. Preparation of Fc-IL2v3

Signal peptide (SEQ ID NO: 1), linker (1) (SEQ ID NO: 18) and IgG4 FcM1 domain (SEQ ID NO: 23), linker (2) (SEQ ID NO: 5) and an IL-2 variant with three amino acids substituted (SEQ ID NO: 6) The polynucleotide containing (SEQ ID NO: 39) was loaded into the pCGS3 vector (Sigma-Aldrich®) using BioXP ^TM 3250 SYSTEM.

Additionally, the vector was introduced into CHO cells (Expi-CHO ^™ , Thermo Fisher Scientific) to express the fusion protein. After introducing the vector, it was cultured for 7 days at 37°C, 127 rpm, 5% CO ₂ , and 80% humidity. Afterwards, the culture medium was collected and the Fc-IL2v3 fusion protein was purified.

Example 2. In vitro environment ( in vitro ) confirmed the ability of GI-106 to inhibit TIGIT/CD155 binding

This experiment evaluated the effect of TIGIT inhibition upon treatment with GI-106 and control anti-TIGIT antibody in an in vitro environment. Specifically, the experiment was conducted using the TIGIT/CD155 blockade bioassay kit (cat. J2205, Promega). TIGIT effector cells and CD155 aAPC/CHO-K1 cells were used in this experiment, and the blocking of TIGIT/CD155 interaction and activation of the CD226 signaling pathway due to anti-TIGIT antibody treatment was measured by quantifying the degree of luminescence. (Figure 5).

TIGIT effector cells stored in liquid nitrogen were dissolved in a constant temperature water bath at 37°C for 3 minutes and then suspended in 0.5 mL of preheated 12 mL assay buffer (90% RPMI 1640 (Promega) + 10% FBS (Promega)). Afterwards, 80 μL of the suspension was added to each well in a 96-well-white cell culture plate (cat. 3917, Corning), and then incubated at 37°C and 5% CO ₂ conditions. It was cultured in an incubator for 20 hours.

After 20 hours, GI-106 and the control anti-TIGIT antibody were diluted using assay buffer, and 20 μL of test substances at various concentrations were added to each well. For the negative control, 20 μL of assay buffer was added. Afterwards, the 96-well plate was left at room temperature until aAPC/CHO-K1 cells expressing CD155 were dispensed.

CD155-expressing aAPC/CHO-K1 cells stored in liquid nitrogen were dissolved in a water bath at 37°C for 3 minutes, then suspended in 0.5 mL of preheated 3 mL assay buffer, followed by TIGIT effector cells and GI-106 or control antibody. 20 μL was dispensed into each well in a 96-well plate. For the negative control, 20 μL of assay buffer was added to make the final volume the same. Afterwards, it was cultured for 6 hours in an incubator under 37°C and 5% CO ₂ conditions.

After 6 hours of reaction, the 96-well plate was removed from the incubator and left at room temperature for 15 minutes. Afterwards, 120 μL of Bio-Glo™ reagent (Promega) pre-dissolved at room temperature was added to each well, and the same amount was also added to the edge wells for background signal correction. After reacting at room temperature for 10 minutes, the degree of luminescence was measured using a Glomax Luminometer (cat. GM3000, Promega), and the results were expressed in a RLU (Relative Light Unit) graph.

As a result, it was confirmed that GI-106 and anti-TIGIT antibodies bind to TIGIT expressed in TIGIT effector cells in a concentration-dependent manner, increasing the luminescence signal. This means that the anti-TIGIT antibody of GI-106 binds to TIGIT expressed on the surface of the TIGIT effector cell, thereby inhibiting the binding to CD155, and as a result, CD155 binds to CD226 to activate signal transduction and increase the luminescent signal ( Figure 6).

Example 3. Confirmation of JAK-STAT pathway activation ability induced by IL-2 variant of GI-106

This experiment is to confirm the activity of the IL-2 variant portion of GI-106. Specifically, the experiment was conducted using HEK-Blue ^TM IL-2 reporter cells (cat. hkb-il2, InvivoGen Inc.). HEK-Blue ^TM IL-2 reporter cells are induced to produce the reporter protein SEAP (secreted embryonic alkaline phosphatase) protein when the JAK-STAT pathway is activated by IL-2. In this experiment, the activation ability by IL-2 was confirmed by detecting SEAP protein.

HEK-Blue ^TM IL-2 reporter cells were incubated with 10% FBS (Gibco), 100 U/mL penicillin (Welgene Inc.), 100 μg/mL streptomycin (Welgene Inc.), and 100 μg/mL Normocin ^TM (cat. Ant. -nr-1, InvivoGen Inc.) was cultured in DMEM medium (Gibco) containing. HEK-Blue ^TM IL-2 reporter cells were subcultured to stabilize the cells, and then the cells were harvested using trypsin (Gibco). Afterwards, dead cells were removed by washing with PBS. The separated cells were created into a cell suspension in the culture medium so that the concentration was about 2.8Х10 ⁵ cells/mL.

GI-106, Aldesleukin (Product name: Proleukin®, Novartis, hereinafter referred to as Proleukin) as a positive control, and recombinant human TGF-β1 (cat. rcyc-htgfb1, InvivoGen Inc.) as a negative control were diluted using PBS and incubated at 96- 20 μL was dispensed into each well of a well-plate (cat. 30096, SPL). 180 μL of the cell suspension prepared in the 96-well plate containing the test substance was added to each well to obtain a cell count of approximately 5Х10 ⁴ and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours.

After 24 hours, the 96-well plate was removed from the incubator, centrifuged at 300Хg for 5 minutes, and 20 μL of the supernatant was transferred to a new 96-well plate. 180 μL of QUANTI-Blue ^TM solution (cat. Rep-qbs, InvivoGen Inc,) dissolved at room temperature was dispensed into each well containing the supernatant, and then reacted in an incubator at 37°C and 5% CO ₂ for 2 hours. . After the reaction, the absorbance was measured at a wavelength of 630 nm using a spectrophotometer (VersaMax ^TM Absorbance Microplate Reader).

As a result, it was confirmed that the absorbance increased in a concentration-dependent manner due to GI-106 and proleukin. Through this, it was confirmed that the JAK-STAT signaling pathway was activated by the IL-2 variant portion of GI-106 (Figure 7). The EC ₅₀ value of GI-106 was confirmed to be 13.13 ng/mL, and the EC ₅₀ value of the positive control proleukin was confirmed to be 0.766 ng/mL.

Example 4. Confirmation of anticancer effect by GI-106 administration in mice implanted with human-derived breast cancer cells

This experiment evaluated the tumor growth inhibition effect after administering the test substance GI-106 in a tumor model in which MDA-MB-231 (human breast cancer cells) cells were transplanted into mice with a human immune system.

To create a mouse model incorporating the human immune system, a suspension of human peripheral blood cells (StemExpress, LLC) was added to NSG-B2m female mice (7 weeks old, The jackson laboratory) at ^1Х107 cells/200 μL per mouse using a disposable syringe ( 31G, cat. 328820, BD medical diabetes care) and administered through the caudal vein. After cell transplantation, general symptoms were observed once daily.

MDA-MB-231 cells were purchased from the Korea Cell Line Bank (KCLB No. 30026) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). Cultured MDA-MB-231 cells were harvested using trypsin (Gibco) and suspended in PBS. To establish a xenograft mouse tumor model, healthy mice 5 days after human peripheral blood cell transplantation were incubated with MDA-MB-231 cell suspension (5Х10 ⁶ cells/0.05 mL) and 0.05 mL BD Matrigel matrix phenol red-free (cat. 356237). , BD Biosciences, USA) was filled into a disposable syringe (31G, cat. 328820, BD medical diabetes care) and administered 0.1 mL per animal subcutaneously to the right back of the animal. After MDA-MB-231 cell transplantation, general symptoms were observed once daily during the engraftment and growth period.

Approximately 20 days after transplanting MDA-MB-231 cells, the tumor volume was measured on mice with no abnormalities in the animal's health, and 12 individuals were selected so that the average of each group reached 40~80 mm ³ . Subjects were selected considering their physiological status (respiration, fur, behavior, tail, posture, body fluids, diet, deformation, metabolism, etc.), weight change, FACS analysis results, and tumor growth rate. The selected animals were divided into 6 animals per group to be as equal as possible based on tumor volume and body weight. As shown in Table 3, the test group was formed and the test substance was administered.

army	administered substance	Route of administration	Dosing cycle	Dosage	population
G1	Vehicle (PBS)	i.v.	BIW (2 times/week)	-mg/kg	6
G2	GI-106	i.v.	BIW (2 times/week)	6mg/kg	6

During the 25-day test period after administration of the test substance, general symptoms such as appearance, behavior, and excrement were observed and recorded for each individual once a day, and dead animals were identified. Tumor volume was measured twice a week during the observation period using a caliper (digital caliper, mitutoyo) to measure the long axis (L, maximum length) and short axis (W, perpendicular width) of the tumor, and substitute them into Equation I below. Tumor volume (TV) and tumor growth inhibition (TGI) were measured. <Equation I>

TV(mm ³ )=(W ² ΥL)/2

TGI=(1-(Ti-T0)/(Vi-V0))Х100

Ti: Tumor volume of the test substance administered group at the end of the test

T0: Tumor volume of the test substance administered group at the time of group separation

Vi: Tumor volume of the negative control group at the end of the test

V0: Tumor volume of the negative control group at the time of group separation

The tumor volume before administration of each subject was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated by comparison with the control group (vehicle (PBS), G1).

All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc.). Comparison of tumor volume measurements was made using two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant.

As a result, tumor growth in the GI-106 administered group was inhibited compared to the control group (vehicle (PBS)) (FIGS. 8 to 11). Looking at the tumor growth inhibition rate, there was only one mouse in the PBS-treated group with a tumor growth inhibition rate of more than 30%, and there were no mice with tumor growth inhibition rates of more than 50% or more than 80%. In the GI-106 administration group, there were 5 mice with a tumor growth inhibition rate of more than 30%, 3 mice with a tumor growth inhibition rate of 50% or more, and 2 mice with a tumor growth inhibition rate of 80% or more (Figure 12).

Example 5. Confirmation of anti-cancer effect by GI-106B7NH06Kv3 administration in mouse-derived breast cancer cell implantation mice

This experiment evaluated the tumor growth inhibition effect after administering the test substance GI-106B7NH06Kv3 in a BALB/c mouse tumor model transplanted with EMT-6 cells, a mouse breast cancer cell line derived from BALB/c mice. .

To create a mouse model transplanted with a cancer cell line, a suspension of EMT-6 cells, a mouse-derived breast cancer cell, was administered to BALB/c female mice (7 weeks old, Orient Bio) at a rate of ^2Х104 cells/40 μL per mouse using a disposable syringe (31G, cat. 328820, BD) was used to open the left side of the animal's abdomen and administer it into the mammalian fat pad.

Specifically, EMT-6 cells were purchased from Orient Bio and cultured in RPMI1640 medium (A1049101, Thermo Fisher Scientific) containing 10% FBS (16000-044, Thermo Fisher Scientific) and 1% antibiotic/antimycotic (15140122, Thermo Fisher Scientific). It was cultured in . Cultured EMT-6 cells were harvested using trypsin-EDTA (Cat. 25200-072, Thermo Fisher Scientific) and then suspended in PBS (Cat. LB 001-04, Welgene Inc.). EMT-6 cell suspension (2Х10 ⁶ cells/0.4 mL) for transplantation of 10 animals was filled into a disposable syringe (31G, cat. 328820, BD medical diabetes care), opened on the left side of the animal's abdomen, and administered into the mammalian fat body. . After cell transplantation, general symptoms were observed once daily.

Approximately 15 days after transplantation of EMT-6 cells, the tumor volume was measured in mice with no abnormalities in the animal's health, and 20 individuals were selected so that the average of each group reached 70~72 mm ³ . Individuals were selected considering their physiological state (respiration, fur, behavior, tail, posture, body fluids, diet, shape deformation, metabolism, etc.), weight change, and tumor growth rate. The selected animals were separated into groups of 10 as evenly as possible based on tumor volume and body weight. As shown in Table 4, the test group was formed and the test substance was administered.

army	administered substance	Route of administration	Dosing cycle	Dosage	Number of individuals (n)
G1	Vehicle (hIgG4)	i.v.	QW (1 time/1 week)	9mg/kg	10
G2	GI-106B7NH06Kv3	i.v.	QW (1 time/1 week)	9mg/kg	10

During the 15-day test period after administration of the test substance, general symptoms such as appearance, behavior, and excrement were observed and recorded for each individual once a day, and dead animals were identified. Tumor volume was measured three times a week during the observation period using calipers to measure the long and short axes of the tumor, and the tumor volume and tumor growth inhibition rate were measured by substituting equation I in Example 4. The tumor volume before administration of each subject was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated by comparison with the control group (vehicle (hIgG4), G1). All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc.). Comparison of tumor volume measurements was done using unpaired t-test. A p value of less than 0.05 was considered significant.

As a result, it was confirmed that the tumor volume of the GI-106B7NH06Kv3 administration group was significantly reduced by about 4 times compared to the control group (vehicle (hIgG4)) (FIG. 13).

In addition, looking at the tumor growth inhibition rate, it was confirmed that the tumor growth of the GI-106B7NH06Kv3 administration group was inhibited compared to the control group (vehicle (hIgG4)). Specifically, in the negative control group, there were 4 mice with a tumor growth inhibition rate of more than 30%, of which 2 mice showed a tumor growth inhibition rate of more than 50%, and among the two mice, one mouse showed a tumor growth inhibition rate of more than 80%. There was 1 animal.

On the other hand, in the GI-106NH06Kv3 administration group, there were 9 mice with a tumor growth inhibition rate of more than 30%, and among them, 7 mice showed a tumor growth inhibition rate of more than 50%, and among the 7 mice, one showed a tumor growth inhibition rate of more than 80%. There were 6 mice. (Figure 14).

Example 6. Confirmation of anti-cancer effect by GI-106B7NH06Kv3 administration in mouse-derived colon cancer cell implantation mice

This experiment evaluated the tumor growth inhibition effect after administering the test substance GI-106B7NH06Kv3 in a tumor model transplanted with MC38 cells, a murine colon cancer cell line derived from C57BL/6 mice.

To create a mouse model transplanted with a cancer cell line, a suspension of MC38 cells, mouse-derived colon cancer cells, was administered to C57BL/6 female mice (6 weeks old, Orient Bio) at 5Х10 ⁵ cells/50 μL using a disposable syringe (31G, cat. 328820, It was filled with BD medical diabetes care and administered subcutaneously to the right back of the animal. After cell transplantation, general symptoms were observed once daily.

MC38 cells were purchased from Applied Stem Cell Inc. and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antimycotic (Gibco). Cultured MC38 cells were harvested using trypsin (Gibco) and suspended in PBS. To establish an allograft mouse tumor model, a solution prepared by mixing MC38 cell suspension (5Х10 ⁵ cells/0.025 mL) and 0.025 mL BD Matrigel matrix phenol red-free (cat. 356231, BD biosciences) was injected into healthy mice using a disposable syringe ( 31G, cat. 328820, BD medical diabetes care) and implanted by administering 0.05 mL per animal subcutaneously on the right back of the animal. After MC38 cell transplantation, general symptoms were observed once daily during the engraftment and growth period.

Approximately 8 days after transplantation of MC38 cells, the tumor volume was measured in mice with no abnormalities in the animal's health, and 40 individuals were selected so that the average of each group reached 60~120 mm ³ . Individuals were selected considering their physiological state (respiration, fur, behavior, tail, posture, body fluids, diet, shape deformation, metabolism, etc.), weight change, and tumor growth rate. The selected animals were separated into groups of 10 as evenly as possible based on tumor volume and body weight. As shown in Table 5, the test group was formed and the test substance was administered.

army	administered substance	Route of administration	Dosing cycle	Dosage	Number of individuals (n)
G1	Vehicle (hIgG4)	i.v.	Q2W (1 time/2 weeks)	9mg/kg	10
G2	GI-106B7NH06Kv3	i.v.	Q2W (1 time/2 weeks)	9mg/kg	10
G3	GI-106B7NH06K-CN	i.v.	Q2W (1 time/2 weeks)	9mg/kg	10
G4	GI-106B7NH06Kv3-CN+Fc-IL2v3	i.v.	Q2W (1 time/2 weeks)	9 mg/kg+3.9 mg/kg	10

During the 24-day test period after administration of the test substance, general symptoms such as appearance, behavior, and excrement were observed and recorded for each individual once a day, and dead animals were identified. Tumor volume was measured three times a week during the observation period, using calipers to measure the long and short axes of the tumor, and the tumor volume (TV) and tumor growth inhibition rate (TGI) were measured by substituting Equation I in Example 4. did. The tumor volume before administration of each subject was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated by comparison with the control group (vehicle (hIgG4), G1). All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc.). Comparison of tumor volume measurements was done using unpaired t-test. A p value of less than 0.05 was considered significant. As a result, it was confirmed that the tumor volume of the GI-106B7NH06Kv3 administration group was significantly reduced by about 3 times compared to the control group (vehicle (hIgG4)). In addition, it was confirmed that the tumor volume of the GI-106B7NH06Kv3 administration group was significantly reduced by about 2.5 times compared to the GI-106NH06K-CN administration group and the GI-106NH06K-CN and Fc-IL2v3 combination administration group (FIG. 15).

Additionally, looking at the tumor growth inhibition rate, there were no mice in the control group (vehicle (hIgG4)) with a tumor growth inhibition rate of more than 30%. In the GI-106NH06K-CN administration group, there were 3 mice with a tumor growth inhibition rate of more than 30%, and among them, 2 mice showed a tumor growth inhibition rate of more than 50%, and among the two mice, one showed a tumor growth inhibition rate of more than 80%. There was only one mouse. Additionally, in the GI-106NH06K-CN and Fc-IL2v3 administration groups, there were no mice with a tumor growth inhibition rate of more than 30%. On the other hand, all groups administered GI-106B7NH06Kv3 showed a tumor growth inhibition rate of more than 30%, of which 6 mice showed a tumor growth inhibition rate of more than 50%, and among the 6 mice, 3 mice showed a tumor growth inhibition rate of more than 80%. It was Marie (Figure 16).

Furthermore, as a result of checking the survival rate of mice in each group, in the control group (vehicle), 7 mice died on the 15th day, and only 3 mice survived until the 25th day, showing a survival rate of 30%. In the case of the GI-106NH06K-CN administration group, 2 mice began to die on the 15th day, and 1 more mouse each died on the 18th, 20th, and 22nd days, and 5 mice survived until the 25th day, resulting in a 50% survival rate. indicated. In the case of the GI-106NH06K-CN and Fc-IL2v3 administration group, mice began to die from day 6 and all mice died on day 22. On the other hand, in the GI-106B7NH06Kv3 administration group, all mice survived until day 25, showing a survival rate of 100% (Figure 17).

Claims (19)

1. An antibody or fragment thereof that specifically binds to TIGIT; and a bispecific antibody containing the IL-2 protein.
2. According to paragraph 1,

   Antibodies or fragments thereof that specifically bind to the TIGIT

   A heavy chain variable region consisting of HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and

   A bispecific antibody comprising a light chain variable region consisting of LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 13, and LCDR3 containing the amino acid sequence of SEQ ID NO: 14.
3. According to paragraph 1,

   The antibody or fragment thereof that specifically binds to TIGIT is a bispecific antibody consisting of a heavy chain variable region including the amino acid sequence of SEQ ID NO: 2 and a light chain variable region including the amino acid sequence of SEQ ID NO: 7.
4. According to paragraph 1,

   A bispecific antibody, wherein the IL-2 protein is an IL-2 variant.
5. According to paragraph 4,

   The IL-2 variant is a bispecific antibody in which amino acids at positions 38, 42, and 61 in the amino acid sequence of SEQ ID NO: 17 are substituted.
6. According to clause 5,

   The IL-2 variant is a bispecific antibody in which R38A, F42A and E61R substitutions occurred in the amino acid sequence of SEQ ID NO: 17.
7. According to paragraph 1,

   An antibody or fragment thereof that specifically binds to the TIGIT; and the IL-2 protein is linked by a linker.
8. According to paragraph 1,

   The bispecific antibody comprises the following structural formulas (I) and (II):

   N'-X-[Linker (1)]o-Fc region fragment or variant thereof-[Linker (2)]p-Y-C' (I) and

   N'-X'-C' (II)

   At this time, in the structural formulas (I) and (II),

   Wherein N' is the N terminus,

   Wherein C' is the C terminus,

   The X is the antigen binding site of the heavy chain of the anti-TIGIT antibody and includes a variable region (VH) and a CH1 region,

   The X' is the light chain antigen binding site of the anti-TIGIT antibody and includes a variable region (VL) and a constant region (CL),

   Y is IL-2 protein or a variant thereof,

   The linker (1) and linker (2) are peptide linkers,

   The o and p are each independently O or 1.
9. According to paragraph 1,

   The bispecific antibody comprises the following structural formulas (III) and (IV):

   N'-X-[Linker (1)]o-Fc region fragment or variant thereof-C' (III) and

   N'-X'-[Linker (3)]q-Y-C' (IV)

   At this time, in the structural formulas (III) and (IV),

   Wherein N' is the N terminus,

   Wherein C' is the C terminus,

   The X is the antigen binding site of the heavy chain of the anti-TIGIT antibody and includes a variable region (VH) and a CH1 region,

   The X' is the light chain antigen binding site of the anti-TIGIT antibody and includes a variable region (VL) and a constant region (CL),

   Y is IL-2 protein or a variant thereof,

   The linker (1) and linker (3) are peptide linkers,

   The o and q are each independently O or 1.
10. According to clause 8 or 9,

    The Fc is a dual specific antibody comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 23.
11. A polynucleotide encoding the bispecific antibody of any one of claims 1 to 10.
12. An expression vector comprising the polynucleotide of claim 11.
13. A transformed cell into which the expression vector of claim 12 has been introduced.
14. i) culturing the transformed cells of claim 13; and

    ii) obtaining a bispecific antibody; an anti-TIGIT antibody or fragment thereof comprising; and a method for producing a bispecific antibody comprising IL-2 protein.
15. A pharmaceutical composition for preventing or treating cancer comprising the bispecific antibody of claim 1 as an active ingredient.
16. According to clause 15,

    The cancer consists of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreas cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. A pharmaceutical composition for preventing or treating cancer, which is selected from the group.
17. Use of the bispecific antibody of claim 1 to prevent or treat cancer.
18. Use of the bispecific antibody of claim 1 for manufacturing a drug for preventing or treating cancer.
19. A method for preventing or treating cancer comprising administering the bispecific antibody of claim 1 to a subject.

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