TW202444743A - Variant of interferon alpha receptor, fusion protein including the same and use thereof - Google Patents

Abstract

The present disclosure relates to an immunocytokine including an interferon protein and an interferon alpha receptor, and a pharmaceutical composition including the immunocytokine as an active ingredient. An immunocytokine according to the present disclosure including an interferon protein or a fragment thereof; an extracellular domain (ECD) fragment of an interferon alpha receptor or a variant thereof; and an antigen binding protein specific to a target protein used the extracellular domain fragment of the interferon alpha receptor or the variant thereof having a reduced binding affinity to an interferon alpha, as a masking moiety. The immunocytokine reduces an off target effect in the body and maintains an efficacy of the interferon alpha, so it is expected to be used as an anticancer agent.

Description

Variants of interferon alpha receptor, fusion proteins thereof and uses thereof

Invention Field

The present disclosure is a novel masking part of interferon α and is related to a variant of interferon α receptor. In addition, the present disclosure is related to an immunocytokine comprising an interferon protein and an interferon α receptor, and a pharmaceutical composition comprising the immunocytokine as an active ingredient.

Invention Background

Multiple myeloma is a blood disease in which malignant plasma cells proliferate abnormally in the bone marrow. Specifically, the disease is characterized by bone invasion and is a difficult-to-treat blood cancer that causes immune, hematopoietic, and renal diseases, accounting for 1.8% of all cancer cases. There are many new drugs on the market to treat these diseases, but these drugs are also known to have many side effects. The biggest problem is that even after treatment, relapses are frequent, and many patients show resistance to existing treatments after relapse.

Interferon alpha (IFNα) is an interferon that regulates the expression of various genes that regulate cancer cell growth, proliferation, death, and immunosuppression. Interferon alpha has been approved by the FDA for the treatment of several cancers, including melanoma, B-cell lymphoma, multiple myeloma, and chronic myeloid leukemia, with interferon alpha 2b (IFNα2b) used to treat multiple myeloma (Xiong et al., Biomark Res. 10:69 (2022)). However, because the interferon alpha receptor (IFNAR) is widely distributed in all cells, including cells in tumor tissue, it exhibits off-target effects of interferon binding to other cells in the body. This phenomenon not only limits the concentration of drug that can be administered but also leads to side effects such as suicidal impulses. Therefore, to overcome this problem, multiple strategies are needed to increase the delivery of interferon α to tumor sites in order to improve efficacy while reducing toxicity.

In general, one of the many ways to reduce the toxicity of cytokines in cancer treatment is to make cytokines act in a cell-specific manner using antigen binding proteins that target and bind to membrane proteins specifically expressed on the surface of cancer. Using substances with such characteristics is a way to maintain the anti-tumor activity of cytokines while minimizing systemic toxicity. Currently, several substances are in the preclinical and clinical stages, but more different substances need to be studied and developed.

Summary of the invention Technical problem

Therefore, when studying a method to minimize systemic toxicity while maintaining the anti-tumor activity of interferon, the inventors developed an immunocytokine, which includes an antigen binding protein, interferon α and interferon α receptor variants, and confirmed that the immunocytokine has an excellent anti-cancer effect, thereby completing the present disclosure. Solution to the problem

To achieve the above objectives, one aspect of the present disclosure provides an immunocytokine comprising an interferon (IFN) protein or a fragment thereof; an extracellular domain (ECD) fragment of an interferon α receptor or a variant thereof; and an antigen binding protein specific to a target protein.

Another aspect of the present disclosure provides a fusion protein comprising an interferon α protein or a fragment thereof; and an extracellular domain fragment of an interferon α receptor or a variant thereof.

Another aspect of the present disclosure provides a polynucleotide encoding an immunocytokine or a fusion protein, an expression vector carrying the polynucleotide, and a cell transformed by the expression vector.

Another aspect of the present disclosure provides a method for producing immunocytokine or fusion protein, which comprises culturing transformed cells and obtaining immunocytokine or fusion protein from a culture medium.

Another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating cancer, which comprises an immunocytokine or fusion protein as an active ingredient.

Another aspect of the present disclosure provides an extracellular domain fragment of interferon α receptor 2 or a variant thereof.

Another aspect of the present disclosure provides the use of immunocytokine or fusion protein for preventing or treating cancer.

Another aspect of the present disclosure provides a method for preventing or treating cancer, which comprises administering immunocytokine and fusion protein to an individual. Effects of the Invention

The immunocytokine according to the present disclosure includes: an interferon protein or a fragment thereof; an extracellular domain (ECD) fragment of an interferon α receptor or a variant thereof; and an antigen binding protein specific to a target protein, wherein the immunocytokine uses the extracellular domain fragment of the interferon α receptor or a variant thereof as a masking portion, and the variant has a reduced binding affinity to interferon α. The immunocytokine reduces off-target effects in vivo and maintains the efficacy of the interferon α itself, and is therefore expected to be used as an anticancer agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definition of Terms Interferon Protein

As used herein, the term "interferon (IFN)" is one of the interferon family with antiviral, antiproliferative and immunomodulatory activities. Human interferons are divided into type 1 (type I) and type 2 (type II) according to the type of receptor they bind to and their mechanism of action. Type 1 is mainly composed of IFN α (alpha) and β (beta), and τ (tau) is also present. Type 1 interferons are composed of a single chain of amino acids and bind to type 1 receptors. Type 2 interferons include interferon γ (gamma), and the two identical proteins form dimers (homodimers) and bind to type 2 receptors to activate intracellular signals.

Interferons (IFNs) may be type 1 (type I) interferons, and as examples, type 1 interferons may be IFN α (alpha) or IFN β (beta). All type 1 interferons bind to a specific cell surface receptor complex, the IFN-α/β receptor (IFNAR). In general, type 1 interferons are produced by viral invasion and are primarily produced by fibroblasts and monocytes. Once secreted, type 1 interferons activate various molecules that prevent viral nucleic acid replication.

The interferon protein of the present disclosure may be a wild-type protein, or may be a variant, a recombinant protein or a fragment thereof that exhibits the same physiological activity as the wild-type interferon protein.

Interferon alpha is a substance secreted by peripheral blood leukocytes or lymphoblasts when exposed to viruses, double-stranded RNA or bacteria, and not only has antiviral effects but also activates natural killer cells. In addition, it is known to regulate the expression of various genes that regulate the growth, proliferation, death and immunosuppression of cancer cells. Therefore, it can be used as an anticancer agent. Interferon alpha has 24 subtypes due to its antigenicity and structural characteristics. Among them, the one with the greatest antiviral effect is interferon alpha 2. Interferon alpha 2a was cloned in myeloid blast cell lines, interferon alpha 2b was cloned in lymphocytes, and interferon alpha 2c was cloned in cancer cells. Among them, interferon alpha 2b is known to constitute the vast majority of human interferons.

In the present disclosure, the interferon α protein may be interferon α2a or interferon α2b. Specifically, the interferon α protein may be interferon α2b. Interferon α2b is a glycoprotein composed of 166 amino acids, wherein the interferon α2b protein may be a wild type, or may be a variant thereof and is preferably a wild type. More specifically, the interferon α2b of the present disclosure may include the amino acid sequence of SEQ ID NO: 2.

In addition, the interferon alpha protein may be composed of a sequence in which one or more amino acids in the protein are added, deleted or substituted, as long as the interferon alpha protein has the same activity as the aforementioned interferon alpha protein or has the same position on the chromosome as the gene encoding interferon alpha. Specifically, the interferon alpha protein may include or consist of an amino acid sequence that is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical or 100% identical to the amino acid sequence of SEQ ID NO: 2.

In addition, in the present disclosure, interferon α protein may include fragments thereof. Interferon α protein fragments refer to fragments in which the N-terminus, C-terminus, or a portion of the N-terminus and/or C-terminus of the wild-type interferon α protein is truncated, and the fragments can exhibit the same physiological activity as the wild-type interferon α protein.

Specifically, the interferon alpha protein fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid sequences are continuously truncated from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 2. In addition, the interferon alpha protein fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid sequences are continuously truncated from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 2. The interferon α protein fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid sequences are continuously truncated from each of the N-terminus and the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 2.

Interferon beta is a protein encoded by the IFNB1 gene and secreted primarily by fibroblasts. Interferon beta balances the expression of pro-inflammatory and anti-inflammatory agents in the brain and reduces the number of inflammatory cells that cross the blood-brain barrier. Interferon beta acts as an immunosuppressant, increasing the activity of inhibitory lymphocytes and inhibiting the activation of other immune cells. Interferon beta is primarily used to treat multiple sclerosis, and two types of interferon beta (interferon beta 1a and interferon beta 1b) can be used to treat multiple sclerosis. In addition to inhibiting inflammation that can damage the myelin sheath, interferon beta 1b can also regulate the production of gamma interferon. Interferon beta (IFN β) is more effective than interferon alpha (IFN α) in inhibiting cell growth. In particular, when used in combination with anticancer agents, synergistic effects can be expected in terms of the range of antiproliferative activity of interferon beta and immune enhancement.

However, therapeutic agents using interferons are known to have high cytotoxicity in treated patients, causing symptoms such as fever (80%), muscle pain (73%), headache (50%), and fatigue (50%). In addition, interferon β is a highly hydrophobic protein and tends to aggregate easily, which reduces biological activity and productivity and has a short half-life. Therefore, in order to develop therapeutic agents using interferons, it is necessary to minimize the limitations caused by interferons.

Information on interferon β can be obtained from known databases, such as Genbank of the National Center for Biotechnology Information (NCBI), and interferon β may have a sequence disclosed in, for example, GenBank: KAI4006768.1; NCBI reference sequence: NP_002167.1; or UniProt reference number P01574.

Interferon β may include or consist of a wild-type amino acid sequence or an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity to the wild-type amino acid sequence. The sequence of wild-type interferon β can be easily confirmed from sequence information and databases known in the art of the present disclosure. In one example, interferon β may have a sequence disclosed in GenBank: KAI4006768.1; NCBI reference sequence: NP_002167.1; or UniProt reference number P01574, or an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the sequence, but is not limited thereto. In another example, interferon β1a may be a sequence disclosed in Drugbank accession number DB00060, or an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the sequence, but is not limited thereto. In another example, interferon β1b may have the sequence disclosed in Drugbank Accession No. DB00068, or an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity, or 100% identity to the sequence, but is not limited thereto.

In addition, in the present disclosure, interferon β protein may include its fragments. Interferon β protein fragments refer to fragments in which the N-terminus, C-terminus, or a portion of the N-terminus and/or C-terminus of the wild-type interferon β protein is truncated, and the fragments can exhibit the same physiological activity as the wild-type interferon β protein.

Specifically, the interferon β protein fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid sequences are continuously truncated from the N-terminus of the protein having the amino acid sequence disclosed in GenBank: KAI4006768.1; NCBI reference sequence: NP_002167.1; or UniProt reference number P01574. In addition, the interferon β1a protein fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid sequences are continuously truncated from the C-terminus of the protein having the amino acid sequence disclosed in Drugbank Accession No. DB00060. In addition, the interferon β1b protein fragment may be in one example in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid sequences are continuously truncated from each of the N-terminus and the C-terminus of the protein having the amino acid sequence disclosed in Drugbank Accession No. DB00068. Interferon α receptor fragment or variant thereof

As used herein, the term "interferon α receptor (interferon α/β receptor, IFNAR or IFNαR)" refers to the cell membrane receptor to which type 1 interferons bind. When type 1 interferons bind to IFNAR, the MAPK, PI3K, Akt and JAK-STAT pathways, which are downstream signal messengers of the receptor, are activated to regulate cell death, autophagy, cell differentiation and proliferation.

Interferon alpha receptors are composed of interferon alpha receptor 1 (IFNAR1) (which is a low affinity subunit) and interferon alpha receptor 2 (IFNAR2) (a high affinity subunit), each of which includes a fiber binding protein type III (FNIII)-like subdomain in the extracellular domain (ECD). Interferon alpha receptor 1 includes four subdomains called SD1 to SD4, and exhibits a binding affinity of about 0.5 μM to about 5 μM for type 1 interferons. Here, it is known that the SD1 to SD3 subdomains are involved in binding to type 1 interferons. On the other hand, interferon alpha receptor 2 includes subdomains called D1 and D2, and exhibits a binding affinity of about 0.4 nM to about 5 nM.

It is known that interferon α or β first binds to interferon α receptor 2, which is a high affinity subunit, and then binds to interferon α receptor 1 to transmit signals into cells. This means that interferon α or β cannot function properly without binding to interferon α receptor 2. Therefore, focusing on these characteristics of interferon α or β, the present disclosure attempts to prevent interferon α or β from acting on cells other than target cells in the bloodstream by using an extracellular domain fragment of interferon α receptor or a variant thereof as a masking moiety, the binding affinity of which is weakened by inducing changes in the extracellular domain of interferon α receptor 2.

In the present disclosure, the interferon alpha receptor may be interferon alpha receptor 1 (IFNAR1) or interferon alpha receptor 2 (IFNAR2). Here, interferon alpha receptor 1 may include the amino acid sequence of SEQ ID NO: 4, and interferon alpha receptor 2 may include the amino acid sequence of SEQ ID NO: 5. In the present disclosure, the extracellular domain fragment of the interferon alpha receptor or a variant thereof may be an extracellular domain fragment of the interferon alpha receptor 2 or a variant thereof.

Specifically, in the present disclosure, the extracellular domain fragment of the interferon α receptor or its variant may include a polypeptide represented by the following structural formula (XV). [N-terminal extension domain] ab-core domain (XV) In the structural formula (XV),

The core domain may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, and the N-terminal extension domain may be a polypeptide comprising 1 to 39 consecutive amino acids starting from position 39 of the amino acid sequence of SEQ ID NO: 9 in the C-terminal to N-terminal direction, and ab may be 0 or 1.

As used herein, the term "core domain" refers to a polypeptide comprising a fragment of the D1 subdomain in the extracellular domain of interferon α receptor 2, which includes the binding site to which interferon α receptor and interferons bind. In one example, the "core domain" may include the site to which interferon α receptor 2 and interferon α2b bind.

The core domain refers to a polypeptide including the amino acid sequence from position 40 to position 106 in the full length of the extracellular domain of interferon α receptor 2. Here, the full length of the extracellular domain of interferon α receptor 2 may include the amino acid sequence of SEQ ID NO: 5, and the D1 subdomain of interferon α receptor 2 may include the amino acid sequence of SEQ ID NO: 6. Specifically, in the present disclosure, the core domain may include the amino acid sequence of SEQ ID NO: 8.

The core domain may be wild type.

Additionally, the core domain may include variants.

As used herein, the term "wild-type" refers to a protein or a nucleic acid encoding the protein as found in nature.

As used herein, the term "variant" refers to a form in which a portion of an amino acid or base sequence is inserted, substituted or deleted when compared to a wild type. In the present disclosure, a protein including a variant may be referred to as a variant.

Therefore, in the present disclosure, a variant of an extracellular domain fragment of an interferon receptor may have an amino acid sequence different from the amino acid sequence of the wild-type. In addition, the variant may have a lower interferon α binding affinity than the wild-type extracellular domain fragment of an interferon receptor.

As used herein, the term "binding affinity" or "binding capacity" refers to the aggregate strength of the non-covalent interaction between a single binding site of a molecule and its binding partner. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant ( _KD ), which is the ratio of the dissociation rate constant and the association rate constant ( _koff and _kon , respectively).

Here, "interferon α binding ability" refers to, for example, the ability of an interferon protein or a fragment thereof to bind to an interferon α receptor, wherein the binding can be measured by methods known to those skilled in the art. In one embodiment, it can be measured by surface plasmon resonance (SPR).

Specifically, the variant of the extracellular domain fragment of the interferon receptor may have a dissociation constant (K _{D ) of ≥1 μM, ≥150 nM, ≥100 nM, ≥50 nM, ≥10 nM or ≥5 nM relative to the interferon protein. In one example, the variant of the extracellular domain fragment of the interferon receptor may have a dissociation constant (K D )} of ≥1 μM, ≥150 nM, ≥100 nM, ≥50 nM, ≥10 nM or ≥5 nM relative to the interferon α2b protein.

In the present disclosure, the change of the core domain can be any amino acid selected from the group consisting of positions 7 to 12, 14, 21, 32, 33, 38 to 40, 42, 44, 60, 62, 64, 65 and combinations thereof in the amino acid sequence of SEQ ID NO: 8, which correspond to positions 46 to 51, 53, 60, 71, 72, 77 to 79, 81, 83, 99, 101, 103 and 104 in the amino acid sequence of the full-length extracellular domain of interferon alpha receptor 2 (SEQ ID NO: 5), respectively.

In the present disclosure, the variation of the core domain may be any amino acid selected from the group consisting of positions 7 to 10, 14, 38 to 40, 42, 62, 65 and combinations thereof in the amino acid sequence of SEQ ID NO: 8, which correspond to positions 46 to 49, 53, 77 to 79, 81, 101 and 104 in the amino acid sequence of the full-length extracellular domain of interferon alpha receptor 2 (SEQ ID NO: 5), respectively.

In the present disclosure, the variation of the core domain may be substitution of at least one amino acid among the amino acid residues at positions 7 to 10, 14, 38 to 40, 42, 62 and 65 of the amino acid sequence of SEQ ID NO: 8 with another amino acid.

In one embodiment, the variation of the core domain may be a substitution of at least one of positions 7 to 10, 14, 38 to 40, 42, 62 and 65 of the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the variation of the core domain may be a substitution of the amino acid at two of positions 7 to 10, 14, 38 to 40, 42, 62 and 65 in the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the variation of the core domain may be a substitution of amino acids at three positions among positions 7 to 10, 14, 38 to 40, 42, 62 and 65 in the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the variation of the core domain may be a substitution of amino acids at four positions among positions 7 to 10, 14, 38 to 40, 42, 62 and 65 in the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the variation of the core domain may be a substitution of amino acids at five positions among positions 7 to 10, 14, 38 to 40, 42, 62 and 65 in the amino acid sequence of SEQ ID NO: 8.

Here, the "amino acid" introduced by substitution may be any one selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, glutamine, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine and proline.

Specifically, regarding the amino acid substitution of the core domain variant, the isoleucine as the amino acid at position 7 and 65 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than isoleucine. Specifically, regarding the amino acid substitution of the core domain variant, the isoleucine as the amino acid at position 7 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (I7A), aspartic acid (I7D), glutamine (I7E), phenylalanine (I7F), glycine (I7G), asparagine (I7N), serine (I7S), tryptophan (I7W) or tyrosine (I7Y). Isoleucine as the amino acid at position 65 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (I65A), glutamine (I65E), glycine (I65G), lysine (I65K), leucine (I65L), methionine (I65M), proline (I65P), glutamine (I65Q), arginine (I65R), serine (I65S) or tyrosine (I65T).

Methionine as the amino acid at position 65 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than methionine. Specifically, with respect to the amino acid substitution of the core domain variant, methionine as the amino acid at position 8 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (M8A), aspartic acid (M8D), glutamine (M8E), glycine (M8G), histidine (M8H), isoleucine (M8I), leucine (M8L), asparagine (M8N), proline (M8P), serine (M8S), threonine (M8T), valine (M8V), tryptophan (M8W) or tyrosine (M8Y).

Serine as the amino acid at position 9 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than serine. Specifically, with respect to the amino acid substitution of the core domain variant, serine as the amino acid at position 9 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (S9A), aspartic acid (S9D), glutamine (S9E), glycine (S9G), leucine (S9L), proline (S9P), valine (S9V) or tryptophan (S9W).

Lysine as the amino acid at position 10 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than lysine. Specifically, with respect to the amino acid substitution of the core domain variant, the lysine as the amino acid at position 10 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (K10A), aspartic acid (K10D), glutamine (K10E), phenylalanine (K10F), glycine (K10G), histidine (K10H), isoleucine (K10I), leucine (K10L), methionine (K10M), asparagine (K10N), proline (K10P), glutamine (K10Q), arginine (K10R), serine (K10S), threonine (K10T), valine (K10V), tryptophan (K10W) or tyrosine (K10Y).

Leucine, which is the amino acid at position 14 of the amino acid sequence of SEQ ID NO: 8, may be substituted with an amino acid other than leucine. Specifically, with respect to the amino acid substitution of the core domain variant, leucine, which is the amino acid at position 14 of the amino acid sequence of SEQ ID NO: 8, may be substituted with alanine (L14A), aspartic acid (L14D), glutamine (L14E), glycine (L14G), lysine (L14K), proline (L14P), glutamine (L14Q) or tyrosine (L14Y).

The histidine as the amino acid at position 38 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than histidine. Specifically, with respect to the amino acid substitution of the core domain variant, histidine as the amino acid at position 38 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (H38A), aspartic acid (H38D), glutamine (H38E), phenylalanine (H38F), glycine (H38G), isoleucine (H38I), lysine (H38K), leucine (H38L), methionine (H38M), asparagine (H38N), proline (H38P), glutamine (H38Q), arginine (H38R), serine (H38S), tyrosine (H38T), valine (H38V), tryptophan (H38W) or tyrosine (H38Y).

Glutamine as the amino acid at position 39 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than glutamine. Specifically, with regard to the amino acid substitution of the core domain variant, glutamine as the amino acid at position 39 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (E39A), aspartic acid (E39D), phenylalanine (E39F), glycine (E39G), histidine (E39H), lysine (E39K), proline (E39P), serine (E39S), tryptophan (E39W) or tyrosine (E39Y).

Alanine, which is the amino acid at position 40 of the amino acid sequence of SEQ ID NO: 8, may be substituted with an amino acid other than alanine. Specifically, with respect to the amino acid substitution of the core domain variant, alanine, which is the amino acid at position 40 of the amino acid sequence of SEQ ID NO: 8, may be substituted with arginine (A40R), aspartic acid (A40D), or glutamine (A40E).

The valine as the amino acid at position 42 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than valine. Specifically, with respect to the amino acid substitution of the core domain variant, valine, which is the amino acid at position 42 of the amino acid sequence of SEQ ID NO: 8, may be substituted with alanine (V42A), aspartic acid (V42D), glutamine (V42E), phenylalanine (V42F), glycine (V42G), histidine (V42H), isoleucine (V42I), lysine (V42K), methionine (V42M), asparagine (V42N), proline (V42P), glutamine (V42Q), arginine (V42R), serine (V37S), tyrosine (V42T), tryptophan (V42W) or tyrosine (V42Y).

Tryptophan as the amino acid at position 62 of the amino acid sequence of SEQ ID NO: 8 may be substituted with an amino acid other than tryptophan. Specifically, with respect to the amino acid substitution of the core domain variant, tryptophan as the amino acid at position 62 of the amino acid sequence of SEQ ID NO: 8 may be substituted with alanine (W62A), aspartic acid (W62D), glutamine (W62E), phenylalanine (W62F), glycine (W62G), histidine (W62H), isoleucine (W62I), lysine (W62K), leucine (W62L), methionine (W62M), asparagine (W62N), proline (W62P), glutamine (W62Q), arginine (W62R), serine (W62S), threonine (W62T), valine (W62V) or tyrosine (W62Y).

As used herein, the term "N-terminal extension domain" is a domain bound to the N-terminus of the core domain and refers to a polypeptide including an amino acid sequence from positions 1 to 39 in the extracellular domain of the full-length interferon α receptor 2 or in the D1 subdomain of the extracellular domain of the interferon α receptor 2. Specifically, the N-terminal extension domain may include the amino acid sequence of SEQ ID NO: 9.

The N-terminal extension domain may be a polypeptide having the amino acid sequence of SEQ ID NO: 9, or a polypeptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 amino acids starting from position 39 of the polypeptide having the amino acid sequence of SEQ ID NO: 9 in the C-terminal to N-terminal direction.

The extracellular domain fragment of interferon α receptor may further include the D2 subdomain of the extracellular domain of interferon α receptor 2. The D2 subdomain may include the amino acid sequence of SEQ ID NO: 7.

In addition, the D2 subdomain of the extracellular domain of interferon alpha receptor 2 may be composed of a sequence in which one or more amino acids of the aforementioned protein are added, deleted or substituted, as long as it has the same activity as the aforementioned protein. Specifically, the D2 subdomain may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 7.

The D2 subdomain of the extracellular domain of interferon alpha receptor 2 may further include variants. In this case, the variants are as described above.

In the present disclosure, the variation of the D2 subdomain may be substitution of any amino acid selected from the group consisting of amino acid residues at positions 28, 31, 33, 35, 48, 54, 81 to 85 and 87 of the amino acid sequence of SEQ ID NO: 7 by another amino acid. In this case, the other amino acid introduced by substitution is as described above. They correspond to positions 134, 137, 139, 141, 154, 160, 187 to 191 and 193 of the amino acid sequence of the full-length extracellular domain of interferon alpha receptor 2 (SEQ ID NO: 5), respectively.

Specifically, the glutamic acid at positions 28, 81, and 85 of the amino acid sequence of SEQ ID NO: 7 may be substituted with an amino acid other than glutamic acid. Specifically, regarding the amino acid substitution of the D2 subdomain, the glutamic acid at positions 28, 81, and 85 of the amino acid sequence of SEQ ID NO: 7 may be substituted with alanine (E28A, E81A, E85A), respectively. The glutamic acid at position 31 of the amino acid sequence of SEQ ID NO: 7 may be substituted with an amino acid other than glutamic acid. Specifically, regarding the amino acid substitution of the D2 subdomain, the glutamic acid at position 31 of the amino acid sequence of SEQ ID NO: 7 may be substituted with alanine (Q31A). Aspartic acid as the amino acid at positions 33 and 84 of the amino acid sequence of SEQ ID NO: 7 may be substituted with an amino acid other than aspartic acid. Specifically, regarding the amino acid substitution of the D2 subdomain, aspartic acid as the amino acid at positions 33 and 84 of the amino acid sequence of SEQ ID NO: 7 may be substituted with alanine (D33A, D84A), respectively. Serine as the amino acid at positions 35 and 83 of the amino acid sequence of SEQ ID NO: 7 may be substituted with an amino acid other than serine. Specifically, regarding the amino acid substitution of the D2 subdomain, serine as the amino acid at positions 35 and 83 of the amino acid sequence of SEQ ID NO: 7 may be substituted with alanine (S35A, S84A), respectively. The lysine as the amino acid at positions 48 and 54 of the amino acid sequence of SEQ ID NO: 7 may be substituted with an amino acid other than lysine. Specifically, regarding the amino acid substitution of the D2 subdomain, the lysine as the amino acid at positions 48 and 54 of the amino acid sequence of SEQ ID NO: 7 may be substituted with alanine (K48A, K54A), respectively. The histidine as the amino acid at position 82 of the amino acid sequence of SEQ ID NO: 7 may be substituted with an amino acid other than histidine. Specifically, regarding the amino acid substitution of the D2 subdomain, the histidine as the amino acid at position 82 of the amino acid sequence of SEQ ID NO: 7 may be substituted with alanine (H82A). Alanine, which is the amino acid at position 87 of the amino acid sequence of SEQ ID NO: 7, may be substituted with an amino acid other than alanine. Specifically, regarding amino acid substitution of the D2 subdomain, alanine, which is the amino acid at position 87 of the amino acid sequence of SEQ ID NO: 7, may be substituted with arginine (A87R).

In one embodiment of the present disclosure, the amino acid sequence of the extracellular domain fragment of interferon α receptor 2 including the wild-type N-terminal extension domain, the wild-type core domain and the wild-type D2 subdomain is listed in Table 1. [Table 1] Extracellular domain fragment of INFAR2 (SEQ ID NO: 5) SEQ ID NO: Sequence Name Amino acid sequence 9 N-terminal extension domain ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTH 8 Core Domain YTLLYTIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTLFSCSHNFWLAIDM 7 D2 subdomain SFEPPEFEIVGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAK Antigen Binding Protein

As used herein, the term "target protein" refers to a protein that is specifically expressed in a target (desired) cell, tissue or disease environment. The fusion protein or immunocytokine according to the present disclosure can be located at a desired location in the body, such as a tumor cell or an inflammatory site, by binding to the target protein.

The target protein may be a protein present on the cell surface or in the cell. In the present disclosure, "antigen binding protein (ABP)" may be an antibody or a fragment thereof, as well as a cell surface molecule binding partner, a fragment thereof or a variant thereof. The cell surface molecule binding partner may include all binding partners that bind to cell surface molecules (such as receptors and ligands). Preferably, it may be an antibody or a fragment thereof that specifically binds to a surface binding protein that is an antigen.

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 an antigen. The antibody may refer to any one selected from IgG, IgE, IgM, IgD, and IgA, and may be IgG1, IgG2, IgG3, or IgG4 as a subclass of IgG, or IgA1 or IgA2 as a subclass of IgA. In addition, the antibody may be an agonist antibody or an antagonist antibody.

Antibodies may include whole antibodies, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof, as well as immunoconjugates. The term "fragment of an antibody" refers to a molecule other than a complete antibody, which includes a portion of a complete antibody that binds to an antigen bound by the complete antibody. Therefore, a fragment of an antibody can bind to the same antigen recognized by the complete antibody, regardless of its structure. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bifunctional antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), single-domain antibodies, and the like.

Immunoglobulins may include a heavy chain and a light chain.

As used herein, the term "heavy chain" refers to a polypeptide chain of about 50 kDa to about 70 kDa. Here, the N-terminal portion includes a variable region of about 120 to 130 or more amino acids, and the C-terminal portion includes a constant region. The constant region can be one of the following five types: α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu). Here, α, δ, and γ include about 450 amino acids and μ and ε include about 550 amino acids. The heavy chain constant regions (CH) are numbered from the N-terminus to the C-terminus (e.g., CH1, CH2, CH3, etc.).

As used herein, the term "light chain" refers to a polypeptide chain of about 25 kDa. Here, the N-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the C-terminal portion includes a constant region. There are two types of light chain constant regions: κ (kappa) and λ (lambda). In addition, the constant region of the light chain is called "CL". Any of the CL and CH1 regions of these antibody classes can be used in the present disclosure. In one embodiment, the CH1 region according to the present disclosure may include the amino acid sequence of SEQ ID NO: 71. In addition, in one embodiment, the CL region may include the amino acid sequence of SEQ ID NO: 70.

The variable regions of the light and heavy chains of immunoglobulins include three hypervariable regions, which are called complementary determining regions (CDRs), and four framework regions (FRs). CDRs are the antigen binding sites that mainly bind to the antigenic determinants of antigens.

As used herein, the term "antigen" refers to a structure that can selectively bind to an antibody. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring compound or synthetic compound. Specifically, the antigen is a polypeptide and can be a protein present on the cell surface or inside the cell.

As used herein, the term "specific binding" refers to binding that is measurably different from non-specific interactions. Specific binding can be determined by competition with a control molecule that is similar to the target but does not have binding activity.

In the present disclosure, the target protein may be an antigen or a cell surface molecule.

The target protein may be a tumor-associated antigen (TAA). The tumor-associated antigen may be, for example, guanosyl cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), carcinoembryonic antigen (CEA), insulin-like growth factor 1 receptor (IGF1R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), phosphatidylinositol proteoglycan 3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), binding protein 4, Liv 1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop 2. Carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), six transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor α (FRα), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblastic glycoprotein (TPBG), CD19, CD22, CD33, CD36, CD38, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein α (SIRPa), PD1, claudin 182, claudin 6, 5T4 BCMA, PD-L1, fibroblast activation protein α (FAPα), melanoma-related chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM), etc., but not limited thereto.

The target protein may be a surface protein of an immune cell. The target protein may be a target protein specific to a T cell. The target protein may be specific to a CD4+ or CD8+ T cell.

The target protein may be an immune checkpoint protein. Immune checkpoint proteins may include, for example, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, TIM-3, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD20, CD24, CD25, CD40, CEACAM1, CD47, CD48, CD70, CD73, SIRPα, A2AR, CD39, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, VISTA, etc., but are not limited thereto.

The target protein may be an interleukin receptor. Interleukin receptors may include, for example, type I interleukin receptors, such as GM-CSF receptor, G-CSF receptor, type I interleukin (IL) receptor, Epo receptor, LIF receptor, CNTF receptor, and TPO receptor; type II interleukin receptors, such as IFN-α receptor (IFNAR1, IFNAR2), IFB-β receptor, IFN-γ receptor (IFNGR1, IFNGR2), and type II interleukin receptor; interleukin receptors, such as CC interleukin receptor, CXC interleukin receptor, , CX3C interleukin receptor and CXC interleukin receptor; tumor necrosis receptor superfamily receptors, such as TNFRSF5/CD40, TNFRSF8/CD30, TNFRSF7/CD27, TNFRSF1A/TNFR1/CD120a and TNFRSF1B/TNFR2/CD120b; TGF-β receptors, such as TGF-β receptor 1 and TGF-β receptor 2; and Ig superfamily receptors, such as IL-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB) and SCFR, but are not limited thereto.

Preferably, the target protein may be an immune checkpoint protein or a tumor-associated antigen. More preferably, it may be selected from any one of the following groups: PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, TIM-3, CD39, CD38, CD73, CD36, CD25, CD47, CD24, CD20, SIPRα, CD40, HER2, CD19, and EGFR. Even more preferably, in one embodiment of the present disclosure, the target protein may be selected from any one of the following groups: PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, CD38, CD47, HER2, CD19, and EGFR.

Specifically, the antigen binding protein may be an anti-PD-1 antibody. Specifically, the antigen binding protein may be an anti-PD-L1 antibody. Specifically, the antigen binding protein may be an anti-TIGIT antibody. Specifically, the antigen binding protein may be an anti-LAG-3 antibody. Specifically, the antigen binding protein may be an anti-CTLA4 antibody. Specifically, the antigen binding protein may be an anti-CD38 antibody. Specifically, the antigen binding protein may be an anti-CD47 antibody. Specifically, the antigen binding protein may be an anti-HER2 antibody. Specifically, the antigen binding protein may be an anti-CD19 antibody. Specifically, the antigen binding protein may be an anti-EGFR antibody.

More specifically, the antigen binding protein can be selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tremelimumab, tiragolumab, relatlimab, daratumumab, isatuximab, mezagitamab, magrolimab, trastuzumab, tafasitamab, cetuximab or variants thereof. Here, variants are as described above. In addition, variants refer to antigen binding proteins that maintain binding affinity and/or specificity compared to the original antigen binding protein.

In one embodiment, the antigen binding protein may include the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of any one antibody selected from the group consisting of nivolumab, pembrolizumab, cemiplizumab, atezolizumab, dotalimumab, durvalumab, avelumab, ipilimumab, tremelimumab, tisleliumab, relalizumab, daratumumab, isatuximab, mazetuzumab, mololimumab, trastuzumab, dafacitinib and cetuximab. In one embodiment, the antigen binding protein may include a heavy chain variable region of an antibody selected from the group consisting of nivolumab (SEQ ID NO: 107), pembrolizumab (SEQ ID NO: 109), cemiplizumab (SEQ ID NO: 111), atezolizumab (SEQ ID NO: 123), dotalimumab (SEQ ID NO: 113), durvalumab (SEQ ID NO: 102), avelumab (SEQ ID NO: 125), ipilimumab (SEQ ID NO: 115), tremelimumab (SEQ ID NO: 117), tisleliumab (SEQ ID NO: 119), relalizumab (SEQ ID NO: 121), daratumumab (SEQ ID NO: 61), isatuximab (SEQ ID NO: 74), mezetumomab (SEQ ID NO: 76), tadalafil (SEQ ID NO: 77), tadalafil (SEQ ID NO: 79), tadalafil (SEQ ID NO: 80), tadalafil (SEQ ID NO: 81), tadalafil (SEQ ID NO: 82), tadalafil (SEQ ID NO: 83), tadalafil (SEQ ID NO: 84), tadalafil (SEQ ID NO: 85), tadalafil (SEQ ID NO: 86), tadalafil (SEQ ID NO: 87), tadalafil (SEQ ID NO: 88), tadalafil (SEQ ID NO: 89), tadalafil (SEQ ID NO: 90), tadalafil (SEQ ID NO: 91), tadalafil (SEQ ID NO: 92), tadalafil (SEQ ID NO: 93), tadalafil (SEQ ID NO: 94), tadalafil (SEQ ID NO: 95), tadalafil (SEQ ID NO: 86), mololimab (SEQ ID NO: 96), trastuzumab (SEQ ID NO: 98), dafacitinib (SEQ ID NO: 100), and cetuximab (SEQ ID NO: 104). In one embodiment, the antigen binding protein may include a light chain variable region of any antibody selected from the group consisting of nivolumab (SEQ ID NO: 108), pembrolizumab (SEQ ID NO: 110), cemiplizumab (SEQ ID NO: 112), atezolizumab (SEQ ID NO: 124), dotalimumab (SEQ ID NO: 114), durvalumab (SEQ ID NO: 103), avelumab (SEQ ID NO: 126), ipilimumab (SEQ ID NO: 116), tremelimumab (SEQ ID NO: 118), tisleliumab (SEQ ID NO: 120), relalizumab (SEQ ID NO: 122), daratumumab (SEQ ID NO: 62), isatuximab (SEQ ID NO: 75), mezetumomab (SEQ ID NO: 76), tadalafil (SEQ ID NO: 77), tadalafil (SEQ ID NO: 79), tadalafil (SEQ ID NO: 80), tadalafil (SEQ ID NO: 81), tadalafil (SEQ ID NO: 82), tadalafil (SEQ ID NO: 83), tadalafil (SEQ ID NO: 84), tadalafil (SEQ ID NO: 85), tadalafil (SEQ ID NO: 86), tadalafil (SEQ ID NO: 87), tadalafil (SEQ ID NO: 88), tadalafil (SEQ ID NO: 89), tadalafil (SEQ ID NO: 90), tadalafil (SEQ ID NO: 91), tadalafil (SEQ ID NO: 92), tadalafil (SEQ ID NO: 93), tadalafil (SEQ ID NO: 94), tadalafil (SEQ ID NO: 87), mololimab (SEQ ID NO: 97), trastuzumab (SEQ ID NO: 99), dafacitinib (SEQ ID NO: 101), and cetuximab (SEQ ID NO: 105). In one embodiment, the antigen binding protein may include a heavy chain variable region and a light chain variable region of any one antibody selected from the group consisting of nivolumab (SEQ ID NO: 107 and SEQ ID NO: 108), pembrolizumab (SEQ ID NO: 109 and SEQ ID NO: 110), cemiplizumab (SEQ ID NO: 111 and SEQ ID NO: 112), atezolizumab (SEQ ID NO: 123 and SEQ ID NO: 124), dotalimumab (SEQ ID NO: 113 and SEQ ID NO: 114), durvalumab (SEQ ID NO: 102 and SEQ ID NO: 103), avelumab (SEQ ID NO: 125 and SEQ ID NO: 126), ipilimumab (SEQ ID NO: 115 and SEQ ID NO: 116), tremelimumab (SEQ ID NO: 117 and SEQ ID NO: 118), and leukopenia (SEQ ID NO: 119). 117 and SEQ ID NO: 118), tisleliumab (SEQ ID NO: 119 and SEQ ID NO: 120), relalizumab (SEQ ID NO: 121 SEQ ID NO: 122), daratumumab (SEQ ID NO: 61 and SEQ ID NO: 62), isatuximab (SEQ ID NO: 74 and SEQ ID NO: 75), mezetumomab (SEQ ID NO: 86 and SEQ ID NO: 87), mololimab (SEQ ID NO: 96 and SEQ ID NO: 97), trastuzumab (SEQ ID NO: 98 and SEQ ID NO: 99), dafacitinib (SEQ ID NO: 100 and SEQ ID NO: 101) and cetuximab (SEQ ID NO: 104 and SEQ ID NO: 105).

Even more preferably, the antigen binding protein may be an antibody that specifically binds to CD38, HER2, PD-L1 or EGFR. CD38 Antigen Binding Site

As used herein, the term "CD38" refers to a cell surface glycoprotein of approximately 46 kDa known as cyclic ADP ribose hydrolase. CD38 breaks down NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide-dependent phosphate) in immune cells to produce NAADP (nicotinic acid adenine dinucleotide phosphate) and cADPR (cyclic ADP ribose), and is involved in cell adhesion, external signal transduction, and regulation of intracellular calcium ion levels in immune cells. CD38 is known to be less expressed in normal lymphoid or bone marrow cells, but is overexpressed in various blood cancers. Therefore, CD38 is used as an antigen target for the diagnosis and treatment of cancer. Among them, Daratumumab (Darzalex®) is the first anti-CD38 antibody-based therapeutic agent approved by the US FDA. It is known that it not only binds to CD38 expressed in multiple myeloma cells to inhibit enzyme activity, but also kills cancer cells through immune-mediated cell death, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity.

In the present disclosure, CD38 may include (but is not limited to) any mammalian CD38, but preferably refers to human CD38. In addition, in the present disclosure, CD38 includes both wild-type and variant CD38, but is not limited thereto. Information on CD38 can be obtained from known databases, such as Genbank of the National Center for Biotechnology Information (NCBI), and may include, for example, the amino acid sequence of GenBank Accession No. NP_001766.2 (SEQ ID NO: 60), but is not limited thereto.

In the present disclosure, "antigen binding protein specific for CD38" may generally refer to a molecule capable of forming an antigen-antibody that specifically binds to CD38. In addition, the antibody or its fragment may be used in any form as long as it includes an antigen binding site that can specifically bind to CD38. The antibody or its fragment may include other amino acids that are not directly involved in binding, or amino acids whose action is blocked by amino acid residues at the antigen binding site.

In one embodiment of the present disclosure, an antigen binding protein that specifically binds to CD38 may include: a heavy chain variable region including a HCDR1 including an amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 76, or SEQ ID NO: 88, a HCDR2 including an amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 77, or SEQ ID NO: 89, and a HCDR3 including an amino acid sequence of SEQ ID NO: 65, SEQ ID NO: 78, or SEQ ID NO: 90; and a light chain variable region including a LCDR1 including an amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 79, or SEQ ID NO: 91, a LCDR2 including an amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 80, or SEQ ID NO: 92, and a HCDR3 including an amino acid sequence of SEQ ID NO: 65, SEQ ID NO: 78, or SEQ ID NO: 90. The antigen binding protein specifically binding to CD38 may include a heavy chain variable region including an amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 74 or SEQ ID NO: 86 and a light chain variable region including an amino acid sequence of SEQ ID NO: 62, SEQ ID NO: 75 or SEQ ID NO: 87.

In one embodiment, an antigen binding protein that specifically binds to CD38 may include: a heavy chain variable region including HCDR1 including the amino acid sequence of SEQ ID NO: 63, HCDR2 including the amino acid sequence of SEQ ID NO: 64, and HCDR3 including the amino acid sequence of SEQ ID NO: 65; and a light chain variable region including LCDR1 including the amino acid sequence of SEQ ID NO: 66, LCDR2 including the amino acid sequence of SEQ ID NO: 67, and LCDR3 including the amino acid sequence of SEQ ID NO: 68. The heavy chain variable region may include the amino acid sequence of SEQ ID NO: 61, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 62.

In one embodiment, an antigen binding protein that specifically binds to CD38 may include: a heavy chain variable region including HCDR1 including the amino acid sequence of SEQ ID NO: 76, HCDR2 including the amino acid sequence of SEQ ID NO: 77, and HCDR3 including the amino acid sequence of SEQ ID NO: 78; and a light chain variable region including LCDR1 including the amino acid sequence of SEQ ID NO: 79, LCDR2 including the amino acid sequence of SEQ ID NO: 80, and LCDR3 including the amino acid sequence of SEQ ID NO: 81. The heavy chain variable region may include the amino acid sequence of SEQ ID NO: 74, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 75.

The antigen binding protein that specifically binds to CD38 may include: a heavy chain variable region including HCDR1 including the amino acid sequence of SEQ ID NO: 88, HCDR2 including the amino acid sequence of SEQ ID NO: 89, and HCDR3 including the amino acid sequence of SEQ ID NO: 90; and a light chain variable region including LCDR1 including the amino acid sequence of SEQ ID NO: 91, LCDR2 including the amino acid sequence of SEQ ID NO: 92, and LCDR3 including the amino acid sequence of SEQ ID NO: 93. The heavy chain variable region may include the amino acid sequence of SEQ ID NO: 86, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 87.

In addition, the heavy chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 74 or SEQ ID NO: 86. In addition, the light chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 62, SEQ ID NO: 75 or SEQ ID NO: 87.

The antigen binding protein that specifically binds to CD38 may include an immunoglobulin heavy chain constant region 1 (CH1) and a light chain constant region (CL). Specifically, it may include a heavy chain constant region 1 including an amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 167, and a light chain constant region including an amino acid sequence of SEQ ID NO: 70. HER2 antigen binding site

As used herein, the term "HER2", also known as ErbB-2 or CD340, is a protein belonging to the ErbB (HER/EGFR/ERBB) family. The ErbB family consists of four types of receptor-binding tyrosine kinase subfamilies: ErbB-1 (HER1, EGFR), ErbB-2 (HER2/neu), ErbB-3 (HER3), and ErbB-4 (HER4). All four types of receptors include an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain. Here, the intracellular domain interacts with multiple signaling molecules in a ligand-dependent or ligand-independent manner. Specifically, the ligand of HER2 is unclear. It is known that HER2 acts by forming a heterodimer with one of three other receptors. Receptors of the ErbB family are known to be involved in regulating cell adhesion, migration, and differentiation in addition to cell proliferation and survival. Specifically, HER2 is known to be the most powerful tumor protein in breast cancer. When HER2 is at normal levels, it is involved in the growth and development of normal breast tissue. However, when HER2 is abnormally overexpressed or amplified, normal cell regulation is disrupted and invasive cancer cells form in breast tissue. HER2 overexpression occurs in approximately 20% to 30% of invasive breast cancers, which are known to be aggressive cancers with a higher degree of malignancy and are associated with a poor prognosis in breast cancer.

In the present disclosure, HER2 may include (but is not limited to) HER2 of any mammal, but preferably refers to HER2 of human. In addition, in the present disclosure, HER2 includes both wild-type and variant HER2, but is not limited thereto. Information about HER2 can be obtained from known databases, such as Genbank of the National Center for Biotechnology Information (NCBI). For example, it may include the amino acid sequence of SEQ ID NO: 127, but is not limited thereto.

In the present disclosure, "antigen binding protein specific for HER2" may generally refer to a molecule capable of forming an antigen-antibody that specifically binds to HER2. In addition, the antibody or its fragment may be used in any form as long as it includes an antigen binding site that can specifically bind to HER2. The antibody or its fragment may include other amino acids that are not directly involved in the binding, or amino acids whose action is blocked by amino acid residues at the antigen binding site.

In one embodiment of the present disclosure, an antigen binding protein that specifically binds to HER2 may include: a heavy chain variable region including HCDR1 including the amino acid sequence of SEQ ID NO: 133, HCDR2 including the amino acid sequence of SEQ ID NO: 134, and HCDR3 including the amino acid sequence of SEQ ID NO: 135; and a light chain variable region including LCDR1 including the amino acid sequence of SEQ ID NO: 136, LCDR2 including the amino acid sequence of SEQ ID NO: 137, and LCDR3 including the amino acid sequence of SEQ ID NO: 138. The heavy chain variable region may include the amino acid sequence of SEQ ID NO: 98, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 99.

In addition, the heavy chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 98. In addition, the light chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 99.

The antigen binding protein that specifically binds to HER2 may include an immunoglobulin heavy chain constant region 1 (CH1) and a light chain constant region (CL). Specifically, it may include a heavy chain constant region 1 including an amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 71, and a light chain constant region including an amino acid sequence of SEQ ID NO: 70. PD-L1 antigen binding site

As used herein, the term "PD-L1 (programmed cell death ligand 1)" is a protein expressed on the surface of tumor cells or hematopoietic cells. It is called CD274 or B7H1. PD-L1 is a ligand of PD-1, and when PD-L1 and PD-1 bind, apoptosis of T cells is induced to weaken the immune activity of the cells.

In the present disclosure, PD-L1 may include, but is not limited to, those derived from mammalian sources, including primates (such as humans and monkeys) and rodents (such as rats and mice). In addition, in the present disclosure, PD-L1 protein may include both wild-type and variant PD-L1 proteins, but is not limited thereto. Wild-type PD-L1 protein generally refers to a polypeptide including the amino acid sequence of wild-type PD-L1 protein. The amino acid sequence and polynucleotide sequence of PD-L1 protein can be obtained from known databases, such as Genbank of the National Center for Biotechnology Information (NCBI). For example, it may include the amino acid sequence of SEQ ID NO: 128, but is not limited thereto.

In the present disclosure, "antigen binding protein specific for PD-L1" may generally refer to antigen-antibody molecules that can specifically bind to PD-L1. In addition, antibodies or fragments thereof may be used in any form as long as they include an antigen binding site that can specifically bind to PD-L1. Antibodies or fragments thereof may include other amino acids that are not directly involved in binding, or amino acids whose action is blocked by amino acid residues at the antigen binding site.

In one embodiment of the present disclosure, an antigen binding protein that specifically binds to PD-L1 may include: a heavy chain variable region including a HCDR1 including an amino acid sequence of SEQ ID NO: 140, SEQ ID NO: 153, or SEQ ID NO: 160, a HCDR2 including an amino acid sequence of SEQ ID NO: 141, SEQ ID NO: 154, or SEQ ID NO: 161, and a HCDR3 including an amino acid sequence of SEQ ID NO: 142, SEQ ID NO: 155, or SEQ ID NO: 162; and a light chain variable region including a LCDR1 including an amino acid sequence of SEQ ID NO: 143, SEQ ID NO: 156, or SEQ ID NO: 163, a HCDR2 including an amino acid sequence of SEQ ID NO: 144, SEQ ID NO: 157, or SEQ ID NO: The antigen binding protein specifically binding to PD-L1 may include a heavy chain variable region including an amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 123 or SEQ ID NO: 125, and a light chain variable region including an amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 124 or SEQ ID NO: 126.

In one embodiment, an antigen binding protein that specifically binds to PD-L1 may include: a heavy chain variable region including HCDR1 including an amino acid sequence of SEQ ID NO: 140, HCDR2 including an amino acid sequence of SEQ ID NO: 141, and HCDR3 including an amino acid sequence of SEQ ID NO: 142; and a light chain variable region including LCDR1 including an amino acid sequence of SEQ ID NO: 143, LCDR2 including an amino acid sequence of SEQ ID NO: 144, and LCDR3 including an amino acid sequence of SEQ ID NO: 145. The heavy chain variable region may include an amino acid sequence of SEQ ID NO: 102, and the light chain variable region may include an amino acid sequence of SEQ ID NO: 103.

In one embodiment, an antigen binding protein that specifically binds to PD-L1 may include: a heavy chain variable region including HCDR1 including an amino acid sequence of SEQ ID NO: 153, HCDR2 including an amino acid sequence of SEQ ID NO: 154, and HCDR3 including an amino acid sequence of SEQ ID NO: 155; and a light chain variable region including LCDR1 including an amino acid sequence of SEQ ID NO: 156, LCDR2 including an amino acid sequence of SEQ ID NO: 157, and LCDR3 including an amino acid sequence of SEQ ID NO: 158. The heavy chain variable region may include an amino acid sequence of SEQ ID NO: 123, and the light chain variable region may include an amino acid sequence of SEQ ID NO: 124.

In one embodiment, an antigen binding protein that specifically binds to PD-L1 may include: a heavy chain variable region including HCDR1 including an amino acid sequence of SEQ ID NO: 160, HCDR2 including an amino acid sequence of SEQ ID NO: 161, and HCDR3 including an amino acid sequence of SEQ ID NO: 162; and a light chain variable region including LCDR1 including an amino acid sequence of SEQ ID NO: 163, LCDR2 including an amino acid sequence of SEQ ID NO: 164, and LCDR3 including an amino acid sequence of SEQ ID NO: 165. The heavy chain variable region may include an amino acid sequence of SEQ ID NO: 125, and the light chain variable region may include an amino acid sequence of SEQ ID NO: 126.

In addition, the heavy chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 123 or SEQ ID NO: 125. In addition, the light chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 124 or SEQ ID NO: 126.

The antigen binding protein that specifically binds to PD-L1 may include an immunoglobulin heavy chain constant region 1 (CH1) and a light chain constant region (CL). Specifically, it may include a heavy chain constant region 1 including an amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 167, and a light chain constant region including an amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 95. EGFR antigen binding site

As used herein, the term "epidermal growth factor receptor (EGFR)" is also referred to as HER1 or ErbB-1 as described above, and is a protein belonging to the ErbB (HER/EGFR/ERBB) family. EGFR regulates cell growth, division, survival, and death. It has been reported that EGFR is overexpressed in tumor tissues of various cancers, and tumor tissues with increased EGFR are known to have invasiveness, cancer metastasis, and high resistance to anticancer agents.

In the present disclosure, "antigen binding protein specific for EGFR" may generally refer to a molecule that can form an antigen-antibody that specifically binds to EGFR. In addition, the antibody or its fragment may be used in any form as long as it includes an antigen binding site that can specifically bind to EGFR. The antibody or its fragment may include other amino acids that are not directly involved in the binding, or amino acids whose action is blocked by amino acid residues in the antigen binding site.

In the present disclosure, EGFR may include, but is not limited to, those derived from mammalian sources, including primates (such as humans and monkeys) and rodents (such as rats and mice). In addition, in the present disclosure, EGFR protein may include both wild-type and variant EGFR proteins, but is not limited thereto. Wild-type EGFR protein generally refers to a polypeptide comprising the amino acid sequence of a wild-type EGFR protein. The amino acid sequence and polynucleotide sequence of EGFR protein can be obtained from known databases, such as Genbank of the National Center for Biotechnology Information (NCBI). For example, it may include the amino acid sequence of SEQ ID NO: 129, but is not limited thereto.

In one embodiment of the present disclosure, an antigen binding protein that specifically binds to EGFR may include: a heavy chain variable region including HCDR1 including the amino acid sequence of SEQ ID NO: 147, HCDR2 including the amino acid sequence of SEQ ID NO: 148, and HCDR3 including the amino acid sequence of SEQ ID NO: 149; and a light chain variable region including LCDR1 including the amino acid sequence of SEQ ID NO: 150, LCDR2 including the amino acid sequence of SEQ ID NO: 151, and LCDR3 including the amino acid sequence of SEQ ID NO: 152. The heavy chain variable region may include the amino acid sequence of SEQ ID NO: 104, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 105.

In addition, the heavy chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity to the amino acid sequence of SEQ ID NO: 104. In addition, the light chain variable region of the antibody may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity to the amino acid sequence of SEQ ID NO: 105.

The antigen binding protein that specifically binds to EGFR may include an immunoglobulin heavy chain constant region 1 (CH1) and a light chain constant region (CL). Specifically, it may include a heavy chain constant region 1 including an amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 71, and a light chain constant region including an amino acid sequence of SEQ ID NO: 70. Fc region or fragment thereof

As used herein, the term "Fc region" refers to the Fc region of an immunoglobulin. The Fc region refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin and does not include the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region 1 (CH1) and the light chain constant region (CL) of the immunoglobulin. The Fc region of an immunoglobulin may be not only a wild-type Fc region, but also a fragment of the Fc region or a variant thereof. For example, a fragment of the Fc region may lack lysine (K) from the C-terminus. The immunoglobulin may be IgG, IgA, IgE, IgD or IgM. Further, it may also be IgG1, IgG2, IgG3 or IgG4 as a subclass of IgG, or IgA1 or IgA2 as a subclass of IgA. Specifically, in the present disclosure, the Fc region may be derived from IgG1 or IgG4. In one embodiment, it may include any amino acid selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 13 and SEQ ID NO: 83. Preferably, it may include the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 83.

As used herein, the term "Fc region variant" may be a form having a glycosylation pattern different from that of a wild-type Fc region, increased glycosylation compared to a wild-type Fc region, decreased glycosylation compared to a wild-type Fc region, or a deglycosylated form. In addition, a deglycosylated Fc region is also included. Fc region variants may have regulated amounts of sialic acid, fucosylation, and glycosylation by regulating culture conditions or genetic manipulation of the host.

In addition, the glycosylation of the Fc region of the immunoglobulin can be modified by known methods such as chemical methods, enzyme methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a mixture of the Fc region of IgG, IgA, IgE, IgD or IgM. In addition, the Fc region variant may be in a form in which some amino acids of the Fc region are substituted by other amino acids. Here, the "amino acid" introduced by substitution and/or addition is as described above.

In one embodiment, the variant may include the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

The variant of the Fc region may further include a variant of the Fc region that promotes heterodimer formation. In one embodiment, the variant of the Fc region that promotes heterodimer formation may include a knob structure or a hole structure, but is not limited thereto, as long as it promotes heterodimer formation.

"Knob-into-hole" is a design strategy used to prepare antibodies that specifically bind to different regions, such as bispecific antibodies, multispecific antibodies or heterodimers. Generally speaking, this technique involves introducing a knob at the interface of a first polypeptide (e.g., the first CH3 region of a first antibody rechain) and a corresponding hole at the interface of a second polypeptide (e.g., the second CH3 region of a second antibody rechain), so that the knob can be positioned in the hole to promote heterodimer formation and hinder homodimer formation.

The 'knob' is constructed by replacing a small amino acid side chain at the interface of a first polypeptide (e.g., the first CH3 region of a first antibody heavy chain) with a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A 'hole' of the same or similar size complementary to the knob is generated by replacing a larger amino acid side chain at the interface of a second polypeptide (e.g., the second CH3 region of a second antibody heavy chain) with a smaller side chain (e.g., alanine, serine, valine, or threonine). The knob and hole can be generated, for example, by inducing site-specific mutations in the nucleic acid encoding the polypeptide or by modification during the peptide synthesis step.

Examples of the knob-and-mortar technique of the Fc region may be variations described in WO2014084607A1 and WO2018059502A1. The disclosures of WO2014084607A1 and WO2018059502A1 are incorporated into the present disclosure by reference.

Specifically, the knob structure may include the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 94. Specifically, the hole structure may include the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 106.

Another example of an Fc region variant that promotes heterodimer formation may include HA-TF variants. Specifically, it may include a variant in which H and A are substituted in the first polypeptide, and a variant in which T and F are substituted at the corresponding positions in the second polypeptide. In one example, in a human IgG1 Fc region, based on EU numbering, the first polypeptide may include S364H and F405A, and the second polypeptide may include Y349T and T394F.

Another example of an Fc region variant that promotes heterodimer formation may include a DD-KK variant. Specifically, it may include a variant in which two amino acids in a first polypeptide are substituted with D, and a variant in which two amino acids at corresponding positions in a second polypeptide are each substituted with K. In one example, in a human IgG1 Fc region, based on EU numbering, the first polypeptide may include K409D and K392D, and the second polypeptide may include D399K and E356K.

Another example of an Fc region variation that promotes heterodimer formation may include an EW-RVT variation. Specifically, it may include a variation in which E and W are substituted in the first polypeptide, and a variation in which R, V, and T are substituted at the corresponding positions in the second polypeptide. In one example, in a human IgG1 Fc region, based on EU numbering, the first polypeptide may include K360E and K409W, and the second polypeptide may include Q347R, D399V, and F405T.

Another example of an Fc region variant that promotes heterodimer formation may include a ZW1 variant. Specifically, it may include a variant that replaces V, Y, A, and V in a first polypeptide, and a variant that replaces V, L, L, and W at the corresponding position in a second polypeptide. In one example, in a human IgG1 Fc region, based on EU numbering, a first polypeptide may include T350V, L351Y, F405A, and Y407V, and a second polypeptide may include T350V, T366L, K392L, and T394W.

In one embodiment, the variation of the Fc region that promotes heterodimer formation may include any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26; and any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27. In one embodiment, the Fc region comprising the amino acid sequence of SEQ ID NO: 20 promotes heterodimer formation with the Fc region comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the Fc region comprising the amino acid sequence of SEQ ID NO: 22 promotes heterodimer formation with the Fc region comprising the amino acid sequence of SEQ ID NO: 23. In one embodiment, an Fc region comprising the amino acid sequence of SEQ ID NO: 24 promotes heterodimer formation with an Fc region comprising the amino acid sequence of SEQ ID NO: 25. In one embodiment, an Fc region comprising the amino acid sequence of SEQ ID NO: 26 promotes heterodimer formation with an Fc region comprising the amino acid sequence of SEQ ID NO: 27. Non-cleavable linkers and cleavable linkers

As used herein, the term "peptide linker" refers to a peptide consisting of one or more amino acids. Typically, it may consist of 1 to 100 consecutive amino acids, 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. Peptide linkers are known in the art or described herein. In one embodiment, the peptide linker may be (G4S)n, (SG4)n, or G4(SG4)n (where n is an integer from 1 to 10). In one embodiment, the peptide linker may be (EAAAK)n (where n is an integer from 1 to 3). Here, n in (G4S)n, (SG4)n, or G4(SG4)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Here, n in (EAAAK)n may be 1, 2 or 3. In one embodiment, the peptide linker may be A(EAAAK) _4ALEA (EAAAK) _4A (SEQ ID NO: 38), PAPAP (SEQ ID NO: 39) or AEAAAKEAAAKA (SEQ ID NO: 40). In one embodiment, the peptide linker may be (Ala-Pro)n(10-33aa) (n is an integer from 5 to 15). Here, n may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In the present disclosure, if a peptide linker does not include a cleavage site cleaved by a protease, it can be used as a non-cleavable linker. Here, the non-cleavable linker can be a simple chemical bond, such as an amide bond. In addition, the non-cleavable linker is not only stable under physiological conditions, but also can be stable in a disease site (e.g., a tumor site or an inflammatory disease site). In one embodiment, the non-cleavable linker can include any one amino acid sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 41.

As used herein, the term "cleavable linker" refers to a polypeptide linker including a cleavable peptide. A cleavable peptide is a polypeptide that includes a protease cleavage site and can be specifically cleaved by a protease (proteinase). A protease is an enzyme that hydrolyzes the peptide bond between two specific amino acid residues in a target substrate protein. Therefore, a "cleavage site" refers to an amino acid sequence in a target substrate protein that is recognized and cleaved by a protease, and a cleavable peptide may include an amino acid sequence of a protease cleavage site. Cleavable peptides are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. In addition, cleavable peptides can be cleaved by changes in environmental signals, such as temperature, pH, or salt concentration.

In the present disclosure, a cleavable peptide may include an amino acid sequence of a cleavage site that is cleaved by a proteolytic enzyme that is overexpressed in a disease site compared to a non-disease site.

In one embodiment, the cleavable peptide may include an amino acid sequence of a tumor-associated proteolytic enzyme cleavage site. Here, the tumor-associated proteolytic enzyme may be a proteolytic enzyme that is overexpressed in tumor cells or tumor environments. In addition, the cleavable peptide may be cleaved by one or more proteolytic enzymes. The tumor-associated proteolytic enzyme may be, for example, a metalloproteinase, a serine protease, a cysteine protease, an aspartic acid protease, and a disintegrin and metalloproteinase (ADAM), but is not limited thereto.

Metalloproteinases may be, for example, matrix metalloproteinases (MMPs) 1 to 28, but are not limited thereto. Serine proteases may be, for example, urokinase-type fibronectin activator (uPA), tissue fibronectin activator (tPA), fibronectin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastic protease (HNE), elastic protease, trypsin (CELA1), interstitial protease (ST14), etc., but are not limited thereto. Cysteine proteases may be, for example, legumin, cathepsin (C, K, L1, S), etc., and aspartic proteases may be, for example, cathepsin D, cathepsin E, secretase (BACE1), etc., but are not limited thereto. ADAM may be, for example, ADAM10, DAMA17, etc., but are not limited thereto. Specifically, the cleavage linker may include an amino acid sequence of a cleavage site of metalloproteinase (MMP) 2/4, uPA and/or matrix protease. In one embodiment, it may include any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO: 59 and combinations thereof. Immunointerferon including an interferon, an extracellular domain fragment of an interferon α receptor or a variant thereof and an antigen binding protein specific for a target protein

In one aspect, the present disclosure provides an immunocytokine comprising an interferon protein or a fragment thereof; an extracellular domain (ECD) fragment of an interferon α receptor or a variant thereof; and an antigen binding protein specific to a target protein.

In one embodiment, the immunocytokine may include an interferon protein or a fragment thereof; an extracellular domain (ECD) fragment of an interferon α receptor or a variant thereof; and an antigen binding protein specific to a target protein.

The immunocytokine may be a dimer formed by: i) a first monomer comprising an antigen binding protein specific for a target protein and an interferon protein or a fragment thereof, and ii) a second monomer comprising an antigen binding protein specific for a target protein and an extracellular domain fragment of an interferon α receptor or a variant thereof. Here, the antigen binding protein specific for a target protein and the interferon α protein or a fragment thereof may be bound by a linker. In addition, the antigen binding protein specific for a target protein and an extracellular domain fragment of an interferon α receptor or a variant thereof may be bound by a linker.

A "linker" connects two proteins. Specific examples of linkers may include 1 to 50 amino acids, or an Fc region of an immunoglobulin or a fragment thereof. Here, the linker may be an immunoglobulin Fc region or a fragment thereof.

The interferon protein or fragment thereof, the extracellular domain fragment of the interferon α receptor or a variant thereof, the target protein, the antigen binding protein and the Fc region or fragment thereof are as described above.

Specifically, the first monomer of the immunocytokine can be composed of the following structural formula (I) and structural formula (II): <First monomer> N'-X'-[Linker (1)]a-Fc region-[Linker (2)]b-Y-C' (I) and N'-X''-C' (II) In structural formula (I) and structural formula (II), N' is the N-terminus of the first monomer, C' is the C-terminus of the first monomer, X is an antigen binding protein representing the antigen binding site of an antibody or a fragment thereof, wherein X includes X' and X'', X' is an antigen binding site including a heavy chain variable region (VH) and a CH1 region, X'' is an antigen binding site including a light chain variable region (VL) and a constant region (CL), Y is an interferon protein or a fragment thereof, The linker (1) and the linker (2) are peptide linkers, and a is 1, and b is 0 or 1.

Specifically, the second monomer of the immunocytokine can be composed of the following structural formula (III) and structural formula (IV): <Second monomer> N'-X'-[Linker (3)]c-Fc region-[Linker (4)]d-Y'-C' (III) and N'-X''-C' (IV) In structural formula (III) and structural formula (IV), N' is the N-terminus of the second monomer, C' is the C-terminus of the second monomer, X is an antigen binding protein representing the antigen binding site of an antibody or a fragment thereof, wherein X includes X' and X'', X' is an antigen binding site including a heavy chain variable region (VH) and a CH1 region, X'' is an antigen binding site including a light chain variable region (VL) and a constant region (CL), Y' is an extracellular domain fragment of an interferon α receptor or a variant thereof, the linker (3) and the linker (4) are peptide linkers, and c is 1, and d is 0 or 1.

The interferon protein or fragment thereof, the extracellular domain fragment of the interferon α receptor or a variant thereof, the target protein, the antigen binding protein and the Fc region or fragment thereof are as described above.

Here, the peptide linker (1) or linker (3) may be composed of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, 10 to 60 consecutive amino acids, or 12 to 50 amino acids. In one embodiment, the peptide linker (1) or linker (3) may be composed of 30 amino acids. In addition, the peptide linker (1) or linker (3) may include at least one cysteine. Specifically, it may include one, two or three cysteine. In addition, the peptide linker (1) or linker (3) may be derived from the hinge of an immunoglobulin. In one embodiment, the peptide linker (1) or linker (3) may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 31 and SEQ ID NO: 166. Preferably, it may include the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 166.

The peptide linker (2) or linker (4) may be independently a cleavable linker or a non-cleavable linker. Here, the cleavable linker and the non-cleavable linker are as described above. In one embodiment, the peptide linker (2) or linker (4) may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 59 and combinations thereof. Preferably, it may include the amino acid sequence of SEQ ID NO: 33.

In the present disclosure, immunocytokines may be heterodimers.

In the present disclosure, when the first monomer and the second monomer form a heterodimer, the immunocytokine can be bound by the cysteine included in the linker that connects the antigen binding protein in the first monomer and the second monomer and the antigen binding site of the Fc region or its fragment. Here, the linker may include the hinge region of an immunoglobulin.

In addition, when the immunocytokine is a heterodimer, the Fc region and its fragments may include variants that promote heterodimer formation. The variants of the Fc region that promote heterodimer formation are as described above.

In the present disclosure, the immunocytokine may be a third monomer, which includes the following structural formula (V) or structural formula (VI); and structural formula (VII) or structural formula (VIII), or a dimer thereof. <Third monomer> N'-X'-[Linker (5)]e-Fc region-[Linker (6)]f-{Y-[Linker (7)]g-Y'}h-C' (V) or N'-X'-[Linker (5)]e-Fc region-[Linker (6)]f-{Y'-[Linker (7)]g-Y}h-C' (VI); and N'-X''-[Linker (8)]i-{Y'-[Linker (9)]j-Y}k-C' (VII) or N'-X''-[Linker (8)]i-{Y-[Linker (9)]j-Y'}k-C' (VIII) In formulas (V) to (VIII), N' is the N-terminus of the third monomer, C' is the C-terminus of the third monomer, X is an antigen binding protein representing an antigen binding site of an antibody or a fragment thereof, wherein X comprises X' and X'', X' is an antigen binding site comprising a heavy chain variable region (VH) and a CH1 region, X'' is an antigen binding site comprising a light chain variable region (VL) and a constant region (CL), Y is an interferon protein or a fragment thereof, Y' is an extracellular domain fragment of an interferon α receptor or a variant thereof, the linker (5) to the linker (9) is a peptide linker, and e, f, g, h, i, j and k are each independently 0 or 1, and one or more of h and k are 1.

The interferon protein or fragment thereof, the extracellular domain fragment of the interferon α receptor or a variant thereof, the target protein, the antigen binding protein and the Fc region or fragment thereof are as described above.

Here, the peptide linker (5) may be composed of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, 10 to 60 consecutive amino acids, or 12 to 50 amino acids. In one embodiment, the peptide linker (5) may be composed of 30 amino acids. In addition, the peptide linker (5) may include at least one cysteine. Specifically, it may include one, two or three cysteine. In addition, the peptide linker (5) may be derived from the hinge of an immunoglobulin. In one embodiment, the peptide linker (5) may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 31 and SEQ ID NO: 166. Preferably, it may include the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 166.

Peptide linker (6) to linker (9) can each independently be a cleavable linker or a non-cleavable linker. Here, the cleavable linker and the non-cleavable linker are as described above. In one embodiment, linker (6) to linker (9) can include any amino acid sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 59 and combinations thereof. Preferably, it can include the amino acid sequence of SEQ ID NO: 33.

Immunointerleukins can exist in monomeric form.

In addition, the immunocytokine may form a dimer in which two third monomers are combined. Here, the dimer may be a homodimer or a heterodimer. When the immunocytokine is a dimer, it may be bound by a cysteine included in a linker that links the antigen binding protein in the third monomer and the antigen binding site of the Fc region or its fragment. Here, the linker may include a hinge region of an immunoglobulin.

When the immunocytokine is a heterodimer, the Fc region and its fragments may include variants that promote heterodimer formation. The variants of the Fc region that promote heterodimer formation are as described above.

In addition, the immunocytokine may be a seventh monomer, which includes the following structural formula (XVI) or structural formula (XVII); and structural formula (VII) or structural formula (VIII). <Seventh monomer> N'-X'-[Linker (17)]s-{Y-[Linker (18)]t-Y'}u-C' (XVI) or N'-X'-[Linker (17)]s-{Y'-[Linker (18)]t-Y}u-C' (XVII); and N'-X''-[Linker (8)]i-{Y'-[Linker (9)]j-Y}k-C' (VII) or N'-X''-[Linker (8)]i-{Y-[Linker (9)]j-Y'}k-C' (VIII) In formula (VII), formula (VIII), formula (XVI) and formula (XVII), N' is the N-terminus of the seventh monomer, C' is the C-terminus of the seventh monomer, X is an antigen binding protein representing an antigen binding site of an antibody or a fragment thereof, wherein X comprises X' and X'', X' is an antigen binding site comprising a heavy chain variable region (VH) and a CH1 region, X'' is an antigen binding site comprising a light chain variable region (VL) and a constant region (CL), Y is an interferon protein or a fragment thereof, Y' is an extracellular domain fragment of an interferon α receptor or a variant thereof, the linker (8), the linker (9), the linker (17) and the linker (18) are peptide linkers, and s, t, u, i, j and k are each independently 0 or 1, and one or more of u and k are 1.

The interferon protein or fragment thereof, the extracellular domain fragment of the interferon α receptor or its variant, the target protein and the antigen-binding protein are as described above.

Here, peptide linker (8), linker (9), linker (17) and linker (18) may each independently be a cleavable linker or a non-cleavable linker. Here, the cleavable linker and the non-cleavable linker are as described above. In one embodiment, linker (8), linker (9), linker (17) and linker (18) may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 59 and combinations thereof. Preferably, it may include the amino acid sequence of SEQ ID NO: 33. Fusion protein comprising an extracellular domain fragment of an interferon protein and an interferon α receptor or a variant thereof

In another aspect, the present disclosure provides a fusion protein comprising an interferon protein or a fragment thereof; and an extracellular domain fragment of an interferon α receptor or a variant thereof. The interferon protein or a fragment thereof; and the extracellular domain fragment of an interferon α receptor or a variant thereof are as described above.

In the present disclosure, the fusion protein may be a dimer.

In the present disclosure, when the fusion protein is a dimer, the fusion protein may be a dimer formed by: i) a fourth monomer including an interferon protein or a fragment thereof, and ii) a fifth monomer including an extracellular domain fragment of an interferon α receptor or a variant thereof. The interferon protein or a fragment thereof; and the extracellular domain fragment of an interferon α receptor or a variant thereof are as described above. Here, the fourth monomer and the fifth monomer may each include a linker. The linker may be an Fc region or a fragment thereof. The linker and the Fc region or a fragment thereof are as described above.

Specifically, the fourth monomer can be composed of the following structural formula (IX) or structural formula (X). <Fourth monomer> N'-Y-[Linker (10)]l-Fc region-C' (IX) or N'-[Linker (11)]m-Fc region-[Linker (12)]n-Y-C' (X) In structural formula (IX) and structural formula (X), N' is the N-terminus of the fourth monomer, C' is the C-terminus of the fourth monomer, Y is an interferon protein or a fragment thereof, The linker (10) to the linker (12) is a peptide linker, and

l and m are 1, and n is 0 or 1.

The fifth monomer may be composed of the following structural formula (XI) or structural formula (XII). <Fifth monomer> N'-Y'-[Linker (13)]o-Fc region-C' (XI) or N'-[Linker (14)]p-Fc region-[Linker (15)]q-Y'-C' (XII) Here, in structural formula (XI) and structural formula (XII), N' is the N-terminus of the fifth monomer, C' is the C-terminus of the fifth monomer, Y is an extracellular domain fragment of the interferon α receptor or a variant thereof, The linker (13) to the linker (15) is a peptide linker, and o and p are 1, and q is 0 or 1.

Here, the interferon protein or a fragment thereof, the extracellular domain fragment of the interferon α receptor or a variant thereof, and the Fc region or a fragment thereof are as described above.

Here, the peptide linker (10), linker (11), linker (13) or linker (14) may consist of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, 10 to 60 consecutive amino acids or 12 to 50 amino acids. In one embodiment, the peptide linker (10), linker (11), linker (13) or linker (14) may consist of 30 amino acids. In addition, the peptide linker (10), linker (11), linker (13) or linker (14) may include at least one cysteine. Specifically, it may include one, two or three cysteine. In addition, the peptide linker (10), linker (11), linker (13) or linker (14) may be derived from the hinge of an immunoglobulin. In one embodiment, the peptide linker (10), linker (11), linker (13) or linker (14) may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 31 and SEQ ID NO: 166. Preferably, it may include the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 166.

The peptide linker (12) or linker (15) can be independently a cleavable linker or a non-cleavable linker. Here, the cleavable linker and the non-cleavable linker are as described above. In one embodiment, the peptide linker (12) or linker (15) can include any one amino acid sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 59 and combinations thereof. Preferably, it can include the amino acid sequence of SEQ ID NO: 33.

More specifically, the fusion protein of the present disclosure may be a heterodimer formed by a fourth monomer (composed of formula (IX)) and a fifth monomer (composed of formula (XI)). The fusion protein of the present disclosure may be a heterodimer formed by a fourth monomer (composed of formula (X)) and a fifth monomer (composed of formula (XII)).

When the fusion protein is a heterodimer, it can be bound by a cysteine included in a linker located at the N-terminus of the Fc region or fragment thereof in the fourth and fifth monomers. Here, the linker may include a hinge region of an immunoglobulin.

In addition, the Fc region or fragment thereof of the fusion protein may include variants for heterodimer formation. Variants of the Fc region that promote heterodimer formation are as described above.

In the present disclosure, the fusion protein may be a monomer.

In the present disclosure, when the fusion protein is a monomer, the fusion protein may be a sixth monomer, wherein i) the interferon protein or a fragment thereof and ii) the extracellular domain fragment of the interferon α receptor or a variant thereof are linked via a linker.

Specifically, the sixth monomer can be composed of the following structural formula (XIII) or structural formula (XIV). ＜Sixth monomer＞ N'-Y-[Linker (16)]r-Y'-C' (XIII) N'-Y'-[Linker (16)]r-Y-C' (XIV) Here, in structural formula (XIII), N' is the N-terminus of the sixth monomer, C' is the C-terminus of the sixth monomer, Y is an interferon protein or a fragment thereof, Y' is an extracellular domain fragment of an interferon α receptor or a variant thereof, the linker (16) is a peptide linker, and

r is 0 or 1.

Here, the interferon protein or a fragment thereof, the extracellular domain fragment of the interferon α receptor or a variant thereof, and the Fc region or a fragment thereof are as described above.

Here, the peptide linker (16) may be a cleavable linker or a non-cleavable linker. Here, the cleavable linker and the non-cleavable linker are as described above. In one embodiment, the linker (16) may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 59 and combinations thereof. Preferably, it may include the amino acid sequence of SEQ ID NO: 33.

The sixth monomer may further include a carrier at the N-terminus or C-terminus. Here, the carrier may be albumin, or the Fc region of an immunoglobulin or a fragment thereof.

In addition, the carrier may further include an antigen binding protein specific for the target protein. The antigen binding protein may be an antibody or a fragment thereof. The target protein and the antigen binding protein are as described above.

In one embodiment, the interferon protein or fragment thereof referred to as Y in the first to sixth monomers may be interferon α or interferon β. More specifically, it may be selected from interferon α2a, interferon α2b, interferon β1a and interferon β1b. Polynucleotides encoding immunocytokine or fusion protein

Another aspect of the present disclosure provides a polynucleotide encoding an interferon α receptor fragment or a variant thereof, an immunocytokine or a fusion protein. Here, the interferon α receptor fragment or a variant thereof, an immunocytokine and a fusion protein are as described above.

Specifically, the polynucleotide encoding the immunocytokine may include the polynucleotide encoding the first monomer and the second monomer. The polynucleotide encoding the immunocytokine may include the polynucleotide encoding the third monomer. The polynucleotide encoding the fusion protein may include the polynucleotide encoding the fourth monomer and the fifth monomer. The polynucleotide encoding the fusion protein may include the polynucleotide encoding the sixth monomer.

In addition, when the polynucleotides encode the same polypeptide, one or more base groups may be modified by substitution, deletion, insertion, or a combination thereof. When the polynucleotide sequence is prepared by chemical synthesis, synthetic methods well known in the art may be used, such as those described in Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988, such as triester, phosphite, phosphoramidite, and H-phosphoester methods, PCR and other autoprimer methods, and synthetic oligonucleotides on solid supports.

The polynucleotide may further include a signal sequence or leader sequence. As used herein, the term "signal sequence" refers to a nucleic acid encoding a signal peptide that directs secretion of a target protein. The signal peptide is cleaved after it is translated in a host cell. Specifically, the signal sequence of the present disclosure is a polynucleotide encoding an amino acid sequence that initiates protein transport across the endoplasmic reticulum (ER) membrane.

Signal sequences are well known in the art and typically include 16 to 30 amino acid residues, but may include more or fewer. 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 includes 4 to 12 hydrophobic residues that anchor the signal sequence throughout the membrane lipid bilayer during transport of the immature polypeptide.

After initiation, the signal sequence is usually cleaved by a cellular enzyme called a signal peptidase in the lumen of the ER. Here, the signal sequence may be a signal sequence of tissue fibronectin lysozyme activator (tPa), herpes simplex virus glycoprotein D (HSV gD), an IgG signal sequence, or a secretory signal sequence of growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells including mammals can be used. Signal sequences that can be used in the present disclosure include antibody light chain signal sequences, such as antibody 14.18 (Gillies et al., J. Immunol. Meth 1989. 125: 191-202), antibody heavy chain signal sequences, such as the MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature, 1980. 286: 676-683) and other signal sequences known in the art (see, e.g., Watson et al., Nucleic Acid Research, 1984. 12: 5145-5164). In one embodiment, the signal sequence may include the amino acid sequence of SEQ ID NO: 32. Vectors carrying polynucleotides

Another aspect of the present disclosure provides a presentation vector carrying a polynucleotide encoding an interferon alpha receptor fragment or variant thereof, an immunocytokine or a fusion protein. Here, the interferon alpha receptor fragment or variant thereof, an immunocytokine, a fusion protein and a polynucleotide are as described above.

As used herein, the term "vector" can be introduced into a host cell and recombined and inserted into the host cell genome. Alternatively, a vector is understood to be a nucleic acid construct comprising a nucleotide sequence configured to replicate spontaneously as a free genome. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, minichromosomes, and the like. 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. In addition, the vector may be a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, pcDNA, etc.), a plasmid derived from E. coli (pYG601BR322, pBR325, pUC118, pUC119, etc.), a plasmid derived from Bacillus subtilis (pUB110, pTP5, etc.), a plasmid derived from yeast (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (bacivirus, etc.), etc. Since the protein expression level 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 can include a selectable marker, such as an antibiotic resistance gene, and the host cells maintaining the plasmid can be cultured under selective conditions.

As used herein, the term "gene expression" or "expression" of the desired protein is understood to mean transcription of the DNA sequence, translation of the mRNA transcript, and secretion of the immunocytokine or fusion protein product or fragments thereof. A suitable expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may include the human cytomegalovirus (CMV) promoter for promoting continuous transcription of the desired gene in mammalian cells, and the bovine growth hormone polyadenylation signal sequence for increasing the steady-state level of the transcribed RNA. Transformed cells

Another aspect of the present disclosure provides a cell transformed by an expression vector, wherein the expression vector carries a polynucleotide encoding an immunocytokine or a fusion protein. Here, the immunocytokine, fusion protein, polynucleotide and expression vector are as described above.

As used herein, the term "transformed cells" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. Transformed cells can be generated by introducing and transforming the vector into a host cell. In addition, the polynucleotides included in the vector can be expressed to produce immunocytokines or fusion proteins according to the present disclosure.

Transformation can be performed by a variety of methods. Such methods are not particularly limited as long as they can produce the immunocytokine or fusion protein according to the present disclosure. Specifically, transformation methods such as _CaCl2 precipitation, Hanahan method (wherein dimethyl sulfoxide (DMSO) is used as a reducing agent in the _CaCl2 precipitation method to increase efficiency), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, agrobacteria-mediated transformation, transformation using PEG, dextran sulfate, lipofectamine and drying/inhibition-mediated transformation can be used. In addition, the desired substance can be delivered to the cell by infection using viral particles. In addition, the vector can be introduced into the host cell by gene bombardment, etc.

In addition, the host cell used to produce the transformed cell is not particularly limited, as long as it can produce the immunocytokine or fusion protein according to the present disclosure. Specifically, the host cell may include prokaryotic cells, eukaryotic cells, mammals, plants, insects, fungi, or cells of cell origin, but is not limited thereto. An example of a prokaryotic cell may be Escherichia coli. In addition, an example of a eukaryotic cell may be yeast. Mammalian cells may include CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2/0 cells, human lymphoblastoid cells, NSO cells, HT-1080 cells, PERC.6 cells, HEK 293 cells, or HEK293T cells, but are not limited thereto, and any cells known to those skilled in the art may be used as long as they can be used as mammalian host cells.

In addition, in order to optimize the characteristics of immunocytokines or fusion proteins as therapeutic agents or for other purposes, the glycosylation pattern of the antibody (e.g., sialic acid, fucosylation, glycosylation) can be modulated by manipulating the glycosylation-related genes of the host cells by methods known to those skilled in the art. Methods for producing immunocytokines or fusion proteins

Another aspect of the present disclosure provides a method for producing an immunocytokine or a fusion protein, comprising i) culturing transformed cells; and ii) obtaining the immunocytokine or the fusion protein from the culture medium. Here, the immunocytokine and the fusion protein are as described above.

As used herein, the term "culturing" refers to growing microorganisms (eg, transformed cells) under appropriate and artificially controlled environmental conditions.

The method of culturing the transformed cells can be carried out using methods widely known in the art. Specifically, the culture is not particularly limited as long as it is configured to express and produce the immunocytokine or fusion protein according to the present disclosure. Specifically, the culture can be carried out continuously in a batch process or in a fed batch or repeated fed batch process.

In addition, obtaining immunocytokines or fusion proteins from culture products can be carried out by methods known in the art. Specifically, the obtaining method is not particularly limited as long as it is configured to produce immunocytokines or fusion proteins according to the present disclosure. Preferably, the obtaining method may be centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion). Uses of immunocytokines or fusion proteins

Another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating cancer, which includes an immunocytokine or fusion protein as an active ingredient. Here, the immunocytokine and fusion protein are as described above.

The cancer may be any one selected from the following: multiple myeloma, gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, esophageal cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, neuroglioma, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, melanoma and lymphoma, but is not limited thereto.

The preferred dosage of the pharmaceutical composition varies according to the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition of the present disclosure for tumor treatment or prevention, the active ingredient may be included in any amount (effective amount) depending on the application, formulation, blending purpose, etc., as long as it exhibits tumor treatment activity or particularly exhibits a therapeutic effect on cancer. Based on the total weight of the composition, a typical effective amount will be determined to be in the range of 0.001% by weight to 20.0% by weight. Here, the term "effective amount" refers to an amount of an active ingredient that can improve disease symptoms or induce therapeutic effects, and in particular improve cancer symptoms or induce cancer therapeutic effects. Such an effective amount can be determined experimentally within the normal ability of those skilled in the art.

As used herein, the term "treatment" may be used to include both therapeutic treatment and preventive treatment. Here, prevention may be used to mean the alleviation or reduction of a pathological condition or disease in an individual. In one embodiment, the term "treatment" includes the administration or any form of administration for the treatment of a disease in a mammal, including humans. In addition, the term includes inhibiting or slowing the progression of a disease; and includes the following meanings: restoring or repairing a damaged or lost function, so that the disease is partially or completely alleviated; or stimulating an inefficient process; or relieving a serious disease.

Pharmacokinetic parameters (such as bioavailability) and basal parameters (such as clearance) can also affect efficacy. Thus, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and improved efficacy, which can be measured by comparing parameters such as clearance and tumor cure or improvement in test animals or human subjects.

At the same time, the pharmaceutical composition of the present disclosure is administered in a therapeutically effective amount.

Herein, "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition that effectively prevents or treats a target disease, which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. The level of an effective amount can be determined by factors such as the patient's health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the mode of administration, the time of administration, the route of administration and the rate of secretion, the duration of treatment, the drugs used in combination or simultaneously, and other factors well known in the medical field. In one embodiment, a therapeutically effective amount refers to an amount of a drug that is effective in treating cancer.

As used herein, the term "administered" means introducing a given substance into an individual by an appropriate method, and the composition can be administered via any common route that allows the substance to reach the desired tissue. Preferably, it can be parenteral administration, which may include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration, but is not limited thereto.

The preferred dosage of the pharmaceutical composition may be in the range of 0.01 μg/kg to 10 g/kg or 0.01 mg/kg to 1 g/kg per day, depending on the patient's condition, weight, sex and age, severity of the patient's condition and route of administration. The dosage may be administered once a day or may be divided into several times a day. Such dosages should not be interpreted in any manner as limiting the scope of the present disclosure.

The subjects to which the (specified) pharmaceutical composition can be administered are mammals and humans, preferably humans.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier that is a non-toxic substance suitable for delivery to a patient. It may include distilled water, alcohol, fat, wax and inert solids as carriers. The pharmaceutical composition may also include pharmacologically acceptable adjuvants (buffers, dispersants).

Specifically, the pharmaceutical composition can be prepared as a parenteral formulation by a known method known in the art depending on the route of administration, which includes a pharmaceutically acceptable carrier and an active ingredient. Here, the term "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have more toxicity than the toxicity that the individual to whom it is administered can tolerate.

When the pharmaceutical composition is formulated as a parenteral formulation, it can be formulated into an injectable preparation, a transdermal preparation, a nasal inhalation and a suppository together with a suitable carrier according to methods known in the art. If formulated as an injection, suitable carriers include sterile water, ethanol, polyols (such as glycerol or propylene glycol) or mixtures thereof. Preferably, isotonic solutions such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection or 5% dextrose can be used. The formulation of the pharmaceutical composition is well known in the art, and specific reference can be made to Remington's Pharmaceutical Sciences (19th edition, 1995) and the like. The above documents are considered part of this specification.

In addition to the active ingredient, the pharmaceutical composition of the present disclosure may further include any compound or natural extract known to have a tumor therapeutic effect, or may be used in combination with a separate composition including the above substances (hereinafter referred to as a therapeutic agent).

The term "combination" includes two or more therapeutic agents included in the same formulation or in separate formulations. When a pharmaceutical composition is prepared in a separate formulation from the therapeutic agent and used in combination, the pharmaceutical composition and the therapeutic agent can be mixed and administered "simultaneously", or each can be administered sequentially. When a pharmaceutical composition is administered sequentially with another therapeutic agent, it can be administered before and/or after the administration of the therapeutic agent. Therapeutic agents include, for example, but are not limited to, cytotoxic agents, interleukins, agents targeting immune checkpoint molecules, agents targeting immune stimulatory molecules, growth inhibitors, immunostimulators, anti-inflammatory agents, or anticancer agents.

Another aspect of the present disclosure provides an immunocytokine or fusion protein for preventing or treating cancer, and a pharmaceutical composition comprising the same for preventing or treating cancer. In this article, immunocytokine, fusion protein, cancer, prevention and treatment are as described above.

Another aspect of the present disclosure provides the use of immunocytokine, fusion protein and pharmaceutical composition comprising the same, which is used to manufacture a medicament for preventing or treating cancer. Here, immunocytokine, fusion protein, cancer, prevention and treatment are as described above.

Another aspect of the present disclosure provides a method for preventing or treating cancer, which includes administering immunocytokine, fusion protein and pharmaceutical composition comprising the same to an individual. Here, immunocytokine, fusion protein, cancer, administration, prevention and treatment are as described above.

The "individual" may be a mammal, preferably a human. In addition, the individual may be a patient suffering from cancer or an individual susceptible to cancer.

Regarding the route of administration, dosage, and frequency of administration, the composition can be administered in various ways and amounts depending on the patient's condition and the presence or absence of side effects. A person skilled in the art can appropriately select the optimal range of the administration method, dosage, and frequency of administration. In addition, the composition can be administered in combination with any compound or natural extract known to have a cancer therapeutic effect, or can be formulated with other drugs into a combined preparation. Extracellular domain fragment of interferon alpha receptor 2 or its variant

Another aspect of the present disclosure provides an extracellular domain fragment of interferon α receptor 2 or a variant thereof.

The extracellular domain fragment of interferon α receptor 2 or its variant may include a polypeptide represented by the following structural formula (XV). [N-terminal extension domain] ab-core domain (XV) In structural formula (XV),

The core domain is a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, the N-terminal extension domain is a polypeptide comprising 1 to 39 consecutive amino acids starting from position 39 of the amino acid sequence of SEQ ID NO: 9 in the C-terminal to N-terminal direction, and ab is 0 or 1.

Here, variants may include variants in the core domain. Specifically, in the variant, any one amino acid selected from the group consisting of positions 7 to 12, 14, 21, 32, 33, 38 to 40, 42, 44, 60, 62, 64, 65 and combinations thereof in the amino acid sequence of SEQ ID NO: 8 may be substituted by another amino acid. They correspond to positions 46 to 51, 53, 60, 71, 72, 77 to 79, 81, 83, 99, 101, 103 and 104 in the amino acid sequence of the full-length extracellular domain of interferon alpha receptor 2 (SEQ ID NO: 5), respectively.

More specifically, in the variant, at least any one or more amino acids selected from the group consisting of positions 7 to 10, 14, 38 to 40, 42, 62, 65 and combinations thereof in the amino acid sequence of SEQ ID NO: 8 may be substituted by another amino acid, which correspond to positions 46 to 49, 53, 77 to 79, 81, 101 and 104 in the amino acid sequence of the full-length extracellular domain of interferon alpha receptor 2 (SEQ ID NO: 5), respectively.

The extracellular domain fragment of interferon α receptor 2 or its variant may further include a D2 subdomain, and the D2 subdomain may include the amino acid sequence of SEQ ID NO: 7.

In addition, the extracellular domain variants of interferon alpha receptor 2 may further include variants in the D2 subdomain. Specifically, the variant of the D2 subdomain may be any amino acid selected from the group consisting of the following amino acid residues at positions 28, 31, 33, 35, 48, 54, 81 to 85 and 87 of the amino acid sequence of SEQ ID NO: 7, which correspond to positions 134, 137, 139, 141, 154, 160, 187 to 191 and 193 in the amino acid sequence of the full-length extracellular domain of interferon alpha receptor 2 (SEQ ID NO: 5), respectively.

In the present disclosure, the extracellular domain fragment of interferon α receptor 2 or its variant may further include a carrier. In addition, the carrier may further include an antigen binding protein.

Interferon α receptor, extracellular domain fragment of interferon α receptor 2 and variants thereof, D2 subdomain and variants thereof, and antigen binding protein are as described above. Mode of the Invention

Hereinafter, the present disclosure will be described in detail with the aid of the following examples. However, the following examples are intended only to illustrate the present disclosure, and the present disclosure is not limited thereto. Example 1. Confirmation of the characteristics of variants of the extracellular domain of IFNAR2 Example 1.1 Generation of variants of the extracellular domain of IFNAR2

Based on the crystal structure (PDB code: 3SE3) and the nuclear magnetic resonance structure (PDB code: 2KZ1), the amino acid residues that are critical for the binding of interferon α receptor 2 (IFNAR2) and interferon α2b (IFNα2b, SEQ ID NO: 2) were selected ( FIG. 2 ).

Alanine scanning was performed to replace the selected 31 amino acid residues with alanine, thereby generating variants of the extracellular domain of interferon alpha receptor 2 (Table 2). The N-terminus of the extracellular domain of IFNAR2 (SEQ ID NO: 5) or its variant was fused to the C-terminus of the hIgG1 Fc region (SEQ ID NO: 10) using a linker (SEQ ID NO: 33), wherein the signal sequence (SEQ ID NO: 32) was located at the N-terminus, thereby generating variants in the form of hIgG1-Fc-IFNAR2 fusion proteins. Example 1.2 Evaluation of the binding affinity of variants of the extracellular domain of IFNAR2 to interferon alpha 2b

The hIgG Fc-IFNR2 fusion protein produced in Example 1.1 was used to confirm its binding strength to IFNα2b by surface plasmon resonance (SPR) analysis.

Specifically, anti-human Fc IgG antibodies were immobilized on CM5 sensor chips. To immobilize anti-human Fc IgG, CM5 sensor chips (Cytiva) were pre-activated with activation buffer including 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Cytiva). Anti-human Fc IgG was diluted to a concentration of 25 μg/ml in 10 mM NaAc (pH 4.5) and immobilized via amine coupling reaction by flowing onto the activated CM5 sensor chip (Cytiva) at a flow rate of 10 μl/min. The test substance IgG1-Fc-IFNAR2 fusion protein was diluted in HBSEP (pH 7.4) and captured by flowing at a flow rate of 10 μl/min onto the sensor chip on which anti-human Fc IgG antibody had been immobilized.

The binding between hIgG1-Fc-IFNAR2 fusion protein and IFNα2b was measured by treating IFNα2b protein at a flow rate of 30 μl/min under the conditions of 2 minutes of binding and 5 minutes of dissociation. Here, IFNα2b protein was untreated (0 nM), or treated by 3-fold serial dilution from 400 nM to a concentration of 0.549 nM, or by 2-fold serial dilution from 10 nM to a concentration of 0.313 nM, depending on the type of hIgG1-Fc-IFNAR2 fusion protein. Between each operation, 10 mM glycine (pH 1.5) was injected to regenerate the sensor chip surface. The binding kinetics were analyzed using data analysis software (version 3.2), and the binding affinity was calculated using a 1:1 binding model and the data were fitted. The results are listed in Table 2. Here, "1.00E-06" was set as the minimum detection limit. [Table 2] Variant number IFNAR2 or its variants produced k _a (1/Ms) k _d (1/s) K _D (M) 1 IgG1 Fc-IFNαR2 wild type 3.90E+06 1.51E-02 3.87E-09 2 IgG1 Fc-IFNαR2 I46A 9.70E+05 1.08E-01 1.11E-07 3 IgG1 Fc-IFNαR2 M47A - - ＞ 1.00E-06 4 IgG1 Fc-IFNαR2 S48A 3.21E+06 3.21E-02 1.00E-08 5 IgG1 Fc-IFNαR2 K49A 3.39E+06 3.82E-02 1.12E-08 6 IgG1 Fc-IFNαR2 P50A 4.42E+06 1.74E-02 3.94E-09 7 IgG1 Fc-IFNαR2 E51A 3.24E+06 1.74E-02 5.38E-09 8 IgG1 Fc-IFNαR2 L53A 1.89E+06 6.96E-02 3.68.E-08 9 IgG1 Fc-IIFNαR2 A60R 3.05E+06 1.34E-02 4.39E-09 10 IgG1 Fc-IFNαR2 D71A 3.13E+06 1.50E-02 4.80E-09 11 IgG1 Fc-IFNαR2 E72A 3.65E+06 1.31E-02 3.59E-09 12 IgG1 Fc-IFNαR2 H77A - - 9.25E-09 13 IgG1 Fc-IFNαR2 E78A 4.48E+05 1.13E-01 2.52E-07 14 IgG1 Fc-IFNαR2 A79R - - ＞ 1.00E-06 15 IgG1 Fc-IFNαR2 V81A - - ＞ 1.00E-06 16 IgG1 Fc-IFNαR2 V83A 3.16E+06 1.39E-02 4.40E-09 17 IgG1 Fc-IFNαR2 N99A 5.19E+06 1.05E-02 2.03E-09 18 IgG1 Fc-IFNαR2 W101A 3.32E+06 3.83E-02 1.16E-08 19 IgG1 Fc-IFNαR2 A103R 2.58E+06 1.12E-02 4.36E-09 20 IgG1 Fc-IFNαR2 I104A 1.16E+06 9.02E-02 7.78.E-08 twenty one IgG1 Fc-IFNαR2 E134A 3.87E+06 1.18E-02 3.04E-09 twenty two IgG1 Fc-IFNαR2 Q137A 3.31E+06 2.14E-02 6.48E-09 twenty three IgG1 Fc-IFNαR2 D139A 3.44E+06 1.12E-02 3.26E-09 twenty four IgG1 Fc-IFNαR2 S141A 3.62E+06 1.37E-02 3.80E-09 25 IgG1 Fc-IFNαR2 K154A 4.04E+06 1.23E-02 3.03E-09 26 IgG1 Fc-IFNαR22 K160A 4.22E+06 1.50E-02 3.55E-09 27 IgG1 Fc-IFNαR2 E187A 3.63E+06 1.16E-02 3.19E-09 28 IgG1 Fc-IFNαR2 H188A 3.63E+06 1.46E-02 4.03E-09 29 IgG1 Fc-IFNαR2 S189A 3.98E+06 1.11E-02 2.79E-09 30 IgG1 Fc-IFNαR2 D190A 3.25E+06 1.23E-02 3.78E-09 31 IgG1 Fc-IFNαR2 E191A 3.26E+06 2.30E-02 7.04E-09 32 IgG1 Fc-IFNαR2 A193R 3.41E+06 2.14E-02 6.27E-09 33 IgG1 Fc-IFNαR2 (2Ser-217Lys) 3.69E+06 1.28E-02 3.48E-09

Therefore, 7 types of variants classified as having no or weak binding strength to IFNα2b and 4 types of variants whose binding strength was reduced by about 3 to 4 times compared with the binding strength to the extracellular domain of the wild-type interferon α receptor 2 were selected.

Various other variants of all 11 amino acid residues selected by the above process were predicted by the Discovery Studio program (DS program). Specifically, the DS program was used to predict the change in Gibbs free energy when each of the 11 amino acid residues of the extracellular domain of interferon alpha receptor 2 was substituted with 19 amino acids other than the wild-type amino acid. Among the prediction results, a total of 123 types of variants (these variants were predicted to have an energy change (ΔE) of 0.5 kcal/mol or more due to the change) were selected (Table 3). Here, "1.00E-06" was set as the minimum detection limit. [Table 3] Variant number IFNAR amino acid residues IFNAR variants produced k _a (1/Ms) k _d (1/s) K _D (M) 34 I46 IgG1 Fc-IFNαR2 I46D - - ＞ 1.00E-06 35 IgG1 Fc-IFNαR2 I46E 4.54E+05 1.01E-01 2.22E-07 36 IgG1 Fc-IFNαR2 I46F 3.01E+06 1.02E-01 3.39E-08 37 IgG1 Fc-IFNαR2 I46G 1.03E+06 1.80E-01 1.75E-07 38 IgG1 Fc-IFNαR2 I46N - - ＞ 1.00E-06 39 IgG1 Fc-IFNαR2 I46S 1.24E+06 1.60E-01 1.29E-07 40 IgG1 Fc-IFNαR2 I46W 1.74E+06 6.43E-02 3.70E-08 41 IgG1 Fc-IFNαR2 I46Y 1.73E+06 5.96E-02 3.45E-08 42 M47 IgG1 Fc-IFNαR2 M47D - - ＞ 1.00E-06 43 IgG1 Fc-IFNαR2 M47E - - ＞ 1.00E-06 44 IgG1 Fc-IFNαR2 M47G - - ＞ 1.00E-06 45 IgG1 Fc-IFNαR2 M47H - - ＞ 1.00E-06 46 IgG1 Fc-IFNαR2 M47I 4.00E+05 3.04E-01 7.61E-07 47 IgG1 Fc-IFNαR2 M47L - - ＞ 1.00E-06 48 IgG1 Fc-IFNαR2 M47N - - ＞ 1.00E-06 49 IgG1 Fc-IFNαR2 M47P - - ＞ 1.00E-06 50 IgG1 Fc-IFNαR2 M47S - - ＞ 1.00E-06 51 IgG1 Fc-IFNαR2 M47T - - ＞ 1.00E-06 52 IgG1 Fc-IFNαR2 M47V 3.80E+05 2.96E-01 7.78E-07 53 IgG1 Fc-IFNαR2 M47W - - ＞ 1.00E-06 54 IgG1 Fc-IFNαR2 M47Y 7.10E+05 2.45E-01 3.45E-07 55 S48 IgG1 Fc-IFNαR2 S48D - - ＞ 1.00E-06 56 IgG1 Fc-IFNαR2 S48E - - ＞ 1.00E-06 57 IgG1 Fc-IFNαR2 S48G - - ＞ 1.00E-06 58 IgG1 Fc-IFNαR2 S48L 9.85E+05 5.49E-01 5.57E-07 59 IgG1 Fc-IFNαR2 S48P - - ＞ 1.00E-06 60 IgG1 Fc-IFNαR2 S48V 5.21E+06 9.50E-02 1.82E-08 61 IgG1 Fc-IFNαR2 S48W 3.03E+06 6.48E-02 2.14E-08 62 K49 IgG1 Fc-IFNαR2 K49D ＞ 1.00E-06 63 IgG1 Fc-IFNαR2 K49E 1.89E+06 1.74E-01 9.21E-08 64 IgG1 Fc-IFNαR2 K49F 3.14E+06 4.51E-02 1.44E-08 65 IgG1 Fc-IFNαR2 K49G 3.72E+06 5.61E-02 1.51E-08 66 IgG1 Fc-IFNαR2 K49H 1.85E+06 4.76E-02 2.58E-08 67 IgG1 Fc-IFNαR2 K49I 4.07E+06 1.16E-02 2.86E-09 68 IgG1 Fc-IFNαR2 K49L 3.65E+06 1.81E-02 4.95E-09 69 IgG1 Fc-IFNαR2 K49M 3.74E+06 3.37E-02 9.02E-09 70 IgG1 Fc-IFNαR2 K49N 1.83E+06 1.05E-01 5.74E-08 71 IgG1 Fc-IFNαR2 K49P - - ＞ 1.00E-06 72 IgG1 Fc-IFNαR2 K49Q 3.86E+06 3.82E-02 9.92E-09 73 IgG1 Fc-IFNαR2 K49R 3.64E+06 1.43E-02 3.93E-09 74 IgG1 Fc-IFNαR2 K49S 4.10E+06 2.75E-02 6.72E-09 75 IgG1 Fc-IFNαR2 K49T 3.80E+06 1.80E-02 4.74E-09 76 IgG1 Fc-IFNαR2 K49V 4.19E+06 1.03E-02 2.45E-09 77 IgG1 Fc-IFNαR2 K49W 2.71E+06 3.82E-02 1.41E-08 78 IgG1 Fc-IFNαR2 K49Y 4.24E+06 3.63E-02 8.55E-09 79 L53 IgG1 Fc-IFNαR2 L53D N/D 80 IgG1 Fc-IFNαR2 L53E 2.12E+06 2.75E-01 1.30E-07 81 IgG1 Fc-IFNαR2 L53G N/D 82 IgG1 Fc-IFNαR2 L53K 1.14E+06 3.35E-01 2.94E-07 83 IgG1 Fc-IFNαR2 L53P 3.60E+06 2.40E-02 6.67E-09 84 IgG1 Fc-IFNαR2 L53Q 1.61E+06 2.69E-01 1.67E-07 85 IgG1 Fc-IFNαR2 L53Y 1.87E+06 1.64E-01 8.77E-08 86 H77 IgG1 Fc-IFNαR2 H77D 1.76E+06 1.51E-01 8.58E-08 87 IgG1 Fc-IFNαR2 H77E 1.69E+06 1.69E-01 1.00E-07 88 IgG1 Fc-IFNαR2 H77F 4.28E+06 2.08E-02 4.86E-09 89 IgG1 Fc-IFNαR2 H77G 1.80E+06 1.59E-01 8.83E-08 90 IgG1 Fc-IFNαR2 H77I 2.32E+06 5.47E-02 2.36E-08 91 IgG1 Fc-IFNαR2 H77K 1.39E+06 1.73E-01 1.24E-07 92 IgG1 Fc-IFNαR2 H77L 2.62E+06 1.55E-01 5.92E-08 93 IgG1 Fc-IFNαR2 H77M 1.97E+06 6.61E-02 3.36E-08 94 IgG1 Fc-IFNαR2 H77N 3.03E+06 5.08E-02 1.68E-08 95 IgG1 Fc-IFNαR2 H77P 8.58E+05 3.86E-02 4.50.E-08 96 IgG1 Fc-IFNαR2 H77Q 1.15E+06 9.81E-02 8.53E-08 97 IgG1 Fc-IFNαR2 H77R 6.50E+05 1.65E-01 2.54E-07 98 IgG1 Fc-IFNαR2 H77S 2.75E+06 4.49E-02 1.63E-08 99 IgG1 Fc-IFNαR2 H77T 1.62E+06 6.67E-02 4.12E-08 100 IgG1 Fc-IFNαR2 H77V 2.97E+06 5.04E-02 1.70E-08 101 IgG1 Fc-IFNαR2 H77W 7.21E+06 4.76E-02 6.60E-09 102 IgG1 Fc-IFNαR2 H77Y 5.73E+06 2.39E-02 4.18E-09 103 E78 IgG1 Fc-IFNαR2 E78D 1.26E+06 8.23E-02 6.53E-08 104 IgG1 Fc-IFNαR2 E78F - - ＞ 1.00E-06 105 IgG1 Fc-IFNαR2 E78G - - ＞ 1.00E-06 106 IgG1 Fc-IFNαR2 E78H 6.23E+05 1.88E-01 3.02E-07 107 IgG1 Fc-IFNαR2 E78K N/D 108 IgG1 Fc-IFNαR2 E78P - - ＞ 1.00E-06 109 IgG1 Fc-IFNαR2 E78S 4.90E+05 3.17E-01 6.47E-07 110 IgG1 Fc-IFNαR2 E78W - - ＞ 1.00E-06 111 IgG1 Fc-IFNαR2 E78Y - - ＞ 1.00E-06 112 A79 IgG1 Fc-IFNαR2 A79D 7.04E+06 3.01E-02 4.27E-09 113 IgG1 Fc-IFNαR2 A79E 4.94E+06 8.70E-02 1.76E-08 114 V81 IgG1 Fc-IFNαR2 V81D - - ＞ 1.00E-06 115 IgG1 Fc-IFNαR2 V81E - - ＞ 1.00E-06 116 IgG1 Fc-IFNαR2 V81F 5.84E+05 2.72E-01 4.66E-07 117 IgG1 Fc-IFNαR2 V81G - - ＞ 1.00E-06 118 IgG1 Fc-IFNαR2 V81H - - ＞ 1.00E-06 119 IgG1 Fc-IFNαR2 V81I 3.41E+06 4.11E-02 1.21E-08 120 IgG1 Fc-IFNαR2 V81K - - ＞ 1.00E-06 121 IgG1 Fc-IFNαR2 V81M - - ＞ 1.00E-06 122 IgG1 Fc-IFNαR2 V81N - - ＞ 1.00E-06 123 IgG1 Fc-IFNαR2 V81P - - ＞ 1.00E-06 124 IgG1 Fc-IFNαR2 V81Q - - ＞ 1.00E-06 125 IgG1 Fc-IFNαR2 V81R - - ＞ 1.00E-06 126 IgG1 Fc-IFNαR2 V81S - - ＞ 1.00E-06 127 IgG1 Fc-IFNαR2 V81T - - ＞ 1.00E-06 128 IgG1 Fc-IFNαR2 V81W - - ＞ 1.00E-06 129 IgG1 Fc-IFNαR2 V81Y - - ＞ 1.00E-06 130 W101 IgG1 Fc-IFNαR2 W101D 9.84E+05 9.40E-02 9.55E-08 131 IgG1 Fc-IFNαR2 W101E 5.40E+05 2.89E-01 5.35E-07 132 IgG1 Fc-IFNαR2 W101F 3.09E+06 1.25E-03 4.06E-10 133 IgG1 Fc-IFNαR2 W101G 8.48E+05 5.48E-02 6.46E-08 134 IgG1 Fc-IFNαR2 W101H 2.04E+06 4.91E-03 2.41E-09 135 IgG1 Fc-IFNαR2 W101I 1.22E+06 9.36E-02 7.67E-08 136 IgG1 Fc-IFNαR2 W101K - - ＞ 1.00E-06 137 IgG1 Fc-IFNαR2 W101L 2.88E+06 2.07E-02 7.18E-09 138 IgG1 Fc-IFNαR2 W101M 2.59E+06 1.09E-03 4.21E-10 139 IgG1 Fc-IFNαR2 W101N 2.55E+06 2.14E-02 8.41E-09 140 IgG1 Fc-IFNαR2 W101P - - ＞ 1.00E-06 141 IgG1 Fc-IFNαR2 W101Q 2.25E+06 8.90E-03 3.95E-09 142 IgG1 Fc-IFNαR2 W101R 4.16E+05 1.02E-01 2.45E-07 143 IgG1 Fc-IFNαR2 W101S - - ＞ 1.00E-06 144 IgG1 Fc-IFNαR2 W101T - - ＞ 1.00E-06 145 IgG1 Fc-IFNαR2 W101V 1.53E+06 5.72E-02 3.74E-08 146 IgG1 Fc-IFNαR2 W101Y 2.06E+06 4.36E-02 2.11E-08 147 I104 IgG1 Fc-IFNαR2 I104E 1.07E+06 1.85E-01 1.73E-07 148 IgG1 Fc-IFNαR2 I104G 1.33E+06 7.16E-02 5.38E-08 149 IgG1 Fc-IFNαR2 I104K 3.88E+05 2.01E-01 5.18E-07 150 IgG1 Fc-IFNαR2 I104L 3.54E+06 5.99E-03 1.69E-09 151 IgG1 Fc-IFNαR2 I104M 2.26E+06 3.58E-02 1.58E-08 152 IgG1 Fc-IFNαR2 I104P 5.38E+05 2.49E-01 4.63E-07 153 IgG1 Fc-IFNαR2 I104Q 1.07E+06 8.59E-02 8.03E-08 154 IgG1 Fc-IFNαR2 I104R 3.65E+05 2.30E-01 6.31E-07 155 IgG1 Fc-IFNαR2 I104S 1.20E+06 1.64E-01 1.37E-07 156 IgG1 Fc-IFNαR2 I104T 1.24E+06 1.27E-01 1.02E-07 * N/D: Not tested Example 2. Evaluation of the activity of immunocytokines including CD38 binding proteins Example 2.1. Preparation of immunocytokines including CD38 binding proteins

An immunocytokine (αCD38-IFNα2b/IFNαR2) comprising the variant of Example 1.1, IFNα2b and anti-CD38 antibody (αCD38) was prepared.

Specifically, the αCD38-IFNα2b/IFNαR2 immunocytokine is prepared by forming a heterodimer of a first monomer and a second monomer, wherein in the first monomer, a fusion protein including the heavy chain of an anti-CD38 antibody and IFNα2b is bound to the light chain of an anti-CD38 antibody, and in the second monomer, a fusion protein including the heavy chain of an anti-CD38 antibody and IFNαR2 or a variant thereof is bound to the anti-CD38 antibody.

Here, a fusion protein comprising the heavy chain of an anti-CD38 antibody and IFNα2b is prepared using a polynucleotide encoding a fusion protein, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-CD38 antibody (SEQ ID NO: 61), a heavy chain constant region 1 (SEQ ID NO: 71), a hinge (linker, SEQ ID NO: 166), an Fc region or a variant thereof (SEQ ID NO: 18), a linker (SEQ ID NO: 33) and IFNα2b (SEQ ID NO: 2).

In addition, a fusion protein comprising the heavy chain of an anti-CD38 antibody and IFNαR2 or a variant thereof was prepared using a polynucleotide encoding a fusion protein, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-CD38 antibody (SEQ ID NO: 61), a heavy chain constant region (SEQ ID NO: 71), a hinge (linker, SEQ ID NO: 166), an Fc region or a variant thereof (SEQ ID NO: 19), a linker (SEQ ID NO: 33), and an extracellular domain of IFNαR2 (SEQ ID NO: 5) or a variant thereof.

In addition, the light chain of the anti-CD38 antibody was prepared using a polynucleotide encoding a fusion protein comprising a signal sequence (SEQ ID NO: 32), a light chain variable region (SEQ ID NO: 62) and a light chain constant region (SEQ ID NO: 70) of the anti-CD38 antibody in order from N-terminus to C-terminus.

Representatively, a first monomer comprising the heavy and light chains of an anti-CD38 antibody and IFNα2b and a second monomer comprising the heavy and light chains of an anti-CD38 antibody and wild-type IFNαR2 were prepared by loading and expressing a polynucleotide comprising the base sequence of SEQ ID NO: 170, a polynucleotide comprising the base sequence of SEQ ID NO: 171, and a polynucleotide comprising the base sequence of SEQ ID NO: 168, respectively, on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The first monomer and the second monomer produced as described above are combined to form a heterodimer, and the heterodimer is named "αCD38-IFNα2b/IFNαR2" or "αCD38-IFNα2b/IFNαR2 variant". Example 2.2. Confirmation of the binding affinity of immunocytokines including CD38 binding proteins to CD38

To determine the kinetic binding affinity of αCD38-IFNα2b/IFNαR2 or its variants prepared in Example 2.1 to CD38, the binding affinity was measured by surface plasmon resonance analysis using Biacore 8K (Cytiva). Surface plasmon resonance analysis was performed in the same manner as in Example 1.2 using αCD38-IFNα2b/IFNαR2 or its variants as test substances. However, instead of IFNα2b, CD38 protein was not treated or treated by serial dilution 2-fold from 200 nM to 6.25 nM, and the results measured under the conditions of 3 minutes of binding and 5 minutes of dissociation are shown in Table 4.

Here, the anti-CD38 antibody produced in-house was used as a control and had a _KD (M) value of 4.48E-08.

The anti-CD38 antibody was produced to include a heavy chain and a light chain, the heavy chain including: a heavy chain variable region including the amino acid sequence of SEQ ID NO: 61, a heavy chain constant region 1 including the amino acid sequence of SEQ ID NO: 71, a hinge including the amino acid sequence of SEQ ID NO: 166, and an Fc region including the amino acid sequence of SEQ ID NO: 13; and the light chain including: a light chain variable region including the amino acid sequence of SEQ ID NO: 62, and a light chain constant region including the amino acid sequence of SEQ ID NO: 70. Specifically, it is produced by loading and expressing a polynucleotide comprising the base sequence of SEQ ID NO: 169 and a polynucleotide comprising the base sequence of SEQ ID NO: 168 on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific). [Table 4] Variant number αCD38-IFNα2b/IFNαR2 or its variants K _D (M) 1 αCD38-IFNα2b/IFNαR2 wild type 4.35E-08 2 αCD38-IFNα2b/IFNαR2 I46A 5.60E-08 3 αCD38-IFNα2b/IFNαR2 I46D 4.92E-08 4 αCD38-IFNα2b/IFNαR2 I46E 5.65E-08 5 αCD38-IFNα2b/IFNαR2 I46F 5.94E-08 6 αCD38-IFNα2b/IFNαR2 I46G 5.65E-08 7 αCD38-IFNα2b/IFNαR2 I46S 5.60E-08 8 αCD38-IFNα2b/IFNαR2 I46W 5.62E-08 9 αCD38-IFNα2b/IFNαR2 I46Y 5.60E-08 10 αCD38-IFNα2b/IFNαR2 M47A 5.43E-08 11 αCD38-IFNα2b/IFNαR2 M47I 5.15E-08 12 αCD38-IFNα2b/IFNαR2 M47V 5.00E-08 13 αCD38-IFNα2b/IFNαR2 M47Y 5.58E-08 14 αCD38-IFNα2b/IFNαR2 S48A 5.47E-08 15 αCD38-IFNα2b/IFNαR2 S48E 5.14E-08 16 αCD38-IFNα2b/IFNαR2 S48G 5.45E-08 17 αCD38-IFNα2b/IFNαR2 S48L 5.38E-08 18 αCD38-IFNα2b/IFNαR2 S48V 5.42E-08 19 αCD38-IFNα2b/IFNαR2 S48W 5.16E-08 20 αCD38-IFNα2b/IFNαR2 K49A 4.22E-08 twenty one αCD38-IFNα2b/IFNαR2 K49D 4.55E-08 twenty two αCD38-IFNα2b/IFNαR2 K49E 5.29E-08 twenty three αCD38-IFNα2b/IFNαR2 K49F 5.50E-08 twenty four αCD38-IFNα2b/IFNαR2 K49G 5.42E-08 25 αCD38-IFNα2b/IFNαR2 K49H 5.51E-08 26 αCD38-IFNα2b/IFNαR2 K49M 5.44E-08 27 αCD38-IFNα2b/IFNαR2 K49N 5.30E-08 28 αCD38-IFNα2b/IFNαR2 K49Q 5.28E-08 29 αCD38-IFNα2b/IFNαR2 K49Y 5.24E-08 30 αCD38-IFNα2b/IFNαR2 L53A 5.16E-08 31 αCD38-IFNα2b/IFNαR2 L53E 5.48E-08 32 αCD38-IFNα2b/IFNαR2 L53K 5.39E-08 33 αCD38-IFNα2b/IFNαR2 L53Q 5.46E-08 34 αCD38-IFNα2b/IFNαR2 L53Y 5.28E-08 35 αCD38-IFNα2b/IFNαR2 H77A 4.04E-08 36 αCD38-IFNα2b/IFNαR2 H77D 4.07E-08 37 αCD38-IFNα2b/IFNαR2 H77E 5.25E-08 38 αCD38-IFNα2b/IFNαR2 H77G 5.10E-08 39 αCD38-IFNα2b/IFNαR2 H77I 5.19E-08 40 αCD38-IFNα2b/IFNαR2 H77K 5.09E-08 41 αCD38-IFNα2b/IFNαR2 H77L 5.41E-08 42 αCD38-IFNα2b/IFNαR2 H77M 5.37E-08 43 αCD38-IFNα2b/IFNαR2 H77N 5.41E-08 44 αCD38-IFNα2b/IFNαR2 H77P 5.51E-08 45 αCD38-IFNα2b/IFNαR2 H77Q 5.26E-08 46 αCD38-IFNα2b/IFNαR2 H77R 5.34E-08 47 αCD38-IFNα2b/IFNαR2 H77S 4.86E-08 48 αCD38-IFNα2b/IFNαR2 H77T 4.95E-08 49 αCD38-IFNα2b/IFNαR2 H77V 5.14E-08 50 αCD38-IFNα2b/IFNαR2 E78A 5.11E-08 51 αCD38-IFNα2b/IFNαR2 E78D 5.29E-08 52 αCD38-IFNα2b/IFNαR2 E78H 5.23E-08 53 αCD38-IFNα2b/IFNαR2 E78S 4.87E-08 54 αCD38-IFNα2b/IFNαR2 E78Y 4.70E-08 55 αCD38-IFNα2b/IFNαR2 A79E 4.81E-08 56 αCD38-IFNα2b/IFNαR2 A79R 4.86E-08 57 αCD38-IFNα2b/IFNαR2 V81F 5.02E-08 58 αCD38-IFNα2b/IFNαR2 V81I 4.74E-08 59 αCD38-IFNα2b/IFNαR2 V81M 4.91E-08 60 αCD38-IFNα2b/IFNαR2 W101A 4.87E-08 61 αCD38-IFNα2b/IFNαR2 W101D 4.97E-08 62 αCD38-IFNα2b/IFNαR2 W101E 5.01E-08 63 αCD38-IFNα2b/IFNαR2 W101G 4.73E-08 64 αCD38-IFNα2b/IFNαR2 W101I 4.70E-08 65 αCD38-IFNα2b/IFNαR2 W101L 4.74E-08 66 αCD38-IFNα2b/IFNαR2 W101N 4.73E-08 67 αCD38-IFNα2b/IFNαR2 W101R 4.71E-08 68 αCD38-IFNα2b/IFNαR2 W101V 4.79E-08 69 αCD38-IFNα2b/IFNαR2 W101Y 4.87E-08 70 αCD38-IFNα2b/IFNαR2 I104A 5.08E-08 71 αCD38-IFNα2b/IFNαR2 I104E 4.79E-08 72 αCD38-IFNα2b/IFNαR2 I104G 4.81E-08 73 αCD38-IFNα2b/IFNαR2 I104K 5.22E-08 74 αCD38-IFNα2b/IFNαR2 I104M 4.75E-08 75 αCD38-IFNα2b/IFNαR2 I104P 5.08E-08 76 αCD38-IFNα2b/IFNαR2 I104R 5.03E-08 77 αCD38-IFNα2b/IFNαR2 I104Q 4.68E-08 78 αCD38-IFNα2b/IFNαR2 I104S 4.79E-08 79 αCD38-IFNα2b/IFNαR2 I104T 5.00E-08 Example 2.3. Evaluation of the IFNAR dimerization-inducing ability of immunoglobulins including CD38 binding proteins

IFNAR dimerization analysis of interferon alpha receptor 2 was performed using PathHunter HEK293 IFNAR1/2 cell line (93-1097C1) and HEK293 IFNAR1/2 CD38 cell line from DiscoverX. Here, HEK293 IFNAR1/2 CD38 cell line is a cell that expresses CD38 in addition to 93-1097C1 cells.

As the cell culture medium, Assay Complete Cell Culture Kit-105 (DiscoverX, 92-3105G) was used, and Assay Complete Hygromycin (DiscoverX, 92-0029) (0.25 μg/ml) and Assay Complete G418 (DiscoverX, 92-0030) (200 μg/ml) were used as antibiotics. Here, HEK293 IFNAR1/2 CD38 cell line was used together with Assay Complete Puromycin (DiscoverX, 92-0028) (800 μg/ml). Each cell was cultured in a 5% CO ₂ incubator, and a constant temperature and humidity condition of 37°C was maintained. During cell subculture, cells were detached from the culture dish by pipetting to minimize cell membrane destabilization caused by the enzyme.

The cells for drug treatment were seeded at 5×10 ⁴ cells/100 μl per well in a 96-well plate and used after culture for about 12 to 16 hours. Here, Assay Complete Cell Seeding Reagent 7 (DiscoverX, 93-0563R7A) was used as the culture solution.

The drugs used to treat cells were dissolved in Hanks' Balanced Salt Solution (HBSS, Gibco, 14175095) and prepared at a concentration 11 times the final treatment concentration. The drug treatment time was set to 16 hours according to the information provided by DiscoverX. After the reaction, the dimerization degree of interferon α receptor on the cell membrane was confirmed by measuring the luminescence reaction using Flexstation 3 (Molecular devices) using the PathHunter detection kit (DiscoverX, 93-0001). Using the above results, concentration response curves of the test substances were prepared by performing 4-parameter logic analysis (4PL analysis) using Graphpad Prism version 7.0, and the maximum response value and _EC50 (treatment concentration of the test substance required to induce half of the maximum response value of the drug) value were calculated.

Using the above method, the immunocytokine (αCD38-IFNα2b/IFNαR2 or its variants) prepared in Example 2.1 was first treated with the HEK293 IFNAR1/2 cell line (93-1097C1) or the HEK293 IFNAR1/2 CD38 cell line at a concentration of 3 nM to select the immunocytokine with excellent dimerization ability of interferon α receptor 2 in each cell (Figure 4A, Figure 4B, Figure 5A and Figure 5B).

The selected immunocytokines were again treated at different concentrations (1000, 166, 27.8, 4.63, 0.772, 0.129, 0.021, 0.004 or 0 nM) in each HEK293 IFNAR1/2 cell or HEK293 IFNAR1/2 CD38 cell, and the results are shown in FIG6 and FIG7.

Therefore, compared with the treatment with IFNα alone, the immunocytokine (αCD38-IFNα2b/IFNαR2 or its variant) showed a high EC ₅₀ value. Based on the above results, it is believed that IFNαR2 of αCD38-IFNα2b/IFNαR2 or its variant shields IFNα2b as a masking part of IFNα2b, thereby preventing it from binding to the receptor (IFNAR) present on the cell membrane. Through the above results, it is confirmed that the shielding ability of αCD38-IFNα2b/IFNαR2 or its variant according to one embodiment of the present disclosure for IFNα2b is well retained.

In addition, it was confirmed that the EC ₅₀ value of αCD38-IFNα2b/IFNαR2 or its variants in cells expressing CD38 was lower. The above results indicate that the binding strength between IFNα2b of the αCD38-IFNα2b/IFNαR2 variant and the receptor (IFNAR) present on the cell membrane is increased. It is believed that immunocytokine first binds to CD38 present on the cell membrane through the anti-CD38 antibody of αCD38-IFNα2b/IFNαR2 or its variants, thereby obtaining the IFNα2b component, which is physically closer to the IFNAR present on the cell membrane to produce the above results.

The above results confirm that αCD38-IFNα2b/IFNαR2 or its variant according to one embodiment of the present disclosure has the ability to shield IFNα2b and has an increased ability to bind to IFNAR at the target (CD38) specific site. Example 2.4. Evaluation of the STAT1 activation ability of immunoglobulins including CD38 binding proteins

The STAT1 activity induction ability (pSTAT1 induction ability) of αCD38-IFNα2b/IFNαR2 or its variants was assessed using the HTRF human and mouse phospho-STAT (1Tyr701) detection kit (revvity, 63ADK026PEH).

Specifically, the same two types of cells as in Example 2.3 were diluted in HBSS (Gibco, 14175095) and seeded at a concentration of 1.0×10 ⁵ cells/8 μl per well in a HTRF low volume 96-well plate (revvity, 66PL96005). The test substance (αCD38-IFNα2b/IFNαR2 or its variant) was prepared at 3 times the final treatment concentration (1000, 166, 27.8, 4.63, 0.772, 0.129, 0.021, 0.004 or 0 nM).

Each cell prepared as above was treated with the test substance and reacted for 20 minutes. After the reaction was completed, the cells were treated with the lysis buffer included in the HTRF Human and Mouse Phospho-STAT 1 (Tyr701) Detection Kit (revvity, 63ADK026PEH), and reacted at room temperature for 30 minutes to lyse the cells. Thereafter, the lysate was treated with a mixture of the anti-phospho-STAT1 antibody-Eu reagent and the anti-pSTAT1 antibody-d2 reagent included in the kit, and reacted overnight at room temperature in dark conditions. After the reaction was completed, the fluorescence value of the reaction product was measured using a Flexstation3 device (HTRF standard analysis Eu protocol, 665 nm/620 nm). Here, human IFNα (hIFNα) was used as a base control to normalize each of the values obtained by treatment with the test substances.

The measured values are used to calculate the final pSTAT1 signal result using the following equation I. < Equation I> pSTAT1 signal result (ratio) = (Signal 665nm)/(Signal 620nm) × 10000

Using the pSTAT1 signal results calculated as above, 4PL analysis was performed using Graphpad Prism version 7.0 to prepare a concentration response curve, which was used to calculate the maximum response value and EC ₅₀ value of the test substance. The maximum response value, minimum response value, Hill slope value and EC ₅₀ value of each αCD38-IFNα2b/IFNαR2 or its variants confirmed in each cell are shown in Table 5. [Table 5] Variant position Values obtained from HEK293 IFNAR1/2 cells Values obtained from HEK293 IFNAR1/2 CD38 cells Minimum response value relative to IFNα2b Relative to the maximum response value of IFNα2b EC ₅₀ (M) Hill slope Minimum response value relative to IFNα2b Relative to the maximum response value of IFNα2b EC ₅₀ (M) Hill slope Wild type 8.15 85.67 1.52E-07 1.38 1.75 77.73 4.06E-08 1.18 K49A 9.35 Unable to obtain maximum response value 2.76E-07 1.57 1.43 76.84 1.43E-08 1.31 K49D 12.51 65.20 1.45E-07 3.53 2.30 89.70 1.44E-08 1.10 H77A 0.26 77.00 2.87E-07 0.82 4.21 77.43 9.33E-09 1.22 H77D 7.79 62.69 1.84E-07 2.11 4.52 80.79 9.94E-09 1.08 I46A 9.61 109.21 1.55E-07 2.39 3.09 73.31 2.54E-08 0.91 I46E 0.09 132.71 3.12E-07 0.90 1.59 68.65 8.47E-09 1.41 I46F 0.16 119.79 1.70E-07 0.62 2.71 85.85 1.77E-08 0.92 I46G 9.90 Unable to obtain maximum response value 5.32E-07 0.95 3.06 82.83 7.31E-09 1.00 I46S 1.73 141.32 2.38E-07 0.77 5.14 80.57 1.21E-08 1.71 I46W 13.04 85.18 1.37E-07 2.41 4.25 101.03 2.33E-08 0.91 I46 6.62 75.80 4.53E-08 2.19 3.08 85.61 1.59E-08 1.52 M47A 7.82 68.85 6.32E-08 1.78 6.49 94.33 8.00E-09 0.80 M47Y 0.23 90.33 1.19E-07 0.95 3.51 87.83 8.42E-09 1.30 S48A 1.81 86.26 2.59E-07 1.20 3.80 92.01 3.63E-08 1.19 S48E 8.70 86.94 7.55E-08 1.90 4.63 94.85 1.05E-08 0.96 S48G 8.15 98.17 1.57E-07 1.55 8.44 92.94 5.45E-09 6.65 S48L 0.22 84.04 8.32E-08 1.64 2.04 103.58 1.45E-08 0.80 S48V 11.12 125.70 1.26E-07 1.57 5.73 102.38 9.42E-09 1.52 S48W 12.26 76.91 1.31E-07 6.22 4.76 93.14 1.73E-08 1.22 K49E 11.01 104.06 2.07E-07 1.28 5.06 96.61 1.98E-08 0.95 K49F 6.25 87.29 2.25E-07 0.86 7.44 89.57 2.68E-08 6.73 K49G 8.42 90.54 1.57E-07 1.03 7.30 87.10 2.44E-08 1.39 K49H -0.02 57.55 5.84E-07 1.88 3.47 84.96 1.92E-08 0.93 K49M -0.03 49.00 3.52E-07 1.41 7.66 81.79 1.93E-08 1.79 K49N -0.02 37.84 1.52E-07 2.36 5.76 70.85 2.23E-08 2.45 K49Q -0.05 50.73 2.46E-07 1.83 8.67 76.79 1.77E-08 1.62 K49Y -0.02 42.38 3.82E-07 1.24 7.31 75.79 1.54E-08 1.59 L53A -0.04 60.19 2.23E-07 2.30 13.25 97.58 1.58E-08 1.63 L53E -0.03 53.65 2.79E-07 0.97 8.61 83.96 6.63E-09 2.54 L53K -0.05 87.28 5.69E-07 1.12 6.90 86.71 5.50E-09 1.50 Q5Q -0.05 49.87 8.11E-08 2.00 8.03 94.46 9.16E-09 0.86 L53 0.01 61.65 3.01E-07 0.83 2.59 92.54 4.90E-09 0.97 H77E 0.00 39.69 8.59E-07 2.14 7.27 70.64 3.58E-08 1.37 H77G -0.04 58.59 2.17E-07 0.94 0.75 117.28 8.25E-08 0.47 H77I -0.08 Unable to obtain maximum response value 4.34E-07 0.82 1.17 Unable to obtain maximum response value 7.86E-08 0.49 H77K 0.01 52.19 1.69E-07 2.59 9.74 97.57 4.63E-08 0.85 H77L -0.04 69.74 3.77E-07 1.17 5.39 95.19 1.76E-08 0.67 H77M -0.04 78.43 1.70E-07 2.87 3.64 85.04 2.07E-08 0.80 H77N -0.03 59.85 1.90E-07 2.26 6.65 93.63 3.36E-08 0.85 H77P 0.01 60.33 3.18E-07 1.75 7.80 Unable to derive maximum response value 1.51E-07 0.52 H77Q 0.14 84.03 5.48E-07 1.80 4.27 100.56 2.47E-08 0.90 H77R 0.04 75.01 2.35E-07 2.27 3.17 105.82 1.87E-08 0.72 H77S -0.04 62.05 3.09E-07 1.52 5.74 82.86 8.71E-09 1.35 H77T 0.08 Unable to obtain maximum response value 4.33E-07 0.88 9.26 94.45 1.06E-08 1.75 H77V 0.03 70.71 1.22E-07 1.30 3.83 95.67 1.01E-08 1.00 E78A 4.95 90.93 1.69E-07 0.90 3.49 102.48 3.59E-08 0.45 E78D 5.61 112.89 2.38E-07 0.89 7.22 76.55 2.01E-08 1.04 E78H 3.52 82.23 1.23E-07 1.28 7.18 81.85 8.29E-09 1.11 E78S 0.02 72.25 1.26E-07 1.11 8.51 79.49 9.73E-09 1.85 E78Y 10.11 109.90 1.18E-07 1.32 8.62 76.83 8.74E-09 1.23 A79E 0.08 75.10 5.64E-08 1.25 10.04 74.00 2.72E-08 28.10 A79R 12.80 109.85 6.19E-08 1.21 2.62 86.03 2.80E-09 0.73 V81I 11.60 132.98 2.73E-07 1.04 6.91 75.73 1.09E-08 1.92 V81M 0.19 84.89 8.54E-08 1.67 10.09 92.01 1.08E-08 0.91 W101A 7.26 Unable to obtain maximum response value 1.30E-07 0.68 5.63 102.39 3.00E-08 1.02 W101D 11.64 115.82 1.28E-07 1.58 9.77 87.29 5.30E-09 6.13 W101G 10.85 109.42 1.25E-07 1.44 6.05 82.69 1.07E-08 0.92 W101I 15.44 104.75 1.00E-07 1.51 4.06 95.39 2.52E-08 0.78 W101L 17.58 83.03 9.03E-08 9.27 2.22 85.93 1.13E-08 0.98 W101N 10.31 114.22 1.93E-07 0.73 5.16 94.48 1.92E-08 0.98 W101R 9.16 84.97 1.34E-07 3.27 8.70 82.97 2.03E-08 1.15 W101V 13.28 117.56 1.44E-07 1.06 3.59 110.57 6.00E-08 0.60 W101Y 0.21 126.98 9.42E-08 0.43 9.66 108.69 1.84E-08 1.29 I104A 13.70 Unable to obtain maximum response value 1.53E-07 0.66 11.88 99.31 1.78E-08 0.94 I104E 6.03 129.58 1.15E-07 0.60 14.40 101.00 1.17E-08 1.07 I104G 12.32 136.59 2.37E-07 1.07 7.65 135.41 8.25E-08 0.47 I104M 18.96 136.88 6.79E-08 0.83 9.08 100.01 9.25E-09 0.88 I104Q 16.26 121.97 1.29E-07 0.81 8.51 102.93 1.19E-08 0.91 I104S 12.73 66.19 1.23E-07 1.56 -1.51 91.22 1.38E-08 0.55 I104T 4.69 64.85 2.47E-08 3.71 3.67 76.29 7.25E-09 1.01 I46D 0.11 77.56 3.48E-07 1.51 2.02 128.89 7.13E-09 0.84 M47I -0.04 99.68 1.91E-07 0.87 7.60 127.00 1.80E-08 0.67 M47V 0.24 128.64 1.76E-07 0.71 14.93 139.97 5.65E-09 1.05 V81F 8.55 135.59 3.50E-07 1.05 4.18 157.92 2.30E-08 0.59 W101E 15.50 110.78 2.45E-07 1.37 18.70 126.25 5.35E-09 1.43 I104K 0.32 118.05 2.77E-07 0.96 10.51 145.50 6.60E-09 1.21 I104P 0.26 137.62 3.20E-07 0.66 16.02 139.64 9.35E-09 0.91 I104R 0.15 96.57 1.43E-07 0.61 13.64 154.49 1.28E-08 0.61 IFNα2b 0.27 96.98 1.21E-08 0.82 2.31 97.81 7.31E-09 0.96

In addition, the ratio of the EC ₅₀ value of each αCD38-IFNα2b/IFNαR2 or its variants in HEK293 IFNAR1/2 cells to the EC ₅₀ value in HEK293 IFNAR1/2 CD38 cells was analyzed and shown in FIG. 8A and FIG. 8B .

Thus, similar to the results obtained in Example 2.3, most αCD38-IFNα2b/IFNαR2 or its variants showed a significant improvement in EC ₅₀ values in cells expressing CD38. Here, IFNα showed similar EC ₅₀ values in both cell lines, regardless of whether CD38 was expressed.

Therefore, through the above results, it was confirmed that αCD38-IFNα2b/IFNαR2 or its variants can induce signal transduction in a target site (CD38)-specific manner through IFNα2b while maintaining the shielding ability. Example 2.5. Evaluation of the binding affinity of immunocytokines including CD38 binding proteins to FcRn

It is known that the binding affinity of antibody-based protein drugs is closely related to their half-life in vivo. Therefore, the binding affinity of αCD38-IFNα2b/IFNαR2 or its variants prepared in Example 2.1 to FcRn was confirmed using a biolayer interferometry (BLI) system (Octet RED96e, Sartorius). Anti-CD38 antibodies were produced in-house and used as a positive control.

Specifically, FAB2G biosensors (Sartorius) were hydrated for 10 minutes in a 96-well plate (Greiner) with Octet operating buffer (100 mM NaPi, 300 mM NaCl, 0.05% Tween20). Test substances (anti-CD38 antibody or αCD38-IFNα2b/IFNαR2 or its variants) were diluted with Octet operating buffer to a final concentration of 5 μg/ml each. Further, soluble FcRn/B2M complex receptor (R&D systems, 8639-FC-050) was prepared by 2-fold serial dilution from 1500 nM to 23.44 nM.

The FAB2G biosensor was loaded with each test substance to the 1.5 nm level, and the baseline (blank) was set by reacting the sensor tip in Octet operating buffer for 5 minutes. Subsequently, the sensor tip loaded with the test substance was reacted with a dilute solution of soluble FcRn/B2M complex receptor. Here, binding and dissociation were measured for 1 minute or until a stable state was reached. The calibration value was calculated by subtracting the baseline (blank) value from the result obtained through the above process. The calibration value was fitted to a 1:1 binding model using Octet BLI analysis 12.2 software to calculate _kon , _koff and _KD values.

Therefore, as shown in FIG. 9A and FIG. 9B , it was confirmed that the binding affinity of the anti-CD38 antibody to FcRn and the binding affinity of αCD38-IFNα2b/IFNαR2 or its variant to FcRn were similar. Through the above results, it is expected that the pharmacokinetic profile of the immunocytokine (αCD38-IFNα2b/IFNαR2 or its variant) according to the present disclosure will have a half-life sufficient to provide a therapeutic effect. Example 2.6. Evaluation of the anti-cancer activity of immunocytokines including CD38 binding proteins at the cellular level Example 2.6.1. Confirmation of cell survival inhibition in tumor cells by treatment with immunocytokines including CD38 binding proteins

The anticancer activity of αCD38-IFNα2b/IFNαR2 or its variants in multiple myeloma cell lines was determined by MTT assay.

Specifically, RPMI8226 cells (ATCC, CCL-155) (which is a multiple myeloma cell line) were seeded at a concentration of 1.0×10 ⁴ cells/100 μl per well in a 96-well plate. Here, RPMI-1640 medium (ATCC, 30-2001) containing 10% FBS and 1% penicillin/streptomycin was used as a cell culture medium. The test substance (αCD38-IFNα2b/IFNαR2 or its variant) was diluted with PBS to a final concentration of 1 nM, treated in the above cells (10 μl for each treatment), and cultured in a 37°C, 5% CO ₂ incubator for 48 hours.

After 48 hours, each well was treated with MTT (Invitrogen, M6494) solution diluted to 5 mg/ml and reacted in an incubator for 2 hours. Thereafter, the plate was centrifuged at 800 rpm (room temperature, 5 minutes) to remove the supernatant, and each well was treated with 100 μl DMSO (MP Bio, 196055) and reacted for 10 minutes. The absorbance (570 nm) of the reaction product was measured using a plate reader (Synergy H4 hybrid microplate reader, BioTek). For each test substance, the above experiment was performed with n=4 (quadruplicate). Cell viability (%) was calculated by dividing the value measured in the test substance group by the value measured in the untreated group and converting it to a percentage (%).

Therefore, as shown in FIG. 10 , it was confirmed that the cell survival rate in the immunocytokine (αCD38-IFNα2b/IFNαR2 or its variant) treated group was significantly decreased compared with the untreated control group or the positive control group treated with anti-CD38 antibody.

The above results demonstrate that the immunocytokine (αCD38-IFNα2b/IFNαR2 or its variant) according to the present disclosure has superior anti-cancer activity compared to anti-CD38 antibodies. Example 2.6.2. Confirmation of apoptosis induction in tumor cells by treatment with immunocytokine including CD38 binding protein

The induction of apoptosis in tumor cells by treatment with immunocytokines including CD38 binding protein was confirmed using the Annexin V apoptosis detection kit (BioLegend, 640914).

Specifically, the test substance was treated and reacted for 48 hours in the same manner as in Example 2.6.1, and then each cell was obtained and washed once with PBS. Thereafter, the cells were resuspended in Annexin V binding buffer (100 μl), treated with FITC-anti-Annexin V antibody and propidium iodide (PI) at 5 μl per 1×10 ⁴ cells, and reacted at room temperature in the dark for 20 minutes. Thereafter, each cell was treated with Annexin V binding buffer (400 μl), and the degree of cell apoptosis was analyzed by measuring the FITC wavelength (Annexin V) and PE wavelength (PI) using a flow cytometer (Cytoflex, Beckman). Here, the degree of cell apoptosis was expressed as the cell apoptosis rate, and the cell apoptosis rate was calculated as a percentage by dividing by the cell apoptosis value of IFNα treatment.

Therefore, as shown in FIG. 11 , it was confirmed that the apoptosis rate of tumor cells in the immunocytokine (αCD38-IFNα2b/IFNαR2 or its variant) treatment group was significantly increased compared to the untreated control group or the positive control group treated with anti-CD38 antibody. Example 3. Evaluation of the activity of immunocytokine including HER2 binding protein Example 3.1. Preparation of immunocytokine including HER2 binding protein

An immunocytokine (αHER2-IFNα2b/IFNαR2) comprising the variant of Example 1.1, IFNα2b and an anti-HER2 antibody (αHER2) was prepared.

Specifically, the αHER2-IFNα2b/IFNαR2 immunocytokine is prepared by forming a heterodimer of a first monomer and a second monomer, wherein in the first monomer, a fusion protein comprising the heavy chain of an anti-HER2 antibody and IFNα2b is bound to the light chain of an anti-HER2 antibody, and in the second monomer, a fusion protein comprising the heavy chain of an anti-HER2 antibody and IFNαR2 or a variant thereof is bound to the anti-HER2 antibody.

Here, a fusion protein comprising the heavy chain of an anti-HER2 antibody and IFNα2b is prepared using a polynucleotide encoding a fusion protein, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-HER2 antibody (SEQ ID NO: 98), a heavy chain constant region 1 (SEQ ID NO: 167), a hinge (linker, SEQ ID NO: 28), an Fc region or a variant thereof (SEQ ID NO: 16), a linker (SEQ ID NO: 33) and IFNα2b (SEQ ID NO: 2).

In addition, a fusion protein comprising the heavy chain of an anti-HER2 antibody and IFNαR2 or a variant thereof is prepared using a polynucleotide encoding a fusion protein, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-HER2 antibody (SEQ ID NO: 98), a heavy chain constant region 1 (SEQ ID NO: 71), a hinge (linker, SEQ ID NO: 28), an Fc region or a variant thereof (SEQ ID NO: 17), a linker (SEQ ID NO: 33), and an extracellular domain of IFNAR2 (SEQ ID NO: 5) or a variant thereof.

In addition, the light chain of the anti-HER2 antibody was prepared using a polynucleotide encoding a fusion protein comprising a signal sequence (SEQ ID NO: 32), a light chain variable region (SEQ ID NO: 99) and a light chain constant region (SEQ ID NO: 70) of the anti-HER2 antibody in order from N-terminus to C-terminus.

Representatively, a first monomer comprising the heavy and light chains of an anti-HER2 antibody and IFNα2b and a second monomer comprising the heavy and light chains of an anti-HER2 antibody and wild-type IFNαR2 were prepared by loading and expressing a polynucleotide comprising the base sequence of SEQ ID NO: 174, a polynucleotide comprising the base sequence of SEQ ID NO: 175, and a polynucleotide comprising the base sequence of SEQ ID NO: 172, respectively, on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The first monomer and the second monomer produced as described above are combined to form a heterodimer, and the heterodimer is named "αHER2-IFNα2b/IFNαR2" or "αHER2-IFNα2b/IFNαR2 variant". Example 3.2. Confirmation of the binding affinity of immunocytokines including HER2 binding proteins to HER2

To determine the kinetic binding affinity of αHER2-IFNα2b/IFNαR2 or its variants prepared in Example 3.1 to HER2 protein, the binding affinity was measured by surface plasmon resonance analysis using Biacore 8K (Cytiva). Surface plasmon resonance analysis was performed in the same manner as in Example 1.2 using αHER2-IFNα2b/IFNαR2 or its variants as test substances. However, instead of IFNα2b, HER2 protein was not treated or treated by serial dilution 2-fold from 120 nM to 3.75 nM, and the results measured under the conditions of binding for 3 minutes and dissociation for 60 minutes are shown in Table 6.

Here, anti-HER2 antibodies were produced in-house and used as a control.

The anti-HER2 antibody was produced to include a heavy chain and a light chain, the heavy chain including: a heavy chain variable region including an amino acid sequence of SEQ ID NO: 98, a heavy chain constant region 1 including an amino acid sequence of SEQ ID NO: 167, a hinge including an amino acid sequence of SEQ ID NO: 28, and an Fc region including an amino acid sequence of SEQ ID NO: 83; and the light chain including: a light chain variable region including an amino acid sequence of SEQ ID NO: 99, and a light chain constant region including an amino acid sequence of SEQ ID NO: 70. Specifically, it was produced by loading and expressing a polynucleotide including a base sequence of SEQ ID NO: 173 and a polynucleotide including a base sequence of SEQ ID NO: 172 on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific), respectively. [Table 6] Variant number αHER2-IFNα2b/IFNαR2 or its variants K _D (M) 1(Control) Anti-HER2 Antibodies 3.58E-10 2 αHER2-IFNα2b/IFNαR2 wild-type 2.68E-10 3 αHER2-IFNα2b/IFNαR2 K49A 2.75E-10 4 αHER2-IFNα2b/IFNαR2 K49D 3.36E-10

Therefore, as shown in Table 6, it was confirmed that the binding affinity of the immunocytokine (αHER2-IFNα2b/IFNαR2 or its variant) to the HER2 protein was at a similar level to that of the anti-HER2 antibody. Example 3.3. Evaluation of the STAT activity inducing ability of immunocytokines including HER2 binding proteins

HEK-Blue™ IFN-α/β cells expressing HER2 protein were used to demonstrate the STAT activity induction ability of immunocytokines including HER2 binding protein. Example 3.3.1. Generation of cell lines expressing HER2 protein

HEK-Blue™ IFN-α/β cells expressing HER2 protein were obtained by expressing HER2 protein in HEK-Blue™ IFN-α/β cells (InvivoGen, hkb-ifnabv2).

HEK-Blue™ IFN-α/β cells (InvivoGen, hkb-ifnabv2) are cells in which interferon α and β receptors are expressed on the basis of HEK293 cells. When the type 1 interferon signaling pathway is activated, the secreted embryonic alkaline phosphatase (SEAP) reporter gene is expressed in a STAT1/2-dependent manner. HEK-Blue™ IFN-α/β cells were cultured in DMEM medium (HyClone, SH30243.01) containing 10% FBS and 2% penicillin. In addition, Normocin, Blasticidin, and Zeocin were used as antibiotics by adding them to the culture medium at final concentrations of 0.2%, 0.3%, and 0.1%, respectively.

To generate cell lines that transiently express HER2 protein, HEK-Blue™ IFN-α/β cells were inoculated into 25T flasks at a concentration of 6.5×10 ⁵ cells/flask and cultured for 24 hours. For cell transfection, plasmid DNA loaded with a polynucleotide encoding HER2 protein (SEQ ID NO: 130) was transfected into the cells using lipofectamine (Invitrogen, L3000001) and cultured for an additional 24 hours. Example 3.3.2. Confirmation of STAT activity induction ability by binding of αHER2-IFNα2b/IFNαR2 or its variants to the interferon α receptor

Using cells transfected as above, the ability of αHER2-IFNα2b/IFNαR2 or its variants according to the present disclosure to activate the interleukin α signaling pathway was confirmed by the SEAP system. Here, the ability of αHER2-IFNα2b/IFNαR2 or its variants to induce STAT activity in HEK-Blue™ IFN-α/β cell lines that do not express HER2 was confirmed using the SEAP system.

Specifically, HEK-Blue™ IFN-α/β cells or HEK-Blue™ IFN-α/β cells expressing HER2 were seeded at a concentration of 5.0×10 ⁴ cells/180 μl/well in a 96-well plate. αHER2-IFNα2b/IFNαR2 or its variants were serially diluted 10-fold with PBS to a final treatment concentration of 10 ^-9 nM to 1 nM, and 20 μl of each was treated in the cells and incubated for another 24 hours.

After the reaction was completed, the culture supernatant (20 μl) and QUANTI-Blue solution (InvivoGen, rep-qbs) (180 μl) were mixed and reacted for 3 hours, after which the absorbance (650 nm) was measured using a plate reader (Synergy H4 hybrid microplate reader, BioTek). Here, a higher absorbance value means that αHER2-IFNα2b/IFNαR2 or its variants bind more strongly to IFNAR. Each treatment group was n=3 (triplicate) and IFNα2b was used as a positive control.

The above results are shown in Table 7 and Figures 12 and 15. [Table 7] Immune interleukins EC ₅₀ (M) HEK-Blue™ IFN-α/β cells expressing HER2 HEK-Blue™ IFN-α/β Cells αHER2-IFNα2b/IFNαR2 wild-type 2.647E-09 1.539E-07 αHER2-IFNα2b/IFNαR2 K49A 1.064E-07 Not calculated* αHER2-IFNα2b/IFNαR2 K49D 1.774E-08 Not calculated*

*EC ₅₀ values were not calculated because the saturated maximum value could not be obtained within the treatment concentration range of the substance, indicating that the EC ₅₀ value when calculated was lower than the EC ₅₀ value when not calculated. Therefore, it was confirmed that immunocytokines (αHER2-IFNα2b/IFNαR2 or variants thereof) were able to bind to HEK-Blue™ IFN-α/β cells expressing HER2, thereby activating STAT in a concentration-dependent manner. In addition, it was confirmed that the EC ₅₀ value in HEK-Blue™ IFN-α/β cells expressing HER2 was further reduced compared to the EC ₅₀ value in HEK-Blue™ IFN-α/β cells (Table 7).

Based on the above results, it is believed that the EC ₅₀ value is decreased due to the increase in the specific binding ability of the HER2 protein binding site of the immunocytokine (αHER2-IFNα2b/IFNαR2 or variant) of the present disclosure to the HER2 protein on the cell surface. Example 4. Evaluation of the activity of immunocytokine containing PD-L1 binding protein Example 4.1. Preparation of immunocytokine containing PD-L1 binding protein

An immunocytokine (αPD-L1-IFNα2b/IFNαR2) comprising the variant of Example 1.1, IFNα2b and an anti-PD-L1 antibody (αPD-L1) was prepared.

Specifically, the αPD-L1-IFNα2b/IFNαR2 immunocytokine is prepared by forming a heterodimer of a first monomer and a second monomer, wherein in the first monomer, a fusion protein including the heavy chain of an anti-PD-L1 antibody and IFNα2b is bound to the light chain of an anti-PD-L1 antibody, and in the second monomer, a fusion protein including the heavy chain of an anti-PD-L1 antibody and IFNαR2 or a variant thereof is bound to the anti-PD-L1 antibody.

Here, a fusion protein comprising the heavy chain of an anti-PD-L1 antibody and IFNα2b is prepared using a polynucleotide encoding a fusion protein, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-PD-L1 antibody (SEQ ID NO: 102), a heavy chain constant region 1 (SEQ ID NO: 167), a hinge (linker, SEQ ID NO: 28), an Fc region or a variant thereof (SEQ ID NO: 94), a linker (SEQ ID NO: 33) and IFNα2b (SEQ ID NO: 2).

In addition, a fusion protein comprising the heavy chain of an anti-PD-L1 antibody and IFNαR2 or a variant thereof is prepared using a polynucleotide encoding a fusion protein, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-PD-L1 antibody (SEQ ID NO: 102), a heavy chain constant region 1 (SEQ ID NO: 167), a hinge (linker, SEQ ID NO: 28), an Fc region or a variant thereof (SEQ ID NO: 106), a linker (SEQ ID NO: 33), and an extracellular domain of IFNαR2 (SEQ ID NO: 5) or a variant thereof.

In addition, the light chain of the anti-PD-L1 antibody was prepared using a polynucleotide encoding a fusion protein, which included a signal sequence (SEQ ID NO: 32), a light chain variable region (SEQ ID NO: 103) and a light chain constant region (SEQ ID NO: 70) of the anti-PD-L1 antibody in order from N-terminus to C-terminus.

Representatively, a first monomer comprising the heavy and light chains of an anti-PD-L1 antibody and IFNα2b and a second monomer comprising the heavy and light chains of an anti-PD-L1 antibody and wild-type IFNαR2 were prepared by loading and expressing a polynucleotide comprising the base sequence of SEQ ID NO: 178, a polynucleotide comprising the base sequence of SEQ ID NO: 179, and a polynucleotide comprising the base sequence of SEQ ID NO: 176, respectively, on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The first monomer and the second monomer generated as described above are combined to form a heterodimer, and the heterodimer is named "αPD-L1-IFNα2b/IFNαR2" or "αPD-L1-IFNα2b/IFNαR2 variant". Example 4.2. Confirmation of the binding affinity of immunocytokines including PD-L1 binding proteins to PD-L1

To determine the kinetic binding affinity of αPD-L1-IFNα2b/IFNαR2 or its variants prepared in Example 4.1 to PD-L1 protein, the binding affinity was measured by surface plasmon resonance analysis using Biacore 8K (Cytiva). Surface plasmon resonance analysis was performed in the same manner as in Example 1.2 using αPD-L1-IFNα2b/IFNαR2 or its variants as test substances. However, instead of IFNα2b, the PD-L1 protein was not treated or treated by serial dilution 2-fold from 15 nM to 0.938 nM, and the results measured under the conditions of binding for 3 minutes and dissociation for 60 minutes are shown in Table 8.

Here, anti-PD-L1 antibody was produced in-house and used as a control.

The anti-PD-L1 antibody was produced to include a heavy chain and a light chain, the heavy chain including: a heavy chain variable region including the amino acid sequence of SEQ ID NO: 102, a heavy chain constant region 1 including the amino acid sequence of SEQ ID NO: 167, a hinge including the amino acid sequence of SEQ ID NO: 28, and an Fc region including the amino acid sequence of SEQ ID NO: 83; and the light chain including: a light chain variable region including the amino acid sequence of SEQ ID NO: 103, and a light chain constant region including the amino acid sequence of SEQ ID NO: 70. Specifically, it was produced by loading and expressing a polynucleotide comprising the base sequence of SEQ ID NO: 177 and a polynucleotide comprising the base sequence of SEQ ID NO: 176 on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific). [Table 8] Variant number αPD-L1-IFNα2b/IFNαR2 or its variants K _D (M) 1(Control) Anti-PD-L1 Antibodies 1.66E-10 2 αPD-L1-IFNα2b/IFNαR2 wild-type 1.29E-10 3 αPD-L1-IFNα2b/IFNαR2 K49A 1.64E-10 4 αPD-L1-IFNα2b/IFNαR2 K49D 1.30E-10

Therefore, as shown in Table 8, it was confirmed that the binding affinity of αPD-L1-IFNα2b/IFNαR2 or its variants to PD-L1 protein was at a similar level to that of the anti-PD-L1 antibody. Example 4.3. Evaluation of STAT activity inducing ability of αPD-L1-IFNα2b/IFNαR2 or variants Example 4.3.1. Producing cell lines expressing PD-L1 protein

HEK-Blue™ IFN-α/β cells expressing PD-L1 protein were generated by transfecting HEK-Blue™ IFN-α/β cells (InvivoGen, hkb-ifnabv2) to express PD-L1 protein. Transfection was performed in the same manner as in Example 3.3.1 using plasmid DNA comprising a polynucleotide encoding PD-L1 protein (SEQ ID NO: 131). Example 4.3.2. SEAP analysis of αPD-L1-IFNα2b/IFNαR2 or its variants

Using cells transfected as above, the ability of αPD-L1-IFNα2b/IFNαR2 or its variants according to the present disclosure to activate the interleukin α signaling pathway was confirmed by the SEAP system. The SEAP analysis was performed in the same manner as in Example 3.3.2.

The above results are shown in Table 9 and Figures 13 and 16. [Table 9] Immune interleukins EC ₅₀ (M) HEK-Blue™ IFN-α/β cell expressing PD-L1 HEK-Blue™ IFN-α/β Cells αPD-L1-IFNα2b/IFNαR2 wild-type 1.0E-05 Not calculated* αPD-L1-IFNα2b/IFNαR2 K49A 1.974E-08 Not calculated* αPD-L1-IFNα2b/IFNαR2 K49D 2.09E-09 Not calculated*

*EC ₅₀ value was not calculated because the saturated maximum value could not be obtained within the treatment concentration range of the substance, indicating that the EC ₅₀ value when calculated was lower than the EC ₅₀ value when not calculated. Therefore, it was confirmed that immunocytokines (αPD-L1-IFNα2b/IFNαR2 or its variants) were able to bind to HEK-Blue™ IFN-α/β cells expressing PD-L1, thereby activating STAT in a concentration-dependent manner. In addition, it was confirmed that the EC ₅₀ value in HEK-Blue™ IFN-α/β cells expressing PD-L1 was further reduced compared with the EC ₅₀ value in HEK-Blue™ IFN-α/β cells (Table 9).

Based on the above results, it is believed that the EC ₅₀ value is decreased due to the increase in the specific binding ability of the PD-L1 protein binding site of the immunocytokine (αPD-L1-IFNα2b/IFNαR2 or variant) of the present disclosure to the PD-L1 protein on the cell surface. Example 5. Evaluation of the activity of immunocytokine containing EGFR binding protein Example 5.1. Preparation of immunocytokine containing EGFR binding protein

An immunocytokine (αEGFR-IFNα2b/IFNαR2) comprising the variant of Example 1.1, IFNα2b and anti-EGFR antibody was prepared.

Specifically, the αEGFR-IFNα2b/IFNαR2 immunocytokine is prepared by forming a heterodimer of a first monomer and a second monomer, wherein in the first monomer, a fusion protein comprising the heavy chain of an anti-EGFR antibody and IFNα2b is bound to the light chain of an anti-EGFR antibody, and in the second monomer, a fusion protein comprising the heavy chain of an anti-EGFR antibody and IFNαR2 or a variant thereof is bound to the anti-EGFR antibody.

Here, a polynucleotide encoding a fusion protein is used to prepare a fusion protein comprising the heavy chain of an anti-EGFR antibody and IFNα2b, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-EGFR antibody (SEQ ID NO: 104), a heavy chain constant region 1 (SEQ ID NO: 167), a hinge (linker, SEQ ID NO: 28), an Fc region or a variant thereof (SEQ ID NO: 16), a linker (SEQ ID NO: 33) and IFNα2b (SEQ ID NO: 2).

In addition, a fusion protein comprising the heavy chain of an anti-EGFR antibody and IFNαR2 or a variant thereof is prepared using a polynucleotide encoding a fusion protein, wherein the fusion protein comprises, in order from N-terminus to C-terminus, a signal sequence (SEQ ID NO: 32), a heavy chain variable region of an anti-EGFR antibody (SEQ ID NO: 104), a heavy chain constant region 1 (SEQ ID NO: 167), a hinge (linker, SEQ ID NO: 28), an Fc region or a variant thereof (SEQ ID NO: 17), a linker (SEQ ID NO: 33), and an extracellular domain of IFNαR2 (SEQ ID NO: 5) or a variant thereof.

In addition, the light chain of the anti-EGFR antibody was prepared using a polynucleotide encoding a fusion protein, which included a signal sequence (SEQ ID NO: 32), a light chain variable region (SEQ ID NO: 105) and a light chain constant region (SEQ ID NO: 70) of the anti-EGFR antibody in order from N-terminus to C-terminus.

Representatively, a first monomer comprising the heavy and light chains of an anti-EGFR antibody and IFNα2b and a second monomer comprising the heavy and light chains of an anti-EGFR antibody and wild-type IFNαR2 were prepared by loading and expressing a polynucleotide comprising the base sequence of SEQ ID NO: 182, a polynucleotide comprising the base sequence of SEQ ID NO: 183, and a polynucleotide comprising the base sequence of SEQ ID NO: 180, respectively, on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The first monomer and the second monomer produced as described above are combined to form a heterodimer, and the heterodimer is named "αEGFR-IFNα2b/IFNαR2" or "αEGFR-IFNα2b/IFNαR2 variant". Example 5.2. Confirmation of the binding affinity of immunocytokines including EGFR binding proteins to EGFR

To determine the kinetic binding affinity of αEGFR-IFNα2b/IFNαR2 or its variants prepared in Example 5.1 to EGFR protein, the binding affinity was measured by surface plasmon resonance analysis using Biacore 8K (Cytiva). Surface plasmon resonance analysis was performed in the same manner as in Example 1.2 using αEGFR-IFNα2b/IFNαR2 or its variants as test substances. However, instead of IFNα2b, the EGFR protein was not treated or treated by serial dilution 2-fold from 30 nM to 0.938 nM, and the results measured under the conditions of binding for 3 minutes and dissociation for 60 minutes are shown in Table 10.

Here, anti-EGFR antibodies were produced in-house and used as a control.

The anti-EGFR antibody is produced to include a heavy chain and a light chain, the heavy chain including: a heavy chain variable region including the amino acid sequence of SEQ ID NO: 104, a heavy chain constant region 1 including the amino acid sequence of SEQ ID NO: 167, a hinge including the amino acid sequence of SEQ ID NO: 28, and an Fc region including the amino acid sequence of SEQ ID NO: 10; and the light chain including: a light chain variable region including the amino acid sequence of SEQ ID NO: 105, and a light chain constant region including the amino acid sequence of SEQ ID NO: 70. Specifically, it is produced by loading and expressing a polynucleotide comprising the base sequence of SEQ ID NO: 181 and a polynucleotide comprising the base sequence of SEQ ID NO: 180 on a pcDNA3.4 TOPO vector (Thermo Fisher Scientific). [Table 10] Variant number αEGFR-IFNα2b/IFNαR2 or its variants K _D (M) 9(Control) Anti-EGFR Antibodies 1.55E-09 10 αEGFR-IFNα2b/IFNαR2 wild type 1.22E-09 11 αEGFR-IFNα2b/IFNαR2 K49D 1.26E-09 12 αEGFR-IFNα2b/IFNαR2 K49D 1.27E-09

Therefore, as shown in Table 10, it was confirmed that the binding affinity of the immunocytokine (αEGFR-IFNα2b/IFNαR2 or its variant) to the EGFR protein was at a similar level to that of the anti-EGFR antibody. Example 5.3. Evaluation of the STAT activity inducing ability of αEGFR-IFNα2b/IFNαR2 or its variant Example 5.3.1. Production of cell lines expressing EGFR protein

HEK-Blue™ IFN-α/β cells expressing EGFR protein were generated by transfecting HEK-Blue™ IFN-α/β cells (InvivoGen, hkb-ifnabv2) to express EGFR protein. Transfection was performed in the same manner as in Example 3.3.1 using plasmid DNA comprising a polynucleotide encoding EGFR protein (SEQ ID NO: 132). Example 5.3.2. SEAP analysis of α EGFR -IFNα2b/IFNαR2 or its variants

Using the cells transfected in Example 5.3.1, the activation ability of αEGFR-L1-IFNα2b/IFNαR2 or its variants according to the present disclosure on the interleukin α signaling pathway was confirmed by the SEAP system. The SEAP analysis was performed in the same manner as in Example 3.3.2.

The above results are shown in Table 11 and Figures 14 and 17. [Table 11] Immune interleukins EC ₅₀ (M) HEK-Blue™ IFN-α/β cells expressing EGFR HEK-Blue™ IFN-α/β Cells αEGFR-IFNα2b/IFNαR2 wild type 5.964E-08 Not calculated* αEGFR-IFNα2b/IFNαR2 K49A 2.776E-08 1.47E-08 αEGFR-IFNα2b/IFNαR2 K49D 3.956E-08 1.077E-07

*EC ₅₀ value was not calculated because the maximum value of saturation could not be obtained within the treatment concentration range of the substance, indicating that the EC ₅₀ value when calculated was lower than the EC ₅₀ value when not calculated. Therefore, it was confirmed that immunocytokines (αEGFR-IFNα2b/IFNαR2 or its variants) were able to bind to HEK-Blue™ IFN-α/β cells expressing EGFR, thereby activating STAT in a concentration-dependent manner. In addition, it was confirmed that the EC ₅₀ value in HEK-Blue™ IFN-α/β cells expressing EGFR was further reduced compared with the EC ₅₀ value in HEK-Blue™ IFN-α/β cells (Table 11). Based on the above results, it is believed that the _EC50 value is decreased due to the increase in the specific binding ability of the EGFR protein binding site of the immunocytokine (αEGFR-IFNα2b/IFNαR2 or variant) of the present disclosure to the EGFR protein on the cell surface.

(without)

FIG. 1 shows the structure of immunocytokines (left) and the mechanism of action of immunocytokines (right), which is an embodiment of the present disclosure.

FIG. 2 is a diagram schematically showing the binding structure of interferon α receptor 2 and interferon α 2b.

FIG3 is a diagram schematically showing the structure of the extracellular domain of interferon α receptor 2.

FIG. 4A and FIG. 4B are graphs showing the results of confirming the dimerization ability of the interferon α receptor after treating a cell line expressing IFNAR1/2 with αCD38 (anti-CD38 antibody)-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure (3 nM).

FIG. 5A and FIG. 5B are graphs showing the results of confirming the dimerization ability of the interferon α receptor after treating a cell line expressing IFNAR1/2 (wherein CD38 is expressed) with αCD38-IFNα2b/IFNαR2 or a variant thereof according to an embodiment of the present disclosure (3 nM).

FIG. 6 is a graph showing the results of confirming the dimerization ability of the interferon α receptor after treating a cell line expressing IFNAR1/2 with αCD38-IFNα2b/IFNαR2 or its variants according to an embodiment of the present disclosure at different concentrations (0.004 nM to 1000 nM).

FIG. 7 is a graph showing the results of confirming the dimerization ability of the interferon α receptor after treating a cell line expressing IFNAR1/2 (wherein CD38 is expressed) with αCD38-IFNα2b/IFNαR2 or its variants according to an embodiment of the present disclosure at different concentrations (0.004 nM to 1000 nM).

FIG8A and FIG8B are graphs showing the ratio of pSTAT1 induction ability (EC 50 value, see Table 5) of αCD38-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure in a cell line expressing IFNAR1/2 and a cell line expressing IFNAR1 _/ 2 in which CD38 is expressed.

FIG. 9A and FIG. 9B are a diagram and a chart showing the results of confirming the binding affinity of the anti-CD38 antibody or αCD38-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure to FcRn by biolayer interferometry (BLI) analysis.

FIG. 10 is a graph showing the results of confirming the cell viability after treating multiple myeloma cell lines (RPMI8226 cells) with αCD38-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure (1 nM).

FIG. 11 is a graph showing the results of confirming cell apoptosis by flow cytometry after treating multiple myeloma cell lines (RPMI8226 cells) with αCD38-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure (1 nM) and staining with Annexin V and propidium iodide.

FIG. 12 is a graph showing the results of measuring STAT activity using the STAT1/2-dependent secretory embryonic alkaline phosphatase (SEAP) system after treating a HEK-Blue™ IFN-α/β cell line expressing HER2 with αHER2 (anti-HER2 antibody)-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure (10 ^-9 nM to 1 nM).

FIG. 13 is a graph showing the results of measuring STAT activity using the SEAP system after treating HEK-Blue™ IFN-α/β cell lines expressing PD-L1 with αPD-L1 (anti-PD-L1 antibody)-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure (10 ^-9 nM to 1 nM).

FIG. 14 is a graph showing the results of measuring STAT activity using the SEAP system after treating HEK-Blue™ IFN-α/β cell lines expressing EGFR2 with αEGFR2 (anti-EGFR antibody)-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure (10 ^-9 nM to 1 nM).

FIG. 15 is a graph showing the results of measuring STAT activity using a STAT1/2-dependent secretory embryonic alkaline phosphatase (SEAP) system after treating HEK-Blue™ IFN-α/β cell line with αHER2 (anti-HER2 antibody)-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure.

FIG. 16 is a graph showing the results of measuring STAT activity using the SEAP system after treating the HEK-Blue™ IFN-α/β cell line with αPD-L1 (anti-PD-L1 antibody)-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure.

FIG. 17 is a graph showing the results of measuring STAT activity using the SEAP system after treating HEK-Blue™ IFN-α/β cell line with αEGFR2 (anti-EGFR antibody)-IFNα2b/IFNαR2 or its variants according to one embodiment of the present disclosure.

TW202444743A_113112108_SEQL.xml

(without)

Claims (20)

An immunocytokine comprising: an interferon alpha (IFNα) protein or a fragment thereof; an extracellular domain (ECD) fragment of an interferon alpha receptor or a variant thereof; and an antigen binding protein (ABP) specific for a target protein. The immunocytokine of claim 1, wherein the extracellular domain fragment of the interferon α receptor or its variant comprises a polypeptide represented by the following structural formula (XV): [N-terminal extension domain] ab-core domain (XV) In the structural formula (XV), the core domain is a polypeptide comprising the amino acid sequence of SEQ ID NO: 8; the N-terminal extension domain is a polypeptide comprising 1 to 39 consecutive amino acids starting from position 39 of the amino acid sequence of SEQ ID NO: 9 in a C-terminal to N-terminal direction; and ab is 0 or 1. The immunocytokine of claim 2, wherein the extracellular domain fragment of the interferon α receptor or its variant further comprises a variant in the core domain, wherein the variant in the core domain is any amino acid selected from the group consisting of positions 7 to 10, 14, 38 to 40, 42, 62, 65 and combinations thereof in the amino acid sequence of SEQ ID NO: 8 replaced by another amino acid. The immunocytokine of claim 2, wherein the extracellular domain fragment of the interferon-α receptor or a variant thereof further comprises a D2 subdomain of the extracellular domain of the interferon-α receptor or a variant thereof. The immunocytokine of claim 4, wherein a variation of the D2 subdomain is substitution of any amino acid selected from the group consisting of the amino acid residues at positions 28, 31, 33, 35, 48, 54, 81 to 85 and 87 of the amino acid sequence of SEQ ID NO: 7 by another amino acid. The immunocytokine of claim 1, wherein the target protein is any one selected from the group consisting of PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, TIM-3, CD39, CD38, CD73, CD36, CD25, CD47, CD24, CD20, SIPRα, CD40, HER2, CD19 and EGFR. An immunoglobulin as claimed in claim 6, wherein an antigen binding protein that specifically binds to CD38 comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 63, HCDR2 comprising the amino acid sequence of SEQ ID NO: 64, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 65, and a light chain variable region comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 67, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 68; a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 76, HCDR2 comprising the amino acid sequence of SEQ ID NO: 77, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 78, and A light chain variable region comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO: 79, LCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 81; or A heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 88, HCDR2 comprising the amino acid sequence of SEQ ID NO: 89, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 90, and A light chain variable region comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO: 91, LCDR2 comprising the amino acid sequence of SEQ ID NO: 92, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 93; wherein an antigen binding protein that specifically binds to HER2 comprises A heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 133, HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 135, and a light chain variable region comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO: 136, LCDR2 comprising the amino acid sequence of SEQ ID NO: 137, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 138; wherein an antigen binding protein that specifically binds to PD-L1 comprises a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 140, HCDR2 comprising the amino acid sequence of SEQ ID NO: 141, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 142, and a light chain variable region comprising: comprising SEQ ID NO: 143, LCDR1 comprising the amino acid sequence of SEQ ID NO: 143, LCDR2 comprising the amino acid sequence of SEQ ID NO: 144, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 145; a heavy chain variable region comprising: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 153, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 154, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 155, and a light chain variable region comprising: a LCDR1 comprising the amino acid sequence of SEQ ID NO: 156, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 157, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 158; or a heavy chain variable region comprising: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 161 and HCDR2 and HCDR3 and SEQ ID NO: 162, and a light chain variable region comprising: LCDR1 and SEQ ID NO: 163, LCDR2 and SEQ ID NO: 164, and LCDR3 and SEQ ID NO: 165; or wherein an antigen binding protein that specifically binds to EGFR comprises a heavy chain variable region comprising: HCDR1 and SEQ ID NO: 147, HCDR2 and SEQ ID NO: 148, and HCDR3 and SEQ ID NO: 149, and a light chain variable region comprising: LCDR1 and SEQ ID NO: 150, HCDR2 and SEQ ID NO: 151, HCDR3 and SEQ ID NO: 152; LCDR2 comprising the amino acid sequence of SEQ ID NO: 151 and LCDR3 comprising the amino acid sequence of SEQ ID NO: 152. As claimed in claim 6, an antigen binding protein that specifically binds to PD-L1 comprises: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 107 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 108, a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 109 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 110, a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 111 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 112, or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 114; wherein an antigen binding protein that specifically binds to CTLA-4 comprises: A heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 116, or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 117 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 118; wherein an antigen binding protein specifically binding to TIGIT comprises: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 120; wherein an antigen binding protein specifically binding to LAG-3 comprises: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 122; An antigen binding protein that specifically binds to CD47 comprises: A heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 96 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 97; and An antigen binding protein that specifically binds to CD19 comprises: A heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 100 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 101. The immunocytokine of claim 1, wherein the immunocytokine is a dimer formed by: i) a first monomer comprising an antigen binding protein and an interferon protein or a fragment thereof, and ii) a second monomer comprising an antigen binding protein and an extracellular domain fragment of an interferon α receptor or a variant thereof. The immunocytokine of claim 9, wherein the first monomer is composed of the following structural formula (I) and structural formula (II), and the second monomer is composed of the following structural formula (III) and structural formula (IV): <First monomer> N'-X'-[Linker (1)]a-Fc region-[Linker (2)]b-Y-C' (I) and N'-X''-C' (II) In the structural formula (I) and the structural formula (II), N' is the N-terminus of the first monomer, C' is the C-terminus of the first monomer, X is an antigen-binding protein representing an antigen-binding site of an antibody or a fragment thereof, wherein X comprises X' and X''; X' is an antigen-binding site comprising a heavy chain variable region (VH) and a CH1 region, X'' is an antigen binding site comprising a light chain variable region (VL) and a constant region (CL), Y is an interferon protein or a fragment thereof, the linker (1) and the linker (2) are peptide linkers, and a is 1, and b is 0 or 1; and <Second Monomer> N'-X'-[Linker (3)]c-Fc Region-[Linker (4)]d-Y'-C' (III) and N'-X''-C' (IV) In the structural formula (III) and the structural formula (IV), N' is the N-terminus of the second monomer, C' is the C-terminus of the second monomer, X is an antigen binding protein representing an antigen binding site of an antibody or a fragment thereof, wherein X comprises X' and X''; X' is an antigen binding site comprising a heavy chain variable region (VH) and a CH1 region, X'' is an antigen binding site comprising a light chain variable region (VL) and a constant region (CL), Y' is an extracellular domain fragment of an interferon α receptor or a variant thereof, the linker (3) and the linker (4) are peptide linkers, and c is 1, and d is 0 or 1. A fusion protein comprising: an interferon alpha protein or a fragment thereof; and an extracellular domain fragment of an interferon alpha receptor or a variant thereof. A fusion protein as claimed in claim 11, wherein the extracellular domain fragment or variant thereof of the interferon α receptor comprises a polypeptide represented by the following structural formula (XV): [N-terminal extension domain] ab-core domain (XV) In the structural formula (XV), the core domain is a polypeptide comprising an amino acid sequence of SEQ ID NO: 8; the N-terminal extension domain is a polypeptide comprising 1 to 39 consecutive amino acids starting from position 39 of the amino acid sequence of SEQ ID NO: 9 in a C-terminal to N-terminal direction; and ab is 0 or 1. The fusion protein of claim 12, wherein the extracellular domain fragment of the interferon α receptor or its variant further comprises a variant in the core domain, wherein the variant in the core domain is any amino acid selected from the group consisting of positions 7 to 10, 14, 38 to 40, 42, 62, 65 and combinations thereof in the amino acid sequence of SEQ ID NO: 8 replaced by another amino acid. The fusion protein of claim 12, wherein the extracellular domain fragment of the interferon-α receptor or its variant further comprises a variant of a D2 subdomain of the extracellular domain of the interferon-α receptor. The fusion protein of claim 14, wherein a variation of the D2 subdomain is substitution of any amino acid selected from the group consisting of the amino acid residues at positions 28, 31, 33, 35, 48, 54, 81 to 85 and 87 of the amino acid sequence of SEQ ID NO: 7 by another amino acid. The fusion protein of claim 11, wherein the fusion protein is a dimer formed by: i) a fourth monomer comprising an interferon protein or a fragment thereof, and ii) a fifth monomer comprising an extracellular domain fragment of an interferon α receptor or a variant thereof. A fusion protein as claimed in claim 16, wherein the fourth monomer is composed of the following structural formula (IX) and structural formula (X), and the fifth monomer is composed of the following structural formula (XI) and structural formula (XII): <Fourth monomer> N'-Y-[Linker (10)]l-Fc region-C' (IX) or N'-[Linker (11)]m-Fc region-[Linker (12)]n-Y-C' (X) In the structural formula (IX) and the structural formula (X), N' is the N-terminus of the fourth monomer, C' is the C-terminus of the fourth monomer, Y is an interferon protein or a fragment thereof, the linker (10) to the linker (12) is a peptide linker, and l and m are 1, and n is 0 or 1; and <Fifth monomer> N'-Y'-[Linker (13)]o-Fc region-C' (XI) or N'-[Linker (14)]p-Fc region-[Linker (15)]q-Y'-C' (XII) In the structural formula (XI) and the structural formula (XII), N' is the N-terminus of the fifth monomer, C' is the C-terminus of the fifth monomer, Y' is an extracellular domain fragment of an interferon α receptor or a variant thereof, the linker (13) to the linker (15) is a peptide linker, and o and p are 1, and q is 0 or 1. A pharmaceutical composition for preventing or treating cancer, comprising the immunocytokine of claim 1 or the fusion protein of claim 11 as an active ingredient. A pharmaceutical composition for preventing or treating cancer as claimed in claim 18, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, esophageal cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, neuroglioma, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, melanoma and lymphoma. A method for preventing or treating cancer, comprising administering the immunocytokine of claim 1 or the fusion protein of claim 11 to a subject.