ICOS-L variants having improved binding force with ICOS

Abstract

The present invention relates to an ICOS-L variant having an increased binding force to ICOS, and the ICOS-L variant of the present invention has a significantly increased binding force to ICOS (up to about 100 times) compared to wild-type ICOS-L and conventional ICOS-L variants, has a significantly small size compared to an IgG antibody that is a macromolecule, is easy to invade into a tumor microenvironment, is easy to produce, and can be used as a novel ICOS-L variant. The compound can be easily used as a therapeutic agent and an imaging molecule by being fused with various immunotherapeutic agents, and can be effectively used in cancer treatment and diagnosis.

Description

Technical Field

[0001] The present invention relates to ICOS-L (T-cell inducible co-stimulatory molecule-ligand) variants having enhanced binding affinity to ICOS. Background Art

[0002] Drugs used for cancer treatment are roughly divided into low-molecular drugs and high-molecular drugs. Compared with low-molecular drugs that have no specificity and relatively large side effects, high-molecular drugs with specificity have attracted much attention as therapeutic agents. In order to evade the killing mechanism by immune cells, cancer cells express immune checkpoint proteins on their cell surfaces that are used by normal cells to inhibit the activation of immune cells. Recently, research on immune checkpoint inhibitors has been actively conducted as a method for treating cancer. It is reported that patients who show a response to immune checkpoint inhibitors have fewer side effects during treatment, and compared with previous anticancer drugs, they have excellent therapeutic effects on various cancers, so their use has exploded. However, more than half of patients still do not respond to clinically approved immune checkpoint inhibitors, and some early responders are still observed to have drug resistance that causes cancer to worsen again after treatment. The causes of resistance to immune checkpoint inhibitors vary depending on the patient's tumor immunological characteristics. Therefore, in order to effectively treat and reduce treatment costs, new biomarkers that can screen patients suitable for treatment are needed. It has been reported that in many preclinical and clinical research pathologies, combination therapy with immunostimulatory drugs improves the patient's response rate. Therefore, effective immunostimulatory therapeutic agents are also needed.

[0003] Recently, with the development of various antibody therapeutics that inhibit the action of immune checkpoint proteins such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1), their clinical needs have exploded, and therefore, the immune checkpoint inhibitor market is expected to continue to grow at an average annual rate of 21.8% from 2020 to 2027. However, since antibodies are giant molecular proteins with a molecular weight of 150,000, it is difficult to invade cancer tissues, so they have the disadvantage of being difficult to inhibit the action of immune checkpoint proteins of tumor cells and immune cells in the tumor microenvironment. For more effective treatment, the demand for protein therapeutics that are much smaller than antibodies and easy to invade cancer tissues is increasing.

[0004] ] On the other hand, the inducible co-stimulatory molecule (ICOS) as a co-stimulatory receptor for T cell activation is a T cell-specific co-stimulatory molecule that activates T cells to respond to external antigens. It is reported to be related to the treatment prognosis of various cancers, revealing that it can not only be used as a therapeutic agent for immunostimulation, but also has the possibility of being used as a biomarker for predicting the treatment response of immune checkpoint inhibitors. In order to observe T cells present in the tumor microenvironment, it is necessary to use real-time imaging of radioactive isotopes or to test whether ICOS is expressed by staining after separating cancer tissues. Preferably, a small protein molecule that is much smaller than a giant molecule antibody and easy to invade cancer tissues is used. In addition, the efficacy of ICOS targeting substances as a combined therapeutic agent with immune checkpoint inhibitors has also been verified, and the development and research of therapeutic agents are carried out from two levels: further activating cytotoxic T cells that play a very important role in killing cancer cells by promoting the activity of ICOS or inducing anti-cancer efficacy by killing regulatory T cells expressing ICOS. However, wild-type ICOS-L has a very low affinity for ICOS (equilibrium dissociation constant = ∼700 nM), and thus has the problem of being difficult to utilize. Summary of the invention

[0005] Technical issues

[0006] An object of the present invention is to provide ICOS-L variants.

[0007] Furthermore, an object of the present invention is to provide an immunomodulatory protein comprising the above ICOS-L variant.

[0008] Furthermore, an object of the present invention is to provide a conjugate comprising the above ICOS-L variant.

[0009] Another object of the present invention is to provide a T cell activator.

[0010] Another object of the present invention is to provide engineered cells.

[0011] Another object of the present invention is to provide an anticancer adjuvant.

[0012] Furthermore, an object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer.

[0013] Another object of the present invention is to provide a composition for cancer diagnosis.

[0014] Furthermore, an object of the present invention is to provide a method for providing information for diagnosing cancer.

[0015] Furthermore, an object of the present invention is to provide a method for predicting therapeutic responsiveness to immune checkpoint inhibitors or for prognostic diagnosis.

[0016] Furthermore, the present invention aims to provide ICOS-L variants, immunomodulatory proteins, conjugates or engineered cells for use in preventing or treating cancer.

[0017] At the same time, the present invention aims to provide a method for treating cancer.

[0018] Technical Solution

[0019] In order to achieve the above object, the present invention provides an ICOS-L variant having an enhanced binding affinity to ICOS.

[0020] Furthermore, the present invention provides an immunomodulatory protein comprising the ICOS-L variant and the Fc domain of an immunoglobulin or a variant thereof.

[0021] Furthermore, the present invention provides a conjugate comprising the ICOS-L variant or immunomodulatory protein and a targeting moiety.

[0022] Furthermore, the present invention includes a T cell activator comprising the above ICOS-L variant, immunomodulatory protein or conjugate.

[0023] Furthermore, the present invention provides an engineered cell comprising the above ICOS-L variant, immunomodulatory protein or conjugate.

[0024] Furthermore, the present invention provides an anti-cancer adjuvant comprising the above ICOS-L variant, immunomodulatory protein, conjugate or engineered cell.

[0025] Furthermore, the present invention provides a pharmaceutical composition for treating or preventing cancer comprising the above ICOS-L variant, immunomodulatory protein, conjugate or engineered cell.

[0026] Furthermore, the present invention provides a composition for cancer diagnosis comprising the ICOS-L variant, immunomodulatory protein, conjugate or engineered cell.

[0027] Furthermore, the present invention provides a method for providing information for diagnosing cancer.

[0028] Furthermore, the present invention provides a method for predicting or prognosticating the therapeutic response to immune checkpoint inhibitors.

[0029] Furthermore, the present invention provides uses of ICOS-L variants, immunomodulatory proteins, conjugates or engineered cells in preventing or treating cancer.

[0030] At the same time, the present invention provides a method for treating cancer, comprising the step of administering an ICOS-L variant, an immunomodulatory protein, a conjugate or an engineered cell in a pharmaceutically effective amount to an individual suffering from cancer.

[0031] Effects of the Invention

[0032] Compared with wild-type ICOS-L and previous ICOS-L variants, the ICOS-L variants of the present invention have significantly increased binding affinity to ICOS (~about 100 times), and are significantly smaller in size than IgG antibodies, which are giant molecules, and are easy to invade the tumor microenvironment. They are easy to produce and can be easily used as therapeutic agents and imaging molecules by fusion with various immunotherapeutic agents, and can be effectively used in cancer treatment and diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The figure shows the expression vector of ICOS-Fc protein and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel photograph of the purified protein.

[0034] Figure 2 The figure shows the binding ability of purified ICOS-Fc to wild-type ICOS-L analyzed by enzyme-linked immunosorbent assay (ELISA). PD-L1 WT: negative control group.

[0035] Figure 3 Graph showing the results of flow cytometric analysis for selecting an ICOS-L display method.

[0036] Figure 4 It is a figure which shows the result of analyzing the amino acid sequence of the constructed initial library.

[0037] Figure 5 The figure shows the results of examining the amplification of the library as the screening progressed using flow cytometry.

[0038] Figure 6 The graph shows the results of analyzing the binding ability of ICOS-L variants to ICOS using flow cytometry.

[0039] Figure 7 The figure shows SDS-PAGE gel photographs of wild-type ICOS-L, control group A184, and six ICOS-L variants (Y3, Y8, Y13, Y20, Y35, and Y48) discovered in the present invention that were expressed and purified.

[0040] Figure 8 The graph shows the ICOS binding ability of wild-type ICOS-L, control group A184, and 6 ICOS-L variants discovered in the present invention analyzed by ELISA.

[0041] Fig. 9 The figure shows the SDS-PAGE gel photograph of ICOS-L variants (Y8_P51Q and Y8_H57N) expressed and purified for key mutation analysis of Y8 variants.

[0042] Fig.10 The graph shows the binding ability of wild-type ICOS-L, control group A184, Y8 variant, Y8_P51Q variant and Y8_H57N variant to ICOS analyzed by enzyme-linked immunosorbent assay.

[0043] Fig.11 The sensorgrams of wild-type ICOS-L, control A184, Y8 variant, Y8_P51Q variant, and Y8_H57N variant measured using Octet are shown.

[0044] Fig.12 The KD values ​​of wild-type ICOS-L, control group A184, Y8 variant, Y8_P51Q variant, and Y8_H57N variant determined using Octet are shown.

[0045] Fig.13 The figure shows a photograph of SDS-PAGE gel of purified ICOS single protein.

[0046] Fig.14 The figure shows the SDS-PAGE gel photographs of wild-type ICOS-L, A184 and variant (Y8 and Y8_H57N) proteins of the present invention fused to expressed and purified Fc.

[0047] Fig.15 The graph shows the binding ability of expressed and purified ICOS-L-Fc (wild-type-Fc, A184-Fc, Y8-Fc and Y8_H57N-Fc) to wild-type ICOS analyzed by ELISA.

[0048] Fig.16 The graph shows the results of examining the degree of T cell differentiation induced by PD-L1-Fc and ICOS-L-Fc (wild-type-Fc, A184-Fc, Y8-Fc, and Y8_H57N-Fc) using flow cytometry.

[0049] Fig.17 The graph shows the results of enzyme-linked immunosorbent assay for the amount of interferon-γ secretion by T cells induced by PD-L1-Fc and ICOS-L-Fc (wild-type-Fc, A184-Fc, Y8-Fc, and Y8_H57N-Fc).

[0050] Fig.18 This is a photograph of SDS-PAGE gel showing atezolizumab (ATZ) with LALAPG mutation introduced into the Fc domain.

[0051] Fig.19The figure shows a photograph of an SDS-PAGE gel of a protein fused with the heavy chain of atezolizumab with the LALAPG mutation introduced into the Fc domain (ICOS-L fused to an antibody) of the wild-type ICOS-L, ICOS-L A184 variant or ICOS-L variant (Y8 and Y8_H57N) protein of the present invention.

[0052] Fig. 20 The figure shows a photograph of an SDS-PAGE gel of a protein fused with the light chain of atezolizumab with the LALAPG mutation introduced into the Fc domain (ICOS-L fused to an antibody) of the wild-type ICOS-L, ICOS-L A184 variant or ICOS-L variant (Y8 and Y8_H57N) protein of the present invention.

[0053] Fig.21 The diagram shows the binding ability of wild-type ICOS-L, ICOS-L A184 variant or ICOS-L variants of the present invention (Y8 and Y8_H57N) fused with the heavy chain of atezolizumab to wild-type ICOS analyzed by enzyme-linked immunosorbent assay. ATZ: Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(H)-Wild-type: a protein in which the wild-type ICOS-L is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(H)-A184: a protein in which the ICOS-LA184 variant is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(H)-Y8: a protein in which the ICOS-L Y8 variant is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; and ATZ(H)-Y8_H57N: a protein in which the ICOS-L Y8_H57N variant is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain.

[0054] Fig. 22Graph showing the binding ability of wild-type ICOS-L, ICOS-L A184 variant, or ICOS-L variants of the present invention (Y8 and Y8_H57N) fused to the light chain of atezolizumab to wild-type ICOS by ELISA. ATZ: Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-Wild-type: a protein in which the wild-type ICOS-L is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-A184: a protein in which the ICOS-LA184 variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-Y8: a protein in which the ICOS-L Y8 variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; and ATZ(L)-Y8_H57N: a protein in which the ICOS-L Y8_H57N variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain.

[0055] Fig.23 The graph shows the results of examining the degree of T cell differentiation induced by proteins (50 nM) fused with wild-type ICOS-L, ICOS-L A184 variant, or ICOS-L variants of the present invention (Y8 and Y8_H57N) to the heavy chain of atezolizumab using flow cytometry. ATZ: Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(H)-Wild-type: a protein in which the wild-type ICOS-L is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(H)-A184: a protein in which the ICOS-L A184 variant is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(H)-Y8: a protein in which the ICOS-L Y8 variant is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; and ATZ(H)-Y8_H57N: a protein in which the ICOS-L Y8_H57N variant is fused to the heavy chain of Atezolizumab with LALAPG mutation introduced into the Fc domain.

[0056] Fig.24The figure shows the results of examining the degree of T cell differentiation induced by the protein (50 nM) fused with the light chain of atezolizumab, wild-type ICOS-L, ICOS-L A184 variant or ICOS-L variants of the present invention (Y8 and Y8_H57N), using flow cytometry. ATZ: Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-Wild-type: a protein in which the wild-type ICOS-L is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-A184: a protein in which the ICOS-L A184 variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-Y8: a protein in which the ICOS-L Y8 variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; and ATZ(L)-Y8_H57N: a protein in which the ICOS-L Y8_H57N variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain.

[0057] Fig.25 The graph shows the results of ELISA testing of the amount of interferon-γ secretion by T cells induced by proteins (100 nM) of wild-type ICOS-L, ICOS-L A184 variant, or ICOS-L variants of the present invention (Y8 and Y8_H57N) fused to the heavy chain of atezolizumab. ATZ(H)-Wild-type: a protein in which the wild-type ICOS-L is fused to the heavy chain of atezolizumab with the LALAPG mutation introduced into the Fc domain; ATZ(H)-A184: a protein in which the ICOS-L A184 variant is fused to the heavy chain of atezolizumab with the LALAPG mutation introduced into the Fc domain; ATZ(H)-Y8: a protein in which the ICOS-L Y8 variant is fused to the heavy chain of atezolizumab with the LALAPG mutation introduced into the Fc domain; and ATZ(H)-Y8_H57N: a protein in which the ICOS-L Y8_H57N variant is fused to the heavy chain of atezolizumab with the LALAPG mutation introduced into the Fc domain.

[0058] Fig.26The figure shows the results of ELISA testing of the amount of interferon-γ secretion by T cells induced by proteins (100 nM) of wild-type ICOS-L, ICOS-L A184 variant, or ICOS-L variants of the present invention (Y8 and Y8_H57N) fused to the light chain of atezolizumab. ATZ: Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-Wild-type: a protein in which the wild-type ICOS-L is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-A184: a protein in which the ICOS-L A184 variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; ATZ(L)-Y8: a protein in which the ICOS-L Y8 variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain; and ATZ(L)-Y8_H57N: a protein in which the ICOS-L Y8_H57N variant is fused to the light chain of Atezolizumab with LALAPG mutation introduced into the Fc domain. DETAILED DESCRIPTION

[0059] Hereinafter, the present invention will be described in detail by referring to the accompanying drawings through examples of the present invention. However, the following examples are merely illustrative of the present invention. When it is judged that the specific description of the technology or structure that is clear in its main purpose to a person skilled in the art of the present invention may unnecessarily confuse the gist of the present invention, its detailed description may be omitted, but the present invention is not limited thereto. The present invention may have various modifications and applications within the description of the scope of the attached invention claims and the equivalent scope interpreted thereby.

[0060] Furthermore, the terminology used in this specification is used to appropriately express the preferred embodiments of the present invention, and may vary according to the intention of the user, operator, or the conventions of the technical field to which the present invention belongs. Therefore, these terms should be defined based on the content of the full text of this specification. In the full text of the specification, when it is mentioned that a part "includes" a certain structural element, if there is no special record to the contrary, it means that other structural elements may also be included, rather than excluding other structural elements.

[0061] Unless otherwise defined, all technical terms used in the present invention are used with the meanings commonly understood by those skilled in the art in the art to which the present invention belongs. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications described as references in this specification are incorporated into the present invention.

[0062] Throughout this specification, the commonly used single-letter or three-letter abbreviations are used for naturally occurring amino acids, and commonly accepted three-letter abbreviations are also used for other amino acids such as α-aminoisobutyric acid (Aib) and N-methylglycine (Sar, N-methylglycine). In addition, the amino acids referred to by abbreviations in the present invention are recorded according to the following IUPAC-IUB nomenclature.

[0063] Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y and Valine: V.

[0064] In one embodiment, the present invention relates to an ICOS-L variant, wherein one or more amino acids selected from the group consisting of amino acids at positions 4, 51, 52, 54, 57, 74, 130 and lower 234 in the amino acid sequence of wild-type T-cell inducible co-stimulatory molecule-ligand (ICOS-L) are replaced by a sequence different from the wild-type amino acid.

[0065] In one embodiment, compared with wild-type ICOS-L, the ICOS-L variant can increase the binding affinity with ICOS.

[0066] In one example, the amino acids of the wild-type ICOS-L may include the amino acid sequence of SEQ ID NO: 1, and the amino acid positions may be based on the amino acid sequence of SEQ ID NO: 1.

[0067] In one example, the ICOS-L variant can consist of an amino acid sequence that shows at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence of SEQ ID NO: 1.

[0068] In one example, the ICOS-L variant of the present invention may comprise one or more amino acid substitutions selected from the group consisting of E4D, Q51P, Q51H, N52S, S54N, N57H, L74R, S130C and K234R, thereby increasing the binding affinity to ICOS.

[0069] In one example, the ICOS-L variant of the present invention may comprise an amino acid substitution N57H, and may be a variant Y8_P51Q comprising the amino acid sequence of SEQ ID NO: 2.

[0070] In one example, the ICOS-L variant of the present invention may comprise the amino acid substitution Q51P, and may be a variant Y8_H51N comprising the amino acid sequence of SEQ ID NO: 3.

[0071] In one example, the ICOS-L variant of the present invention may comprise amino acid substitutions Q51P and N57H, and may be variant Y8 comprising the amino acid sequence of sequence 4.

[0072] In one example, the ICOS-L variant of the present invention may comprise amino acid substitutions Q51H, S54N and S130C, and may be variant Y13 comprising the amino acid sequence of SEQ ID NO: 5.

[0073] In one example, the ICOS-L variant of the present invention may comprise amino acid substitutions N52S and K234R, and may be variant Y20 comprising the amino acid sequence of SEQ ID NO: 6.

[0074] In one example, the ICOS-L variant of the present invention may comprise amino acid substitutions E4D and Q51P, and may be variant Y35 comprising the amino acid sequence of SEQ ID NO: 7.

[0075] In one example, the ICOS-L variant of the present invention may comprise amino acid substitutions Q51P and L74R, and may be variant Y48 comprising the amino acid sequence of SEQ ID NO: 8.

[0076] In one example, the ICOS-L variant of the present invention may further comprise a transmembrane domain.

[0077] In one embodiment, the ICOS-L variant of the present invention may further comprise a cytoplasmic signaling domain connected to the transmembrane domain.

[0078] In one embodiment, the ICOS-L variant of the present invention can be labeled by a marker selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromophore, a luminescent substance and a fluorescent substance. The fluorescent substance can be a cyanine (Cy, cyanine) series, a rhodamine (Rhodamine) series, an Alexa series, a BODIPY series or a ROX series fluorescent substance, and can be Nile Red, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine or Alexa.

[0079] The ICOS-L variant of the present invention has a portion of the amino acid sequence substituted in the wild-type ICOS-L protein (or peptide). The term "variant" used in the present invention refers to a corresponding amino acid sequence that contains at least one amino acid difference (substitution, insertion or deletion) when compared with a standard substance. In a specific embodiment, the "variant" has high amino acid sequence homology (homolog) and / or protective amino acid substitutions, deletions and / or insertions when compared with a standard sequence. In addition, the term "ICOS-L variant" used in the present invention refers to an ICOS-L variant protein in which one or more amino acids are mutated in order to regulate its binding activity with ICOS.

[0080] Specifically, the ICOS-L variants of the present invention can be prepared by standard synthesis methods, recombinant expression systems or any other methods in the art to which the present invention belongs. Therefore, the peptides of the present invention can be synthesized by many methods including, for example, the following methods: method (a), synthesizing the peptide in a step or fragment assembly manner by means of a solid phase or liquid phase method, and then isolating and purifying the final peptide product; or method (b), after expressing a nucleic acid construct encoding the peptide in a host cell, the expression product is recovered from the host cell culture; or method (c), after expressing a nucleic acid construct encoding the peptide in a cell-free laboratory, the expression product is recovered; or after obtaining peptide fragments by any combination of method (a), method (b) and method (c), the peptide is obtained by connecting the fragments to obtain the peptide, thereby recovering the relevant peptide.

[0081] In a more specific example, a gene encoding the ICOS-L variant of the present invention can be prepared by genetic engineering and transformed into a host cell, and then expressed to produce the ICOS-L variant of the present invention.

[0082] In one embodiment, the invention relates to an immunomodulatory protein comprising an ICOS-L variant of the invention and a half-life extending moiety.

[0083] In one example, the ICOS-L variant can be linked to the half-life extending moiety directly or indirectly through a linker.

[0084] In one example, the half-life extending portion can comprise a multimerization domain, albumin, an albumin binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide (CTP) of the beta isoform of human chorionic gonadotropin, polyethylene glycol (PEG), a structured long hydrophilic sequence of unformed amino acids (XTEN), hydroxyethyl starch (HES), an albumin binding small molecule, or a combination thereof.

[0085] In one example, the multimerization domain can be an Fc domain of an immunoglobulin, a leucine zipper, a leucine zipper, or a zinc finger protein.

[0086] In one example, the immunomodulatory protein of the present invention may comprise the ICOS-L variant and the Fc domain of an immunoglobulin or a variant thereof.

[0087] In one example, the Fc domain variant may comprise one or more amino acid substitutions in the Fc domain of wild-type human IgG1.

[0088] In one example, compared with the Fc of wild-type human IgG1, the Fc domain variant may exhibit reduced effector function, and may be an inactivated or effectorless Fc.

[0089] In one example, the effector function can be antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity, programmed apoptosis, or cellular phagocytosis.

[0090] In one example, the Fc domain variant may replace the amino acid at position 234, 235 or 329 numbered according to the Kabat numbering system in the Fc domain of wild-type human IgG1 with a sequence different from the wild-type amino acid, and may include amino acid substitutions of L234A, L235A and P329G.

[0091] In one embodiment, the invention relates to a conjugate comprising an ICOS-L variant or an immunomodulatory protein of the invention and a targeting moiety.

[0092] In one example, the ICOS-L variant or immunomodulatory protein can be linked to the targeting moiety directly or indirectly via a linker.

[0093] In one example, the targeting moiety can specifically bind to a molecule on the surface of an immune cell, which can be an antigen presenting cell or a lymphocyte.

[0094] In one example, the targeting moiety can be a tumor localizing moiety that binds to a molecule on the surface of a tumor.

[0095] In one example, the targeting moiety can be directed against molecules HER1 / EGFR, HER2 / ERBB2, CD20, CD25 (IL-2Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, tag 72, Caprin-1, mesothelin, PDGF receptor (PDGFR; e.g., PDGF-Rα), PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF) , CD30, EpCAM, EphA2, Glypican-3, gpA33, mucin, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM1, and GM2), VEGF receptor (VEGFR), VEGFR2, VEGF-A, integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10β, HLA-DR antigen, IgE, MUC-1, nuC242, PEM antigen, metalloproteinase, ephrin receptor, ephrin ligand, HGF receptor, CXCR4, CXCR4, bombesin receptor, SK-1 antigen, Bcr-abl, RET, MET, TRKB, TIE2, ALK, ROS, EML4-ALK, ROS1, BRAFV600E, SRC, c-KIT, mTOR, TSC1, TSC2, BTK, KIT, BRCA, CDK 4 / 6, JAK1, JAK2, BRAF, FLT-3, MEK1, MEK2, SMO or B7-H6 (NCR3LG1) are specific for or bind to the above components.

[0096] In one example, the targeting moiety is a protein, a peptide, a nucleic acid, a small molecule or a nanoparticle, more preferably, an antibody or an active fragment thereof.

[0097] In one embodiment, the antibody can be cetuximab, panitumumab, zalutumumab, inoluzumab, trastuzumab, ado-trastuzumab, emtansine, tositumomab Rituximab (retuximab, MabThera), ibritumomab tiuxetan (Zevalin), daclizumab (ZENAPAX), gemtuzumab tuzumab (Mylotarg), alemtuzumab, CEA scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab (Avastin), afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, denosumab, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparib Panib, olaratumab, palbociclib, pazopanib, pertuzumab, ramucirumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, vismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), AMP-224, MSB001078C, MEDI4736, BMS-935559, LY3300054, atezolizumab, avelumab or durvalumab or a variant of the antibody may be atezolizumab or a variant thereof.

[0098] In one example, the fragment having immunological activity can be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and bispecific antibody (diabody).

[0099] In one example, the ICOS-L variant of the present invention can be directly linked to the C-terminus or N-terminus of the heavy chain or light chain of an antibody or an immunologically active fragment thereof, or indirectly linked via a linker.

[0100] In one example, the conjugate of the present invention may be a fusion protein.

[0101] In one example, the conjugate of the present invention may further comprise a label for detection or purification.

[0102] In one example, the conjugate of the present invention specifically binds to cancer cell surface molecules (e.g., PD-L1) through the antibody of the conjugate or a fragment thereof having immunological activity, and the ICOS-L variant of the conjugate binds to the ICOS of T cells present in the tumor microenvironment to activate T cells, thereby having anti-cancer activity through apoptosis of cancer cells caused by immune cells. Since it specifically binds to cancer cells, it can be used as a therapeutic diagnostic preparation that can also be used in the diagnosis of cancer. In addition, chimeric antigen receptor (CAR) T cells can also be prepared for use as anticancer agents, and can also be used as anticancer adjuvants administered simultaneously, separately, or sequentially with anticancer agents.

[0103] The above antibodies include not only complete (whole) antibody forms, but also functional fragments of antibody molecules. A complete antibody has a structure with two full-length light chains (light chain) and two full-length heavy chains (heavy chain), and each light chain is connected to the heavy chain by a disulfide bond. The functional fragment of an antibody molecule refers to a fragment that retains the antigen binding function. Examples of antibody fragments include: (i) Fab fragments formed by the variable region (VL) of the light chain and the variable region (VH) of the heavy chain and the constant region (CL) of the light chain and the first constant region (CH1) of the heavy chain; (ii) Fd fragments formed by the VH and CH1 domains; (iii) Fv fragments formed by the VL and VH domains of a single-chain antibody; (iv) dAb fragments formed by the VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) isolated CDR regions; (vi) F(ab')2 fragments as bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv) bound by a peptide linker in which the VH domain and the VL domain form an antigen binding site; (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965); and (ix) bispecific antibodies as multivalent or multispecific fragments prepared by gene fusion (WO94 / 13804).

[0104] The antibody or immunologically active fragment thereof of the present invention can be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof. The above-mentioned antibody can be produced by recombination or synthesis.

[0105] Animal-derived antibodies produced by immunizing animals with desired antigens usually cause immune rejection when administered to humans for therapeutic purposes. Chimeric antibodies have been developed to suppress such immune rejection. Chimeric antibodies are antibodies in which the constant region of animal-derived antibodies, which is the cause of anti-isotype reactions, is replaced with the constant region of human antibodies using genetic engineering. Although chimeric antibodies can achieve considerable improvements in anti-isotype reactions compared to animal-derived antibodies, animal-derived amino acids are still present in the variable region, thus containing potential side effects of anti-idiotypic reactions. Humanized antibodies have been developed to improve such side effects. These are prepared by transplanting the CDR (complementarity determining regions) site in the variable region of chimeric antibodies, which plays an important role in antigen binding, into the human antibody framework.

[0106] The most important thing in the CDR grafting technology for preparing humanized antibodies is to select the most optimized human antibody that can best accept the CDR site of the animal-derived antibody, and for this purpose, antibody databases, crystal structure analysis, molecular modeling technology, etc. can be used. However, even if the CDR site of the animal-derived antibody is transplanted into the optimized human antibody framework, there are still amino acids located in the framework of the animal-derived antibody that affect antigen binding, and there are still cases where the antigen binding ability is not preserved in a considerable part, so it can be said that the application of additional antibody engineering technology for restoring the antigen binding ability is necessary.

[0107] The above-mentioned antibody or fragment having immunological activity thereof may be isolated from an organism (not present in the organism) or non-naturally produced (non-naturally occurring), for example, may be produced by synthesis or recombination.

[0108] In the present invention, "antibody" refers to a substance produced by the stimulation of an antigen in the immune system, and its type is not particularly limited and can be obtained by natural or non-natural (e.g., synthetic or recombinant) methods. Not only in vitro, antibodies are also very stable and have a long half-life in vivo, so they are conducive to mass expression and production. In addition, antibodies have a dimer structure in nature, so the affinity is very high. A complete antibody is a structure having only two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), muon (μ), alpha (α), delta (δ) and epsilon (ε) types, and subtypes include gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

[0109] The term "heavy chain" in the present invention is interpreted as comprising a variable region domain VH having an amino acid sequence sufficient to confer specificity to an antigen and three constant region domains C. H1 , C H2 and C H3 The term "light chain" is defined as a full-length heavy chain and fragments thereof including a hinge. Furthermore, the term "light chain" is defined as a variable region domain V having an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and constant region domain C L The meaning includes the full-length light chain and its fragments.

[0110] The term "variable region" or "variable site" in the present invention refers to a portion of an antibody molecule that shows many variations in sequence while performing the function of specifically binding to an antigen, and the variable region contains CDR1, CDR2, and CDR3 as complementary determining regions. A framework region (FR) exists between the CDRs to support the CDR loop. The "complementarity determining region" is a circular site associated with antigen recognition, and the specificity of the antibody to the antigen is determined by changes in the sequence of the site.

[0111] The term "single chain variable region (scFv, single chain fragment variable)" in the present invention refers to a single chain antibody prepared by gene recombination in a manner that only expresses the variable region of an antibody, and refers to an antibody in a single chain form in which the VH region and the VL region of the antibody are connected by a short peptide chain. If there is no other explicit indication or no other understanding in the context, the above term "scFv" includes scFv fragments such as antigen binding fragments. For ordinary technicians in the technical field to which the present invention belongs, this is self-evident.

[0112] The term "complementarity determining region (CDR)" in the present invention refers to the amino acid sequence of the hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy chain and the light chain may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The above CDRs may provide the main contact residues in the binding of an antibody to an antigen or an antigenic determinant.

[0113] In the present invention, the term "specific binding" or "specific recognition" has the same meaning as commonly known to those of ordinary skill in the art to which the present invention belongs, and refers to inducing an immune response through the specific interaction between antigen and antibody.

[0114] The term "antigen-binding fragment" in the present invention refers to a part of a polypeptide that contains a portion that can bind to an antigen as a fragment relative to the entire structure of an immunoglobulin. For example, it may be scFv, (scFv)2, scFv-Fc, Fab, Fab' or F(ab')2, but is not limited thereto. In the above antigen-binding fragment, Fab is a fragment having the variable regions of the light chain and the heavy chain, the constant region of the light chain and the first constant region of the heavy chain (C H1 ) structure, with one antigen binding site. H1The C-terminus of the domain has a hinge region containing more than one cysteine ​​residue, which is different from Fab. F(ab')2 antibodies are generated by forming disulfide bonds between the cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only the heavy chain variable region and the light chain variable region, and the recombinant technology for generating Fv fragments is well known in the technical field to which the present invention belongs. Double-chain Fv (two-chain Fv) connects the heavy chain variable region and the light chain variable region by non-covalent bonds, and single-chain Fv (single-chain Fv) usually connects the heavy chain variable region and the single chain variable region by covalent bonds or directly connects at the C-terminus through a peptide linker, thereby forming a dimer-like structure like a double-chain Fv. The above-mentioned linker can be a peptide linker formed by 1 to 100 or 2 to 50 arbitrary amino acids, and the appropriate sequence is well known in the technical field to which the present invention belongs. The above antigen-binding fragments can be obtained by using proteolytic enzymes (for example, limited cleavage of intact antibodies using papain can obtain Fab, and cleavage using pepsin can obtain F(ab')2 fragments), and can be prepared by genetic recombination technology.

[0115] The term "hinge region" in the present invention refers to a region included in the heavy chain of an antibody, existing between the CH1 and CH2 regions, and refers to a region that provides flexibility to the antigen-binding site in the antibody. For example, the hinge may be derived from a human antibody, specifically, from IgA, IgE or IgG, for example, from IgG1, IgG2, IgG3 or IgG4.

[0116] In one embodiment, the present invention relates to nucleic acid molecules encoding ICOS-L variants, immunomodulatory proteins or conjugates of the present invention, vectors comprising the same, and host cells comprising the above vectors.

[0117] The term "nucleic acid molecule" used in the present invention refers to deoxyribonucleic acid or ribonucleic acid in single-stranded or double-stranded form, and unless otherwise specified, includes natural nucleic acid analogs (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

[0118] The term "vector" used in the present invention refers to any nucleic acid that is inserted into a host cell, recombines with the cell genome and is inserted therein, or contains a competent nucleotide sequence that replicates spontaneously as an episome. The above-mentioned vectors include linear nucleic acids, plasmids, phagemids, cosmids, ribonucleic acid (RNA) vectors, viral vectors, etc.

[0119] The term "host cell" used in the present invention refers to a eukaryotic or prokaryotic cell into which one or more deoxyribonucleic acids (DNA) or vectors are introduced, and should not be understood to refer only to specific target cells, but should also be understood to refer to their offspring or potential offspring. Although the above-mentioned offspring may be different from the parent cell due to sudden changes in the offspring or environmental influences, they are still included in the scope of the above-mentioned term used in the present invention.

[0120] In one embodiment, the present invention relates to a T cell activator comprising an ICOS-L variant, immunomodulatory protein or conjugate of the present invention.

[0121] In one example, the T cell can be a cytotoxic T cell or a CAR-T cell.

[0122] ICOS is a T cell-specific co-stimulatory molecule that activates T cells to respond to external antigens and is reported to be associated with the treatment prognosis of various cancers. The T cell activator of the present invention can be used not only as a therapeutic agent to enhance immune stimulation, but also as a biomarker for predicting the treatment response of immune checkpoint inhibitors.

[0123] In one embodiment, the present invention relates to a composition for detecting ICOS comprising the ICOS-L variant, immunomodulatory protein or conjugate of the present invention.

[0124] In one example, the ICOS detection composition can be used to observe T cells present in the tumor microenvironment.

[0125] In one example, T cells present in the tumor microenvironment can be observed by examining whether ICOS is expressed in cancer tissues.

[0126] In one example, the composition can detect and quantify the protein expression of ICOS.

[0127] In one embodiment, the present invention relates to a composition for biological imaging comprising the ICOS-L variant, immunomodulatory protein or conjugate of the present invention.

[0128] In one embodiment, the present invention relates to an ICOS-L variant comprising a transmembrane domain or a nucleic acid encoding an ICOS-L variant further comprising a cytoplasmic signaling domain connected to the above transmembrane domain and a polynucleotide comprising more than one nucleic acid encoding more than one chain of a recombinant antigen receptor.

[0129] In one example, the recombinant antigen receptor can be a chimeric antigen receptor or an engineered T cell receptor (T cell receptor, TCR).

[0130] In one embodiment, the nucleic acid encoding the ICOS-L variant and the nucleic acid encoding one or more chains of the recombinant receptor can be separated by nucleic acids encoding a peptide causing ribosome skipping or a self-cleaving peptide, respectively.

[0131] In one example, the polynucleotides may include a nucleic acid encoding an ICOS-L variant, a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, and a nucleic acid encoding a CAR.

[0132] In one example, the polynucleotides may include a nucleic acid encoding an ICOS-L variant, a nucleic acid encoding a first self-cleaving peptide or a peptide that causes ribosome skipping, a nucleic acid encoding an α chain of an engineered TCR or one of the β chains of an engineered TCR, a nucleic acid encoding a second self-cleaving peptide or a peptide that causes ribosome skipping, and a nucleic acid encoding the other chain of the α chain of an engineered TCR or the β chain of an engineered TCR, wherein the self-cleaving peptide or the peptide that causes ribosome skipping may be T2A, P2A, E2A or F2A.

[0133] The term "chimeric antigen receptor (CAR)" in the present invention refers to a non-natural receptor that can confer specificity to a specific antigen to immune effector cells. Typically, the above-mentioned CAR refers to a receptor used to transplant the specificity of a monoclonal antibody to T cells. CAR is generally composed of an extracellular domain (Ectodomain), a transmembrane domain (transmembrane domain) and an intracellular domain (Ectodomain).

[0134] The extracellular domain includes an antigen binding site (antigen recognition region), and the transmembrane domain of CAR is a form connected to the extracellular domain, which can be of natural or synthetic origin. In the case of being derived from nature, it can be derived from membrane-bound or transmembrane proteins, and can be derived from α, beta or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or CD8 and other proteins. The sequence of the transmembrane domain can be obtained from the commonly known technical field of the transmembrane region of the known transmembrane protein, etc., but is not limited thereto.

[0135] Furthermore, when the above-mentioned transmembrane domain is synthesized, it can mainly contain hydrophobic amino acid residues such as leucine and valine. For example, a transmembrane domain of a triplet of phenylalanine, tryptophan and valine can be synthesized, but it is not limited thereto. Sequence information about the above-mentioned transmembrane domain can be obtained from the literature known in the general technical field related to the synthetic transmembrane domain, but it is not limited thereto.

[0136] In the CAR of the present invention, the intracellular domain is a part of the domain of the CAR present in the cell, and is in a form connected to the transmembrane domain. The intracellular domain of the present invention may include an intracellular signaling domain, and the intracellular signaling domain is characterized in that when the antigen binding site of the CAR is combined with the antigen, the T cell is activated, preferably, causing T cell proliferation. As long as the intracellular signaling domain is a part that conducts a signal that can cause T cell activation when the antibody binds to the antigen binding site present outside the cell, its type is not particularly limited, and a variety of intracellular signaling domains can be used, for example, it can be an immunoreceptor tyrosine-based activation motif, i.e., ITAM, and the ITAM includes those derived from CD3ζ (zeta), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD66d or FcεRIγ, but is not limited thereto.

[0137] CAR includes a single-chain variable region (scFv) of an antibody specific for a tumor-associated antigen (TAA) coupled to a T cell signaling molecule cytoplasmic domain via a hinge and a transmembrane region. Most common lymphocyte activation portions include T cell co-stimulatory (e.g., CD28, CD137, OX40, ICOS, and CD27) domains in a tandem with a T cell trigger (e.g., CD3ζ) portion. In CAR-mediated adoptive immunotherapy, cells transplanted with CAR directly recognize TAAs on tumor cells in a non-HLA-restricted manner.

[0138] In one embodiment, the present invention relates to a vector comprising the above-mentioned polynucleotide.

[0139] In one example, the vector may be a viral vector, and the viral vector may be a retroviral vector or a lentiviral vector.

[0140] In one embodiment, the present invention relates to an engineered cell comprising the above-mentioned polynucleotide or vector.

[0141] In one embodiment, the invention relates to an engineered cell comprising an ICOS-L variant, immunomodulatory protein or conjugate of the invention.

[0142] In one example, the engineered cells may further comprise a chimeric antigen receptor or an engineered T cell receptor.

[0143] In one embodiment, the ICOS-L variant, immunomodulatory protein or conjugate of the present invention can be secreted from an engineered cell, and the engineered cell can contain an ICOS-L variant containing a transmembrane domain.

[0144] In one embodiment, the ICOS-L variants of the present invention can be expressed on the surface of a cell.

[0145] In one example, the cell may be an immune cell, which may be an antigen presenting cell (APC) or a lymphocyte, which may be a T cell. The T cell may be a CD4 + T cells (helper T cells, TH cells), CD8 + T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Treg cells), natural killer T cells, etc. Preferably, the T cells introduced with CAR in the present invention are CD8 + T cells, but not limited thereto.

[0146] In one example, the engineered cells may be primary cells. Depending on the circumstances, the cells may be mammalian cells or human cells.

[0147] In one example, the engineered cells may be chimeric antigen receptor expressing cells, which may be chimeric antigen receptor expressing macrophages (CAR-macrophage), chimeric antigen receptor expressing T (CAR-T) cells, chimeric antigen receptor expressing-γ-δT (CAR-Gamma-delta T) cells or natural killer (CAR-NK) cells.

[0148] The term "chimeric antigen receptor expressing T (CAR-T) cell" in the present invention refers to a T cell expressing CAR. The chimeric antigen receptor expressing T cell has the following advantages: i) it recognizes cancer antigens in a manner independent of human leukocyte antigen (HLA, human leukocyte antigen), and can treat cancers that escape the effects of anticancer agents by reducing the expression of HLA on the cell surface, ii) it can be used in treatment regardless of the HLA type and the patient's HLA type, and iii) a large number of T cells specific to cancer can be generated in a short period of time, thereby showing excellent anticancer effects.

[0149] In one embodiment, the invention relates to an infectious agent comprising a nucleic acid molecule encoding an ICOS-L variant, immunomodulatory protein or conjugate of the invention.

[0150] In one embodiment, the ICOS-L variant, immunomodulatory protein or conjugate is secreted from the infectious agent when expressed. In some cases, the encoded ICOS-L variant may include a transmembrane domain.

[0151] In one example, the encoded ICOS-L variant may be barely expressed on the surface of the cell in which it is expressed.

[0152] In one example, the infectious agent can be bacteria or virus, and the virus can be an oncolytic virus. The oncolytic virus can be an adenovirus, an adeno-associated virus, a herpes virus, a herpes zoster virus, a vesicular stomatitis virus, a reovirus, a Newcastle disease virus, a parvovirus, a measles virus, a vesicular stomatitis virus (VSV), a coxsackie virus, or a vaccinia virus.

[0153] In one example, the virus can be specifically targeted to dendritic cells (DC) and / or be dendritic cell-tropic, and the virus can be a lentiviral vector camouflaged by a Sindbis virus envelope.

[0154] In one example, the infectious material may also include nucleic acid molecules encoding other gene products that cause apoptosis of the targeted cells or that can initiate or promote an immune response. The additional gene products may be selected from anticancer agents, antimetabolites, antiangiogenic agents, immunomodulatory molecules, immune checkpoint inhibitors, antibodies, cytokines, growth factors, antigens, cytotoxic gene products, pro-apoptotic gene products, anti-apoptotic gene products, cell matrix decomposition genes, genes for tissue regeneration, or cells that reprogram human somatic cells with pluripotency.

[0155] In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating inflammation or autoimmune diseases, comprising the ICOS-L variant or immunomodulatory protein of the present invention as an active ingredient.

[0156] In one example, the inflammatory disease can be anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplantation, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory neurological disease, inflammatory lung disease, inflammatory endocrine disease or autoimmune blood disease.

[0157] In one example, the autoimmune disease may be inflammatory bowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis or psoriasis.

[0158] In one embodiment, the present invention relates to an anti-cancer adjuvant comprising the ICOS-L variant, immunomodulatory protein, conjugate or engineered cell of the present invention.

[0159] In one example, the above-mentioned adjuvant can enhance the anti-cancer effect of immune checkpoint inhibitors, and can be administered simultaneously, separately or sequentially with the immune checkpoint inhibitors.

[0160] In one example, the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a variant thereof.

[0161] In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the ICOS-L variant, immunomodulatory protein, conjugate or engineered cell of the present invention as an active ingredient.

[0162] In one example, the cancer can be one or more selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma.

[0163] The pharmaceutical composition of the present invention can be used as a single treatment method or in combination with other common biological therapies, chemotherapy methods or radiotherapy. When such a combination therapy is implemented, cancer can be treated more effectively. When the present invention is used in the prevention and treatment of cancer, the chemotherapeutic agents that can be used together with the above composition include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin D, and dactinomycin D. The invention also includes the following: omycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin, and methotrexate, etc. Radiotherapy that can be used together with the composition of the present invention is X-ray irradiation and gamma ray irradiation.

[0164] The term "prevention" in the present invention refers to all actions to inhibit or delay the onset, spread and recurrence of cancer by administering the ICOS-L variant of the present invention or a composition comprising the same.

[0165] The therapeutically effective amount of the composition of the present invention will vary with various factors, such as the method of administration, the target site, the patient's condition, etc. Therefore, when used in humans, the dosage should be determined by considering both safety and efficiency. The amount used in humans can be deduced from the effective amount determined by animal experiments. Matters to be considered when determining the effective amount are described in, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EWMartin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0166] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" used in the present invention refers to a sufficient and side-effect-free amount for treating a disease at a reasonable benefit / risk ratio that can be used in medical treatment, and the effective dose level can be determined based on factors including the patient's health status, the type of disease, the severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the metabolic ratio, the treatment period, the drugs used in conjunction or simultaneously, and other factors well known in the medical field. The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered once or multiple times. The key point is that after considering all the above factors, the minimum amount without side effects is used to obtain the maximum effect, which can be easily determined by a person of ordinary skill in the art to which the present invention belongs.

[0167] The pharmaceutical composition of the present invention may contain a carrier, a diluent, an excipient or a combination of two or more of the carriers commonly used in biological preparations. The term "pharmaceutically acceptable" used in the present invention refers to a property of being non-toxic to normal cells or humans exposed to the above-mentioned composition. The above-mentioned carrier is not particularly limited as long as it can transport the composition into the body. For example, the compounds recorded in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered physiological saline, glucose solution, maltodextrin solution, glycerol, ethanol and a mixture of one or more of the above components can be used, and antioxidants, buffers, antibacterial agents and other common additives can also be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants can also be added to prepare injection dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, the drug can be preferably formulated according to each disease or component using an appropriate method in the technical field to which the present invention pertains or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0168] In one example, the pharmaceutical composition can be in one or more dosage forms selected from the group consisting of oral dosage forms, external preparations, suppositories, sterile injection solutions and sprays.

[0169] In addition, the composition of the present invention may include a carrier, a diluent, an excipient or a combination of two or more of the carriers commonly used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they can transport the composition into the body. For example, compounds recorded in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered physiological saline, glucose solution, maltodextrin solution, glycerol, ethanol and a mixture of one or more of the above components can be used, and antioxidants, buffers, antibacterial agents and other common additives can also be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants can also be added to prepare injection dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, the appropriate method in the technical field to which the present invention belongs or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) can be preferably prepared according to each disease or component.

[0170] The composition of the present invention may also include one or more active ingredients showing the same or similar functions. Relative to the total weight of the composition, the composition of the present invention includes 0.0001 to 10 weight percent of the above protein, preferably, 0.001 to 1 weight percent.

[0171] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives. In this case, the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium phosphate, lactose, mannitol, starch syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, palm wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, glucose, sorbitol, talc, etc. Relative to the above composition, preferably, the pharmaceutically acceptable additive of the present invention is included in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0172] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) or orally according to the target method, and the dosage range can vary according to the patient's weight, age, sex, health status, diet, administration time, administration method, metabolic rate and severity of the disease. The daily dosage of the composition of the present invention can be 0.0001 to 10 mg / ml, preferably, 0.0001 to 5 mg / ml, more preferably, once a day or divided into several times.

[0173] Liquid preparations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., which may contain, in addition to water and liquid paraffin, which are usually used as simple diluents, various excipients, such as wetting agents, sweeteners, aromatics, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, freeze-dried preparations, suppositories, etc.

[0174] In one example, the composition of the present invention can be used as a combined therapeutic agent with an immune checkpoint inhibitor.

[0175] In one embodiment, the present invention relates to a composition for cancer diagnosis comprising the ICOS-L variant, immunomodulatory protein, conjugate or engineered cell of the present invention.

[0176] In one example, the composition may further include one selected from the group consisting of a color-developing enzyme, a radioisotope, a chromophore, a luminescent substance, and a fluorescent substance.

[0177] In one embodiment, the present invention relates to a cancer diagnosis kit comprising the cancer diagnosis composition of the present invention.

[0178] In one example, the above-mentioned kit may include not only tools and / or reagents for collecting biological samples from subjects or patients, but also tools and / or reagents for preparing genomic deoxyribonucleic acid, complementary deoxyribonucleic acid (cDNA), ribonucleic acid or protein from the sample.

[0179] The term "cancer diagnostic kit" in the present invention refers to a kit comprising the composition for cancer diagnosis of the present invention. Therefore, the above expression "cancer diagnostic kit" can be used interchangeably or mixed with "composition for cancer diagnosis". The term "diagnosis" in this specification includes: determining the sensitivity of an individual to a specific disease or illness; determining whether an individual currently suffers from a specific disease or illness; determining the prognosis of an individual who has suffered from a specific disease or illness (e.g., testing for pre-metastatic or metastatic cancer status, determining the stage of cancer, or determining the responsiveness of cancer to treatment); or therametrics (e.g., monitoring the status of an individual in order to provide information about the efficacy of a treatment).

[0180] The ICOS-L variant of the present invention specifically binds to ICOS with significantly increased binding force, and exhibits anti-cancer activity by killing cancer cells by acting as an immunostimulatory drug that promotes the activation of T cells, or has anti-cancer activity by killing regulatory T cells that express ICOS to induce anti-cancer efficacy. Since it specifically binds to cancer cells, it can be used as a therapeutic diagnostic agent that can also be used in the diagnosis of cancer.

[0181] In one embodiment, the present invention relates to a method for providing information for diagnosing cancer, comprising: a step of contacting a biological sample isolated from a subject with the cancer diagnostic composition of the present invention; a step of confirming the binding level of ICOS-L variants to ICOS; and a step of comparing the binding level of the ICOS-L variants to ICOS in a normal control sample.

[0182] In one example, the method may further include the following step: if the binding level of ICOS-L variant and ICOS in the biological sample isolated from the subject is higher than the binding level of ICOS-L variant and ICOS in the normal control sample, then determining that the subject of examination has cancer.

[0183] The term "sample" as used in the present invention refers to a biological sample obtained from a subject or patient. The source of the biological sample can be: fresh, frozen and / or preserved organ or tissue samples, or solid tissue from a biopsy or aspiration; blood or any structural component of blood; cells from the subject's body or at any point in development.

[0184] In one embodiment, the present invention relates to a method for predicting or prognosticating treatment responsiveness to an immune checkpoint inhibitor, comprising the step of confirming the expression level of ICOS in a biological sample isolated from a subject treated with an immune checkpoint inhibitor using the ICOS-L variant, immunomodulatory protein, conjugate or engineered cell of the present invention.

[0185] In one embodiment, the present invention relates to the use of the ICOS-L variant, immunomodulatory protein, conjugate or engineered cell of the present invention in preventing or treating cancer.

[0186] In one embodiment, the present invention relates to a method for treating cancer, comprising the step of administering a pharmaceutically effective amount of the ICOS-L variant, immunomodulatory protein, conjugate or engineered cell of the present invention to an individual suffering from cancer.

[0187] The present invention is described in more detail by the following examples, but the following examples are only used to make the present invention specific, rather than to limit the present invention.

[0188] Embodiments of the present invention

[0189] Example 1. Preparation of dimeric human ICOS (ICOS-Fc) for exploring ICOS-L variants

[0190] 1-1. Cloning of dimeric human ICOS

[0191] In order to more efficiently screen ICOS-L variants with strong binding ability to ICOS by improving binding activity (Avidity), the Fc region (domain) of human IgG antibody was expressed at the C-terminal part of ICOS to induce dimerization reaction (dimerization), and a GS linker composed of glycine and serine was inserted between Fc and ICOS to ensure the fluidity of each protein. Specifically, the DNA of the amino acid sequence E21-K140, which is the ICOS extracellular region, was synthesized at Twist Bioscience (USA) and the gene was amplified using the designed primers and Vent polymerase (New England Biolab). The Fc gene was also amplified using the designed primers and Vent polymerase, and the amplified ICOS and Fc genes were subjected to assembly polymerase chain reaction (assembly PCR), and then treated with restriction enzymes using BssHII and XbaI (New England Biolab). The ICOS-Fc gene treated with restriction enzymes was ligated to the pMAZ vector, which is a vector for animal cells treated with the same restriction enzymes. The ligated plasmid was transformed into Jude1 Escherichia coli, and a single clone was obtained. The successful insertion of ICOS-Fc into the pMAZ vector was confirmed by base sequence analysis.

[0192] 1-2. Expression and purification of dimeric human ICOS (ICOS-Fc)

[0193] Expi293F cells were cultured at 2 × 10 6300 ml of cells / ml were subcultured. One day later, the ICOS-Fc expression vector prepared in Example 1 was mixed with polyethyleneimine (PEI, Polyehylenimine, Polyscience, 23966) in a ratio of 1:4 in 30 ml of Freestyle293 expression culture medium (Gibco.12338-018) and placed at room temperature for 20 minutes before transfection into the Expi293F animal cells. After culturing the cells for 7 days at 37°C, 125 rpm, and 8% CO2 in a CO2 shaking incubator, only the supernatant was collected by centrifugation. Then, after balancing with 25x phosphate buffered saline (PBS), the cells were filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. 1 ml of protein A resin (Protein Aresin) was added to the filtered culture solution and stirred at 4°C for 16 hours. The resin was recovered by passing it through a column and then washed with 10 ml of phosphate buffer solution. After eluting the washed resin with 100 mM glycine (pH 2.7) buffer, it was neutralized with 1 M Tris-HCl (pH 8.0) to obtain the purified ICOS-Fc dimer protein and then replaced with 1x phosphate buffer solution using a centrifugal filter unit 3K (Centrifugal filter units 3K, Merck Millipore). The expressed and purified ICOS-Fc dimer protein ( Figure 1 ).

[0194] 1-3. Testing the binding ability of purified ICOS-Fc and ICOS-L and fluorescent substance labeling

[0195] In order to analyze the binding ability of ICOS-Fc purified in the above Example 1-2 to ICOS-L, an enzyme-linked immunosorbent assay was performed. Specifically, 50 μl of ICOS-Fc diluted to 4 μg / ml in 0.05 M sodium carbonate (Na2CO3) (pH 9.6) was dispensed into each well of a flat bottom polystyrene high bind (Flat Bottom Polystyrene High Bind) 96-well ELISA plate (Costar Company), fixed at 4°C for 16 hours, and then blocked with 100 μl of 0.5% bovine serum albumin (BSA, Bovineserum albumin) (Gibco) (contained in phosphate buffer solution (in PBS), pH 7.4) at room temperature for 2 hours. After washing 4 times with 180 μl of 0.05% PBST (pH 7.4), 50 μl of wild-type ICOS-L serially diluted with 0.5% bovine serum albumin (in phosphate buffered saline, pH 7.4) was dispensed into each well and reacted at room temperature for 1 hour. After washing 4 times, 50 μl of anti-His-HRP conjugate was added and reacted at room temperature for 1 hour and then washed 4 times. Then, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Substrate S olution) (Thermo Fisher Scientific) was added to each well for color development and 50 μl of 2M sulfuric acid (H2SO4) was used to terminate the reaction and analyzed using an Epoch Microplate Spectrophotometer (Microplate Spectrophotometer) (BioTek Instrument Co., Ltd.).

[0196] As a result, it was confirmed that ICOS-Fc bound to wild-type ICOS-L ( Figure 2 ), in order to be used as a probe, ICOS dimer protein was fluorescently labeled using Alexa488 labeling kit.

[0197] Example 2. Selection of screening method

[0198] 2-1. Cloning of ICOS-L for selection of yeast surface display method

[0199] For efficient variant screening, it was decided to identify the yeast surface anchoring motif, and ICOS-L was cloned to compare the system in which the N-terminus of ICOS-L is anchored to Aga2 (pCTCON-Aga2-ICOS-L-FLAG) and the system in which the C-terminus is anchored to Aga2 (pCTCON-ICOS-L-Aga2-FLAG). Specifically, the wild-type ICOS-L gene and the existing variant A184 (N52H, N57Y and Q100P) were synthesized at Twist Bioscience (USA) and the genes were amplified using designed primers and Vent polymerase (New England Biolabs). The amplified wild-type ICOS-L and A184 genes were treated with restriction enzymes using SfiI (New England Biolabs), and the genes treated with restriction enzymes were ligated to the pCTCON vector treated with the same restriction enzymes. After the ligated plasmid was transformed into Jude1 Escherichia coli, a single clone was obtained and base sequence analysis confirmed that four plasmids, pCTCON-Aga2-ICOS-L_WT-FLAG, pCTCON-Aga2-ICOS-L_A184-FLAG, pCTCON-ICOS-L_WT-Aga2-FLAG and pCTCON-ICOS-L_A184-Aga2-FLAG, were successfully cloned.

[0200] 2-2. Choose the display method

[0201] The yeast display method was selected by testing the binding ability with ICOS. The four plasmids prepared in the above Example 2-1 were transformed into the AWY101 (Trp-) strain respectively. In order to confirm the binding ability of ICOS-L with ICOS using flow cytometry, 5 ml of SDCAA (20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L disodium hydrogen phosphate (Na2HPO4), 8.56 g / L sodium dihydrogen phosphate (NaH2PO4)) medium supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol was cultured at 30°C and 225 rpm for 16 hours. The cultured cells were centrifuged (2500g, 5 minutes, 4°C) to obtain 5×10 7After 2×10 cells were grown, they were induced in 5 ml of SGCAA (20 g / L galactose, 6.7 g / L yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L disodium hydrogen phosphate, 8.56 g / L sodium dihydrogen phosphate) medium supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol at 20°C and 225 rpm for 24 hours. 7 The cells were centrifuged (14000 g, 30 seconds, 4°C) and collected in e-tubes. 1 ml of PBSB (phosphate buffered saline containing 0.1% bovine serum albumin (0.1% BSA in PBS)) was added to each e-tube to collect the cells, resuspended and centrifuged (14000 g, 30 seconds, 4°C) to reaggregate the cells, and then resuspended in 0.5 ml of PBSB to prepare 4×10 7 After transferring 25 μl of cells to a new e-tube, add 25 μl of PBSB, ICOS-Fc-Alexa488 (200 nM) and Anti-FLAG-iFluor647 (1000:

[0202] 1) Probe and incubate at room temperature for 30 minutes to label cells with fluorescent probe. Then, centrifuge (14000g, 30 seconds, 4°C) to remove the supernatant and resuspend and wash with 200μl PBSB, centrifuge again (14000g, 1 minute, 4°C) and resuspend with 200μl PBSB to prepare the sample. Use FACSLyric (BD Biosciences) equipment to measure the fluorescence signal value of the prepared sample to analyze the expression level of wild-type ICOS-L and A184 and indirectly analyze the binding force with ICOS.

[0203] As a result, although the above plasmids were all expressed well, it was confirmed that ICOS-L expressed in yeast with its C-terminal part anchored to Aga2 had significantly increased binding ability to ICOS compared to wild-type ICOS-L expressed in yeast with its N-terminal part anchored to Aga2, so it was decided to perform screening in the form anchored to the C-terminus ( Figure 3 ).

[0204] Example 3. Preparation of a huge ICOS-L error-prone library for ultra-high-speed screening

[0205] In order to quickly explore ICOS-L variants with increased binding affinity to ICOS, primers containing SfiI sites on both sides were designed based on pCTCON-ICOS-L_WT-Aga2-FLAG so that all sites of ICOS-L could be randomly mutated. DNA was amplified once using the designed primers and Taq polymerase (TAKARA), dNTPs (Invitrogen), magnesium chloride (MgCl2) and manganese chloride (MnCl2) (SIGMA) by error-prone PCR. The amplified gene was prepared by secondary amplification using Vent polymerase (24 μg), and the relevant vector was treated with SfiI restriction enzyme (8 μg). The prepared two genes were transformed into the AWY101 strain to construct a library by homologous recombination. The size of the constructed library was 4.5×10 7 Sequence analysis confirmed that the error rate was 0.89% based on DNA (average 6.3 mutations / 714 bp) and 2.05% based on amino acids (average 4.8 mutations / 238 amino acids) ( Figure 4 ).

[0206] Example 4. Screening of ICOS-L variants

[0207] 4-1. Screening of ICOS-L variants

[0208] The initial library transformed into AWY101 was cultured in 500 ml of SDCAA (20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L disodium hydrogen phosphate, 8.56 g / L sodium dihydrogen phosphate) medium supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol at 30°C and 225 rpm for 16 hours. In order to remove dead cells, the cultured cells were centrifuged at OD 450 =0.7 was inoculated into 100 ml of SDCAA medium and cultured at 30°C and 225 rpm for 16 hours. 450 =0.7 was inoculated into 100 ml of SGCAA medium and cultured at 20°C and 225 rpm for 2 days. 8The cells were centrifuged (14000g, 30 seconds, 4°C) and collected in an e-tube. 1 ml of PBSB (phosphate buffer solution containing 0.1% bovine serum albumin) was added to the e-tube to collect the cells, resuspended and centrifuged (14000g, 30 seconds, 4°C) to reaggregate the cells, and then resuspended in 1 ml of PBSB containing Anti-FLAG-iFluor647 (1000:1) and ICOS-Fc-Alexa488 (200nM) probes and incubated at room temperature for 1 hour for labeling. Then, the supernatant was removed by centrifugation (14000g, 30 seconds, 4°C), and then resuspended in 1 ml of PBSB and washed, and then centrifuged again (14000g, 1 minute, 4°C) and resuspended in 1 ml of PBSB to prepare a sample. The fluorescent signal value of the prepared library sample was measured using an S3sorter (Bio-Rad) device to recover yeast with high binding affinity to ICOS, and the recovered yeast was cultured in 20 ml of SDCAA at 30°C and 225 rpm. The next day, the cultured cells were recovered and cultured in 100 ml of SGCAA at 20°C and 225 rpm for 2-3 days for induction, and then the next round was performed. The above screening process was performed a total of 4 times by reducing the concentration of the probe.

[0209] 4-2. Confirmation of the abundance of ICOS-L variants with enhanced binding ability to ICOS

[0210] The initial, first, second, third and fourth round libraries were inoculated into 100 ml of SDCAA (20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L disodium hydrogen phosphate, 8.56 g / L sodium dihydrogen phosphate) medium supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol and cultured at 30°C and 225 rpm for 16 hours. In order to remove dead cells, the cultured cells were measured at OD 450 =0.7 was inoculated into 100 ml of SDCAA medium and cultured at 30°C and 225 rpm for 16 hours. 450 =0.7 was inoculated into 100 ml of SGCAA medium and cultured at 20°C and 225 rpm for 2 days. 7 After centrifugation (14000g, 30 seconds, 4°C) of the library, 1 ml of PBSB (phosphate buffer solution containing 0.1% bovine serum albumin) was added to each e-tube to collect the cells, resuspended and centrifuged (14000g, 30 seconds, 4°C) to reaggregate the cells, and then resuspended in 0.5 ml of PBSB to prepare 4×10 7The density of cell / ml. After transferring 25μl of cells to a new e-tube, add 25μl of PBSB, ICOS-Fc-Alexa488 (200nM) and Anti-FLAG-iFluor647 (1000:1) probes respectively and incubate at room temperature for 30 minutes to label the cells with fluorescent probes. Then, centrifuge (14000g, 30 seconds, 4°C) to remove the supernatant and resuspend and wash with 200μl of PBSB, centrifuge again (14000g, 1 minute, 4°C) and resuspend with 200μl of PBSB to prepare the sample. Use FACSLyric (BD Biosciences) equipment to measure the fluorescence signal value of the prepared sample to indirectly analyze the binding force of each library with ICOS.

[0211] As a result, it was confirmed that as the screening progressed, variants with improved binding ability to ICOS were gradually enriched ( Figure 5 ).

[0212] Example 5. Analysis of amino acid sequences of ICOS-L variants and screening of ICOS-L variants with enhanced binding affinity to ICOS

[0213] After obtaining DNA from four rounds of libraries using Zymoprep yeast plasmid miniprep kits (ZymoResearch), the DNA was transformed into Jude1 Escherichia coli and 50 clones were sequenced. As a result, 18 variants were screened, and in order to analyze their binding ability to ICOS, the plasmids of the 18 variants were transformed into the AWY101 (Trp-) strain. In order to confirm the binding ability of variants to ICOS by flow cytometry, wild-type ICOS-L, A184 and 18 variants were cultured in 5 ml of SDCAA (20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L disodium hydrogen phosphate, 8.56 g / L sodium dihydrogen phosphate) medium supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol at 30°C and 225 rpm for 16 hours. The cultured cells were centrifuged (2500 g, 5 minutes, 4°C) to obtain 5×10 7 After 2×10 cells were grown, they were induced in 5 ml of SGCAA (20 g / L galactose, 6.7 g / L yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L disodium hydrogen phosphate, 8.56 g / L sodium dihydrogen phosphate) medium supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol at 20°C and 225 rpm for 24 hours.7 The cells were centrifuged (14000 g, 30 seconds, 4°C) and collected in e-tubes. 1 ml of PBSB (phosphate buffer solution containing 0.1% bovine serum albumin) was added to each e-tube to collect the cells, resuspended and centrifuged (14000 g, 30 seconds, 4°C) to reaggregate the cells, and then resuspended in 0.5 ml of PBSB to prepare 4×10 7 cell / ml density. After transferring 25 μl of cells to a new e-tube, add 25 μl of PBSB, ICOS-Fc-Alexa488 (67.7 nM) and Anti-FLAG-iFluor647 (1000:1) probes respectively and incubate at room temperature for 30 minutes to label the cells with fluorescent probes. Then, centrifuge (14000 g, 30 seconds, 4°C) to remove the supernatant, resuspend and wash with 200 μl of PBSB, centrifuge again (14000 g, 1 minute, 4°C) and resuspend with 200 μl of PBSB to prepare the sample. Use FACSLyric (BD Biosciences) equipment to measure the fluorescence signal value of the prepared sample to analyze the expression level of the variant and indirectly analyze the binding ability with ICOS. Through this, 6 ICOS-L variants with improved binding ability with ICOS, Y3, Y8, Y13, Y20, Y35 and Y48, were screened ( Figure 6 ).

[0214] Example 6. Expression and purification of ICOS-L variants with enhanced binding affinity to ICOS

[0215] In order to prepare the animal cell expression vector of ICOS-L variant, Y3, Y13, Y20, Y35 and Y48 of the wild-type ICOS-L, ICOS-L_A184 as a control group, and the 6 variant genes obtained in the above-mentioned Example 5 were amplified using designed primers and Vent Polymerase (Vent Polymerase, New England Biolabs). The amplified gene was treated with restriction enzymes using BssHII and XbaI (New England Biolabs). The ICOS-Fc gene treated with restriction enzymes was connected to the pMAZ vector as an animal cell vector treated with the same restriction enzymes. After the connected plasmid was transformed into Jude1 Escherichia coli, the result of base sequence analysis after obtaining a single clone was confirmed to confirm that the gene of the variant was successfully inserted into the vector. In addition, since the XbaI restriction enzyme position exists in the gene sequence of the Y8 variant, after amplification using designed primers and then Vent Polymerase, 2X Gibson assembly master mix (assembly master mix) (New England Biolabs) was used to insert into the pMAZ vector. After the reaction was completed, the gene sample was transformed into Jude1 Escherichia coli, and a single clone was obtained. The base sequence analysis confirmed that the Y8 gene was also successfully inserted into the vector. The prepared ICOS-L variant expression vectors (pMAZ-ICOS-L_WT, pMAZ-ICOS-L_A184, pMAZ-ICOS-L_Y3, pMAZ-ICOS-L_Y8, pMAZ-ICOS-L_Y13, pMAZ-ICOS-L_Y20, pMAZ-ICOS-L_Y35 and pMAZ-ICOS-L_Y48) were transfected into Expi293F animal cells and cultured in a CO2 shaking incubator at 37°C, 125 rpm and 8% CO2 for 7 days. After centrifugation, only the supernatant was separated. Then, after equilibration with 25x phosphate buffer solution, it was filtered using a 0.2μm syringe filter (Sartorius, S6634). 0.15 ml of Ni-NTA resin was added to the filtered culture solution and stirred at room temperature for 1 hour to recover the resin. Then, the resin was washed twice with 1 ml of phosphate buffer solution containing 10 mM imidazole (Sigma), and then washed twice with 1 ml of phosphate buffer solution containing 20 mM imidazole. After elution with phosphate buffer solution containing 250 mM imidazole, the buffer was replaced with 1X phosphate buffer solution (pH 7.4) using centrifugal filter units 10K (Merck Millipore). Then, the expressed and purified wild-type ICOS-L, the control group A184, and the 6 variants discovered in the present invention ( Figure 7 ).

[0216] Example 7. Testing the binding ability of ICOS-L variants to ICOS

[0217] In order to analyze the binding ability of the purified ICOS-L variants to ICOS, an enzyme-linked immunosorbent assay was performed. Specifically, 50 μl of ICOS-Fc diluted to 4 μg / ml in 0.05 M sodium carbonate (pH 9.6) was dispensed into each well of a flat-bottomed polystyrene high-binding 96-well ELISA plate (Costar), fixed at 4°C for 16 hours, and then blocked at room temperature for 2 hours with 100 μl of 0.5% bovine serum albumin (Gibco) (contained in phosphate buffer solution, pH 7.4). After washing four times with 180 μl of 0.05% PBST (pH 7.4), 50 μl of ICOS-L variants serially diluted with 0.5% bovine serum albumin (contained in phosphate buffer solution, pH 7.4) were dispensed into each well and reacted at room temperature for 1 hour. After washing four times, 50 μl of anti-His-HRP conjugate was added and reacted at room temperature for 1 hour, and then washed four times. Then, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to develop color, and the reaction was terminated with 50 μl of 2 M sulfuric acid, followed by analysis using an Epoch microplate spectrophotometer (Biotin Instruments).

[0218] As a result, all variants except the Y3 variant showed higher binding affinity to ICOS than the wild-type ICOS-L, and in particular, the Y8 variant showed higher binding affinity than the existing A184 ( Figure 8 ).

[0219] Example 8. Exploration of the decisive mutation of ICOS-L that improves the binding ability to ICOS

[0220] 8-1. Preparation of mutation recovery variants

[0221] In order to explore what the critical mutation of the Y8 variant with improved binding ability compared to A184 as a control group is, the two mutations possessed by Y8 were prepared as variants Y8_P51Q (Q substituted for P51 of the Y8 variant) and Y8_H57N (N substituted for H57 of the Y8 variant) substituted by the amino acid sequence of wild-type ICOS-L. In order to prepare animal cell expression vectors of Y8_P51Q and Y8_H57N variants, the genome of the pMAZ-ICOS-L_Y8 plasmid was amplified by the Quikchange polymerase chain reaction method using designed primers and Pfu turbo polymerase (Pfu turbo polymerase, Agilent). The amplified gene was transformed into Jude1 to confirm the sequence. The prepared ICOS-L variant expression vectors (pMAZ-ICOS-L_WT, pMAZ-ICOS-L_A184, pMAZ-ICOS-L_Y8, pMAZ-ICOS-L_Y8_P51Q and pMAZ-ICOS-L_Y8_H57N) were transfected into Expi293F animal cells and cultured for 7 days, followed by centrifugation to separate only the supernatant. Then, after equilibration with 25x phosphate buffer solution (PBS), the supernatant was filtered through a 0.2 μm filter (Thermo Fisher Scientific) using a bottle top filter. 0.5 ml of Ni-NTA resin was added to the filtered culture solution and stirred at 4°C for 16 hours. After the resin was recovered using a column, it was washed with 20 CV (column volume) of a phosphate buffer solution containing 10 mM imidazole (Sigma), and then washed again with 20 CV of a phosphate buffer solution containing 20 mM imidazole. Then, after elution with a phosphate buffer solution containing 250 mM imidazole, the buffer was replaced with a 1X phosphate buffer solution (pH 7.4) using a centrifugal filter unit 10K (Merck Millipore). As a result, more than 1 mg of purified 4 variants were obtained. In order to improve the purity of the purified ICOS-L variants, size exclusion chromatography (NGC Chromatography, Biorad) was performed. Aggregated proteins were removed using a Superdex 200 10 / 300GL column (Cytiva), and only fractions 21 and 22 were recovered. Then, SDS-PAGE confirmed that the purity of the 5 ICOS-L variants was improved ( Fig. 9 ).

[0222] 8-2. Exploring the decisive mutations of the Y8 variant

[0223] In order to explore the decisive mutation of the Y8 variant, an enzyme-linked immunosorbent assay was performed. Specifically, 50 μl of ICOS-Fc diluted to 4 μg / ml in 0.05 M sodium carbonate (pH 9.6) was dispensed into each well of a flat-bottomed polystyrene high-binding 96-well ELISA plate (Costar), fixed at 4°C for 16 hours, and then blocked at room temperature for 2 hours with 100 μl of 0.5% bovine serum albumin (Gibco) (contained in phosphate buffer solution, pH 7.4). After washing 4 times with 180 μl of 0.05% PBST (pH 7.4), 50 μl of each purified ICOS-L variant prepared in Example 8-1 above, which was serially diluted with 0.5% bovine serum albumin (contained in phosphate buffer solution, pH 7.4), was dispensed into each well, and reacted at room temperature for 1 hour. After washing 4 times, 50 μl of anti-His-HRP conjugate was added and reacted at room temperature for 1 hour and then washed 4 times. Then, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to develop the color and 50 μl of 2M sulfuric acid was used to terminate the reaction and analyzed using Epoch microplate spectrophotometer (Benton Instrument Co., Ltd.).

[0224] The results showed that the P51 mutation had a greater effect on the improvement of binding ability than the H57 mutation of Y8, and the best binding ability was shown when the P51 and H57 mutations existed together ( Fig.10 ).

[0225] Example 9. Comparison of ICOS-L variants containing critical mutations in their binding to ICOS

[0226] The binding ability of wild-type ICOS-L, existing A184 variant, and ICOS-L variants (Y8, Y8_P51Q, and Y8_H57N) to ICOS was compared by bio-layer interferometry assay (Octet R8, Sartorius). Specifically, 5 μg / ml ICOS-Fc was immobilized for 1 minute in a Protein A biosensor (Sartorius) equilibrated with 1X phosphate buffer (pH 7.4) for 1 minute, and a baseline was drawn with 1X phosphate buffer (pH 7.4) for 1 minute. After allowing ICOS-L variants in the concentration range of 3.13 nM to 500 nM to associate for 10 minutes, 1X phosphate buffer (pH 7.4) was passed for 10 minutes for dissociation. Thus, the sensorgrams of different concentrations of each variant were analyzed ( Fig.11 ), thereby analyzing the final equilibrium dissociation constant (KD) value ( Fig.12 ).

[0227] Example 10. Preparation of ICOS Monomers for Determination of Binding Ability of ICOS-L Variants

[0228] 10-1. Cloning of ICOS monomer

[0229] In order to measure the ICOS binding ability of various ICOS-L variants, the designed primers and Vent polymerase (New England Biolabs) were used to amplify the deoxyribonucleic acid encoding the amino acid sequence E21-H129 of the ICOS extracellular region that eliminates the possibility of ICOS dimer formation. The amplified gene was treated with restriction enzymes using BssHII and XbaI (New England Biolabs), and the ICOS (E21-H129) gene treated with restriction enzymes was ligated to the pMAZ vector, which is a vector for animal cells, treated with the same restriction enzymes. After the ligated plasmid was transformed into Jude1 Escherichia coli, a single clone was obtained and the base sequence analysis confirmed that ICOS (E21-H129) was successfully inserted into the pMAZ vector.

[0230] 10-2. Expression and purification of ICOS single protein

[0231] After the prepared ICOS expression vector was transfected into Expi293F animal cells, the cells were cultured in a CO2 shaking incubator at 37°C, 125 rpm and 8% CO2 for 7 days and then centrifuged to separate only the supernatant. Then, the cells were balanced with 25× phosphate buffer solution and filtered using a 0.2μm bottle top filter (Merck Millipore). 0.5 ml of Ni-NTA resin was added to the filtered culture solution and stirred at room temperature for 1 hour to recover the resin. After washing with 10 ml of phosphate buffer solution containing 10 mM imidazole (Sigma), the cells were washed again with 10 ml of phosphate buffer solution containing 20 mM imidazole. After elution with a phosphate buffer solution containing 250 mM imidazole, the buffer was replaced with a 1× phosphate buffer solution (pH 7.4) using centrifugal filter units 10K (Merck Millipore). Then, the expressed and purified ICOS single protein ( Fig.13 ).

[0232] Example 11. Preparation of ICOS-L variants fused to antibody Fc domain

[0233] 11-1. Cloning of ICOS-L variants fused to antibody Fc domain

[0234] In order to evaluate the T cell activation ability of the discovered ICOS-L variant, it was fused with LALAPG (L234A / L235A / P329G), which is a silent Fc variant of human IgG1 antibody. In order to fuse the Fc domain of human IgG1 antibody to the C-terminal part of wild-type ICOS-L, existing A184 variant, and ICOS-L variants of the present invention (Y8 and Y8_H57N), ICOS-L and Fc (LALAPG) genes were amplified using designed primers and Vent polymerase (New England Biolabs). After assembly polymerase chain reaction of the amplified genes, they were inserted into pMAZ vectors (pMAZ-ICOS-L_WT-Fc, pMAZ-ICOS-L_A184-Fc, pMAZ-ICOS-L_Y8-Fc, and pMAZ-ICOS-L_Y8_H57N-Fc) using 2X Gibson assembly premix (New England Biolabs). The gene sample after the reaction was transformed into Jude1 Escherichia coli, and a single clone was obtained and subjected to base sequence analysis. It was confirmed that four ICOS-L variants fused with Fc (LALAPG) were successfully inserted into the pMAZ vector.

[0235] 11-2. Expression and purification of ICOS-L variants fused to antibody Fc domain

[0236] Expi293F cells were cultured at 2 × 10 6The cells were subcultured at a density of 100 ml cells / ml. One day later, the ICOS-L variant expression vectors fused with Fc (pMAZ-ICOS-L_WT-Fc, pMAZ-ICOS-L_A184-Fc, pMAZ-ICOS-L_Y8-Fc and pMAZ-ICOS-L_Y8_H57N-Fc) prepared in the above Example 11-1 and polyethyleneimine (Polyscience, 23966) were mixed in a ratio of 1:4 in 10 ml of Freestyle293 expression culture medium (Gibco.12338-018) and placed at room temperature for 20 minutes before transfection into the above Expi293F animal cells. After culturing the cells for 7 days at 37°C, 125 rpm and 8% CO2 in a CO2 shaking incubator, only the supernatant was collected by centrifugation, equilibrated with 25× phosphate buffer solution, and filtered using a bottle top filter with a 0.2μm filter (Merck Millipore). 0.5 ml of protein A resin was added to the filtered culture solution and passed through the column to recover the resin, and then washed with 10 ml of phosphate buffer solution. After eluting the washed resin with 100 mM glycine (pH 2.7) buffer, it was neutralized with 1 M Tris-HCl (pH 8.0) to obtain the purified ICOS-L variant fusion protein, and the buffer was replaced with 1× phosphate buffer solution using a centrifugal filter device 10K (Merck Millipore). The expression and purification of the ICOS-L variant fused to the antibody Fc domain (LALAPG) was confirmed by SDS-PAGE gel. Fig.14 ).

[0237] 11-3. Examination of the binding ability of ICOS-L variants fused to antibody Fc domain to ICOS

[0238] In order to analyze the binding ability of ICOS-L variant fused with antibody Fc domain (LALAPG) purified in Example 11-2 above to ICOS, an enzyme-linked immunosorbent assay was performed. Specifically, 50 μl of ICOS-L-Fc diluted to 4 μg / ml in 0.05 M sodium carbonate (pH 9.6) was dispensed into each well of a flat-bottomed polystyrene high-binding 96-well ELISA plate (Costar), fixed at 4°C for 16 hours, and then blocked at room temperature for 2 hours using 100 μl of 0.5% bovine serum albumin (Gibco) (contained in phosphate buffer solution, pH 7.4). After washing four times with 180 μl of 1× phosphate buffer solution (pH 7.4) (PBST) containing 0.05% Tween 20, 50 μl of wild-type ICOS serially diluted with phosphate buffer solution (pH 7.4) containing 0.5% bovine serum albumin was dispensed into each well and reacted at room temperature for 1 hour. After washing four times, 50 μl of anti-His-HRP conjugate was added and reacted at room temperature for 1 hour and then washed four times. Then, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well for color development and the reaction was terminated with 50 μl of 2M sulfuric acid and analyzed using an Epoch microplate spectrophotometer (Berteng Instruments Co., Ltd.). As a result, it was confirmed that the ICOS-L variant fused to the antibody Fc domain (LALAPG) bound to the wild-type ICOS in the same order as before the Fc fusion ( Fig.15 ).

[0239] Example 12. Confirmation of T cell activation of ICOS-L variants fused to antibody Fc domain

[0240] 12-1. Examination of T cell activity by T cell differentiation analysis

[0241] In order to confirm the T cell activation ability of ICOS-L variants fused to the antibody Fc domain by co-stimulation signaling, the degree of T cell differentiation was confirmed. Specifically, 1.5 μg / ml of anti-CD3 antibody (Biolegend) and 50 μl of ICOS-L variants fused with 50 nM Fc were dispensed into each well of a Flat Bottom Cell Culture Plate (SPL) and fixed at 4°C for 20 hours, followed by washing three times with 200 μl of phosphate buffer solution (pH 7.4). T cells were isolated from human peripheral blood mononuclear cells (PBMCs) using PanT Cell Isolation Kit (Miltenyi Biotec), stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) and resuspended in AIM-V medium (Gibco). 5×10 cells were dispensed into each well of a protein-fixed culture plate. 4 cells. After culturing for 3 days in a CO2 cell culture incubator at 37°C and 5% CO2, the cultured T cell culture solution was centrifuged at 400×g for 5 minutes, resuspended with 100μl of phosphate buffer solution and recovered using a V-bottom cell culture plate (Corning). The recovered cells were centrifuged and resuspended with 50μl of phosphate buffer solution, and 50μl of anti-CD4-APC probe or anti-CD8-APC probe (200:1) was placed in each well and cultured at room temperature for 20 minutes to label the cells with fluorescent probes. Then, the cells were washed with 100μl of phosphate buffer solution, centrifuged (400×g, 5 minutes) and resuspended with 200μl of phosphate buffer solution to prepare the sample, and the reduction in the CFSE fluorescence signal value of the prepared sample was measured using a FACSLyric (BD Biosciences) device. As a result, it was confirmed that the ICOS-L variant fused to the antibody Fc domain induced the activation of T cells, thereby causing the differentiation of T cells ( Fig.16 ). Thus, it was confirmed that the ICOS-L variant of the present invention, which has an improved binding ability to ICOS, successfully induced CD4 + T cells and CD8 + Differentiation of T cells.

[0242] 12-2. Examination of T cell activity by cytokine secretion analysis

[0243] In order to confirm the T cell activation ability caused by the costimulatory signaling of the ICOS-L variant fused to the antibody Fc domain, the degree of cytokine (γ-interferon) secretion was confirmed. Specifically, T cells were isolated in the same manner as in Example 12-1, stained with CFSE (Invitrogen) and resuspended in AIM-V medium (Gibco), and 5×10 cells were dispensed into each well of the protein-fixed culture plate. 4 After culturing for 3 days in a CO2 cell culture incubator at 37°C and 5% CO2, the cultured T cell culture medium was centrifuged at 400×g for 5 minutes, and the supernatant was obtained and used Human IFN-γ ELISA Kit (R&D Systems) was used to test the T cell activation ability of ICOS-L variants fused to the Fc domain of an antibody. The results showed that the variants fused to the Fc domain of an antibody showed a significant increase in cytokine secretion ( Fig.17 ).

[0244] Example 13. Preparation of ICOS-L variants fused to antibodies

[0245] 13-1. Preparation of anti-PD-L1 antibody (Atezolizumab)

[0246] 13-1-1. Cloning of atezolizumab expression vector

[0247] As an example of ICOS-L variant application, in order to confirm whether the efficacy of the antibody can be maximized by fusing the antibody with the ICOS-L variant, atezolizumab was prepared as an anti-PD-L1 antibody. In order to improve the purification yield and protein stability compared with existing antibodies, the LALAPG mutation was introduced into the Fc domain of atezolizumab instead of the existing IgG1N297A mutation to reduce the effector function of the antibody. Specifically, the variable region gene of the heavy chain of atezolizumab and the constant region gene of the heavy chain introduced with the LALAPG mutation were respectively amplified using the designed primers and Vent polymerase (New England Biolabs) to perform assembly polymerase chain reaction, and the obtained genes were treated with restriction enzymes using BssHII and XbaI (New England Biolabs). In addition, the variable region gene of the light chain of atezolizumab was also assembled with the constant region gene of the light chain to treat the same restriction enzyme. The heavy chain and light chain genes treated with restriction enzymes were ligated to the pMAZ vector, which is an animal cell vector treated with the same restriction enzyme. The ligated plasmid was transformed into Jude1 Escherichia coli to obtain a single clone, and then base sequence analysis was performed to confirm that the heavy chain and light chain genes of atezolizumab with the effector function removed were successfully inserted into the pMAZ vector.

[0248] 13-1-2. Expression and purification of atezolizumab

[0249] The heavy chain and light chain genes of atezolizumab prepared in the above Example 13-1-1 were mixed in a ratio of 1:1, and then mixed with polyethyleneimine (Polyscience, 23966) in a ratio of 1:4 and placed at room temperature for 20 minutes before transfection into Expi293F animal cells. After culturing for 7 days in a CO2 shaking incubator at 37°C, 125rpm and 8% CO2, centrifugation was performed to separate only the supernatant. Then, after balancing with 25x phosphate buffer solution, the supernatant was filtered using a bottle top filter with a 0.2μm filter (Merck Millipore). After adding 1ml of protein A resin to the filtered culture solution and passing it through the column to recover the resin, it was washed with 10ml of phosphate buffer solution. After eluting the washed resin with 100 mM glycine (pH 2.7) buffer, the purified atezolizumab was obtained by neutralization with 1 M Tris-HCl (pH 8.0), and then the buffer was replaced with 1× phosphate buffer solution using a centrifugal filter unit 30K (Centrifugal filter units 30K, Merck Millipore). Then, the expressed and purified atezolizumab antibody (ATZ) was confirmed using SDS-PAGE gel ( Fig.18 ).

[0250] 13-2. Preparation of ICOS-L variants fused to anti-PD-L1 antibodies

[0251] 13-2-1. Cloning of ICOS-L variants fused to atezolizumab

[0252] In order to fuse the ICOS-L variant of the present invention with atezolizumab (IgG1 Fc-LALAPG), which is an anti-PD-L1 antibody, cloned. Specifically, in order to fuse wild-type ICOS-L, existing ICOS-L A184 variants, and ICOS-L variants of the present invention (Y8 and Y8_H57N) to the C-terminus of the heavy chain or the C-terminus of the light chain of atezolizumab, ICOS-L and the heavy and light chain genes of atezolizumab were amplified using designed primers and Vent polymerase (New England Biolabs). After assembling the amplified gene by polymerase chain reaction, 2XGibson assembly premix (New England Biolabs) was used to insert it into the pMAZ vector. After the gene sample of the reaction technology was transformed into Jude1 Escherichia coli, a monoclonal clone was obtained and the results of base sequence analysis were confirmed, confirming that the ICOS-L variant fused to the C-terminus of the heavy chain or light chain of atezolizumab was successfully inserted into the pMAZ vector.

[0253] 13-2-2. Expression and purification of ICOS-L variants fused to atezolizumab

[0254] Expi293F cells were cultured at 2 × 10 6The cells were subcultured at a density of cells / ml in 100 ml. One day later, the ICOS-L variant expression vectors (pMAZ-Atezolizumab-IgH-ICOS-L_WT, pMAZ-Atezolizumab-IgH-ICOS-L_A184, pMAZ-Atezolizumab-IgH-ICOS-L_Y8 or pMAZ-Atezolizumab-IgH-ICOS-L_Y8_H57N) prepared in Example 13-2-1 and expressing the heavy chain of atezolizumab were cultured in 10 ml of Freestyle 293 expression medium (Gibco.12338-018). b-IgL) and ICOS-L variant expression vectors fused with the light chain of atezolizumab (pMAZ-Atezolizumab-IgL-ICOS-L_WT, pMAZ-Atezolizumab-IgL-ICOS-L_A184, pMAZ-Atezolizumab-IgL-ICOS-L_Y8 or pMAZ-Atezolizumab-IgL-ICOS-L_Y8_H57N) (+pMAZ-Atezolizumab-IgH) were mixed with polyethyleneimine (Polyscience, 23966) at a ratio of 1:4 and placed at room temperature for 20 minutes before transfection into the above-mentioned Expi293F animal cells. After culturing the cells for 7 days in a CO2 shaking incubator at 37°C, 125 rpm, and 8% CO2, only the supernatant was collected by centrifugation. Then, after balancing with 25x phosphate buffer solution, filter using a bottle top filter with a 0.2μm filter (Merck Millipore). The filtered culture solution was first placed in 0.5ml of protein A resin and passed through the column to recover the resin, and then washed with 10ml of phosphate buffer solution. After eluting the washed resin with 100mM glycine (pH2.7) buffer, it was neutralized with 1M Tris-HCl (pH8.0) to recover the ICOS-L variant fused with the purified heavy chain or light chain of atezolizumab, and then the buffer was replaced with 1× phosphate buffer solution using a centrifugal filter device 10K (Merck Millipore). In addition, the expressed and purified ICOS-L variant fused with the heavy chain or light chain of atezolizumab ( Fig.19 and Fig. 20 ).

[0255] 13-3. Examination of the binding ability of ICOS-L variants fused to anti-PD-L1 antibodies to ICOS

[0256] In order to analyze the binding ability of the ICOS-L variant fused with the heavy chain or light chain of atezolizumab purified in the above Example 13-2 to ICOS, an enzyme-linked immunosorbent assay was performed. Specifically, 50 μl of ICOS-Fc diluted to 4 μg / ml in 0.05 M sodium carbonate (pH 9.6) was dispensed into each well of a flat-bottomed polystyrene high-binding 96-well ELISA plate (Costar Company), fixed at 4°C for 16 hours, and then blocked at room temperature for 2 hours using 100 μl of phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin (Gibco). After washing 4 times with 180 μl of 0.05% PBST (pH 7.4), 200 nM of ICOS-L variant fused with the heavy chain or light chain of atezolizumab diluted with phosphate buffer solution (pH 7.4) containing 0.5% bovine serum albumin was reacted at room temperature for 1 hour. After washing 4 times, 50 μl of wild-type ICOS serially diluted with 0.5% bovine serum albumin (in phosphate buffer solution, pH 7.4) was dispensed into each well and reacted at room temperature for 1 hour. After washing 4 times, 50 μl of anti-His-HRP conjugate was added and reacted at room temperature for 1 hour and then washed 4 times. Then, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well for color development and 50 μl of 2M sulfuric acid was used to terminate the reaction and analyzed using an Epoch microplate spectrophotometer (Benton Instrument Co., Ltd.). The results showed that ICOS-L variants fused to the heavy chain of atezolizumab (ATZ(H)-A184, ATZ(H)-Y8, and ATZ(H)-Y8_H57N) and ICOS-L variants fused to the light chain (ATZ(L)-A184, ATZ(L)-Y8, and ATZ(L)-Y8_H57N) bound to wild-type ICOS in the same order as before fusion with atezolizumab ( Fig.21 and Fig. 22 ).

[0257] Example 14. Testing the T cell activation ability of ICOS-L variants fused to antibodies

[0258] 14-1. Examination of T cell activity by T cell differentiation analysis

[0259] In order to examine the T cell activation ability of ICOS-L variants fused to the heavy chain or light chain of atezolizumab caused by co-stimulatory signaling, the degree of T cell differentiation was confirmed in the same manner as in Example 12-1. As a result, it was confirmed that the ICOS-L variants fused to the heavy chain or light chain of atezolizumab induced T cell activation, thereby causing T cell differentiation ( Fig.23 and Fig.24). This confirmed that ICOS-L variants successfully induced CD4 + T cells and CD8 + Differentiation of T cells.

[0260] 14-2. Examination of T cell activity by cytokine secretion analysis

[0261] In order to examine the T cell activation ability caused by costimulatory signaling of ICOS-L variants fused to the heavy chain or light chain of atezolizumab, the degree of cytokine (γ-interferon) secretion was confirmed in the same manner as in Example 12-2 above. The results showed that the cytokine secretion of T cells treated with atezolizumab fused with ICOS-L was significantly increased compared with atezolizumab antibody ( Fig.25 and Fig.26 ). Through this confirmation, ICOS-L maximizes the efficacy of atezolizumab by successfully inducing the activation of T cells.

Claims

1. An ICOS-L variant, characterized in that One or more amino acids selected from the group consisting of amino acids at positions 4, 51, 52, 54, 57, 74, 130 and lower 234 in the amino acid sequence of wild-type ICOS-L are substituted with a sequence different from the wild-type amino acid.

2. The ICOS-L variant according to claim 1, characterized in that The amino acid sequence of wild-type ICOS-L comprises the amino acid sequence of SEQ ID NO:

1.

3. The ICOS-L variant according to claim 1, characterized in that It comprises one or more amino acid substitutions selected from the group consisting of E4D, Q51P, Q51H, N52S, S54N, N57H, L74R, S130C and K234R.

4. The ICOS-L variant according to claim 1, characterized in that Contains amino acid substitutions Q51P and N57H.

5. The ICOS-L variant according to claim 1, characterized in that Contains amino acid substitutions of Q51H, S54N and S130C.

6. The ICOS-L variant according to claim 1, characterized in that Contains amino acid substitutions N52S and K234R.

7. The ICOS-L variant according to claim 1, characterized in that Contains amino acid substitutions E4D and Q51P.

8. The ICOS-L variant according to claim 1, characterized in that Contains amino acid substitutions Q51P and L74R.

9. The ICOS-L variant according to claim 1, characterized in that It also contains a transmembrane domain.

10. The ICOS-L variant according to claim 9, characterized in that It also contains a cytoplasmic signaling domain connected to the transmembrane domain.

11. An immunomodulatory protein, characterized in that Comprising the ICOS-L variant according to claim 1 and an Fc domain of an immunoglobulin or a variant thereof.

12. The immunomodulatory protein according to claim 11, characterized in that The Fc domain variants contain one or more amino acid substitutions in the Fc domain of wild-type human IgG1.

13. The immunomodulatory protein according to claim 11, characterized in that The Fc domain variant is an Fc domain variant that exhibits reduced effector function compared to the Fc of wild-type human IgG1.

14. The immunomodulatory protein according to claim 11, characterized in that The Fc domain variant is an Fc domain variant in which the amino acid 234, 235 or 329 numbered according to the Kabat numbering system in the Fc domain of wild-type human IgG1 is substituted with a sequence different from the wild-type amino acid.

15. The immunomodulatory protein according to claim 11, characterized in that The Fc domain variants comprised amino acid substitutions of L234A, L235A and P329G.

16. A conjugate, characterized in that: It comprises the ICOS-L variant of claim 1 or the immunomodulatory protein of claim 11 and a targeting portion.

17. The conjugate according to claim 16, characterized in that The targeting moiety binds specifically to molecules on the surface of immune cells.

18. The conjugate according to claim 17, characterized in that Immune cells are antigen presenting cells or lymphocytes.

19. The conjugate according to claim 16, characterized in that A targeting moiety is a tumor localizing moiety that binds to a molecule on the surface of the tumor.

20. The conjugate according to claim 16, characterized in that The targeting moiety is an antibody or a fragment thereof having immunological activity.

21. The conjugate according to claim 20, characterized in that The antibodies are cetuximab, panitumumab, zalutumumab, inolizumab, trastuzumab, ado-trastuzumab, emtansine, tositumomab, rituximab, ibritumomab tiuxetan, daclizumab, gemtuzumab, alemtuzumab, CEA scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab, afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, denosumab, erlotinib, everolimus, ibrutinib, imatinib, la Patinib, lenvatinib, nilotinib, olaparib, olaratumab, palbociclib, pazopanib, pertuzumab, ramucirumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, vismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab, AMP-224, MSB001078C, MEDI4736, BMS-935559, LY3300054, atezolizumab, avelumab, or durvalumab.

22. A T cell activator, characterized in that It comprises the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11 or the conjugate of claim 16.

23. The T cell activator according to claim 22, characterized in that The T cells are cytotoxic T cells or chimeric antigen receptor T cells.

24. A polynucleotide, characterized in that The method comprises a nucleic acid encoding the ICOS-L variant of claim 9 and one or more nucleic acids encoding one or more chains of a recombinant antigen receptor.

25. The polynucleotide according to claim 24, characterized in that Recombinant antigen receptors are chimeric antigen receptors or engineered T cell receptors.

26. An engineered cell, characterized in that It comprises the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11 or the conjugate of claim 16.

27. The engineered cell according to claim 26, characterized in that Also included are chimeric antigen receptors or engineered T cell receptors.

28. An anticancer adjuvant, characterized in that: It comprises the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11, the conjugate of claim 16 or the engineered cell of claim 26.

29. The anticancer adjuvant according to claim 28, characterized in that The above-mentioned anticancer adjuvant is an immune anticancer adjuvant that enhances the anticancer effect of the immune checkpoint inhibitor.

30. The anticancer adjuvant according to claim 28, characterized in that The above-mentioned anticancer adjuvants are administered simultaneously, separately or sequentially in combination with immune checkpoint inhibitors.

31. The anticancer adjuvant according to claim 29, characterized in that Immune checkpoint inhibitors are anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, or their variants.

32. A pharmaceutical composition for preventing or treating cancer, characterized in that: The invention comprises the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11, the conjugate of claim 16 or the engineered cell of claim 26 as an active ingredient.

33. The pharmaceutical composition for preventing or treating cancer according to claim 32, characterized in that: The cancer is one or more selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma.

34. A composition for cancer diagnosis, characterized in that It comprises the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11, the conjugate of claim 16 or the engineered cell of claim 26.

35. The cancer diagnosis composition according to claim 34, characterized in that It further comprises one selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromophore, a luminescent substance, and a fluorescent substance.

36. A method for providing information for diagnosing cancer, characterized in that include: Step a), contacting a biological sample separated from a subject with the composition for cancer diagnosis according to claim 34; Step b), confirming the expression of ICOS in the sample; as well as Step c), comparing the expression of ICOS in the samples with that in the normal control group.

37. A method for predicting the therapeutic responsiveness or prognostic diagnosis of immune checkpoint inhibitors, characterized in that: The method comprises the step of confirming the expression level of ICOS in a biological sample isolated from a subject treated with an immune checkpoint inhibitor using the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11, the conjugate of claim 16 or the engineered cell of claim 26.

38. Use of the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11, the conjugate of claim 16 or the engineered cell of claim 26 in preventing or treating cancer.

39. A method for treating cancer, characterized in that: The method comprises administering the ICOS-L variant of claim 1, the immunomodulatory protein of claim 11, the conjugate of claim 16 or the engineered cell of claim 26 to an individual suffering from cancer in a pharmaceutically effective amount.

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