WO2025127849A1 - Anticancer composition for co-administration with anticancer agent, comprising novel peptide as active ingredient - Google Patents

Abstract

The present invention relates to a combination therapy of a TB511 peptide having an anticancer effect by specifically binding to CD18, and a cancer immunotherapy agent. The TB511 of the present invention specifically binds to CD18 having an activated form of M2-tumor-associated macrophages (M2-TAMs) contributing to tumor progression in the tumor microenvironment (TME), and thus induces apoptosis, does not exhibit toxicity against other tissue-resident macrophages that are not M2-TAMs, and, in various cancer models and humanized mouse models, increases the infiltration of tumor-killing cells and exhibits an antitumor effect, and particularly, exhibits a synergistic antitumor effect when used in combination with another cancer immunotherapy agent, and thus may be used as a cancer immunotherapy agent or a combination therewith.

Description

Anticancer composition for co-administration with anticancer agent containing novel peptide as active ingredient

The present invention relates to a combination therapy of a TB511 peptide that specifically binds to CD18 and has an anticancer effect and an anticancer agent.

Existing cancer treatments have been studied in the direction of directly attacking cancer cells or enhancing the activity of the body's immune cells that attack cancer cells. However, these anticancer drugs attack normal cells in addition to cancer cells, resulting in many side effects such as hair loss, nausea, and vomiting, and causing additional reactions due to excessive increase in immune cells. Compared to existing chemotherapy or radiation therapy, anticancer immunotherapy is a method of treating cancer using the body's immune system, which can minimize side effects. Among these anticancer immunotherapy techniques, there are cell therapy methods that activate therapeutic immune cells such as T cells (including CAR-T), dendritic cells, and natural killer cells outside the body and then directly inject them into the body, and methods for anticancer vaccines that directly activate immune cells existing in the body by injecting cancer antigens and immune-activating substances into the body, thereby increasing anticancer efficacy. However, these cell therapy agents or cancer vaccines are mainly used for diseases related to blood cancer, and most of them have a disadvantage in that their therapeutic efficacy is very low for solid cancers. One of the reasons for this is due to microenvironmental factors that suppress immune function around solid cancers. In fact, cells that reduce the function of immune cells in the tumor microenvironment (MDSC: myeoloid-derived stromal cells, Treg: regulatory T cells, TAM: tumor-assocaited macrophages) or immunosuppressive cytokines and metabolites actively work, rapidly reducing the activity of immune-activating substances and therapeutic immune cells. Therefore, it is becoming important to develop a treatment that has an anticancer effect by controlling only the microenvironment around tumor cells without directly affecting tumor cells and immune cells, thereby blocking the supply of nutrients to tumor cells and angiogenesis around tumor cells. The tumor microenvironment is greatly considered as a treatment target because it contributes to the proliferation and survival of malignant cells, angiogenesis, metastasis, abnormal adaptive immunity, and decreased response to hormones and chemotherapeutics.

The microenvironment around the tumor is composed of endothelial cells, inflammatory cells, and fibroblasts, and in the 1970s, tumor-associated macrophages (TAMs) were found to play a critical role in tumor growth. TAMs play an important role in the overall tumor microenvironment, including cancer growth and metastasis, and are classified into two phenotypes: tumor-suppressive M1 macrophages and tumor-supportive M2 macrophages. M1 macrophages have a potent antigen-presenting ability, are generally activated by interferon-γ, lipopolysaccharide (LPS), and tumor necrosis factor (TNF)-α, and exhibit proinflammatory and bactericidal effects. M2 macrophages are known to promote immunosuppression, tumorigenesis, and angiogenesis by releasing various extracellular matrix components, angiogenic and chemotactic factors. In general, they are induced by IL-4 and IL-13, and are distinguished from M1 type tumor-associated macrophages by expressing markers such as arginase-1, mannose (MMR, CD206), scavenger receptors (SR-A, CD204), CD163, and IL-10. Tumor-associated macrophages present around tumors are closely related to the growth and metastasis of tumor cells, and it has been reported that when a large number of M2 type tumor-associated macrophages exist around tumors in cancer patients, the prognosis and survival rate of the patients are poor. M2 type macrophages produce cytokines such as IL-10, TGFβ, and CCL18 that promote cancer growth, and receptors such as PDL1 and B7-1/2 present on the surface of M2 type tumor-associated macrophages have been reported to suppress the antitumor activity of T cells and NK cells. Since tumor growth, differentiation, and metastasis occur actively in a microenvironment where M2 type tumor-associated macrophages exist in large numbers, development of targeted therapeutics targeting M2 type tumor-associated macrophages is necessary.

Meanwhile, cancer cells express immune checkpoint proteins on their cell surface, which are used by normal cells to suppress immune cell activation in order to avoid the killing mechanism by immune cells. Recently, research on immune checkpoint inhibitor proteins as a method for treating cancer has been actively conducted. It has been reported in academic circles that among immune checkpoint inhibitor proteins, blocking PD-1/PD-L1 binding is particularly effective in cancer treatment and has fewer side effects than other immune checkpoint inhibitor proteins (J. Naidoo et al. (2015) Annals of Oncology, Lucia Gelao et al. (2014) Toxins, Gorge K. Philips et al (2015) International Immunology). The PD-1 receptor is expressed on the surface of activated immune cell types, including T cells, B cells, and natural killer (NK)/natural killer T (NKT) cells (Goodman, Patel & Kurzrock, PD-1-PD-L1 immune-checkpoint blockade in B-cell lymphomas, Nature Reviews Clinical Oncology, 14:203-220, 2017.). PD-1 is a negative regulator of T cell activation, and the interaction of PD-1 with one of its ligands, PD-L1, on the tumor surface represents an immune checkpoint blockade that reduces the ability of activated T cells to generate effective immune responses. Therapeutic antibodies targeting PD-1 or PD-L1 block the ligand-receptor interaction and restore immune function to the tumor microenvironment. Several therapeutic monoclonal antibodies (mAbs) targeting PD-1 and PD-L1 are on the market, and anti-PD1 antibody treatments that inhibit the binding of PD-1 and PD-L1, such as Opdivo (nivolumab) from BMS, Keytruda (Pembrolizumab) from Merck, and Libtayo (Cemiplimab) developed by Regeneron, and anti-PD-L1 antibody treatments such as Tecentriq (Atezolizumab) from Roche, Imfinzi (Durvalumab) from AstraZeneca, and Bavencio (Avelumab) from Merck Sereno, have received US FDA approval and are revolutionizing the treatment of intractable cancers in clinical trials.

An object of the present invention is to provide a chimeric antigen receptor comprising a peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an antigen binding domain.

It is also an object of the present invention to provide a recombinant vector comprising a gene encoding a chimeric antigen receptor.

Furthermore, it is an object of the present invention to provide a chimeric antigen receptor expressing cell transformed with a recombinant vector.

Furthermore, it is an object of the present invention to provide a T cell engager comprising a peptide of sequence number 1 or sequence number 2.

In addition, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer.

Furthermore, it is an object of the present invention to provide a combination for preventing or treating cancer.

In addition, an object of the present invention is to provide a composition for cancer diagnosis.

In addition, the purpose of the present invention is to provide a method for preventing or treating cancer.

To solve the above problem, the present invention provides a chimeric antigen receptor comprising a peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an antigen binding domain.

Additionally, the present invention provides a recombinant vector comprising a gene encoding the chimeric antigen receptor.

In addition, the present invention provides a chimeric antigen receptor expressing cell transformed with the recombinant vector.

Additionally, the present invention provides a T cell engager comprising a peptide of sequence number 1 or sequence number 2.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a peptide of the sequence number 1 or sequence number 2, a chimeric antigen receptor, a chimeric antigen receptor expressing cell, or a T cell engager as an active ingredient.

In addition, the present invention provides a combination for preventing or treating cancer, comprising a peptide of the above sequence number 1 or sequence number 2, a chimeric antigen receptor, a chimeric antigen receptor expressing cell, or a T cell engager; and an immunotherapy agent, a chemotherapy agent, or an antibody-drug conjugate (ADC).

In addition, the present invention provides a composition for diagnosing cancer, comprising a peptide of the sequence number 1 or sequence number 2, a chimeric antigen receptor, a chimeric antigen receptor expressing cell, or a T cell engager as an effective ingredient.

In addition, the present invention provides a method for preventing or treating cancer, comprising a step of administering the pharmaceutical composition or the combination to a subject.

TB511 of the present invention induces apoptosis by specifically binding to CD18 of activated form of M2-Tumor-associated macrophages (M2-TAMs) that contribute to tumor progression in the tumor microenvironment (TME), and does not exhibit toxicity to other tissue-resident macrophages other than M2-TAM, and increases the infiltration of tumor killer cells and exhibits antitumor effects in various cancer models and humanized mouse models, and in particular, exhibits synergistic antitumor effects when used in combination with other immunotherapy drugs, and therefore can be utilized as an immunotherapy drug or a combination thereof.

Figures 1 to 11 are diagrams analyzing the target of TB511 and analyzing the relationship between CD18, the target of TB511, and the progression of solid tumors:

Figure 1: Target protein analysis process of TB511;

Figure 2: Validation results of CD18 knockdown cells;

Figure 3: Cell viability analysis results;

Figure 4: Results of direct binding analysis of TB511 and CD18;

Figure 5: Localization analysis of TAMpep and CD18 in M2 macrophages;

Figures 6 and 7: Results of CD18 expression analysis in solid tumors; and

Figures 8 to 11: Results of CD18 expression analysis according to macrophage subtype.

Figures 12 to 15 are diagrams showing the results of analysis of membrane proteins of M0, M1, and M2 macrophages binding to TAMpep.

Figure 12: Proteins extracted from M0 macrophages;

Figure 13: Proteins extracted from M1 macrophages;

Figure 14: Proteins extracted from M2 macrophages; and

Figure 15: Ratios of M2/M0 and M2/M1.

Figures 16 and 17 are diagrams showing the results of analyzing the binding affinity of TAMpep to CD18:

a: Binding affinity of TAMpep to CD18; and

b: Binding affinity of anti-CD18 antibody to CD18.

Figures 18 to 26 are diagrams analyzing the cytotoxicity induction effect through CD18 structure-specific targeting of TB511:

Figure 18: Results of protein-peptide interaction modeling analysis;

Figure 19: Results of CD18 binding site analysis of TB511;

Figures 20 and 21: Cytotoxicity analysis results of TB511 depending on CD18 activation;

Figure 22: Results of analysis of CD18 activation level according to tumor microenvironment induction;

Figure 23: Results of analysis of CD18 activation levels according to macrophage subtypes;

Figure 24 and Figure 25: Results of apoptosis analysis of TB511 according to macrophage subtype; and

Figure 26: Results of analysis of CD18 activation level in tumor tissue.

Figures 27 to 34 show the analysis of internalization and endosomal escape of TB511 in M2 macrophages:

Figure 27: Results of analysis of the distribution of TB511 in M2 macrophages;

Figures 28 to 30: Results of 3D spatial trajectory analysis of TB511

Figure 31: Results of analysis of the localization of early endosome antigen 1 (EEA1) and TB511-Cy5 in endosomes following treatment with CPZ (chlorpromazine), M*?*CD (methyl-_-cyclodextrin), or CyD (cytochalasin D);

Figure 32: Results of TB511-induced apoptosis analysis according to treatment with CPZ (chlorpromazine), M_CD (methyl-_-cyclodextrin) or CyD (cytochalasin D);

Figure 33: Results of localization analysis of early endosome antigen 1 and TB511-Cy5 in endosomes according to treatment; and

Figure 34: Results of TB511-induced apoptosis analysis following Baf-A1 (bafilomycin A1) or CQ (chloroquine) treatment.

Figures 35 to 53 are diagrams analyzing the antitumor effect of TB511:

Figures 35-37: Antitumor effect of TB511 in a mouse model of cold tumors that are nonresponsive to immunotherapy;

Figures 38-41: Antitumor effect of TB511 in a mouse model of immunotherapy-responsive tumors (hot tumors);

Figures 42 to 47: Results of selective targeting analysis of TB511 on macrophage subtypes in tumor tissue; and

Figures 49 to 51: Results of macrophage count analysis in normal and tumor tissues.

Figures 52 to 55 are diagrams analyzing the tumor targeting of TB511 through the biological distribution analysis of TB511 in a breast cancer mouse model.

Figures 56 to 72 are diagrams analyzing immune cell changes caused by TB511 in TME:

Figures 56 and 57: Results of analysis of changes in CD8+ T and NK cells in prostate cancer (PC) specimens;

Figures 58 and 59: Results of analysis of the effects of CD8+ T and NK cells on the antitumor effect of TB511 in prostate cancer (PC) specimens;

Figure 60: Process for preparing NK or CD8 deficient colon cancer mouse model;

Figures 61 to 63: Results of cell viability analysis in the control group (isotype control), CD8+ T cell depletion group (CD8 depletion), and NK cell depletion group (NK depletion) in a prostate cancer cell line injection mouse model;

Figures 64 and 65: Analysis of the number of tumor-infiltrating CD8+ T cells and the number of exhausted CD8+ T cells in a lung cancer cells (LLC) injection mouse model;

Figures 66 and 67: Results of analysis of the number of anti-tumor immune cells in colorectal cancer (CRC) cells;

Figures 68 and 69: Results of CD8+ T and Granzyme B+ cell count analysis;

Figure 70: Manufacturing process of CD8 deficient colon cancer mouse model;

Fig. 71: Antitumor effect of TB511 in control mice; and

Figure 72: Antitumor effect of TB511 in a CD8 deficient colon cancer mouse model.

Figures 73 to 76 are diagrams analyzing the number of CD8+ T and NK cells in the blood of a CD8 deficient mouse model (a) or an NK deficient mouse model (b).

Figures 77 to 93 are diagrams evaluating the clinical potential of TB511:

Figure 77: Process for manufacturing a humanized prostate cancer mouse model;

Figure 78: Antitumor effect of TB511 in immunodeficient mice;

Figure 79: Antitumor effect of TB511 in humanized prostate cancer mice;

Figures 80 to 82: Analysis of human immune cells infiltrating the TME;

Figure 83: Process for manufacturing a humanized lung cancer mouse model;

Figure 84: Antitumor effect of TB511 in immunodeficient mice;

Figure 85: Antitumor effects of TB511 alone, anti-PD-1 antibody alone, and TB511 and anti-PD-1 antibody combination in humanized lung cancer mice;

Figures 86 to 88: Cell count analysis results of M2-TAMs (CD86+CD11b+/CD45+ cells);

Figures 89 to 91: Cell count analysis results of exhausted CD8+ T-cells (PD-1+CD8+/CD45+ cells); and

Figure 92 and Figure 93: CD8+ cell counts in each group determined by immunohistochemical staining in humanized non-small cell lung cancer (NSCLC) tumor tissues.

Figures 94 to 99 are diagrams analyzing the synergistic effect of combined administration of TB511 and an immune checkpoint inhibitor.

Figure 94: Process for manufacturing hPD-L1 MC38 tumor-humanized PD-1 mouse model;

Figure 95: Tumor volume in the control group;

Figure 96: Tumor volume in the anti-PD-1 monotherapy group;

Figure 97: Tumor volume in the TB511 monotherapy group;

Figure 98: Tumor volume of the group administered with combination of TB511 and anti-PD-1 antibody; and

Figure 99: Survival rate of mice in each group.

Figures 100 to 105 are diagrams analyzing the anticancer effect of combined administration of TB511 and oxaliplatin.

Figure 100: Manufacturing process of colon cancer mouse model and drug administration;

Figure 101: Tumor volume in the control group;

Figure 102: Tumor volume of the control group;

Figure 103: Tumor volume in the TB511 monotherapy group;

Figure 104: Tumor volume in the oxaliplatin monotherapy group; and

Figure 105: Tumor volume of the combination therapy group of TB511 and oxaliplatin.

Figures 106 to 108 are diagrams analyzing tumor weight and body weight changes according to combined administration of TB511 and oxaliplatin.

Fig. 106: Size of tumor tissue;

Fig. 107: Changes in the weight of tumor tissue; and

Fig. 108: Weight change of the mouse.

Figures 109 to 113 are diagrams analyzing the decrease in M2-like tumor associated macrophages (TAMs) in the tumor microenvironment (TME) following combined administration of TB511 and oxaliplatin.

Figures 109 and 110: Flow cytometry analysis results for macrophage populations within the TME;

Figure 111: M1-like TAMs (CD86+ F4/80+ cells within CD45+ CD11b+ cells);

Figure 112: M2-like TAMs (CD206+ F4/80+ cells within CD45+ CD11b+); and

Fig. 113: M1/M2 ratio.

Figures 114 to 117 are diagrams analyzing the induction of infiltration of cytotoxic T cells into the TME by combined administration of TB511 and oxaliplatin.

Figure 114: Flow cytometry analysis results for lymphocyte distribution within the TME;

Figure 115: CD4 T cells (CD45+ CD3+ CD4+ cells);

Figure 116: CD8 T cells (CD45+ CD3+ CD8+ cells); and

Figure 117: NK cells (CD45+ CD3+ NKp46+ cells).

Figure 118 is a diagram showing the triple-negative breast cancer mouse model setup and drug process.

Figure 119 is a diagram showing the results of an analysis of the anticancer effect according to combined administration of TB511 and paclitaxel.

Figures 120 to 125 are diagrams showing the results of tumor inhibition analysis according to combined administration of TB511 and PADCEV in a human pancreatic cancer mouse model.

Figure 120: Tumor sample 43 days after tumor inoculation;

Figure 121: Tumor volume over time after tumor inoculation;

Figures 122 and 123: Graphs showing immunohistochemical staining of Ki67 for tumor tissue and quantification of Ki67 positive cells; and

Figures 124 and 125: Results of immunofluorescence staining analysis of E-cadherin and Vimentin in tumor tissues.

Figures 126 to 131 are diagrams showing the results of analysis of the reduction of M2 macrophages in tumor tissues following combined administration of TB511 and PADCEV.

Figures 126 and 127: Graphs showing the results of immunohistochemical staining of CD163 and actin in tumor tissue and quantification of CD163 positive cells;

Figure 128 and Figure 129: Results of immunohistochemical staining of CD18 in tumor tissue and quantification of CD18 positive cells; and

Figures 130 and 131: Graphs showing the results of immunohistochemical staining of Kim127 and CD11b in tumor tissue and the quantification of Kim127 and CD11b positive cells.

Figures 132 and 133 are diagrams showing the results of analysis of the increase in CD8 T cells in tumor tissues following combined administration of TB511 and PADCEV.

Figure 132: Results of immunofluorescence staining analysis of CD8 in tumor tissue; and

Figure 133: Graph quantifying the number of CD8 positive cells.

Figures 134 to 141 are diagrams showing the results of an association analysis of TB511 with macrophages and CD8 T cells expressing activated CD18 in normal tissues.

Figures 134 to 137: Flow cytometry analysis results for macrophages using anti-Kim127, anti-CD18, anti-CD11b and anti-CD45 antibodies; and

Figures 138 to 141: Flow cytometry analysis of CD8 T cells using anti-Kim127, anti-CD18, anti-CD8, and anti-CD45 antibodies.

Figures 142 to 156 are diagrams showing the results of analysis on immune cell regulation within the tumor microenvironment following combined administration of TB511 and PADCEV.

Figures 142 to 145: Cell clusters within tumor tissue; and

Figures 146 to 149: UMAP analysis results for cell clusters within tumor tissue.

Figures 150 to 153: Pie charts showing the proportion of cell types in cell clusters within tumor tissue; and

Figures 154 and 155: Cell population (%) by each cell type.

Figures 156 to 159 illustrate the results of analysis of the effect of TB511 on the activation of CD8 T cells within a tumor microenvironment.

Figures 156 and 157: CD8 T cell exhausted genes (white arrows) and activator genes (red arrows) in tumor tissue; and

Figures 158 and 159: CTLA4, FOXP3, LAG3, GZMB, IFNG and PDCD1 gene expression levels.

Hereinafter, the present invention will be described in detail with reference to the attached drawings as embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description thereof may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

In addition, the terminology used in this specification is a terminology used to appropriately express the preferred embodiment of the present invention, and this may vary depending on the intention of the user or operator, or the custom of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless specifically stated otherwise.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in this specification, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications mentioned as references in this specification are incorporated into the present invention.

Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as the generally accepted three-letter codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. In addition, amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:

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.

In one aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising a peptide of SEQ ID NO: 1 or SEQ ID NO: 2 as an antigen binding domain.

In one embodiment, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 can specifically bind to M2 macrophages or M2 tumor associated macrophages (M2 TAM).

In one embodiment, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 can specifically bind to CD18.

In one embodiment, the peptide may comprise a variant thereof or an analog thereof.

In the present invention, it is preferable that the peptide has the amino acid sequence described above, but is not limited thereto. According to a preferred embodiment of the present invention, it is preferable that the peptide has a high ratio of the amino acid of 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 100%.

In the present invention, the peptide may also include additional amino acid sequences designed for a specific purpose, such as a targeting sequence, a tag, a labeled residue, a half-life or to increase the stability of the peptide. In addition, the peptide of the present invention may be linked to coupling partners, such as effectors, drugs, prodrugs, toxins, peptides, delivery molecules, etc.

In the present invention, the peptide can be prepared in the form of a pharmaceutically acceptable salt. Specifically, a salt can be formed by adding an acid, for example, an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), an organic carboxylic acid (e.g., acetic acid, haloacetic acids such as trifluoroacetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), and an organic sulfonic acid including an acidic sugar (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), an acidic polysaccharide (e.g., hyaluronic acid, chondroitin sulfate, arginic acid), a sulfonic acid sugar ester such as chondroitin sulfate (e.g., methanesulfonic acid, p-toluene sulfonic acid), etc.

The term "peptide" used in the present invention refers to an amino acid polymer, which may include not only natural amino acids but also non-protein amino acids as components.

The term "peptide variant" as used herein refers to a corresponding amino acid sequence that contains at least one amino acid difference (substitution, insertion or deletion) compared to a reference sequence. In certain embodiments, a "variant" has high amino acid sequence homology and/or conservative amino acid substitutions, deletions and/or insertions compared to a reference sequence.

The term "peptide analogue" as used herein may include analogues in which the side chain or the alpha-amino acid backbone of an amino acid is substituted with one or more other functional groups. Examples of side chain or backbone modified peptide analogues include, but are not limited to, hydroxyproline in which the pyrrolidine ring is substituted with a hydroxy group, or N-methyl glycine "peptoids". Types of peptide analogues are well known in the art.

As used herein, the term "conservative amino acid substitution" refers to a modification of a variant that includes replacing one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the peptide. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well known and defined in the art. These classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Peptides according to the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, peptides according to the present invention can be synthesized by a number of methods, including, for example, methods including:

(a) a method of synthesizing a peptide stepwise or by fragment assembly by means of solid-phase or liquid-phase methods, and isolating and purifying the final peptide product; or

(b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

(c) a method of performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

A method of obtaining fragments of a peptide by any combination of (a), (b), and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

The term "CAR (chimeric antigen receptor)" used in the present invention refers to a non-naturally occurring receptor capable of conferring specificity for a specific antigen to immune effector cells. CAR is usually composed of an extracellular domain (Ectodomain), a transmembrane domain, and an intracellular domain (Ectodomain).

The above extracellular domain includes an antigen recognition region, and the transmembrane domain of the CAR is in a form connected to the extracellular domain, and may be derived from natural or synthetic sources. If derived from a naturally existing source, it may be derived from a membrane-bound or membrane-permeable protein, and may be a portion derived from a membrane-permeable region of various proteins such as the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154 or CD8. The sequence of such a transmembrane domain can be obtained from, but is not limited to, documents known in the art that disclose membrane-permeable region portions of membrane-permeable proteins.

In addition, when the above-mentioned transmembrane domain is synthetic, it may mainly include hydrophobic amino acid residues such as leucine and valine, and for example, a triplet of phenylalanine, tryptophan and valine may be present in the synthetic transmembrane domain, but is not limited thereto. Sequence information on such transmembrane domain can be obtained from literature known in the art regarding synthetic transmembrane domains, but is not limited thereto.

In the CAR of the present invention, the intracellular domain is a part of the domain of the CAR existing within a cell, and is connected to the transmembrane domain. The intracellular domain of the present invention may include an intracellular signaling domain which is characterized by causing T cell activation, preferably T cell proliferation, when an antigen binds to the antigen binding site of the CAR. The intracellular signaling domain is not particularly limited in its type as long as it is a part that transmits a signal capable of causing T cell activation when an antibody binds to the antigen binding site existing outside the cell, and various types of intracellular signaling domains may be used. Examples of the intracellular signaling domain include an immunoreceptor tyrosine-based activation motif or ITAM, and the ITAM includes, but is not limited to, those derived from CD3 zeta (ξ, zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d or FcεRIγ.

CARs comprise a single-chain fragment variable region (scFv) of an antibody specific for a tumor-associated antigen (TAA) that is coupled via the hinge and transmembrane regions to the cytoplasmic domain of a T-cell signaling molecule. Most common lymphocyte activating moieties comprise a T-cell costimulatory (e.g., CD28, CD137, OX40, ICOS, and CD27) domain in tandem with a T-cell triggering (e.g., CD3ζ) moiety. CAR-mediated adoptive immunotherapy allows CAR-engrafted cells to directly recognize TAAs on target tumor cells in a non-HLA-restricted manner.

In one aspect, the present invention relates to a recombinant vector comprising a gene encoding the chimeric antigen receptor.

In one aspect, the present invention relates to a chimeric antigen receptor expressing cell transformed with the recombinant vector.

In one embodiment, the chimeric antigen receptor expressing cell can be a chimeric antigen receptor expressing macrophage (CAR-macrophage), a chimeric antigen receptor expressing T (CAR-T) cell, a chimeric antigen receptor expressing-gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.

The term "chimeric antigen receptor expressing T (CAR-T) cell" used in the present invention refers to a T cell expressing a CAR. The chimeric antigen receptor expressing T (CAR-T) cell has the advantage of: i) recognizing a cancer antigen in a manner independent of HLA (human leukocyte antigen), and thus capable of treating cancers that evade the action of anticancer agents by reducing HLA expression on the cell surface; ii) being independent of the HLA type, and thus capable of being used for treatment regardless of the patient's HLA type; and iii) capable of producing a large amount of cancer-specific T cells in a short period of time, and thus capable of exhibiting an excellent anticancer effect.

The above T cells include CD4 ⁺ T cells (helper T cells, TH cells), CD8 ⁺ T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells), natural killer T cells, etc., and the T cells into which the CAR is introduced in the present invention are preferably CD8 ⁺ T cells, but are not limited thereto.

In one aspect, the present invention relates to a T-cell engager comprising a peptide having sequence number 1 or sequence number 2.

In one embodiment, the T cell engager can be, for example, a bispecific T-cell engager (BiTE). The BiTE is a class of artificial bispecific monoclonal antibodies, which are fusion proteins consisting of amino acid sequences from four different genes or two single chain variable region fragments (scFv) of different antibodies on a single peptide chain of about 55 kilodaltons. One of the scFv binds to the T cell via the CD3 receptor, and the other binds to the tumor cell via a tumor-specific molecule. Similar to other bispecific antibodies, and unlike conventional monoclonal antibodies, the BiTE forms a link between the T cell and the tumor cell. It causes the T cell to exert cytotoxic activity on the tumor cell independently of the presence of MHC I or costimulatory molecules by producing proteins such as perforin and granzymes. These proteins enter the tumor cell and initiate apoptosis of the cell.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a peptide of SEQ ID NO: 1 or SEQ ID NO: 2, a chimeric antigen receptor of the present invention, a cell expressing a chimeric antigen receptor of the present invention, or a T cell engager of the present invention as an active ingredient.

In one embodiment, the cancer can be a non-responsive or responsive cancer to an immunotherapy agent, can be a solid cancer, and is preferably, but not limited to, breast cancer, colon cancer, or pancreatic cancer.

In one embodiment, the composition may be an immunotherapy agent.

In one embodiment, the composition may be characterized by, but is not limited to, an enhanced anticancer effect when co-administered with a chemotherapeutic agent or an antibody-drug conjugate (ADC).

In one embodiment, the antibody-drug conjugate (ADC) may be, but is not limited to, an anti-Nectin-4 antibody drug conjugate (PADCEV).

The pharmaceutical composition of the present invention can be used as a single therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy, and when such combination therapy is performed, cancer can be treated more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapeutic agents that can be used together with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, etc. Radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and γ-ray irradiation, etc.

In one embodiment, the composition of the present invention may further comprise an immunogenic apoptosis inducer, wherein the immunogenic apoptosis inducer may be at least one selected from the group consisting of an anthracycline series anticancer agent, a taxane series anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide series anticancer agent, a GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin, and the anthracycline series anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or It could be valrubicin, and the taxane family of chemotherapy agents could be paclitaxel or docetaxel.

The pharmaceutical composition for preventing or treating cancer of the present invention can increase the cancer treatment effect of conventional anticancer drugs through the cancer cell killing effect by administering it together with a chemical anticancer drug (anticancer agent), etc. The combined administration can be performed simultaneously with or sequentially with the anticancer agent. Examples of the anticancer agent include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; Anti-cancer antibiotics include, but are not limited to, dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids include, but are not limited to, vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan.

In the present invention, the term “prevention” means any act of inhibiting or delaying the occurrence, spread, and recurrence of cancer by administering a pharmaceutical composition according to the present invention.

The term "treatment" used in the present invention means any act of killing cancer cells or improving or beneficially changing the symptoms of cancer by administering the composition of the present invention. Anyone with ordinary knowledge in the technical field to which the present invention pertains will be able to refer to materials presented by the Korean Medical Association, etc. to know the exact criteria for diseases to which the composition of the present invention is effective, and to determine the degree of improvement, enhancement, and treatment.

The term "therapeutically effective amount" used in combination with the active ingredient in the present invention means the amount of a pharmaceutically acceptable salt of the composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage when used in humans should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. Such considerations 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 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used in the present invention means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined according to factors including the patient's health condition, the type and severity of cancer, the activity of the drug, the sensitivity to the drug, the administration method, the administration time, the administration route and the excretion rate, the treatment period, the drug used in combination or simultaneously, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

The composition of the present invention may also include a carrier, a diluent, an excipient or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, and for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components may be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, a diluent, a dispersant, a surfactant, a binder and a lubricant may be additionally added to formulate the composition into a main-use dosage form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule or a tablet. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention can be administered non-orally (for example, intravenously, subcutaneously, intraperitoneally, or locally as an injection formulation) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to administer once a day or in divided doses several times.

Liquid preparations for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

In one aspect, the present invention relates to a combination for preventing or treating cancer, comprising a peptide having sequence number 1 or sequence number 2 of the present invention, a chimeric antigen receptor of the present invention, a cell expressing a chimeric antigen receptor of the present invention, or a T cell engager of the present invention; and an immunotherapy agent, a chemotherapeutic agent, or an antibody-drug conjugate (ADC).

In one embodiment, the immunotherapy agent may be an immune checkpoint inhibitor, an immunosuppressant modulating drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine required for maintaining the activity of an immune cell for cancer treatment.

In one embodiment, the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-CTLA4, or an antigen-binding fragment of such an antibody that specifically binds to PD-1, PDL-1, or CTLA4, or a combination thereof.

In one embodiment, the immune checkpoint inhibitor can be an anti-PD1 antibody nivolumab or pembrolizumab, an anti-PD-L1 antibody atezolizumab, avelumab or durvalumab, an anti-CTLA-4 antibody tremelimumab or ipilimumab, or a combination thereof.

In one embodiment, the combination may additionally comprise a chemotherapeutic agent, or radiation.

In one embodiment, the chemotherapeutic agent may be a pro-apoptotic peptide, an immunogenic apoptosis inducer or an anticancer agent, and may be selected from the group consisting of SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, Ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, Duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, It may be selected from the group consisting of izabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof, preferably oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, It may be one or more selected from the group consisting of Vincristine, Etoposide and Doxorubicin, most preferably Oxaliplatin or Paclitaxel.

In one embodiment, the antibody-drug conjugate may be an anti-Nectin-4 antibody drug conjugate (PADCEV).

The PADCEV of the present invention is an antibody-drug conjugate (ADC). Specifically, PADCEV is a drug called enfortumab vedotin, which is approved by the FDA and is used to treat urothelial carcinoma, including bladder cancer.

Nectin-4 is a protein that is overexpressed in cancer cells such as bladder cancer cells, and when anti-Nectin-4 antibodies bind to Nectin-4, the drug enters the cancer cells. MMAE is released inside the cells, destroying microtubules and killing cancer cells. It is usually used for patients with urothelial cancer, and is especially common for patients who do not respond to previous immunotherapy (e.g., PD-1/PD-L1 inhibitors) or chemotherapy.

In one embodiment, the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention, the chimeric antigen receptor of the present invention, the chimeric antigen receptor expressing cell of the present invention, or the T cell engager of the present invention; and an immunotherapy agent, a chemotherapeutic agent, or an antibody-drug conjugate (ADC); may be administered simultaneously, separately, or sequentially.

In one aspect, the present invention relates to an anticancer adjuvant comprising a peptide of the present invention having sequence number 1 or sequence number 2, a chimeric antigen receptor of the present invention, a cell expressing the chimeric antigen receptor of the present invention, or a T cell engager of the present invention as an active ingredient.

In one embodiment, the anticancer adjuvant of the present invention may be administered concurrently, separately, or sequentially with an immunotherapy agent.

In one embodiment, the immunotherapy agent may be an immune checkpoint inhibitor, an immunosuppressant modulating drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine required for maintaining the activity of an immune cell for cancer treatment.

In one embodiment, the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-CTLA4, or an antigen-binding fragment of such an antibody that specifically binds to PD-1, PDL-1, or CTLA4, or a combination thereof.

In one embodiment, the immune checkpoint inhibitor can be an anti-PD1 antibody nivolumab or pembrolizumab, an anti-PD-L1 antibody atezolizumab, avelumab or durvalumab, an anti-CTLA-4 antibody tremelimumab or ipilimumab, or a combination thereof.

In one embodiment, the cancer targeted by the pharmaceutical composition or combination of the present invention may be selected from the group consisting of ovarian cancer, breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, liver cancer, prostate cancer, head and neck cancer, bladder cancer, colon cancer, colon cancer, pancreatic cancer, kidney cancer, bone marrow cancer, uterine cancer, melanoma, brain cancer and thyroid cancer, but is preferably selected from the group consisting of breast cancer, lung cancer, non-small cell lung cancer, liver cancer, colon cancer, colon cancer, pancreatic cancer and brain cancer, and is most preferably breast cancer, colon cancer or breast cancer, but is not limited thereto.

In one embodiment, the pharmaceutical composition or combination of the present invention may be administered at a concentration of TB511 of 100 to 300 nmol/kg, and oxaliplatin may be administered at a concentration of 1 to 2 mg/kg, but is not limited thereto.

In one embodiment, the pharmaceutical composition or combination of the present invention may be administered at a concentration of TB511 of 100 to 300 nmol/kg, and paclitaxel may be administered at a concentration of 3 to 7 mg/kg, but is not limited thereto.

In one embodiment, the pharmaceutical composition or combination of the present invention may be administered at a concentration of TB511 of 100 to 300 nmol/kg, and PADCEV (anti-Nectin-4 antibody drug conjugate) may be administered at a concentration of 3 to 7 mg/kg, but is not limited thereto.

In one aspect, the present invention relates to a composition for diagnosing cancer, comprising a peptide of sequence number 1 or sequence number 2 of the present invention, a chimeric antigen receptor of the present invention, a cell expressing a chimeric antigen receptor of the present invention, or a T cell engager of the present invention as an active ingredient.

In one embodiment, the composition can further comprise a label, and the label can be a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, a fluorescent substance, a probe or a tag, and the fluorescent substance can be a fluorescent substance of the Cy (cyanine) series, Rhodamine series, Alexa series, BODIPY series or ROX series, and can be Nile Red, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine or Alexa.

In one aspect, the present invention relates to a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition or combination of the present invention.

The term "subject" used in the present invention means a subject that requires a method for preventing, controlling or treating a disease, and can be used without limitation as a human, dog, monkey, cat, rodent, such as a mouse, a genetically engineered mouse, etc. More specifically, it means a mammal, such as a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to concretize the contents of the present invention and the present invention is not limited thereto.

Example 1. Synthesis of TB511 peptide

To synthesize a peptide that targets and kills tumor-associated macrophages (TAMs), which mainly exist in an M2-like phenotype and contribute to tumor progression by establishing an immunosuppressive environment in the tumor microenvironment (TME), a TAM-targeting peptide, TAMpep (SEQ ID NO: 2) and a pro-apoptotic peptide, dKLA (SEQ ID NO: 3), with a linker consisting of four glycines and one serine to minimize their interaction and folding, TB511 peptide (SEQ ID NO: 1) was synthesized and purified by GenScript (Piscataway, NJ, USA). In this case, the D-isomer, not the L-form, of KLA was used to minimize degradation in the body. In addition, FITC or Cy5 (cyanine 5) was conjugated via an amide bond at the N-terminus of the peptide, and biotin was conjugated via an amide bond at the lysine residue of the C-terminus of the peptide. In addition, PEGylation was performed at the amino terminus of the peptide. All peptides were purified with a purity of more than 95%. The payload of TB511, d(KLA), is a cationic and amphipathic α-helical peptide that can bind to and disrupt negatively charged mitochondrial membranes, and cannot pass through zwitterionic eukaryotic cells, so it does not exhibit toxicity to eukaryotic cells.

designation order Sequence number TB511 [PEG2]VLTTGLPALISWIKRKRQQ-GGGGS-d[KLAKLAKKLAKLAK] 1 TAMpep VLTTGLPALISWIKRKRQQ 2 d(KLA) d(KLAKLAKLAKLAKLAK) 3 Cy5 conjugated TB511 Cy5-[PEG2] VLTTGLPALISWIKRKRQQGGGGS d[KLAKLAKKLAKLAK] Biotinylated TAMpep VLTTGLPALISWIKRKRQQK-biotin FITC conjugated TAMpep FITC-VLTTGLPALISWIKRKRQQ

Example 2. Target protein analysis of TB511

To identify the target protein of TB511, THP-1 monocyte cells were differentiated into M0, M1, or M2 macrophages, and proteomic analysis was performed. Specifically, THP-1 was differentiated into M0 macrophages by culturing in complete media with 100 nM PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Additionally, to polarize into M1 or M2 cells, M0 cells were cultured with 20 ng/mL recombinant human interferon-gamma (rhIFN-γ; Prospec, Rehovot, Israel) and 100 ng/mL lipopolysaccharides (LPS; Sigma-Aldrich), or 20 ng/mL recombinant human interleukin-4 (rhIL-4; Prospec) and 20 μg/mL rhIL-13 (Prospec) in RPMI 1640 medium supplemented with 5% FBS for 72 h. Additionally, PC3 or A549 cells were grown in complete medium to 80% confluence, washed and cultured in serum-free medium for 24 h, and then filtered through a 0.2 μm syringe filter (Sartorius, Göttingen, Germany) to prepare tumor-conditioned medium (TCM), and M0 cells were cultured with 10% or 50% tumor-conditioned medium for 72 h to polarize to M2-TAM. Afterwards, THP-1 macrophages differentiated into M0, M1, and M2 were disrupted using Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the cell lysates were incubated with biotinylated TAMpep for 1 h at room temperature. After incubation with streptavidin agarose resin (Thermo Fisher Scientific) at room temperature for 30 min, the resin was washed, and the membrane-bound proteins were eluted from the resin. The eluted protein samples were analyzed by LC-MS/MS using a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The LC-MS/MS data of each analyzed sample were analyzed using Proteome Discoverer, and the UniProt human database was used for label-free quantification. Proteomic analysis was performed using the ExDEGA (Excel-based Differentially Expressed Gene Analysis) tool (eBiogen, Seoul, Korea) (Fig. 1).

As a result, a total of 1,450 proteins were identified in M0, M1, and M2 macrophages, and 54 of them preferentially bound to biotin-labeled TAMpep in M2 macrophages compared to M0 and M1 macrophages (Figs. 12 to 14). Among these proteins, CD18 was particularly highly expressed in myeloid lineage cell lines such as macrophages, and was weakly expressed only in lymphoid (Fig. 15).

Example 3. CD18 targeting analysis of TB511

To confirm that CD18 is the target protein of TB511, CD18-targeting sgRNA (sgRNA/CD18; GTTCAACGTGACCTTCCGGC) was transfected into M2-differentiated cas9 stable THP-1 cells to perform CD18/sgRNA knockdown, and then CD18 mRNA levels were analyzed by qRT-PCR to verify the generation of knockdown cells (Fig. 2). After that, the cells were treated with TB511 (3 μM) for 24 h, and cell viability was confirmed by MTS assay. Additionally, direct binding of TB511 to CD18 was analyzed by incubating cell lysates with scrambled or TAMpep peptide (0, 0.001, 0.01, 0.1, 1, or 10 μg), followed by incubation with biotinylated TAMpep, and performing PAGE analysis of the eluted proteins to separate peptide-bound membrane proteins using Streptavidin Dynabeads (Thermo Fisher Scientific). In addition, immunofluorescence staining analysis was performed using FITC-labeled TAMpep and APC-conjugated anti-rabbit IgG secondary antibody (1:500, Invitrogen). In addition, the binding affinity of TAMpep to CD18 was analyzed.

Cell viability analysis results showed that cell viability was significantly reduced in CD18/sgRNA knockdown cells treated with TB511 compared to mock-transfected cells (Fig. 3). In addition, immunoprecipitation analysis results showed that CD18 was immunoprecipitated with biotinylated TAMpep, and this binding was blocked in a concentration-dependent manner when pre-incubated with non-biotinylated TAMpep (Fig. 4), confirming the direct binding of TB511 to CD18. In addition, immunofluorescence staining analysis results confirmed that TAMpep was present at the same location as CD18 in M2 macrophages (Fig. 5). In addition, analysis of the binding affinity of TAMpep to CD18 showed a K _D value of 1.963 Х 10 ^-6 (Figs. 16 and 17), confirming that it is at a similar level to the binding affinity of anti-CD18 antibody.

Example 4. Analysis of tumor relevance of CD18

To determine whether CD18 is associated with the progression of solid tumors, especially with the infiltration of M2 macrophages into tumor tissues, the correlation between CD18 expression and tumor progression was determined using tumor tissue arrays. Specifically, paraffin was removed from normal and tumor tissue slides of breast, colon, lung, liver, kidney, and skin using EZ prep (Ventana Medical Systems, #950-102) at 76°C for 4 min, followed by incubation with cell conditioning solution 1 (Ventana Medical Systems, #950-124) at 100°C for 24 min for antigen retrieval. The slides were treated with OptiView peroxidase inhibitor (Ventana Medical Systems, #760-700) for 4 min at 37°C, followed by incubation with anti-CD18 antibody (1:500, LSbio). Slides were then visualized using the OptiView DAB IHC Detection Kit (Ventana Medical Systems, #760-700), and images were scanned and analyzed using Aperio ImageScope software (Leica, Wetzlar, Germany).

As a result, the expression level of CD18 was found to be significantly higher in breast, colon, lung, liver, kidney, and skin tumor tissues than in breast, colon, lung, liver, kidney, and skin normal tissues (Figs. 6 and 7).

Example 5. Analysis of CD18 expression according to macrophage subtype

As in Example 2 above, THP-1 monocytes were differentiated into M0, M1, and M2 macrophages, and polarized with M2-TAM. The relative mRNA expression level of CD18 was analyzed by qRT-PCR, and the protein expression level was confirmed by Western blot analysis and immunofluorescence staining.

As a result, the expression levels of CD18 genes and proteins were found to be significantly higher in M2 macrophages and M2-TAM than in other subtypes of macrophages (Figs. 8 to 11).

Example 6. Binding and effect analysis of TB511 to CD18 in activated conformation

6-1. Combination modeling of TB511 and CD18

The binding of TB511 to CD18 was analyzed using protein-peptide interaction modeling. Specifically, based on the sequence of the cysteine-rich region of CD18 (Uniport _p05107, cysteine-rich tandem repeat region: 449-617), the 3D structure was constructed using the trRosetta algorithm ( https:/yanglab.nankai.edu.cn/trRosetta/ ). Using the CD18 PDB file generated by trRosetta and the amino acid sequence of TB511, docking simulations of TB511 and CD18 were performed using the CABS-dock server ( http:/biocomp.chem.uw.edu.pl/CABSdock/ ). Ten models were generated. Additionally, 5 μg/mL CD18 protein (Abbexa) and FITC-conjugated TAMpep and alanine-substituted library peptides of TAMpep were reacted at room temperature for 2 hours, and then the binding affinity between the peptides was analyzed using ELISA. Based on the results of the above binding affinity analysis, a representative model was selected from among the 10 models generated through the above docking simulation.

Protein-peptide interaction modeling analysis results showed that TB511 binding to total CD18 in the bent (closed) conformation was not predicted, whereas the open conformation cysteine-rich domain was strongly predicted to bind to TB511 (Fig. 18), suggesting that lysine and tryptophan residues of TB511 are the major binding elements. In addition, binding affinity analysis using the alanine substitution library of TAMpep showed that the A6 peptide, in which lysine was substituted with alanine, and the A12 peptide, in which tryptophan was substituted with alanine, exhibited significantly reduced binding to CD18 protein (Fig. 19).

6-2. Analysis of CD18-specific binding and apoptosis through activation of TB511

To determine whether TB511 recognizes the activated open conformation of CD18 and induces cytotoxicity through this, CD18 was activated in THP-1 cells and the cytotoxicity induced by TB511 treatment was compared with that of the inactivated case. Specifically, THP-1 cells were activated in the resting state by treating them with 1 mM manganese chloride tetrahydrate (Mn ²⁺ , Sigma-Aldrich) for 1 h. Then, TB511 (1 μM) was treated to the activated THP-1 cells and the inactivated control THP-1 cells, respectively. Cell viability was determined by staining with Annexin V (APC) and 7AAD and analyzing the stained cells with a FACS Lyric System (BD Bioscience, NJ, USA).

As a result, it was shown that apoptosis significantly increased in CD18-activated THP-1 cells compared to the control cells when treated with TB511 (Figs. 20 and 21). Therefore, it was confirmed that TB511 recognizes the activated open structure of CD18 and induces cytotoxicity.

6-3. Analysis of CD18 activation according to tumor microenvironment and macrophage subtype

To determine whether CD18 maintains the activated structure in M2-TAM of the tumor microenvironment, THP-1 cells were treated with A549 tumor-conditioned medium, and the activation level of CD18 was analyzed by FACS using FITC-conjugated mAb KIM127 (Leinco Technologies, Pantone, MO, USA). In addition, THP-1 cells were differentiated into M0, M1, M2, and M2-TAM subtypes, respectively, and the degree of CD18 activation was analyzed.

As a result, the activation level of CD18 was found to be significantly increased in a concentration-dependent manner by A549 tumor-conditioned medium treatment (Fig. 22). In addition, among the subtypes of macrophages, the activation level of CD18 was found to be the highest in polarized M2-TAM cultured with tumor-conditioned medium (Fig. 23), confirming that M2-TAM maintains the activated form of CD18 in the TME.

6-4. Analysis of cell death by TB511 according to macrophage subtype

To compare the extent of TB511-induced apoptosis in each macrophage subtype, THP-1 cells were differentiated into M0, M1, M2, or M2-TAM and treated with MitoTracker RedROX (Invitrogen) and caspase 3/7 reagents for 30 min, followed by TB511 (1 μM), and live-cell imaging was performed immediately using restoration fluorescence microscopy (DeltaVison, NJ, USA).

As a result, caspase 3/7 activation by TB511 started in M2-TAM within 5 min of incubation with TB511 and reached a peak at 15 min. M2 macrophages were affected by TB511 to a lesser extent, but no caspase 3/7 activation was observed in M0 or M1 macrophages (Figs. 24 and 25). This confirmed that the conformational-specific binding between CD18 and TB511 resulted in selective cytotoxicity toward M2-TAM among macrophage subtypes.

6-5. Analysis of tumor relevance of activated CD18

Analysis of the level of activated open conformation of CD18 using tumor tissue array revealed that the level of CD18 activation (pan CD18 and KIM127-double positive) was significantly increased in tumor tissues compared to normal tissues of prostate, liver, breast, lung, and colon (Fig. 26), indicating that CD18 exists in an inactive form in most tissue-resident macrophages in normal cells, but changes to an activated form in the TME.

Example 7. Analysis of M2 macrophage membrane dynamics and intracellular transport of TB511

To track the membrane dynamics and intracellular transport of TB511, TB511 was conjugated to carboxylic acid-functionalized gold nanorods (AuNRs; Nanopartz, Salt Lake City, UT, USA) using 1-ethyl-3-3-(dimethylaminopropyl)carbodiimide hydrochloride (Pierce, Rockford, IL, USA) and sulfo-N-hydroxysulfosuccinimide (Pierce). THP-1-differentiated M2 macrophages were treated with 20 μL of TB511-AuNRs (about 1.5 Х 10 ⁹ particles/mL) and incubated with MitoTracker Green (Invitrogen) and Hoechst 33342 (Invitrogen) for 0.5 h. Single-particle tracking images were acquired at 10-ms intervals using iMLSM (integrated multi-dimensional light-sheet microscopy). The acquired images were analyzed using ImageJ (NIH) and MATLAB (Mathworks, Torrance, CA, USA), and the 3D spatial trajectories (x, y, z) of individual peptide-AuNRs were super-localized by the astigmatism method. Additionally, the rotation and orientation of individual peptide-AuNR were calculated by fitting the transverse surface plasmon resonance (TSPR) with cos2φ and the longitudinal surface plasmon resonance (LSPR) with sin2θ. In addition, for endocytosis inhibition, CPZ (chlorpromazine), an inhibitor of clathrin-mediated endocytosis (CME), MβCD (methyl-β-cyclodextrin), an inhibitor that inhibits both clathrin-mediated endocytosis and caveolae-mediated endocytosis, or CyD (cytochalasin D), an inhibitor that inhibits macropinocytosis (MP), or Baf-A1 (bafilomycin A1) or CQ (chloroquine) were treated to inhibit acidification of early endosomes, or maturation and association of endosomes with lysosomes, and the locations of early endosome antigen 1 (EEA1) and TB511-Cy5 in endosomes were confirmed by immunofluorescence staining analysis.

As a result, DIC (differential interference contrast) and SRRF (super-resolution radial fluctuations) images of M2 showed that TB511-AuNRs were distributed in the mitochondria and nucleus within the cells (Fig. 27). Analysis of the 3D spatial trajectories of individual peptide-AuNRs showed that displacements and scattering intensity fluctuations in the x, y, and z directions were gradually lost (Figs. 28 to 30), confirming the binding of TB511-AuNRs to the membrane receptors of M2 macrophages. In addition, at a specific time of approximately 20-41 s, TB511-AuNRs showed less spatial movement and slower directional changes, confirming that they were packed into the membrane and assembled into endocytosis apparatuses, after which clear movements in the x, y, and z directions were observed, confirming that TB511-AuNRs were separated from the membrane and transported into the cell. In addition, when treated with inhibitors that inhibit cellular uptake, CPZ and CyD inhibited the uptake of TB511 and reduced TB511-induced apoptosis, whereas MβCD did not cause apoptosis (Figs. 31 and 32). In addition, treatment with Baf-A1 and CQ increased the co-localization of EEA1 and TB511-Cy5 in endosomes, and significantly reduced TB511-induced apoptosis (Figs. 33 and 34).

This suggests that TB511 is internalized into M2 macrophages through endocytosis, exits the endosomes, and enters the cytoplasm.

Example 8. TB511 Antitumor effect analysis

8-1. Antitumor effect on cold tumors that are not responsive to immunotherapy

To determine the antitumor effect of TB511 on Cold tumors, which have low responsiveness to immunotherapy and lack tumor antigens, a subcutaneous prostate cancer (PC) mouse model and an orthotopic hepatocellular carcinoma (HCC) mouse model were prepared and the antitumor effect of TB511 was evaluated. Specifically, to prepare a subcutaneous tumor mouse model, PC tumor cells were mixed 1:1 with Matrigel (Corning, NY, USA) and then subcutaneously injected into the right flank of 6-8 week-old C57BL/6 or BALB/c mice (Taconic Biosciences, NY, USA). When the tumor volume reached 50-100 mm ³ , the tumor-bearing mice were divided into two groups, and TB511 was injected subcutaneously into the TB511 group at a dose of 200 nmol/kg every 3 days. To prepare orthotopic tumor mouse models, the abdomens of mice were incised, and hepa1-6-Luc cells were injected into the cecum or the subserosal plane of the liver, and then sutured. Seven or 14 days after tumor injection, the mice were divided into two groups and injected subcutaneously with TB511 every 3 days, and D-luciferin (BioVision, Milpitas, CA, USA) was injected intraperitoneally at a dose of 3 mg per mouse to track tumor growth. Tumor diameters were measured using a digital caliper, and tumor volumes were calculated by the following mathematical formula 1. Mice were evaluated once or twice a week using a NightOWL LB 983 in vivo imaging system (Berthold Technologies, Bad Wildbad, Germany), and 30 days after tumor injection, they were euthanized and tumor tissues were isolated.

As a result, TB511 was shown to significantly inhibit tumor growth in a mouse model of immunotherapy-unresponsive tumors (Cold tumors) (Figs. 35 to 37).

8-2. Antitumor effect on hot tumors responsive to immunotherapy

To confirm the antitumor effect of TB511 on hot tumors, which exhibit high responsiveness to immunotherapy and are characterized by immune activation of tumor antigens and infiltrated T cells, subcutaneous non-small cell lung cancer (NSCLC) or renal cell carcinoma (RCC) mouse models and orthotopic colorectal cancer (CRC) mouse models were prepared as in Example 8-1, and TB511 was administered thereto, and the antitumor effect was analyzed.

As a result, TB511 was shown to significantly inhibit tumor growth in a mouse model of immunotherapy-responsive tumors (Hot tumors) (Figs. 38 to 41).

Through this, it was confirmed that TB511 exhibited antitumor effects regardless of responsiveness to immunotherapy.

8-3. Analysis of M2-TAM targeting of TB511 in tumor microenvironment

To determine whether the antitumor effect of TB511 was caused by targeting M2-TAM in the TME, the cell counts of M2-TAM (the percentage of F4/80 ⁺ CD206 ⁺ among the CD45 ⁺ gated cells) and M1-TAM (the percentage of CD206 ⁺ F4/80 ⁺ among the CD45 ⁺ gated cells) in PC and CRC tumor tissues were analyzed by FACS, and the M1/M2 ratio was calculated. In addition, to confirm the selective targeting of TB511 to macrophage subtypes, the cell counts of M1-TAM and M2-TAM in CRC tumor tissues, and the number of tissue-resident macrophages present in normal tissues including brain, skin, kidney, and liver after treatment with TB511 were analyzed by immunohistochemistry (IHC).

As a result, the number of M2-TAM cells in PC and CRC tumor tissues was significantly reduced in the TB511-administered group compared to the control group, whereas the number of M1-TAM cells did not change significantly, indicating that the M1/M2 ratio was significantly increased by TB511 administration (Figs. 42 to 47). In addition, the number of M2-TAM cells was reduced and the number of M1-TAM cells was not changed by TB511 treatment in tumor tissues, whereas the tissue-resident macrophage population (F4/80 ⁺ ) in normal tissues was not affected by TB511 treatment (Figs. 48 to 51).

8-4. Tumor targeting analysis of TB511

Cy5-labeled TB511 was injected into an orthotopic breast cancer mouse model, and the bio-distribution of TB511 was investigated. Specifically, 4T1 breast cancer cells were injected into the mammary fat pad of mice at a density of 5Х10 ⁵ cells/mouse to prepare an orthotopic breast cancer mouse model, and 7 days later, Cy5-labeled TB511 or scrambled peptides (control) were intravenously injected at 2.5 mg/kg. The mice were anesthetized, and the distribution of TB511 in vivo was photographed and analyzed using IVIS Lumina II (PerkinElmer, Waltham, MA, USA).

As a result, 48 hours after injection, the fluorescent signal of TB511 was observed only in the injected breast tissue, not in the liver, lung, heart, kidney, or muscle, and showed a higher biological distribution in the tumor tissue compared to the control group (Figs. 52 to 55).

Through this, we confirmed that TB511 specifically accumulates at tumor sites and selectively removes M2-TAM in the TME, but does not affect tissue-resident macrophages within normal tissues.

Example 9. Analysis of immune cell changes in TME caused by TB511

9-1. Immune cell reprogramming analysis

To determine whether the removal of M2-TAM by TB511 reprograms immune cells, PC specimens were treated with TB511, and changes in anti-tumor immune cell populations within the TME, such as CD4 ⁺ , CD8 ⁺ , and NK cells, were analyzed by FACS, and the data were confirmed by t-SNE analysis. In addition, to confirm the influence of CD8 ⁺ T and NK cells on the antitumor effect of TB511, anti-NK1. 1 antibody (clone PK136; BioXcell) or anti-CD8 antibody (clone 2.43; BioXcell, Lebanon, NH, USA) was injected intraperitoneally 3 days and 1 day before tumor injection, and TRAMP-C2 cells, which are colon cancer cells, were mixed with Matrigel and injected as in Example 8-1, and then the antibodies were injected weekly (control group: injection of IgGa isotype (C1.18.4, BioXcell)) to prepare NK or CD8 deficient mouse models (Fig. 60). Thereafter, the numbers of CD8 ⁺ T and NK cells in the peripheral blood mononuclear cells of the mice were analyzed. In addition, the antitumor effect of TB511 was analyzed in the NK or CD8 deficient mouse models.

As a result, the NK cell population in the PC was significantly increased, whereas the CD8 ⁺ T cell population did not change statistically significantly (Figs. 56 and 57). In addition, the number of NCAM1 ⁺ NK cells was found to increase in the PC specimens after TB511 treatment (Figs. 58 and 59). In addition, the numbers of CD8 ⁺ T and NK cells in the blood of NK or CD8 deficient mouse models were significantly decreased by antibody treatment (Figs. 73 to 76). In addition, TB511 showed an antitumor effect in both the control group and the CD8 ⁺ T cell deficient group, whereas it did not show an antitumor effect in the NK cell deficient group (Figs. 61 to 63).

9-2. Analysis of tumor-infiltrating cell counts

The number of tumor-infiltrated CD8 ⁺ T cells (CD45 ⁺ CD8 ⁺ ) and exhausted CD8 ⁺ T cells (PD-1 ⁺ CD8 ⁺ T) by TB511 treatment in immunotherapy-responsive tumors such as NSCLC and CRC were determined by FACS analysis, and the number of CD8 ⁺ T and Granzyme B ⁺ cells were determined by IHC analysis. In addition, a CRC mouse model was created by injecting CT26 cells, a colon cancer cell line, and anti-CD8 antibody (Fig. 70), and the antitumor effect by TB511 treatment was analyzed.

As a result, the number of tumor-infiltrating CD8 ⁺ T cells in NSCLC increased in the group administered TB511, while the number of depleted CD8 ⁺ T cells decreased (Figs. 64 and 65). In addition, among the antitumor immune cells, only CD8 ⁺ T cells were significantly increased in CRC (Figs. 66 and 67). In addition, the IHC analysis results showed that the number of CD8 ⁺ T and Granzyme B ⁺ cells increased by TB511 treatment (Figs. 68 and 69), and the antitumor effect of TB511 was not observed only in CD8 ⁺ -depleted cells in the CRC mouse model (Figs. 70 to 72).

Through this, we confirmed that tumor-infiltrating cells in the TME significantly changed after TB511 treatment in both immunotherapy-responsive and non-responsive tumors, but the types of infiltrating cells differed depending on each tumor type.

Example 10. Clinical feasibility assessment of TB511

10-1. Analysis of antitumor effect of TB511 through immune system

To evaluate the clinical potential of TB511, 4-week-old SID mice (NOD-Prkdc ^em1Beak IL2rg ^em1Break ), which are immunodeficient mice, were irradiated with 1.5 Gy of gamma irradiation and intravenously injected with human CD34 ⁺ hematopoietic stem cells (Lonza, Basel, Switzerland) at 2Х10 ⁵ cells/mouse to generate a humanized mouse model. Eleven weeks after stem cell injection, peripheral blood was collected, and flow cytometry was used to confirm the engraftment of mature human leukocytes (human CD45 ⁺ cells). The hCD45 ⁺ cell population accounted for 37.32 ± 13.44% in male mice (n = 16) and 42.63 ± 10.41% in female mice (n = 15) 11 weeks after transplantation, which met the condition of a humanized mouse model with more than 25% human CD45 cells in the peripheral blood. Then, PC3 (human prostate cancer cell line) and A549 (human lung cancer cell line) were injected subcutaneously into the humanized mouse model, and the volume was measured every 3 days. TB511 was administered every 3 days from day 11 after tumor cell injection (Figs. 77 and 85), and the tumor growth in the mice and the changes in human immune cells infiltrating the TME were investigated by t-SNE analysis.

As a result, the antitumor effect of TB511 was completely lost in immunodeficient mice injected with PC3 tumor cells or A549 cells (Figs. 78 and 84), whereas TB511 inhibited tumor growth in CD34 ⁺ transplanted mice (Figs. 79 and 85), confirming that an intact immune system is essential for the antitumor effect of TB511. In addition, as a result of analyzing human immune cells infiltrating the TME, the number of M2-TAM (CD11b ⁺ CD206 ⁺ /CD45 ⁺ cells) was significantly reduced after TB511 treatment, while the number of M1-TAM (CD11b ⁺ CD86 ⁺ /CD45 ⁺ cells) did not change, indicating that the M1/M2 ratio increased (Figs. 80 to 82). Additionally, tumor-killer cells, including CD335 ⁺ NK and CD8 ⁺ T cells, were shown to significantly infiltrate the TME after TB511 treatment (Figs. 80 to 82).

10-2. Analysis of the effects of TB511 and immune checkpoint inhibitors alone or in combination

To determine the effect of combined treatment with immune checkpoint inhibitors of TB511, humanized mice injected with A549 cells were administered TB511 alone, anti-PD-1 antibody (pembrolizumab) alone, or a combination of TB511 and anti-PD-1 antibody, and the antitumor effect was analyzed by measuring tumor volume and analyzing changes in tumor-infiltrating immune cell populations.

As a result of analyzing the antitumor effect, the antitumor effects of the TB511 monotherapy group and the anti-PD-1 antibody monotherapy group were found to be similar, while the antitumor effect of the group administered in combination was found to be significantly increased (Fig. 85). In addition, as a result of analyzing the change in the tumor-infiltrating immune cell population, the cell number of M1-TAMs (CD206 ⁺ CD11b ⁺ /CD45 ⁺ cells) was found to be significantly increased in the group administered in combination with TB511 and anti-PD-1 antibody, while the cell number of M2-TAMs (CD86 ⁺ CD11b ⁺ /CD45 ⁺ cells) was found to be significantly decreased (Figs. 86 to 88). In addition, Granzyme B-positive CD8 ⁺ T cells were significantly increased by co-administration of TB511 and anti-PD-1 antibody, indicating increased infiltration into the TME, while the depleted CD8 ⁺ T-cell (PD-1 ⁺ CD8 ⁺ /CD45 ⁺ cell) population was significantly decreased in the TB511 monotherapy group and the TB511 and anti-PD-1 antibody co-administration group (Figs. 89 to 91).

10-3. Analysis of synergistic effects by combined administration of TB511 and immune checkpoint inhibitors

To confirm the synergistic effect of the combination of TB511 and anti-PD-1 antibody (pembrolizumab), a hPD-L1 MC38 tumor-humanized PD-1 mouse model was generated by subcutaneously injecting human PD-L1-expressing mouse colon cancer MC38 cells (hPD-L1 MC38 cells) (Shanghai Model Organisms Center, Shanghai, China) stably expressing human PD-L1 into the dorsal skin of genetically modified C57BL/6J mice (humanized PD-1 knock-in mice) (Shanghai Model Organisms Center, Shanghai, China) (Figure 96). Mice were divided into groups administered PBS, TB511, anti-PD-1 antibody (Selleckchem), or TB511 and anti-PD-1 antibody, and then PBS or TB511 (200 nmol/kg) was administered subcutaneously, and anti-PD-1 antibody (2.5 mg/kg) was administered intraperitoneally twice a week for 32 days to confirm the synergistic effect of co-administration of TB511 and anti-PD-1 antibody (Figs. 94 to 99).

Example 11. Analysis of anticancer effects of combined administration of TB511 and chemotherapy (oxaliplatin) in a colon cancer animal model

11-1. Cell culture

Mouse colon cancer cells (CT-26) were cultured in RPMI1640 medium (Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene, Gyeongsan, Korea), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, CA, USA). Cells were seeded every 3 days when 80% confluent was reached, and cultured at 37°C and 5% CO ₂ .

11-2. Establishment of colon cancer mouse model

To establish a mouse model of colon cancer, CT-26 cells were harvested and mixed with Matrigel at a 1:1 ratio. The mixed cells were injected subcutaneously into the right flank of the mice at a concentration of 3 Х 10^5 cells/mouse. When the tumor volume reached 50-100 mm³, the mice were divided into four groups: control, TB511, Oxaliplatin (Oxp), and combination treatment group. The mice in each group were injected subcutaneously with PBS or TB511 (200 nmol/kg) every 3 days, and Oxp (1.5 mg/kg) was injected intraperitoneally as a single dose. The tumor volume was measured every 3 days using a digital caliper. The formula for calculating the tumor volume is V = (width Х width Х length) / 2. The animal experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHSAP(SE)-24-332). All mice were housed in a specific pathogen-free environment with free access to food and water under a 12-h light/dark cycle. After the analysis, all animals were euthanized using isoflurane and cervical dislocation. Tumor tissue was isolated and used for further analysis.

11-3. Flow cytometry

Tumor tissues were minced and digested in serum-free cell culture medium containing DNase I (1 U/mL) and collagenase D (1 mg/mL). The samples were incubated for 1 h at 37°C with gentle agitation. The samples were filtered through a 100-μm nylon mesh sieve. Red blood cells were lysed with Pharmlyse buffer (BD Bioscience, CA, USA). Single cells were filtered through a 40-μm nylon mesh sieve and stained with the following antibodies. For mouse lymphocytes, anti-CD45-APCcy7 (clone 30-F11; BD Bioscience, #557659), anti-CD4-BB700 (clone RM4-5; BD Bioscience, #566407), anti-CD8-PE-cy7 (clone 53-6.7; BD Bioscience, #552877), anti-NKp46-BV711 (clone 29A1.4; BD Bioscience, #740822); For mouse macrophages, the following antibodies were used: anti-CD45-APCcy7 (clone 30-F11; BD Bioscience, #557659), anti-CD11b-BV510 (clone M1/70; BD Bioscience, #566117), anti-F4/80-PE (clone BM8; Biolegend, #123110), anti-CD206-APC (clone C068C2; Biolegend, #141708), anti-CD86-BV786 (clone GL1; BD Bioscience, #740900). Cells were detected on a FACS Lyric system (BD Bioscience) and analyzed using FlowJo software (BD Bioscience).

11-4. Statistical Analysis

All data are presented as means ± standard errors of means (SEMs). Statistical analyses were performed using Prism 10.1.2 software (GraphPad Software Inc., CA, USA) using one-way ANOVA followed by Tukey's post hoc analysis for group comparisons or two-way ANOVA followed by Bonferroni's post hoc analysis.

11-5. Analysis of anticancer effects of combined administration of TB511 and oxaliplatin in a colon cancer mouse model

According to the above Example 11-2, TB511 and oxaliplatin were administered alone or in combination to a colon cancer mouse model. The mice were injected intraperitoneally with a single dose of oxaliplatin on the 7th day, and TB511 was injected every 3 days (Fig. 100). Compared to the control group, the tumor volume was significantly reduced in the TB511 and oxaliplatin groups, and the combination group showed the highest anticancer efficacy compared to the other groups, and tumor growth was significantly inhibited compared to the control and TB511 groups (Figs. 101 to 105).

11-6. Analysis of changes in tumor weight and mouse body weight by combined administration of TB511 and oxaliplatin

The weight changes of tumor tissues isolated from euthanized mice were measured according to TB511 and oxaliplatin treatment. As a result, it was shown that the tumor size and weight were significantly reduced in the combined treatment group compared to the control group (Fig. 106 and Fig. 108). In addition, when the weight change due to TB511 or oxaliplatin administration was checked, no weight change was observed in any group (Fig. 109).

11-7. Reduction of M2-like TAM (Tumor Associated Macrophage) in the Tumor Microenvironment (TME) by Combination Administration of TB511 and Oxaliplatin

We confirmed that TB511 selectively targets M2-like TAM among macrophage subtypes in the TME. After making tumor tissues into single cells, the distribution of macrophage subtypes in the TME was analyzed by flow cytometry. The flow cytometry results showed that the distribution of M1-like TAM (CD86 ⁺ F4/80 ⁺ cells within CD45 ⁺ CD11b ⁺ cells) did not show significant changes in any group. However, the distribution of M2-like TAM (CD206 ⁺ F4/80 ⁺ cells within CD45 ⁺ CD11b ⁺ ) significantly decreased in the TB511 group compared to the control group. In addition, M2-like TAM significantly decreased in the combination treatment group compared to the control group (Figs. 109 to 112). In addition, in the combination treatment group, it was confirmed that the decrease in M2-like TAM in the TME increased the M1/M2 ratio (Fig. 113). Through the above results, we confirmed that combined administration of TB511 and chemotherapy eliminated M2-like TAMs in the TME, thereby inducing a change to an anti-tumor environment.

11-7. Analysis of induction of cytotoxic T cell infiltration into TME by combined administration of TB511 and oxaliplatin

We analyzed the changes in the distribution of lymphocytes in the TME due to the removal of TB511-induced M2-like TAMs. The distribution of lymphocytes, including CD4 T cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ cells), CD8 T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ cells), and NK cells (CD45 ⁺ CD3 ⁺ NKp46 ⁺ cells), was analyzed by flow cytometry. The distribution of CD4 T cells and NK cells did not show significant differences in any group. However, CD8 T cells significantly increased in the combination treatment group. Through the above results, we confirmed that the decrease in M2-like TAMs due to TB511 treatment increased the infiltration of CD8 T cells into tumors, thereby exhibiting the activity of directly removing tumor cells.

In summary of the above examples, it was shown that M2-like TAM elimination by TB511 enhanced the anticancer efficacy of oxaliplatin, and that combined administration of TB511 and oxaliplatin promoted an antitumor immune environment by increasing tumor infiltration of T cells compared to when each was administered alone, confirming the synergistic effect of combined administration of TB511 and oxaliplatin.

Example 12. Analysis of anticancer effects of combined administration of TB511 and chemotherapy (paclitaxel) in a triple-negative breast cancer animal model

12-1. Establishment of triple-negative breast cancer mouse model

The following analyses were performed using Balb/c (BALB/cAnNTac, Taconic Bioscience) female mice (6–8 weeks old). Balb/c is a strain suitable for transplantation of the triple-negative breast cancer cell line 4T1 and was selected because it is widely used in immunology and tumor experiments. Mice were housed in an SPF environment at 22±1℃, 55±15% relative humidity, and 12-h day/night lighting cycle (lights on at 07:00, lights off at 19:00). Up to five mice were housed in cages (W 260 X L 420 X H 180 mm), and individual animals were identified with a permanent marker on the tail. Food and water were provided ad libitum through solid food for laboratory animals and water bottles, respectively. This experiment was approved by the Animal Ethics Committee of Kyung Hee University (KHUAP(SE)-20-398) in accordance with the Animal Protection Act.

12-2. Tumor volume measurement

Female Balb/c mice (BALB/cAnNTac, Taconic Bioscience) were injected with triple-negative breast cancer cells, 4T1 (ATCC), at a density of 1Х10 cells per fourth nipple. The 4T1 cells were suspended in serum-free media and mixed with Matrigel (Corning) to promote tumor formation. A group treated with PBS was set as a control group, and drug administration and analysis were performed on 18 mice per group. TB511 and paclitaxel were administered as a single administration or in combination at 3-day intervals starting 5 days after tumor cell injection. TB511 was administered by subcutaneous injection, and paclitaxel was administered by intraperitoneal injection (Fig. 118). The tumor size was measured using a digital caliper to calculate the volume.

12-3. Statistical Methods

The significance difference analysis for the measurement results was performed by analyzing the mean values using Graphpad Prism software (Version 5, CA, USA), and a P-value less than 0.05 was considered statistically significant according to the unpaired T test. All experiments were performed blindly and were repeated independently under the same conditions.

12-4. Analysis of anticancer effects of combined administration of TB511 and chemotherapy (paclitaxel)

According to the above Example 12-2, when TB511 or paclitaxel was administered, the change in tumor size was measured, and through this, the anticancer effect of combined administration of TB511 and paclitaxel was confirmed. The analysis results are shown in Fig. 119. As can be seen in Fig. 119, the tumor size in the group administered combined administration of TB511 and paclitaxel was significantly smaller compared to the group administered alone of TB511 and paclitaxel and the control group. Through the above results, it was confirmed that when TB511 and paclitaxel were administered combinedly, an enhanced anticancer effect was exhibited.

Example 13. Analysis of anticancer effects of combined administration of TB511 and PADCEV (anti-Nectin-4 antibody antibody drug conjugate) in a humanized pancreatic cancer mouse model

13-1. Setting up the animal model

SID mice (NOD-Prkdcem1BeakIL2rgem1Break, 4 weeks old) were purchased from GemBioscience (Chungbuk, South Korea). Mice were irradiated with 1.5 Gy of γ-rays and then intravenously injected with human CD34+ hematopoietic stem cell 2Х10 cells (Lonza, Basel, Switzerland). SID mice were selected because they are a suitable humanized mouse model that can express humanized immune cells, are small in size and easy to handle, are convenient for experiments, and have immunodeficiency characteristics that do not generate an immune response for transplantation of human-derived hHSC cells. Mice were 16 weeks old at the start of administration, and a total of 20 female humanized mice were used. Animals were housed in polycarbonate cages measuring W 200 X L 260 X H 130 (mm) using irradiated bedding, with a maximum of five animals per cage. Individuals were identified by marking their tails, and cages were labeled with the test number and group information. The housing conditions were maintained under 12-h light/dark cycles with 100% HEPA-filtered air, at least 10 air changes per hour, a temperature of 20.2-23.8°C, a relative humidity of 41.9-55.4%, and a light intensity of 150-300 Lux. All animals were provided with standard irradiated pelleted experimental diet (Purina Rodent Chow 38057) and water ad libitum. The analyses were performed in compliance with the Animal Protection Act and related regulations with the approval of the Animal Ethics Committee of Kyung Hee University (KHUASP(SE)-20-308).

13-2. Setting up a humanized mouse model

To establish a humanized mouse model, 4-week-old male immunodeficient mice (SID) were irradiated with 100 cGy for 1 minute, and human hematopoietic stem cells (hHSCs) were injected into the tail vein using a 30G insulin syringe within one hour. The expression of human immune cells was confirmed by flow cytometry at 10 and 16 weeks, and individuals with hCD45+ cell expression rate of 25% or higher were used in the experiment. Table 2 shows the immune cell expression rate of the mice. The humanized mice used in the analysis were supplied by Lonza.

No hCD45+ % in hCD45+ population CD19 (B cell) CD3 (T cell) 1 37.5 94 0.8 2 29.4 89 1.8 3 34.6 92.1 0.4 4 43.7 90.5 2.2 5 43.9 95.3 0.3 6 42.1 93.4 1.3 7 59.9 91.9 0.8 8 34.5 93.9 0.4 9 48.6 95.4 1 10 68.2 90.5 1.6 11 42 94.4 0.4 12 29.1 92.8 0.5 13 41.4 93.5 0.6 14 40.3 93 2.4 15 44.2 93.3 1.3 16 34.8 88.9 2.6 17 46.4 91.5 2.6 18 29 89.3 2.6 19 67.3 88.5 3.2 20 33.4 89.3 3.8 21 34.6 88.9 2.8 22 41.9 88 3.6 23 35.9 87.7 4.6 24 33.7 80.8 11.4 25 34.8 88.9 2.6 26 53.6 88.7 3.3 27 40.8 81.8 7.8 28 33.1 86.6 3.5 29 34.3 90.4 2 30 41 87.4 2.9

In addition, to establish a humanized mouse model of lung cancer, human lung cancer cell lines (PANC1; ATCC) were prepared at a concentration of 2Х10 cells per mouse, mixed 1:1 with Matrigel (Corning), and 100 μL of the mixture was injected subcutaneously into the right dorsal area of the mice using a 30G syringe.

13-3. Drug administration method

The dosage form of TB511 was prepared by the sponsor at a concentration of 5 mg/ml in D-PBS and diluted to 200 nmol/kg for administration. The administration dose of TB511 was set to 200 nmol/kg based on the results of the previous efficacy evaluation in a lung cancer mouse model, and PADCEV (anti-Nectin-4 antibody drug conjugate) was set to 5 mg/kg with reference to external data. The mice were divided into four groups for the study: G1 (PBS administration group), G2 (anti-nectin-4 administration group, 5 mg/kg), G3 (TB511 administration group, 200 nmol/kg), and G4 (combination administration group, PADCEV 5 mg/kg + TB511 200 nmol/kg).

Group Gender No. of Animals Dose leverl G1 F 5 0 G2 F 5 5 mg/kg G3 F 5 200 nmol G4 F 5 5 mg/kg, 200 nmol/kg

TB511 was administered subcutaneously into the back of mice using an insulin syringe after disinfecting with a 70% alcohol swab, and PADCEV was injected intraperitoneally using the same method. The adjuvant volume for both drugs was the same at 100 μL, and the administration interval was every 3 days.

13-4. Tumor volume measurement

Tumor volume was calculated using the formula V = (width (2) Х length)/2. According to the guidelines, mice were sacrificed when tumor size reached a maximum diameter of 1–1.5 cm after inoculation.

13-5. Flow Cytometry Analysis

Brain, breast, colon, kidney, liver, lung, skin, and tumor tissues were cut into thin pieces using a MACS digester (Milteny Biotec, Auburn, CA, USA) and incubated in RPMI 1640 serum-free medium containing 1 U/mL DNase (Cincinnati, Indianapolis, IN, USA) and 1 mg/mL collagenase D (Roche) at 37°C for 30 min. Tissues were filtered through a 100 μm nylon mesh filter. Red blood cells in the filtered single cells were lysed with BD Pharm lyse buffer (BD Bioscience, CA, USA). Single cells were then stained with the following antibodies in BD Pharmingen Stain Buffer (BD Bioscience): anti-CD45-FITC, anti-CD11b-BV510, anti-CD18-PE, and anti-KIM127-BV786 (BD Bioscience) for human macrophages. Cells were detected using a FACS Lyric system (BD Bioscience) and analyzed using FlowJo software (BD Bioscience).

13-6. Immunohistochemistry (IHC)

For histological analysis, tumor tissues were fixed overnight in 10% neutral buffered formalin. The tumor tissues embedded in paraffin were then sectioned at 5 μm thickness. The slides were deparaffinized with xylene and dehydrated with ethanol and deionized water. For antigen retrieval, the slides were incubated in sodium citrate buffer (pH 6.0) for 15 min in a microwave oven. The slides were then treated with peroxidase blocking solution (Dako, Glostrup, Denmark) for 15 min and blocked with 5% bovine serum albumin. The slides were incubated with primary antibodies overnight, washed with TBS containing 0.1% Tween 20, and incubated with an avidin-biotin complex kit (Vector Laboratories, Burlingame, CA, USA). The slides were visualized with diaminobenzidine-HCL (Vector Laboratories). Nuclei were counterstained with hematoxylin. Slides were observed under a bright-field microscope (Nikon, Tokyo, Japan) and analyzed using ImageJ software (NIH). Antibodies used for IHC staining were anti-KI67 and anti-CD18 antibodies (1:200; Abcam, Cambridge, UK).

13-7. Immunofluorescence staining

After embedding in paraffin, tumor tissues were sectioned at 5 μm thickness. The slides were deparaffinized with xylene and dehydrated with ethanol and deionized water. For antigen retrieval, the slides were incubated in sodium citrate buffer (pH 6.0) for 15 min in a microwave oven. The slides were then treated with peroxidase-blocking solution (Dako, Glostrup, Denmark) for 15 min and blocked with 5% bovine serum albumin. The slides were incubated with primary antibodies overnight and then washed with TBS containing 0.1% Tween 20. The slides were incubated overnight at 4°C with anti-vimentin, anti-E-cadherin, anti-actin, anti-CD163, anti-CD11b, anti-Kim127, and anti-CD8 antibodies (1:500; Abcam). Afterwards, FITC-labeled TAMpep and APC-conjugated anti-rabbit IgG secondary antibody (1:500, Invitrogen) were stained for 1 h at 37°C. To visualize nuclei, coverslips were mounted with Vectashield mounting medium (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using an LSM 800 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

13-8. Spatial Transcriptomics

Paraffin-embedded blocks were precisely sectioned into 5 μm thickness and mounted on Xenium slides. The sectioned slides were dried in a thermal cycler (C1000 96-well, Bio-Rad) at 42°C for 3 h. Deparaffinization and de-crosslinking were then performed. After prime hybridization and RNase treatment, the sectioned slides were hybridized overnight (~24 h) at 50°C with Xenium human 5k pan-tissue probe (10x Genomics). The slides were then washed and ligated at 42°C for 30 min. Amplification enhancement and amplification were immediately processed in a thermal cycler (C1000 96-well, Bio-Rad). The slides were washed again, and the cell division staining probe was applied overnight (~24 h) at 4°C. After staining enhancement and washing, autofluorescence suppression and nuclear staining were performed.

The cut slides were then loaded onto the Xenium analyzer (XOA v3.0.2, 10x Genomics) for fluorescence signal detection. After running Xenium, the raw data were preprocessed with xeniumranger v3.0.0.

13-9. Statistical Analysis

All data are expressed as the mean ± standard deviation of three or four independent experiments. Comparisons were performed using the two-sample Mann-Whitney U-test, one- and two-way analysis of variance (ANOVA) (IBM SPSS Statistics version 21; IBM Inc., Chicago, IL, USA), and a P value < 0.05 was considered statistically significant.

13-10. Tumor suppression analysis of combined administration of TB511 and PADCEV in a human pancreatic cancer mouse model

To determine whether combined administration of TB511 and PADCEV (anti-nectin4 antibody) could inhibit tumor growth in a humanized pancreatic cancer mouse model, a tumor mouse model was established by subcutaneously injecting human pancreatic cancer cell line (PANC1) into humanized mice, and the drug was administered to the mice twice a week. The tumor size was significantly reduced in the TB511 or PADCEV administration groups compared to the PBS group, and in particular, the combination administration group showed the most significant reduction (Figs. 120 and 121). In tumor tissues, the expression level of PCNA, a proliferation factor, was significantly reduced in the TB511, PADCEV, and combination groups compared to the PBS group (Figs. 122 and 123). In addition, the expression of E-cadherin, an epithelial cell marker, was significantly increased in the TB511, PADCEV, and combination groups compared to the PBS group, and the expression of vimentin, a mesenchymal cell marker, showed an opposite pattern to E-cadherin and was significantly decreased in the TB511, PADCEV, and combination groups compared to the PBS group (Figs. 124 and 125). Through the above results, it was confirmed that when TB511 and PADCEV were administered together, they exhibited an activity of inhibiting tumor progression in a humanized pancreatic cancer mouse model compared to when each was administered separately.

13-11. Reduction of M2 macrophages in tumor tissues following combined administration of TB511 and PADCEV

To determine whether TB511 reduces M2 macrophages in the tumor microenvironment, tumor tissues were analyzed using immunohistochemical staining and flow cytometry. The results showed that the TB511 and combination treatment groups significantly decreased cells expressing CD163, a marker of M2 macrophages, in tumor tissues compared to the PBS group, whereas no effect was observed in the PADCEV group (Figs. 126 and 127). In particular, TB511 was shown to selectively bind to activated CD18 in M2 macrophages. CD18-positive cells in tumor tissues were shown to significantly decrease in the TB511 and combination treatment groups (Figs. 128 and 129). In addition, it was shown that TB511 reduced M2 macrophages using the Kim127 antibody that binds to activated CD18 (Figs. 130 to 133). Through the above results, we confirmed that TB511 selectively targets activated CD18 of M2 macrophages in tumor tissues and induces a decrease in these cells.

13-12. Increase in CD8 T cells in tumor tissues following combined administration of TB511 and PADCEV

It has been reported that M2 macrophages in the tumor microenvironment play an immunosuppressive role and inhibit the influx of CD8 T cells (doi: 10.1186/s12964-023-01424-6). To confirm whether TB511 targeting M2 macrophages increases the influx of CD8 T cells in the tumor microenvironment, tumor tissues were analyzed by immunofluorescence staining. As a result of the analysis, CD8 T cells in the tumor tissues significantly increased in the TB511 and combination treatment groups compared to the PBS group, and no significant change was observed in the PADCEV group (Figs. 132 and 133). Through the above results, it was confirmed that TB511 induces a decrease in M2 macrophages and promotes the influx of CD8 T cells. However, since PADCEV directly targets tumor cells by targeting nectin-4 expressed on tumor cells, it did not show significant changes in immune cells such as macrophages and CD8 T cells in the tumor microenvironment.

13-13. Association of TB511 with activated CD18-expressing macrophages and CD8 T cells in normal tissues

The association of TB511 with activated CD18-expressing macrophages and CD8 T cells in normal tissues was shown in Fig. 20, where TB511 decreased activated CD18-expressing macrophages. To investigate the effect of TB511 on macrophages and CD8 T cells in normal tissues, single cells were obtained from spleen, liver, lung, and brain tissues, stained for activated CD18 using Kim127 antibody, and analyzed by flow cytometry. TB511, PADCEV, and combination treatment groups showed no effect on Kim127-positive macrophages and CD8 T cells compared to the PBS group (Figs. 134 to 141). These results confirmed that TB511 could specifically target activated CD18-expressing M2 macrophages in tumor tissues.

13-14. Regulation of immune cells in the tumor microenvironment by combined administration of TB511 and PADCEV

To investigate the changes in immune cells in the tumor microenvironment of pancreatic cancer after combined administration of TB511 and PADCEV, genetic analysis of tumor tissues was performed using spatial transcriptomics. Figures 142 to 145 show the cell distribution of the PBS, TB511, PADCEV, and combination treatment groups. In the PBS group, epithelial cells, endothelial cells, stromal cells, fibroblasts, and neural cells were evenly distributed near the tumor cells, whereas in the TB511, PADCEV, and combination treatment groups, the cell clusters were reduced to 4 or 3, respectively (Figures 142 to 145).

The above cell populations were analyzed using UMAP. As a result of the analysis, the proportions of immune cells in the PBS, TB511, PADCEV, and combination administration groups were 18%, 23%, 17%, and 26%, respectively. That is, it can be confirmed that the groups treated with TB511 showed a higher proportion of immune cells compared to the other groups (Figs. 146 to 153). As a result of analyzing the above immune cells, the proportion of epithelial cells, which are pancreatic cancer cells, decreased in the TB511, PADCEV, and combination administration groups, and M1 macrophages increased, M2 macrophages decreased, and CD8 T cells and NK cells increased in the TB511 and combination administration groups. Treg cells did not appear in any drug group (Figs. 154 and 155). Through the above results, it was confirmed that TB511 and PADCEV induced a decrease in tumor cells, and TB511 specifically targeted M2 macrophages to induce changes in immune cells within the microenvironment. However, in the case of PADCEV, since it directly targets pancreatic cancer cells, it did not show any induction or changes in immune cells.

13-15. Role of TB511 in CD8 T cell activation in the tumor microenvironment

To confirm whether TB511 targets M2 macrophages and induces CD8 T cell activation, we analyzed the gene expression levels of exhausted markers PDCD1, CTLA4, FOXP3, LAG3 in CD8 T cells, and the gene expression levels of activation markers GZMB and IFNG. CD8 T cells expressing exhausted T cell genes (brown cluster cells) are indicated by white arrows, and cells expressing activated T cell genes are indicated by red arrows. The analysis results showed that the number of activated T cells increased in the TB511 and combination treatment groups compared to the PBS and PADCEV groups (Figs. 156 and 157). In addition, the analysis of the expression levels of each gene showed that there was no significant change in PDCD1 (i.e., PD-1 gene) in any group, but the exhausted marker genes CTLA4, FOXP3, LAG3 were significantly decreased in the combination treatment group. , the expression of inflammatory cytokines such as Granzyme B and interferon gamma significantly increased in the TB511 or combination group. On the other hand, in the PADCEV group, the effect of inducing CD8 T cell activation was lower than that of TB511 (Figs. 158 and 159). Through the above results, it was confirmed that TB511 induces the infiltration and activation of CD8 T cells by targeting and reducing M2 macrophages in pancreatic cancer tumor tissues, and PADCEV directly targets nectin-4 of tumor cells to reduce tumor growth, but does not contribute to the regulation of immune cells in the tumor microenvironment.

In summary of the above examples, when TB511 and PADCEV were administered together, compared to when each was administered alone, tumor size and proliferation were reduced, the proportions of M1 macrophages, CD8 T cells, and NK cells were increased, and the proportion of M2 macrophages was reduced, confirming the synergistic effect of combined administration of TB511 and PADCEV.

Through the results confirmed from the above humanized mouse model, the excellent efficacy of TB511 monotherapy and combination therapy with anticancer drugs was confirmed in various solid cancers.

Claims (22)

A chimeric antigen receptor (CAR) comprising a peptide of sequence number 1 or sequence number 2 as an antigen binding domain. A recombinant vector comprising a gene encoding the chimeric antigen receptor of claim 1. A chimeric antigen receptor expressing cell transformed with the recombinant vector of claim 2. In claim 3, the chimeric antigen receptor expressing cell is a chimeric antigen receptor expressing T (CAR-T) cell or a natural killer (CAR-NK) cell. A T-cell engager comprising a peptide having sequence number 1 or sequence number 2. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a peptide of sequence number 1 or sequence number 2, a chimeric antigen receptor of claim 1, a chimeric antigen receptor expressing cell of claim 3, or a T cell engager of claim 5. A pharmaceutical composition for preventing or treating cancer, wherein the cancer in claim 6 is a non-responsive or responsive cancer to an immunotherapy agent. A pharmaceutical composition for preventing or treating cancer, wherein the cancer is a solid cancer in claim 6. A pharmaceutical composition for preventing or treating cancer, which is an immunotherapy agent in claim 6. In paragraph 6, A pharmaceutical composition for preventing or treating cancer, characterized in that the anticancer effect is enhanced when the composition is administered in combination with a chemotherapeutic anticancer agent or an antibody-drug conjugate (ADC). In Article 10, A pharmaceutical composition for preventing or treating cancer, wherein the chemotherapeutic anticancer agent is at least one selected from the group consisting of Oxaliplatin, Pemetrexed, Cisplatin, Gemcitabine, Carboplatin, Fluorouracil (5-FU), Cyclophosphamide, Paclitaxel, Vincristine, Etoposide, and Doxorubicin. In Article 10, A pharmaceutical composition for preventing or treating cancer, wherein the antibody-drug conjugate is PADCEV (anti-Nectin-4 antibody drug conjugate). 1) a peptide of sequence number 1 or sequence number 2, a chimeric antigen receptor of claim 1, a cell expressing a chimeric antigen receptor of claim 3, or a T cell engager of claim 5; and 2) A combination for the prevention or treatment of cancer, comprising an immunotherapy agent, a chemotherapy agent or an antibody-drug conjugate (ADC). In claim 13, the immunotherapy agent is an immune checkpoint inhibitor, an immunosuppressant controlling drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine necessary for maintaining the activity of an immune cell for cancer treatment, a combination for preventing or treating cancer. A combination for the prevention or treatment of cancer, wherein in claim 14, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR. A combination for the prevention or treatment of cancer, wherein the combination is an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-CTLA4, or an antigen-binding fragment of the antibody that specifically binds to PD-1, PDL-1, or CTLA4, or a combination thereof, in claim 15. A combination for the prevention or treatment of cancer, wherein the combination comprises at least one selected from the group consisting of Oxaliplatin, Pemetrexed, Cisplatin, Gemcitabine, Carboplatin, Fluorouracil (5-FU), Cyclophosphamide, Paclitaxel, Vincristine, Etoposide, and Doxorubicin. A combination for preventing or treating cancer, wherein the antibody-drug conjugate in claim 13 is PADCEV (anti-Nectin-4 antibody drug conjugate). A composition for diagnosing cancer, comprising a peptide of sequence number 1 or sequence number 2, a chimeric antigen receptor of claim 1, a chimeric antigen receptor expressing cell of claim 3, or a T cell engager of claim 5 as an active ingredient. A composition for diagnosing cancer, further comprising a label in claim 19. A composition for diagnosing cancer, wherein the label is a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, a fluorescent substance, a probe or a tag in claim 20. A method for preventing or treating cancer, comprising administering to a subject the pharmaceutical composition of claim 6 or the combination of claim 13.

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