Therapeutic single-domain antibody

Abstract

This invention provides a method for preventing abnormal cell proliferation. [Solution] The present invention provides a composition and method for preventing abnormal cell proliferation in a target using a single-domain antibody (sdAb) made against an intracellular component, wherein the abnormal cell proliferation may be cancer. The sdAb is synergistic with one or more chemotherapeutic agents and improves the therapeutic effect of one or more chemotherapeutic agents against cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 237,987, filed on 27 August 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application is filed together with an electronic sequence listing. The sequence listing is provided as a file titled "seq listing_ST26," created on August 19, 2022, and has a size of 3,000 bytes. The electronic information of the sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Activator of signaling and transcription 3 (STAT3) is a signaling molecule and transcription factor, while Kirsten RAS's mammalian homolog (KRAS) is a signaling molecule that relays GTP-binding proteins. Both regulate cell proliferation, survival, differentiation, apoptosis, cell migration, stem cell regeneration, inflammation, and angiogenesis. Abnormal expression of mutant active forms of STAT3 and KRAS (e.g., G12D, G12C, G12V) is well-established in the induction and maintenance of several cancers. Due to their central roles in tumorigenesis, STAT3 and KRAS mutant proteins have been considered anti-cancer targets. However, STAT3 and KRAS mutant proteins are also considered intracellular molecules that are clinically “unsuitable as drugs” due to pharmacokinetic and toxicity barriers. Inhibition of these proteins in cancer cells would likely reduce tumor burden and enhance the clinical management of cancer, but this important challenge remains an unmet need.

[0004] Traditional chemotherapy, when used alone, is often insufficient for treatment and can be highly toxic, making it difficult to treat many human malignancies, including pancreatic cancer, triple-negative breast cancer (TNBC), glioblastoma, and sarcoma. Furthermore, novel targeted therapies can ultimately become ineffective due to the development of drug resistance in cancer cells, often associated with the blockade of critical signaling pathways. Oncogenic mutations, loss of tumor suppressor genes, overexpression of normal proteins, or some combination of these events also contribute to drug resistance. The Janus kinase (JAK) / STAT pathway is a key regulator of cell proliferation, differentiation, and inflammatory responses. Elevated phosphorylated STAT3 (p-STAT3) has been associated with poor prognosis in cancers with solid tumors. Activated STAT3 forms homodimers that translocate to the nucleus, where it binds DNA and initiates transcription of target genes related to cell proliferation, growth, anti-apoptosis, angiogenesis, immunosuppression, and invasion / migration. Due to its central role in tumor processes, STAT3 has been considered a potential anti-cancer target since its initial description as an oncogene in 1998, leading to evaluations of STAT3 inhibitors for their antitumor activity in in vitro and in vivo experimental tumor models. However, primarily due to pharmacokinetic, efficacy, and safety concerns, most of these inhibitors have not progressed to clinical use for cancer treatment. KRAS variants have been shown to be driver mutations for approximately 25% of human cancers, but are most frequently found in pancreatic cancer (98%) and colorectal cancer (53%). The variant retains GDP without hydrolyzing GTP, thereby becoming constitutively active. Many researchers have focused on developing small molecule targets for KRAS variants for decades. However, this has been a nearly impossible challenge due to problems in detecting the binding pocket for these small molecules to bind to KRAS, and to date, no inhibitory drugs have been approved for use in treatment.

[0005] Cancer cells utilize p-STAT3 as an evasion mechanism to become resistant to chemotherapy and radiotherapy. Inhibiting STAT3 with small molecule inhibitors has been shown to suppress cancer growth, activate apoptosis, inhibit angiogenesis, and even reconstruct the pancreatic cancer stroma. Inhibiting STAT3 in human patients during a Phase 1 trial demonstrated that this is a safe and well-tolerated approach. Research has focused on identifying novel small molecule inhibitors of STAT3 that act by inhibiting STAT3 phosphorylation, inhibiting DNA binding, or preventing the formation of functional STAT3 dimers.

[0006] Antibodies in camelids consist of two heavy-chain immunoglobulins (VHHs), each having one variable domain per heavy chain. Camelid VHHs have been used to target multiple extracellular targets (e.g., IL-6R, IL-17, TNF-α, VWF, etc.), and several VHHs are in various stages of human clinical trials (Phase II and Phase III), with no major side effects or toxicity reported. One VHH, caplacizumab, has been approved by the FDA and is successfully marketed for the treatment of acquired thrombotic thrombocytopenic purpura in adults.

[0007] This invention relates to the therapeutic use of a 15kDa single-domain antibody (sdAb), SBT-100 (SEQ ID NO: 1). SBT-100 (SEQ ID NO: 1) binds to both STAT3 and KRAS proteins with nanomolar affinity, permeates into the cytoplasm of tumor cells, impairs STAT3 phosphorylation and nuclear translocation, and can ultimately lead to decreased VEGF levels and PD-L1 expression, reduced viral replication, and significant inhibition of cancer cell proliferation. Furthermore, SBT-100 (SEQ ID NO: 1) inhibits KRAS GTPase activity and downstream phosphorylation of ERK in vitro. In addition to inhibiting the proliferation of multiple human cancer cell lines in vitro, SBT-100 (SEQ ID NO: 1) treatment reduces tumor volume without observable toxicity in both athymic xenograft mouse models of triple-negative breast cancer cell line with KRAS(G13D) mutation (MDA-MB-231) and pancreatic cancer cell line with KRAS(G12D) mutation (PANC-1). SBT-100 (SEQ ID NO: 1) also appears to be unparalleled in its ability to cross the blood-brain barrier (BBB). These results demonstrate that it is possible to simultaneously target hard-to-reach abnormal intracellular transcription factors and signaling proteins with a single VHH to improve cancer treatment. [Overview of the project]

[0008] The present invention includes a method for preventing abnormal cell proliferation in a subject using a single-domain antibody (sdAb) produced against an intracellular component. In one aspect, the abnormal cell proliferation is, for example, cancer such as osteosarcoma, fibrosarcoma, glioblastoma, leukemia, pancreatic cancer, breast cancer, and prostate cancer. In another aspect, the sdAb is synergistic with one or more chemotherapeutic agents, improving the therapeutic effect of one or more chemotherapeutic agents against cancer, such as doxorubicin and gemcitabine. In another aspect, the sbAb reduces the toxicity of one or more chemotherapeutic agents and improves the survival of the treated subject. In another aspect, the sbAb is used in combination with one or more compounds. In one aspect, the intracellular component includes, for example, proteins such as STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, or STAT6. The method according to claim 1, wherein the sdAb comprises SBT-100 (SEQ ID NO: 1). A method for inhibiting the phosphorylation of STAT3, comprising administering an sdAb, such as SBT-100 (SEQ ID NO: 1), to a patient who requires inhibition of STAT3 phosphorylation.

[0009] In another embodiment, the present invention includes a method for inhibiting the activation of STAT3, comprising administering an sdAb to a patient who requires inhibition of STAT3 activation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0010] In another embodiment, the present invention includes a method for inhibiting T cell proliferation, comprising administering an sdAb to a patient who requires inhibition of T cell proliferation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0011] In another embodiment, the present invention includes a method for maintaining visual acuity, comprising administering an sdAb to a patient who requires the maintenance of visual acuity. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0012] In another embodiment, the present invention includes a method for inhibiting the proliferation of CD4+IL-17+ T cells, comprising administering an sdAb to a patient who requires inhibition of CD4+IL-17+ T cell proliferation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0013] In another embodiment, the present invention includes a method for inhibiting a disease caused by CD4+IL-17+T cells, comprising administering an sdAb to a patient who requires inhibition of a disease caused by CD4+IL-17+T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0014] In another embodiment, the present invention includes a method for inhibiting the proliferation of CD4+IFN-γ+T cells, comprising administering an sdAb to a patient who requires inhibition of CD4+IFN-γ+T cell proliferation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0015] In another embodiment, the present invention includes a method for inhibiting a disease caused by CD4+IFN-γ+T cell proliferation, comprising administering an sdAb to a patient who requires inhibition of the disease caused by CD4+IFN-γ+T cell proliferation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1) (SEQ ID NO: 1).

[0016] In another embodiment, the present invention includes a method for inhibiting the proliferation of CD4+IL-17+IFN-γ+T cells, comprising administering an sdAb to a patient who requires inhibition of CD4+IL-17+IFN-γ+T cell proliferation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1) (SEQ ID NO: 1).

[0017] In another embodiment, the present invention includes a method for inhibiting a disease caused by the proliferation of CD4+IL-17+IFN-γ+T cells, comprising administering an sdAb to a patient who requires inhibition of a disease caused by the proliferation of CD4+IL-17+IFN-γ+T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0018] In another embodiment, the present invention includes a method for inhibiting the proliferation of CD4+RORγT+T cells, comprising administering an sdAb to a patient who requires inhibition of CD4+RORγT+T cell proliferation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0019] In another embodiment, the present invention includes a method for inhibiting a disease caused by the proliferation of CD4+RORγT+T cells, comprising administering an sdAb to a patient who requires inhibition of a disease caused by the proliferation of CD4+RORγT+T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0020] In another embodiment, the present invention includes a method for inhibiting the proliferation of CD4+ granzyme-B+ T cells, comprising administering an sdAb to a patient who requires inhibition of CD4+ granzyme-B+ T cell proliferation. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0021] In another embodiment, the present invention includes a method for inhibiting a disease caused by CD4+ granzyme-B+ T cells, comprising administering an sdAb to a patient who requires inhibition of a disease caused by CD4+ granzyme-B+ T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0022] In another aspect, the present invention includes a method for inhibiting the proliferation of CD4+Foxp3+ T cells, the method comprising administering sdAb to a patient in need of inhibiting the proliferation of CD4+Foxp3+ T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0023] In another aspect, the present invention includes a method for inhibiting the proliferation of CD25+Foxp3+ T cells, the method comprising administering sdAb to a patient in need of inhibiting the proliferation of CD25+Foxp3+ T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0024] In another aspect, the present invention includes a method for inhibiting a disease caused by CD25+Foxp3+ T cells, the method comprising administering sdAb to a patient in need of inhibiting the disease caused by CD25+Foxp3+ T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0025] In another aspect, the present invention includes a method for inhibiting the proliferation of CD4+IL-10+ T cells, the method comprising administering sdAb to a patient in need of inhibiting the proliferation of CD4+IL-10+ T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0026] In another aspect, the present invention includes a method for inhibiting a disease caused by CD4+IL-10+ T cells, the method comprising administering sdAb to a patient in need of inhibiting the disease caused by CD4+IL-10+ T cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0027] In another embodiment, the present invention includes a method for inhibiting a disease caused by one or more cytokines selected from the group comprising IL-17, IFN-γ, IL-23, GM-CSF, and IL-1α, comprising administering an sdAb to a patient who requires inhibition of a disease caused by one or more cytokines selected from the group comprising IL-17, IFN-γ, IL-23, GM-CSF, and IL-1α. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0028] In another embodiment, the present invention includes a method for inhibiting the proliferation of Th1, Treg, and Th17 pathogenic cells, comprising administering an sdAb to a patient who requires inhibition of the proliferation of Th1, Treg, and Th17 pathogenic cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0029] In another aspect, the present invention includes a method for inhibiting diseases caused by Th1, Treg, and Th17 pathogenic cells, comprising administering an sdAb to a patient who requires inhibition of a disease caused by Th1, Treg, and Th17 pathogenic cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1). In another aspect, the disease is selected from the group comprising rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, atopic dermatitis, and type 1 diabetes.

[0030] In another embodiment, the present invention includes a method for inhibiting a disease caused by an autoimmune disease, comprising administering an sdAb to a patient who requires inhibition of a disease caused by an autoimmune disease. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0031] In another embodiment, the present invention includes a method for inhibiting VEGF production by retinal epithelial cells in an in vitro model of age-related macular degeneration (AMD), comprising administering an sdAb to a patient who requires inhibition of VEGF production by retinal epithelial cells in an in vitro model of age-related macular degeneration (AMD). In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0032] In another embodiment, the present invention includes a method for inhibiting diseases caused by AMD and neovascular disease of the eye, comprising administering an sdAb to a patient who requires inhibition of diseases caused by AMD and neovascular disease of the eye. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0033] In another embodiment, the present invention includes a method for inhibiting a disease caused by VEGF, comprising administering an sdAb to a patient who requires inhibition of a disease caused by VEGF. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0034] In another embodiment, the present invention includes a method for downregulating PD-L1 expression, comprising administering an sdAb to a patient requiring downregulation of PD-L1 expression. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0035] In another embodiment, the present invention includes a method for inhibiting the translocation of STAT3 into the nucleus of a cell, comprising administering an sdAb to a patient who requires inhibition of the translocation of STAT3 into the nucleus of a cell. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0036] In another embodiment, the present invention includes a method for inhibiting the action of IL-6, comprising administering an sdAb to a patient who requires inhibition of the action of IL-6. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0037] In another embodiment, the present invention includes a method for enhancing the efficacy of a chemotherapeutic agent, comprising administering an sdAb to a patient who requires enhanced efficacy of the chemotherapeutic agent. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1). In another aspect, the chemotherapeutic agent comprises gemcitabine.

[0038] In another embodiment, the present invention includes a method for permeating cell membranes, the blood-brain barrier, and the blood-retinal barrier, comprising administering an sdAb to a patient who requires permeation of cell membranes, the blood-brain barrier, and the blood-retinal barrier. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0039] In another embodiment, the present invention includes a method for reducing the toxicity of a chemotherapeutic agent, comprising administering an sdAb to a patient who requires a reduction in the toxicity of the chemotherapeutic agent. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1). In one aspect, the chemotherapeutic agent is doxorubicin. In another aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0040] In another embodiment, the present invention includes a method for inhibiting the function of STAT3, comprising administering an sdAb to a patient who requires inhibition of the function of STAT3. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0041] In another embodiment, the present invention includes a method for inhibiting the function of KRAS and mutant KRAS in cancer cells, comprising administering an sdAb to a patient who requires inhibition of the function of KRAS and mutant KRAS in cancer cells. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0042] In another embodiment, the present invention includes a method for inhibiting VEGF production, comprising administering an sdAb to a patient who requires inhibition of VEGF production. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0043] In another embodiment, the present invention includes a method for inhibiting PD-L1 expression, comprising administering an sdAb to a patient who requires inhibition of PD-L1 expression. In one aspect, the sdAb comprises SBT-100 (SEQ ID NO: 1).

[0044] These and other features, aspects and advantages of the present invention will be better understood with reference to the following description, the appended claims and drawings. [Brief explanation of the drawing]

[0045] [Figure 1] Figure 1 shows the permeability of the cell membrane of MDA-MD-231 cells with SBT-100 (SEQ ID NO: 1) detection, as indicated by immunofluorescence staining of MDA-MB-231 cells incubated with A) SBT-100 (SEQ ID NO: 1) antibody, B) no VHH antibody, and C) negative control, and confocal images of SBT-100 (SEQ ID NO: 1) detection.

[0046] [Figure 2] Figure 2 shows fluorescence images of intracellular levels of both phosphorylated STAT3 and total STAT3 in MDA-MB-231 cells A) untreated and B) treated with SBT-100 (SEQ ID NO: 1) for 6 hours; C) confocal images of tSTAT3 in MDA-MB-231 cells untreated or D) after 6 hours of SBT-100 (SEQ ID NO: 1) treatment; E) fluorescence images of pSTAT3 in MDA-MB-231 cells either untreated or F) after 6 hours of SBT-100 (SEQ ID NO: 1) treatment; G) confocal images of pSTAT3 in MDA-MB-231 cells either untreated or H) after 6 hours of SBT-100 (SEQ ID NO: 1) treatment.

[0047] [Figure 3A] Figure 3A shows immunoblots of MDA-MB-231 protein extracts for pSTAT3, tSTAT3, and PD-L1 with and without SBT-100 (SEQ ID NO: 1). [Figure 3B] Figure 3B shows the quantification of the immunoblot from Figure 3A.

[0048] [Figure 4] Figure 4 shows fluorescence microscopy images of phosphorylated STAT3 in Hep-2 cells under A) normal conditions, B) IL-6 stimulation, and C) both IL-6 stimulation and SBT-100 (SEQ ID NO: 1) treatment, and in Panc-1 cells under D) normal conditions, E) IL-6 stimulation, and F) both IL-6 stimulation and SBT-100 (SEQ ID NO: 1) treatment.

[0049] [Figure 5] Figure 5 shows the KRAS GTPase activity of phosphorylated ERK1 / 2 in various KRAS-mutated cancer cells, measured by A) luminescence (RLU) and B) Western blot analysis, with or without SBT-100 (SEQ ID NO: 1). Lanes 1 and 2 are MDA-MB-231 cells, lanes 3 and 4 are PANC-1 cells, and lanes 5 and 6 are BxPC3 cancer cells incubated with vehicle only.

[0050] [Figure 6] Figure 6 shows a graph of tumor volume over time in athymic nude mice treated with SBT-100 (SEQ ID NO: 1) and untreated.

[0051] [Figure 7] Figure 7 shows images of immunohistochemistry of SBT-100 (SEQ ID NO: 1) in athymic nude mice with large, established MDA-MB-231 tumors, A) tumor cells and B) brain cells.

[0052] [Figure 8] Figure 8 shows the fundus examination scores for mice treated with and without SBT-100 (SEQ ID NO: 1).

[0053] [Figure 9] Figure 9 shows the results from optical coherence tomography with and without SBT-100 (SEQ ID NO: 1) treatment. Legend: GC / IPL: ganglion cells - inner plexiform layer; OPL: outer plexiform layer; INL: inner granular layer; ONL: outer granular layer; OLM: ocular larval migrans; RPE / CC: retinal pigment epithelium / choroidal capillary.

[0054] [Figure 10] Figure 10 shows the electroretinogram (ERG) of EAU mice with and without SBT-100 (SEQ ID NO: 1).

[0055] [Figure 11] Figure 11 shows, A) FACS analysis of EAU mice with and without SBT-100 (SEQ ID NO: 1). B) Percentage of IFN-γ+IL-17A+ cells in EAU mice with and without SBT-100 (SEQ ID NO: 1).

[0056] [Figure 12] Figure 12 shows immunoprecipitation studies in cell lines. M = marker, Lane 1 = PANC-1 STAT3; Lane 2 = DU145; Lane 3 = HeLa+IFN-γ(P-STAT3); Lane 4 = 4T1; Lane 5 = PANC-1 KRAS; Lane 6 = PC-3.

[0057] [Figure 13A] Figure 13A shows Ebola virus replication in HeLa cells treated with SBT-100 (SEQ ID NO: 1). [Figure 13B] Figure 13B shows Ebola virus replication in HFF cells.

[0058] [Figure 14]Figure 14 shows Zika virus replication in Vero cells, and Figure 14B shows EC50 in Vero and HFF cells infected with Zika virus.

[0059] [Figure 15] Figure 15 shows the EC50 in HeLa, BE2M17, and U87MG cells infected with VEE virus.

[0060] [Figure 16] Figure 16 shows the EC50 in U205 cells infected with chikungunya virus. [Modes for carrying out the invention]

[0061] As used herein, the following terms and their variations shall have the meanings given below unless explicitly intended to have different meanings depending on the context in which such terms are used.

[0062] As used herein, the terms “a,” “an,” and “the,” and similar referents, should be interpreted as encompassing both singular and plural nouns unless their use in context indicates otherwise.

[0063] The term “antigenic determinant” refers to an epitope on an antigen that is recognized by an antigen-binding molecule (such as the sdAb or polypeptide of the present invention), more specifically by the antigen-binding site of the antigen-binding molecule. The terms “antigenic determinant” and “epitope” may also be used interchangeably. An amino acid sequence that has affinity and / or specificity, can bind to a particular antigenic determinant, epitope, antigen, or protein, is said to be “made for” or “made for” an antigenic determinant, epitope, antigen, or protein.

[0064] As used herein, the term “comprise” and variations thereof such as “comprising” and “comprises” are not intended to exclude any other additional elements, components, integers, or processes.

[0065] The sdAbs, polypeptides, and proteins described herein may contain so-called “conservative” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is substituted with another amino acid residue of a similar chemical structure and has little or no effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. A conservative substitution is a substitution in which one amino acid from the following groups (a) to (e) is substituted with another amino acid from the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar positively charged residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Other conservative substitutions include those from Ala to Gly or Ser; from Arg to Lys; from Asn to Gln or His; from Asp to Glu; from Cys to Ser; from Gln to Asn; from Glu to Asp; from Gly to Ala or Pro; from His to Asn or Gln; from Ile to Leu or Val; from Leu to Ile or Val; from Lys to Arg, Gln or Glu; from Met to Leu, Tyr or Ile; from Phe to Met, Leu or Tyr; from Ser to Thr; from Thr to Ser; from Trp to Tyr; from Tyr to Trp; and / or from Phe to Val, Ile or Leu.

[0066] As used herein, “domain” generally refers to a globular region of an antibody chain, particularly a globular region of a heavy-chain antibody, or a polypeptide essentially derived from such a globular region.

[0067] The amino acid sequence and structure of sdAb typically consist of four framework regions or "FRs," referred to as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively. Each framework region is interrupted by three complementarity-determining regions or "CDRs," referred to as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively.

[0068] As used herein, the term “humanized sdAb” means an sdAb in which one or more amino acid residues in the amino acid sequence of a naturally occurring VHH sequence are replaced by one or more amino acid residues located at the corresponding positions in the VH domain of a conventional human-derived four-chain antibody. This can be done by methods well known in the art. For example, the FR of an sdAb may be replaced by a human variable FR.

[0069] As used herein, “isolated” nucleic acids or amino acids are those that have been separated from at least one other component normally associated with that nucleic acid or amino acid, such as its source or culture medium, another nucleic acid, another protein / polypeptide, another biological component or macromolecule or contaminant, impurities or trace components.

[0070] The term "mammal" is defined as an individual belonging to the class Mammalia, and includes, but is not limited to, livestock and farm animals such as humans, cattle, horses, sheep, dogs and cats, as well as animals in zoos, sports and pets.

[0071] As used herein, “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent and absorption retarder, etc., that is suitable for pharmaceutically acceptable administration. Suitable carriers are listed in the latest edition of Remington's Pharmaceutical Sciences, the standard reference text in the art. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, PBS (phosphate-buffered saline), and 5% human serum albumin. Non-aqueous vehicles such as liposomes, cationic lipids, and non-volatile oils may also be used. The use of such media and active substances for pharmaceutically active substances is well known in the art. Unless any conventional media or active substance is incompatible with the therapeutic agent as defined above, its use in the compositions of the present invention is intended.

[0072] A "quantitative immunoassay" refers to any method of measuring the amount of antigen present in a sample using antibodies. Methods for performing quantitative immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), specific analyte labeling and recapture assay (SALRA), liquid chromatography, mass spectrometry, and fluorescence-activated cell sorting.

[0073] The term "solution" refers to a composition containing a solvent and a solute, and includes true solutions and suspensions. Examples of solutions include solids, liquids, or gases dissolved in a liquid, and fine particles or micelles suspended in a liquid.

[0074] The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity. Affinity, expressed by the equilibrium constant (KD) of dissociation between the antigen and the antigen-binding protein, is a measure of the binding strength between the antigenic determinant and the antigen-binding site on the antigen-binding protein; a lower KD value indicates a stronger binding strength between the antigenic determinant and the antigen-binding molecule (or affinity can also be expressed as an affinity constant (KA) of 1 / KD). As will be apparent to those skilled in the art, affinity can be determined depending on the specific antigen of interest. Avidity is a measure of the strength of binding between the antigen-binding molecule and the antigen. Avidity relates to both the affinity between the antigenic determinant and its antigen-binding site on the antigen-binding molecule and the number of relevant binding sites present on the antigen-binding molecule. The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any known method, such as scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and sandwich competition assays.

[0075] As used herein, the term “recombinant” refers to the use of genetic engineering methods (e.g., cloning and amplification) used to produce the sdAb of the present invention.

[0076] A “single-domain antibody,” “sdAb,” or “VHH” can generally be defined as a polypeptide or protein containing an amino acid sequence consisting of four framework regions interrupted by three complementarity-determining regions. This is represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The sdAb of this invention also contains a polypeptide or protein containing the sdAb amino acid sequence. Typically, sdAbs are produced in camelid animals such as llamas, but they can also be produced synthetically using techniques well known in the art. As used herein, the variable domain present in naturally occurring heavy-chain antibodies is also called the “VHH domain” to distinguish it from the heavy-chain variable domain present in conventional four-chain antibodies, called the “VH domain,” and the light-chain variable domain present in conventional four-chain antibodies, called the “VL domain.” “VHH” and “sdAb” are used interchangeably herein. The numbering of amino acid residues in sdAb or polypeptides follows the general numbering for the VH domain provided by Kabat et al. ("Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, MD, Publication No. 91). According to this numbering, sdAb FR1 contains amino acid residues at positions 1-30, sdAb CDR1 contains amino acid residues at positions 31-36, sdAb FR2 contains amino acid residues at positions 36-49, sdAb CDR2 contains amino acid residues at positions 50-65, sdAb FR3 contains amino acid residues at positions 66-94, sdAb CDR3 contains amino acid residues at positions 95-102, and sdAb FR4 contains amino acid residues at positions 103-113.

[0077] The term "synthesis" refers to production by in vitro chemical synthesis or enzymatic synthesis.

[0078] As used herein, the term “target” refers to any component, antigen, or part recognized by sdAb. The term “intracellular target” refers to any component, antigen, or part located within a cell. A “transmembrane target” is a component, antigen, or part located within the cell membrane. An “extracellular target” refers to a component, antigen, or part located outside the cell.

[0079] As used herein, “therapeutic composition” means a substance intended to have a therapeutic effect, such as a pharmaceutical composition, genetic material, a biologic, and other substances. Genetic material includes substances intended to have a direct or indirect gene-therapeutic effect, such as gene vectors, gene regulatory elements, gene structural elements, DNA, and RNA. Biologics include substances that are living organisms or derived from living organisms and are intended to have a therapeutic effect.

[0080] As used herein, the terms “therapeutic dose” and “preventive dose” refer to the amount that provides therapeutic benefit in the treatment, prevention, or management of a disease or its apparent symptoms. A therapeutic dose is an amount that can treat a disease or symptoms, symptoms of a disease or predisposition to a disease, for the purpose of curing, resolving, alleviating, reducing, altering, treating, relieving, improving, or influencing the disease, symptoms of a disease or predisposition to a disease. A specific therapeutically effective dose can be readily determined by a typical healthcare professional and may vary depending on factors known in the art, such as the type of disease, the patient’s medical history and age, the stage of the disease, and the administration of other therapeutic agents.

[0081] STAT3 is an intracellular transcription factor activated by IL-6, other cytokines, and intracellular kinases. As a result, P-STAT3 activates genes such as vascular endothelial growth factor (VEGF), promoting the differentiation of TH17 cells necessary for CNV and inflammatory diseases. Inhibiting STAT3 stops VEGF production and prevents the generation of TH17 cells.

[0082] Despite the short half-life of VHH in serum, camelid VHH is being increasingly considered for clinical use due to its ability to target antigens present in tissues with insufficient angiogenesis and that are not easily accessible. Furthermore, VHH is stable at room temperature and in a reductive cytoplasmic environment. Described herein is a cell-permeable VHH, SBT-100 (SEQ ID NO: 1), capable of binding to two different non-homologous intracellular targets (STAT3 and KRAS) involved in tumorigenesis. SBT-100 (SEQ ID NO: 1) (1) binds to intracellular STAT3 across the cell membrane, (2) inhibits the phosphorylation of STAT3, (3) reduces total STAT3, (4) blocks the IL-6-mediated translocation of activated STAT3 into the nucleus, preventing pSTAT3 dimers from binding to their target genes, (5) inhibits the expression of vascular endothelial growth factor (VEGF), a key angiogenic factor and a known regulator of tumor cells, and (6) inhibits the cell-permeable translocation of the checkpoint inhibitor PD-L1 on the surface of tumor cells. It inhibits surface expression, thereby improving antitumor immunity in immunonormal mice; (7) inhibits cell proliferation by inhibiting KRAS-GTPase activity and downstream ERK phosphorylation; (8) shows broad antitumor cell proliferation in vitro against 11 human cancers; and (9) induces tumor regression (human cancer with activated KRAS mutations) without observable toxicity in a thymic xenograft mouse model for triple-negative breast cancer cell line (MDA-MB-231) and pancreatic cancer (PANC-1). The biological effects of SBT-100 (SEQ ID NO: 1) are reversible and last for at least 72 hours in vitro and 7 days in vivo. SBT-100 (SEQ ID NO: 1) also appears to be unparalleled in its ability to permeate the BBB. The ability of SBT-100 (SEQ ID NO: 1) to permeate the cell membrane and bind intracellular KRAS and STAT3 leads to functional suppression of cancer growth and proliferation in vitro and in vivo. This was demonstrated using multiple human cancers to show the broad applicability of SBT-100 (SEQ ID NO: 1), which inhibits human cancer.

[0083] This invention relates to the capabilities of SBT-100 (SEQ ID NO: 1), which acts as a bispecific antibody with nM binding affinity, possessing the ability to permeate the cell membrane, bind to STAT3, cross-react with KRAS (mutant and non-mutant), and (1) cross the cell membrane and bind to intracellular STAT3, (2) inhibit the phosphorylation of STAT3, (3) reduce total STAT3, (4) block the IL-6-mediated translocation of activated STAT3 into the nucleus, preventing the p-STAT3 dimer from binding to its target gene, (5) inhibit the expression of vascular endothelial growth factor (VEGF), an important angiogenic factor and a known regulator of tumor cells, and (6) inhibit the cell surface expression of the checkpoint inhibitor PD-L1 on the surface of tumor cells, thereby providing antitumor immunity in immunonormal mice. SBT-100 (SEQ ID NO: 1) can be administered therapeutically as an anticancer drug because it (7) inhibits KRAS-GTPase activity and downstream ERK phosphorylation, thereby inhibiting cell proliferation, (8) exhibits broad antitumor cell proliferation in vitro, and (9) induces tumor regression without observable toxicity in athymic xenograft mouse models of triple-negative breast cancer cell lines with KRAS(G13D) mutations (MDA-MB-231) and pancreatic cancer cell lines with KRAS(G12D) mutations (PANC-1). Furthermore, the biological effects of SBT-100 (SEQ ID NO: 1) persist for 72 hours in vitro and 7 days in vivo.

[0084] The current standard of treatment for age-related macular degeneration (AMD) involves targeting VEGF and requires intravitreal injections every 3-4 weeks. This anti-VEGF antibody therapy targets only one cytokine pathway, leaving other cytokine pathways unaffected.

[0085] Higher levels of systemic inflammatory markers, CRP and IL-6, are independently associated with the progression of AMD. Choroidal neovascularization (CNV) membranes in AMD are associated with increased IL-6. Systemic IL-6 levels have been shown to correlate with the onset and progression of AMD. IL-6 signaling may contribute to the pathogenesis of subretinal fibrosis in late neovascular AMD.

[0086] Increased IL-10 in aging eyes activates STAT3 signaling, which induces macrophage activation and angiogenesis. Targeted inhibition of both IL-10 receptor-mediated signaling and STAT3 activation in macrophages reverses the aging phenotype.

[0087] IL-17 is involved in the pathogenic inflammation of AMD. IL-17 has a strong potential to stimulate angiogenesis independently of VEGF. IL-17A reduces cell viability, alters cellular metabolism, and induces apoptosis in ARPE-19 cells. Aging and AMD-like degeneration are associated with increased IL-17 expression in the eyes of mice.

[0088] Genetic deletion of SOCS3 in myeloid cells resulted in spontaneous STAT3 activation and accelerated CNV formation. Inhibition of STAT3 activation using the small peptide LLL12 suppressed laser-induced CNVs. STAT3 activation in circulating immune cells is associated with neovascular age-related macular degeneration.

[0089] STAT3 is a transcription factor that transcribes VEGF, IL-6, IL-10, and IL-17. All of these cytokines cause inflammatory changes in the retina that promote AMD. Therefore, inhibition of STAT3 would stop the production of these AMD-causing cytokines.

[0090] SBT-100 (SEQ ID NO: 1) translocates into cells within 6 hours, as demonstrated by immunohistochemistry (IHC) staining. SBT-100 (SEQ ID NO: 1) inhibits VEGF protein production by human retinal epithelial cells to near zero within 12 hours. SBT-100 (SEQ ID NO: 1) inhibits PD-L1 cell surface expression to 1 / 10 within 24–48 hours. Both VEGF and PD-L1 are genetic targets of the STAT3 transcription factor inhibited by SBT-100 (SEQ ID NO: 1).

[0091] SBT-100 (SEQ ID NO: 1) crosses the blood-brain barrier (BBB) ​​in 15 minutes and can stain neurons and glial cells in the mouse brain. The serum half-life of SBT-100 (SEQ ID NO: 1) in mice and rats is 1 hour. The biological half-life of SBT-100 (SEQ ID NO: 1) in cancer xenograft models is 12–24 hours. The biological half-life in the retina is at least 24–48 hours. SBT-100 (SEQ ID NO: 1) permeates the cell membranes of 11 different types of human cancer and retinal cells, crosses the BBB, and also crosses the blood-retinal barrier.

[0092] Inhibiting STAT3 by blocking the inflammatory cascade and vascular signaling pathways may be effective in treating many inflammatory and neovascular conditions of the eye, such as corneal neovascularization, proliferative diabetic retinopathy, keratoconjunctivitis sicca, AMD, and uveitis. Corneal neovascularization is caused by an imbalance between angiogenic and anti-angiogenic factors that maintain corneal transparency. Proliferative diabetic retinopathy (PDR) occurs primarily when blood vessels in the retina close, obstructing blood flow. In an attempt to supply blood to the area where the original vessels have closed, the retina responds by growing new blood vessels (neovascularization). These new vessels are abnormal, supplying inadequate blood flow to the retina, and the new vessels are often accompanied by scar tissue that causes wrinkles or detachment of the retina. Retinal ischemia promotes vascular growth factors that induce the growth of fibrous and neovascularization, which subsequently leads to retinal damage. Keratoconjunctivitis sicca is a destruction of the ocular surface, with the lacrimal film leading to inflammation and damage of the cornea. Chronic bilateral dryness of the conjunctiva and cornea due to insufficient lacrimal film (dryness). Subretinal neovascularization from choroidal neovascularization (CNVM) is new blood vessels that grow beneath the retina and disrupt vision. CNVM is associated with many serious eye diseases, most commonly "exudative" age-related macular degeneration (AMD).

[0093] Central nervous system (CNS) autoimmune diseases such as uveitis and multiple sclerosis result from the disruption of immune privileges in the brain, spinal cord, or neuroretina, which are maintained by the blood-retinal barrier (BRB), blood-brain barrier (BBB), and neurovascular units (NVUs) composed of pericytes, perivascular macrophages, tightly bound endothelial cells, and the Müller / microglia glial boundary membrane. These structures isolate CNS tissue from the peripheral immune system, and granzyme B-producing Th17 cells are involved in the initial events that initiate CNS autoimmune diseases by promoting the disruption of the BBB or BRB. However, sustained activation of microglial cells and recruitment of other inflammatory cells amplify the inflammatory response and are responsible for the pathology characteristic of chronic uveitis or multiple sclerosis. Nevertheless, intervention studies using biologics such as cytokines or immunosuppressive compounds to suppress uveitis in mice consistently show a strong correlation between disease improvement and suppression of pathogenic Th17 cells. Subsequent research revealed the necessity of STAT3 for Th17 differentiation and development, while other studies showed that targeted deletion of STAT3 inhibits EAE or EAU development. These studies led to the now-established concept that targeting Th17 cells is a viable therapeutic approach for suppressing and mitigating autoimmune and autoinflammatory diseases.

[0094] Uveitis is a diverse group of potentially vision-threatening intraocular inflammatory diseases characterized by recurrent cycles of remission and relapsing endophthalmoinflammatory disease, and visual impairment is of paramount public health importance as it impacts patients' quality of life. Increased recruitment of Th17 cells to the retina is involved in the pathophysiology of uveitis, and current treatments include periorbital or intravitreal corticosteroids. However, long-term use of periorbital or intravitreal corticosteroids to treat chronic uveitis is associated with the development of serious side effects such as glaucoma, which has driven the development of alternative therapies. Genetically modified mice that cannot induce Th17 cells are resistant to the development of uveitis, so targeting the STAT3 pathway, which is necessary for the differentiation and expansion of Th17 cells, has been proposed as a potential treatment to alleviate uveitis. However, the main obstacles to targeting the STAT3 pathway are that STAT3 is an intracellular protein, making it inaccessible to STAT3-specific antibodies, as well as the unpredictable pharmacokinetic characteristics of low molecular weight STAT3 inhibitory peptides or mimics.

[0095] Uveitis is an inflammation of the iris, ciliary body, or choroid of the eye, which can result from autoimmune symptoms, trauma, or infection. It is a general term for a group of inflammatory diseases that cause swelling and destroy the intermediate layer of tissue within the eye wall (uvea). These diseases include sympathetic ophthalmia, shotgun chorioretinopathy, Behçet's disease, Vogt-Koyanagi-Harada disease, and ocular sarcoidosis. Uveitis affects not only the uvea but also the lens, retina, optic nerve, and vitreous humor, leading to decreased vision or blindness. Uveitis is a group of intraocular inflammatory diseases that account for 10% of vision loss in the United States. A subset of Th17 T helper cells is involved in the pathogenesis of uveitis in mice and humans. STAT3 plays a crucial role in the differentiation of Th17 cells, and mice with targeted deletion of Th17 cells do not develop experimental autoimmune uveitis (EAU), a mouse model of human uveitis. As a result, there is great interest in developing drugs and biologics that target the STAT3 pathway as treatments for uveitis and other inflammatory diseases.

[0096] SBT-100 (SEQ ID NO: 1) rapidly crosses the cell membrane in less than 6 hours in vitro and crosses the blood-brain barrier (BBB) ​​in less than 15 minutes in vivo. Once inside the cell, SBT-100 (SEQ ID NO: 1) non-covalently binds to KRAS and STAT3 with nanomolar affinity. Unlike small molecule inhibitors that form irreversible covalent bonds, SBT-100 (SEQ ID NO: 1) is less likely to cause toxicity due to its non-covalent, reversible binding to KRAS and STAT3. Blocking the GTPase activity of KRAS and the subsequent reduction in pERK1 / 2 levels inhibits the KRAS pathway's ability to promote cell proliferation, survival, and apoptosis evasion. Simultaneously, SBT-100 (SEQ ID NO: 1) also binds to STAT3, causing inhibition of STAT3 phosphorylation, preventing STAT3 from translocating into the nucleus and preventing STAT3 from binding to its DNA promoter. A prime example of SBT-100's (SEQ ID NO: 1) inhibitory and anti-inflammatory capabilities is also demonstrated by its ability to block the effects of IL-6 on cancer and normal cells in vitro by preventing STAT3 from transcribing nuclear target genes such as VEGF and PD-L1.

[0097] VEGF plays a crucial role in tumor growth and metastasis by inducing the development of new blood vessels. SBT-100 (SEQ ID NO: 1) significantly reduces VEGF production by retinal epithelial cells in vitro with a rapid 12-hour response, and the biological effect of a single dose lasts for at least 48 hours. This suggests that SBT-100 (SEQ ID NO: 1) may reduce antitumor effects in cancer and reduce blindness in neovascular conditions such as age-related macular degeneration (AMD). In an in vivo model for blindness, SBT-100 (SEQ ID NO: 1) has been shown to provide significant improvement in visual acuity. Other gene targets of STAT3 are PD-1 and PD-L1. IFA demonstrates that SBT-100 (SEQ ID NO: 1) reduces PD-L1 expression on TNBC (MDA-MB-231) within 24 hours. Similar results were obtained for osteosarcoma (SJSA-1), and FACS analysis showed that SBT-100 (SEQ ID NO: 1) reduced PD-L1 expression within 48 hours. This represents a novel approach to immunotherapy by downregulating checkpoint inhibitor genes. This strategy using SBT-100 (SEQ ID NO: 1) may reduce the number of PD-L1 molecules and possibly PD-1 molecules by reducing the availability of STAT3, thus resulting in a smaller presence of cell surface targets blocked by nivolumab and pembrolizumab. This may enhance the checkpoint inhibitor response or allow for a reduction in checkpoint inhibitor dosage. Since STAT3 is a pro-inflammatory mediator, STAT3 inhibition by SBT-100 (SEQ ID NO: 1) may also reduce some of the inflammatory complications associated with checkpoint inhibitor therapy, such as pneumonitis and severe COVID-19 pathophysiology.

[0098] These results indicate that TNBC tumors with KRAS(G13D) mutations (at least 50-100 mm) 3The efficacy of SBT-100 (SEQ ID NO: 1) was confirmed in the form of tumor regression in xenografts of athymic nude mice having ). The therapeutic effect of SBT-100 (SEQ ID NO: 1) lasted for at least 7 days after the last dose. Similarly, SBT-100 (SEQ ID NO: 1) enhanced the suppression of tumor growth when combined with gemcitabine. PANC-1 is known to be a difficult malignancy to treat because it is KRAS-independent. These experiments suggest that SBT-100 (SEQ ID NO: 1), alone or in combination with other chemotherapeutic agents, induces significant suppression of tumor growth in vivo.

[0099] The most distinctive aspect of SBT-100 (SEQ ID NO: 1) as described herein is its intracellular permeability and cross-reactivity with non-homologous KRAS. The novel properties of SBT-100 (SEQ ID NO: 1), including its ability to permeate the cell membrane and blood-brain barrier (BBB), give it significant clinical potential for targeting STAT3-mediated diseases or cancers with KRAS mutations.

[0100] example Example 1: Materials and Methods

[0101] Development of SBT-100 (SEQ ID NO: 1): Recombinant full-length human STAT3 (STAT3-1496H) with a GST tag fused to its N-terminus was provided by Creative BioMart (Shirley, NY). Briefly, Camelus bactrianus was used for immunization with recombinant human STAT3. Generation of SBT-100 (SEQ ID NO: 1) VHH: Camelids were immunized with the relevant antigen. After the immunization period, peripheral leukocytes (PWBCs) were collected and a phage display library was constructed to search for VHHs of interest. After the panning process was complete, VHHs were identified by their binding affinity to STAT3 and KRAS. Final endotoxin levels were less than 1 EU / mg. The amino acid sequence of SBT-100 (SEQ ID NO: 1) is HVQLVESGGGSVQAGGSLRLSCAASGANGGRSCMGWFRQVPGKEREGVSGISTGGLITYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSRFDCYRGSWFNRYMYNSWGQGTQVTVSS). SBT-100 (SEQ ID NO: 1) was previously described in U.S. Patent No. 14,922,093, the contents of which are incorporated herein by reference.

[0102] Cell lines and cell cultures: Cell lines PANC-1, BxPC3, MDA-MB-231, MDA-468, MCF-7, BT474, U87, SJSA-1, HT-1080, HEp2, DU-145, and retinal epithelial cells (ARPE-19) were all obtained from the American Type Culture Collection (ATCC) (Manassas, VA). All cells were grown at 37°C and 5% CO2 in either DMEM or RPMI medium, with or without fetal bovine serum.

[0103] Immunofluorescence and Immunohistochemical Staining: Standard procedures were used for immunohistochemical and immunofluorescence assay (IFA) staining. Primary antibodies for IF were anti-t-STAT3 (Cell Signaling Technology), anti-p-STAT3 (Cell Signaling Technology), anti-PD-L1 (Cell Signaling Technology), anti-VHH antibody (Rockland), and Alexa Fluor 488-anti-rabbit IgG (AF anti-rab IgG, Jackson ImmunoResearch). Blocking solution, 1° and 2° antibody dilutions were 1% BP (1% BSA in PBS). All cell incubation was performed in culture medium in a 5% CO2 incubator at 37°C. 4,500 cells / well were seeded onto chamber slides and allowed to adhere overnight. Cells were treated with SBT-100 (SEQ ID NO: 1) at various time points and then fixed with 100% methanol at -20°C.

[0104] IFA: Wells were blocked for at least 30 minutes, the blocking agent was removed, and primary antibodies were added at the following dilutions and incubated overnight at 5°C: anti-VHH=1:500, anti-t-STAT3=1:300, anti-P-STAT3=1:125, anti-PD-L1=1:300. Wells were washed with PBS, incubated with AF anti-rabIgG (1:300) for at least 1 hour, washed with PBS, covered with a coverslip, and examined by fluorescence microscopy. For DAPI staining, wells were incubated with 0.143 mM DAPI for 7 minutes and washed with PBS before applying a coverslip. Conventional fluorescence microscopy was performed using a Nikon 80i microscope and appropriate wavelength filters. Images were taken using the attached Spot RT3 camera (model 25.4, 2Mp slider) and the accompanying Spot 5.1 software.

[0105] Confocal microscopy: Confocal images were obtained using a Leica TCS SP8 confocal microscope. Images were quantified using Fiji software.

[0106] IHC staining for intratumoral and BBB methodologies: Athymic nude mice (n=3) with established MDA-MB-231 tumors were intravenously injected with SBT-100 (SEQ ID NO: 1) (1 mg / kg). After 15 minutes, the mice were sacrificed and their brains and tumors were collected. The tissues were placed in 10% formalin for 24 hours and then transferred to 70% ethanol. The tissues were sectioned using a dermatome (AML Laboratories, Baltimore, MD). Goat anti-Ilama conjugate (Bethyl Laboratories, Montgomery, TX) secondary antibody (1:10,000) was incubated with these tissue sections at room temperature for 10 minutes and washed twice with PBS-Tween 20 for 3 minutes each. The tissue sections were incubated in streptavidin / peroxidase complex at room temperature for 5 minutes and then washed with PBS for 5 minutes. Next, the tissue sections were incubated in peroxidase substrate solution (AEC) for 15 minutes, washed with tap water for 5 minutes, counterstained with hematoxylin QS (one drop on each section), and incubated for 30 seconds. Then, the tissue sections were rinsed with tap water until the water was colorless. The sections were mounted in aqueous mounting medium, and after 15 minutes, these slides were observed under an Olympus BX51 fluorescence microscope.

[0107] Western Blot (Slot Blot): Standard procedure was used for immunoblotting. Primary antibodies were anti-β-actin (Cell Signaling Technology), anti-t-STAT3 (Cell Signaling Technology), anti-P-STAT3 (Cell Signaling Technology), anti-PD-L1 (Cell Signaling Technology), and HRP-anti-rabbit IgG (Jackson ImmunoResearch). Blocking buffer, primary and secondary antibody diluents = 5% BT: (5% BSA in TBS) TBS = Tris-buffered saline: 25mM Tris, 150mM NaCl, pH 7.5 TBST = TBS + 0.1% Tween-20. Briefly, 2 × 10 5100 MDA cells / well were seeded into each well of a 6-well plate and adhered overnight in culture medium at 37°C in a 5% CO2 incubator. The medium was removed and SBT-100 (SEQ ID NO: 1) was added to the medium. After incubation for the indicated time, the medium was removed and the adherent monolayer of cells was washed with ice-cold PBS, and then the cells were lysed by scraping them off in TBS + 0.05% SDS supplemented with EDTA, a protease inhibitor, and a phosphatase inhibitor. At a subsequent point in the process when non-adherent cells were evident, these cells were collected by centrifugation, lysed, and combined with the solubilized adherent cell population.

[0108] Western blot quantification: Protein concentrations in each fraction were determined using a BCA protein assay (ThermoFisher Scientific). Equal amounts of protein from each cell fraction (typically about 10 μg / slot) were diluted in 200 μl / slot with TBS and loaded onto PVDF membranes pre-activated in 100% methanol and then equilibrated in TBS via a slot blotting instrument. The blots were blocked in 5% BT for at least 1 hour and then incubated overnight at 5°C in the following antibody dilutions: anti-β-actin = 1:1000, anti-t-STAT3 = 1:1000, anti-P-STAT3 = 1:750, anti-PD-L1 = 1:1000. The blots were washed three times with TBST, briefly equilibrated in TBS, and then pre-incubated with HRP-anti-rabIgG (1:5000) for at least 1 hour. The washing step was repeated, and the blots were incubated with chemiluminescent HRP substrate according to the manufacturer's protocol. The reaction was visualized using a chemiimager. Quantification was performed using ImageJ software included in the Fiji image processing package.

[0109] IL-6 stimulation and inhibition of p-STAT3 nuclear translocation: Cells (HEp-2 and PANC-1) were grown on four Permanox chamber slides. SBT-100 (SEQ ID NO: 1) antibody was added overnight (diluted 1-10 times in medium), and the slides were kept at 37°C. SBT-100 (SEQ ID NO: 1) antibody was not added to the negative control sample. The following day, the cells were stimulated with IL-6 (Peprotech, 100 ng / ml) for 15 minutes. After stimulation, the chamber slides were immediately fixed in ice-cold 100% methanol at 20°C for 10 minutes. The slides were dried, and the IFA process described above was carried out. The slides were blocked with 3% BSA in PBS at room temperature for 1 hour, and then with the primary antibody Stat3 (Cell Signaling Technology) overnight at 4°C. Secondary antibodies anti-mouse IgG (H&L) and Alexa Fluor 488 (Cell Signaling Technology) were added at room temperature for 1 hour. Finally, the chamber slide was cleaned, mounted with mounting medium, and observed under a Nikon fluorescence microscope.

[0110] Promega Dual Luciferase Reporter Assay System (GTPase-Glo® Assay): This assay measured STAT3 transcriptional activity using the HEK 293 IL-6 STAT3 reporter cell line (Promega, Madison WI). In this cell line, induction with 40 ng / ml of IL-6 activated the STAT3 transcription factor, promoting luciferase reporter expression, which could then be measured using a standard luminometer. 10 5 Cells / wells were incubated for 48 hours with or without SBT-100 (SEQ ID NO: 1) antibody. To test IC50 values, 8-point 2x titrations starting at 100 ug / ml were performed. IL-6 was added during the last 18 hours of incubation. All time points are timed from the addition of the SBT-100 (SEQ ID NO: 1) inhibitor. After 48 hours, cells were lysed and luminescence measurements were performed using a BMG Labtech microplate reader. Results are expressed as a percentage of the control well (cells + IL-6).

[0111] Human VEGF-A ELISA Assay: The human VEGF-A ELISA (ThermoFisher Scientific) assay was modified from a 24-well format to a 96-well plate format. ARPE-19 cells were serum-starved with 10% FBS in DMEM and incubated overnight. The medium was then replaced with a medium containing 100, 10, 1, or 0.1 μg / ml of SBT-100 (SEQ ID NO: 1), anti-EMP 2 antibody (Abcam), or medium alone, and incubated for 12, 24, or 48 hours. At appropriate time points, the supernatant was removed and stored at ≤-65°C. Cells were lysed using RIPA lysis buffer (ThermoFisher Scientific), and the protein content of the cell solubil was measured by BCA assay (ThermoFisher Scientific). VEGF-A was measured using the Human VEGF-A ELISA Kit (ThermoFisher Scientific) according to the kit instructions. Experimental statistical analysis was performed using ANOVA in Dunnett's multiple comparison test with a negative control as the control column.

[0112] Flow cytometry analysis: SJSA-1 cells were incubated with 50 ng / ml recombinant human IFN-γ (Peprotech) for 24 hours, then the medium was replaced with a medium containing 50 μg / ml SBT-100 (SEQ ID NO: 1), and incubated for 48 hours. Cells were then harvested and stained with the following antibodies: CD276 (Clone MIH-42, Biolegend), CD274 (Clone 29E.2A3, Billegend), and CD200 (Clone OX-104, Billegend). After staining, the samples were passed through a BD Celesta Flow Cytometer, and the data were analyzed using FlowJo software.

[0113] MTT Assay: For these experiments, cancer cells were grown until they reached a 90% density. The cells were washed, trypsinized, and counted using a Coulter Counter (Beckman, Brea, CA). Growth tests were performed using the 3-[4,5-dimethylthiarolyl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (Roche Diagnostics Corporation, catalog 11465007001, Sigma-Aldrich). For this, 5 × 10⁶ cells were placed in a 96-well plate. 3 Cells were seeded at the specified density. Cells were allowed to adhere for 24 hours and treated with appropriate concentrations (serial dilutions starting at 100 ug / ml) as described in Table 2. On day 3, 10 ul of MTT reagent (0.5 mg / ml) was added to each well as indicated by the manufacturer. After a 4-hour incubation period, 100 ul of solubilizing solution was added, and the plates were left in the incubator overnight. All plates were read at a wavelength of 570 nm using a Biotek plate reader (Winooski, VT). All data were analyzed using GraphPad InStat3 (GraphPad Software, Inc., La Jolla, CA). The treatment group was compared to the vehicle control group using one-way ANOVA. If a significant difference (p<0.05) was observed, a Tukey-Kramer multiple comparison test was performed.

[0114] Measurement of KRAS inhibitory activity in enzyme assays: KRAS GTPase activity converts GTP to GDP. The GTPase-Glo reagent kit (Promega, Madison WI) is designed to measure this activity. The Glo reagent converts unhydrolyzed GTP to ATP, producing a luminescence signal. When KRAS activity is inhibited, GTP remains unhydrolyzed, and a high luminescence signal is expected. If KRAS is not inhibited, GTP is converted to GDP, and a low signal is observed. Luminescence was measured using a PHERAstar plate reader (BMG Labtech). The activity of mature, active KRAS (SignalChem, Richmond, BC Canada) supplied in manufacturer's buffer was tested in the presence of a dilution series of inhibitors. Commercial KRAS GTPase activity was titrated in Promega GTPase / GAP buffer and SBT-100 (SEQ ID NO: 1) buffer in the presence of several inhibitors. The inhibition and effect of buffer conditions on KRAS GTPase activity were compared.

[0115] Animals: All animals were kept under pathogen-free conditions, and experiments were conducted in accordance with the Illinois Institute of Technology (IIT) Research Institute Animal Use and Care Committee (IACUC), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Five-to-six-week-old athymoid nude-Foxn1nu female mice were purchased from ENVIGO Laboratories (Indianapolis, IN). The animals were isolated for one week and housed at 20°C–26°C and 50% relative humidity, with five mice per cage in a 12-hour light-dark cycle. Drinking water and diet (PicoLab Rodent Diet 20 Irradiated, consisting of 20% crude protein, 4.5% crude fat, and 6.0% crude fiber) were freely supplied to the animals.

[0116] Mouse xenograft model: Tumor cells at the fifth passage were used for transplantation and harvested during logarithmic growth. 5 × 10 per 100 μl of medium 6 PANC-1 cells or MDA-MB-231 cells at the cell concentration were injected subcutaneously into the right flank. Tumor measurement was started immediately after the tumor became palpable. Thereafter, tumors were measured twice a week. Tumors were measured in two dimensions using vernier calipers, and the volume was calculated using the following formula: Tumor volume (mm 3 ) = (w 2 × l) / 2; wherein w = the width of the tumor, and l = the length of the tumor in mm. For PANC-1 tumors, when the tumor reached 79~172 mm 3 , and for MDA-MB-231 tumors, when the tumor reached 55~150 mm 3 in size range, animals were randomized using a stratified random sampling algorithm. Treatment of animals with SBT-100 (SEQ ID NO: 1) or vehicle injected via the intraperitoneal route was started the day after randomization, designated as day 1. Animals were randomized, data (e.g., dosing, body weight, tumor measurements, clinical observations) were collected, and statistical data analysis was performed using Study Log Study Director Animal Study Management Software (San Francisco, CA).

[0117] Example 2: Binding properties of anti-STAT3 and anti-KRAS camelid VHH: SBT-100 (SEQ ID NO: 1)

[0118] Recombinant full-length human STAT3 (STAT3-1496H) with a GST tag fused to its N-terminus was provided by Creative BioMart (Shirley, NY, USA). Briefly, Camelus bactrianus was used for immunization with recombinant human STAT3. After the immunization protocol was completed, peripheral blood cells were collected for total RNA isolation and two rounds of nested PCR to construct a VHH library. Next, clones that showed the best cell permeability and binding to human STAT3 and human KRAS proteins were selected. In this screening, high affinity binding of SBT-100 (SEQ ID NO: 1) to recombinant STAT3 and KRAS, both wild-type and mutant (G12D), was observed using Biacore 3000 (Table 1). For reconnaissance, samples were flowed onto the chip and the binding of samples to ligands was monitored in real time. The affinity constant (KD) was determined as the ratio of the dissociation rate to the association rate. SBT-100 (SEQ ID NO: 1) reduces wild-type KRAS by KD = 4.20 × 10⁻¹⁰ -9 The two are joined together, and KRAS(G12D) is expressed as KD=1.50×10 -8 Combine them and set STAT3 to KD=2.24×10 -8It binds to STAT3. SBT-100 (SEQ ID NO: 1) does not bind to unrelated antigens (12-lipoxygenase). Although STAT3 and KRAS molecules are highly conserved in nature, they do not share significant homology in their protein sequences. Binding of SBT-100 (SEQ ID NO: 1) to its immunogen, STAT3, was expected, but cross-reactivity to KRAS was not. The sequence of the anti-KRAS VHH (SBT-102) (SEQ ID NO: 2) used was EVQLVESGGGSVQTGGSLRLSCAVSGNIGSSYCMGWFRQAPGKKREAVARIVRDGATGYADYVKGRFTISRDSAKNTLYLQMNRLIPEDTAIYYCAADLPPGCLTQAIWNFGYRGQGTLVTVSS. Therefore, SBT-100 (SEQ ID NO: 1) can bind to and inhibit cancers with wild-type KRAS, KRAS(G12D) (the most common variant), and KRAS(G13D) mutations. SBT-100 (SEQ ID NO: 1) likely binds to a common epitope near the KRAS GTPase active site, thereby making SBT-100 (SEQ ID NO: 1) a pan-KRAS inhibitor. The bispecific binding of SBT-100 (SEQ ID NO: 1) sdAb to KRAS and STAT3 may allow SBT-100 (SEQ ID NO: 1) sdAb to concentrate inside the cell along with high concentrations of STAT3, which then brings SBT-100 (SEQ ID NO: 1) closer to KRAS.

[0119] Table 1 also shows the binding of anti-Ebola VP24 VHH (SBT-106) (SEQ ID NO: EVQLVESGGGSVQAGGSLRLSCAASVYSYNTNCMGWFRQAPGKEREGVAVIYAAGGLTYYADSVKGRFTISQENGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS) and anti-HIV-1 reverse transcriptase (RT) VHH (SBT-107) (SEQ ID NO: MGDVQLVESGGDSVRAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTLVTVSSSSGLE) to Ebola VP24 and HIV-1 RT, respectively. [Table 1]

[0120] Example 3: Cell permeability by SBT-100 (SEQ ID NO: 1)

[0121] Immunofluorescence analysis (IFA) of the MDA-MD-231 TNBC cell line was used to determine the ability of SBT-100(SEQ ID NO: 1)VHH to permeate the cell membrane and bind intracellular STAT3. Cells treated with SBT-100(SEQ ID NO: 1) not only showed intracellular localization of SBT-100(SEQ ID NO: 1), but also showed some membrane association using an anti-GST tag antibody in the TNBC cell line MDA-MD-231. Figure 1A shows immunofluorescence staining of MDA-MB-231 cells incubated with SBT-100(SEQ ID NO: 1) antibody, with positive cytoplasmic staining using an anti-His tag antibody in the green channel. Figure 1B shows a negative control of MDA-MB-231 cells treated with vehicle alone. As expected, the vehicle-treated cells showed no staining. As a further negative control, MDA-MB-231 cells were cultured with anti-HIV-1 reverse transcriptase VHH (Figure 1C); since MDA-MB-231 cells do not produce HIV-1 virus, no intracellular staining was observed in these cells using conventional fluorescence microscopy. Figure 1D shows a confocal image of SBT-100 (SEQ ID NO: 1) detection via anti-VHH antibody, showing granular staining located throughout the cytoplasm of MDA-MB-231 cells. Confocal fluorescence microscopy revealed that SBT-100 (SEQ ID NO: 1) is present as granular staining dispersed throughout the cytoplasm of MDA-MB-231 cells after treatment with SBT-100 (SEQ ID NO: 1) (Figure 1D). The observed staining of SBT-100 (SEQ ID NO: 1) in Figures 1A and 1D is cytosolic, not endocytic.

[0122] The effect of SBT-100 (SEQ ID NO: 1) on intracellular levels of STAT3 was determined. As shown in Figures 2A and 2B, IFAs indicate that SBT-100 (SEQ ID NO: 1) reduces intracellular levels of both phosphorylated STAT3 and total (t)STAT3. MDA-MB-231 cells were either untreated (Figure 2A) or treated with 150 μg / ml of SBT-100 (SEQ ID NO: 1) for 6 hours (Figure 2B). There is a redistribution of tSTAT3, primarily from the nucleus and perinuclear to the entire cell, indicating that nuclear-localized t-STAT3 is reduced in MDA-MB-231 cells treated with SBT-100 (SEQ ID NO: 1). Confocal images of tSTAT3 in MDA-MB-231 cells either untreated (Figure 2C) or 6 hours after treatment with 150 μg / ml SBT-100 (SEQ ID NO: 1) (Figure 2D) demonstrate that treatment with SBT-100 (SEQ ID NO: 1) reduces intracellular levels of phosphorylated (p)STAT3 in MDA-MB-231 cells. Furthermore, confocal fluorescence imaging analysis of both tSTAT3-stained and DAPI (4',6-diamidino-2-phenylindole)-stained nuclei further supports this redistribution of SBT-100 (SEQ ID NO: 1)-induced tSTAT3 staining from the nuclear / cellular center to the entire cytoplasm of the cell (Figures 2C and 2D). Protein sequence analysis using the online tool "Prot Pi" (https: / / www.protpi.ch / Calculator / ProteinTool#Results) yielded a theoretical net charge of only +2.34 at physiological pH, a significant deviation from the suggested optimal net charge of +14. Therefore, SBT-100 (SEQ ID NO: 1) appears unparalleled in its ability to permeate cells, bind non-homologous proteins (STAT3 and KRAS), and cross the blood-brain barrier (BBB). While cell permeability via VHH has been demonstrated by others, this was only observed after cationic resurfacing of the molecule. The ability of SBT-100 (SEQ ID NO: 1) to permeate the cell membrane does not involve polycationic resurfacing, genetic engineering, or monomer conjugation.

[0123] Example 4: Inhibition of STAT3 phosphorylation and nuclear translocation, followed by a decrease in checkpoint molecules.

[0124] Following the demonstration of the cell permeability of SBT-100 (SEQ ID NO: 1), its effect on pSTAT3 in vitro was evaluated. IFA showed that SBT-100 (SEQ ID NO: 1) reduced intracellular levels of tSTAT3. Furthermore, there was a redistribution of tSTAT3 from primarily the nucleus and perinuclear to dispersed throughout the cell, indicating that nuclear-localized tSTAT3 was reduced in MDA-MB-231 cells treated with SBT-100 (SEQ ID NO: 1). Confocal fluorescence imaging analysis of both tSTAT3-stained and DAPI-stained nuclei further supported this redistribution of SBT-100 (SEQ ID NO: 1)-induced tSTAT3 staining from the nucleus / central cell to staining throughout the cytoplasm of the cell (Figures 2C and 2D). IFA studies demonstrated that MDA-MB-231 cells incubated with SBT-100 (SEQ ID NO: 1) showed reduced pSTAT3 levels within 6 hours. Microscopic observations were performed on untreated MDA-MB-231 cells (Figures 2E and 2G) or after 6 hours of treatment with 150 μg / ml SBT-100 (SEQ ID NO: 1) (Figures 2F and 2H). Figures 2E, 2F, 2G, and 2H show the reduced pSTAT3 levels after treatment. The changes in the cellular localization of pSTAT3 induced by SBT-100 (SEQ ID NO: 1) were remarkably similar to the changes in tSTAT3. These data suggest that SBT-100 (SEQ ID NO: 1) inhibits the translocation of both total STAT3 and phosphorylated STAT3 from the cytoplasm to the nucleus. Thus, both conventional fluorescence imaging and confocal fluorescence imaging revealed that SBT-100 (SEQ ID NO: 1) reduces intracellular levels of pSTAT3 in MDA-MB-231 cells.

[0125] The effects of SBT-100 (SEQ ID NO: 1) on immune checkpoint inhibitors in cancer cells, such as PD-L1, which is a transcriptional gene target of STAT3, were investigated. Checkpoint molecules are expressed on the surface of tumor cells and ultimately suppress the activation function of T cells, contributing to an immunosuppressive tumor microenvironment. Inflammatory cytokines such as IL-6 and IFN-γ promote the expression of checkpoint molecules such as PD-L1 (CD274), B7-H3 (CD276), and OX-2 (CD200). By culturing the human osteosarcoma cell line SJSA-1 with SBT-100 (SEQ ID NO: 1), stimulation with 75 ug / ml IFN-γ in the presence or absence of SBT-100 (SEQ ID NO: 1) resulted in a decrease in the cell surface expression of B7-H3, OX-2, and PD-L1 in SJSA-1 osteosarcoma cells (data not shown). B7-H3 decreased to approximately 1 / 3, OX-2 decreased to approximately 1 / 4, and PD-L1 decreased to approximately 1 / 4. MDA-MB-231 cells were analyzed after 24 hours of treatment with SBT-100 (SEQ ID NO: 1) to investigate potential changes in PD-L1 protein synthesis levels in other cell types. Fluorescence microscopy observations of PD-L1 expression were shown in MDA-MB-231 cells stimulated with 75 ug / ml IFN-γ for 24 hours, either with or without SBT-100 (SEQ ID NO: 1) (data not shown). Confocal fluorescence microscopy observations of PD-L1 expression in MDA-MB-231 cells were shown after 24 hours of stimulation with 75 ug / ml IFN-γ, either with or without SBT-100 (SEQ ID NO: 1) (data not shown). PD-L1 staining using IFA showed a decrease in membrane and intracellular protein levels mediated by SBT-100 (SEQ ID NO: 1), consistent with published data on the localization of PD-L1 expression in cells. Interferon (IFN)-γ is known to induce PD-L1 expression, and both intracellular and membrane staining increased after treatment with IFN-γ (data not shown). This is the first demonstration of downregulation of checkpoint inhibitors at the gene expression level by an antibody.

[0126] Changes in protein levels were quantified using immunoblotting techniques. Figure 3A shows representative images of immunoblotting of MDA-MB-231 protein extracts for pSTAT3, tSTAT3, and PD-L1 with and without SBT-100 (SEQ ID NO: 1) at 24 hours. Changes in tSTAT3, pSTAT3, and PD-L1 protein levels induced by SBT-100 (SEQ ID NO: 1) treatment of MDA-MB-231 cells were quantified by immunoblotting of protein extracts from untreated MDA-MB-231 cells and cells treated with SBT-100 (SEQ ID NO: 1) for 3 hours, 6 hours, and 24 hours using the STAT3 inhibitor S31-201 as a positive control (Figure 3B and data not shown). Each data point represents the mean ± standard deviation of values ​​normalized to β-actin, obtained from 3–5 independent experiments in which MDA-MB-231 cells were treated with four different SBT-100 (SEQ ID NO: 1) preparations, the final concentrations of which varied between 50 μg / ml and 100 μg / ml. Replicated immunoblots were reacted with antibodies against each of the three proteins under investigation. After chemiluminescence detection, the signal intensity of each protein in each extract was normalized to the intensity of β-actin present in that sample. The differences in induced tSTAT3, pSTAT3, and PD-L1 levels at the time of SBT-100 (SEQ ID NO: 1) administration were then quantified by comparing the levels in each treated sample to the levels observed in untreated cells (set to 1). S3I-201, a known STAT3 phosphorylation inhibitor shown to reduce PD-L1 levels, was used as a positive control. Treatment with SBT-100 (SEQ ID NO: 1) reduced the levels of tSTAT3, p-STAT3, and PD-L1, with the most significant decrease observed in all three proteins over 24 hours. Thus, quantitative analysis confirmed the data observed by immunofluorescence.

[0127] The nuclear region of cells stained with commercially available tSTAT3 and pSTAT3 antibodies was determined from two-dimensional images by quantifying the green antibody-specific fluorescence that occurred simultaneously with nuclear (blue DAPI) staining. After 6 hours of treatment with SBT-100 (SEQ ID NO: 1), 41% of the original tSTAT3 stain and 70% of the original pSTAT3 stain remained in the nucleus. Based on the three-dimensional projection of the corresponding Z-stack images obtained by confocal microscopy, none of the remaining tSTAT3 remained in the nucleus. All tSTAT3 co-stained with DAPI in the two-dimensional images were located in the cytoplasm surrounding the nuclear membrane. This analysis using the pSTAT3 antibody is impossible due to its extremely low staining intensity in the IFA system. These data indicate that SBT-100 (SEQ ID NO: 1), like S3I-201, reduced tSTAT3, pSTAT3, and PD-L1 during 24 hours of treatment. Prior to the reduction, there appeared to be an early (3-hour) increase in all three protein species. The decrease in tSTAT3 was evident at 6 hours, preceding the decrease in pSTAT3, whereas the decrease in PD-L1 levels was evident 24 hours after treatment with SBT-100 (SEQ ID NO: 1). Taken together, these data support the hypothesis that the binding of STAT-3 to SBT-100 (SEQ ID NO: 1) reduces PD-L1 translation by decreasing the level of the pSTAT3 transcription factor.

[0128] Example 5: Inhibition of IL-6-induced STAT3 nuclear translocation and suppression of STAT3-mediated VEGF production.

[0129] Since IL-6 plays a crucial role in regulating the nuclear translocation of activated pSTAT3, experiments were conducted to determine whether SBT-100 (SEQ ID NO: 1) mediates this activity by inhibiting IL-6 activity. As shown in Figure 4, IL-6 stimulation of HEp-2 and PANC-1 cells with SBT-100 (SEQ ID NO: 1) inhibited the nuclear translocation of pSTAT3 and subsequently reduced the proliferation of HEp-2 and PANC-1 cells. Fluorescence microscopy observations of phosphorylated STAT3 in Hep-2 cells are shown under normal conditions (Figure 4A), with IL-6 stimulation (Figure 4B), and with both IL-6 stimulation and SBT-100 (SEQ ID NO: 1) treatment (Figure 4C). Fluorescence microscopy observations of phosphorylated STAT3 in Panc-1 cells are shown under normal conditions (Figure 4D), with IL-6 stimulation (Figure 4E), and with both IL-6 stimulation and SBT-100 (SEQ ID NO: 1) treatment (Figure 4F). A reporter cell assay was performed to measure luciferase expression from HEK 293 cells transfected with a construct linking the STAT3 promoter to the luciferase gene. The STAT3 luciferase reporter assay in HEK 293 T cells treated with SBT-100 (SEQ ID NO: 1) after IL-6 stimulation showed that STAT3 dimers translocate to the nucleus, bind to the STAT3 promoter, and induce luciferase activity. Addition of SBT-100 (SEQ ID NO: 1) dose-dependently abolished this effect. Therefore, the effect of IL-6 on STAT3 activity was significantly inhibited by SBT-100 (SEQ ID NO: 1). This assay showed that treatment with SBT-100 (SEQ ID NO: 1) at a dose of 100 μg / mL reduced IL-6-inducible binding of pSTAT3 to its DNA promoter by almost 100%, and IC at 2.68 μg / mL (0.18 μM) 50 This was demonstrated (without showing the data).

[0130] At 12, 24, and 48 hours, 100 ug / ml of SBT-100 (SEQ ID NO: 1) suppressed VEGF protein production by more than 99% (p<0.01).

[0131] In cancer cells, STAT3 transcribes many genes necessary for cell proliferation and survival, including VEGF. To determine the ability of SBT-100 (SEQ ID NO: 1) to inhibit VEGF production, retinal epithelial cells that produce large amounts of VEGF were used. VEGF levels were measured by ELISA in retinal epithelial cells stimulated with IL-6 in the presence of gradually increasing concentrations of SBT-100 (SEQ ID NO: 1). At 12, 24, and 48 hours, 100 μg / mL of SBT-100 (SEQ ID NO: 1) resulted in over 99% suppression of VEGF protein production (data not shown). No cytotoxicity was observed at any of the concentrations tested in this experiment (data not shown). VEGF is a cytokine crucial for the proliferation and survival of cancer cells, and it is well known that inhibiting VEGF function can significantly improve overall survival in cancer patients (colorectal cancer, lung cancer, glioblastoma, renal cancer, cervical cancer, and ovarian cancer). For example, bevacizumab inhibits the action of VEGF by binding it in the extracellular space. SBT-100 (SEQ ID NO: 1) permeates the cell membrane and inhibits STAT3 function, resulting in a significantly reduced production of VEGF protein. This is a completely unique method of inhibiting VEGF compared to bevacizumab.

[0132] High levels of IL-6 are present in the blood of patients with severe COVID-19 infection. IL-6 plays a crucial role in the tumor microenvironment of many cancers and promotes STAT3-mediated inflammation in the eye, leading to macular degeneration and uveitis. SBT-100 (SEQ ID NO: 1) may reduce SARS-CoV-2 replication in patients by binding to and inhibiting STAT3. By blocking the action of IL-6, SBT-100 (SEQ ID NO: 1) may help reduce lung inflammation, which may subsequently improve lung compliance and oxygenation in patients.

[0133] Example 6: SBT-100 (SEQ ID NO: 1) inhibits the growth of human cancers with KRAS mutations and constitutive expression of pSTAT3 in vitro.

[0134] SBT-100 (SEQ ID NO: 1) inhibited proliferation in 11 human cell lines derived from various cancers, including pancreatic cancer (PANC-1 and BxPC3), TNBC (MDA-MB-231, MDA-MB-468, MDA-MB-453), ER+PR+ breast cancer (MCF-7), HER-2+ amplified breast cancer (BT474), glioblastoma (U87), osteosarcoma (SJSA-1), fibrosarcoma (HT-1080), and metastatic chemotherapy-resistant prostate cancer (DU-145) (Table 2). MDA-MB-231 cells possessed KRAS(G13D), and PANC-1 cells had KRAS(G12D) activating mutations. Most of these cancer cells constitutively expressed pSTAT3. These results demonstrate that SBT-100 (SEQ ID NO: 1) has broad-spectrum anticancer activity. This data suggests that SBT-100 (SEQ ID NO: 1) has a significant (p<0.001) tumor cell inhibitory effect (85-93%) against human malignancies with or without activating KRAS mutations and constitutive pSTAT3 expression. [Table 2]

[0135] Example 7: Inhibition of KRAS GTPase activity and suppression of downstream pERK signaling.

[0136] Binding data, as determined using KRAS and STAT3 proteins and lipoxygenase, demonstrate that SBT-100 (SEQ ID NO: 1) is bispecific to KRAS and STAT3, while SBT-102 is monospecific to human KRAS and its most common mutant KRAS (G12D) at nanomolar affinity, but does not bind to STAT3 (Table 1). The biochemical effects of SBT-100 (SEQ ID NO: 1) and SBT-102 (SEQ ID NO: 2) on KRAS were demonstrated using GTPase activity. RLU was measured as a read for KRAS GTPase activity. Reagents were incubated with either SBT-100 (SEQ ID NO: 1), SBT-102 (SEQ ID NO: 2), or an anti-KRAS polyclonal antibody, and RLU was measured. KRAS GTPase activity was inhibited by escalating doses of SBT-100 (SEQ ID NO: 1) and SBT-102 (SEQ ID NO: 2), demonstrating their inhibitory binding activity (Figure 5A). Here, an anti-KRAS polyclonal antibody was used as a positive control. Inhibition of KRAS GTPase activity by SBT-100 (SEQ ID NO: 1), SBT-102 (SEQ ID NO: 2), and the anti-KRAS polyclonal antibody occurred in a dose-dependent manner, and the results were comparable among the three. Western blot analysis was performed on phosphorylated ERK1 / 2 in various KRAS-mutated cancer cells with or without SBT-100 (SEQ ID NO: 1) treatment (Figure 5B). In lanes 1 (MDA-MB-231), 3 (PANC-1), and 5 (BxPC3), cancer cells were incubated with the vehicle alone. In lanes 2 (MDA-MB-231), 4 (PANC-1), and 6 (BxPC3), cancer cells were incubated with 100 ug / ml of SBT-100 (SEQ ID NO: 1) for 72 hours. The results show that downstream phosphorylation of ERK (pERK) is inhibited in both MDA-MD-231 and PANC-1 cancer cell lines with KRAS mutations. By binding to KRAS and inhibiting its GTPase activity, SBT-100 (SEQ ID NO: 1) blocks the KRAS signaling pathway.Cancer cells with greater basal STAT3 activity may trap SBT-100 (SEQ ID NO: 1) in the cytoplasm to a greater extent, thereby enhancing SBT-100's ability to inhibit KRAS. This likely explains the differing pERK levels among these cancer cell lines. MDA-MB-231 has been shown to have much higher levels of STAT3 than PANC-1. This may result in a shorter time for SBT-100 (SEQ ID NO: 1) to bind to and inhibit KRAS. Furthermore, the literature indicates that PANC-1 is KRAS-independent, while other cancers with KRAS mutations are KRAS-dependent. This may also explain the less pronounced decrease in pERK levels in PANC-1 compared to MDA-MB-231. Therefore, SBT-100 (SEQ ID NO: 1) binds to KRAS, and subsequently, inhibition of KRAS GTPase activity and suppression of downstream KRAS signaling reduce pERK levels, which in turn inhibits the proliferation of cancer cells with activating KRAS mutations.

[0137] Example 8: Reduction of tumor volume in tumor-bearing mice with SBT-100 (SEQ ID NO: 1)

[0138] Athymic mice were subcutaneously injected with either the TNBC cell line (MDA-MB-231) or pancreatic cancer cells (PANC-1), treated with SBT-100 (SEQ ID NO: 1) for 14 days, and after a 7-day recovery period, tumor volume was measured. MDA-MB-231 tumors were 50-100 mm before the start of treatment. 3 (Figure 6). Tumor-bearing mice were randomly divided into a control group, which received PBS by intraperitoneal injection for 14 days until sacrifice, and a treatment group, which received 5 mg / kg of SBT-100 (SEQ ID NO: 1) twice daily. The treatment group showed rapid suppression of tumor growth, which was maintained after the 14-day treatment period. During the 7-day post-treatment observation period, there was no rebound in tumor growth, and all mice survived the treatment and maintained normal body weight. After the final dose of SBT-100 (SEQ ID NO: 1), there was no significant proliferation of MDA-MB-231 during the observation period.

[0139] PANC-1 tumors were 100-150 mm before treatment. 3 The mice were then randomized to one of four groups, all administered intraperitoneally for 14 days: control (PBS), gemcitabine alone (20 mg / kg, once daily), SBT-100 (SEQ ID NO: 1) alone (100 mg / kg, twice daily), and gemcitabine and SBT-100 (SEQ ID NO: 1). After the 14-day treatment period, there was a 7-day observation period. At the end of the study, the gemcitabine-only group had a 14.93% reduction in tumor growth compared to the control group (Table 3). The SBT-100 (SEQ ID NO: 1)-only group had a 19.17% reduction in tumor growth compared to the control group. Finally, the group receiving gemcitabine and SBT-100 (SEQ ID NO: 1) showed a 31.52% reduction compared to the control group (p<0.05). None of the treated mice died or experienced weight loss due to SBT-100 (SEQ ID NO: 1). To determine the potential toxicity of SBT-100 (SEQ ID NO: 1), body weight data from all xenotransplant studies in which mice were administered SBT-100 (SEQ ID NO: 1) were combined. During the 3-week xenotransplant studies, there was no significant weight loss in the group that received SBT-100 (SEQ ID NO: 1) for treatment (data not shown). Possible reasons for the absence of observable toxicity include the fact that the reversible biological effects last for up to 7 days, while the serum half-life is short. Furthermore, the role of STAT3 in normal adult tissues is limited. Conditional disruption of STAT3 in adult mice has been shown to affect different systems to varying degrees without being lethal, unlike embryonic targeting. Due to abnormal levels of intracellular STAT3 expression in cancer cells, it is possible that injected SBT-100 (SEQ ID NO: 1) preferentially accumulates within cancerous lesions.

[0140] [Table 3]

[0141] Another important in vivo finding was that SBT-100 (SEQ ID NO: 1) crossed the blood-brain barrier (BBB) ​​in large, established MDA-MB-231 tumors (>200mm). 3 Athymoid nude mice possessing ) were given a single IP injection of 5 mg / kg of SBT-100 (SEQ ID NO: 1) for 15 minutes and then sacrificed. Immunohistochemical analysis demonstrates the localization of SBT-100 (SEQ ID NO: 1) inside cancer cells, crossing the blood-brain barrier (BBB). (Figure 7A) This representative section of the tumor mass shows intracellular staining within TNBC cells, as indicated by the arrows. These cells are surrounded by dense tumor stroma. (Figure 7B) Arrows point to a collection of intracellular staining of neurons and glial cells in the brain of tumor-bearing mice. It has been demonstrated that VHH with a basic pI can spontaneously cross the BBB (can cross at 9.3 but not at 7.7). SBT-100 (SEQ ID NO: 1) has a basic pI of 8.22 (calculated using the Prot Pi online tool), which is outside the range of reported pIs that enable BBB crossing.

[0142] Example 9: Eye disease

[0143] Experimental autoimmune uveitis (EAU) was induced in mice. To do this, mice were immunized with photoreceptor-retinoid-binding protein (IRBP) in an emulsion of cfacilitator effusion (CFA) and pertussis toxin. Once EAU was induced, SBT-100 was then injected into the eye. The effect of SBT-100 (SEQ ID NO: 1) on uveitis was examined by fundus examination on days 15 and 18, optical coherence tomography (OCT) on day 15, and electroretinography (ERG) on day 14. Fundus examination is an examination that uses a magnifying lens and light to examine the fundus of the eye (the posterior medial part of the eye, including the retina and optic nerve). Mice were sacrificed on day 18, eyes were collected for histology, and intracellular cytokine analysis was performed by FACS. As shown in Figure 8, the EAU clinical score, as measured by fundus examination, was reduced in SBT-100 (SEQ ID NO: 1) treated mice. Fundus examination images showed that SBT-100 (SEQ ID NO: 1) improved uveitis (data not shown). OCT results showed reduced recruitment of inflammatory cells in the optic disc and vitreous humor (Figure 9). Figures 10A–10D show ERG results demonstrating improved visual function in SBT-100 (SEQ ID NO: 1) treated EAU mice. Figure 11 shows that cytokine staining analysis of cells treated with SBT-100 (SEQ ID NO: 1) resulted in a significant reduction in pathogenic Th17 cells secreting IL-17. Therefore, SBT-100 (SEQ ID NO: 1) attenuated the severity of uveitis in a mouse model of human uveitis, suggesting that single-domain SBT-100 (SEQ ID NO: 1) could be used as a treatment for uveitis and other inflammatory diseases.

[0144] Example 10: SBT-100 (SEQ ID NO: 1) binds STAT3 from lysed cancer cell lines.

[0145] It has been previously shown that SPT-100 can bind to unphosphorylated STAT3. Figure 12 demonstrates the binding of phosphorylated STAT3 to SBT-100 (SEQ ID NO: 1). Cell solubilizers were incubated at 4°C for 1 hour with Dynabeads pre-loaded with SBT-100 (SEQ ID NO: 1), a positive control (commercially available STAT3), or a negative control (commercially available STAT1). After separating the bound proteins by SDS-PAGE, Western blot analysis was performed using STAT3 (79D7) rabbit mAb #4904. Figure 12 shows the amount of STAT3 cells: lane 1 is PANC-1 cells, lane 2 is DU145 cells, lane 3 is HeLa cells, lane 4 is mouse STAT3 (99% homologous to human STAT3) and 4T1 cells, lane 5 is PANC-1 and a negative control with KRAS, and lane 6 is PC-3 cells. Immunoblotting shows that PANC-1 cells have low intracellular STAT3 levels, while DU145 prostate cancer cells have high concentrations of STAT3. This may explain why STAT3 is more effectively suppressed compared to other cells with lower STAT3 levels.

[0146] Example 11: STAT3 in viral infections

[0147] Ebola virus infection impairs the host immune response and triggers a cytokine storm. The Ebola virus VP24 protein binds to caryopherin α1 and blocks the nuclear accumulation of STAT1. The Ebola virus interferon antagonist VP24 directly binds to STAT1. In the absence of STAT1, an antiviral state cannot be established. STAT3 signaling is activated by DNA and RNA viruses, including EBV, MCMV, HCMV, HSV, VZV, and HCV. Activation or increased expression of STAT3 is required for massive viral replication by suppressing type I IFN-mediated antiviral responses or by controlling microtubule dynamics. STAT3 inhibitors significantly reduce viral replication.

[0148] SBT-100 (SEQ ID NO: 1) inhibits Ebola virus replication in HeLa cells (Figure 13A) and HFF cells (Figure 13B). Furthermore, SBT-100 (SEQ ID NO: 1) inhibits Zika virus (Dakar, Senegal) replication in Vero cells (Figure 14A) and HFF cells (Figure 14B). SBT-100 (SEQ ID NO: 1) inhibits Venezuelan encephalitis (TC83) virus replication in different cell lines (Figure 15). SBT-100 (SEQ ID NO: 1) inhibits Chikungunya virus replication (Figure 16).

[0149] [Table 4]

[0150] SBT-100 (SEQ ID NO: 1) may inhibit the replication of other viruses, including hemorrhagic fever viruses such as dengue, Marburg, arenavirus (Lassa and Junin viruses), and bunyavirus; togaviruses (alphaviruses) such as mosquito-borne encephalitis virus, West Nile virus (WNV), Venezuelan encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), and Western equine encephalitis virus (WEE); chikungunya virus, and coronaviruses, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).

[0151] As shown above, SBT-100 (SEQ ID NO: 1) inhibits IL-6. IL-6 is a key mediator of inflammation and uses the STAT3 pathway to mediate inflammation. High levels of IL-6 are present in the blood of patients with severe COVID-19 infection. IL-6 plays a crucial role in the tumor microenvironment of many cancers and promotes STAT3-mediated inflammation in the eye, leading to macular degeneration and uveitis. By binding to and inhibiting STAT3, SBT-100 (SEQ ID NO: 1) may reduce SARS-CoV-2 replication in patients. By blocking the action of IL-6, SBT-100 (SEQ ID NO: 1) may help reduce lung inflammation, which may subsequently improve lung compliance and oxygenation in patients.

[0152] While the present invention is described in considerable detail with reference to certain preferred embodiments, other embodiments are also possible. The steps disclosed for this method are merely illustrative steps and are not intended to limit, for example, any step to indicate that each step is essential to the method. Accordingly, the appended claims should not be limited to the description of preferred embodiments contained herein. All references cited herein are incorporated by reference in their entirety.

[0153] To the extent that the above description discloses additional subject matter not included in the following claims, the present invention is not made available to the public, and the right to file one or more applications to claim such additional inventions is reserved.

Claims

1. A method for preventing abnormal cell proliferation in a target organism using a single-domain antibody (sdAb) produced against intracellular components.

2. The method according to claim 2, wherein the abnormal cell proliferation is cancer.

3. The method according to claim 2, wherein the cancer is selected from the group including osteosarcoma, fibrosarcoma, glioblastoma, leukemia, pancreatic cancer, breast cancer, and prostate cancer.

4. The method according to claim 2, wherein the sdAb is synergistic with one or more chemotherapy agents and improves the therapeutic effect of the one or more chemotherapy agents on cancer.

5. The method according to claim 4, wherein the one or more chemotherapeutic agents include doxorubicin.

6. The method according to claim 4, wherein the one or more chemotherapeutic agents include gemcitabine.

7. The method according to claim 4, wherein the sbAb reduces the toxicity of one or more chemotherapeutic agents and improves the survival of the treated subject.

8. The method according to claim 1, wherein the sbAb is used in combination with one or more compounds.

9. The method according to claim 1, wherein the intracellular component includes a protein.

10. The method according to claim 8, wherein the protein comprises STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, or STAT6.

11. The method according to claim 1, wherein sdAb includes sequence number 1.

12. A method for inhibiting the phosphorylation of STAT3, comprising administering sdAb to a patient who requires inhibition of STAT3 phosphorylation.

13. The method according to claim 12, wherein sdAb includes sequence number 1.

14. A method for inhibiting the activation of STAT3, comprising administering sdAb to a patient for whom inhibition of STAT3 activation is required.

15. The method according to claim 14, wherein sdAb includes sequence number 1.

16. A method for inhibiting T cell proliferation, comprising administering sdAb to a patient who requires inhibition of T cell proliferation.

17. The method according to claim 16, wherein sdAb includes sequence number 1.

18. A method for maintaining visual acuity, comprising administering sdAb to a patient who requires the maintenance of visual acuity.

19. The method according to claim 18, wherein sdAb includes sequence number 1.

20. A method for inhibiting the proliferation of CD4+ IL-17+ T cells, comprising administering sdAb to a patient who requires inhibition of CD4+ IL-17+ T cell proliferation.

21. The method according to claim 20, wherein sdAb includes sequence number 1.

22. A method for inhibiting a disease caused by CD4+ IL-17+ T cells, comprising administering sdAb to a patient who requires inhibition of a disease caused by CD4+ IL-17+ T cells.

23. The method according to claim 22, wherein sdAb includes sequence number 1.

24. A method for inhibiting the proliferation of CD4+IFN-γ+ T cells, comprising administering sdAb to a patient who requires inhibition of CD4+IFN-γ+ T cell proliferation.

25. The method according to claim 24, wherein sdAb includes sequence number 1.

26. A method for inhibiting a disease caused by CD4+IFN-γ+ T cell proliferation, comprising administering sdAb to a patient who requires inhibition of a disease caused by CD4+IFN-γ+ T cell proliferation.

27. The method according to claim 26, wherein sdAb includes sequence number 1.

28. A method for inhibiting the proliferation of CD4+IL-17+IFN-γ+ T cells, comprising administering sdAb to a patient who requires inhibition of the proliferation of CD4+IL-17+IFN-γ+ T cells.

29. The method according to claim 28, wherein sdAb includes sequence number 1.

30. A method for inhibiting a disease caused by the proliferation of CD4+IL-17+IFN-γ+ T cells, comprising administering sdAb to a patient who requires inhibition of a disease caused by the proliferation of CD4+IL-17+IFN-γ+ T cells.

31. The method according to claim 30, wherein sdAb includes sequence number 1.

32. A method for inhibiting the proliferation of CD4+RORγT+ T cells, comprising administering sdAb to a patient who requires inhibition of CD4+RORγT+ T cell proliferation.

33. The method according to claim 32, wherein sdAb includes sequence number 1.

34. A method for inhibiting a disease caused by proliferation of CD4+ RORγT+ T cells, comprising administering sdAb to a patient who requires inhibition of a disease caused by proliferation of CD4+ RORγT+ T cells.

35. The method according to claim 34, wherein sdAb includes sequence number 1.

36. A method for inhibiting the proliferation of CD4+ granzyme-B+ T cells, comprising administering sdAb to a patient who requires inhibition of CD4+ granzyme-B+ T cell proliferation.

37. The method according to claim 36, wherein sdAb includes sequence number 1.

38. A method for inhibiting a disease caused by CD4+ granzyme-B+ T cells, comprising administering sdAb to a patient who requires inhibition of a disease caused by CD4+ granzyme-B+ T cells.

39. The method according to claim 38, wherein sdAb includes sequence number 1.

40. A method for inhibiting the proliferation of CD4+Foxp3+ T cells, comprising administering sdAb to a patient who requires inhibition of CD4+Foxp3+ T cell proliferation.

41. The method according to claim 40, wherein sdAb includes sequence number 1.

42. A method for inhibiting the proliferation of CD25+Foxp3+ T cells, comprising administering sdAb to a patient who requires inhibition of CD25+Foxp3+ T cell proliferation.

43. The method according to claim 42, wherein sdAb includes sequence number 1.

44. A method for inhibiting a disease caused by CD25+Foxp3+ T cells, comprising administering sdAb to a patient who requires inhibition of a disease caused by CD25+Foxp3+ T cells.

45. The method according to claim 44, wherein sdAb includes sequence number 1.

46. A method for inhibiting the proliferation of CD4+ IL-10+ T cells, comprising administering sdAb to a patient who requires inhibition of CD4+ IL-10+ T cell proliferation.

47. The method according to claim 46, wherein sdAb includes sequence number 1.

48. A method for inhibiting a disease caused by CD4+ IL-10+ T cells, comprising administering sdAb to a patient who requires inhibition of a disease caused by CD4+ IL-10+ T cells.

49. The method according to the claim, wherein sdAb includes sequence number 1.

50. A method for inhibiting a disease caused by one or more cytokines selected from the group comprising IL-17, IFN-γ, IL-23, GM-CSF, and IL-1α, comprising administering sdAb to a patient who requires inhibition of a disease caused by one or more cytokines selected from the group comprising IL-17, IFN-γ, IL-23, GM-CSF, and IL-1α.

51. The method according to claim 50, wherein sdAb includes sequence number 1.

52. A method for inhibiting the proliferation of Th1, Treg and Th17 pathogenic cells, comprising administering sdAb to a patient who requires inhibition of the proliferation of Th1, Treg and Th17 pathogenic cells.

53. The method according to claim 52, wherein sdAb includes sequence number 1.

54. A method for inhibiting a disease caused by Th1, Treg and Th17 pathogenic cells, comprising administering sdAb to a patient who requires inhibition of a disease caused by Th1, Treg and Th17 pathogenic cells.

55. The method according to claim 54, wherein sdAb includes sequence number 1.

56. The method according to claim 54, wherein the disease is selected from the group including rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, atopic dermatitis, and type 1 diabetes.

57. A method for inhibiting a disease caused by an autoimmune disease, comprising administering sdAb to a patient who requires inhibition of a disease caused by an autoimmune disease.

58. The method according to claim 57, wherein sdAb includes sequence number 1.

59. A method for inhibiting VEGF production by retinal epithelial cells in an in vitro model of age-related macular degeneration (AMD), comprising administering sdAb to a patient who requires inhibition of VEGF production by retinal epithelial cells in an in vitro model of age-related macular degeneration (AMD).

60. The method according to claim 59, wherein sdAb includes sequence number 1.

61. A method for inhibiting diseases caused by AMD and neovascular disease of the eye, comprising administering sdAb to a patient who requires inhibition of diseases caused by AMD and neovascular disease of the eye.

62. The method according to claim 61, wherein sdAb includes sequence number 1.

63. A method for inhibiting a disease caused by VEGF, comprising administering sdAb to a patient who requires inhibition of a disease caused by VEGF.

64. The method according to claim 63, wherein sdAb includes sequence number 1.

65. A method for downregulating PD-L1 expression, comprising administering sdAb to a patient requiring downregulation of PD-L1 expression.

66. The method according to claim 65, wherein sdAb includes sequence number 1.

67. A method for inhibiting the translocation of STAT3 into the nucleus of a cell, comprising administering sdAb to a patient who requires inhibition of the translocation of STAT3 into the nucleus of a cell.

68. The method according to claim 67, wherein sdAb includes sequence number 1.

69. A method for inhibiting the action of IL-6, comprising administering sdAb to a patient who requires inhibition of the action of IL-6.

70. The method according to claim 69, wherein sdAb includes sequence number 1.

71. A method for enhancing the efficacy of a chemotherapy drug, comprising administering sdAb to a patient who requires enhanced efficacy of the chemotherapy drug.

72. The method according to claim 71, wherein sdAb includes sequence number 1.

73. The method according to claim 71, wherein the chemotherapeutic agent comprises gemcitabine.

74. A method for permeating cell membranes, the blood-brain barrier, and the blood-retinal barrier, comprising administering sdAb to a patient who requires permeation of cell membranes, the blood-brain barrier, and the blood-retinal barrier.

75. The method according to claim 74, wherein sdAb includes sequence number 1.

76. A method for reducing the toxicity of a chemotherapy drug, comprising administering sdAb to a patient who requires a reduction in the toxicity of the chemotherapy drug.

77. The method according to the claim, wherein sdAb includes sequence number 1.

78. The method according to claim 76, wherein the chemotherapeutic agent is doxorubicin.

79. A method for inhibiting the function of STAT3, comprising administering sdAb to a patient for whom inhibition of STAT3 function is required.

80. The method according to claim 79, wherein sdAb includes sequence number 1.

81. A method for inhibiting the function of KRAS and mutant KRAS in cancer cells, comprising administering sdAb to a patient who requires inhibition of the function of KRAS and mutant KRAS in cancer cells.

82. The method according to claim 81, wherein sdAb includes sequence number 1.

83. A method for inhibiting VEGF production, comprising administering sdAb to a patient who requires inhibition of VEGF production.

84. The method according to claim 83, wherein sdAb includes sequence number 1.

85. A method for inhibiting PD-L1 expression, comprising administering sdAb to a patient who requires inhibition of PD-L1 expression.

86. The method according to claim 85, wherein sdAb includes sequence number 1.

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