Single domain antibodies that specifically bind to globulin series glycans

Abstract

The present invention relates to the field of single domain antibodies (sdAbs) against Globo series glycans, in particular Globo H. In more detail, the present invention relates to sdAbs that specifically bind to one or more glycans selected from Globo H, Gb3, Gb4 and Gb5. The present invention also provides polypeptides comprising multimeric single domain antibodies, and T cell chimeric antigen receptors comprising the anti-glycan sdAbs. Therefore, the present invention provides polypeptides that can be used to target and / or treat several types of cancers associated with cells that overexpress the Globo H and / or Gb3, Gb4, Gb5. The present invention also relates to recombinant nucleic acid sequences encoding the polypeptides, and expression vectors and host cells comprising the recombinant nucleic acid sequences.

Description

Technical Field

[0001] This invention relates to the field of single-domain antibodies (sdAbs) targeting globular series glycans (particularly Globo H). More specifically, this invention relates to sdAbs that specifically bind to one or more glycans selected from Globo H, Gb3, Gb4, and Gb5. The invention also provides polypeptides comprising multimeric single-domain antibodies, and T-cell chimeric antigen receptors comprising said anti-glycan sdAbs. Therefore, the invention provides polypeptides that can be used to target and / or treat several types of cancer associated with cells overexpressing said Globo H and / or Gb3, Gb4, Gb5. The invention also relates to recombinant nucleic acid sequences encoding said polypeptides, and expression vectors and host cells comprising said recombinant nucleic acid sequences. Background Technology

[0002] Globo H contains a group of neutral glycosphingolipids in which ceramides are linked to glycans with a GalNAcβ3Galα4Galβ4Glc root structure. Normally, these glycans are retained on the plasma membrane and aggregate into lipid rafts. The endogenous function of this glycan family is largely unknown. However, their expression does occur in early stages of development and is thought to mediate cell contact and adhesion. Importantly, changes in these glycans have been observed during differentiation and tumorigenesis. Two notable hexasaccharide members of this family are stage-specific embryonic antigen-4 (SSEA-4) and Globo H (…). Figure 2 These glycans share a common precursor, SSEA-3 (Galβ3GalNAcβ3Galα4Galβ4Glc), but differ in their terminal monosaccharides: β3-linked N-acetylneuraminic acid in SSEA-4 and α2-linked L-fucose in Globo H.

[0003] SSEA-4 is expressed in many stem cell types, including induced pluripotent stem cells, embryonic cancer cells, breast cancer cells, and malignant glioma cells, which form the most aggressive and common brain tumors in adults. Therefore, antibodies against SSEA-4 could support targeting SSEA-4 in cancer vaccines.

[0004] Globo H is a hexasaccharide with the chemical formula Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1→O-Cer. Globo H expression has been found in several epithelial cancers, such as endometrial colon cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, prostate cancer, and breast cancer. In normal tissues, it is moderately present in the breast, colon, esophagus, small intestine, prostate, rectum, testis, and cervix, but only on the apical epithelial cells at the luminal boundary. Wang et al. (PNAS, 2008) found, using glycan arrays, that both normal donors and breast cancer patients expressed high levels of antibodies against SSEA-3, but the level of antibodies against Globo H expressed by breast cancer patients was significantly higher than that of normal donors. Because the sites where SSEA-3 or Globo H can be found in normal tissues are generally considered to be inaccessible to immune cells, both SSEA-3 and Globo H are attractive targets for cancer vaccines.

[0005] However, most carbohydrate antigens are generally tolerated by the immune system, thus the immunogenicity induced by them is limited. Furthermore, the production of antibodies against specific immunogens typically involves a synergistic interaction between two types of lymphocytes: B cells and helper T cells. Globo H alone cannot activate helper T cells, also attributed to its poor immunogenicity. Therefore, immunization with Globo H alone usually results in low immunoglobulin M (IgM) titers, failure to classify and convert to immunoglobulin G (IgG), and ineffective antibody affinity maturation. Recently, it has been demonstrated that antibodies against Globo H, including class conversion from IgM to IgG, can be induced by adding appropriate adjuvants. Therefore, Globo H is a promising therapeutic target for cancer vaccination. This approach has been tested in clinical trials targeting different stages of various cancers, including breast, ovarian, prostate, and lung cancer.

[0006] Previous studies have shown that human anti-polysaccharide responses are predominantly dominated by IgM and IgG1 types, while heavy chain antibodies from camels belong to IgG2 and IgG3 classes (Daley et al., Clin Vaccine Immunol. 2010), supporting the insufficient production of single-domain antibodies responding to glycans. Furthermore, it is known that the interaction between polysaccharides and individual binding sites in proteins is generally weak, and that binding strength and specificity are enhanced through polymerization interactions between polysaccharides and oligosaccharide-binding proteins. VHHs, essentially acting as strict monomeric binders, are not well-suited for binding polysaccharides in this respect. Therefore, those skilled in the art hypothesize that enhancing the binding of light-chain-deficient immunoglobulins against globular series glycans would be extremely difficult.

[0007] While conventional antibodies against Globo H have shown promise in cancer diagnosis and treatment (WO 2015 / 143123 A), there remains a need for better anti-Globo H antibodies. For the intended use, monoclonal antibodies are required, preferably single-domain antibodies. To date, no single-domain antibodies (sdAbs or VHHs) specifically binding to globular glycosphingolipids (e.g., Globo H or fragments thereof) have been disclosed in the art, although attempts have been made to prepare such antibodies.

[0008] Patent application WO 2015 / 143123 A discloses conventional anti-Globo H antibodies for the prevention or treatment of Globo H-positive cancers and pharmaceutical compositions comprising these antibodies. However, these antibodies possess CDR sequences that can confer chemical instability, making them unsuitable for further production scale-up and clinical studies.

[0009] International patent application WO 2018 / 054353 A discloses a therapeutic human conventional monoclonal antibody that binds to Globo H and has a CDR sequence, which is engineered to improve stability to resist undesirable modifications and the formation of large aggregates that may occur under high-expression manufacturing conditions.

[0010] Unlike conventional mammalian IgG antibodies, sdAbs contain only heavy chains and lack light chains. The antigen-binding domain of a single-domain antibody is called VHH or VHHs offer several general advantages over conventional antibodies. First, they are only 13-15 kDa in size, approximately 10 times smaller than conventional IgG antibodies (150 kDa). Even in crowded cellular environments, they acquire better epitopes and penetrate tissues, organs, and animals more effectively. Compared to conventional antibodies, they exhibit significantly higher chemical stability (e.g., up to 8 M urea) and thermal stability (up to 83°C), as well as a longer shelf life.

[0011] Therefore, the object of the present invention is to provide single-domain antibodies that specifically bind to Globo H and globulin series glycans, such as Gb3 (globulin trisaccharide), Gb4 (globulin tetrasaccharide), and Gb5 (globulin pentasaccharide Gb5).

[0012] This objective is achieved through the teachings of the independent claims. Further advantageous features, aspects, and details of the invention will be apparent from the dependent claims, description, drawings, and embodiments of this application.

[0013] Invention Summary

[0014] This application relates to polypeptides that specifically bind globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in the light chain and naturally lacking in constant region 1, or a variant of the variable domain, and wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5.

[0015] In some embodiments, the polypeptide that specifically binds to globo-series glycans comprises at least one single-domain antibody having at least 90% sequence identity with a sequence selected from SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 136, SEQ ID NO 137, and SEQ ID NO 138, wherein the single-domain antibody binds at least one globo-series glycan selected from Globo H, Gb3, Gb4, and Gb5. In some embodiments, the polypeptide that specifically binds to globo-series glycans comprises at least one single-domain antibody that binds at least one Globo-series glycan selected from Globo H, Gb3, Gb4, and Gb5, and has at least 90% sequence identity with SEQ ID NO 5.

[0016] In a particular embodiment, the polypeptide that specifically binds to globular series glycans comprises at least one single-domain antibody disclosed herein, and at least one effector molecule linked to said at least one single-domain antibody, wherein the effector molecule is selected from anticancer peptides, cleavage peptides, L-rhamnose, galactose-α-1,3-galactose, dinitrophenyl, serum-stabilizing molecules, fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, radioisotopes, chromophores, disuccinimide adipic acid, and human Fc antibody fragments. In a more specific embodiment, the polypeptide that specifically binds to globular series glycans comprises at least one single-domain antibody that binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and at least one cleavage peptide selected from the group comprising modified cysteine-deficient horseshoe crab antimicrobial peptide-I (Tachyplesin-I), BMAP28A, and Polybia-MP1. In a more specific embodiment, the polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody and further comprising at least one cleavage peptide linked to at least one single-domain antibody, has at least 85% sequence identity with a sequence selected from SEQ ID NOs:19-63, 142-156, 179-226.

[0017] This invention also relates to polypeptides that specifically bind globular series glycans, comprising at least one single-domain antibody disclosed herein, wherein said single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region. The extracellular hinge region, transmembrane domain, co-stimulatory domain, and intracellular activation domain represent modules or elements of a chimeric antigen receptor (CAR) for T cells. The chimeric antigen receptor disclosed herein can be used to generate CAR T cells that specifically recognize globular series glycans selected from Globo H, Gb3, Gb4, and Gb5. Therefore, said CAR T cells can be used for cancer therapy, wherein cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0018] The present invention also provides a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the at least one single-domain antibody is a humanized single-domain antibody, and wherein the at least one single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5. Furthermore, the present invention provides a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the at least one single-domain antibody is a humanized single-domain antibody, and further comprising at least one effector molecule linked to the at least one humanized single-domain antibody, wherein the at least one humanized single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5. According to the present invention, at least one single-domain antibody is humanized according to Kabat numbering by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4.

[0019] This invention also provides recombinant nucleic acid molecules encoding the polypeptides of this invention, and vectors comprising said recombinant nucleic acid molecules. Host cells comprising the recombinant nucleic acid molecules or vectors of this invention are also provided herein.

[0020] Furthermore, the present invention also relates to a therapeutically effective amount of the present invention's polypeptide pharmaceutical composition, the polypeptide specifically binding to globular polysaccharides, and comprising at least one single-domain antibody, and at least one pharmaceutically acceptable mediator, excipient, and / or diluent.

[0021] The present invention also provides the use of the polypeptides of the present invention or the pharmaceutical compositions of the present invention in the treatment and / or diagnosis of cancer, wherein the cancer cells of said cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5 on their surface. In other words, the present invention provides the use of the polypeptides of the present invention or the pharmaceutical compositions of the present invention in the treatment and / or diagnosis of cancer, wherein said cancer is characterized by cells expressing at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5 on their surface.

[0022] The present invention also provides the use of the polypeptides of the present invention or the pharmaceutical compositions of the present invention in the treatment and / or diagnosis of cancer, wherein the cancer cells of said cancer express at least one globular polysaccharide selected from Globo H, Gb3, Gb4 and Gb5 on their surface, and wherein said cancer is selected from brain cancer, liver cancer, bile duct cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer and colorectal cancer.

[0023] The present invention also provides a diagnostic kit comprising a polypeptide containing at least one single-domain antibody that specifically binds to globoside, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant thereof, wherein the at least one single-domain antibody binds to at least one globoside selected from Globo H, Gb3, Gb4, and Gb5 for screening cancers characterized by cells expressing at least one globoside selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0024] Another embodiment of the invention provides a polypeptide as described above, wherein the number of single-domain antibodies binding to at least two globular series glycans selected from Globo H, Gb3, Gb4, and Gb5 is at least two. The two or more single-domain antibodies may be sequence-different or sequence-identical.

[0025] One embodiment of the present invention provides a polypeptide as described above, wherein the number of single-domain antibodies binding at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5 is three. Another embodiment of the present invention provides a polypeptide as described above, wherein the number of single-domain antibodies binding at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5 is three, and wherein said single-domain antibodies have at least 90% sequence identity with SEQ ID NO 5. Invention Details

[0027] definition

[0028] Tumor-associated antigens (TACAs) and types

[0029] Cancer cells can be distinguished from normal cells because they exhibit abnormal levels and types of carbohydrate structures on their surface. These carbohydrate structures are called tumor-associated carbohydrate antigens (TACAs). TACAs are considered promising targets for designing anti-cancer vaccines. Unfortunately, carbohydrates themselves only elicit poor immunogenicity because they cannot induce T-cell-dependent immune responses, which are crucial for cancer treatment.

[0030] Glycosphingolipids (GSLs) represent a group of complex lipids composed of glycan structures linked to lipid tails containing sphingolipid ceramides. The basic structure of GSLs is a monosaccharide, typically glucose or galactose, directly linked to a ceramide molecule, producing glucocerebroside (GlcCer) or galactocerebroside (GalCer), respectively. GSLs are ubiquitous in cell membranes and are known to participate in cellular processes such as signal transduction, adhesion, and cell differentiation, among other functions. Certain GSLs highly expressed in tumor cells or tissues have been defined by specific monoclonal antibodies and thus identified as tumor-associated carbohydrate antigens (TACAs), derived from aberrant GSL synthesis in tumors. Tumor cells express aberrant glycosylation in GSLs, manifesting as incomplete synthesis leading to precursor accumulation or further addition of glycan residues to form new structures.

[0031] Although globulin trisaccharide ceramide (Gb3Cer) and globulin glycoside (Gb4Cer) form the basis of the P blood group system, galactosyl globulin glycoside (Gb5Cer) and sialic acid galactosyl globulin glycoside (sialic acid Gb5Cer) are also known as stage-specific embryonic antigen-3 (SSEA-3) and SSEA-4, respectively, and are widely used as cell surface markers for defining human embryonic stem cells.

[0032] Globo series GSLs have also been observed to be overexpressed in tumors, particularly in many epithelial cancers such as endometrial cancer, colon cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, prostate cancer, and breast cancer.

[0033] As used in this article, "Globo H" refers to a hexasaccharide with the formula Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1→O-cer, which has the following structure:

[0034]

[0035] "Globo H positive cancer" refers to cancer containing cancer cells that express Globo H on their surface.

[0036] The study also reported the presence of truncated forms or fragments of Globo H on the surface of cancer cells. These truncated forms are Gb3 (globulotrisaccharide), Gb4 (globulotetrasaccharide), and Gb5 (globulopentasaccharide). Figure 2 ).

[0037] "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins with the same structural features. While antibodies exhibit binding specificity to a particular antigen, immunoglobulins include antibodies and other antibody-like molecules that typically lack antigen specificity. The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, human antibodies, multispecific (or heteroconjugated) antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, antigen-binding antibody fragments (e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, single-chain Fv fragments, sdAbs, VHH), antibody fusions, and synthetic antibodies (or antibody mimics). Antibodies can be chimeric, human, humanized, and / or affinity-matured.

[0038] As used in this article, the term "antigen" is defined as any substance that can elicit an immune response.

[0039] As used in this article, the term "immunogenicity" refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0040] As used herein, the term “epitope” is defined as the portion of an antigen molecule that comes into contact with the antigen-binding site of an antibody or T-cell receptor.

[0041] As used herein, the terms "specific binding," "specifically binding," or "binds specifically" refer to the interaction between binding pairs (e.g., antibody and antigen). In various cases, specific binding can occur through approximately 10... -6 moles per liter, approximately 10 -7 moles per liter or about 10 -8 The affinity constant is expressed in moles per liter or less.

[0042] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Low-affinity antibodies typically bind antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind antigens more quickly and tend to remain bound for longer periods. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this invention. Specific illustrative examples are described below.

[0043] An antibody's "functional antigen-binding site" is the site at which it can bind to a target antigen. The antigen-binding affinity of an antigen-binding site is not necessarily as strong as that of the parent antibody derived from the antigen-binding site, but the ability to bind to the antigen must be measured using any of the many methods known for assessing antibody-antigen binding.

[0044] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid it is linked to. One type of vector is the "plasmid," which is a circular double-stranded DNA loop capable of linking to an additional DNA segment. Another type of vector is the bacteriophage vector. Yet another type of vector is the viral vector, in which an additional DNA segment can be linked to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced. Other vectors (e.g., non-attachment mammalian vectors) can integrate into the host cell's genome after introduction and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are effectively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors" or "recombinant vectors"). Generally, expression vectors used in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector.

[0045] In this document, the terms "recombinant polynucleotide" or "recombinant nucleic acid molecule" used interchangeably refer to nucleotide polymers of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases or through a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence can be broken down by non-nucleotide components. Polynucleotides can be further modified post-synthetic, for example, by conjugation with tags. Other types of modifications include, for example, "capping," replacing one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, triphosphates, phosphatases, carbamates, etc.) and charged bonds (e.g., thiophosphates, dithiophosphates, etc.); modifications containing side chain moieties, such as those of proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.); those with intercalating agents (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); those containing alkylating agents; those with modifying bonds (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. Furthermore, any hydroxyl groups normally present in sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages with additional nucleotides, or can be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH groups can be phosphorylated or partially substituted with amines or organic end-capping groups of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived as standard protecting groups.

[0046] As used in this article, "oligonucleotide" generally refers to a short, typically single-stranded, typically synthetic polynucleotide whose length is typically, but not necessarily, less than about 200 nucleotides. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides applies equally and fully to oligonucleotides.

[0047] "Globular glycan-binding single-domain antibody (sdAb)" or "sdAb binding to globo H, Gb3, Gb4, and Gb5" refers to an sdAb that binds to globo H, Gb3, Gb4, and Gb5 globo Glycans with sufficient affinity, such that the sdAb can be used as a diagnostic and / or therapeutic agent by binding to the target glycan. "Globo H-binding single-domain antibody (sdAb)" or "Globo H-binding sdAb" refers to an sdAb that binds to Globo H with sufficient affinity, thereby making the sdAb usable as a diagnostic and / or therapeutic agent by binding to Globo H. "Gb3-binding single-domain antibody (sdAb)" or "Gb3-binding sdAb" refers to an sdAb that binds to Gb3 with sufficient affinity, such that the sdAb can be used as a diagnostic and / or therapeutic agent by binding to Gb3. "Gb4-binding single-domain antibody (sdAb)" or "Gb4-binding sdAb" refers to an sdAb that binds to Gb4 with sufficient affinity, such that the sdAb can be used as a diagnostic and / or therapeutic agent by binding to Gb4. "Gb5-binding single-domain antibody (sdAb)" or "Gb5-binding sdAb" refers to an sdAb that binds to Gb5 with sufficient affinity, such that the sdAb can be used as a diagnostic and / or therapeutic agent by binding to Gb5.

[0048] The terms "full-length antibody," "complete antibody," or "all antibody" are used interchangeably in this article and refer to antibodies with a structure that is substantially similar to that of natural antibodies.

[0049] An "antibody fragment" refers to a portion of a full-length antibody that is capable of binding to the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., single-chain Fv); single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0050] The "variable region" or "variable domain" of an antibody refers to the N-terminal domain of either the heavy or light chain. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Specifically, the "variable region" or "variable domain" of the single-domain antibody disclosed herein refers to the N-terminal domain of the heavy chain.

[0051] The term "variable" refers to the fact that certain portions of the variable domain differ widely in sequence between antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, the variability is concentrated in three segments in the variable domains of the light and heavy chains, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, predominantly in a β-sheet configuration, linked by three CDRs that form loops connecting the β-sheet structure and, in some cases, part of the β-sheet structure. The CDRs in each chain are held together closely by the FR regions and, together with CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as participating as antibodies in antibody-dependent cytotoxicity.

[0052] As used herein, a “hypervariant region” or “HVR” refers to each region in the variable domain of an antibody that exhibits sequence hypervariability and / or forms a structurally defined loop. Typically, natural antibodies contain four chains with six HVRs: three in the heavy chain variable domain, VH (H1, H2, H3), and three in the light chain variable domain, VL (L1, L2, L3). Single-domain antibodies contain only three HVRs in the heavy chain variable domain. HVRs typically contain amino acid residues from the hypervariant loop and / or from complementarity-determining regions (CDRs). Unless otherwise stated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., 1991.

[0053] As used herein, a “complementarity-determining region” or “CDR” refers to the region within the hypervariable domain of a variable domain that has the highest complementarity-determining region. Sequence variability and / or participation in antigen recognition are crucial. Typically, natural antibodies comprise four chains with six CDRs: three in the heavy chain variable domain, VH (H1, H2, H3), and three in the light chain variable domain, VL (L1, L2, L3). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35 of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3 (Kabat et al., 1991). The single-domain antibodies disclosed herein contain only CDR-H1 or CDR1, CDR-H2 or CDR2 and CDR-H3 or CDR3 because they lack light chains.

[0054] "Natural antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light (CL) domain. Based on the amino acid sequence of their constant domains, antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ).

[0055] As used herein, "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting that population are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., variant antibodies containing mutations that are naturally present or that occur during the production of the monoclonal antibody, typically present in small amounts). Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the term "monoclonal" indicates the characteristic of an antibody derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method.

[0056] A "chimeric antibody" is an antibody in which a portion of the heavy chain and / or light chain comes from a specific source or species, while the remainder of the heavy chain and / or light chain comes from a different source or species.

[0057] "Humanized antibody" refers to a chimeric antibody comprising an amino acid sequence from non-human HVRs and an amino acid sequence from human FRs. In some embodiments, the humanized antibody will substantially comprise a single-domain antibody, wherein all or substantially all CDRs correspond to the CDRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region from the human antibody. The "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

[0058] "Human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an amino acid sequence derived from an antibody of non-human origin that utilizes the encoding sequences of the complete set of human antibodies or other human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0059] The "human common framework" is a framework that represents the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Typically, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, this sequence subgroup is as described in Kabat et al., 1991.

[0060] As used herein, a "multivalent antibody" is an antibody containing three or more antigen-binding sites. Multivalent antibodies are preferably engineered to have three or more antigen-binding sites and are typically not naturally occurring IgM or IgA antibodies.

[0061] A "multispecific antibody" is an antibody with at least two different binding sites, each with different binding specificities. Multispecific antibodies can be full-length antibodies or antibody fragments, and the different binding sites can each bind to different antigens, or the different binding sites can bind to two different epitopes of the same antigen.

[0062] The "Fc region" or "Fc antibody fragment" refers to a dimeric complex containing the C-terminal polypeptide sequence of an immunoglobulin heavy chain, where the C-terminal polypeptide sequence is a sequence obtainable by digesting an intact antibody with papain. The Fc region may contain native or variant Fc sequences.

[0063] "Fab fragment" refers to an antibody fragment containing the variable and constant regions of the light chain and the variable and first constant region (CH1) of the heavy chain. Papain digestion of the antibody produces two identical "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its ease of crystallization. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking the antigen. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. The F(ab')2 antibody fragment is initially produced as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known in the art.

[0064] A “Fv fragment” refers to an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly bound heavy-chain variable domain and a light-chain variable domain, which can be covalently related, as in a single-chain Fv. It is in this configuration that the three HVRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. The six HVRs or a subset thereof collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigens, although its affinity is generally lower than that of the entire binding site.

[0065] "Single-chain Fv" or "single-chain Fv" refers to an antibody fragment containing the VH and VL domains of the antibody, where these domains are contained within a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the VH and VL domains, which allows the single-chain Fv to form the desired antigen-binding structure.

[0066] "Dual antibody" refers to a small antibody fragment with two antigen-binding sites, containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, creating two antigen-binding sites.

[0067] "Naked antibody" refers to an antibody that has not been bound to a heterologous part (such as a cytotoxic part) or a radioactive label.

[0068] "Isolated" antibodies are antibodies that have been identified, isolated, and / or recovered from components of their natural environment. Contaminating components of their natural environment are materials that can interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. Typically, isolated antibodies are prepared through at least one purification step.

[0069] As used herein, the terms "substantially similar," "substantially identical," "equivalent," or "substantially equivalent" refer to a sufficiently high degree of similarity between two values ​​(e.g., one related to a test antibody and the other to a reference antibody) such that a person skilled in the art would consider the difference between the two values ​​to be of little or no biological and / or statistical significance in the context of the biological characteristics measured by said values ​​(e.g., KD values). The difference between said two values ​​is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10%, as a function of the reference / comparison molecule value.

[0070] As used herein, "substantially different" means a sufficiently high degree of difference between two values ​​(typically one related to a molecule and the other to a reference molecule) such that a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by said value (e.g., KD value). The difference between said two values, as a function of the value of the reference / comparison molecule, is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50%.

[0071] This invention relates to polypeptides that specifically bind globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, and wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5.

[0072] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globular series glycans, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and wherein the at least one single-domain antibody has at least 90% sequence identity with a sequence selected from SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 136, SEQ ID NO 137, and SEQ ID NO 138 (Table 4). A preferred embodiment is a polypeptide comprising at least one single-domain antibody that specifically binds to globular series glycans, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 5 (Table 4).

[0073] A preferred embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 1.

[0074] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globo series glycans, wherein the single-domain antibody binds at least one globo series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 3.

[0075] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globular series glycans, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 7.

[0076] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globo series glycans, wherein the single-domain antibody binds at least one globo series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 9.

[0077] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globo series glycans, wherein the single-domain antibody binds at least one globo series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 11.

[0078] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globo series glycans, wherein the single-domain antibody binds at least one globo series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 13.

[0079] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globo series glycans, wherein the single-domain antibody binds at least one globo series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 15.

[0080] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globo series glycans, wherein the single-domain antibody binds at least one globo series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 17.

[0081] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globosaccharides, wherein the single-domain antibody binds at least one globosaccharide selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 136.

[0082] Another embodiment of the invention is a polypeptide comprising at least one single domain that specifically binds to globular series glycans. The single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO:137.

[0083] Another embodiment of the present invention is a polypeptide comprising at least one single-domain antibody that specifically binds to Globo series glycans, wherein the single-domain antibody binds at least one Globo series glycan selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody has at least 90% sequence identity with SEQ ID NO 138.

[0084] The sequences SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 136, SEQ ID NO 137 and SEQ ID NO 138 (sdAb or VHH) are heavy chain antibodies derived from alpaca (Vicugna pacos) and have been used for immunization against Globo H glycan.

[0085] The present invention also relates to a recombinant nucleic acid molecule capable of encoding the polypeptide, and a vector and a host cell comprising the recombinant nucleic acid molecule.

[0086] A single-domain antibody is an antibody whose complementation-determining region is part of a single-domain polypeptide. Examples include the single-domain antibody disclosed in WO1994004678A1. For clarity, the variable domains from naturally occurring heavy-chain antibodies lacking the light chain are referred to herein as VHHs or sdAbs (single-domain antibodies) to distinguish them from the conventional VHs of four-chain immunoglobulins. Such VHH molecules can originate from antibodies produced in camel species, such as camels, dromedary camels, llamas, alpacas, and guanacos. Other species besides camels may produce naturally occurring heavy-chain antibodies lacking the light chain.

[0087] According to one aspect of the invention, the single-domain antibody, as used herein, is a naturally occurring single-domain antibody called a heavy chain antibody, which is naturally lacking a light chain and naturally lacking constant region 1, or a variant of the variable domain.

[0088] sdAbs are approximately 13-15 kDa, thus almost ten times smaller than IgG molecules. They are single polypeptides and are highly stable during storage and use, meaning the integrity of the antigen-binding protein is maintained under storage and / or use conditions that may include elevated temperatures, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, the presence of harmful chemicals, etc. Furthermore, they are resistant to protease activity, unlike conventional antibodies. Further, the single-domain antibodies according to the invention are readily prepared in vitro with high yields, proper folding, and functionality. Additionally, antibodies produced in alpacas or more generally camelids will recognize epitopes other than those recognized by antibodies produced in vitro using an antibody library or by antibodies produced by immunizing mammals other than camelids (WO2005044858A1). Therefore, sdAbs targeting GloboH, Gb3, Gb4, or Gb5 can interact with the target antigen more effectively than conventional antibodies, allowing for the detection or treatment of cancers characterized by cells expressing GloboH, Gb3, Gb4, or Gb5 with significantly higher efficiency. Since sdAbs are known to bind to aberrant epitopes such as cavities or grooves (WO2005044858A1), the affinity of such antibodies may be applicable to therapeutic therapies.

[0089] Therefore, a single-domain antibody can generally be defined as an amino acid sequence having the following (general) structure:

[0090] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0091] FR1 to FR4 refer to frame regions 1 to 4, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3.

[0092] Therefore, the "single-domain antibody" according to the present invention comprises amino acid sequences containing FR1-FR4 and CDR1-CDR3 or variant sequences thereof, and their binding specificity to sphingolipids is conferred by the amino acid sequences of three complementarity-determining regions CDR1, CDR2, and CDR3.

[0093] The sequences of FR1-FR4 and CDR1-CDR3 of the single-domain antibody disclosed in this invention are SEQ ID NO: 64-126, 157-177. The annotations of the FR and CDR regions are based on Kabat's unique numbering system.

[0094] Therefore, one embodiment of the present invention also relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, and wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the single-domain antibody comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0095] (a) CDR1 contains an amino acid sequence selected from SEQ ID NO:64-72, 157-159 or variants thereof;

[0096] (b) CDR2 contains an amino acid sequence selected from SEQ ID NO:73-81, 160-162 or variants thereof;

[0097] (c)CDR3 contains an amino acid sequence selected from SEQ ID NO:82-90, 163-165 or variants thereof.

[0098] Another embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, and wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the single-domain antibody comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0099] (a) CDR1 contains an amino acid sequence that has at least 90% sequence identity with sequences selected from SEQ ID NO:64-72, 157-159;

[0100] (b) CDR2 contains an amino acid sequence that has at least 90% sequence identity with the sequences selected from SEQ ID NO:73-81, 160-162;

[0101] (c)CDR3 contains an amino acid sequence that has at least 90% sequence identity with the sequences selected from SEQ ID NO:82-90, 163-165.

[0102] GloboH is the primary target according to the present invention. Single-domain antibody targeting the target can achieve a concentration better than 10... -6 M binds to the single-domain antibody targeting the target due to its affinity.

[0103] The target site can also be a fragment of the primary target. Therefore, the target site is also a fragment of the primary target that can elicit an immune response. The target site is also a fragment of the primary target that can bind to single-domain antibodies generated against the full-length target. Preferred fragments of the primary target Globo H are Gb3, Gb4, and Gb5.

[0104] The binding of single-domain antibodies to glycosphingolipids selected from GloboH, Gb3, Gb4, and Gb5 preferably occurs with high affinity: typically, the dissociation constant between single-domain antibodies and globular target glycans is less than 10. -5 M, more preferably, has a dissociation constant less than 10. - 6 M, or even more preferably, a dissociation constant less than 10 -7 M, most preferably, has a dissociation constant less than 10. -8 M. Alternatively, the dissociation constant of the binding between the single-domain antibody and Globo H is less than 10. -5 M, more preferably, has a dissociation constant less than 10. -6 M, or even more preferably, a dissociation constant less than 10 -7 M, most preferably, has a dissociation constant less than 10. -8 M. Alternatively, the dissociation constant of the binding between the single-domain antibody and Gb3 is less than 10. -5 M, more preferably, has a dissociation constant less than 10. -6 M, or even more preferably, a dissociation constant less than 10 -7 M, most preferably, has a dissociation constant less than 10. -8 M. Alternatively, the dissociation constant of the binding between a single-domain antibody and Gb4 is less than 10. -5 M, more preferably, has a dissociation constant less than 10. -6 M, or even more preferably, a dissociation constant less than 10 -7 M, most preferably, has a dissociation constant less than 10. -8 M. Alternatively, the dissociation constant of the binding between a single-domain antibody and Gb5 is less than 10. -5 M, more preferably, has a dissociation constant less than 10. -6 M, or even more preferably, a dissociation constant less than 10 -7 M, most preferably, has a dissociation constant less than 10. -8 M.

[0105] "Variant sequence"

[0106] According to one aspect of the invention, the polypeptide binding to a glycan selected from GloboH, Gb3, Gb4, and Gb5 can be a variant sequence of a full-length polypeptide bound to a glycan selected from GloboH, Gb3, Gb4, and Gb5. According to another aspect of the invention, the polypeptide binding to a glycan selected from GloboH, Gb3, Gb4, and Gb5 can comprise the sequence of a full-length polypeptide bound to a glycan selected from GloboH, Gb3, Gb4, and Gb5.

[0107] According to one aspect of the invention, the single-domain antibody contained in a polypeptide that binds to a glycan selected from GloboH, Gb3, Gb4 and Gb5 can be a complete single-domain antibody (e.g., sdAb or VHH) or a variant sequence thereof.

[0108] As used herein, the variant sequences of the present invention may include the addition, deletion or substitution of one or more amino acids, which substantially do not alter the functional characteristics of the polypeptides of the present invention compared to the unmodified parent polypeptide (also known as the reference polypeptide).

[0109] The variant sequences according to the present invention can be sequences found in other camelid species, such as camels, dromedary camels, llamas, alpacas, guanacos, etc.

[0110] Compared with the parental sequence, the variant sequence preferably has as many as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids deleted or substituted.

[0111] When a variant sequence indicates sequence identity, it refers to a variant sequence that exhibits high sequence identity, for example, a sequence identity with the parent sequence exceeding 70%, 75%, 80%, 85%, 90%, 95%, or 98%, and preferably is characterized by having similar properties to the parent sequence, i.e., affinity, which is calculated as described below.

[0112] The percentage of “sequence identity” is determined by comparing two optimally aligned nucleic acid or polypeptide sequences over a “comparison window” of the full length of a reference sequence. As used herein, a “comparison window” refers to the optimal alignment between a reference sequence and a variant sequence after optimal alignment, where the variant nucleic acid or polypeptide sequence in the comparison window may contain 20% or less, typically 5% to 15%, or 10% to 12% of additions or deletions (i.e., vacancies), compared to the reference sequence used for optimal alignment (which contains no additions or deletions). The percentage of identity is calculated as follows: the number of positions in both sequences where the same nucleic acid base or amino acid residue appears is determined to obtain the number of matching positions; this number is divided by the total number of positions in the reference sequence (i.e., the full length of the amino acid or nucleotide); and the result is multiplied by 100 to obtain the percentage of sequence identity. Two nucleic acid or polypeptide sequences are said to be “identical” if the nucleotide or amino acid sequences in the two sequences are identical when optimally aligned as described above.

[0113] Alternatively, the variant sequence can also be any amino acid sequence resulting from allowed substitutions at any number of positions of the parental sequence according to the following formula:

[0114] Ser is replaced by Ser, Thr, Gly, and Asn;

[0115] Arg is replaced by one of Arg, His, Gin, Lys, and Glu;

[0116] Leu is replaced by one of Leu, Ile, Phe, Tyr, Met, and Val;

[0117] Pro is replaced by one of Pro, Gly, Ala, and Thr;

[0118] Thr is replaced by one of Thr, Pro, Ser, Ala, Gly, His, and Gin;

[0119] Ala was replaced by one of Ala, Gly, Thr, and Pro;

[0120] Val is replaced by one of Val, Met, Tyr, Phe, Ile, and Leu;

[0121] Gly is replaced by one of Gly, Ala, Thr, Pro, and Ser;

[0122] Ile was replaced by one of Ile, Met, Tyr, Phe, Val, and Leu;

[0123] Phe is replaced by one of Phe, Trp, Met, Tyr, lie, Val, and Leu;

[0124] Tyr is replaced by one of Tyr, Trp, Met, Phe, Ile, Val, and Leu;

[0125] His is replaced by one of His, Glu, Lys, Gin, Thr, and Arg;

[0126] Gln is replaced by one of Gin, Glu, Lys, Asn, His, Thr and Arg;

[0127] Asn is replaced by one of Asn, Glu, Asp, Gin and Ser;

[0128] Lys is replaced by one of Lys, Glu, Gln, His, and Arg;

[0129] Asp is replaced by one of Asp, Glu, and Asn;

[0130] Glu is replaced by one of Glu, Asp, Lys, Asn, Gln, His, and Arg;

[0131] Met was replaced by one of Met, Phe, Ile, Val, Leu, and Tyr.

[0132] As used herein, variants can also be sequences in which each frame region (FR) and each complementarity-determining region (CDR) shows at least 80% identity with the corresponding region in the reference sequence, preferably at least 85% identity, more preferably 90% identity, and even more preferably 95% identity. In this case, the sequence identity of the FR1 variant with the FR1 reference, the CDR1 variant with the CDR1 reference, the FR2 variant with the FR2 reference, the CDR2 variant with the CDR2 reference, the FR3 variant with the FR3 reference, the CDR3 variant with the CDR3 reference, and the FR4 variant with the FR4 reference is determined as described above.

[0133] In another embodiment, a recombinant nucleic acid sequence encoding any of the above-described antigen-binding proteins or variants thereof is also part of the present invention.

[0134] The present invention provides recombinant nucleic acid molecules encoding one or more polypeptides, said polypeptides comprising amino acid sequences selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 136, SEQ ID NO 137 and SEQ ID NO 138.

[0135] Therefore, the present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence, or a variant thereof, as shown in one or more of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140 and SEQ ID NO 141 (Table 4). The present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence, or a variant thereof, as shown in SEQ ID NO:2. The present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence, or a variant thereof, as shown in SEQ ID NO:4. The present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence, or a variant thereof, as shown in SEQ ID NO:6. The present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence, or a variant thereof, as shown in SEQ ID NO:8. The present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence, or a variant thereof, as shown in SEQ ID NO:10. The present invention provides a recombinant nucleic acid molecule comprising a nucleic acid sequence, or a variant thereof, as shown in SEQ ID NO:12. This invention provides recombinant nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID NO:14 or a variant thereof. This invention provides recombinant nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID NO:16 or a variant thereof. This invention provides recombinant nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID NO:18 or a variant thereof. This invention provides recombinant nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID NO:139 or a variant thereof. This invention provides recombinant nucleic acids in some embodiments, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO:140 or a variant thereof. This invention provides recombinant nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID NO:141 or a variant thereof.

[0136] Furthermore, the present invention provides a host cell comprising one or more recombinant nucleic acid molecules, or variants thereof, as shown in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140, and SEQ ID NO 141. Specifically, the present invention provides a cell comprising one or more recombinant nucleic acid molecules, or variants thereof, as shown in SEQ ID NO: 6.

[0137] On the other hand, the present invention provides recombinant nucleic acid molecules and variants thereof encoding polypeptides that specifically bind to at least one globular series glycan selected from GloboH, Gb3, Gb4, and Gb5, wherein such variant recombinant nucleic acid molecules share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the following sequences selected from SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140, and SEQ ID NO 141, wherein the percentage of sequence identity is determined as described above. These amounts are not intended to be limiting, and the increments between the percentages are specifically contemplated as part of the disclosure herein. Specifically, the present invention provides recombinant nucleic acid molecules and variants thereof encoding polypeptides that specifically bind to at least one globular series glycan selected from GloboH, Gb3, Gb4 and Gb5, wherein such variant recombinant nucleic acid molecules share at least 80% sequence identity with any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140 and SEQ ID NO 141. More specifically, the present invention provides recombinant nucleic acid molecules and variants thereof encoding polypeptides that specifically bind to at least one globular series glycan selected from GloboH, Gb3, Gb4 and Gb5, wherein such variant recombinant nucleic acid molecules share at least 85% sequence identity with any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140 and SEQ ID NO 141.More specifically, the present invention provides recombinant nucleic acid molecules and variants thereof encoding polypeptides that specifically bind to at least one globular series glycan selected from GloboH, Gb3, Gb4 and Gb5, wherein such variant recombinant nucleic acid molecules share at least 90% sequence identity with any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140 and SEQ ID NO 141. More specifically, the present invention provides recombinant nucleic acid molecules and variants thereof encoding polypeptides that specifically bind to at least one globular series glycan selected from GloboH, Gb3, Gb4 and Gb5, wherein such variant recombinant nucleic acid molecules share at least 95% sequence identity with any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140 and SEQ ID NO 141. More specifically, the present invention provides recombinant nucleic acid molecules and variants thereof encoding polypeptides that specifically bind to at least one globular series glycan selected from GloboH, Gb3, Gb4 and Gb5, wherein such variant recombinant nucleic acid molecules share at least 98% sequence identity with any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 139, SEQ ID NO 140 and SEQ ID NO 141.

[0138] This disclosure also covers polynucleotide or nucleic acid molecules complementary to any such sequence. Nucleic acid molecules can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules containing introns and corresponding one-to-one with DNA molecules, and mRNA molecules without introns. Additional coding or non-coding sequences may, but do not need to, be present within the polynucleotides disclosed herein, and the polynucleotides may, but do not need to, be linked to other molecules and / or supporting material.

[0139] Nucleic acid molecules may contain natural sequences (i.e., endogenous sequences encoding antibodies or portions thereof) or variants of such sequences.

[0140] The nucleic acid variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide specifically binding to at least one globular series glycan selected from GloboH, Gb3, Gb4, and Gb5 is substantially indistinguishable from that of a reference naturally immunoreactive polypeptide. The effect on the immunoreactivity of the encoded polypeptide can typically be assessed as described herein. In some embodiments, the nucleic acid variant exhibits at least about 70% identity with the nucleic acid sequence encoding a reference naturally occurring single-domain antibody or a portion thereof; in some embodiments, at least about 80% identity; in some embodiments, at least about 85% identity; in some embodiments, at least about 90% identity; and in some embodiments, at least about 95% identity, wherein the percentage of sequence identity is determined as described above. These amounts are not intended to be limiting, and increments between said percentages are specifically contemplated as part of the disclosure herein.

[0141] The recombinant nucleic acid molecules disclosed herein can be obtained using chemical synthesis, recombination methods, or polymerase chain reaction (PCR). Methods for the chemical synthesis of polynucleotides are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercial DNA synthesizers to generate the desired DNA sequences. To prepare polynucleotides using recombination methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides can be inserted into host cells using any method known in the art. Cells are transformed by introducing exogenous polynucleotides through direct uptake, endocytosis, transfection, F-crossing, or electroporation. Once introduced, the exogenous polynucleotide can be retained within the cell as a non-integrating vector (e.g., a plasmid) or integrated into the host cell genome. The polynucleotides thus amplified can be isolated from the host cell using methods well known in the art.

[0142] Suitable cloning and expression vectors can include a variety of components, such as promoters, enhancers, and other transcriptional regulatory sequences. Vectors can also be constructed to allow for the subsequent cloning of antibody variable domains into different vectors. Suitable cloning vectors can be constructed according to standard techniques, or suitable vectors can be selected from a large number of cloning vectors available in the art. While the chosen cloning vector may vary depending on the intended host cell, useful cloning vectors are generally self-replicating, may have a single target site for a specific restriction endonuclease, and / or may carry a gene that can be used to select clones containing that vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, and Bluescript (e.g., pBS SK).+ ) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as Merck, BioRad, Stratogene, and Invitrogen.

[0143] Furthermore, the present invention envisions expression vectors comprising nucleic acid sequences encoding any single-domain antibody or variant thereof disclosed herein, and host cells comprising such expression vectors. This means that the expression vector must be replicable in the host cell, whether as an episome or as an integrated portion of chromosomal DNA. Vector components may typically include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements are typically also required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0144] The vector containing the recombinant nucleic acid molecule of interest and / or the recombinant nucleic acid molecule itself can be introduced into host cells by any of a variety of appropriate methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan or other substances; microblasting; fat infection; and infection (e.g., when the vector is an infectious agent, such as vaccinia virus).

[0145] The choice of a specific program often depends on the characteristics of the host cell.

[0146] Suitable expression systems include constitutive and inducible expression systems in bacteria or yeast, viral expression systems such as baculoviruses, semliki forest viruses, and lentiviruses, or transient transfection in insect or mammalian cells. Particularly preferred are pET expression vectors (Novagen) for cloning and expressing recombinant proteins in *E. coli*. In the pET system, the target gene is cloned into the pET plasmid under the control of strong phage T7 transcription and selective translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. The T7 RNA polymerase is selective and active, such that upon complete induction, almost all cellular resources are converted to target gene expression; the desired product can account for more than 50% of total cellular protein several hours after induction. The pET-22b(+) vector carries an N-terminal pelB signal sequence for potential periplasmic localization, and optionally a C-terminal... sequence.

[0147] Suitable host cells include *Escherichia coli*, *Lactococcus lactis*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Pichia pastoris*. Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, and DT40. Particularly preferred are… Escherichia coli cells. Cloning, expression, and / or purification of antigen-binding proteins can be performed using techniques known to those skilled in the art, including baculovirus-infected insect cell systems.

[0148] Functionalization of single-domain antibodies.

[0149] In addition to their selectable multivalent and multispecific properties, a significant advantage of single-domain antibodies (sdAbs) is their ability to be readily cloned and expressed together with effector molecules on a single DNA plasmid. These extensions enable sdAbs to be used in a wide range of applications beyond antigen binding itself. Due to their single-domain nature, sdAbs are easily equipped with a variety of effector functions through molecular or biochemical engineering. Suitable effector molecules possess many different functions, such as activating antibody-dependent cytotoxicity, lysing parasite and cancer cell membranes, in vitro / in vivo imaging, and diagnostics. Particularly preferred effector molecules in this invention are anticancer peptides, cleavage peptides, L-rhamnose, galactose-α-1,3-galactose, dinitrophenyl, serum-stabilizing molecules, fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, radioisotopes, chromophores, disuccinimide adipic acid, and human Fc antibody fragments.

[0150] Therefore, one embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is selected from anticancer peptides, cleavage peptides, L-rhamnose, galactose-α-1,3-galactose, dinitrophenyl, serum-stabilizing molecules, fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, radioisotopes, chromophores, disuccinimide adipic acid, and human Fc antibody fragments.

[0151] Specifically, the effector molecule is preferably linked to the C-terminus of the single-domain antibody. Furthermore, the effector molecule is preferably linked to the C-terminus of the single-domain antibody via a linker. Preferred linkers are selected from GS_linker (SEQ ID NO: 127: GGGGSGGGGS), GSAA_linker (SEQ ID NO: 128: GGGGSEAAAKGGGGS), SRGS_linker (SEQ ID NO: 178: SRGSSGSSSSGSSGGSG), and GGGGS_linker (SEQ ID NO: 228: GGGGSGGGGSGGGGS). Preferably, a "short linker sequence" (SEQ ID NO: 227: AAXX, where X can be any amino acid) comprising two alanine amino acids and two amino acid "XX" corresponding to the enzyme restriction site is linked to the C-terminus of the single-domain antibody before the linker sequence, where X can be any amino acid.

[0152] "Anticancer peptides" are peptides found in plants, animals, and other organisms, initially discovered for their antimicrobial activity. These peptides can kill bacteria and cancer cells through membrane interactions. The difference between bacterial or cancer cells and healthy mammalian cells lies in the charge of their membranes. Both bacteria and cancer cells have membranes that are negatively charged overall. Due to the similarity between the membranes of bacteria and cancer cells, the mechanisms by which membrane rupture occurs and induces cell death are also thought to be similar. Exemplary antimicrobial peptides with anticancer activity are the C-terminus of LL-37, lactoferrin, or platelet factor 4 (PF4). Single-domain antibodies (sdAbs) and anticancer peptides are preferably expressed with linkers, which are preferably different repeats of (Gly4Ser)n or (Gly2Ser)n units, or different repeats of the sequence GGGGSEAAAKGGGGS. Preferred linkers are GS_linker (SEQ ID NO: 127: GGGGSGGGGS), GSAA_linker (SEQ ID NO: 128: GGGGSEAAAKGGGGS), and SRGS_linker (SEQ ID NO: 178: SRGSSGSSSSGSSGGSG). More specifically, the linker and the anticancer peptide are linked to the C-terminus of the sdAb. Furthermore, a short linker sequence (SEQ ID NO: 227: AAXX, where X can be any amino acid) containing two alanine residues and two amino acid sequences “XX” corresponding to the enzyme restriction site is preferably linked to the C-terminus of the single-domain antibody before the linker sequence.

[0153] The “cleavage peptides” of the present invention comprise modified cysteine-deficient horseshoe crab antimicrobial peptide-I (KWFRVYRGIYR, SEQ ID NO: 130), BMAP28A (GGLRSLGRKILRAWKKYGPIIVPIIRIG, SEQ ID NO: 131), and Polybia-MP1 (IDWKKLLDAAKQIL, SEQ ID NO: 132).

[0154] Horseshoe crab antimicrobial peptide-I is an antimicrobial peptide isolated from horseshoe crabs that inhibits the growth of many different types of bacteria through its ability to penetrate cell membranes. Starting with previously reported linear antimicrobial peptide analogs lacking cysteine ​​(cysteine-deficient antimicrobial peptide, CDT, KWFRVYRGIYRRR-CONH2), the study by Wang et al. (Int J Pept Res Ther. 2014) showed that removing two C-terminal arginine residues from CDT yields a peptide ([des-Arg... 12,13 [CDT], which retains antimicrobial activity but with reduced hemolysis and the selectivity required for therapeutic agents. As used herein, the modified cysteine-deficient horseshoe crab antimicrobial peptide-I refers to the sequence KWFRVYRGIYR.

[0155] Polybia-MP1 is a cleavage peptide derived from the venom of the Brazilian wasp and possesses known anticancer properties. In vitro studies of Polybia-MP1 have provided evidence of cytotoxicity caused by necrosis resulting from acute cell damage, swelling, and rupture through membrane disruption or the formation of transmembrane pores.

[0156] BMAP-28 is a bovine antimicrobial peptide of the cathelicidin family that induces membrane permeability and cell death in human tumor cell lines and activated but non-quiescent human lymphocytes.

[0157] L-rhamnose, galactose-α-1,3-galactose (αGal), and dinitrophenyl (DNP) can successfully induce antibody-dependent cytotoxicity regardless of the lack of a fragment crystallizable (Fc) region in the single-domain antibody. In fact, L-rhamnose, galactose-α-1,3-galactose (αGal), and dinitrophenyl (DNP) can recruit naturally occurring antibodies to tumor cells. Cells targeted in this manner can be recognized as foreign by the immune system and marked as potentially destructive. Recruiting endogenous antibodies to tumor cells can be disrupted through two antibody effector mechanisms: complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0158]

[0159]

[0160] Therefore, a preferred embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in the light chain and naturally lacking in constant region 1, or a variant of the variable domain, and wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1.

[0161] A particularly preferred embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from cysteine-deficient Limulus amebocyte lysate antimicrobial peptide-1, BMAP28A, and Polybia-MP1, wherein the cleavage peptide is linked to the C-terminus of the single-domain antibody via a linker. Preferred linkers are selected from GS_linker (SEQ ID NO: 127: GGGGSGGGGS), GSAA_linker (SEQ ID NO: 128: GGGGSEAAAKGGGGS), and SRGS_linker (SEQ ID NO: 178: SRGSSGSSSSGSSGGSG). Preferably, the short linker sequence (SEQ ID NO: 227: AAXX, where X can be any amino acid) is linked to the C-terminus of the single-domain antibody before the linker sequence.

[0162] A particularly preferred embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, and wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from cysteine-deficient Limulus amebocyte lysate antimicrobial peptide-1, BMAP28A, and Polybia-MP1, wherein the cleavage peptide is linked to the C-terminus of the single-domain antibody via a linker. Preferred linkers are selected from GS_linker (SEQ ID NO: 127: GGGGSGGGGS), GSAA_linker (SEQ ID NO: 128: GGGGSEAAAKGGGGS), and SRGS_linker (SEQ ID NO: 178: SRGSSGSSSSGSSGGSG). Preferably, the short linker sequence (SEQ ID NO: 227: AAXX, where X can be any amino acid) is linked to the C-terminus of the single-domain antibody before the linker sequence.

[0163] Another preferred embodiment of the invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and polyBia-MP1, and the polypeptide has at least 85% sequence identity with sequences selected from SEQ ID NO: 19-63, 142-156, and 179-226.

[0164] Another particularly preferred embodiment of the invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking both a light chain and a constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from the group comprising cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1, wherein the cleavage peptide is linked to the C-terminus of the single-domain antibody by a linker selected from the group comprising GS_linker (SEQ ID NO: 127: GGGGSGGGGS), GSAA_linker (SEQ ID NO: 128: GGGGSEAAAKGGGGS), and SRGS_linker (SEQ ID NO: 178: SRGSSGSSSSGSSGGSG), and the polypeptide is linked to the C-terminus of the single-domain antibody by a linker selected from the group comprising SEQ ID NO: 127. The sequences NO: 19-63, 142-156, and 179-226 have at least 85% sequence identity. Preferably, the short linker sequence (SEQ ID NO: 227: AAXX, where X can be any amino acid) is linked to the C-terminus of the single-domain antibody before the linker sequence.

[0165] In other words, a preferred embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from the group comprising cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1, wherein the cleavage peptide is linked to the C-terminus of the single-domain antibody via a linker selected from the group comprising GS_linker (SEQ ID NO: 127: GGGGSGGGGS), GSAA_linker (SEQ ID NO: 128: GGGGSEAAAKGGGGS), and SRGS_linker (SEQ ID NO: 178: SRGSSGSSSSGSSGGSG), wherein the short linker sequence (SEQ ID NO: 127: GGGGSGGGGS) is connected to the C-terminus of the single-domain antibody. NO: 227, where X can be any amino acid) is attached to the C-terminus of the single-domain antibody prior to the adapter sequence, the polypeptide having at least 85% sequence identity with sequences selected from SEQ ID NO: 19-63, 142-156, 179-226.

[0166] sdAbs lack the Fc region and therefore cannot induce Fc receptor-dependent effector functions. However, human Fc antibody fragments can be linked or fused with single-domain antibodies to restore their ability to interact with neonatal Fc receptors or FcRn. A significant advantage of sdAb-Fc fusion structures is the introduction of Fc receptor-dependent effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). The Fc regions (CH2-CH3) of different human and mouse antibody subclasses have been genetically fused with sdAbs. The linking or fusion of sdAbs with Fc antibody fragments also has the advantage of significantly increasing serum half-life and enhancing target antigen binding through affinity effects.

[0167] Stabilization of single-domain antibodies.

[0168] Another strategy to extend the half-life of sdAbs is to couple them to long-lived serum proteins or structural units that target these proteins. For example, serum albumin's long serum half-life is due to its ability to evade catabolism after cellular uptake. Another approach involves fusing sdAbs with albumin that binds to them.

[0169] Therefore, the present invention also relates to a polypeptide that further comprises an antibody (e.g., a single-domain antibody) targeting one or more serum proteins of a subject, which significantly prolongs the half-life of the polypeptide during circulation. The serum protein can be any suitable protein or fragment thereof found in the serum of a subject. In one aspect of the invention, the serum protein is serum albumin, serum immunoglobulin, thyroxine-binding protein, transferrin, or fibrinogen. Depending on the intended use, such as the half-life required for effective treatment and / or compartmentalization of the target antigen, the antibody may target one of the aforementioned serum proteins.

[0170] CAR-T cell therapy based on single-domain antibodies

[0171] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid protein or polypeptide containing an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (single-chain Fv) or sdAb) that is linked to a transmembrane domain of a T cell, which in turn links to an intracellular signaling or activation domain of the T cell; the connection between the antigen-binding domain and the transmembrane domain occurs via a hinge region. Domains or other functional or structural sequences of the CAR, such as the transmembrane domain or the intracellular activation or signaling domain, are referred to as elements or modules of the CAR.

[0172] CARs can utilize the antigen-binding properties of monoclonal antibodies to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner.

[0173] The antigen recognition module (also known as the extracellular domain) of CAR T cells is typically a single-chain Fv, which is connected to hinge regions, transmembrane regions, co-stimulatory domains, and cytoplasmic activation domains (such as the intracellular signaling domains of CD3-ζ or FcRγ). However, single-chain Fvs are not always efficiently folded and readily aggregate. In contrast, the variable regions of antibodies with only heavy chains (VHHs or sdAbs) are small, stable single-domain antibody fragments with affinity comparable to conventional single-chain Fvs.

[0174] A major challenge in developing CAR T cells for cancer treatment is the lack of targetable antigens (Xie et al., 2018). Most antigens proposed as targets for CAR T cells to treat cancer are limited to specific cancer types, and limited information on cancer-specific antigens for the vast majority of cancers makes CAR T cell therapy unsuitable.

[0175] Therefore, one particular advantage of the present invention is that it provides CARs that specifically target globular series glycans, such as Globo H, Gb3, Gb4, or Gb5, which are expressed in a variety of cancers and can be used to design anti-glycan CAR T cells, which can then be used to treat several different cancers.

[0176] Therefore, one embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, said single-domain antibody being a variable domain of a heavy chain antibody naturally lacking both a light chain and a constant region 1, or a variant of said variable domain, wherein said single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprising at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one intracellular activation domain, wherein said single-domain antibody is linked to the extracellular hinge region. The polypeptide is a T-cell chimeric antigen receptor comprising, in the N-terminal to C-terminal direction, said at least one single-domain antibody binding to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one intracellular activation domain.

[0177] In other words, one embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one CAR extracellular hinge region, at least one CAR transmembrane domain, at least one CAR co-stimulatory domain, and at least one CAR intracellular activation domain in the N-terminal to C-terminal direction, wherein the single-domain antibody is linked to the CAR extracellular hinge region.

[0178] More specifically, one embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and the constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular CD8-α hinge region, at least one CD8-α or CD28 transmembrane domain, at least one CD28, 4-IBB, ICOS co-stimulatory domain, and at least one CD3-ζ intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region.

[0179] Another embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region, and wherein the at least one single-domain antibody has at least 90% sequence identity with a sequence selected from SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 136, SEQ ID NO 137, and SEQ ID NO 138.

[0180] Another preferred embodiment of the invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking both a light chain and a constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one co-stimulatory domain, wherein the single-domain antibody is linked to the extracellular hinge region, and wherein the at least one single-domain antibody is a humanized single-domain antibody. It is also disclosed that, according to Kabat designations, single-domain antibodies can be humanized by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4.

[0181] The present invention also relates to recombinant nucleic acid molecules encoding polypeptides that specifically bind globular series glycans, said polypeptide comprising at least one single-domain antibody, wherein said single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, or a variant of said variable domain, wherein said single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5, and further comprises at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain and at least one intracellular activation domain, wherein said single-domain antibody is linked to said extracellular hinge region. Furthermore, the present invention provides a vector and a host cell comprising a recombinant nucleic acid molecule encoding a polypeptide comprising at least one single-domain antibody that specifically binds to globulin series glycans, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globulin series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one intracellular activation domain, wherein the single-domain antibody is connected to the extracellular hinge region.

[0182] A more preferred embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, at least one extracellular hinge region, and at least one intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region for treating cancer, wherein the cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0183] A more preferred embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that naturally lacks a light chain and naturally lacks constant region 1, or a variant of the variable domain, the single-domain antibody binding to at least one globular series glycan selected from GloboH, Gb3, Gb4, and Gb5, and further comprising at least one extracellular hinge region, at least one transmembrane region, at least one co-stimulatory region, and at least one intracellular activation region, wherein the single-domain antibody is linked to the extracellular hinge region for cancer treatment, wherein the cancer cells of the cancer express at least one globular series glycan selected from globoH, Gb3, Gb4, and Gb5 on their surface, wherein the cancer is selected from brain cancer, liver cancer, bile duct cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0184] The hinge region of a chimeric antigen receptor is typically derived from the human CD8-α chain (exemplary sequence SEQ ID NO:4 in WO2016019300A1, or a fragment thereof as described in US20160303166 A1).

[0185] The transmembrane domain of a chimeric antigen receptor crosses the plasma membrane and connects the extracellular structural domain and the intracellular signaling structural domain. Examples of transmembrane domains include, but are not limited to, human CD28 (exemplary sequence: residues 153-179 of SEQ ID NO:4 in US20160303166A1) and CD8-α (exemplary sequence: SEQ ID NO:12 in WO2016019300A1).

[0186] The intracellular activation domain transmits signals required for the effector function of CAR T cells. More specifically, the intracellular activation domain transmits signals required for cell activation when the extracellular domain binds to target Globo H series glycans. The intracellular activation domain is typically a human CD3-ζ (exemplary sequence SEQ ID NO: 18 or 20 in WO2016019300A1).

[0187] As used herein, a “co-stimulatory domain” (CSD) refers to the portion of a CAR that enhances the proliferation, survival, and / or development of memory cells. The CARs of this invention may comprise one or more co-stimulatory domains. Each co-stimulatory domain may comprise, for example, a co-stimulatory domain of CD28 (exemplary sequence SEQ ID NO:44 in WO2016019300A1), 4-1BB (CD137, exemplary sequence SEQ ID NO:14 in WO2016019300A1), and ICOS (exemplary sequence SEQ ID NO:263 in WO2016014553A1).

[0188] Methods for constructing chimeric antigen receptors have been described in the prior art, such as US 20160303166A1, WO2016014553 A1, and Xie et al. in PNAS2018.

[0189] Furthermore, the present invention also describes a method for generating genetically engineered chimeric antigen receptor T cells (CAR T cells), comprising:

[0190] a) Producing a chimeric antigen receptor (CAR) construct having at least one single-domain antibody, wherein the single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region.

[0191] b) Transfect T cells taken from the subject's blood.

[0192] c) Expressing a CAR construct to generate a functional CAR in T cells to produce CAR T cells that specifically recognize at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5.

[0193] Genetically engineered T cells that may contain and express the CARs of the present invention include, but are not limited to, T lymphocytes (T cells), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (TEM)), natural killer cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells capable of producing treatment-related offspring. In one embodiment, the genetically engineered cell is an autologous cell. For example, a single T cell of the present invention may be a CD4+ T cell. + / CD8 - CD4 - / CD8 + CD4 - / CD8 - or CD4 + / CD8 + T cells can be CD4+. + / CD8 - and CD4 - / CD8 + The cells can be a mixture or a population of single clones. When co-cultured in vitro with cells expressing the target antigen (i.e., cancer cells expressing Globo H-series glycans), the T cells CD4 of this invention... + It can produce IL-2, IFNγ, TNFα, and other T cell effector cytokines. When co-cultured in vitro with target cells (i.e., cancer cells expressing Globo H series glycans), the CD8 T cells of this invention... + It can lyse antigen-specific target cells.

[0194] Finally, the present invention also describes a method for treating a subject suffering from cancer, wherein the cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to the subject suffering from said cancer chimeric antigen receptor T cells (CAR T cells) that specifically recognize at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein said CAR T cells express a functional CAR polypeptide comprising a single-domain antibody that binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, at least one extracellular hinge region, at least one transmembrane domain, at least one co-stimulatory domain, and at least one intracellular activation domain, in an effective amount for treating the subject suffering from said cancer.

[0195] Humanization of single-domain antibodies

[0196] Variant sequences of the present invention may include a humanized single-domain antibody that specifically binds to at least one glycan selected from GloboH, Gb3, Gb4, and Gb5. Humanization of the sdAb aims to further reduce the likelihood of unwanted immune responses in human individuals after administration. One embodiment of the invention relates to a modified single-domain antibody based on an alpaca antibody, wherein amino acid residues of the sdAb that do not affect the natural affinity of the domain for the target are modified to reduce the immunogenicity of the sdAb relative to humans.

[0197] More specifically, the present invention relates to modified sdAbs for administration to humans, and the use of such "humanized" sdAbs in the treatment of human diseases. The humanization of the single-domain antibody of the present invention comprises the step of replacing one or more alpaca amino acids with human counterparts found in human common sequences, without causing the single-domain antibody to lose its characteristic features, i.e., the humanization does not significantly affect the antigen-binding ability of the resulting single-domain antibody and the polypeptide comprising it. Such methods are known to those skilled in the art.

[0198] Humanization of alpaca single-domain antibodies, or more generally camel single-domain antibodies, requires the introduction and mutagenesis of a limited number of amino acids into a single polypeptide chain.

[0199] Therefore, one embodiment of the present invention is a polypeptide that specifically binds to globosaccharides, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in the light chain and naturally lacking in constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globosaccharide selected from Globo H, Gb3, Gb4 and Gb5, wherein the at least one single-domain antibody is a humanized single-domain antibody.

[0200] Typically, as described in EP 1687338 B1, humanized single-domain antibodies are obtained by replacing any of the following residues in an alpaca single-domain antibody, either alone or in combination:

[0201] The marker amino acids for FR1 located at positions 1, 5, 11, 28, and 30.

[0202] The marker amino acids for FR2 located at positions 44 and 45,

[0203] The marker amino acids for FR3 located at positions 74, 75, 76, 83, 84, 93, and 94.

[0204] The marker amino acids for FR4 located at positions 103, 104, 108, and 111;

[0205] The numbering mentioned therein is based on the Kabat numbering.

[0206] Humanization of residues in FR1, FR3, and FR4 has minimal impact on the function and stability of single-domain antibodies, while mutations at FR2 positions 44 and 45 can even stabilize single-domain antibodies (Vincke et al., J. Biol. Chem. 2009). However, EP1687338 B1 reported that mutagenesis of Q108L in FR4 resulted in lower production levels in *E. coli*. Position 108 is exposed to solvents in *VHH* (cameloid) antibodies, while in human antibodies, this position is buried at the VH-VL interface. Introducing nonpolar hydrophobic Leu, rather than polar uncharged Gln, can have a significant impact on the intrinsic folding / stability of the molecule. Single-domain antibody marker residues at positions 37 and 47 in FR2 have a major impact on the integrity of antigen-antigen interactions and therefore cannot be humanized (Vincke et al., 2009).

[0207] As described in US 7807162 B2, Table 1 reports the marker residues in the alpaca sdAbs of the present invention and the corresponding amino acid residues at the corresponding positions of the most closely related human VH domain VH3. The table shows that the marker amino acids at positions 103 and 111 of the alpaca single-domain antibodies disclosed herein are identical to the most common residues of human VH3.

[0208] Therefore, another embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in the light chain and naturally lacking in constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5, and, according to the Kabat numbering, wherein the at least one single-domain antibody is humanized by replacing one or more amino acid residues located at positions 1, 5, 11, 28 and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93 and 94 in FR3, and positions 104 and 108 in FR4.

[0209] Therefore, the humanized peptide exhibits high amino acid sequence homology with the human VH framework region, and the peptide can be directly administered to humans without the desired generation of an unwanted immune response, and without the burden of further humanization. The present invention also relates to recombinant nucleic acids capable of encoding said humanized peptides.

[0210] A preferred embodiment of the present invention is a globular polysaccharide-binding polypeptide comprising at least one single-domain antibody specifically binding to at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5, wherein the at least one single-domain antibody is formed by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, and further comprising at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is selected from anticancer peptides, cleavage peptides, L-rhamnose, galactose-α-1,3-galactose, dinitrophenyl, serum-stabilizing molecules, fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, radioisotopes, chromophores, disuccinimide adipate, and human Fc antibody fragments.

[0211] A more preferred embodiment of the present invention is a globular polysaccharide-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5, wherein the at least one single-domain antibody is humanized by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, and further comprising at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from modified cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1.

[0212]

[0213] Amino acids are listed by single-letter codes; bold residues: the most common residues in humans; underlined residues: residues in alpaca sdAbs, corresponding to residues in human VH3.

[0214] Pharmaceutical Composition

[0215] "Pharmaceutical composition" means a formulation which enables the bioactivity of an active ingredient to be effective and which does not contain any additional components that would be toxic to a subject who is given the formulation.

[0216] As used herein, the terms “pharmaceutically acceptable,” “physiologically tolerable,” and their grammatical variations are used interchangeably and indicate that a material can be administered to mammals or to mammals without producing undesirable physiological effects, such as nausea, dizziness, stomach upset, etc.

[0217] Design pharmaceutical compositions to facilitate the effective administration of the present invention's polypeptides containing single-domain antibodies.

[0218] "Pharmaceutically acceptable mediators" refer to components in a pharmaceutical preparation other than the active ingredient that are non-toxic to the subject to which the drug is administered. Pharmaceutically acceptable mediators include, but are not limited to, buffers, stabilizers, or preservatives.

[0219] Examples of suitable mediators or excipients include, but are not limited to, lactose, dextrose, sucrose, glucose, powdered sugar, sorbitol, mannitol, xylitol, starch, gum arabic, xanthan gum, guar gum, tara gum, mesquitegum, fenugreek gum, locust bean gum, ghatti gum, astragalus gum, inositol, molasses, maltodextrin, Irish moss extract, panval gum, and mucliage of isapol. husks), magnesium aluminum silicate (Veegum), larch arabinogalactan, calcium silicate, calcium phosphate, dicalcium phosphate, calcium sulfate, kaolin, sodium chloride, polyethylene glycol, alginate, gelatin, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, brine, syrup, methylcellulose, ethylcellulose, hydroxypropyl ethylcellulose, carboxymethylcellulose, polyacrylic acid such as carbomer, such as carbomer 941, carbomer 980, carbomer 981, and pharmacogen. TM (SPIPharma Group; Newcastle, NJ) and similar resin bases; generally, the compositions of the present invention comprise about 10% to about 90% by weight of a medium, excipients or a combination thereof.

[0220] Preferably, the pharmaceutical composition contains about 0.001% to about 90% by weight, preferably about 0.01% to about 75%, more preferably about 0.1% to 50%, and even more preferably about 0.1% to 10% of the polypeptide of the present invention, the remainder consisting of a suitable pharmaceutical carrier, excipient and / or diluent.

[0221] Pharmaceutical compositions can be formulated as powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral dosage forms, topical preparations, suppositories, or sterile injectable solutions, for example, by conventional nebulization. During formulation, diluents or excipients can be used, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0222] In pharmaceutical compositions, solid dosage forms for oral administration can be tablets, pills, powders, granules, or capsules. Solid dosage forms may further contain excipients. Excipients may be such as starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, solid dosage forms may contain lubricants, such as magnesium stearate or talc. In pharmaceutical compositions, liquid dosage forms for oral administration are preferably suspensions, solutions, emulsions, or syrups. Liquid dosage forms may contain water or liquid paraffin. As excipients, liquid dosage forms may include wetting agents, sweeteners, flavoring agents, or preservatives. For parenteral administration purposes, compositions containing the polypeptides of the present invention are preferably dissolved in distilled water, and the pH is preferably adjusted to about 6 to 8.

[0223] Useful formulations of the compositions of the present invention for parenteral administration also include sterile aqueous and non-aqueous solvents, suspensions, and emulsions. Examples of useful non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, fish oils, and injectable organic esters.

[0224] One embodiment of the present invention is a pharmaceutical composition comprising a polypeptide that specifically binds to globular series glycans, the polypeptide comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and at least one pharmaceutically acceptable mediator, excipient, and / or diluent.

[0225] Another embodiment of the present invention is a pharmaceutical composition comprising a globular polysaccharide-binding polypeptide, the globular polysaccharide-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5, and further comprising at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is selected from anticancer peptides, cleavage peptides, L-rhamnose, galactose-α-1,3-galactose, dinitrophenyl, serum-stabilizing molecules, fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, radioisotopes, chromophores, disuccinimide adipate, human Fc antibody fragments, and at least one pharmaceutically acceptable mediator, excipient, and / or diluent.

[0226] Another specific embodiment of the present invention is a pharmaceutical composition comprising a globular glycan-binding polypeptide and at least one pharmaceutically acceptable mediator, excipient, and / or diluent, wherein the globular glycan-binding polypeptide comprises at least one single-domain antibody that specifically binds to at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1.

[0227] A more specific embodiment of the present invention is a pharmaceutical composition comprising a globular glycan-binding polypeptide, the globular glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprising at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1, the polypeptide having at least 85% sequence identity with a sequence selected from SEQ ID NO:19-63, 142-156, and at least one pharmaceutically acceptable mediator, excipient, and / or diluent.

[0228] Another embodiment of the present invention is a pharmaceutical composition comprising a globular glycan-binding polypeptide, said globular glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, and, according to the Kabat number, said at least one single-domain antibody by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4; and at least one pharmaceutically acceptable mediator, excipient, and / or diluent. One embodiment of the present invention is a pharmaceutical composition comprising a polypeptide comprising at least one humanized single-domain antibody that specifically binds to at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, and at least one pharmaceutically acceptable mediator, excipient, and / or diluent.

[0229] Uses of single-domain antibodies against cancer

[0230] Another embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5, or a pharmaceutical composition comprising the polypeptide for treating and / or diagnosing cancer, wherein the cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5 on their surface.

[0231] Another embodiment of the invention relates to a polypeptide that specifically binds to a globular series glycan comprising at least one single-domain antibody or a variant thereof, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, or a pharmaceutical composition comprising said polypeptide for treating and / or diagnosing cancer, wherein the cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, and wherein said cancer is selected from brain cancer, liver cancer, bile duct cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0232] Furthermore, one aspect of the present invention relates to a globular glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, and, according to the Kabat number, wherein the at least one single-domain antibody is obtained by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4; wherein the cancer cells of the cancer express at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface. One embodiment of the invention, slightly modified, relates to a globular glycan-binding polypeptide for the treatment and / or diagnosis of cancer, comprising at least one humanized single-domain antibody that specifically binds at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein cancer cells express at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0233] Furthermore, one aspect of the present invention relates to a pharmaceutical composition comprising a globular glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein the at least one single-domain antibody is used for the treatment and / or diagnosis of cancer by replacing one or more amino acid residues located at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, wherein the cancer cells of the cancer express at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0234] A preferred embodiment of the present invention is a globular polysaccharide-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein the at least one single-domain antibody is used for the treatment and / or diagnosis of cancer by replacing one or more amino acid residues located at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, or a pharmaceutical composition comprising the polypeptide. The cancer cells of the cancer express at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5 on their surface, and the cancer is selected from brain cancer, liver cancer, cholangiocarcinoma, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0235] A more preferred embodiment of the present invention is a globular polysaccharide-binding polypeptide comprising at least one single-domain antibody specifically binding to at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein the at least one single-domain antibody is formed by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, and further comprising at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from modified cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, Polybia-MP1, or a pharmaceutical composition comprising the polypeptide, for treating cancer, wherein the cancer cells express at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0236] A more preferred embodiment of the present invention is a globular series glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein the at least one single-domain antibody is formed by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, and further comprising at least one effector molecule linked to said single-domain antibody, wherein said effector molecule It is a cleavage peptide selected from the group consisting of modified cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1, or a pharmaceutical composition containing said polypeptide, for the treatment of cancer, wherein the cancer cells express at least one globular series polysaccharide selected from GloboH, Gb3, Gb4, and Gb5 on their surface, wherein said cancer is selected from brain cancer, liver cancer, bile duct cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0237] Another embodiment of the invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one CAR extracellular hinge region, at least one CAR transmembrane domain, at least one CAR co-stimulatory domain, and at least one CAR intracellular activation domain, wherein the single-domain antibody is linked to the CAR extracellular hinge region for treating cancer, wherein the cancer cells of the cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0238] Another embodiment of the invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and the constant region 1, or a variant of the variable domain, and wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular CD8-α hinge region, at least one CD8-α or CD28 transmembrane domain, at least one CD28, 4-IBB, ICOS co-stimulatory domain, and at least one CD3-ζ intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region of a CAR for the treatment of cancer, wherein the cancer cells of the cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0239] Another embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one CAR extracellular hinge region, at least one CAR transmembrane domain, at least one CAR co-stimulatory domain, and at least one CAR intracellular activation domain, wherein the single-domain antibody is linked to the CAR extracellular hinge region for treating cancer, wherein the cancer cells of the cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, wherein the cancer is selected from brain cancer, liver cancer, cholangiocarcinoma, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0240] Another embodiment of the present invention relates to a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprises at least one extracellular CD8-α hinge region, at least one CD8-α or CD28 transmembrane domain, at least one CD28, 4-IBB, ICOS co-stimulatory domain, and at least one CD3-ζ intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region of a CAR for the treatment of cancer, wherein the cancer cells of the cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, wherein the cancer is selected from brain cancer, liver cancer, cholangiocarcinoma, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0241] This document also describes a method for treating cancer, wherein cancer cells express at least one globular polysaccharide selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to a patient suffering from said cancer a therapeutically effective amount of a polypeptide as disclosed herein or a therapeutically effective amount of a pharmaceutical composition comprising said polypeptide.

[0242] Specifically, the present invention describes a method for treating cancer, wherein cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to a patient suffering from said cancer a therapeutically effective amount of a polypeptide specifically binding to a globular series glycan comprising at least one single-domain antibody, wherein said single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking a constant region 1, or a variant of said variable domain, wherein said single-domain antibody binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, or a therapeutically effective amount of a pharmaceutical composition comprising said polypeptide.

[0243] This document also describes a method for treating cancer, wherein the cancer cells of said cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to a patient suffering from said cancer a therapeutically effective amount of a polypeptide as disclosed herein or a therapeutically effective amount of a pharmaceutical composition comprising said polypeptide, wherein said cancer is selected from the group comprising brain cancer, liver cancer, bile duct cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0244] Specifically, the present invention describes a method for treating cancer, wherein cancer cells of the cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to a patient suffering from the cancer a therapeutically effective amount of a globular series glycan-binding polypeptide, the globular series glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein the at least one single-domain antibody is humanized by replacing one or more amino acid residues located at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, or a therapeutically effective amount of a pharmaceutical composition comprising the polypeptide.

[0245] Furthermore, the present invention describes a method for treating cancer, wherein cancer cells of said cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to a patient suffering from said cancer a therapeutically effective amount of a globular series glycan-binding polypeptide, said globular series glycan-binding polypeptide comprising at least one single-domain antibody specifically binding to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein said at least one single-domain antibody is humanized by replacing one or more amino acid residues located at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, or a therapeutically effective amount of a pharmaceutical composition comprising said polypeptide, wherein said cancer is selected from brain cancer, liver cancer, bile duct cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0246] More specifically, the present invention describes a method for treating cancer, wherein cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to a patient a therapeutically effective amount of a globular series glycan-binding polypeptide, the globular series glycan-binding polypeptide comprising at least one single-domain antibody specifically binding to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein the at least one single-domain antibody is humanized by replacing one or more amino acid residues located at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, and further comprising at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from the group comprising modified cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1, or a therapeutically effective amount of a pharmaceutical composition comprising the polypeptide.

[0247] More specifically, the present invention describes a method for treating cancer, wherein cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering to a patient suffering from said cancer a therapeutically effective amount of a globular series glycan-binding polypeptide, the globular series glycan-binding polypeptide comprising at least one specific binding to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5. Single-domain antibodies of globular series glycans of H, Gb3, Gb4, and Gb5, according to Kabat numbering, wherein at least one single-domain antibody is humanized by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4; and further comprising at least one effector molecule linked to the single-domain antibody, wherein the effector molecule is a cleavage peptide selected from the group comprising modified cysteine-deficient horseshoe crab antimicrobial peptide-1, BMAP28A, and Polybia-MP1, or a therapeutically effective amount of a pharmaceutical composition comprising the polypeptide, wherein the cancer is selected from the group comprising brain cancer, liver cancer, cholangiocarcinoma, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0248] Furthermore, the present invention describes a method for treating cancer, wherein cancer cells of the cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering a therapeutically effective amount of a globular series glycan-binding polypeptide to a patient suffering from the cancer, the globular series glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprising at least one CAR extracellular hinge region, at least one CAR transmembrane domain, at least one CAR co-stimulatory domain, and at least one CAR intracellular activation domain, wherein the single-domain antibody is linked to the CAR extracellular hinge region.

[0249] Furthermore, the present invention describes a method for treating cancer, wherein cancer cells of the cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering a therapeutically effective amount of a globular series glycan-binding polypeptide to a patient suffering from the cancer, the globular series glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprising at least one CAR extracellular hinge region, at least one CAR transmembrane domain, at least one CAR costimulatory domain, and at least one CAR intracellular activation domain, wherein the single-domain antibody is linked to the CAR extracellular hinge region, and further comprising at least one extracellular CD8-α hinge region, at least one CD8-α or CD28 transmembrane domain, at least one CD28, 4-IBB, ICOS costimulatory domain, and at least one CD3-ζ intracellular activation domain, wherein the single-domain antibody is linked to the CAR extracellular hinge region.

[0250] More specifically, the present invention describes a method for treating cancer, wherein cancer cells of said cancer express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering a therapeutically effective amount of a globular series glycan-binding polypeptide to a patient suffering from said cancer, said globular series glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprising at least one CAR extracellular hinge region, at least one CAR transmembrane domain, at least one Globo series glycan-binding polypeptide, at least one CAR co-stimulatory domain, and at least one CAR intracellular activation domain, wherein said single-domain antibody is linked to the CAR extracellular hinge region, wherein said cancer is selected from brain cancer, liver cancer, cholangiocarcinoma, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0251] Furthermore, the present invention describes a method for treating cancer, wherein cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising administering a therapeutically effective amount of a globular series glycan-binding polypeptide to a patient suffering from said cancer, the globular series glycan-binding polypeptide comprising at least one specific binding to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5. A single-domain antibody comprising a globular series of H, Gb3, Gb4, and Gb5 globular glycans, and further comprising at least one extracellular hinge region of a CAR, at least one transmembrane domain of a CAR, at least one costimulatory domain of a CAR, and at least one intracellular activation domain of a CAR, wherein the single-domain antibody is linked to the extracellular hinge region of the CAR, and further comprising at least one extracellular CD8-α hinge region, at least one CD8-α or CD28 transmembrane domain, at least one CD28, 4-IBB, ICOS costimulatory domain, and at least one CD3-ζ intracellular activation domain, wherein the single-domain antibody is linked to the extracellular hinge region of the CAR, and wherein the cancer is selected from brain cancer, liver cancer, cholangiocarcinoma, kidney cancer, breast cancer, prostate cancer, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer.

[0252] Another embodiment describes a method for diagnosing cancer, characterized in that cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, the method comprising the following steps:

[0253] a) Contact the sample with a polypeptide that specifically binds to at least one globular series glycan selected from Globo H, Gb3, Gb4 and Gb5 as disclosed herein.

[0254] b) Detect the binding of the polypeptide to the sample.

[0255] c) Compare the binding detected in step b) with a standard, wherein the difference in binding relative to the sample is a characteristic for diagnosing cancer, characterized in that the cancer cells of the cancer express at least one globular polysaccharide selected from Globo H, Gb3, Gb4 and Gb5 on their surface.

[0256] "Disorder" is any condition that will benefit from treatment with the substances / molecules or methods described in this article.

[0257] "Cellular proliferation disorders" and "proliferative disorders" refer to conditions associated with a certain degree of abnormal cell proliferation, such as cancer.

[0258] "Cancer" and "cancerous" refer to or describe a physiological state in mammals typically characterized by a proliferative disorder of cells. Cancer generally includes, but is not limited to, epithelial carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of cancer may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancer.

[0259] "Tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. As discussed in this article, the terms "cancer," "carcinogenesis," "proliferative disorder," "proliferative lesion," and "tumor" are not mutually exclusive.

[0260] "Metastasis" refers to the spread of cancer and / or tumors from their primary site to other locations within an individual's body.

[0261] "Treatment," "treat," or "treating" refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated, and may be used for prevention or in the course of clinicopathological processes. Desired therapeutic outcomes include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of progression, improvement or mitigation of the disease state, and relief or improvement of prognosis. For example, treatment may include administering to a subject a therapeutically effective amount of a pharmaceutical preparation containing an anti-Globo H antibody to delay the development or slow the progression of cancer, wherein the cancer cells express at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0262] "Pharmaceutical formulation" refers to a formulation in which the biological activity of the active ingredient is effective and which does not contain any additional components that would be toxic to the subject administering the formulation.

[0263] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation other than the active ingredient that are non-toxic to the subject to which they are administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0264] "Therapeutic effective amount" refers to the amount of an active ingredient or agent (e.g., a pharmaceutical preparation) that achieves the desired therapeutic or preventative outcome, such as treating or preventing a disease or condition in a subject. In the case of cancer, a therapeutically effective amount of a therapeutic agent is the amount that reduces the number of cancer cells; reduces the size of the primary tumor; inhibits (i.e., to some extent slows and preferably stops) the infiltration of cancer cells into peripheral organs; inhibits (i.e., to some extent slows and preferably stops) tumor metastasis; inhibits tumor growth to some extent; and / or alleviates one or more symptoms associated with cancer to some extent. It may have cytotoxic and / or inhibitory properties, depending on the extent to which the drug can stop growth and / or kill existing cancer cells. For cancer treatment, efficacy in vivo can be assessed by, for example, duration of survival, time to progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0265] "Individual" or "subject" refers to mammals, including but not limited to domesticated animals (such as cattle, sheep, cats, dogs, and horses), primates (such as humans and non-human primates such as monkeys), rabbits, and rodents (such as mice and rats).

[0266] "Anticancer therapeutic agents" refers to drugs used to treat cancer. Exemplary anticancer therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitors, cytotoxic agents, drugs used in radiotherapy, anti-angiogenic agents, apoptosis agents, anti-microtubule agents and other drugs for treating cancer, anti-CD20 antibodies, platelet-derived growth factor inhibitors, COX-2 inhibitors, interferons, cytokines, antagonists that bind to one or more targets (e.g., PDGFR-β, APRIL, BCMA receptor, TRAIL / Apo2), other bioactive agents and organic chemical agents, and combinations thereof.

[0267] Uses of single-domain antibodies in cancer diagnosis

[0268] Another embodiment of the present invention is a diagnostic kit for detecting cells expressing at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface. The diagnostic kit comprises a globular series glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds to at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the single-domain antibody is a variable domain of a heavy chain antibody that naturally lacks a light chain and naturally lacks constant region 1, or a variant of the variable domain. In addition to the polypeptide binding to one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, the kit may also contain reagents required for labeling and / or detecting and / or quantifying antigen-binding cells.

[0269] With slight modification, the present invention also relates to a diagnostic kit comprising a globular glycan-binding polypeptide, the globular glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, or a variant of the variable domain, the diagnostic kit being used to screen for cancers characterized by cells expressing at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface.

[0270] Another aspect of the invention is a diagnostic kit comprising a globular glycan-binding polypeptide comprising at least one single-domain antibody that specifically binds at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, or a variant of the variable domain, wherein the at least one single-domain antibody is humanized by replacing one or more amino acid residues at positions 1, 5, 11, 28, and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93, and 94 in FR3, and positions 104 and 108 in FR4, for screening cancers characterized by the expression of at least one globular glycan selected from Globo H, Gb3, Gb4, and Gb5 on their cell surface.

[0271] As described above, when linked to effector molecules that serve as detectable markers, the single-domain antibodies of the present invention can be used for diagnostic purposes.

[0272] Suitable detectable markers and techniques for attaching, using, and detecting them will be apparent to those skilled in the art, and include, for example, but not limited to, fluorescent molecules (e.g., fluorescein, isothiocyanates, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescein, as well as fluorescent metals, such as Eu or other metals derived from the lanthanides), phosphorescent molecules, chemiluminescent molecules, or bioluminescent molecules (e.g., luminol, isoluminol, thermoacridone esters, imidazole, acridine salts, oxalates, ethylene dioxide, or GFP and their analogues), radioisotopes, metals, metal chelates, or metal cations, or other metals or metal cations particularly suitable for in vivo, in vitro, or in situ diagnostic and imaging purposes, as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, biotin-avidin protein peroxidase). Horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0273] Therefore, another embodiment of the present invention is a diagnostic kit comprising a polypeptide that specifically binds to globular series glycans, the polypeptide comprising at least one single-domain antibody, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, and further comprising at least one effector molecule linked to the single-domain antibody, the diagnostic kit being used to screen for cancers characterized by cells expressing at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 on their surface, wherein the effector molecule is selected from fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, radioisotopes, and chromophores.

[0274] A polypeptide containing a multimeric single-domain antibody.

[0275] One embodiment of the present invention is a polypeptide that specifically binds to globosaccharides, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant thereof, wherein the single-domain antibody binds at least one globosaccharide selected from Globo H, Gb3, Gb4, and Gb5, and wherein the number of single-domain antibodies binding at least one globosaccharide selected from Globo H, Gb3, Gb4, and Gb5 is at least two.

[0276] Such peptides containing at least two single-domain antibodies have the advantage of higher affinity for the target, as exemplified by the sdAb46 trimer.

[0277] A particular embodiment of the present invention is a polypeptide that specifically binds to globular series glycans, comprising at least one single-domain antibody, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the number of single-domain antibodies binding at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 is at least two, and wherein the two or more single-domain antibodies are sequence-distinct.

[0278] A more specific embodiment of the invention is a polypeptide that specifically binds to globosyl series glycans, comprising at least one single-domain antibody, said single-domain antibody being a variable domain of a heavy chain antibody that is naturally lacking a light chain and naturally lacking constant region 1, or a variant of said variable domain, wherein said single-domain antibody binds at least one globosyl series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the number of single-domain antibodies binding at least one globosyl series glycan selected from Globo H, Gb3, Gb4, and Gb5 is at least two, and wherein said two or more single-domain antibodies are sequence-identical.

[0279] A more specific embodiment of the invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globular series glycans, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the number of single-domain antibodies binding at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 is 3, and wherein the two or more single-domain antibodies are sequence-identical.

[0280] Another more specific embodiment of the invention is a polypeptide comprising at least one single-domain antibody that specifically binds to globular series glycans, wherein the single-domain antibody is a variable domain of a heavy chain antibody that is naturally lacking in both the light chain and constant region 1, or a variant of the variable domain, wherein the single-domain antibody binds at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5, wherein the number of single-domain antibodies binding at least one globular series glycan selected from Globo H, Gb3, Gb4, and Gb5 is three, wherein the single-domain antibody has at least 90% sequence identity with SEQ ID NO 5.

[0281] Single-domain antibodies can be linked using methods known in the art or any future methods to form any polypeptide of the specific binding globular series of glycans disclosed herein and comprising more than one single-domain antibody. Single-domain antibodies can be linked non-covalently (e.g., using streptomycin / biotin combinations, antibody / tag combinations) or covalently. They can also be fused via chemical crosslinking of amino acid residues with an organic derivatizing agent.

[0282] Alternatively, single-domain antibodies can be genetically fused at the DNA level. One way to link single-domain antibodies is through a genetic pathway, directly or via peptide linkers, connecting the single-domain antibody coding sequence. For example, the C-terminus of a single-domain antibody can be linked to the N-terminus of another single-domain antibody. This linking pattern can be extended to link additional single-domain antibodies for the construction and generation of tri-, tetra-, and other functional structures. Furthermore, single-domain antibodies can be linked via glutathione S-transferase (GST) to form dimers (e.g., as described in Smith DB, Johnson KS, Gene 1988; Tudyka T, Skerra A, Protein Sci 2008), or via dodecanoic acid, a scaffold protein from Mycobacterium tuberculosis mtDod, to form dodecomers (Bordeaux et al., Sci Rep 2020; CA3076791 A1). sequence list <110> Max Planck Society for the Advancement of Science (Max-Planck-Gesellschaft zurFoerderung der Wissenschaften eV) <120> Single-domain antibodies that specifically bind to globular series glycans <130> GAR-P04166WO <160> 228 <170> BiSSAP 1.3.6 <210> 1 <211> 126 <212> PRT <213> Alpaca (Vicugna pacos) <400> 1 Met Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe 20 25 30 Ser Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg 35 40 45 Glu Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met 65 70 75 80 Val Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr 100 105 110 Ile Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 2 <211> 379 <212> DNA <213> Alpaca (Vicugna pacos) <220> <223> Nucleic acid <400> 2 atgggagcac acgtccagct tgtagagtca gggggcggct tagtccagcc aggtggaagt 60 ttacgtctta gttgtgaagg gtcggggatg accttctcta tatacgactt cggttggtat 120 cgtcaggtgc cagggaatca gcgcgagtta gttgcaagca ttggctttgg gggcaacatt 180 gattacgcaa actctgttaa aggacgcttc acgatctccc gtgatggtga caaaaacatg 240 gttagtctgc aaatgaatac gctgcgccca gaggatacag caatttatta ttgcgtggct 300 cgttggacgt tcggaataca gagaggagat tactacatat ggtcgtggaa gcaaggtaca 360 caagtaacag taagttccg 379 <210> 3 <211> 125 <212> PRT <213> Alpaca (Vicugna pacos) <400> 3 Met Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser​​​​​​​​​​​​​​​Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr 85 90 95 Tyr Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp 100 105 110 His Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 4 <211> 375 <212> DNA <213> Alpaca (Vicugna pacos) <400> 4 atgggagacg ttcaactgca ggaatcgggg ggcggactgg cacagccggg aggctcttta 60 agactgagct gcgccgcctc tggcttcact ttctcctcct atgcgatgag ctgggcacgc 120 caggctccag gaaagggcct ggagtgggta tcgggtatat accgggatgg gatagacact 180 tactacgcgg atagcgtcaa ggggcggttt acgatctctc gtgataatgc caaaaatacc​​​​​​​​​​​​​<213> Alpaca (Vicugna pacos) <400> 5 Met Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp 20 25 30 Asp Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln 50 55 60 Ser Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr [[ID=,26]]85 90 95 Phe Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly 100 105 110 Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 378 <212> DNA <213> Alpaca (Vicugna pacos) <400> 6 atgggagatg ttcaactgca agaatctggc ggcggtctgg ttcatccggg cggtagtctg 60 cgtctgtctt gcgttgcgag cggctttacc tttgatgatt atgcgctgac ctgggttcgt 120 caagcaccgg gtaaaggtct ggagtgggta tctggtatca actggaacgg cgacgatacc 180 tactacaccc aaagcatgaa aggccgcttt accatcagtc gcgataacgc gaaaaacacc 240 ctgtacctgc agatgaacag cctgaaaagc gaagacaccg cggtttactt ctgcaccaaa 300 aacgtcggcg gtaattgggt ttctggggat tacggtctgg attattgggg tcagggtacc 360 caagttaccg ttagttct 378 <210> 7 <211> 123 <212> PRT <213> Alpaca (Vicugna pacos) <400> 7 Met Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly 20 25 30 Asn His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 8 <211> 371 <212> DNA <213> Alpaca (Vicugna pacos) <400> 8 atgggagatg ttcaactggt tgaatctggc ggcggtctgg ttcaaccggg cggtagtctg 60 cgtctgtctt gtaccgcaag cggttttacc tttggcaacc actggatgta ctgggttcgt 120 caaaccccgg gtaaaggtct ggagtgggtt gcaaccgcaa ataccggcgg tcgcgatacc 180 tattatgcag atagcgtctg gggtcgtttt accatttctg gcgataacgc gaaaaacacc 240 ctgtacctgc agatgaacaa cctgaaaagc gaggacaccg cgatctatta ttgcgcacgc 300 gaagttaacg actacctgga atacgcgctg ggttattggg gtcaaggtac ccaagttacc 360 gttagctctg g 371 <210> 9 <211> 125 <212> PRT <213> Alpaca (Vicugna pacos) <400> 9 Met Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Cys Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 375 <212> DNA <213> Alpaca (Vicugna pacos) <400> 10 atgggtgacg ttcaactggt tgaatctggc ggcggtctgg ttcaaccggg cggtagtctg 60 cgtctgagtt gcgcagcatc tggttttacc ttctcctcct actggatgca ttgggttcgt 120 caagcaccgg gtaaaggtct ggagtgcgtc agcgttatta ataccggcaa cggtagtccg 180 tactacgcgg atagcgttaa aggccgtttc accatcagca ccgataacgc gaaaaacacc 240 ctgtacctgc agatgaacag cctgaaaagc gaagataccg cggtttatta ttgcgcacgc 300 gatattccgg atccggcagg tcgcgctggc ggtatggatt attggggtaa aggcacccag 360 gttaccgtta gcagc 375 <210> 11 [[ID=*27]]<211> 121 <212> PRT <213> Alpaca (Vicugna pacos) <400> 11 Met Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30<Ser Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45 Glu Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn 65 70 75 80 Val Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys 85 90 95 Tyr Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly 100 105 110 Gln Glu Thr Gln Val Thr Val Ser Ser 115 120 <210> 12 <211> 365 <212> DNA <213> Alpaca (Vicugna pacos) <400> 12 atgggtgcgc aagttcagct ggttgaatct ggcggtggtc tggttcaacc gggcggtagt 60 ctgaccgtta gttgcgcagc atctggtttt acctttagcc gttacggcgt tacctggcat 120 cgtcaagcac cgggtaaaga acgcgaactg gttgcggata tcatctttac cggcctgaac 180 ccgacctata gcgatagcgt taaaggtcgt ttcaccatca gccgcgataa cgacaaaaac 240 gtcgcctacc tgcagatgaa cagcctgaaa ccggaagata ccgcgaaata ctactgcaac 300 gtcaacgata tcgtcgttcg cggcaactat tggggtcaag aaacccaggt taccgtttct 360 agtgg 365 <210> 13 <211> 122 <212> PRT <213> Alpaca (Vicugna pacos) <400> 13 Met Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu 20 25 30 Gly Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg 35 40 45 Glu Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met 65 70 75 80 Val Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 14 <211> 368 <212> DNA <213> Alpaca (Vicugna pacos) <400> 14 atgggcgcgg acgttaaact ggttgaatct ggcggtggtc tggttcaacc gggcggtagt 60 ctgcgtctga gttgcgcagt ttctggtacc attctgggcc tgaacatgat gggttggtat 120 cgtcaagcac cgggtaaaca acgcgaactg gttgcgagca ttggctttgg cggtaacatt 180 gattacgcga acagcgtcaa aggccgtttt accattagtc gcgacggcga caaaaacatg 240 gtctctctgc agatgaatac cctgcgtccg gaagataccg cgatttatta ctgcgtcgcg 300 cgttggacct ttggtattca acgcggcgat tattggggtc aaggtaccca ggttaccgtt 360 agcagtgg 368 <210> 15 <211> 131 <212> PRT <213> Alpaca (Vicugna pacos) <400> 15 Met Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln 1 5 1'0 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe 20 25 30 Ser Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro 35 40 45 Glu Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val 50 55 60 Asp Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr 100 105 110 Gly Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr 115 120 125 Val Ser Ser 130 <210> 16 <211> 393 <212> DNA <213> Alpaca (Vicugna pacos) <400> 16 atgggcgcag acgttcaact ggttgaatct ggcggtggta tggttcaacc gggcggtagt 60 ctgcgtctga gttgcgcagc atctggtttt atcttctccc gccacgatat gcattgggta 120 cgtcaaggtc cgggtaaagg tccggaatgg gttagtctga ttaccaccgg cggcgaaggt 180 acctggtacg ttgatagcgt caaaggtcgt ttcagcatca gccgcgataa cgcgaaaaac 240 accgtctacc tgcagatgaa cgcactgaaa ccggaagata ccgcagttta ctattgcgca 300 gcacatcgcg cagaaattcc gcgcgaaatt tgctacggcg atagcctgag ctacaactat 360 tggggtcagg gtacccaagt taccgtttct agt 393 <210> 17 <211> 128 <212> PRT <213> Alpaca (Vicugna pacos) <400> 17 Met Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Leu Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu 20 25 30 Gln Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45 Glu Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro 100 105 110 Ala His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 18 <211> 386 <212> DNA <213> Alpaca (Vicugna pacos) <400> 18 atgggtgcgc aagttcagct ggttgaatct ggcggtggtc tggttcaact gggcggtagt 60 ctgcgtctga gttgtaccgc aagcgaaatt gacctgcagt actacccgat tgcttggttt 120 cgtcaagctc cgggtaaaga acgcgaaggc gttagttgta ttcgcgcaac cgatagcggt 180 acctattacg cggatagcgt caaaggtcgt ttcaccattg cgcgcgataa cgcgaaaaac 240 accgtctacc tgcagatgaa caatctggca ccggaagata ccgctgttta ctattgcggt 300 accgacgata gcgaagattg caccgattac gcaccggcac attacggtta ttggggtcgc 360 ggtacccaag ttaccgttag tagtgg 386 <210> 19 <211> 167[[ID=#39]] <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 19 Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser 130 135 140 Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile 145 150 155 160 Val Pro Ile Ile Arg Ile Gly 165 <210> 20 <211> 172 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 20 Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser 130 135 140 Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys 145 150 155 160 Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> twenty one <211> 150 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> twenty one Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val 130 135 140 Tyr Arg Gly Ile Tyr Arg 145 150 <210> 22 <211> 155 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> twenty two Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser 130 135 140 Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> twenty three <211> 158 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to the cleavage peptide Polybia-MP1 <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> twenty three Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser 130 135 140 Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> twenty four <211> 166 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> twenty four Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser Leu 130 135 140 Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val 145 150 155 160 Pro Ile Ile Arg Ile Gly 165 <210> 25 <211> 171 <212> PRT <213> artificial sequence <220> <223> sdAb37 linked by GSAA_linker to BMAP28A peptide <220> <221> Site <222> 127, 128 <223> Xaa can be any amino acid <400> 25 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30<0​​​​​​​​​​​​​​​​​​​​​​​​​​​ Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr 145 150 155 160 Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 26 <211> 149 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 26 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val Tyr 130 135 140 Arg Gly Ile Tyr Arg 145 <210> 27 <211> 154 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 27 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys 130 135 140 Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 28 <211> 157 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 28 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Ile 130 135 140 Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 29 <211> 167 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 29 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser 130 135 140 Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile 145 150 155 160 Val Pro Ile Ile Arg Ile Gly 165 <210> 30 <211> 172 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 30 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser 130 135 140 Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys 145 150 155 160 Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 31 <211> 150 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 31 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val 130 135 140 Tyr Arg Gly Ile Tyr Arg 145 150 <210> 32 <211> 155 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 32 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser 130 135 140 Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 33 <211> 158 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 33 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser 130 135 140 Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 34 <211> 164 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 125, 126 <223> Xaa can be any amino acid. <400> 34 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95[[ID=​​​​​​​​​​​​​​​​​​​​​​<221> Site <222> 125, 126 <223> Xaa can be any amino acid <400> 35 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly 115 120 125 Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Leu 130 135 140 Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro 145 150 155 160 Ile Ile Val Pro Ile Ile Arg Ile Gly 165 <210> 36 <211> 147 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 125, 126 <223> Xaa can be any amino acid. <400> 36 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val Tyr Arg Gly 130 135 140 Ile Tyr Arg 145 <210> 37 <211> 152 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 125, 126 <223> Xaa can be any amino acid. <400> 37 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly 115 120 125 Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys Trp Phe 130 135 140 Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 38 <211> 155 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 125, 126 <223> Xaa can be any amino acid. <400> 38 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly 115 120 125 Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Ile Asp Trp 130 135 140 Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 39 <211> 166 <212> PRT <213> Synthetic Sequence <220> <223> sdAb56 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 39 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser Leu 130 135 140 Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val 145 150 155 160 Pro Ile Ile Arg Ile Gly 165 <210> 40 <211> 171 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 40 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly 130 135 140 Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr 145 150 155 160 Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 41 <211> 149 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 41 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val Tyr 130 135 140 Arg Gly Ile Tyr Arg 145 <210> 42 <211> 154 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GSAA_linker <220> <221> Site <222> 127, 128 <223> Xaa can be any amino acid <400> 42 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys 130 135 140 Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 43 <211> 157 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 43 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Ile 130 135 140 Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 44 <211> 162 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 44 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile 130 135 140 Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg 145 150 155 160 Ile Gly <210> 45 <211> 167 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 45 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser 130 135 140 Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile 145 150 155 160 Val Pro Ile Ile Arg Ile Gly 165 <210> 46 <211> 145 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 46 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr 130 135 140 Arg 145 <210> 47 <211> 150 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 47 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys Trp Phe Arg Val 130 135 140 Tyr Arg Gly Ile Tyr Arg 145 150 <210> 48 <211> 153 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to the cleavage peptide Polybia-MP1 <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 48 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys 130 135 140 Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 <210> 49 <211> 163 <212> PRT <213> Artificial sequence <220> <223> sdAb62 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 49 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser Leu Gly Arg Lys 130 135 140 Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile 145 150 155 160 Arg Ile Gly <210> 50 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> sdAb62 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 50 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly 115 120 125 Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Leu Arg 130 135 140 Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile 145 150 155 160 Ile Val Pro Ile Ile Arg Ile Gly 165 <210> 51 <211> 146 <212> PRT <213> Artificial sequence <220> <223> sdAb62 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 51 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val Tyr Arg Gly Ile 130 135 140 Tyr Arg 145 <210> 52 <211> 151 <212> PRT <213> Artificial sequence <220> <223> sdAb62 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 52 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly 115 120 125 Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys Trp Phe Arg 130 135 140 Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 53 <211> 154 <212> PRT <213> Artificial sequence <220> <223> sdAb62 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 53 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly 115 120 125 Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Ile Asp Trp Lys 130 135 140 Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 <210> 54 <211> 172 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 133,134 <223> Xaa can be any amino acid. <400> 54 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys 145 150 155 160 Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 55 <211> 177 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 133,134 <223> Xaa can be any amino acid. <400> 55 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys 130 135 140 Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu 145 150 155 160 Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile 165 170 175 Gly <210> 56 <211> 155 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> Site <222> 133,134 <223> Xaa can be any amino acid <400> 56 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 57 <211> 160 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GSAA_linker <220> <221> site <222> 133,134 <223> Xaa can be any amino acid. <400> 57 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys 130 135 140 Gly Gly Gly Gly Ser Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 160 <210> 58 <211> 163 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 133,134 <223> Xaa can be any amino acid. <400> 58 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys 130 135 140 Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys 145 150 155 160 Gln Ile Leu <210> 59 <211> 169 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 59 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu 130 135 140 Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro 145 150 155 160 Ile Ile Val Pro Ile Ile Arg Ile Gly 165 <210> 60 <211> 174 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 60 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly 130 135 140 Gly Ser Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp 145 150 155 160 Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 61 <211> 152 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 61 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe 130 135 140 Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 62 <211> 157 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 62 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly 130 135 140 Gly Ser Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 63 <211> 160 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 63 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly 130 135 140 Gly Ser Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 160 <210> 64 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb26 <400> 64 Ile Tyr Asp Phe Gly 1 5 <210> 65 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb37 <400> 65 Ser Tyr Ala Met Ser 1 5 <210> 66 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb46 <400> 66 Asp Tyr Ala Leu Thr 1 5 <210> 67 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb47 <400> 67 Asn His Trp Met Tyr 1 5 <210> 68 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb56 <400> 68 Ser Tyr Trp Met His 1 5 <210> 69 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb59 <400> 69 Arg Tyr Gly Val Thr 1 5 <210> 70 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb62 <400> 70 Leu Asn Met Met Gly 1 5 <210> 71 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb63 <400> 71 Arg His Asp Met His 1 5 <210> 72 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb65 <400> 72 Tyr Tyr Pro Ile Ala 1 5 <210> 73 <211> 16 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb26 <400> 73 Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser Val Lys Gly 1 5 10 15 <210> 74 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb37 <400> 74 Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 75 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb46 <400> 75 Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser Met Lys 1 5 10 15 Gly <210> 76 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb47 <400> 76 Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Tyr Ala Asp Ser Val Trp 1 5 10 15 Gly <210> 77 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb56 <400> 77 Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 78 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb59 <400> 78 Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 79 <211> 16 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb62 <400> 79 Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser Val Lys Gly 1 5 10 15 <210> 80 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb63 <400> 80 Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp Ser Val Lys 1 5 10 15 Gly <210> 81 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb65 <400> 81 Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 82 <211> 15 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR3 of sdAb26 <400> 82 Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile Trp Ser 1 5 10 15 <210> 83 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR3 of sdAb37 <400> 83 Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His Tyr 1 5 10 <210> 84 <211> 15 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAb46's CDR3 <400> 84 Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu Asp Tyr 1 5 10 15 <210> 85 <211> 12 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR3 of sdAb47 <400> 85 Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr 1 5 10 <210> 86 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAb56's CDR3 <400> 86 Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp Tyr 1 5 10 <210> 87 <211> 9 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR3 of sdAb59 <400> 87 Asn Asp Ile Val Val Arg Gly Asn Tyr 1 5 <210> 88 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAb62's CDR3 <400> 88 Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr 1 5 10 <210> 89 <211> 19 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAb63 CDR3 <400> 89 His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly Asp Ser Leu Ser 1 5 10 15 Tyr Asn Tyr <210> 90 <211> 16 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR3 of sdAb65 <400> 90 Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala His Tyr Gly Tyr 1 5 10 15 <210> 91 <211> 33 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb26 <400> 91 Met Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe 20 25 30 Ser <210> 92 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb37 <400> 92 Met Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro 1 5 10 15<​​​​​​​​​​​ <220> <223> FR1 of sdAb46 <400> 93 Met Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp 20 25 30 <210> 94 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb47 <400> 94<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<210> 96 <211> 33 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb59 <400> 96 Met Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser <210> 97 <211> 33 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb62 <400> 97 Met Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu 20 25 30 Gly <210> 98 <211> 33 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb63 <400> 98 Met Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe 20 25 30 Ser <210> 99 <211> 33 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb65 <400> 99 Met Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Leu Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu 20 25 30 Gln <210> 100 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb26 <400> 100<00​​​​​​​​​​​​​​​​​​​ 1 5 10 <210> 102 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb46 <400> 102 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 103 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb47 <400> 103 Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 <210> 104 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb56 <400> 104 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys Val Ser 1 5 10 <210> 105 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb59 <400> 105 Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 106 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb62 <400> 106 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 107 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb63 <400> 107 Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu Trp Val Ser 1 5 10 <210> 108 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb65 <400> 108 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 <210> 109 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb26 <400> 109 Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val Ser Leu Gln 1 5 10 15 Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Val Ala 20 25 30 <210> 110 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb37 <400> 110 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys 20 25 30 <210> 111 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb46 <400> 111 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe Cys Thr Lys 20 25 30 <210> 112 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb47 <400> 112 Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg 20 25 30 <210> 113 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb56 <400> 113 Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 114 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb59 <400> 114 Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Tyr Cys Asn Val 20 25 30 <210> 115 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb62 <400> 115 Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val Ser Leu Gln 1 5 10 15 Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Val Ala 20 25 30 <210> 116 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb63 <400> 116 Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 117 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb65 <400> 117 Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr Tyr Cys Gly Thr 20 25 30 <210> 118 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb26 <400> 118 Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 119 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb37 <400> 119 Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 120 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb46 <400> 120 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 121 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb47 <400> 121 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 122 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb56 <400> 122 Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 123 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb59 <400> 123 Trp Gly Gln Glu Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 124 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb62 <400> 124 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 125 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb63 <400> 125 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 126 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb65 <400> 126 Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 127 <211> 10 <212> PRT <213> Artificial sequence <220> <223> GS_linker used as a linker between sdAb and cleaved peptides <220> <221> site <222> 3,4 <223> Xaa can be any amino acid. <400> 127 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 128 <211> 15 <212> PRT <213> Artificial sequence <220> <223> GSAA_linker used as a linker between sdAb and cleaved peptides <220> <221> site <222> 3,4 <223> Xaa can be any amino acid. <400> 128 Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser 1 5 10 15 <210> 129 <211> 28 <212> PRT <213> Cow (Bos taurus) <220> <223> BMAP28A <400> 129 Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys 1 5 10 15 Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 20 25 <210> 130 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Modified cysteine-deficient horseshoe crab antimicrobial peptide-I <400> 130 Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 1 5 10 <210> 131 <211> 14 <212> PRT <213> Wasp (Polybia paulista) <220> <223> Polybia-MP1 <400> 131 Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 1 5 10 <210> 132 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primer CALL001 for PCR amplification of the VHH variable region <400> 132 gtcctggctg ctcttctaca agg 23 <210> 133 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primer CALL002 for PCR amplification of the VHH variable region <400> 133 ggtacgtgct gttgaactgt tcc 23 <210> 134 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers VHH_back used for PCR amplification of the VHH variable region <400> 134 gatgtgcagc tgcaggagtc tggrggagg 29 <210> 135 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primers used for PCR amplification of the VHH variable region: VHH_for <400> 135 ggactagtgc ggccgctgga gacggtgacc tgg 33 <210> 136 <211> 121 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAb14 amino acid sequence <400> 136 Met Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala 1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val 20 25 30 Phe Ser Ser Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ser Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 65 70 75 80 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala 85 90 95 Val Tyr Phe Cys Leu His Ser Gly His Thr His Thr Arg Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser[[ID=二十九]] 115 120 <210> 137 <211> 129 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAb 60 amino acid sequence <400> 137 Met Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Pro Gly Leu Val 1 5 10 15 Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser 20 25 30 Ile Thr Thr Thr Phe Ser His Trp Thr Trp Ile Arg Gln Ser Pro Glu 35 40 45 Lys Gly Leu Glu Trp Met Gly Ala Ile Gly Tyr Gly Gly Ser Ile Tyr 50 55 60 Tyr Ser Pro Ser Phe Lys Ser Arg Thr Ser Ile Ser Arg Asp Thr Ser 65 70 75 80 Arg Asn Gln Phe Thr Leu His Leu Ser Ser Val Thr Pro Glu Asp Thr 85 90 95 Ala Val Tyr Tyr Cys Ala Arg Leu Pro His Ser Ser Gly Tyr Ala Lys 100 105 110 Phe Gly His Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 138 <211> 124 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAbS amino acid sequence <400> 138 Met Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Gly Leu Val 1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 20 25 30 Phe Ser Asn Tyr Trp Met Tyr Trp Ala Arg Gln Ala Pro Gly Lys Gly 35 40 45 Leu Glu Trp Val Ser Ala Ile Asp Gln Ser Gly Thr Thr Thr Arg Tyr 50 55 60 Ala Asp Ser Val Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys 65 70 75 80 His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 85 90 95 Leu Tyr Tyr Cys Thr Thr Ser Gly Arg Tyr Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Lys Gly Thr Gln Val Thr Val Ser Thr Ser Ser 115 120 <210> 139 <211> 363 <212> DNA <213> Alpaca (Vicugna pacos) <220> <223> sdAb14 nucleic acid sequence <400> 139 atgggaatgg ccgaggtgga ggtggtggcg tctgggggag acttggcgca gcctgggggg 60 tctctgagac tctcctgtgc agcctctgga ttcgtcttca gtagctatgc catgatctgg 120 gcccgccagg ctccaggaaa ggggctcgag tgggtgtccg gtattaatag tgatgctagt 180 ggcacatggt atgctgactc cgtgaagggc cgattcacta tctccagaga caacgccaag 240 aacacgttgt atctgcaaat gaacagcctg aaacctgatg acacggccgt gtatttctgt 300 cttcatagtg gtcatactca cacgcgctac tggggccagg ggacccaggt caccgtctcc 360 tcg 363 <210> 140 <211> 387 <212> DNA <213> Alpaca (Vicugna pacos) <220> <223> sdAb60 nucleic acid sequence <400> 140 atgggaatgg ccgaggtgga ggtggtggcg tcgggcccag gcctggtgaa accctcgcag 60 acactctccc tcacctgcac tgtctctggt gactccatca caaccacatt ttctcactgg 120 acctggatcc gccagtcccc agagaagggg ctggagtgga tgggagccat aggttatggt 180 ggcagtattt attacagccc atccttcaag agccgcactt ccatctccag ggacacgtcc 240 aggaaccaat ttaccctgca cctgtcatca gtgactcctg aagacacagc cgtctattac 300 tgtgccagac tcccccatag tagtggctat gccaagttcg gccaccttga ctactggggc 360 caggggaccc aggtcactgtctcctcc 387 <210> 141 <211> 372 <212> DNA <213> Alpaca (Vicugna pacos) <220> <223> sdAbS nucleic acid sequence <400> 141 atgggaatgg ccgaggtgga ggtggtggcg tctggggggag gcttggtgca gcctgggggg 60 tctctaagac tctcctgtgc agcctctgga ttcaccttca gtaactactg gatgtattgg 120 gcccgtcagg ctccagggaa ggggctcgag tgggtgtcag ctattagatca gagtggtact 180 accacaagat atgcagactc cgtgaagggc caattcacca tctctagaga caacgccaag 240 cacacgctgt atctgcaaat gaacagcctg aaacctgagg acacggccct gtattactgt 300 acgacctcag gcaggtacta cgccatggac tactggggca aagggaccca ggtcactgtc 360 tccacatcgt cg 372 <210> 142 <211> 162 <212> PRT <213> Artificial sequence <220> <223> sdAb 14 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 142 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe 20 25 30 Ser Ser Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val 85 90 95 Tyr Phe Cys Leu His Ser Gly His Thr His Thr Arg Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile 130 135 140 Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg 145 150 155 160 Ile Gly <210> 143 <211> 167 <212> PRT <213> Artificial sequence <220> <223> sdAb 14 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 143 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe 20 25 30 Ser Ser Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val 85 90 95 Tyr Phe Cys Leu His Ser Gly His Thr His Thr Arg Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser 130 135 140 Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile 145 150 155 160 Val Pro Ile Ile Arg Ile Gly 165 <210> 144 <211> 150 <212> PRT <213> Artificial sequence <220> <223> sdAb14 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 144 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe 20 25 30 Ser Ser Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val 85 90 95 Tyr Phe Cys Leu His Ser Gly His Thr His Thr Arg Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys Trp Phe Arg Val 130 135 140 Tyr Arg Gly Ile Tyr Arg 145 150 <210> 145 <211> 150 <212> PRT <213> Artificial sequence <220> <223> sdAb14 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via GSAA_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 145 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe 20 25 30 Ser Ser Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val 85 90 95 Tyr Phe Cys Leu His Ser Gly His Thr His Thr Arg Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys Trp Phe Arg Val 130 135 14"0 Tyr Arg Gly Ile Tyr Arg 145 150 <210> 146 <211> 153 <212> PRT <213> Artificial Sequence <220> [[ID=**46**]]<223> sdAb 14 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> Site <222> 123, 124 <223> Xaa can be any amino acid <400> 146 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe 20 25 30 Ser Ser Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val 85 90 95<00033​​​​​​​​​​​ Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 <210> 147 <211> 170 <212> PRT <213> Artificial sequence <220> <223> sdAb60 linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 131,132 <223> Xaa can be any amino acid. <400> 147 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile 20 25 30 Thr Thr Thr Phe Ser His Trp Thr Trp Ile Arg Gln Ser Pro Glu Lys 35 40 45 Gly Leu Glu Trp Met Gly Ala Ile Gly Tyr Gly Gly Ser Ile Tyr Tyr 50 55 60 Ser Pro Ser Phe Lys Ser Arg Thr Ser Ile Ser Arg Asp Thr Ser Arg 65 70 75 80 Asn Gln Phe Thr Leu His Leu Ser Ser Val Thr Pro Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Leu Pro His Ser Ser Gly Tyr Ala Lys Phe 100 105 110 Gly His Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Xaa Xaa Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly 145 150 155 160 Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 148 <211> 175 <212> PRT <213> Artificial sequence <220> <223> sdAb60 linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 131,132 <223> Xaa can be any amino acid. <400> 148 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile 20 25 30 Thr Thr Thr Phe Ser His Trp Thr Trp Ile Arg Gln Ser Pro Glu Lys 35 40 45 Gly Leu Glu Trp Met Gly Ala Ile Gly Tyr Gly Gly Ser Ile Tyr Tyr 50 55 60 Ser Pro Ser Phe Lys Ser Arg Thr Ser Ile Ser Arg Asp Thr Ser Arg 65 70 75 80 Asn Gln Phe Thr Leu His Leu Ser Ser Val Thr Pro Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Leu Pro His Ser Ser Gly Tyr Ala Lys Phe 100 105 110 Gly His Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly 130 135 140 Gly Gly Ser Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala 145 150 155 160 Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 175 <210> 149 <211> 153 <212> PRT <213> Synthetic Sequence <220> <223> sdAb60 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 131,132 <223> Xaa can be any amino acid. <400> 149 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile 20 25 30 Thr Thr Thr Phe Ser His Trp Thr Trp Ile Arg Gln Ser Pro Glu Lys 35 40 45 Gly Leu Glu Trp Met Gly Ala Ile Gly Tyr Gly Gly Ser Ile Tyr Tyr 50 55 60 Ser Pro Ser Phe Lys Ser Arg Thr Ser Ile Ser Arg Asp Thr Ser Arg 65 70 75 80 Asn Gln Phe Thr Leu His Leu Ser Ser Val Thr Pro Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Leu Pro His Ser Ser Gly Tyr Ala Lys Phe 100 105 110 Gly His Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp 130 135 140 Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 150 <211> 158 <212> PRT <213> Artificial sequence <220> <223> sdAb60 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GSAA_linker <220> <221> site <222> 131,132 <223> Xaa can be any amino acid. <400> 150 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile 20 25 30 Thr Thr Thr Phe Ser His Trp Thr Trp Ile Arg Gln Ser Pro Glu Lys 35 40 45 Gly Leu Glu Trp Met Gly Ala Ile Gly Tyr Gly Gly Ser Ile Tyr Tyr 50 55 60 Ser Pro Ser Phe Lys Ser Arg Thr Ser Ile Ser Arg Asp Thr Ser Arg 65 70 75 80 Asn Gln Phe Thr Leu His Leu Ser Ser Val Thr Pro Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Leu Pro His Ser Ser Gly Tyr Ala Lys Phe 100 105 110 Gly His Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly 130 135 140 Gly Gly Ser Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 151 <211> 161 <212> PRT <213> Artificial sequence <220> <223> sdAb60 linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 131,132 <223> Xaa can be any amino acid. <400> 151 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile 20 25 30 Thr Thr Thr Phe Ser His Trp Thr Trp Ile Arg Gln Ser Pro Glu Lys 35 40 45 Gly Leu Glu Trp Met Gly Ala Ile Gly Tyr Gly Gly Ser Ile Tyr Tyr 50 55 60 Ser Pro Ser Phe Lys Ser Arg Thr Ser Ile Ser Arg Asp Thr Ser Arg 65 70 75 80 Asn Gln Phe Thr Leu His Leu Ser Ser Val Thr Pro Glu Asp Thr Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Leu Pro His Ser Ser Gly Tyr Ala Lys Phe 100 105 110 Gly His Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly 130 135 140 Gly Gly Ser Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile 145 150 155 160 Leu <210> 152 <211> 165 <212> PRT <213> Artificial sequence <220> <223> sdAbS_ linked to the cleavage peptide BMAP28A via GS_linker <220> <221> site <222> 126,127 <223> Xaa can be any amino acid <400> 152 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Asn Tyr Trp Met Tyr Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Ala Ile Asp Gln Ser Gly Thr Thr Thr Arg Tyr Ala 50 55 60 Asp Ser Val Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys His 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu 85 90 95 Tyr Tyr Cys Thr Thr Ser Gly Arg Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Lys Gly Thr Gln Val Thr Val Ser Thr Ser Ser Ala Ala Xaa Xaa Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Arg Ser Leu Gly 130 135 140 Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro 145 150 155 160 Ile Ile Arg Ile Gly 165 <210> 153 <211> 170 <212> PRT <213> Artificial sequence <220> <223> sdAbS_ linked to the cleavage peptide BMAP28A via GSAA_linker <220> <221> site <222> 126,127 <223> Xaa can be any amino acid. <400> 153 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Asn Tyr Trp Met Tyr Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Ala Ile Asp Gln Ser Gly Thr Thr Thr Arg Tyr Ala 50 55 60 Asp Ser Val Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys His 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu 85 90 95 Tyr Tyr Cys Thr Thr Ser Gly Arg Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Lys Gly Thr Gln Val Thr Val Ser Thr Ser Ser Ala Ala Xaa Xaa Gly 115 120 125 Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Gly Gly 130 135 140 Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly 145 150 155 160 Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 154 <211> 148 <212> PRT <213> Artificial sequence <220> <223> sdAbS linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GS_linker <220> <221> site <222> 126,127 <223> Xaa can be any amino acid. <400> 154 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Asn Tyr Trp Met Tyr Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Ala Ile Asp Gln Ser Gly Thr Thr Thr Arg Tyr Ala 50 55 60 Asp Ser Val Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys His 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu 85 90 95 Tyr Tyr Cys Thr Thr Ser Gly Arg Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Lys Gly Thr Gln Val Thr Val Ser Thr Ser Ser Ala Ala Xaa Xaa Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Trp Phe Arg Val Tyr Arg 130 135 140 Gly Ile Tyr Arg 145 <210> 155 <211> 153 <212> PRT <213> Artificial sequence <220> <223> sdAbS linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with GSAA_linker <220> <221> site <222> 126,127 <223> Xaa can be any amino acid. <400> 155 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Asn Tyr Trp Met Tyr Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Ala Ile Asp Gln Ser Gly Thr Thr Thr Arg Tyr Ala 50 55 60 Asp Ser Val Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys His 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu 85 90 95 Tyr Tyr Cys Thr Thr Ser Gly Arg Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Lys Gly Thr Gln Val Thr Val Ser Thr Ser Ser Ala Ala Xaa Xaa Gly 115 120 125 Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Lys Trp 130 135 140 Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 156 <211> 156 <212> PRT <213> Artificial sequence <220> <223> sdAbS_ linked to the cleavage peptide Polybia-MP1 via GSAA_linker <220> <221> site <222> 126,127 <223> Xaa can be any amino acid. <400> 156 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Asn Tyr Trp Met Tyr Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Ala Ile Asp Gln Ser Gly Thr Thr Thr Arg Tyr Ala 50 55 60 Asp Ser Val Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys His 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu 85 90 95 Tyr Tyr Cys Thr Thr Ser Gly Arg Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Lys Gly Thr Gln Val Thr Val Ser Thr Ser Ser Ala Ala Xaa Xaa Gly 115 120 125 Gly Gly Gly Ser Glu Ala Ala Ala Lys Gly Gly Gly Gly Ser Ile Asp 130 135 140 Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 157 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb14 <400> 157 Ser Tyr Ala Met Ile 1 5 <210> 158 <211> 7 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR1 of sdAb60 <400> 158 Thr Thr Phe Ser His Trp Thr 1 5 <210> 159 <211> 5 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAbS's CDR1 <400> 159 Asn Tyr Trp Met Tyr 1 5 <210> 160 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb14 <400> 160 Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 161 <211> 16 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR2 of sdAb60 <400> 161 Ala Ile Gly Tyr Gly Gly Ser Ile Tyr Tyr Ser Pro Ser Phe Lys Ser 1 5 10 15 <210> 162 <211> 17 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAbS's CDR2 <400> 162 Ala Ile Asp Gln Ser Gly Thr Thr Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 163 <211> 8 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> CDR3 of sdAb14 <400> 163 Ser Gly His Thr His Thr Arg Tyr 1 5 <210> 164 <211> 13 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAb60's CDR3 <400> 164 Leu Pro His Ser Ser Gly Tyr Ala Lys Phe Gly His Leu 1 5 10 <210> 165 <211> 7 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAbS's CDR3 <400> 165 Ser Gly Arg Tyr Tyr Ala Met 1 5 <210> 166 <211> 34 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb14 <400> 166 Met Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala 1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val 20 25 30 Phe Ser <210> 167 <211> 35 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR1 of sdAb60 <400> 167 Met Gly Met Ala Glu Glu Val Glu Val Val Ala Ser Gly Pro Gly Leu 1 5 10 15 Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp 20 25 30 Ser Ile Thr 35 <210> 168 <211> 34 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> sdAbS <400> 168 Met Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Gly Leu Val 1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 20 25 30 Phe Ser <210> 169 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb14 <400> 169 Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10​​​​​​​<213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAb60 <400> 170 Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Met Gly 1 5 10 <210> 171 <211> 14 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR2 of sdAbS <400> 171 Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 172 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb14 <400> 172 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Phe Cys Leu His 20 25 30 <210> 173 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAb60 <400> 173 Arg Thr Ser Ile Ser Arg Asp Thr Ser Arg Asn Gln Phe Thr Leu His 1 5 10 15 Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 174 <211> 32 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR3 of sdAbS <400> 174 Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys His Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Thr 20 25 30 <210> 175 <211> 11 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb14 <400> 175 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 176 <211> 13 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAb60 <400> 176 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 177 <211> 15 <212> PRT <213> Alpaca (Vicugna pacos) <220> <223> FR4 of sdAbS <400> 177 Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Thr Ser Ser 1 5 10 15 <210> 178 <211> 17 <212> PRT <213> Artificial sequence <220> <223> SRGS_linker used as a linker between sdAb and cleaved peptides <220> <221> site <222> 3,4 <223> Xaa can be any amino acid. <400> 178 Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser 1 5 10 15 Gly <210> 179 <211> 174 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 179 Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly 130 135 140 Ser Gly Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp 145 150 155 160 Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 180 <211> 157 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via SRGS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 180 Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly 130 135 140 Ser Gly Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 181 <211> 153 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to the cleavage peptide Polybia-MP1 via GS linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 181 Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys 130 135 140 Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 <210> 182 <211> 160 <212> PRT <213> Artificial sequence <220> <223> sdAb26 linked to the cleavage peptide Polybia-MP1 via SRGS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 182 Gly Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Met Thr Phe Ser 20 25 30 Ile Tyr Asp Phe Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Tyr Ile 100 105 110 Trp Ser Trp Lys Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly 130 135 140 Ser Gly Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 160 <210> 183 <211> 173 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 183 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser 130 135 140 Gly Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys 145 150 155 160 Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 184 <211> 156 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via SRGS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 184 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser 130 135 140 Gly Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 185 <211> 152 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 185 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys Leu 130 135 140 Leu Asp Ala Ala Lys Gln Ile Leu 145 150 <210> 186 <211> 159 <212> PRT <213> Artificial sequence <220> <223> sdAb37 linked to the cleavage peptide Polybia-MP1 via SRGS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 186 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Tyr Arg Asp Gly Ile Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Leu Leu Ala Gly Trp Gly Pro Leu Ile Ser Trp His 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser 130 135 140 Gly Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 187 <211> 174 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 187 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly 130 135 140 Ser Gly Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp 145 150 155 160 Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 188 <211> 157 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via SRGS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 188 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly 130 135 140 Ser Gly Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 189 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> sdAb46 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 189 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys 130 135 140 Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 <210> 190 <211> 160 <212> PRT <213> Artificial sequence <220> <223> sdAb46 linked to the cleavage peptide Polybia-MP1 via SRGS_linker <220> <221> site <222> 128,129 <223> Xaa can be any amino acid. <400> 190 Gly Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Gly Ile Asn Trp Asn Gly Asp Asp Thr Tyr Tyr Thr Gln Ser 50 55 60 Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Phe 85 90 95 Cys Thr Lys Asn Val Gly Gly Asn Trp Val Ser Gly Asp Tyr Gly Leu 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa 115 120 125 Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly 130 135 140 Ser Gly Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 160 <210> 191 <211> 171 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 125, 126 <223> Xaa can be any amino acid. <400> 191 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg 115 120 125 Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Gly 130 135 140 Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr 145 150 155 160 Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 192 <211> 154 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via SRGS_linker <220> <221> site <222> 125,126 <223> Xaa can be any amino acid <400> 192 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg 115 120 125 Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Lys 130 135 140 Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 193 <211> 150 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 125, 126 <223> Xaa can be any amino acid. <400> 193 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys Leu Leu Asp 130 135 140 Ala Ala Lys Gln Ile Leu 145 150 <210> 194 <211> 157 <212> PRT <213> Artificial sequence <220> <223> sdAb47 linked to the cleavage peptide Polybia-MP1 via SRGS_linker <220> <221> site <222> 125, 126 <223> Xaa can be any amino acid. <400> 194 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asn 20 25 30 His Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Thr Ala Asn Thr Gly Gly Arg Asp Thr Tyr Tyr Ala Asp Ser 50 55 60 Val Trp Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Glu Val Asn Asp Tyr Leu Glu Tyr Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg 115 120 125 Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Ile 130 135 140 Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 195 <211> 173 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 195 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser 130 135 140 Gly Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys 145 150 155 160 Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 196 <211> 156 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with SRGS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 196 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser 130 135 140 Gly Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 197 <211> 152 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 197 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys Leu 130 135 140 Leu Asp Ala Ala Lys Gln Ile Leu 145 150 <210> 198 <211> 159 <212> PRT <213> Artificial sequence <220> <223> sdAb56 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 127,128 <223> Xaa can be any amino acid. <400> 198 Gly Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys 35 40 45 Val Ser Val Ile Asn Thr Gly Asn Gly Ser Pro Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Ile Pro Asp Pro Ala Gly Arg Ala Gly Gly Met Asp 100 105 110 Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa 115 120 125 Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser 130 135 140 Gly Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 199 <211> 169 <212> PRT <213> artificial sequence <220> <223> sdAb59 linked by SRGS_linker to BMAP28A peptide <220> <221> Site <222> 123,124 <223> Xaa can be any amino acid <400> 199 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg Gly Ser [[ID=第37行]] 115 120 125 Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly Leu 130 135 140 Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro 145 150 155 160 Ile Ile Val Pro Ile Ile Arg Ile Gly 165 <210> 200 <211> 152 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with SRGS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 200 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg Gly Ser 115 120 125 Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Lys Trp Phe 130 135 140 Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 201 <211> 148 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 201 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala 130 135 140 Lys Gln Ile Leu 145 <210> 202 <211> 155 <212> PRT <213> Artificial sequence <220> <223> sdAb59 linked to the cleavage peptide Polybia-MP1 via SRGS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 202 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Thr Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Arg Tyr Gly Val Thr Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Leu Val Ala Asp Ile Ile Phe Thr Gly Leu Asn Pro Thr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Val 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr 85 90 95 Tyr Cys Asn Val Asn Asp Ile Val Val Arg Gly Asn Tyr Trp Gly Gln 100 105 110 Glu Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg Gly Ser 115 120 125 Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Ile Asp Trp 130 135 140 Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 203 <211> 170 <212> PRT <213> Artificial sequence <220> <223> sdAb62 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 203 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg Gly 115 120 125 Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly 130 135 140 Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys Tyr Gly 145 150 155 160 Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 <210> 204 <211> 153 <212> PRT <213> Artificial sequence <220> <223> sdAb62 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via SRGS_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 204 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg Gly 115 120 125 Ser Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Lys Trp 130 135 140 Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 <210> 205 <211> 149 <212> PRT <213> Artificial sequence <220> <223> sdAb62 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 124,125 <223> Xaa can be any amino acid. <400> 205 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Ile Asp Trp Lys Lys Leu Leu Asp Ala 130 135 140 Ala Lys Gln Ile Leu 145 <210> 206 <211> 156 <212> PRT <213> artificial sequence <220> <223> sdAb62 linked by SRGS_linker to Polybia-MP1 peptide <220> <221> Site <222> 124, 125 <223> Xaa can be any amino acid <400> 206 Gly Ala Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Leu Gly 20 25 30 Leu Asn Met Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40 45 Leu Val Ala Ser Ile Gly Phe Gly Gly Asn Ile Asp Tyr Ala Asn Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Asp Lys Asn Met Val 65 70 75 80 Ser Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Ala Arg Trp Thr Phe Gly Ile Gln Arg Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg Gly 115 120 125 Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Ser Gly Ile Asp 130 135 140 Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 207 <211> 179 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 133,134 <223> Xaa can be any amino acid. <400> 207 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser 130 135 140 Gly Ser Ser Gly Gly Ser Gly Gly Gly Leu Arg Ser Leu Gly Arg Lys 145 150 155 160 Ile Leu Arg Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile 165 170 175 Arg Ile Gly <210> 208 <211> 162 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via SRGS_linker <220> <221> site <222> 133,134 <223> Xaa can be any amino acid. <400> 208 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser 130 135 140 Gly Ser Ser Gly Gly Ser Gly Lys Trp Phe Arg Val Tyr Arg Gly Ile 145 150 155 160 Tyr Arg <210> 209 <211> 158 <212> PRT <213> Artificial Sequence <220> <223> sdAb63 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> Site <222> 133, 134 <223> Xaa can be any amino acid <400> 209 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 210 <211> 165 <212> PRT <213> Artificial sequence <220> <223> sdAb63 linked to the cleavage peptide Polybia-MP1 via SRGS_linker <220> <221> site <222> 133,134 <223> Xaa can be any amino acid. <400> 210 Gly Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg His Asp Met His Trp Val Arg Gln Gly Pro Gly Lys Gly Pro Glu 35 40 45 Trp Val Ser Leu Ile Thr Thr Gly Gly Glu Gly Thr Trp Tyr Val Asp 50 55 60 Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Arg Ala Glu Ile Pro Arg Glu Ile Cys Tyr Gly 100 105 110 Asp Ser Leu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Ala Ala Xaa Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser 130 135 140 Gly Ser Ser Gly Gly Ser Gly Ile Asp Trp Lys Lys Leu Leu Asp Ala 145 150 155 160 Ala Lys Gln Ile Leu 165 <210> 211 <211> 176 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 211 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser 130 135 140 Gly Gly Ser Gly Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg 145 150 155 160 Ala Trp Lys Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly 165 170 175 <210> 212 <211> 159 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to cysteine-deficient horseshoe crab antimicrobial peptide-I modified with cleavage peptide via SRGS_linker <220> <221> Site <222> 130, 131 <223> Xaa can be any amino acid <400> 212 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser 130 135 140 Gly Gly Ser Gly Lys Trp Phe Arg Val Tyr Arg Gly Ile Tyr Arg 145 150 155 <210> 213 <211> 155 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to the cleavage peptide Polybia-MP1 via GS_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 213 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asp Trp 130 135 140 Lys Lys Leu Leu Asp Ala Ala Lys Gln Ile Leu 145 150 155 <210> 214 <211> 162 <212> PRT <213> Artificial sequence <220> <223> sdAb65 linked to the cleavage peptide Polybia-MP1 via SRGS_linker <220> <221> site <222> 130,131 <223> Xaa can be any amino acid. <400> 214 Gly Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Leu 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Glu Ile Asp Leu Gln 20 25 30 Tyr Tyr Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Arg Ala Thr Asp Ser Gly Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Gly Thr Asp Asp Ser Glu Asp Cys Thr Asp Tyr Ala Pro Ala 100 105 110 His Tyr Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala 115 120 125 Ala Xaa Xaa Ser Arg Gly Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser 130 135 140 Gly Gly Ser Gly Ile Asp Trp Lys Lys Leu Leu Asp Ala Ala Lys Gln 145 150 155 160 Ile Leu <210> 215 <211> 169 <212> PRT <213> Artificial sequence <220> <223> sdAb14 linked to the cleavage peptide BMAP28A via SRGS_linker <220> <221> site <222> 123,124 <223> Xaa can be any amino acid. <400> 215 Gly Met Ala Glu Val Glu Val Val Ala Ser Gly Gly Asp Leu Ala Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe 20 25 30 Ser Ser Tyr Ala Met Ile Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ser Gly Ile Asn Ser Asp Ala Ser Gly Thr Trp Tyr Ala 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val 85 90 95 Tyr Phe Cys Leu His Ser Gly His Thr His Thr Arg Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ala Xaa Xaa Ser Arg Gly Ser 115 120 125 Ser Gly Ser Ser Ser Ser Gly Ser Ser Gly Gly Se...

Claims

1. A polypeptide that specifically binds to Globo H, wherein the polypeptide is a monomer or trimer of a single-domain antibody, the single-domain antibody is a variable domain of a heavy chain antibody that is naturally free of light chains and naturally free of constant region 1, or a variant of the variable domain, and wherein at least one complementarity determining region of the single-domain antibody is: (a) CDR1 is the amino acid sequence of SEQ ID NO: 66; (b) CDR2 is the amino acid sequence of SEQ ID NO: 75; (c) CDR3 has the amino acid sequence of SEQ ID NO:

84. 2 . The polypeptide according to claim 1 , wherein the amino acid sequence of the at least one single domain antibody is the sequence of SEQ ID NO:

5. 3 .

3. The polypeptide of claim 1, wherein the at least one single domain antibody is a humanized single domain antibody.

4. The polypeptide according to claim 3, wherein the single domain antibody is humanized by replacing one or more amino acid residues located at positions 1, 5, 11, 28 and 30 in FR1, positions 44 and 45 in FR2, positions 74, 75, 76, 83, 84, 93 and 94 in FR3; positions 104 and 108 in FR4 according to Kabat numbering.

5. A recombinant nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 4. A vector comprising the recombinant nucleic acid molecule according to claim 5 . A host cell comprising the recombinant nucleic acid molecule according to claim 5 .

8. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 4 and at least one pharmaceutically acceptable medium, excipient and / or diluent.

9. Use of the polypeptide according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating and / or diagnosing breast cancer, wherein the breast cancer cells express Globo H on their surface.

10. A diagnostic kit comprising the polypeptide according to any one of claims 1 to 4, for screening cancer characterized by expression of Globo H on the cell surface.

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