Circular epitope tag

Abstract

This invention relates to cyclic peptides and compositions thereof that are useful as epitope tags. Furthermore, this invention relates to methods for using them in complex formation.

Description

[Technical field]

[0001] The present invention relates to cyclic peptides useful as epitope tags and compositions thereof, and further to methods of their use in complex formation. [Background technology]

[0002] Epitope tagging is a technique that links epitopes to compounds, such as proteins. Epitope-tagged compounds, such as fusion proteins, can be labeled using an antibody specific to the epitope tag. The antibody may be linked to an additional compound, thus resulting in an interaction between the two compounds. Epitope tags are widely used as tools to detect, purify and manipulate compounds, such as proteins. Epitope tagging has proven to be an efficient method that allows immunochemical and immunocytochemical methods for target compounds, such as proteins. Epitope tagging can be used for a variety of applications, including purification, Western blotting, immunoprecipitation, flow cytometry, and immunofluorescence. Epitope tagging can also be used for diagnostic or therapeutic purposes.

[0003] Recently, an epitope tag / binder system (ALFA system) has been described that includes the ALFA tag and an ALFA-specific single domain antibody (sdAb), the NbALFA nanobody (Gotzke, H. et al, Nat Commun 10, 4403 (2019)). The ALFA system is suitable for a wide range of applications and offers an excellent and versatile alternative to the most common epitope tag systems. Summary of the Invention [Problem to be solved by the invention]

[0004] However, the stability of the ALFA tag was found to be compromised, as it is susceptible to enzymatic cleavage in serum and plasma. This may limit the application of the ALFA system, especially in in vivo settings. Therefore, we attempted to modify the ALFA system by adding a cyclic ring to the ALFA tag that would protect the peptide from enzymatic cleavage while maintaining the specificity and favorable biochemical properties of the ALFA system. [Means for solving the problem]

[0005] Disclosed is a peptide moiety that includes the following cyclized amino acid sequence: -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, (wherein any two side chains of the amino acids AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 (X1, X2) are covalently bonded; and The amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 that are not X1 and X2 are: AA0 is Pro or deleted; AA1 is Ser, Gly, Thr or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln or Glu; AA9 is Arg, Ala, Gln or Glu; AA10 is Arg; AA11 is Leu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or is deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; and AA14 is Pro or deleted)

[0006] The peptides described herein are useful as epitope tags. In particular, the peptides described herein are useful as epitope tags in the ALFA system-epitope tag / binder system, including ALFA-specific single domain antibodies (sdAbs), NbALFA nanobodies. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows embodiments of cyclic ALFA peptides that exhibit longer half-lives in human and mouse plasma in in vitro assays. SD 14316: Ac-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2; SD 14626: X-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2; SD 15194: Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2; SD 15196: Ac-Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2; SD 15192:Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-(cyclo8)Lys-Arg-Arg-Leu-Thr-Glu-NH2; SD 15193:Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-(cyclo13)Glu-NH2;

[0008] [Diagram 2] FIG. 2 shows an embodiment of a cyclic peptide that retains binding affinity to anti-ALFA VHH with an EC50 of 6.0 nM compared to the parent linear ALFA peptide of 2.6 nM. SD 15197: Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2

[0009] [Diagram 3] Figure 3: UPLC-UV214 chromatogram of peptide Ac-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-NH2.

[0010] [Figure 4] FIG. 4 is a mass spectrometer spectrograph of the peptide Ac-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-NH2.

[0011] [Diagram 5] FIG. 5 shows FACS data demonstrating that both linear and cyclic ALFA peptides bind only to target-expressing cells that were pre-incubated with NbALFA×anti-target adaptor protein.

[0012] [Figure 6] FIG. 6 is a graph showing the NbALFA binding behavior of linear alpha tag, Ac-ALFA-amide 2 (SD15197).

[0013] [Figure 7] FIG. 7 is a graph showing the NbALFA binding behavior of Ac-5-8Cyclo (SD15192), a cyclic alpha tag.

[0014] [Figure 8]FIG. 8 is a graph showing the NbALFA binding behavior of the cyclic alpha tag, Ac-9-13Cyclo (SD15193).

[0015] [Figure 9] FIG. 9 is a graph showing the NbALFA binding behavior of cyclic alpha tag, Ac-5E-9Cyclo (SD15194).

[0016] [Figure 10] FIG. 10 is a graph showing the NbALFA binding behavior of cyclic alpha tag, Ac-5D-9Cyclo (SD15196).

[0017] [Figure 11] FIG. 11 is a graph showing the NbALFA binding behavior of cyclic alpha tag SD15341: SD 15341: Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclo10)Lys-Arg-Leu-Thr-Glu-NH2.

[0018] [Figure 12] FIG. 12 is a graph showing the NbALFA binding behavior of cyclic alpha tag, SD15805: SD 15805: Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Lys-Glu-Leu-Arg-(cyclo10)Glu-Arg-Leu-Thr-Glu-NH2.

[0019] [Figure 13] FIG. 13 is a graph showing the NbALFA binding behavior of cyclic alpha tag SD15806: SD 15806: Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2;

[0020] [Figure 14]FIG. 14 is a graph showing the NbALFA binding behavior of cyclic alpha tag, SD15807: SD 15807: Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Cys-Glu-Leu-(cyclo9)dCys-Arg-Arg-Leu-Thr-Glu-NH2.

[0021] [Figure 15] FIG. 15 is a graph showing the NbALFA binding behavior of cyclic alpha tag, SD15808: SD 15808: Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclo10)dLys-Arg-Leu-Thr-Glu-NH2.

[0022] [Figure 16] FIG. 16 is a graph showing comparative plasma concentrations (in mice) and PK parameters for linear and cyclic alpha tag.

[0023] [Figure 17] FIG. 17 is a graph showing comparative plasma concentrations (in mice) and PK parameters for linear and cyclic alpha tag. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present disclosure will be described in more detail below, but it should be understood that the disclosure is not limited to the specific methods, protocols and reagents described herein, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0025] In the following, the elements of the present disclosure are described in more detail. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present disclosure to only the embodiments explicitly described. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0026] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques described in the art.

[0027] Throughout this specification and the claims, unless otherwise required by context, the word "comprises" and variations such as "comprising" are understood to mean the inclusion of a described feature, element, member, integer or step or group of features, elements, members, integers or steps, but not the exclusion of any other feature, element, member, integer or step or group of features, elements, members, integers or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified feature, element, member, integer or step, as well as those that do not materially affect the basic and novel feature(s) of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified feature, element, member, integer or step. The term "comprising" encompasses the term "consisting essentially of", which in turn encompasses the term "consisting of". Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of". Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of".

[0028] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the," and similar references, unless otherwise indicated herein or clearly contradicted by context, should be construed to encompass both the singular and the plural.

[0029] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0030] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0031] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0032] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0033] In the context of the present disclosure, the term "about" denotes an interval of accuracy that a person skilled in the art would understand to still guarantee the technical effect of the feature in question. The term typically denotes a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, e.g., ±0.01% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±10% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±5% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±4% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±3% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±2% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±1% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.9% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.8% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.7% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.6% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.5% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.4% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.3% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.2% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.1% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.05% from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.01% from the indicated numerical value. As will be appreciated by those skilled in the art, such specific deviations for the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than artificial or engineered technical effects.

[0034] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein.

[0035] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0036] Disclosed herein are peptides containing cyclized peptide sequences for use as epitope tags, and compounds containing such peptides.

[0037] The term "peptide" refers to a substance that contains about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules. Thus, the terms "peptide", "protein" and "polypeptide" are generally used synonymously herein.

[0038] In some embodiments, a peptide comprising a cyclized peptide sequence disclosed herein has a length of about 11 to about 100 amino acids, e.g., about 12 to about 50 amino acids, e.g., about 13 to about 25 amino acids, e.g., about 13, about 14, about 15, about 16, about 17, about 18, or about 19 amino acids.

[0039] In some embodiments, the peptides disclosed herein are composed of naturally occurring amino acids, non-naturally occurring amino acids, amino acid derivatives and non-amino acid components, or mixtures thereof. In some embodiments, the peptides disclosed herein include amino acid mimetics and amino acid analogs. In some embodiments, the peptides disclosed herein include non-natural amino acid sequences that are resistant to enzymatic cleavage.

[0040] In some embodiments, one or more positions of the peptides disclosed herein are substituted with a non-naturally occurring amino acid, in some embodiments, the substituted amino acid is chemically related to the original residue (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic) or an isostere of the original residue.

[0041] In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a peptide, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including the carboxy- and / or amino-terminal amino acids in a peptide, may include structural modifications compared to the general structures described above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a peptide containing the modified amino acid, compared to a peptide containing an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a peptide containing the modified amino acid, compared to a peptide containing an otherwise identical unmodified amino acid. As is clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments, it may be used to refer to an amino acid residue of a peptide.

[0042] The following table lists the 20 naturally occurring amino acids and their abbreviations. [Table 1]

[0043] Generally, amino acids are L-amino acids, with D-amino acids designated by the prefix "D." The prefix "homo" or "h" denotes an α-amino acid that is otherwise similar to the common one but contains another methylene group in the carbon chain.

[0044] As used herein, "Orn" means ornithine or 2,5-diaminopentanoic acid, "Dab" means 2,4-diaminobutanoic acid, "Dap" means 2,3-diaminopropanoic acid, "hLys" means 2,7-diaminoheptanoic acid, "hCys" means 2-amino-4-mercaptobutanoic acid, and "Pen" means penicillamine or 2-amino-3-methyl-3-sulfanylbutanoic acid.

[0045] It may also be possible to include non-peptide bonds and other chemical modifications.For example, part or all of a peptide can be synthesized as a peptide mimetic, such as a peptoid (see, for example, Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends Biotechnol.13:132-4).A peptide can include one or more (e.g., all) non-hydrolyzable bonds.Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesizing peptides that contain such bonds. Exemplary non-hydrolyzable bonds include, -[CH2NH]-reduced amide peptide bond, -[COCH2]-ketomethylene peptide bond, -[CH(CN)NH]-(cyanomethylene)amino peptide bond, -[CH2CH(OH)]-hydroxyethylene peptide bond, -[CHO]-oxymethylene peptide bond, and -[CH2S]-thiomethylene peptide bond (see, e.g., U.S. Pat. No. 6,172,043).

[0046] As used herein, "epitope tag" refers to a peptide to which an antibody or a proteinaceous molecule with antibody-like function can bind.

[0047] In some embodiments, the peptides, compounds or complexes described herein are isolated.

[0048] "Isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition from a manufacturing process. For example, a peptide or polypeptide that is naturally present in a living animal is not "isolated," but the same peptide or polypeptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated peptide or polypeptide may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0049] The peptides disclosed herein include at least one cyclic portion, i.e., a polypeptide chain that includes a cyclic sequence of bonds, referred to herein as a "cyclic peptide." The cyclic sequence may result from a bond between the amino and carboxyl termini of the peptide; a bond between the amino terminus and a side chain; a bond between the carboxyl terminus and a side chain; or a bond between two side chains that include the sulfur groups of two cysteine ​​amino acids by forming a disulfide bond, or a more complex arrangement.

[0050] The term "amide" as used herein refers to a group of the formula "-NHC(O)-".

[0051] The term "thioamide" refers to a group of the formula "-NHC(S)-".

[0052] As used herein, the term "disulfide bond," "disulfide bridge," or "disulfide" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0053] The term "ether" refers to a group or compound that contains an oxygen between two carbon atoms.

[0054] The term "thioether" refers to a group or compound that has a sulfur between two carbon atoms.

[0055] The term "ester" refers to a compound derived from a carboxylic acid and an alcohol by linking the hydroxyl group of the -C(=O)OH group of the carboxylic acid with a hydroxyl group of the alcohol with the formal loss of water. The term thus refers to the group -C(O)O-.

[0056] The term "thioester" refers to the group -C(O)S-.

[0057] The term "alkylene" refers to a saturated linear or branched divalent hydrocarbon moiety which can have 2 to 30, typically 3 to 20, and often 4 to 18 carbon atoms.

[0058] The term "alkenylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond and which may contain a total of 2 to 30, typically 3 to 20, and often 4 to 18 carbon atoms.

[0059] The term "alkynylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon triple bond and which may contain a total of 2 to 30 carbon atoms, typically 3 to 20, and often 4 to 18. An alkynyl group may optionally have one or more carbon-carbon double bonds.

[0060] The term "triazole" refers to a chemical compound that incorporates into its structure any heterocyclic structure having a five-membered ring of two carbon atoms and three nitrogen atoms (eg, 1,2,3-triazole). In one aspect, disclosed herein is a peptide comprising the following cyclized amino acid sequence: -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, (wherein any two side chains of the amino acids AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 (X1, X2) are covalently bonded; and The amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 that are not X1 and X2 are: AA0 is Pro or deleted; AA1 is Ser, Gly, Thr or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln or Glu; AA9 is Arg, Ala, Gln or Glu; AA10 is Arg; AA11 is Leu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or is deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; and AA14 is Pro or deleted.

[0061] In some embodiments, X1 and X2 are separated by two or three amino acids.

[0062] In some embodiments, AA5 is X1 and AA9 is X2, AA5 is X1 and AA8 is X2, AA9 is X1 and AA13 is X2, AA6 is X1 and AA9 is X2, AA9 is X1 and AA12 is X2, AA10 is X1 and AA13 is X2, AA6 is X1 and AA10 is X2, or AA4 is X1 and AA8 is X2.

[0063] In one aspect, disclosed herein is a peptide comprising a cyclized amino acid sequence selected from the group consisting of: TIFF2025505164000002.tif47147 where the side chains of the X1 and X2 amino acid residues are covalently linked; AA0 is Pro or deleted; AA1 is Ser, Gly, Thr or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln or Glu; AA9 is Arg, Ala, Gln or Glu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or is deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; and AA14 is Pro or deleted.

[0064] In some embodiments, X1 and X2 in the peptides disclosed herein are covalently linked via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

[0065] In some embodiments, the cyclized amino acid sequences described herein are produced by linking the amino group on the side chain of one of X1 and X2 to the carboxyl group on the side chain of the other of X1 and X2 via an amide bond. An amino group on the side chain of an amino acid having a pendant amine group, such as lysine or a lysine derivative, and a carboxyl group on the side chain of an acidic amino acid, such as aspartic acid, glutamic acid or derivatives thereof, can be used to produce a cyclized amino acid sequence via an amide bond.

[0066] In some embodiments, the cyclized amino acid sequences described herein are generated by linking a sulfhydryl group on the side chain of one of X1 and X2 to a sulfhydryl group on the side chain of the other of X1 and X2 via a disulfide bond. Sulfhydryl-containing amino acids include cysteine ​​and other sulfhydryl-containing amino acids such as Pen.

[0067] In some embodiments, X1 and X2 are independently selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, provided that when X1 is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen. When X1 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap or DDap, X2 is Glu, DGlu, Asp or DAsp; when X1 is Cys, DCys, hCys, DhCys, Pen or DPen, X2 is Cys, DCys, hCys, DhCys, Pen or DPen.

[0068] In some embodiments, X1 is Glu and X2 is Lys. In some embodiments, -cyclo(Glu--------Lys)-, -c(Glu-------Lys)-, -cyclo(E-------K)-, -c(E---------K)-, -E------K-cyclo, or -cycloE----cycloK- include the following structures: [ka]

[0069] In some embodiments, X1 is Lys and X2 is Glu. In some embodiments, -cyclo(Lys-----Glu)-, -c(Lys------Glu)-, -cyclo(K------E)-, -c(K-------E)-, -K----E-cyclo or cycloK------cycloE- include the following structures: [ka]

[0070] In some embodiments, X1 is Cys and X2 is Cys. In some embodiments, -cyclo(Cys------Cys)-, c(Cys------Cys)-, -cyclo(C------C)-, -c(C--------C)-, -C----C-cyclo, or -cycloC-----cycloC- comprises the following structures: [ka]

[0071] Specific cyclized amino acid sequences of the general formula identified above include, for example, the following: TIFF2025505164000006.tif80147TIFF2025505164000007.tif177147TIFF2025505164000008.ti f176147TIFF2025505164000009.tif176147TIFF2025505164000010.tif176147TIFF20255051640 00011.tif176147TIFF2025505164000012.tif176147TIFF2025505164000013.tif176147TIFF202 5505164000014.tif176147TIFF2025505164000015.tif176147TIFF2025505164000016.tif121147

[0072] In some embodiments, the cyclic peptide is linked to a 3-mercaptopropionyl moiety via the α-amine moiety of the left-most amino acid of the cyclic peptide, hi some embodiments, the right-most amino acid of the cyclic peptide comprises an amide.

[0073] In some embodiments, the cyclized amino acid sequence is selected from the group consisting of: TIFF2025505164000017.tif126147

[0074] In some embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In yet some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-. In yet some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.

[0075] Cyclic peptides can have a variety of cyclic bridging moieties that form ring structures. Preferably, the ring structure contains a chemically stable bridging moiety, such as an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole group. The following are examples that show the variability of bridging moieties in peptides: [ka]

[0076] In some embodiments, a peptide comprising a cyclized amino acid sequence disclosed herein is bound to a compound comprising a moiety that binds to a peptide comprising a cyclized amino acid sequence disclosed herein. In some embodiments, the moiety that binds to a peptide comprising a cyclized amino acid sequence binds to the peptide by binding to the cyclized amino acid sequence. In some embodiments, the moiety that binds to a peptide comprising a cyclized amino acid sequence comprises an antibody or antibody fragment, for example an antibody disclosed herein, such as a camelid VHH domain disclosed herein.

[0077] Linear ALFA SMThe peptide was found to be prone to reduced stability in serum and plasma due to enzymatic cleavage at the peptide bond between the Arg-Leu residues, as shown below. [ka]

[0078] Without intending to be bound by any particular theory, it is believed that the cyclic ring can protect the peptide bonds inside or near it from enzymatic cleavage. In addition, the cyclic structure can enhance the α-helical structure and thus retain binding affinity for anti-ALFA VHHs that recognize the α-helical structure of the peptide. For example, referring to FIG. 1, the illustrated cyclic ALFA peptide embodiment showed a longer half-life in human and mouse plasma in an in vitro assay. Among the cyclic ALFA peptides, the H-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-OH peptide, which is "Cyclo1" in FIG. 1 and "Ac_5E-9Cyclo_ALFA" in Table 1, has a half-life of more than 24 hours in both human and mouse plasma (FIG. 1). Importantly, this cyclic peptide retains binding affinity for the anti-ALFA VHH with an EC50 of 6.0 nM compared to 2.6 nM for the parent linear ALFA peptide (Figure 2 and Table 1). [Table 2]

[0079] Peptides containing the cyclized amino acid sequences disclosed herein can be components of a larger entity, such as a compound or complex of compounds.

[0080] In some embodiments, such larger units may be compounds that include one or more peptides that include a cyclized amino acid sequence disclosed herein and (i) one or more peptide moieties other than the peptides that include a cyclized amino acid sequence disclosed herein, (ii) one or more non-peptide moieties, or (iii) a combination of (i) and (ii). In some embodiments, one or more peptides that include a cyclized amino acid sequence disclosed herein may be linked to one or more peptide moieties other than the peptides that include a cyclized amino acid sequence disclosed herein and / or one or more non-peptide moieties by direct fusion or via a linker. A "linker" as used herein links together, for example, two or more subunits of a fusion protein. The bond may be a covalent bond. In some embodiments, the covalent bond is via a peptide bond, such as a peptide bond between amino acids. In some embodiments, the linker is a peptide linker. In some embodiments, the linker includes one or more amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Peptide linkers include glycine-serine (GS) linkers, glycosylated GS linkers, and polymeric proline-alanine-serine (PAS) linkers.

[0081] In some embodiments, such a larger unit may be a complex of two or more compounds, where at least one compound comprises one or more peptides that include a cyclized amino acid sequence disclosed herein.

[0082] In some embodiments, a peptide comprising a cyclized amino acid sequence disclosed herein can be a component of a fusion protein. In some embodiments, the fusion protein is a recombinant protein.

[0083] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the association of two or more elements or components or domains.

[0084] The term "fusion protein" as used herein refers to a polypeptide or protein that comprises two or more subunits. Preferably, the fusion protein is a translational fusion between two or more subunits. A translational fusion can be generated by genetically engineering the coding nucleotide sequence of one subunit in a reading frame with the coding nucleotide sequence of an additional subunit. The subunits can be interspersed by linkers.

[0085] In the fusion protein disclosed herein, the peptide disclosed herein, i.e., epitope tag, can be located at any position of the fusion protein.The peptide can be fused to the N-terminus or C-terminus of the polypeptide to be fused.Alternatively, the peptide can be fused internally to the polypeptide at a position between the N-terminus and C-terminus of the polypeptide.As an illustrative example, the peptide can be fused between two domains of the polypeptide.

[0086] The term "recombinant" in the context of this disclosure means "created through genetic engineering." In some embodiments, "recombinant" in the context of this disclosure is not naturally occurring.

[0087] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a source in nature, and has not been intentionally modified by humans in a laboratory, is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0088] In some embodiments, peptides comprising cyclized amino acid sequences disclosed herein can be components of functionalized stealth lipids, such as those disclosed in U.S. Provisional Patent Application No. 63 / 305,905, filed February 2, 2022, the disclosure of which is incorporated herein by reference. Stealth lipids are lipids that increase the length of time that nanoparticles can exist in vivo (e.g., in the blood).

[0089] The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules that contain one or more hydrophobic moieties or groups, and optionally one or more hydrophilic groups attached directly or indirectly to the lipid moiety. As used herein, the term "lipid" should be construed to encompass both lipids and lipid-like substances, unless otherwise indicated herein or clearly contradicted by context.

[0090] Molecules that contain hydrophobic and hydrophilic portions are often also called amphiphiles. In an aqueous environment, the amphiphilic nature allows such molecules to self-assemble into organized structures and various phases. One of those phases consists of lipid bilayers when they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0091] As used herein, the term "amphiphilic" refers to a molecule that has both polar and non-polar portions. Often, amphipathic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Furthermore, the polar portion may have either a formal positive or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a formal negative charge and may be a zwitterion or an inner salt. For purposes of this disclosure, an amphipathic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0092] Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids. In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. The term "lipid" is sometimes used as a synonym for fat, which is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0093] The disclosed self-assembling amphiphiles are referred to herein as "lipid nanoparticles". Thus, "lipid nanoparticles" refers to particles that contain a plurality (i.e., more than one) of lipid molecules physically associated with each other by intermolecular forces. Nanoparticles can be, for example, microspheres (unilamellar and multilamellar vesicles, such as "liposomes" - in some embodiments, containing lamellar phase lipid bilayers that are substantially spherical (and may contain non-spherical morphologies), and in more specific embodiments, can contain a core that is aqueous or non-aqueous, for example, the dispersed phase in an emulsion, a micelle, or the internal phase in a suspension.

[0094] In some embodiments, the conjugate comprising a peptide comprising a cyclized amino acid sequence disclosed herein comprises a compound comprising a peptide comprising a cyclized amino acid sequence disclosed herein and a compound comprising a moiety that binds to the peptide comprising the cyclized amino acid sequence. In some embodiments, the moiety that binds to the peptide comprising the cyclized amino acid sequence comprises an antibody or an antibody fragment. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a single domain antibody. In some embodiments, the antibody comprises or consists of a VHH domain, for example a camelid VHH domain.

[0095] In some embodiments, the moiety that binds to a peptide comprising a cyclized amino acid sequence comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTX1SALNAMAMG (X1 is I or V), the CDR2 sequence AVSX2RGNAM (X2 is E, H, N, D or S), and the CDR3 sequence LEDRVDSFHDY.

[0096] In some embodiments, the moiety that binds to a peptide comprising a cyclized amino acid sequence comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTX1SALNAMAMG (X1 is I or V), the CDR2 sequence AVSX2RGNAM (X2 is E, H, N, D or S), and the CDR3 sequence LEDRVDSFHDY.

[0097] In some embodiments the moiety that binds to a peptide comprising a cyclized amino acid sequence comprises a single domain antibody, for example a camelid VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0098] In some embodiments the moiety that binds to a peptide comprising a cyclized amino acid sequence comprises a single domain antibody, for example a camelid VHH domain comprising the CDR1 sequence GVTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0099] In some embodiments, the moiety that binds to a peptide comprising a cyclized amino acid sequence is a single domain antibody, e.g., EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence, or a fragment of said amino acid sequence or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence. In some embodiments, the amino acid sequence comprises the CDR1, CDR2 and CDR3 sequences set out above.

[0100] The term "bind" or "binding" refers to a non-covalent interaction with a target. In some embodiments, the term "bind" or "binding" refers to specific binding. As used herein, the term "specific binding" or "specifically binds" refers to a molecule, such as an antibody, that recognizes a specific target molecule but does not substantially recognize or bind other molecules in a sample or subject. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such species cross-reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific.

[0101] In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, antibodies recognize and bind to specific protein structures rather than proteins in general. If an antibody is specific for epitope "A", then in a reaction involving labeled "A" and an antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0102] As used herein, the terms "binding" or "capable of binding" typically refer to binding to a molecule that binds to a molecule of interest within about 10, as determined using biolayer interferometry (BLI) or, for example, as determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K below M D In some embodiments, the binding moiety or binder has a K that is at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, such as at least 100,000 times lower, than its affinity for binding to a non-specific target (e.g., BSA, casein). D The β-amino acid binds to a given target with an affinity corresponding to

[0103] As used herein, "k d ”(seconds -1 The term k ) refers to the dissociation rate constant of a particular interaction, e.g., an antibody-antigen interaction. off Also called value.

[0104] As used herein, "K D The term "M" refers to the dissociation equilibrium constant of a particular interaction, e.g., an antibody-antigen interaction.

[0105] Disclosed herein are binding moieties and binding agents that bind to peptides that include the cyclized amino acid sequences disclosed herein. Such binding moieties and binding agents can form conjugates with the peptides.

[0106] The term "binding agent" as used herein refers to any agent capable of binding to a desired antigen. In certain embodiments, the binding agent is or comprises an antibody, an antibody fragment, or any other binding protein, or any combination thereof.

[0107] In some embodiments, the binding agents disclosed herein include bispecific or multispecific binding agents, such as bispecific antibodies comprising a first and a second binding domain, where the first binding domain is capable of binding to a peptide comprising a cyclized amino acid sequence disclosed herein and the second binding domain is capable of binding to a different target.

[0108] The term "binding moiety" as used herein refers to any moiety, group or domain capable of binding to a desired antigen. In certain embodiments, the binding moiety is or comprises an antibody, an antibody fragment, or any other binding protein, or any combination thereof.

[0109] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically comprises a heavy chain variable region (referred to herein as V H or VH) and a heavy chain constant region (herein referred to as C HThe heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically comprises a light chain variable region (herein referred to as V L or VL) and a light chain constant region (abbreviated herein as C L or CL). The light chain constant region is typically composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or configuration of structurally defined loops), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise indicated or contradicted by context, references to amino acid positions of constant regions in this disclosure are according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1): 78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). In general, the CDRs described herein are defined according to Kabat.

[0110] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, that has the ability to bind, preferably specifically, to an antigen or epitope. The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domain that interacts with the antigen or epitope.

[0111] As used herein, the term "antigen-binding region," "binding region," or "binding domain" refers to the region or domain that interacts with an antigen and typically comprises both the VH and VL regions.

[0112] The term antibody as used herein includes not only monospecific antibodies but also multispecific antibodies that contain multiple, e.g., two or more, e.g., three or more, different antigen-binding regions. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. As mentioned above, the term antibody as used herein includes antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to antigens, and antibody derivatives, i.e., constructs derived from antibodies, unless otherwise indicated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed by the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO2007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody, (v) dAb fragments consisting essentially of the VH domain, also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(11):484-90) (Ward et al., Nature 341,544-546(1989)); (vi) camelid or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1):111-24) and (vii) isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be made, using recombinant techniques, as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs), see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term antibody unless otherwise stated or clearly indicated by the context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention and exhibit different biological properties and usefulness. These and other useful antibody fragments in the context of the present invention, as well as bispecific formats of such fragments, are discussed further herein. The term antibody, unless otherwise specified, should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0113] The phrase "single-chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy and light chains (VH and VL) of a traditional two-chain antibody are combined to form a single chain. Optionally, a linker (usually a peptide) is inserted between the two chains to allow proper folding and creation of an active binding site.

[0114] Single domain antibodies, also known as nanobodies, are antibody fragments that consist of a single monomeric variable antibody domain. In one embodiment, single domain antibodies consist of the variable domain (V) of a heavy chain antibody. H). These are called VHH fragments. Like whole antibodies, single domain antibodies can selectively bind to a specific antigen. The first single domain antibodies were engineered from heavy chain antibodies found in camelids. Cartilaginous fish also have heavy chain antibodies (IgNAR, "immunoglobulin novel antigen receptor"), from which single domain antibodies called VNAR fragments can be derived. Another approach is to split the dimeric variable domain from common immunoglobulin G (IgG) from human or mouse into monomers. Most work on single domain antibodies is currently based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind target epitopes.

[0115] An antibody can have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgG1, than to other isotypes. Thus, for example, an IgG1 antibody can be a sequence variant of a naturally occurring IgG1 antibody, including mutations in the constant region.

[0116] In various embodiments, the antibody is an IgG1 antibody, more specifically an IgG1 kappa or IgG1 lambda isotype (i.e. IgG1κ, IgG1λ), an IgG2a antibody (e.g. IgG2aκ, IgG2aλ), an IgG2b antibody (e.g. IgG2bκ, IgG2bλ), an IgG3 antibody (e.g. IgG3κ, IgG3λ) or an IgG4 antibody (e.g. IgG4κ, IgG4λ).

[0117] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas containing B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, whose genome includes human heavy and light chain transgenes fused to an immortalized cell.

[0118] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, such as human. Chimeric monoclonal antibodies for therapeutic use have been developed to reduce antibody immunogenicity. The term "variable region" or "variable domain" as used in the context of a chimeric antibody refers to the region that includes the CDR and framework regions of both the heavy and light chains of an immunoglobulin. Chimeric antibodies can be produced by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Chimeric antibodies can be genetically engineered or enzymatically engineered recombinant antibodies. It is within the knowledge of one of skill in the art to produce chimeric antibodies, and therefore production of chimeric antibodies can be performed by methods other than those described herein.

[0119] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen binding site into a homologous human acceptor framework region (FR) (see WO 92 / 22653 and EP 0629240). Substitution (backmutation) of framework residues from the parent antibody (i.e., non-human antibody) into the human framework region may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions that optionally contain one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications, not necessarily back mutations, can be applied to obtain humanized antibodies with favorable characteristics, such as affinity and biochemical properties.

[0120] The term "human antibody" as used herein refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse or rat, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody techniques, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are generally preferred, other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of human antibody genes. A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the mouse system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g. mouse myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be made using, for example, transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat system. Thus, in some embodiments, human antibodies are obtained from transgenic animals, such as mice or rats, carrying human germline immunoglobulin sequences instead of animal immunoglobulin sequences.In such embodiments, the antibodies are derived from human germline immunoglobulin sequences introduced into the animal, with the final antibody sequence being the result of further modification of said human germline immunoglobulin sequences by somatic hypermutations and affinity maturation by the endogenous animal antibody machinery, see e.g., Mendez et al. 1997 Nat Genet. 15(2):146-56.

[0121] As used herein, unless contradicted by context, the terms "Fab arm," "binding arm" or "arm" include one heavy-light chain pair and are used interchangeably herein with "half molecule."

[0122] The term "full length" when used in reference to an antibody indicates that the antibody is not a fragment, but includes all of the domains of a particular isotype that are normally found for that isotype in nature, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains of an IgG1 antibody.

[0123] As used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least the hinge region, CH2 domain, and CH3 domain.

[0124] The present disclosure also contemplates antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies described herein. A functional variant of a VL, VH, or CDR used in connection with an antibody still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or specificity / selectivity of the "reference" or "parent" antibody, and in some cases, such an antibody may be associated with higher affinity, selectivity, and / or specificity than the parent antibody.

[0125] Such functional variants typically retain significant sequence identity to the parent antibody.

[0126] Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of a parent antibody sequence primarily by conservative substitutions; for example, up to 10, e.g., 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue substitutions.

[0127] Functional variants of the antibody sequences described herein, such as the VL or VH regions, or antibody sequences having a degree of homology or identity to the antibody sequences described herein, such as the VL or VH regions, preferably contain modifications or mutations of non-CDR sequences, while the CDR sequences preferably remain unchanged.

[0128] The term "specificity", as used herein, unless contradicted by context, is intended to have the following meaning: Two antibodies have the "same specificity" if they bind to the same antigen and the same epitope.

[0129] Antibodies or fragments useful herein may compete with the specific antibodies or fragments described herein.

[0130] The terms "compete" and "competition" may refer to the competition between a first antibody and a second antibody for the same antigen. Methods for testing the competition of antibodies for binding to target antigen are well known to those skilled in the art. One example of such a method is the so-called cross-competition assay, which can be carried out, for example, as ELISA or by flow cytometry. Alternatively, biolayer interferometry can be used to determine competition.

[0131] Antibodies that compete for binding to a target antigen can bind to different epitopes on the antigen, and the epitopes are so close to each other that a first antibody that binds to one epitope prevents a second antibody from binding to the other epitope. However, in other situations, two different antibodies can bind to the same epitope on an antigen and compete for binding in a competitive binding assay. Such antibodies that bind to the same epitope are considered to have the same specificity herein. Thus, in some embodiments, antibodies that bind to the same epitope are considered to bind to the same amino acid on the target molecule. The binding of antibodies to the same epitope on a target antigen can be determined by standard alanine scanning experiments or antibody-antigen crystallization experiments known to those skilled in the art. Preferably, antibodies or binding domains that bind to different epitopes do not compete with each other for binding to their respective epitopes. EXAMPLES

[0132] Example 1: Cyclic ALFA Peptide Synthesis Materials and Equipment Liberty Blue HT24 synthesizer (CEM Corporation) ·CEM Corporation L-Amino Acid: Fmoc-Lys(Mmt)-OH ·Sigma / Millipore L-amino acid:Fmoc-Glu(O-2-PhiPr)-OH Advanced Chemtech Amino Acid: Mpa(Trt)-OH ·Matrix Innovations L-amino acids: Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH Advanced Chemtech: Sieber amide resin, 100-200 mesh · Total and semi-protected cleavage reagents: TFA (JT Baker W729-07 Baker Analytical Reagents), triisopropylsilane (Sigma-Aldrich), ethanedithiol (Sigma-Aldrich), thioanisole (Sigma-Aldrich), diethyl ether (Fischer Scientific), HPLC water (HoneyWell), dichloromethane (Sigma-Aldrich), t-butanol (Sigma-Aldrich), acetonitrile (The Lab Depot), hexane (VWR Chemicals).

[0133] Synthesis: The synthesis of three replicate intermediate peptide sequences is carried out on a 0.1 mmol scale using a Liberty Blue HT24 synthesizer. For preswelling and resin transfer, the Sieber amide resin is loaded onto a Liberty HT suspended in 10 mL of 1:1 dichloromethane / dimethylformamide (DCM:DMF). The synthesis method starts with deprotection of the N-terminal α Fmoc protecting group using 4 mL of 20% piperidine in DMF heated at 60° C. for 3 min in a microwave with nitrogen dispenses and mixing every 3 s. After draining, the resin is washed three times with 5 mL of DMF for 5 s per wash. Then, 2.5 mL of the next 0.3 M amino acid solution (7.5 eq.), 1 mL of 1 M DIC (10 eq.), and 0.5 mL of 1 M Oxyma+0.1 M diisopropylethylamine (DIEA) (5 eq.) are added to the reaction vessel (RV) for the coupling reaction. The double coupling step proceeds with an initial 6 min cycle at 50 °C and an additional 10 min cycle at 50 °C with nitrogen dispensed and mixed every 3 sec during both coupling cycles. After the second coupling time is complete, the resin is drained and 20% piperidine in DMF is added to the RV to initiate the deprotection of the next incoming amino acid. This cycle of Fmoc removal and coupling is repeated successively for each amino acid. As shown in Table 2, the external position #6 of Liberty Blue is used for the addition of Fmoc-Glu(O-2-PhiPr)-OH to the peptide sequence and #7 is used for the addition of Fmoc-Lys(Mmt)-OH amino acid. [Table 3] Table 2. List of lactam peptides used; amino acids (E) and (K) in the sequences indicate the position of the lactam bridge.

[0134] Due to the absence of Fmoc protection of the Mpa(Trt)-OH amino acid, final deprotection of the peptide was not necessary and the peptide resin was washed four times with 4 mL of DMF before being transferred from the RV back to the starting HT position and the next peptide in the queue began to be synthesized.

[0135] Combined protected peptide cleavage and orthogonal deprotection of lysine and glutamic acid side chains: After the instrumental synthesis run of each peptide is completed, the resin is transferred to a 24 mL fritted syringe using DCM and rinsed three times with DCM to remove excess DMF. After washing, the resin is treated six times with 5 mL of 2% trifluoroacetic acid (TFA), 1% triisopropylsilane (TIS) in DCM for each reaction for approximately 5 min. The flow-through solution from each reaction containing the semi-protected peptide is then collected in a separate 50 mL conical tube for each peptide. The semi-protected peptide solution is then evaporated under a gentle nitrogen (N2) atmosphere until less than 5 mL of total volume remains in each tube. Once the volume in each conical tube is less than 5 mL, the peptide is resuspended in 20 mL of 1:1 acetonitrile:t-butanol (MeCN:tBu-OH) and sonicated until the solution is homogenous. The peptide tubes are then frozen at -80 °C and placed on a lyophilizer for drying.

[0136] Lactam cyclization via side chain amide bond formation: Once the semi-protected peptide is dry, weigh out approximately 5 equivalents for a 0.1 mmol scale synthesis of (7-azabenzotriazol-1-yloxy)trispyrrolizinophosphonium hexafluorophosphate (PyAOP) and 1-hydroxy-7-azabenzotriazole (HOAt) and dissolve in 4 mL of DMF. Once the reagents are completely dissolved, add this solution to each peptide tube. After the semi-protected peptide material has dissolved, add approximately 10 equivalents for a 0.1 mmol scale synthesis of DIEA to each solution and observe a slight color change where the solutions turn yellow. The solutions are then left to react at room temperature for 2-3 hours.

[0137] Precipitation of protected lactam peptides: After 2-3 h reaction time at room temperature, precipitate the protected peptides by adding approximately 45 mL of chilled HPLC water to each 50 mL conical tube, then centrifuge the tubes at 1000-1200 rpm for approximately 5 min. This is repeated twice to remove excess DMF, and after the second precipitation, resuspend the peptide pellet in frozen 1:1 (ACN:tBu-OH) at -80 °C and lyophilize overnight. This is done to assess whether there is any unexpected loss of peptide during the precipitation step above.

[0138] Global peptide cleavage: After the peptides have completed drying by lyophilization, add approximately 10 mL of cleavage cocktail containing 92.5% (TFA), 2.5% water (HO), 2.5% (TIS) and 2.5% thioanisole to each 50 mL conical tube and leave shaking at room temperature for 2-3 h.

[0139] Final precipitation: 10 mL of cleavage cocktail is divided into two 50 mL conical tubes at 5 mL each. Approximately 45 mL of cold (-20 °C) 1:1 hexane:diethyl ether is added to each tube. The mixture is then centrifuged at 3500 rpm for 10 min. After the ether is decanted, another 40 mL of cold 1:1 hexane:diethyl ether is added to further wash the peptide pellet. The vessel is agitated to break up the peptide pellet, then centrifuged again at 3500 rpm for 10 min. After the ether is decanted a second time, the tube is placed on its side in a fume hood to dry. The peptide pellet is then reconstituted in approximately 10 mL of 1:1 acetonitrile / water (ACN:H2O), frozen at -80 °C, and lyophilized.

[0140] UPLC-MS analysis and peptide QC sample preparation: A small sample of the dried crude peptide powder is transferred to a 1.5 mL Eppendorf tube and then dissolved with enough 3:1 dimethyl sulfoxide / water (DMSO:HO) to obtain a peptide sample concentration of approximately 2 mg / mL. 50 μl of the peptide sample is then transferred to a plastic vial and loaded onto a Waters Acuity UPLC-MS for crude analysis. The method used to analyze the crude peptides utilized a flow rate of 0.5 mL / min on a Waters BEH 1.7 μm x 100 mm column. An ACN:HO gradient was performed for 10 min (10% to 80% ACN in 8 min), increasing to 90% ACN from 10-11 min. Once the retention time and mass of the target peptide have been confirmed, the peptide powder is then stored at -20 °C until required for further reactions / conjugations or reverse phase purification.

[0141] Example 2: Purification of cyclized alpha tag Materials and Equipment Preparative HPLC: Automated purification system with 2767 sample manager, 2545 binary gradient manager, 515 column dilution pump, 2489 UV detector, and SQD2 mass spectrometer (Waters Corporation) UPLC-MS: Waters Acquity UPLC-MS equipped with an H-Class FTN sample manager, an H-Class quaternary solvent manager, a column manager, a photodiode array UV detector, and a QDa mass detector. Purification column: Phenomenex Luna C18(2) LC column, 100Å pore size, 10μm particle size, 30mm x 250mm (part number: 00G-4253-U0-AX) Salt exchange preparative column: Waters XSelect Peptide CSH C 18 OBD Prep Column, 130Å pore size, 5μm particle size, 19×100mm (Part Number: 186007020) Analytical column: Waters Acquity UPLC Peptide BEH C18 Column, 130Å pore size, 1.7μm particle size, 2.1mm x 100mm (part number 186003555) Water: Honeywell HPLC grade water (catalog no. AH365-4); or in-house Sartorius Arium Pro Water Purification System Acetonitrile: Sigma-Aldrich HPLC grade acetonitrile (catalog no. 34851-4L); or Lab Depot LABSOLV LC / MS acetonitrile (catalog no. LSL1140) Trifluoroacetic acid (TFA): Sigma-Aldrich HPLC grade TFA (Cat. no. 302031-1L) Dimethyl sulfoxide (DMSO): Sigma-Aldrich dimethyl sulfoxide (catalog number D1435-500ML) Ammonium Acetate: Sigma Life Science Ammonium Acetate (Cat. No. A1542-250G) Acetic acid: Mallinckrodt Chemicals glacial acetic acid (V193-46)

[0142] Crude sample preparation for purification: Lyophilized crude peptides were dissolved in DMSO or 50 / 50 water / acetonitrile to approximately 50 mg / mL. They were vortexed and sonicated until completely dissolved (no solid material was visibly present). The dissolved peptides were pipetted into the sample plate for HPLC injection.

[0143] Peptide purification: Peptides were loaded onto a preparative HPLC column with an autoinjector via a 10 mL sample loop. Sample loading was performed with 95% mobile phase A, 5% mobile phase B. Peptide elution was performed in a 36 min gradient with 15% mobile phase B increment (0.4% / min). The composition of the gradient mobile phase B was determined by crude peptide UPLC retention time correlation coefficient. Fraction collection was automatically triggered based on the M+1H, M+2H, M+3H values ​​of the target peptides using an SQD2 mass spectrometer. UV 214 The absorption of 1000 ppm was observed but was not used for fraction triggering. The purification HPLC conditions are defined in Table 3. [Table 4]

[0144] In-process purification QC: Aliquots of selected peptide fractions were pipetted into a 96-well plate and injected into the UPLC-MS. The UPLC-MS conditions used for in-process fraction QC are listed in Table 4. For some peptides, preparative UV chromatography was performed to estimate which fractions were of high purity. 214 In-process fraction QC was omitted by visual observation of absorbance and MS. [Table 5]

[0145] Fraction pooling: Fractions with purity > 90% were combined into a tared 50mL polypropylene tube and lyophilized. If the pure fraction volume exceeded the tube capacity, multiple polypropylene tubes were filled and lyophilized. Each peptide was then resuspended in 50 / 50 water / acetonitrile + 0.05% TFA, combined into one tared polypropylene tube and lyophilized.

[0146] Salt exchange: For in vivo applications, peptide counterions were exchanged from trifluoroacetate to acetate. In this case, peptides were dissolved in ammonium bicarbonate (10 g / L), vortexed until completely dissolved (no visible solid material), sonicated, and pipetted into the sample plate for HPLC injection. Peptides were loaded onto the salt-exchanged HPLC column using an autoinjector via a 10 mL sample loop containing 100% ammonium bicarbonate (10 g / L). Ammonium bicarbonate was run through the column at 15 mL / min for 20 min. Then, 1% v / v acetic acid in water was run through the column for 20 min. Finally, peptides were eluted with a steep gradient from 0 to 60% acetonitrile in 15 min. Peptide recovery was triggered by a mass spectrometer that was used to observe the M+1H, M+2H, M+3H values ​​of the target peptides. Fractions were combined into tared polypropylene tubes and lyophilized.

[0147] Lyophilization: The peptide solutions were frozen on dry ice for at least 2 hours. They were then lyophilized at a pressure of <150 mTorr. After at least 48 hours, the peptides were removed from the lyophilizer and visually inspected to ensure they were a white powder with no liquid present.

[0148] Peptide Weighing: After lyophilization, the polypropylene tubes containing the peptide powder were weighed on an analytical balance. The tare weight of the tube was subtracted and the weight was recorded to the nearest tenth of a milligram.

[0149] Final QC: Final QC of peptides was performed on H-Class UPLC-MS. Each peptide was dissolved at approximately 1 mg / mL in 0.05% TFA in water and injected into the UPLC. The UPLC / MS conditions were the same as those used for the in-process purification QC and are defined in Table 4. Peptide purity was determined by relative UV area at 214 nm and identity was determined by mass spectrometry. The acceptance criteria was that each peptide had a UV 214 Purity was > 90% and molecular weight was within 1 AMU of the theoretical MW. The results for each peptide are shown in Table 5. [Table 6]

[0150] UPLC-UV of peptide Ac-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-NH2(MW:1778.8) 214 and UV 214 The purity (100%) is shown in Figures 3 and 4, respectively.

[0151] Example 3: Cell-binding analysis of linear and cyclic ALFA peptides The following example shows Fluorescence Activated Cell Sorting (FACS) data for linear and cyclic ALFA peptides (conjugated to AlexaFluor680) on cells.

[0152] Cells were plated in 96-well microtiter plates (round button) to a final density of 2 × 10 5Cells were then washed and incubated for 30 min at 4 °C with different concentrations (4 nM, 16 nM, 64 nM, 256 nM and 1024 nM) of linear [Ac(C-Alexa680)(PEG6)PSRLEEELRRRLTE-NH2] or cyclic [Ac(C-Alexa680)(PEG6)PSRLE(E)ELR(K)RLTE-NH2] ALFA peptide. After washing again, cells were measured on a FACS Canto II (BD Biosciences). Binding of ALFA peptide was detected using AlexaFluor680 signal. Therefore, an excitation laser line of 633 nm was used to detect the AlexaFluor 680 signal.

[0153] Referring now to Figure 5, the FACS data show that both linear and cyclic ALFA peptides bind only to target-expressing cells that have been pre-incubated with NbALFAx anti-target adaptor protein. No binding was observed in controls (binding to target-negative cells and pre-incubation with PBS or NbALFAx non-binder). Thus, binding of ALFA peptides to target cells does not occur non-specifically, but only in dependence on the pre-administered NbALFAx anti-target adaptor.

[0154] Example 4: SPR measurements of NbALFA binding to (cyclic)ALFA Affinity measurements were performed using a Biacore T200 (GE Healthcare, Biacore T200 control software 3.2). For affinity measurements, Biotin CAPture kit, series S (Cytiva Europe, Cat. No. 28920234) was utilized. Freshly docked Biotin CAPture chips were rehydrated overnight in the instrument (standby mode). The next day, the chip surface was conditioned three times with regeneration solution (6M guanidine-HCl, 0.25M NaOH) on flow cells (FC) 1 and FC2 at 10 μL / min for 60 s. Biotin CAPture reagent (Cytiva Europe, Cat. No. 29423383) was then applied to FC1 and FC2 at 2 μL / min for 300 s, followed by capture of 25 nM biotinylated NbALFA (NanoTag). Immobilization of nanobodies was performed on FC2 for 180 s at 10 μL / min, followed by quenching of unbound streptavidin with 0.1 mM biotin for 60 s and 30 μL / min on both flow cells. Five injections of cyclic ALFA variants at 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM and 0 nM (blank control) were performed on both flow cells for 120 s at a flow rate of 40 μL / min, followed by dissociation for 1500 s. After each cycle, the surface was regenerated with regeneration solution for 120 s at 10 μL / min. All measurements were performed at 25 °C with HBS-EP+ buffer (Cytiva Europe, Cat. No. BR100669). The control response from FC1 was subtracted by the sample measurement on FC2 (FC2-FC1) followed by subtraction of the 0 nM blank injection. The resulting binding curves of this multi-cycle kinetic experiment were fitted using Biacore T200 evaluation software 3.2 and a 1:1 binding model.

[0155] The results are shown in Figures 6-15 and Tables 6 and 7, and show that samples SD15194, SD15341 and SD15808 show very good binding to NbALFA. Samples SD15194, SD15341 and SD15808 utilize E5-K9 or E6-K10 cyclization. Table 8 shows the structure of each sample tested. [Table 7] [Table 8] [Table 9]

[0156] Example 5: Pharmacokinetic (PK) study (mouse) Show me how Animals and animal housing C57BL / 6NRj mice were obtained from Janvier Labs, France. Female mice approximately 8 weeks old were used in this study. Animal housing conditions are listed in Table 9. [Table 10]

[0157] Animal protocols The studies were carried out in strict accordance with national and European guidelines for the care and use of laboratory animals (Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes).

[0158] All procedures in this study were carried out in accordance with the German Animal Welfare Act and German regulations (TierSchG / TierSchVersV) and were approved by the local authorities (Regierung von Oberbayern, Munchen).

[0159] PK study Compounds used for intravenous bolus injections were formulated in aqueous glucose solutions (SD14316 in 4% glucose, SD15341 in 5% glucose). Before administration into the lateral tail vein, the animal's body weight was determined to calculate the individual volume of injected compound (5 mL / kg body weight). Injections and blood sampling were performed under short-term anesthesia (0.7 L / min O2, 1.7 L / min N2O, 2% isoflurane). At the time points indicated in Table 10, blood was collected from the retro-orbital plexus using microhematocrit capillary tubes (0.8 mm diameter) and collected in K3EDTA tubes. [Table 11]

[0160] Plasma was separated by centrifugation (10,000 xg; 10 min; 4°C), aliquoted into microtubes, and stored at -80°C.

[0161] LC-MS / MS quantification 20 μl of each plasma sample was transferred to a PCR tube. 80 μl of IS (internal standard) solution in acetonitrile + 1% FA was added for protein precipitation.

[0162] The sample tubes were sealed with a puncturable cap mat and vortexed. After homogenization in an ultrasonic bath for 5 min, the samples were centrifuged at 3000 rpm for 15 min at ambient temperature. 1 μl of the supernatant was injected into the instrument for LC-MS / MS analysis. For LC-MS / MS quantification, an ExionLC™ UHPLC coupled to a QTRAP® 6500+ mass spectrometer (Sciex) was used in ESI positive mode. Mass spectrometry parameters are listed in the table below. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (Waters, 1.7 μm, 2.1×50 mm). The mobile phase composition, gradient information, mass transitions and spectrometer settings are tabulated below.

[0163] Compound concentrations in plasma samples were calculated using a 10-point calibration curve in plasma over the concentration range of 1-1000 ng / ml. QC samples of 1, 10, 100, 1000 ng / ml were analyzed before and after each dose group. Samples above the upper limit of quantification (1000 ng / ml) were reanalyzed after 10-fold dilution with blank plasma. All calculations were performed with Sciex OS version 1.7.0.36606 and MS-EXCEL®.

[0164] Pharmacokinetic parameters were calculated for both compounds using the software Kinetica (version 4.4.1, Thermo Scientific) applying the noncompartmental model (NCA). [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17]

[0165] The results are shown in Figures 16 and 17 and Tables 17 and 18, and demonstrate that the cyclic alpha compounds have a much higher area under the curve (AUC) and dramatically reduced clearance compared to the linear alpha compounds. [Table 18] [Table 19]

[0166] Example 6: Plasma stability - human and mouse Plasma stability measurements were performed by spiking 5 μl of 1 mM compound stock solution in DMSO into 495 μl of human or mouse Li-heparin plasma (Innovative Research, Cat. No. IPLALIH5OML (human) and IMS-BC-N (Balb C mouse)). The spiked plasma was distributed in 50 μl aliquots into capped PCR tubes for incubation at 37 °C. At each time point (0, 2, 6, 24 h*), 3× aliquots were withdrawn from the incubator. The enzymatic reaction was quenched immediately upon withdrawal by adding 100 μl of acetonitrile / formic acid = 99 / 1 (v / v). The quenched samples were stored at ambient temperature until the end of the incubation period (24 h*). After centrifugation, the supernatant was transferred to a new vial and analyzed by LC-MS / MS in ESI positive mode (Waters MassLynx with a Xevo TQD triple quadrupole mass spectrometer and control software Waters MassLynx). ACQUITY UPLC I-Class System (SM-FTN). Chromatographic separation was performed on a Waters Cortex T3 analytical column (1.6 μm particle size, 2.1 × 30 mm). Mobile phase composition, gradient information, mass transitions and spectrometer settings are tabulated below. Compound concentrations in assay samples after incubation were calculated using a three-point calibration curve in acetonitrile / water = 2 / 1 (v / v) over the concentration range of 0.1-10 μM. Values ​​below the limit of quantification (0.1 μM) and outliers from triplicate results were excluded from further calculations. An exponential regression curve was fitted to the concentration results. Half-life times were calculated from the slope of the regression curve (t 1 / 2 = ln(2) / slope).

[0167] For some samples, the incubation time was 6 hours, as indicated below: 24 hour data were measured in triplicate, 6 hour data were measured in duplicate.

[0168] Tables 19-21 provide the LC-MS / MS instrument settings for each sample tested. [Table 20] [Table 21] [Table 22]

[0169] Table 22 provides the half-life times in human plasma. [Table 23]

[0170] Table 23 provides the half-life times in mouse plasma. [Table 24]

[0171] The stability results in human plasma for each sample tested are listed in Tables 24-32. [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33]

[0172] The stability results in mouse plasma for each sample tested are listed in Tables 33 to 41. [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41] [Table 42]

[0173] As disclosed herein, several embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the embodiments disclosed herein. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A compound comprising a peptide comprising the following cyclized amino acid sequence: -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, (wherein any two side chains of amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 (X1, X2) are covalently linked; and The amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 which are not X1 and X2 are: AA0 is Pro or deleted; AA1 is Ser, Gly, Thr or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, He, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, He, or Val; AA8 is Arg, Ala, Gin or Glu; AA9 is Arg, Ala, Gin or Glu; AA10 is Arg; AA11 is Leu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; and AA14 is Pro or deleted

2. The compound of claim 1, wherein X1 and X2 are separated by 2 or 3 amino acids.

3. 3. The compound according to claim 1 or 2, wherein AA5 is X1 and AA9 is X2, AA5 is X1 and AA8 is X2, AA9 is X1 and AA13 is X2, AA6 is X1 and AA9 is X2, AA9 is X1 and AA12 is X2, AA10 is X1 and AA13 is X2, AA6 is X1 and AA10 is X2, or AA4 is X1 and AA8 is X2.

4. The compound according to any one of claims 1 to 3, wherein the cyclized amino acid sequence is selected from the group consisting of: (Here, X １ and X ２ the side chains of the amino acid residues are covalently linked; and AA0 is Pro or deleted; AA1 is Ser, Gly, Thr or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, He, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, He, or Val; AA8 is Arg, Ala, Gin or Glu; AA9 is Arg, Ala, Gin or Glu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; and AA14 is Pro or deleted.

5. X １ and X ２ is covalently attached via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

6. X １ and X ２ is independently selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that X １ is Glu, DGlu, Asp or DAsp; ２ is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap or DDap; When X1 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap or DDap, X2 is Glu, DGlu, Asp or DAsp; When X1 is Cys, DCys, hCys, DhCys, Pen or DPen, X2 is Cys, DCys, hCys, DhCys, Pen or DPen; The compound according to any one of claims 1 to 5.

7. The compound according to any one of claims 1 to 6, wherein the cyclized amino acid sequence is selected from the group consisting of:

8. The compound according to any one of claims 1 to 6, wherein the cyclized amino acid sequence is selected from the group consisting of:

9. The compound according to any one of claims 1 to 6, wherein the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-.

10. 7. The compound according to any one of claims 1 to 6, wherein the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-.

11. 7. The compound according to any one of claims 1 to 6, wherein the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.

12. The compound according to any one of claims 1 to 6, wherein the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.

13. The compound according to any one of claims 1 to 6, wherein the cyclized amino acid sequence is selected from the group consisting of: 【Chemistry 1】

14. The compound according to any one of claims 1 to 13, wherein the peptide consists of the cyclized amino acid sequence.

15. The compound according to any one of claims 1 to 14, which is a fusion protein.

16. The compound of claim 15 , wherein the fusion protein comprises the peptide and an additional peptide or polypeptide.

17. 17. The compound of claim 15 or 16, which is free of non-peptide components.

18. A compound according to any one of claims 1 to 14, comprising one or more non-peptide components.

19. 20. A conjugate comprising a compound according to any one of claims 1 to 18 and a compound comprising a moiety that binds to said peptide comprising a cyclized amino acid sequence.

20. 20. The conjugate of claim 19, wherein the moiety that binds the peptide comprising a cyclized amino acid sequence comprises an antibody or an antibody fragment.

21. 20. A method of forming a complex comprising contacting a compound according to any one of claims 1 to 18 with a compound comprising a moiety that binds to said peptide comprising a cyclized amino acid sequence.

22. 22. The method of claim 21, wherein the moiety that binds the peptide comprising a cyclized amino acid sequence comprises an antibody or an antibody fragment.

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