Composite polypeptides having metal-binding motifs and molecular constructs comprising the same

Abstract

To provide a method for effectively conjugating a functional element to a specific site of a biomolecule, particularly, a peptide-based molecule.SOLUTION: Provided is a linker unit in which a center core comprises 2 to 10 lysine (K) residues, and any two of the K residues are adjacent to each other or are separated by a filler; an SH-reactive group is linked to the first or last K residue of the center core by forming an amide bond therewith; in other cases, the center core further comprises a terminal spacer, and the SH-reactive group is linked to a terminal amino acid residue of the terminal spacer by forming the amide bond therewith; the terminal spacers may be an N-terminal spacer linked to the N-terminus of the first K residue or a C-terminal spacer linked to the C-terminus of the last K residue; and each of the filler and the terminal spacer comprises, independently, (1) 1 to 12 non-K amino acid residues, or (2) a PEGylated amino acid having 1 to 12 repeats of ethylene glycol unit.SELECTED DRAWING: Figure 7B

Description

[Technical Field]

[0001] The present disclosure relates to composite polypeptides having metal binding motifs that are short oligopeptides capable of complexing with zinc cations. [Background technology]

[0002] Bioconjugation is a technique used to form covalent bonds between biomolecules and other molecules, which may or may not be biomolecules. Biomolecules generally refer to molecules that are present in organisms and essential for biological processes. Biomolecules include natural and synthetic polypeptides or proteins, carbohydrates, lipids, nucleic acids, and metabolites.

[0003] The concept of arming antibodies or antibody fragments with toxins (i.e., antibody-toxin conjugates, or "ATCs"), cytotoxic drugs (i.e., antibody-drug conjugates, or "ADCs"), and radionuclides (i.e., antibody-radionuclide conjugates, or "ARCs") arose in the 1970s. The synthesis of such bioconjugates often involves conjugation reactions such as coupling of surface-accessible lysine, cysteine, or tyrosine residues and modification of surface-accessible lysine or tryptophan residues or the N- and C-termini. Nevertheless, these amino acid residues are abundant in parent antibodies, and conjugation reactions are typically random processes. As a result, conjugation reactions often lack chemoselectivity and efficiency and result in heterogeneous products. For example, previous studies on the maytansinoid-monoclonal antibody immunoconjugate huN901-DM1 revealed that lysine-conjugated ADC samples potentially contained over 4.5 million unique molecules, with ADCs at different drug-to-antibody ratios (DARs) ranging from 0 to 6.

[0004] In view of the foregoing, there is an increasing need for conjugation strategies that can effectively attach functional elements to specific sites on biomolecules, particularly peptide-based molecules, and the present invention is believed to be an answer to that need. Summary of the Invention

[0005] The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an extensive overview of the disclosure, and it does not identify key / critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented below.

[0006] In one aspect, the present disclosure is directed to metal binding motifs that are capable of forming complexes with zinc ions under appropriate conditions.

[0007] According to one embodiment of the present disclosure, the metal binding motif comprises, in N-terminal to C-terminal order or C-terminal to N-terminal order, the amino acid sequence of CX1X2HA (SEQ ID No: (SEQ ID NO:) 1), where X1 is glycine or proline and X2 is glycine or alanine, and the metal binding motif is located at the N- or C-terminus of the parent polypeptide. Exemplary metal binding motifs include, but are not limited to, CGGHA (SEQ ID NO: 2), CPGHA (SEQ ID NO: 3), CGAHA (SEQ ID NO: 4), CPAHA (SEQ ID NO: 5), GCGGHA (SEQ ID NO: 6), ACPGHA (SEQ ID NO: 7), and GCPGHA (SEQ ID NO: 8).

[0008] In another aspect, the present disclosure is directed to a composite polypeptide comprising a metal-binding motif according to the above aspect / embodiment. Thus, the composite polypeptide can form a complex with a metal ion (e.g., zinc ion) via the metal-binding motif under appropriate conditions. Thus, the sulfhydryl group of the cysteine ​​residue in the complex with the metal ion is more reactive than the sulfhydryl group of the cysteine ​​residue not complexed with the metal ion. As can be seen, the complex can undergo chemical bonding with other chemical moieties or biomolecules in a site-specific manner to form a bioconjugate.

[0009] According to one embodiment of the present disclosure, a composite polypeptide comprises a parent polypeptide and a metal binding motif located at the N- or C-terminus of the parent polypeptide, in certain alternative embodiments, there is an intervening sequence of 1-10 glycine residues between the parent polypeptide and the metal binding motif, while in other embodiments, the metal binding motif immediately precedes or follows the parent polypeptide.

[0010] In yet another aspect, one or more functional elements can be covalently attached to a composite polypeptide according to the above aspects / embodiments of the present disclosure by reacting with sulfhydryl (SH) groups of cysteine ​​residues in the metal binding motif of the composite polypeptide to form a molecular construct (or bioconjugate).

[0011] According to alternative embodiments of the present disclosure, the SH-reactive group is a maleimide, iodoacetyl, bromoacetyl, vinylsulfone, monosulfone, methylsulfonylbenzothiazole, or 2-pyridyldithiol group.

[0012] In certain alternative embodiments, the composite polypeptide is in the form of a dimer, e.g., the composite polypeptide may comprise an Fc region, an F(ab')2, or a portion of an antibody, in which case each composite polypeptide chain has one functional element attached thereto.

[0013] According to various embodiments of the present disclosure, the functional element is a small molecule moiety capable of eliciting a therapeutic effect. For example, the small molecule moiety can be a cytotoxic drug, a Toll-like receptor agonist, or a chelator complexed with a radionuclide. Alternatively, the functional element can be a fatty acid chain that can alter the pharmacokinetic profile of the parent polypeptide or molecular construct as a whole.

[0014] Alternatively, the functional element is in the form of a linker unit (also called a "bundle"), which, according to certain embodiments of the invention, comprises a central core and a plurality of targeting, effector or pharmacokinetic elements.

[0015] Specifically, the central core comprises 2 to 10 lysine (K) residues. Any two of the K residues may be adjacent to each other or separated by a filler. In some cases, the SH-reactive group is linked to the first or last K residue of the central core by forming an amide bond with it. In other cases, the central core further comprises a terminal spacer, and the SH-reactive group is linked to the terminal amino acid residue of the terminal spacer by forming an amide bond with it. The terminal spacer may be an N-terminal spacer linked to the N-terminus of the first K residue or a C-terminal spacer linked to the C-terminus of the last K residue. Each of the filler and terminal spacer independently comprises (1) 1 to 12 non-K amino acid residues or (2) a PEGylated amino acid having 1 to 12 repeats of ethylene glycol (EG) units.

[0016] According to certain embodiments of the present disclosure, the central core carries a localized negative charge at or near the amino acid residue linked to the SH-reactive group. For example, the localized negative charge is present within the first 5 to 15 amino acid residues, starting from the amino acid residue linked to the SH-reactive group. Specifically, the localized negative charge is present within the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, starting from the amino acid residue linked to the SH-reactive group. The localized negative charge can be imparted by including one or more amino acid residues that are negatively charged at pH 7, such as aspartic acid (D) and glutamic acid (E) residues. Alternatively, the core can be bound to multiple negatively charged effector elements.

[0017] In some embodiments, each targeting element, effector element, or pharmacokinetic element is linked to the K residue of the core via an amide bond with the ε-amino group of the K residue. In other cases, the molecular construct further comprises 2-10 linking arms, one end of which is linked to the K residue of the core via an amide bond with the ε-amino group of the K residue, and the other end of which is linked to each targeting element, effector element, or pharmacokinetic element. For example, the linking arms can be peptides comprising 2-12 non-K amino acid residues or polyethylene glycol (PEG) chains having 2-24 repeats of EG units.

[0018] According to certain embodiments of the present invention, the above-described composite polypeptides act as targeting elements that can direct molecular constructs to a site of interest or increase the relative levels of molecular constructs at a site of interest. In these cases, the linker unit (or bundle) may carry multiple effector elements, such as the small molecule components described above.

[0019] According to some embodiments of the present invention, the above-described composite polypeptides act as effector elements capable of eliciting a desired therapeutic effect in a subject. In these cases, the linker unit (or bundle) may be selected from the group consisting of C, D, E, F, G, H, I, I, M, I, M, M, N, O ... 8-28 Fatty acid derivatives or C 8-28 According to various embodiments of the present invention, the ε-amino group of the K residue may be C 8-28 Fatty acid derivatives or C 8-28 The modified parent polypeptide is linked to a diacid fatty acid derivative, which is capable of binding to serum albumin and thus improving the kinetic properties of the modified parent polypeptide in vivo.

[0020] Also within the scope of the present invention are the linker units described above, which are capable of forming molecular constructs with the present composite polypeptides.

[0021] In yet another aspect, the present invention is directed to a linker unit capable of carrying multiple composite polypeptides according to the above aspects / embodiments of the present disclosure. According to embodiments of the present disclosure, a linker unit having multiple composite polypeptides covalently linked to its central core may also be referred to as a polypeptide bundle.

[0022] According to certain embodiments of the present invention, the linker unit comprises a central core and a plurality of linking arms.

[0023] Specifically, the central core comprises 2 to 10 lysine (K) residues and a linking group. Any two of the K residues may be adjacent to each other or separated by a filler. In some cases, the linking group is linked to the first or last K residue of the central core by forming an amide bond with it. Examples of linking groups include, but are not limited to, azide, alkyne, tetrazine, cyclooctene, and cyclooctyne groups. In other examples, the central core further comprises a terminal spacer, and the linking group is linked to the terminal amino acid residue of the terminal spacer by forming an amide bond with it. The terminal spacer can be an N-terminal spacer linked to the N-terminus of the first K residue or a C-terminal spacer linked to the C-terminus of the last K residue. The filler and the terminal spacer each independently comprise (1) 1 to 12 non-K amino acid residues or (2) a PEGylated amino acid having 1 to 12 repeats of ethylene glycol (EG) units.

[0024] According to some embodiments, one end of each linking arm is linked to a K residue of the core via forming an amide bond with the ε-amino group of the K residue, while the other end of each linking arm has an SH-reactive group. For example, the linking arm can be a peptide comprising 2-12 non-K amino acid residues or a polyethylene glycol (PEG) chain having 2-24 repeats of EG units.

[0025] According to one embodiment of the present invention, the linker unit further comprises 2 to 10 composite polypeptides according to the above aspects / embodiments and one functional element, each composite polypeptide being linked to the thiol group of a cysteine ​​residue in the metal binding motif via SH-reactive crosslinking chemistry.

[0026] According to an alternative embodiment of the present invention, each linking arm carries a localized negative charge at or near the amino acid residue linked to the SH-reactive group. Specifically, the localized negative charge is present within the first 5 to 15 amino acid residues, starting from the amino acid residue linked to the SH-reactive group. For example, the localized negative charge is present within the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, starting from the amino acid residue linked to the SH-reactive group. The localized negative charge can be imparted by including one or more amino acid residues that are negatively charged at pH 7, such as aspartic acid (D) and glutamic acid (E) residues.

[0027] Many of the attendant features and advantages of the present disclosure will become better understood by reference to the following detailed description considered in connection with the accompanying drawings.

[0028] The present description will be better understood from the following detailed description read in light of the accompanying drawings. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 shows the results of MALDI-TOF of the Mal-peptide 1 central core according to an effective embodiment of the present invention. [Figure 2] FIG. 2 shows the structure of a Mal-peptide2-DOTA bundle according to one effective embodiment of the present invention. [Figure 3A] FIG. 3A shows the reversed-phase analytical HPLC elution profile of the Mal-peptide2-DOTA bundle. [Figure 3B] FIG. 3B shows the MALDI-TOF results of the Mal-peptide2-DOTA bundle according to one effective embodiment of the present invention. [Figure 4A] FIG. 4A shows the structure of recombinant two-chain (scFv α CD19)-Fc-MBM-1 according to one effective embodiment of the present invention. [Figure 4B] FIG. 4B shows a non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CD19)-Fc-MBM-1 according to an effective embodiment of the present invention. [Figure 5] FIG. 5 shows non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CD19)-Fc-MBM-2 according to one effective embodiment of the present invention. [Figure 6] FIG. 6 shows a non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CD19)-Fc-MBM-3 according to one effective embodiment of the present invention. [Figure 7A] FIG. 7A shows a reducing SDS-PAGE analysis of anti-CD19 antibody-MBM-1 or anti-CD19 antibody-MBM-2 according to an effective embodiment of the present invention. [Figure 7B] FIG. 7B shows a reducing SDS-PAGE analysis of anti-CD19 antibody-MBM-1 or anti-CD19 antibody-MBM-2 according to an effective embodiment of the present invention. [Figure 8] FIG. 8 shows a non-reducing SDS-PAGE analysis of four (scFv α CD33)-Fc-MBM molecule constructs according to one effective embodiment of the present invention. [Figure 9] FIG. 9 shows non-reducing SDS-PAGE analysis of recombinant (scFv α CD20)-Fc-MBM-1 in both VL-VH and VH-VL configurations according to an effective embodiment of the present invention. [Figure 10A] FIG. 10A shows a non-reducing SDS-PAGE analysis of recombinant two-chain (VH-VL scFv α CD20)-Fc-MBM-2 according to one effective embodiment of the present invention. [Figure 10B] FIG. 10B shows non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CD20)-Fc-MBM-3 according to one effective embodiment of the present invention. [Figure 11A]FIG. 11A shows a non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CA19-9)-Fc-MBM-1 according to one effective embodiment of the present invention. [Figure 11B] FIG. 11B shows non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CA19-9)-Fc-MBM-2 according to one effective embodiment of the present invention. [Figure 12A] FIG. 12A shows a non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CC38)-Fc-MBM-1 according to one effective embodiment of the present invention. [Figure 12B] FIG. 12B shows a non-reducing SDS-PAGE analysis of recombinant two-chain (VL-VH scFv α CC38)-Fc-MBM-2 according to an effective embodiment of the present invention. [Figure 13A] FIG. 13A shows the structure of a recombinant two-chain (scFv α CD19)-Fc-MBM-1×2DOTA bundle according to one effective embodiment of the present invention. [Figure 13B] FIG. 13B shows a non-reducing SDS-PAGE analysis of a recombinant two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 14] FIG. 14 shows a non-reducing SDS-PAGE analysis of a recombinant two-chain (scFv α CD19)-Fc-MBM-2×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 15] FIG. 15 shows non-reducing SDS-PAGE analysis of recombinant two-chain (scFv α CD19)-Fc-MBM-3×2DOTA bundle. [Figure 16] FIG. 16 shows a reducing SDS-PAGE analysis of an intact anti-CD19 antibody-MBM-1×2DOTA bundle according to one effective embodiment of the present invention. [Figure 17] FIG. 17 shows a reducing SDS-PAGE analysis of an intact anti-CD19 antibody-MBM-2×2 DOTA bundle f according to an effective embodiment of the present invention. [Figure 18]FIG. 18 shows a non-reducing SDS-PAGE analysis of a recombinant two-chain (scFv α CD33)-Fc-MBM-1×2DOTA bundle according to one effective embodiment of the present invention. [Figure 19] FIG. 19 shows a non-reducing SDS-PAGE analysis of a recombinant two-chain (scFv α CD20)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 20] FIG. 20 shows the FPLC elution profile of an anion exchange column for a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 21] FIG. 21 shows SDS-PAGE analysis of fractions collected from an anion exchange column for a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 22] FIG. 22 shows SDS-PAGE analysis of fractions collected from an anion exchange column for a stabilized two-chain (scFv α CD20)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 23] FIG. 23 shows the results of MALDI-TOF of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 24] FIG. 24 shows the MALDI-TOF results of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle chelated with 89Y 3+ ions according to one effective embodiment of the present invention. [Figure 25] FIG. 25 shows the MALDI-TOF results of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle chelated with 175Lu 3+ ions according to one effective embodiment of the present invention. [Figure 26] FIG. 26 shows a TLC analysis of the radiochemical purity of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle labeled with 111In according to one effective embodiment of the present invention. [Figure 27]FIG. 27 shows the results of a stability study analyzed by Western blotting of an 111In-labeled stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 28A] FIG. 28A shows the results of a staining analysis of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 28B] FIG. 28B shows the results of a staining analysis of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 28C] FIG. 28C shows the results of a staining analysis of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 28D] FIG. 28D shows the results of a staining analysis of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 29] FIG. 29 shows the results of a stability study analyzed by SDS-PAGE of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 30] FIG. 30 shows the results of a stability study analyzed by TLC of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 31] FIG. 31 shows the half-life of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 32] FIG. 32 shows the targeting effect of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to an effective embodiment of the present invention on CD19-expressing xenograft tumors. [Figure 33]FIG. 33 shows the results of a biodistribution analysis of a stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle according to an effective embodiment of the present invention in CD19-expressing xenograft tumors. [Figure 34] FIG. 34 is a schematic diagram showing the structure of Aib-GLP-1 agonist-Cys according to one effective embodiment of the present invention. [Figure 35] FIG. 35 is a schematic diagram showing the structure of Cys-octreotide according to one advantageous embodiment of the present invention. [Figure 36] FIG. 36 shows the results of MALDI-TOF / TOF of Cys-octreotide according to one effective embodiment of the present invention. [Figure 37] FIG. 37 is a schematic diagram showing the structure of a Cys-PSMA ligand according to one effective embodiment of the present invention. [Figure 38] FIG. 38 shows the results of ESI-MS of a Cys-PSMA ligand according to one effective embodiment of the present invention. [Figure 39] FIG. 39 is a schematic diagram showing the structure of calcitonin-MBM-1 according to one advantageous embodiment of the present invention. [Figure 40] FIG. 40 is a schematic diagram showing the structure of a Mal-peptide 3-lenalidomide bundle according to one effective embodiment of the present invention. [Figure 41] FIG. 41 shows the results of ESI-MS of the Mal-peptide 3-lenalidomide bundle according to one effective embodiment of the present invention. [Figure 42] FIG. 42 is a schematic diagram showing the structure of a Mal-peptide 4-lenalidomide bundle according to one effective embodiment of the present invention. [Figure 43] FIG. 43 is a schematic diagram showing the structure of a Mal-peptide 5-fatty acid bundle according to one effective embodiment of the present invention. [Figure 44] FIG. 44 shows the results of ESI-MS of Mal-peptide 5-fatty acid bundle according to one effective embodiment of the present invention. [Figure 45] FIG. 45 is a schematic diagram showing the structure of a 3-DOTA arm linker unit according to one effective embodiment of the present invention. [Figure 46] FIG. 46 shows the results of ESI-MS of the 3-DOTA arm linker unit according to one effective embodiment of the present invention. [Figure 47] FIG. 47 shows an SDS-PAGE analysis of MBM-1-IL-2 according to one effective embodiment of the present invention. [Figure 48] FIG. 48 shows an SDS-PAGE analysis of a two-chain (scFv α CD38)-Fc-MBM-1×2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 49] FIG. 49 is a schematic diagram showing the structure of an octreotide x fatty acid bundle according to one effective embodiment of the present invention. [Figure 50A] FIG. 50A is a schematic diagram showing the structure of an Aib-GLP-1 agonist x fatty acid bundle according to one effective embodiment of the present invention. [Figure 50B] FIG. 50B is a schematic diagram showing the structure of a teriparatide-MBM-1 x fatty acid bundle according to one effective embodiment of the present invention. [Figure 51A] FIG. 51A is a schematic diagram showing the structure of a leuprolide-MBM-3×fatty acid bundle according to one effective embodiment of the present invention. [Figure 51B] FIG. 51B shows MALDI-TOF results of leuprolide-MBM-3×fatty acid bundles according to one effective embodiment of the present invention. [Figure 52A] FIG. 52A is a schematic diagram showing the structure of a 3-DOTA arm linker unit x 3 PSMA ligand according to one effective embodiment of the present invention. [Figure 52B] FIG. 52B shows the results of ESI-MS of a 3-DOTA arm linker unit x 3 PSMA ligand according to one effective embodiment of the present invention. [Figure 53] FIG. 53 shows an SDS-PAGE analysis of a purified two-chain (scFv α CA19-9)-Fc-MBM-1×2 DOTA bundle according to one effective embodiment of the present invention. [Figure 54] Figure 54 shows an SDS-PAGE analysis of a purified two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 55] Figure 55 shows MALDI-TOF results of a purified two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 56A] Figure 56A shows the half-life of a parent anti-CD38hIgG1.Fc antibody according to one effective embodiment of the present invention. [Figure 56B] Figure 56B shows the half-life of a two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 57A] Figure 57A shows the targeting effect of a two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 57B] Figure 57B shows the targeting effect of a two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 57C] Figure 57C shows the targeting effect of a two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 58A] Figure 58A shows the ADCC effect of a two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. [Figure 58B] Figure 58B shows the ADCC effect of a two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to one effective embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] The detailed description provided below in connection with the accompanying drawings is intended as a description of the present embodiment and not the only manner in which the present embodiment may be constructed or utilized. The description sets forth the functions of the embodiment and the sequence of steps for constructing and operating the embodiment, although the same or equivalent functions and sequences may be accomplished by different embodiments.

[0031] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless otherwise defined herein, scientific and technical terms employed in this disclosure shall have the meanings commonly understood and used by those of ordinary skill in the art.

[0032] Also, unless otherwise required by context, singular terms shall be understood to include the plural and plural terms shall include the singular. Also, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more. Still further, as used throughout this specification and the appended claims, the phrases "at least one of A, B, and C," "at least one of A, B, or C," and "at least one of A, B, and / or C" are intended to include A only, B only, C only, both A and B, both B and C, both A and C, and all of A, B, and C.

[0033] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within the acceptable standard error of the mean as considered by one of ordinary skill in the art. Other than in the context of effective / effective examples, unless expressly stated otherwise, all numerical ranges, amounts, values, and ratios, such as those relating to amounts of materials, durations, temperatures, operating conditions, ratios of amounts, and the like, disclosed herein should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this disclosure and the appended claims are approximations that may be varied as desired. At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Ranges may be expressed herein as from one endpoint to the other endpoint, or between two endpoints. All ranges disclosed herein include the endpoints unless otherwise specified.

[0034] As used herein, the term "targeting element" refers to a portion of a molecular construct that facilitates delivery of the molecular construct to a target of interest (e.g., a cell surface receptor or a protein within a tissue) by directly or indirectly binding to the target of interest. In some embodiments, the targeting element can direct the molecular construct to the vicinity of a target cell. In other cases, the targeting element specifically binds to a molecule present on the surface of the target cell or to a second molecule that specifically binds to a molecule present on the cell surface. In some cases, the targeting element, once bound to the target of interest, can be internalized along with the molecular construct, thereby transporting it into the cytosol of the target cell. The targeting element can be an antibody or ligand for a cell surface receptor, or a molecule that indirectly targets the molecular construct to a target site (e.g., the surface of a selected cell) by binding to such an antibody or ligand. The localization of the effector (therapeutic agent) at the disease site is enhanced or optimized with the molecular construct compared to a therapeutic agent lacking targeting functionality. Localization is a matter of degree or relative proportion, and does not imply absolute or total localization of the effector to the disease site.

[0035] According to the present invention, the term "effector element" refers to a portion of a molecular construct that, when directed to its target site, causes a biological activity (e.g., inducing or suppressing immune activity, exerting a cytotoxic effect, inhibiting an enzyme, etc.) or other functional activity (e.g., delivering immune cells or other therapeutic molecules). The "effect" can be therapeutic or diagnostic. Effector elements include those that bind to cellular and / or extracellular immunoregulatory factors. Effector elements comprise agents such as proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties (both small molecule drugs and biologics), compounds, elements and isotopes, and fragments thereof.

[0036] According to the present invention, a "pharmacokinetic element" is intended to mean an element capable of modifying at least one of the following properties of a molecular construct: solubility, clearance rate, half-life, and bioavailability. For example, the pharmacokinetic element may comprise a long PEG chain having a molecular weight of about 20,000 to 50,000 daltons. Alternatively, the pharmacokinetic element may comprise a C 8-28 Fatty acid chain or C 8-28 It may comprise a diacidic fatty acid chain.

[0037] The term "bioconjugate" according to the present invention refers to a molecular construct comprising a composite polypeptide of the invention and one or more functional elements.

[0038] Terms such as first, second, third, etc. may be used herein to describe various elements, components, regions, and / or portions, but these elements (and components, regions, and / or portions) are not limited by these terms. Furthermore, the use of such ordinal numbers does not imply a permutation or order unless clearly indicated by context. On the contrary, these terms are merely used to distinguish one element from another. Thus, a first element described below may be referred to as a second element without departing from the teachings of the exemplary embodiments.

[0039] As used herein, the terms "link," "couple," and "conjugate" are used interchangeably to refer to any means of connecting two components through a direct or indirect connection between the two components.

[0040] As used herein, the term "polypeptide" refers to a polymer having at least two amino acid residues. Typically, polypeptides comprise amino acid residues ranging in length from 2 to about 200 residues, although it also encompasses polymers having more than 200 amino acid residues. Where amino acid sequences are given herein, L-, D-, or beta-amino acid versions of the sequence are contemplated. Polypeptides also include amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids and naturally occurring amino acid polymers. Furthermore, the term applies to amino acids joined by peptide bonds or other "modified linkages," for example, in which the peptide linkage is replaced by an α-ester, β-ester, thioamide, phosphoramide, carbomate, hydroxylate, etc.

[0041] In certain embodiments, conservative substitutions of amino acids comprising any of the sequences described herein are contemplated. In various embodiments, 1, 2, 3, 4, or 5 different residues are substituted. The term "conservative substitution" is used to reflect amino acid substitutions that do not substantially alter the activity of a molecule (e.g., biological or functional activity and / or specificity). Typically, conservative amino acid substitutions involve replacing one amino acid with another amino acid with similar chemical properties (e.g., charge or hydrophobicity). Some conservative substitutions include "analogous substitutions," in which a standard amino acid is replaced with a non-standard (e.g., rare, synthetic, etc.) amino acid that differs minimally from the parent residue. Amino acid analogs are synthetically derived from standard amino acids without significant changes to the parent structure, are isomers, or are metabolic precursors. In this application, the amino acid residues (1) lysine, which contains an amine group in its side chain; (2) cysteine, which contains a thiol group in its side chain; (3) serine and threonine, which contain hydroxyl groups in their side chains; and (4) aspartic acid and glutamic acid, which contain carboxyl groups in their side chains, are considered to be the four characteristic groups of amino acids. Each of these four groups of amino acids contains a unique functional group in its side chain that can be adapted for attachment to various chemical moieties. Non-natural amino acids containing the same functional group in their side chains may be substituted for similar purposes.

[0042] In certain embodiments, polypeptides with at least 80%, preferably at least 85% or 90%, and more preferably at least 95% or 98% sequence identity to any of the sequences described herein are also contemplated.

[0043] "Percentage (%) amino acid sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of polypeptide residues in a candidate sequence that are identical to amino acid residues in a particular peptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be accomplished in a variety of ways within the skill of the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For purposes herein, sequence comparison between two polypeptide sequences was performed using the computer program Blastp (protein-protein BLAST), provided online by the National Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given polypeptide acid sequence A to a given polypeptide acid sequence B (alternatively, a given polypeptide acid sequence A has a given % amino acid sequence identity to a given polypeptide acid sequence B) is calculated according to the following formula: X / Y×100% where X is the number of amino acid residues scored as perfect matches by the sequence alignment program BLAST in that program's alignment of A and B, and Y is the total number of amino acid residues in A or B, whichever is shorter.

[0044] As used herein, the term "PEGylated amino acid" refers to a polyethylene glycol (PEG) chain having one amino group and one carboxyl group. Generally, a PEGylated amino acid is a PEG chain having the structure NH2-(CH2CHO) n It has the formula -CO2H. In the present disclosure, the value of n ranges from 1 to 20, preferably from 2 to 12.

[0045] The term "binding moiety," as used herein, refers to a molecule having one or more functional groups (also referred to as "linking groups") that are chemically reactive and capable of covalently binding to other chemical units. Non-limiting examples of functional groups include hydroxyl, carbonyl, carboxyl, thiol, amine, tert-butyldimethylsilyl (TBDMS), N-hydroxysuccinimidyl (NHS), SH-reactive groups (e.g., maleimide, haloacetyl, sulfone, or pyridyl disulfide), iodo, iodoacetamide azide, alkyne, tetrazine, cyclooctene, and cyclooctyne groups. According to embodiments of the present disclosure, the binding moiety of the molecular construct has two functional groups: one is a carboxyl or amine group for binding to the alpha amino or carboxyl group of the terminal amino acid residue of the core via forming an amide bond therebetween, such that the binding moiety is attached to the N- or C-terminal amino acid residue of the core, and the other is an SH-reactive group for binding to a second element via SH-reactive crosslinking chemistry.

[0046] As used herein, the term "terminus" with respect to a polypeptide refers to an amino acid residue at the N-terminus or C-terminus of the polypeptide. With respect to a polymer, the term "terminus" refers to a building block of a polymer (e.g., polyethylene glycol of the present disclosure) that is located at the end of the polymer backbone. In this specification and claims, the term "free end" is used to mean that the terminal amino acid residue or building block is not chemically bonded to any other molecule.

[0047] The terms "antigen" or "Ag" are used interchangeably and refer to a molecule that elicits an immune response. This immune response may involve a secretory, humoral, and / or cellular antigen-specific response. In the present disclosure, the term "antigen" may be any of a protein, polypeptide (including mutants or biologically active fragments thereof), polysaccharide, glycoprotein, glycolipid, nucleic acid, or combinations thereof.

[0048] In the present specification and claims, the terms "antibody" and "antibody fragment" are used broadly to encompass antigen-binding antibody fragments, such as fully assembled antibodies, antigen-binding fragments (Fab / Fab'), F(ab')2 fragments (having two antigen-binding Fab portions interlinked by disulfide bonds), variable fragments (Fv), single-chain variable fragments (scFv), bispecific single-chain variable fragments (bi-scFv), nanobodies (also known as single-domain antibodies (sdAbs)), unibodies, and diabodies. An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. An antibody fragment may comprise a pair of scFvs fused to the N- or C-terminus of a pair of CH2-CH3 segments derived from human γ4 or γ1 immunoglobulin. Typically, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Well-known immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The standard immunoglobulin (antibody) structural unit is known to consist of a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. According to embodiments of the present disclosure, antibody fragments can be produced by modifying natural antibodies or by de novo synthesis using recombinant DNA methodologies. According to certain embodiments of the present disclosure, antibodies and / or antibody fragments can be bispecific and can be of various configurations. For example, bispecific antibodies can comprise two different antigen-binding portions (variable regions).In various embodiments, bispecific antibodies can be produced by hybridoma technology or recombinant DNA technology. In certain embodiments, bispecific antibodies have binding specificities for at least two different epitopes. In many molecular constructs employing antibody fragments, the antibody fragments can be replaced with antibody mimetics that bind to the same antigenic component as the antibody fragment. Antibody mimetics include anticalins, DARPins, affibodies, phylomers, ankyrins, avimers, and others.

[0049] As used herein, the term "specifically binds" means to bind to a cell that binds to a specific -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 The ability of an antibody or antigen-binding fragment thereof to bind to an antigen with a dissociation constant (Kd) of M or less and / or to bind to an antigen with an affinity at least two-fold higher than its affinity for a non-specific antigen.

[0050] The term "treatment / therapy / treatment" as used herein includes preventative (e.g., prophylactic), curative, or palliative treatment / therapy / treatment, and the term "treatment / treating" as used herein also includes preventative (e.g., preventative), curative, or palliative treatment / therapy / treatment. In particular, the term "treatment / therapy / treating" as used herein refers to the application or administration of the molecular construct or a pharmaceutical composition comprising the molecular construct to a subject having a medical condition, a symptom associated with a medical condition, a disease or disorder secondary to a medical condition, or a predisposition to a medical condition, with the aim of partially or completely alleviating, ameliorating, relieving, delaying the onset of, inhibiting progression of, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of the particular disease, disorder, and / or condition. Treatment / therapy / treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition, or who exhibits only early signs of a disease, disorder, and / or condition, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0051] As used herein, the term "effective amount" refers to a quantity of the molecular construct sufficient to produce the desired therapeutic response. An effective amount of a drug is not required to cure a disease or condition, but rather provides treatment for a disease or condition such that the onset of the disease or condition is delayed, inhibited, or prevented, or the symptoms of the disease or condition are ameliorated. An effective amount may be divided into one, two, or more doses in an appropriate form to be administered one, two, or more times over a specified period of time. The specific effective or sufficient amount will vary depending on factors such as the particular condition being treated, the patient's physical condition (e.g., the patient's weight, age, or sex), the type of subject being treated, the duration of treatment, the nature of concomitant therapy (if any), and the specific formulation and structure of the compound or its derivative employed. An effective amount may be expressed, for example, as the total mass (e.g., grams, milligrams, or micrograms) of the active ingredient or as a ratio of the mass of the active ingredient to body weight, e.g., milligrams per kilogram (mg / kg).

[0052] The terms "application" and "administration" are used interchangeably herein to mean the application of a molecular construct or pharmaceutical composition of the present invention to a subject in need of such treatment.

[0053] The terms "subject" and "patient" are used interchangeably herein and are intended to refer to animals, including the human species, treatable by the molecular constructs, pharmaceutical compositions, and / or methods of the present invention. The term "subject" or "patient" refers to both male and female genders unless one gender is specifically indicated. Accordingly, the term "subject" or "patient" includes any mammal that may benefit from the treatment methods of the present disclosure. Examples of "subject" or "patient" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry. In exemplary embodiments, the patient is a human. The term "mammal" refers to all members of the mammalian genus, including humans, primates, domestic and farm animals such as rabbits, pigs, sheep, and cattle, as well as zoo, sport, or pet animals, and rodents such as mice and rats. The term "non-human mammal" refers to all members of the mammalian genus other than humans.

[0054] According to various embodiments of the present invention, the term "metal binding motif" refers to Zn 2+ , Ni 2+ , Co 2+ , Fe 2+ , Mn 2+ or Cu 2+ In certain embodiments, a metal binding motif capable of binding zinc ions is also referred to as a "zinc binding motif," although one of skill in the art will appreciate that such tags may also bind other metal ions with similar physical and / or chemical properties to zinc ions.

[0055] The present disclosure is based on the design of several novel metal-binding motifs that can be fused to at least the N- or C-terminus of a parent polypeptide. The metal-binding motifs are advantageous for the construction of bioconjugates in several aspects. First, because the metal-binding motifs are sequences of human origin, they are less immunogenic than conventional metal-binding motifs that are not part of the human genome. Second, the expression yield of composite polypeptides having the metal-binding motifs is desirable, and the resulting expressed composite polypeptides are highly stable during storage, binding, and purification processes. Third, the metal-binding motifs allow for facile site-specific conjugation reactions between the SH groups of the cysteine ​​residues of the metal-binding motifs and the SH-reactive groups of functional elements under conditions where the cysteine ​​residues of the parent polypeptides are largely inert. Thus, the present invention provides a variety of means for constructing multifunctional bioconjugates. Aspects and embodiments of the present invention are provided below.

[0056] (I) Metal-binding motif The first aspect of the present disclosure is a method for producing Zn under appropriate conditions. 2+ , Ni 2+ , Co 2+ , Fe 2+ , Mn 2+ or Cu 2+ The present invention is directed to metal binding motifs capable of forming complexes with metal ions such as

[0057] According to one embodiment of the present disclosure, the metal binding motif comprises the amino acid sequence, in N-terminal to C-terminal order or C-terminal to N-terminal order, of CX1X2HA (SEQ ID NO: 1), where X1 is glycine or proline and X2 is glycine or alanine. Exemplary metal binding motifs include, but are not limited to, CGGHA (SEQ ID NO: 2), CPGHA (SEQ ID NO: 3), CGAHA (SEQ ID NO: 4), CPAHA (SEQ ID NO: 5), GCGGHA (SEQ ID NO: 6), ACPGHA (SEQ ID NO: 7), and GCPGHA (SEQ ID NO: 8).

[0058] (II) Complex polypeptides containing metal-binding motifs In another aspect, the present disclosure is directed to a composite polypeptide comprising a metal-binding motif according to the above aspects / embodiments, such that the composite polypeptide is capable of forming a complex with a metal ion via the metal-binding motif under appropriate conditions.

[0059] According to one embodiment of the present disclosure, a composite polypeptide comprises a parent polypeptide according to the above aspects / embodiments of the invention and a metal-binding motif. In various embodiments, the metal-binding motif is fused to the N- or C-terminus of the parent polypeptide, either directly or with one or more intervening amino acid residues. In alternative embodiments, the intervening sequence may have 2 to 10 glycine residues.

[0060] According to various embodiments of the present disclosure, when the metal binding motif is located at the N-terminus of the parent polypeptide, the metal binding motif can have the sequence CX1X2HA (SEQ ID NO: 1) in N-terminal to C-terminal order or C-terminal to N-terminal order, where X1 is glycine or proline and X2 is glycine or alanine. Similarly, when the metal binding motif is located at the C-terminus of the parent polypeptide, the metal binding motif can have the sequence CX1X2HA (SEQ ID NO: 1) in N-terminal to C-terminal order or C-terminal to N-terminal order, where X1 is glycine or proline and X2 is glycine or alanine.

[0061] According to various embodiments of the present disclosure, the parent polypeptide can be a peptide hormone or its equivalent. Equivalents of peptide hormones include functional fragments, precursors, analogs, or derivatives known to those skilled in the art. Non-limiting examples of peptide hormones suitable for use herein include adrenocorticotropic hormone (ACTH), amylin, angiotensin, atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormone, follicle-stimulating hormone (FSH), insulin, leptin, melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, and vasoactive intestinal peptide. For example, equivalents of insulin include, but are not limited to, lispro, aspart, glulisine, detemir, degludec, and glargine. Equivalents of calcitonin include, but are not limited to, procalcitonin and adrenomedullin. Equivalents of somatostatin include, but are not limited to, octreotide and lanreotide.

[0062] In certain embodiments, the parent polypeptide can be a peptidomimetic ligand that binds to cell surface receptors.Peptidomimetics are small protein-like chains designed to mimic peptides.For example, a series of glutamic acid-urea-lysine-based peptidomimetics with PSMA-binding affinity have been designed.

[0063] In alternative embodiments, the parent polypeptide may be an antibody, such as a monoclonal antibody, immunoglobulin A (IgA), IgD, IgE, IgG, or IgM. Functional fragments or derivatives of whole antibodies are also encompassed by the scope of the present disclosure, examples of which include, but are not limited to, bispecific antibodies, chimeric antibodies, human antibodies, humanized antibodies, single-chain antibodies (scAb), single-chain variable fragments (scFv), tandem di-scFv, tandem tri-scFv, diabodies, triabodies, tetrabodies, Fab fragments, F(ab')2 fragments, Fd, domain antibodies, and minibodies.

[0064] Alternatively, the parent polypeptide may be a cytokine or its equivalent. Cytokine equivalents include functional fragments, precursors, analogs, or derivatives well known to those skilled in the art. Exemplary cytokines include, but are not limited to, interleukin-2 (IL-2), IL-10, IL-12, interferon alpha (IFN-α), IFN-γ, transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α).

[0065] In some embodiments, the parent polypeptide can be a single-chain antibody fragment, such as a single-domain antibody (sdAb), a single-chain antibody (scAb), a single-chain variable fragment (scFv), a bispecific T cell-triggering antibody (BiTE), a tandem di-scFv, or a tandem tri-scFv. In other embodiments, the parent polypeptide can be an antibody fragment derived from an immunoglobulin heavy or light chain, and the composite polypeptide comprises a portion of a full-length antibody, a fragment crystallizable region (Fc region), a fragment antigen-binding fragment (Fab), Fab', F(ab')2, Fab', Fv, (scFv)2, sc(Fv)2, a diabody, Fd, Fd', or a minibody. The immunoglobulin can be derived from immunoglobulin D (IgD), IgE, or IgG. The antibody can be a bispecific antibody, a chimeric antibody, a human antibody, or a humanized antibody.

[0066] In certain embodiments, the antibodies (and functional fragments thereof) are specific for cell surface antigens associated with and / or overexpressed on diffuse tumors. Examples of such cell surface antigens include CD5, CD19, CD20, CD22, CD23, CD27, CD30, CD33, CD34, CD37, CD38, CD43, CD72a, CD78, CD79a, CD79b, CD86, CD134, CD137, CD138, and CD319. Antibodies (or fragments or derivatives thereof) against these cell surface antigens can be used as targeting elements that can direct molecular constructs comprising them to disease sites. In other alternative embodiments, the antibodies (and functional fragments or derivatives thereof) are specific for other cell surface antigens, such antibodies being capable of eliciting a therapeutic effect in the human body, examples of such cell surface antigens include CD3, CD16a, CD28, CD134, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death 1 ligand 1 (PD-L1). Alternatively, the antibody (or fragment thereof) may be specific for a tumor-associated antigen (TAA), such as human epidermal growth factor receptor (HER1), HER2, HER3, HER4, carbohydrate antigen 19-9 (CA19-9), CA125, carcinoembryonic antigen (CEA), mucin 1 (MUC1), ganglioside GD2, melanoma-associated antigen (MAGE), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, mucin-associated Tn, sialyl Tn, globo H, stage-specific embryonic antigen-4 (SSEA-4), and epithelial cell adhesion molecule (EpCAM). Each TAA is often overexpressed on the cell surface of one or more types of solid tumors, and antibodies against such TAA can be used as targeting elements as part of the molecular constructs proposed herein. In certain other alternative embodiments, the antibody (or fragment thereof) is specific to a growth factor selected from the group consisting of epidermal growth factor (EGF), mutant EGF, epiregulin, heparin-binding epidermal growth factor (HB-EGF), vascular endothelial growth factor A (VEGF-A), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF).In certain embodiments, the antibody (or fragment thereof) is specific for the above cytokines.

[0067] (III) Molecular architecture of composite polypeptides containing functional elements and metal-binding motifs In yet another aspect, one or more functional elements can be covalently attached to a composite polypeptide according to the above aspects / embodiments of the present disclosure by reacting with the sulfhydryl (SH) groups of cysteine ​​residues in the metal-binding motif of the composite polypeptide, thereby forming a molecular construct (or bioconjugate) according to the invention. In particular, the metal-binding activity of the composite polypeptide allows for site-specific attachment, thereby resulting in a homogeneous molecular construct.

[0068] According to alternative embodiments of the present disclosure, the SH-reactive group is a maleimide, iodoacetyl, bromoacetyl, vinylsulfone, monosulfone, methylsulfonylbenzothiazole, or 2-pyridyldithiol group.

[0069] As can be understood, the functional element can be an effector molecule capable of inducing a desired therapeutic effect in the body of a subject. Alternatively, the functional element can be a targeting element capable of directing the molecular construct to a target site in the body of a subject. Alternatively, the functional element can be a pharmacokinetic element capable of modifying the pharmacokinetic profile of the molecular construct in the body of a subject. Alternatively, the functional element is in the form of a linker unit or bundle. According to certain embodiments of the present invention, the linker unit comprises a central core and multiple targeting elements, effector elements or pharmacokinetic elements, and the structure of said linker unit is described in Section (IV) below.

[0070] Examples of effector molecules that can be used in the present invention include, but are not limited to, cytotoxic drugs (alone or complexed with a radionuclide), Toll-like receptor (TLR) agonists or chelators.

[0071] As can be appreciated, cytotoxic drugs that exhibit a cytotoxic effect on specific cells include antiestrogens (e.g., tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g., goscurculin and leuprolide), antiandrogens (e.g., flutamide and bicalutamide), photodynamic therapy (e.g., vertoporphine, phthalocyanines, photosensitizer Pc4, and demethoxy-hypocrelin A), nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and megestrol), and the like. flufenaconazole), nitrosoureas (e.g., carmustine and lomustine), alkylsulfonates (e.g., busulfan and treosulfan), triazenes (e.g., dacarbazine, temozolomide), platinum-containing compounds (e.g., cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g., paclitaxel, docetaxel, and taxol), epipodophyllins (e.g., etoposide, etoposide phosphate, teniposide, topotheca camptothecin, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mitomycin C), antimetabolites, DHFR inhibitors (e.g., methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g., hydroxyurea and deferoxamine), uracil analogs (e.g., 5-fluorouracil (5-FU), floxuridine, doxifluridine, latitrexate, sed, tegafur-uracil, capecitabine), cytosine analogs (e.g., cytarabine (araC), cytosine arabinoside, and fludarabine), purine analogs (e.g., mercaptopurine and thioguanine), vitamin D3 analogs (e.g., EB1089, CB1093, and KH1060), isoprenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin),Bleomycin (e.g., bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g., verapamil), Ca2+ ATPase inhibitors (e.g., thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, neratinib, nilotinib, semaxanib, sunitinib, toceranib, vandetanib, vatalanib, rituximab, nilotinib, sorafenib, everolimus, temsirolimus), proteasome inhibitors (e.g., bortezomib), mTOR inhibitors (e.g., rapamycin, temsirolimus), The cytotoxic drug may be musirolimus, everolimus, and ridaforolimus), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbidine, prednisolone, dexamethasone, campatethecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurocidin, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, or hexamethylmelamine. According to a specific embodiment of the present disclosure, the cytotoxic drug is mertansine, auristatin, maytansine, doxorubicin, calicheamicin, or camptothecin.

[0072] Examples of Toll-like receptor agonists include lipoteichoic acid, glucan, motolimod, imiquimod, resiquimod, gardiquimod, CpG oligodeoxynucleotides (CpG DON), lipopolysaccharide (LPS), monophosphoryl lipid A, and zymosan.

[0073] In various embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), N,N''-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclo-nonane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 1,4,7-triazacyclo-nonanephosphinic acid (TRAP), 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[ 9.3.1.]Pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (=PCTA), N'{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA) ), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (oxo-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-Bz-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA). In certain embodiments, the radionuclide is 111 In, 131 I or 177 Lu, and in other embodiments, the radionuclide is 90 Y, 68 Ga, 99m Tc, 64 Cu, 153 Gd, 155 Gd, 157Gd, 213 Bi, 225 It can be Ac or Fe.

[0074] In other embodiments, the choice of effector element for the molecular constructs of this invention also encompasses a wide range of peptide-based molecules, including: (1) antibody fragments specific for inflammatory cytokines (such as TNF-α, IL-12 / IL-23, IL-17, IL-1, IL-6, BAFF), (2) antibody fragments specific for RANKL, (3) antibody fragments against CD3 and CD16a expressed on T cells and NK cells, (4) antibody fragments specific for PD-1, PD-L1, CTLA-4 and other immune checkpoints, and (5) immune-enhancing cytokines (IFN-α, IFN-γ, IL-2, TNF-α).

[0075] Targeting moieties for use in embodiments of the present invention can be selected depending on the disease to be treated. For example, targeting moieties for treating various diseases include: (1) antibody fragments specific for components of the extracellular matrix of joints, skin, or bone, such as type I collagen, type II collagen, type III collagen, type V collagen, type VII collagen, type IX collagen, type XI collagen, α-aggrecan, and osteonectin; (2) antibodies specific for CD19, CD20, CD22, CD30, CD52, CD79a, CD79b; CD38, CD56, CD74, CD78, CD138; CD319, CD5, CD4, CD7, CD8, CD30; or CD13 (3) antibody fragments specific for clusters of differentiation markers such as CD14, CD15, CD33, CD34, CD36, CD37, CD41, CD61, CD64, CD65, and CD11c, and other surface antigens of cells of lymphoid and myeloid lineages and plasma cells, or (4) antibody fragments specific for receptors or antigens overexpressed on the cell surface of solid tumors, such as human epidermal growth factor receptor (HER1), HER2 / Neu, HER3, Tn, globo H, ganglioside GD-2, CA125, CA19-9, and carcinoembryonic antigen (CEA).

[0076] The targeting element may also be an antibody to a hormone, growth factor, or cytokine whose receptor is expressed on tumor cells or other diseased cells, hi some alternative embodiments, the targeting element of the molecular construct is a growth factor.

[0077] According to certain embodiments of the present disclosure, at least one of the parent polypeptide and the functional element is an antibody fragment specific for a growth factor. In certain embodiments, the growth factor is selected from the group consisting of epidermal growth factor (EGF), mutant EGF, epiregulin, heparin-binding epidermal growth factor (HB-EGF), vascular endothelial growth factor A (VEGF-A), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF). Similar to the above concept, when the targeting element is a growth factor (e.g., EGF), the molecular construct can specifically target cells / tissues / organs expressing the receptor (e.g., tumor cells on which EGF receptors are expressed). When the effector element is an antibody fragment specific for a growth factor (e.g., VEGF-A), it can capture and neutralize growth factor-related signaling pathways (e.g., VEGF-A-induced angiogenesis). According to an effective embodiment, the molecular construct is useful for treating solid tumors when the effector element is an antibody fragment specific for VEGF-A.

[0078] Examples of pharmacokinetic factors include C 8-28 Fatty acid derivatives or C 8-28The functional elements are diacid fatty acid derivatives. Each pharmacokinetic element is linked to one of the K residues via the ε-amino acid group of the K residue. According to various embodiments of the present disclosure, the functional elements are fatty acid derivatives derived from octanoic acid, pelargonic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanoic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ricinoleic acid, or vaccenic acid, eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA). According to certain embodiments of the present disclosure, the functional element is a diacid fatty acid derivative derived from suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, brassylic acid, tetradecanedioic acid, pentadecanedioic acid, thapsic acid, heptadecanedioic acid, or octadecanedioic acid. In some embodiments, the functional element is derived from myristic acid or palmitic acid. In other embodiments, the functional element is derived from tetradecanedioic acid or thapsic acid.

[0079] (IV) a linker unit for binding to a composite polypeptide containing a metal-binding motif In an alternative embodiment, the functional element attached to the composite polypeptide is in the form of a bundle or linker unit. According to a particular embodiment of the present invention, the linker unit comprises a central core and multiple targeting elements, effector elements or pharmacokinetic elements. As can be understood, such linker units are also included in the scope of protection of the present disclosure.

[0080] According to a particular embodiment of the invention, the linker unit comprises a central core according to the above aspects / embodiments and a plurality of targeting, effector or pharmacokinetic elements.

[0081] Specifically, the central core is a polypeptide having a length of 3 to 120 amino acid residues and comprising 2 to 10 lysine (K) residues. For example, the core may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 K residues. Any two of the K residues may be adjacent to each other or separated by a filler. In some cases, the SH-reactive group is linked to the first or last K residue of the central core by forming an amide bond with it. In other cases, the central core further comprises a terminal spacer, and the SH-reactive group is linked to the terminal amino acid residue of the terminal spacer by forming an amide bond with it. In this way, it is feasible to combine the terminal SH-reactive group of the linker unit with the SH group of the cysteine ​​residue of the metal-binding motif of the composite polypeptide under conditions where the cysteine ​​residue of the parent polypeptide is largely inert, thereby achieving site-specific conjugation to produce a homogeneous molecular construct.

[0082] The terminal spacer can be an N-terminal spacer linked to the N-terminus of the first K residue or a C-terminal spacer linked to the C-terminus of the last K residue. The filler and terminal spacer each independently comprise (1) 1 to 12 non-K amino acid residues or (2) a PEGylated amino acid having 1 to 12 repeats of ethylene glycol (EG) units.

[0083] When the linker unit does not have a linking arm, each targeting element, effector element, or pharmacokinetic element is linked to the K residue of the core through the formation of an amide bond with the ε-amino group of the K residue. On the other hand, when the linker unit of the molecular construct further comprises 2 to 10 linking arms, one end of each linking arm is linked to the K residue of the core through the formation of an amide bond with the ε-amino group of the K residue, and the other end of each linking arm is linked to each targeting element, effector element, or pharmacokinetic element. For example, the linking arm can be a peptide comprising 2 to 10 non-K amino acid residues or a polyethylene glycol (PEG) chain having 2 to 24 repeats of EG units.

[0084] According to alternative embodiments of the present disclosure, prior to being conjugated with a targeting element, effector element, or pharmacokinetic element, the free end of the linking arm (i.e., the end not linked to the lysine residue of the central core) may bear a linking group selected from the group consisting of an amine, carboxyl, N-hydroxysuccinimidyl (NHS), azide, alkyne, cyclooctyne, tetrazine, and cyclooctene group. Depending on the linking group (i.e., amine, carboxyl, NHS, azide, alkyne, cyclooctyne, tetrazine, or cyclooctene group) that modifies the side chain amino group of the lysine residue (in the absence of a linking arm) or that is present at the free end of the linking arm, it is feasible to design a functional element (such as a targeting element, therapeutic effector element, or pharmacokinetic element) with a corresponding functional group such that the functional element can be linked to the free end of the linking arm via any of the following chemical reactions: (1) Formation of an amide bond between them: in this case, the linking group is an amine, carboxyl or NHS group and the functional element has an amine or carboxyl group; (2) Copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC reaction): one of the linking group and the functional group has an azide or picolyl azide group, while the other has an alkyne group. (3) Inverse electron demand Diels-Alder (iEDDA) reaction: one of the linking group and the functional group has a tetrazine group, while the other has a cyclooctene group (e.g., TCO or norbornene group), or (4) Strain-promoted azide-alkyne click chemistry (SPAAC) reaction: One of the linking group and the functional group has an azide group, while the other has a cyclooctyne group.

[0085] According to various embodiments of the present disclosure, the tetrazine group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, 1,2,4,5-tetrazine or a derivative thereof, the cyclooctene group is a norbornene or transcyclooctene (TCO) group, and the cyclooctyne group is selected from the group consisting of dibenzocyclooctyne (DIBO), difluorocyclooctyne (DIFO), bicyclononyne (BCN), and dibenzoazacyclooctyne (DIBAC or DBCO). According to one embodiment of the present disclosure, the tetrazine group is 6-methyltetrazine.

[0086] According to an alternative embodiment of the present invention, the central core carries a localized negative charge at or near the amino acid residue linked to the SH-reactive group. Specifically, the localized negative charge is present within the first 5 to 15 amino acid residues starting from the amino acid residue linked to the SH-reactive group. For example, the localized negative charge is present within the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues starting from the amino acid residue linked to the SH-reactive group. The localized negative charge can be imparted by including one or more amino acid residues that are negatively charged at pH 7, such as aspartic acid (D) and glutamic acid (E) residues.

[0087] Alternatively, the core can be coupled to multiple negatively charged effector elements such that the central core carries a localized negative charge at or near the SH-reactive group. For example, the effector elements can comprise chelators (e.g., DOTA, DOTAGA, etc.) that can increase the net negative charge of the linker units as a whole.

[0088] The targeting elements, effector elements, and pharmacokinetic elements described in Section (III) above are applicable to the embodiments in Section (IV), and for the sake of brevity, a detailed description thereof will not be provided here. In various embodiments, a linker unit carrying multiple effector elements is referred to as an "effector bundle" or "drug bundle," a linker unit carrying multiple chelators is referred to as a "chelator bundle," a linker unit carrying multiple targeting elements is referred to as a "targeting bundle," a linker unit carrying multiple scFvs is referred to as an "scFv bundle," a linker unit carrying multiple pharmacokinetic elements is referred to as a "pharmacokinetic (PK) bundle," and a linker unit carrying multiple fatty acids and / or diacid fatty acid chains is referred to as a "fatty acid (FA) bundle."

[0089] In certain embodiments, the fatty acid (or diacid fatty acid) derivative is a chemically modified fatty acid molecule (or diacid fatty acid). For example, the carboxyl group of the fatty acid molecule (or one of the carboxyl groups of the diacid fatty acid molecule) is reacted with a chemical moiety having two functional groups, one functional group being carboxyl-reactive (thereby forming a covalent bond with the (diacid) fatty acid molecule) and the other functional group being capable of reacting with the side chain amino group of a lysine residue. According to alternative embodiments of the present disclosure, the chemical moiety modifying the (diacid) fatty acid molecule is a glutamic acid residue, an aspartic acid residue, amino-EG2-acid, or gamma-aminobutyric acid, etc., but the present disclosure is not limited thereto.

[0090] As can be appreciated, in cases where there are no cysteine ​​residues or other amino acid residues containing sulfhydryl groups in the parent polypeptide, a terminal cysteine ​​residue may be introduced in place of the terminal metal binding motif to form a composite polypeptide, and the linker unit can form a bond with such a composite polypeptide via the terminal cysteine ​​residue.

[0091] (V) a linker unit carrying multiple composite polypeptides containing metal-binding motifs; In yet another aspect, the present invention is directed to a linker unit capable of carrying multiple composite polypeptides according to the above aspects / embodiments of the present disclosure. In certain embodiments, such a linker unit is referred to as a "peptide bundle."

[0092] According to a particular embodiment of the present invention, the linker unit comprises a central core according to the above aspect / embodiment, 2 to 10 linking arms, and 2 to 10 composite polypeptides. The linking arms of such a linker unit have an SH-reactive group at their free ends, and each composite polypeptide has the present metal-binding motif, so that the composite polypeptides can be attached to the linking arms in a site-specific manner under conditions where the cysteine ​​residues of the parent polypeptides are largely inactive.

[0093] Specifically, the central core is a polypeptide having a length of 3 to 120 amino acid residues, and the core comprises 2 to 10 lysine (K) residues, for example, the core may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 K residues and a linking group, where any two of the K residues are adjacent to each other or separated by a filler.

[0094] In some cases, the central core further comprises a linking group that is connected to the first or last K residue of the central core by forming an amide bond therewith. Examples of linking groups include, but are not limited to, azide, alkyne, tetrazine, cyclooctene, and cyclooctyne groups.

[0095] In other cases, the central core further comprises a terminal spacer, and the linking group is linked to the terminal amino acid residue of the terminal spacer by forming an amide bond therewith. The terminal spacer can be an N-terminal spacer linked to the N-terminus of the first K residue or a C-terminal spacer linked to the C-terminus of the last K residue.

[0096] The filler and terminal spacer each independently comprise (1) 1 to 12 non-K amino acid residues or (2) a PEGylated amino acid having 1 to 12 repeats of ethylene glycol (EG) units. Generally, the terminal spacer or filler can be (1) an oligopeptide having 1 to 12 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) non-K amino acid residues or (2) a PEGylated amino acid having 1 to 12 EG units (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 EG units). Specifically, when the core comprises multiple K residues, the terminal spacer or filler may comprise a PEGylated amino acid having 2 to 12 EG units, or may comprise 1 to 12 non-K amino acid residues, each of which is selected from the group consisting of glycine (G), aspartic acid (D), glutamic acid (E), serine (S), arginine (R), histidine (H), threonine (T), asparagine (N), glutamine (Q), proline (P), and the like. ), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W) residues, preferably each of the non-K amino acid residues is selected from the group consisting of G, S, R, H, T, N, Q, P, A, V, I, L, M, F, Y and W, more preferably each of the non-K amino acid residues is a G and / or S residue, respectively.

[0097] According to some embodiments, one end of each linking arm is linked to a K residue of the core via an amide bond with the ε-amino group of the K residue, while the other end of each linking arm has a sulfhydryl (SH)-reactive group. For example, the linking arms can be peptides comprising 2-12 non-K amino acid residues or polyethylene glycol (PEG) chains comprising 2-24 repeats of EG units.

[0098] Exemplary SH-reactive groups include maleimide, haloacetyl (eg, iodoacetyl or bromoacetyl), sulfone (eg, vinylsulfone, monosulfone, methylsulfonylbenzothiazole), and pyridyl disulfide (eg, 2-pyridyldithiol) groups.

[0099] According to certain embodiments of the present disclosure, the linking arm is a peptide comprising 2 to 12 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues (each of which can be a natural or unnatural amino acid residue), other than the K amino acid residue. For example, the linking arm is a peptide comprising 2 to 12 amino acid residues independently selected from the group consisting of G, E, D, S, R, H, T, N, Q, P, A, V, I, L, M, F, Y, and W residues. According to one advantageous embodiment, the linking arm is a peptide comprising 5 to 10 amino acid residues independently selected from the group consisting of G, S, E, and R residues. Alternatively, the linking arm can be a PEG chain having 2 to 24 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) repeats of EG units, preferably 5 to 15 repeats of EG units, more preferably 6 to 12 repeats of EG units. As can be appreciated, the peptide or PEG chain of the linking arm can be replaced with a polymer of approximately the same length. Polymers comprising carbohydrate or other hydrophilic building blocks are suitable for use as the linking arm.

[0100] According to one embodiment of the present invention, each linking arm carries a localized negative charge at or near the amino acid residue linked to the SH-reactive group. Specifically, the localized negative charge is present within the first 5 to 15 amino acid residues starting from the amino acid residue linked to the SH-reactive group. For example, the localized negative charge is present within the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues starting from the amino acid residue linked to the SH-reactive group. The localized negative charge can be imparted by including one or more amino acid residues that are negatively charged at pH 7, such as aspartic acid (D) and glutamic acid (E) residues.

[0101] As can be understood, when the core comprises at least two terminal spacers or fillers, each of the terminal spacers or fillers may be the same or different, i.e., each terminal spacer or filler may comprise the same or different amino acid sequence and / or EG unit. According to one embodiment of the present disclosure, the core comprises three fillers, one of which is a PEGylated amino acid having eight repeats of EG units, and the other two spacers each comprise one S residue and one G residue. According to another embodiment of the present disclosure, the core comprises one terminal spacer and four fillers, one of which is composed of four G and two S residues, while the other four spacers each comprise one G and one S residue. According to yet another embodiment of the present disclosure, the core comprises two terminal spacers and three fillers, each of which is a PEGylated amino acid having four repeats of EG units.

[0102] According to an alternative embodiment of the present invention, the above-mentioned linker units (or peptide bundles) comprising multiple composite polypeptides can be linked with additional linker units by reacting with the linking group of the central core to form a molecular construct. For example, the additional linker units can be effector bundles, targeting bundles, or PK bundles. For example, the additional linker units can adopt structures similar to those described in the applicant's previously filed patent applications. Briefly, the additional linker units can have structures similar to those described in Section (III) above, except that the terminal SH-reactive groups are replaced with linking groups corresponding to the linking groups of the peptide bundles.

[0103] In embodiments where the targeting or effector element carried by the additional linker unit is a peptide-based element, the peptide-based element may have the zinc-binding motif described above, and the additional linker unit may adopt the structure of this peptide bundle, thereby forming a molecular construct composed of two peptide bundles carrying different types of functional elements.

[0104] In an alternative embodiment, the linking group is part of a linking moiety that has a functional group capable of forming a covalent bond with the α-amino group (—NH) of the terminal amino acid residue (i.e., the first linked or N-terminal amino acid residue of the N-terminal spacer) or the carboxyl group (—COOH) of the terminal amino acid residue (i.e., the last linked or C-terminal amino acid residue of the C-terminal spacer), thereby linking the linking moiety thereto. In certain embodiments, the core may have only one of the N- and C-terminal spacers, with both a first and a second linking moiety linked to the two terminal amino acid residues, respectively (which may be the terminal linked or terminal amino acid residues of the terminal spacers). There are also embodiments in which the core comprises both an N- and a C-terminal spacer, and two linking moieties are linked to the terminal amino acid residues of the two terminal spacers, respectively. In a preferred embodiment, the covalent bond formed between the linking moiety and the terminal amino acid residue is an amide bond. As can be appreciated, to ensure the homogeneity of the resulting bundle, it is important that one linking moiety has only one functional group capable of reacting with either the α-amino group or the carboxyl group.

[0105] Optionally, the linking moiety further comprises a PEG chain having 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10) repeats of EG units connecting the carboxyl or amino group and the linking group; for example, the PEG chain can have 4, 6, 7, or 8 repeats of EG units.

[0106] According to various embodiments of the present disclosure, the two linking moieties can have the same or different reactivities. Preferably, when the second linking group of the linking moiety is an azide, alkyne, or cyclooctyne group, the linking group of the linking arm is not an azide, alkyne, or cyclooctyne group to avoid reaction between the second linking group and the linking group; rather, the linking group can be a tetrazine, cyclooctene, amine, carboxyl, or N-hydroxysuccinimidyl (NHS) group. Alternatively, when the second linking group is a tetrazine or cyclooctene group, the linking group is not a tetrazine or cyclooctene group; instead, the linking group can be an azide, alkyne, cyclooctyne, amine, carboxyl, or N-hydroxysuccinimidyl (NHS) group.

[0107] As can be appreciated, the number of optional linking arms attached to a core is determined primarily by the number of linking amino acid residues (i.e., K residues) provided on the core. Since there are at least two linking amino acid residues provided on the core, the targeting bundle or effector bundle can comprise multiple linking arms.

[0108] As can be appreciated, a description of an effector element, targeting element, or pharmacokinetic element is applicable to all aspects / embodiments involving such elements, unless the context clearly indicates otherwise.

[0109] As can be appreciated, during solid-phase synthesis of the peptide central core, it is also feasible to incorporate K amino acid residues modified with specific functional elements into the peptide chain, instead of attaching the functional element to the core after the core has been synthesized. Thus, according to various embodiments, K residues modified with different functional elements may be added sequentially during solid-phase synthesis to give a batch of homogeneous pharmacokinetic bundles, each of which may have two or more different functional elements linked thereto.

[0110] The following examples are provided to clarify certain aspects of the present invention and to assist those skilled in the art in practicing the present invention. These examples should not be construed as limiting the scope of the invention in any way. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. [Example]

[0111] Synthesis of Mal-peptide 1 core In this example, a peptide with five lysine residues was designed by the present inventors and its production was outsourced to KareBay Co., Ltd. (Monmans Junction, USA). This peptide can be used as a linker unit or a central core for constructing functional bundles. Mal-peptide 1 (maleimido-ethyl-GGSGGSKGSKGSKGSKGSK; SEQ ID NO: 11) was synthesized using standard Fmoc-based solid-phase methods. Mal-peptide 1 had a purity of 95.67%.

[0112] The resulting peptides were identified using mass spectrometry MALDI-TOF. Mass spectrometry analysis was performed by the Mass Core Facility of the Institute of Molecular Biology (IMB), Academia Sinica, Taipei, Taiwan. Measurements were performed on a Bruker Autoflex III MALDI-TOF / TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).

[0113] FIG. 1 shows the results of mass spectroscopy MALDI-TOF, which shows that Mal-peptide 1 has a molecular weight of 1790.916 daltons. [Example]

[0114] Synthesis of Mal-peptide2-DOTA bundles In this example, a chelator bundle (see Figure 2) having a maleimide-containing peptide with three lysine residues as a central core and three DOTA molecules bound to the central core (Mal-peptide2-DOTA bundle) was designed by the inventors, and its production was outsourced to KareBay Co., Ltd. (Montmans Junction, USA).

[0115] Mal-peptide 2 (maleimido-ethyl-GGSGGSGKGGSGGSGKGGSGGSGK, SEQ ID NO: 12), used as the central core, was synthesized using standard Fmoc-based solid-phase synthesis, and then the DOTA molecule was attached to the central core using solution-phase synthesis.

[0116] The resulting purified sample of the synthesized chelator bundle was analyzed by reversed-phase analytical high-performance liquid chromatography (HPLC) on a Supelco C18 column (250 mm × 4.6 mm; 5 μm) using a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, a linear gradient of 0% to 100% acetonitrile, over 30 min at a flow rate of 1.0 ml / min and a column temperature of 35° C. Figure 3A shows the reversed-phase HPLC profile of the chelator bundle of Example 2, showing that the chelator bundle peak has a retention time of 7.06 min.

[0117] The identification of the Mal-peptide2-DOTA bundle was carried out by mass spectrometry MALDI-TOF. Figure 3B shows that the results of mass spectrometry MALDI-TOF indicated that the chelating bundle had a molecular weight of 3093.454 Daltons. [Example]

[0118] Construction, expression, and purification of recombinant two-chain (scFv α CD19)-Fc-MBM-1 V of scFv specific for human CD19 L and V HThe present study was based on the monoclonal antibody RB4v1.2. A genetic sequence encoding a recombinant scFv-CH2-CH3 (human γ1) chain was constructed by fusing a genetic sequence encoding a human CD19-specific scFv upstream of the genetic sequence encoding the flexible hinge region and CH2 domain of IgG1.Fc, and a genetic sequence encoding a metal binding motif (MBM)-1, ACPGHA (SEQ ID NO: 7), downstream of the genetic sequence encoding the CH3 domain of IgG1.Fc and a short flexible linker, GGGG (SEQ ID NO: 9).

[0119] The scFv was synthesized in two orientations: V L -Linker-V H and V H -Linker-V L Consists of V L and V H and were connected by a hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10). L -Linker-V H and V H -Linker-V L The amino acid sequences of the recombinant chains of this (scFv α CD19)-Fc-MBM-1 in configuration are shown in SEQ ID NOs: 13 and 14, respectively, and the general structure for these two-chain (scFv α CD19)-Fc-MBM-1 molecular constructs is shown in Figure 4A.

[0120] To prepare the recombinant proteins, a mammalian overexpression system based on the Expi293F™ cell line was used, employing the ExpiFectamine™ 293 Transfection Kit (Life Technologies, Carlsbad, USA), which consists of the Expi293F™ cell line, the cationic lipid-based ExpiFectamine™ 293 Reagent, ExpiFectamine™ 293 Transfection Enhancers 1 and 2, and the medium that was part of the expression system (Gibco, New York, USA).

[0121] The gene sequence constructed as described above was placed into a pcDNA3 expression cassette. Expi293F cells were cultured in Expi293F expression medium at 2.0 × 10 6 Cells were seeded at a density of 7.5 x 10 viable cells / ml and left for 18-24 hours before transfection to ensure that the cells were actively dividing at the time of transfection. For transfection, 7.5 x 10 cells were seeded in 255 ml of medium in a 2-liter Erlenmeyer shaker flask. 8 Cells were transfected using ExpiFectamine™ 293 transfection reagent according to the manufacturer's instructions. The transfected cells were incubated at 37°C on an orbital shaker (125 rpm) for 16–18 hours post-transfection. The incubated cells were then added to a shaker flask with ExpiFectamine™ 293 Transfection Enhancer 1 and Enhancer 2 and incubated for an additional 5–6 days. The culture supernatant was harvested, and the expressed hIgG1.Fc fusion recombinant protein in the medium was purified using protein A affinity chromatography.

[0122] After buffer exchange into phosphate-buffered saline (PBS), the concentration of the (scFv α CD19)-Fc-MBM-1 was determined and analyzed using 10% SDS-PAGE. The results showed that the (scFv α CD19)-Fc-MBM-1 had a molecular weight of approximately 120 kDa in non-reducing SDS-PAGE, which is somewhat larger than the expected size of 110 kDa. Figure 4B shows the two-chain (V L -V H The results of non-reducing SDS-PAGE of scFv α (CD19)-Fc-MBM-1 (see the major band indicated by the arrow) are shown as an example. As can be understood, the SDS-PAGE results are used here to provide a preliminary confirmation of the approximate molecular weight of the resulting synthesized molecular construct, and more precise analysis of the molecular weight was determined using mass spectrometry. For example, the MALDI-TOF results of this molecular construct are shown in Figure 23. [Example]

[0123] Construction, expression, and purification of recombinant two-chain (scFv α CD19)-Fc-MBM-2 In this example, V L -Linker-V H Recombinant two-chain (scFv α CD19)-Fc-MBM-2 in the construct was constructed, expressed, and purified using a protocol similar to that described in the working example above, except that a different metal binding motif, GCPGHA (SEQ ID NO: 8, hereafter MBM-2), was used. L -V H The amino acid sequence of the recombinant chain of scFv α(CD19)-Fc-MBM-2 is shown in SEQ ID NO:15.

[0124] The SDA-PAGE results in Figure 5 show that the recombinant strand of this molecular construct has a size of approximately 120 kDa (indicated by an arrow), which is somewhat larger than expected. [Example]

[0125] Construction, expression, and purification of recombinant two-chain (scFv α CD19)-Fc-MBM-3 In this example, V L -Linker-V H Recombinant two-chain (scFv α CD19)-Fc-MBM-3 in the construct was constructed, expressed, and purified using a protocol similar to that described in the working example above, except that a different metal binding motif, GCGGHA (SEQ ID NO: 6, hereafter MBM-3), was used. L -Linker-V H The amino acid sequence of this (scFv α CD19)-Fc-MBM-3 recombinant chain in the construct is shown in SEQ ID NO:16.

[0126] (V L -V H The scFv α (CD19)-Fc-MBM-3 molecular construct was characterized using SDS-PAGE. The SDS-PAGE results in Figure 6 show that the recombinant chain of the new construct has a size of approximately 110 kDa (indicated by an arrow), which is consistent with the expected size. [Example]

[0127] Construction, expression and purification of recombinant anti-CD19 antibody MBM A recombinant IgG (human gamma 1) molecular construct was constructed by fusing a short flexible linker, GGGG (SEQ ID NO: 9), and an MBM sequence (in this example, MBM-1 or MBM-2) to the C-terminus of the heavy chain of an intact antibody specific for CD19. The two gene sequences were inserted into a pG1K expression cassette with multiple cloning sites. Expression and purification of this recombinant anti-CD19 antibody, MBM, was carried out using a protocol similar to that described in the working example above.

[0128] The amino acid sequences of the heavy chains of antibodies specific for the human CD19 antibodies MBM-1 or MBM-2 are shown in SEQ ID NOs: 17 and 18, respectively, and the amino acid sequence of the light chain of an intact anti-CD19 antibody is shown in SEQ ID NO: 19.

[0129] The molecular constructs were characterized using SDS-PAGE. The results of reducing SDS-PAGE in Figure 7A show that the control protein (lane 1), human CD19 antibody MBM-1 (lane 2), and human CD19 antibody MBM-2 (lane 3) have a high MW band of approximately 55 kDa and a low MW band of approximately 25 kDa, which are consistent with the predicted sizes of the heavy and light chains of the constructs, respectively.

[0130] The general structure for the anti-CD19 antibody MBM-1 molecule construct is shown in Figure 7B. [Example]

[0131] Construction, expression, and purification of recombinant two-chain (scFv α CD33)-Fc-MBM V of scFv specific for human CD33 L and V HThe monoclonal antibody huMy9-6 was used. A gene sequence encoding the (scFv α CD33)-CH2-CH3 (human γ1) recombinant chain was constructed by fusing a gene sequence encoding a human CD33-specific scFv upstream of a gene sequence encoding the flexible hinge region and CH2 domain of IgG1.Fc, and a gene sequence encoding MBM-1 or MBM-2 downstream of a gene sequence encoding the CH3 domain of IgG1.Fc and a short flexible linker GGGG (SEQ ID NO: 9).

[0132] The scFv was synthesized in two orientations: V L -Linker-V H and V H -Linker-V L Consists of V L and V H and were connected by a hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10). L -Linker-V H and V H -Linker-V L The amino acid sequences of the recombinant chains of this (scFv α CD33)-Fc-MBM-1 in the configuration are shown in SEQ ID NOs: 20 and 21, respectively, while V L -Linker-V H and V H -Linker-V L The amino acid sequences of the recombinant chains of this (scFv α CD33)-Fc-MBM-2 in the construct are shown in SEQ ID NOs: 22 and 23, respectively.

[0133] Expression and purification of this recombinant two-chain (scFv α CD33)-Fc-MBM was performed using a protocol similar to that described in the working example above.

[0134] Characterization of the molecular constructs was performed using non-reducing SDS-PAGE. The SDS-PAGE results in Figure 8 show (V L -V H scFv α CD33)-Fc-MBM-1 (lane 1), (V H -V LscFv α CD33)-Fc-MBM-1 (lane 2), (V L -V H scFv α CD33)-Fc-MBM-2 (lane 3) and (V H -V L Each of the scFv αCD33)-Fc-MBM-2 (lane 4) was shown to have a size of approximately 120 kDa, which is somewhat larger than the predicted size of 110 kDa. [Example]

[0135] Construction, expression, and purification of recombinant two-chain (scFv α CD20)-Fc-MBM V of scFv specific for human CD20 L and V H The metal binding motifs MBM-1, MBM-2, and MBM-3 were each fused to the C-terminus of the CH3 domain of the (scFv α CD20)-CH2-CH3(human γ1) recombinant chain via a short flexible linker GGGG (SEQ ID NO: 9). L -Linker-V H or V H -Linker-V L The orientation of V L and V H were connected by a hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10).

[0136] Expression and purification of the recombinant two-chain (scFv α CD20)-Fc-MBM-1, -MBM-2, or -MBM-3 was carried out using a protocol similar to that described in the working example above. L -V H scFv α CD20)-Fc-MBM-1, (V H -V L scFv α CD20)-Fc-MBM-1, (V H -V L scFv α CD20)-Fc-MBM-2, (V L -V H scFv α CD20)-Fc-MBM-3 and (VH -V L The sequences of the recombinant chains of scFv α(CD20)-Fc-MBM-3 are set forth in SEQ ID NOs: 24, 25, 26, 27 and 28, respectively.

[0137] Expression and purification of this recombinant two-chain (scFv α CD20)-Fc-MBM was performed using a protocol similar to that described in the working example above.

[0138] Characterization of the molecular constructs was performed using non-reducing SDS-PAGE. The SDS-PAGE results in Figure 9 show (V L -V H scFv α CD20)-Fc-MBM-1 (lane 1) and (V H -V L The SDA-PAGE results in Figures 10A and 10B show that both molecular constructs, scFv α CD20)-Fc-MBM-1 (lane 2), have a size of approximately 120 kDa, which is somewhat larger than the expected size of 110 kDa. H -V L scFv α CD20)-Fc-MBM-2 and (V L -V H Both molecular constructs, scFv α)-Fc-MBM-3, were shown to have a size of approximately 120 kDa, which is somewhat larger than the predicted size of 110 kDa. [Example]

[0139] Construction, expression, and purification of recombinant two-chain (scFv α CA19-9)-Fc-MBM The mouse B cell hybridoma HB8059, which produces anti-CA19-9 antibodies, was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa. Poly(A)+ RNA was reverse transcribed using the SuperScript III RT-PCR system (Invitrogen, Waltham, USA) to synthesize first-strand cDNA. H and V LThe nucleotide and amino acid sequences have not been published. H and V L The cDNA of the HB8059 monoclonal antibody specific for human CA19-9 was amplified by PCR using a set of DNA primers provided in the Ig primer set (Novagen, Madison, USA) according to the manufacturer's instructions. H and V L The amino acid sequences of these are set forth in SEQ ID NOs: 29 and 30.

[0140] MBM-1 or MBM-3 was fused to the CH3 domain (scFv α CA19-9)-CH2-CH3 (human γ1) recombinant chain via a short flexible linker GGGG (SEQ ID NO: 9). L -Linker-V H The orientation of V L and V H and were connected by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10). The sequences of the recombinant chains of (scFv α CA19-9)-Fc-MBM-1 and (scFv α CA19-9)-Fc-MBM-3 are shown as SEQ ID NOs: 31 and 32, respectively.

[0141] Expression and purification of this recombinant two-chain (scFv α CA19-9)-Fc-MBM was performed using a protocol similar to that described in the working example above.

[0142] The molecular constructs were characterized using SDS-PAGE. The SDS-PAGE results in Figures 11A and 11B show that the recombinant chains of both the (scFv α CA19-9)-Fc-MBM-1 and (scFv α CA19-9)-Fc-MBM-3 molecular constructs have a size of approximately 120 kDa (lane 1), which is somewhat larger than the expected size of 110 kDa. [Example]

[0143] Construction, expression, and purification of recombinant two-chain (scFv α CD38)-Fc-MBM V of scFv specific for human CD38 L and V H The metal binding motifs MBM-1 and MBM-2 were fused to the C-terminus of the CH3 domain of the (scFv α CD38)-CH2-CH3(human γ1) recombinant chain via the short flexible linker GGGG (SEQ ID NO: 9), respectively. The scFvs were L -Linker-V H or V H -Linker-V L The orientation of V L and V H and were connected by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10). Expression and purification of the recombinant two-chain (scFv α CD38)-Fc-MBM-1 or -MBM-2 was carried out using the same protocol as described in the working example above. L -V H scFv α CD38)-Fc-MBM-1, (V H -V L scFv α CD38)-Fc-MBM-1, (V L -V H scFv α CD38)-Fc-MBM-2 and (V H -V L The sequences of the recombinant chains of scFv α(CD38)-Fc-MBM-2 are set forth in SEQ ID NOs: 33, 34, 35 and 36, respectively.

[0144] The molecular constructs were characterized using SDS-PAGE. The SDS-PAGE results in Figure 12A show (V L -V H The SDA-PAGE results in Figure 12B show that the recombinant chain of scFv α CD38)-Fc-MBM-1 (lane 1) has a size of approximately 120 kDa, which is somewhat larger than the expected size of 110 kDa. L -V HThe recombinant chain of scFv α(CD38)-Fc-MBM-2 was shown to have a size of approximately 120 kDa, which is somewhat larger than the predicted size of 110 kDa. [Example]

[0145] Preparation of recombinant two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle In this example, a molecular construct was prepared with two DOTA bundles attached to two cysteine ​​residues in the metal binding motif of two-chain (scFv α CD19)-Fc-MBM-1. A schematic diagram showing the structure of this molecular construct is shown in Figure 13A. The purified recombinant two-chain (V L -V H To reduce the cysteine ​​residue at the C-terminus of scFv α (CD19)-Fc-MBM-1, the recombinant protein in sodium succinate buffer (10 mM sodium succinate, pH 6.0, and 30 mM sucrose) was incubated with 45 μM tris(2-carboxyethyl)phosphine (TCEP) at room temperature for 30 minutes with gentle shaking. After the reduction reaction, excess TCEP was removed by dialysis against 10 mM sodium succinate buffer (pH 6.0 with 30 mM sucrose) containing 60 μM Zn(II) ions. The reduced protein sample was then treated with 15 μM Mal-peptide2-DOTA bundle from Example 2 and incubated at room temperature for 1 hour. Unreacted DOTA bundle was removed using a desalting column, and the product was analyzed by SDS-PAGE.

[0146] Figure 13B shows the results of SDS-PAGE analysis of this molecular construct (shown below), indicating that the molecular construct has a molecular weight of approximately 120 kDa (lane 2, indicated by an arrow), which is slightly larger than expected. The unbound fusion protein appears as lane 1. As can be seen in Figure 13B, the yield of DOTA bundle binding to the recombinant two-chain (scFv α CD19)-Fc-MBM-1 is approximately 90%. These protein bands on SDS-PAGE were quantified by Image J. [Example]

[0147] Preparation of recombinant two-chain (scFv α CD19)-Fc-MBM-2×2 DOTA bundle In this example, recombinant two chains (V L -V H Conjugation of two DOTA bundles to scFv αCD19)-Fc-MBM-2 was performed as described in the previous example.

[0148] Figure 14 shows the results of SDS-PAGE analysis of this molecular construct, which showed that it had a molecular weight of approximately 120 kDa (arrowed lane 2), which is consistent with or slightly larger than the predicted size. The unbound fusion protein resembled lane 1. As can be seen in Figure 14, the conjugation yield of the DOTA bundle to the recombinant two-chain (scFv α CD19)-Fc-MBM-2 was approximately 90%. [Example]

[0149] Preparation of recombinant two-chain (scFv α CD19)-Fc-MBM-3×2 DOTA bundle In this example, recombinant two chains (V L -V H Conjugation of two DOTA bundles to scFv αCD19)-Fc-MBM-3 was performed as described in the previous example.

[0150] Figure 15 shows the results of SDS-PAGE analysis of this molecular construct, which showed that it had a molecular weight of approximately 120 kDa (arrowed lane 2), which is consistent with or slightly larger than the predicted size. The unbound fusion protein resembled lane 1. As can be seen in Figure 15, the conjugation yield of the DOTA bundle to the recombinant two-chain (scFv α CD19)-Fc-MBM-3 was approximately 90%. [Example]

[0151] Preparation of anti-CD19 antibody MBM-1x2DOTA bundle In this example, conjugation of two DOTA bundles to an intact human anti-CD19 antibody fused to C-terminal MBM-1 according to Example 6 was performed as described in the previous example.

[0152] Figure 16 shows an SDS-PAGE analysis of this molecular construct of the anti-CD19 antibody MBM-1x2DOTA bundle. As shown in Figure 16, the heavy chain molecular weight is approximately 54 kDa (indicated by arrow #1 in lane 2), which is consistent with the expected size. Arrow #2 in lane 2 indicates the unbound heavy chain of anti-CD19 antibody MBM-1, and arrow #3 indicates the light chain of anti-CD19 antibody MBM-1. [Example]

[0153] Preparation of anti-CD19 antibody MBM-2x2DOTA bundle In this example, conjugation of two DOTA bundles to an intact anti-CD19 antibody fused to C-terminal MBM-2 according to Example 6 was performed as described in the previous example.

[0154] Figure 17 shows an SDS-PAGE analysis of this molecular construct. As shown in Figure 17, the molecular weight of the heavy chain is approximately 54 kDa (indicated by arrow #1 in lane 2), which is consistent with the expected size. Arrow #2 in lane 2 indicates the unbound heavy chain of the anti-CD19 antibody MBM-2, and arrow #3 indicates the light chain of the anti-CD19 antibody MBM-2. [Example]

[0155] Preparation of recombinant two-chain (scFv α CD33)-Fc-MBM×2DOTA bundle In this example, the recombinant two chains (V L -V H Conjugation of two DOTA bundles to scFv α(CD33)-Fc-MBM-1 or -MBM-2 was performed as described in the previous examples.

[0156] Figure 18 shows the results of SDS-PAGE analysis of the binding of the DOTA bundle to the recombinant two-chain(scFv α CD33)-Fc-MBM-1. As shown in Figure 18, this molecular construct has a molecular weight of approximately 120 kDa (indicated by arrow #1 in lane 2), which is consistent with or slightly larger than the expected size. Arrow #2 in lane 1 indicates the unbound fusion protein. The SDS-PAGE results indicate that the yield of the DOTA bundle binding to the recombinant two-chain(scFv α CD33)-hIgG1.Fc-MBM-1 is approximately 65%. [Example]

[0157] Preparation of recombinant two-chain (scFv α CD20)-Fc-MBM-1x2 DOTA bundle In this example, recombinant two chains (V L -V H Conjugation of two DOTA bundles to scFv α(CD20)-Fc-MBM-1 was performed as described in the previous example.

[0158] Figure 19 shows the results of SDS-PAGE analysis of the binding of the DOTA bundle to recombinant two-chain (scFv α CD20)-Fc-MBM-1. As shown in Figure 19, this molecular construct has a molecular weight of approximately 120 kDa (indicated by arrow #1 in lane 2), which is consistent with or slightly larger than the expected size. Arrow #2 in lane 2 indicates the unbound fusion protein. The SDS-PAGE results in Figure 19 also show that the binding yield of the DOTA bundle to recombinant two-chain (scFv α CD20)-Fc-MBM-1 is approximately 90%. [Example]

[0159] Preparation of recombinant two-chain (scFv α CA19-9)-Fc-MBM×2DOTA In this example, conjugation of two DOTA bundles to recombinant two-chain (scFv α CA19-9)-Fc-MBM-1 or -MBM-3 according to Example 9 was performed as described in the previous example. SDS-PAGE results show that the conjugation yields of DOTA bundles to recombinant two-chain (scFv α CA19-9)-Fc-MBM-1 and -MBM-3 were approximately 65% ​​and 70%, respectively (data not shown). [Example]

[0160] Stabilization of two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle To stabilize the two-chain (scFv α CD19)-Fc-MBM-1×2DOTA bundle, a ring-opening reaction was carried out using the following protocol.

[0161] The reaction product of the molecular construct obtained in Example 14 was buffer-exchanged into Tris buffer (100 mM Tris, 100 mM L-arginine, and 100 mM sodium chloride, pH 9.0) using a NAP-10 Sephadex G-25 column (GE Healthcare). The resulting solution was then heated to 37°C for 5 hours. The solution was cooled and buffer-exchanged into 50 mM Bis-Tris buffer, pH 5.5, by centrifugation. The final sample was concentrated to approximately 1-3 mg / mL of protein.

[0162] For purification, the stabilized product was adjusted to pH 5.5 and then applied to a pre-equilibrated (50 mM Bis-Tris buffer, pH 5.5) anion-exchange column, Hi-Trap™ Q HP (GE Healthcare). The stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle was eluted using a step elution of 250 mM NaCl for 15 min and a linear gradient from 250 mM to 324 mM NaCl for 50 min at a flow rate of 1.0 ml / min.

[0163] The stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle was then separated from free two-chain fusion protein, two-chain fusion protein bound to three or four DOTA bundles, and aggregated material using an anion exchange column, Q HP. The purified two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle was concentrated and buffer-exchanged into Tris buffer (50 mM Tris buffer, pH 7.0, and 290 mM NaCl).

[0164] Figure 20 shows the FPLC elution profile of the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle from an anion exchange column Q HP. As shown in Figure 20, the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle eluted in a peak at 69.91 ml. Figure 21 shows the results of SDS-PAGE analysis of fractions collected from the anion exchange column Q HP. Lanes A, B, C, and D correspond to the unbound molecular construct, the molecular construct bound to a DOTA bundle, the stabilized molecular construct bound to two DOTA bundles in the storage solution, and the stabilized molecular construct bound to two DOTA bundles before loading onto the column, respectively.

[0165] The pure product, stabilized two-chain (scFv α CD19)-Fc-MBM-1x2DOTA bundle, was collected from fractions #53, #54 and #55 shown in FIG. [Example]

[0166] Stabilization of two-chain (scFv α CD20)-Fc-MBM-1×2 DOTA bundles Purification of stabilized two-chain (scFv α CD20)-Fc-MBM-1x2 DOTA bundle The procedure for stabilizing the two-chain (scFv α CD20)-Fc-MBM-1×2DOTA bundle is similar to that described in Example 19.

[0167] For purification, the stabilized product was adjusted to pH 5.1 and then applied to a pre-equilibrated (50 mM acetic acid, pH 5.1) anion-exchange column, Hi-Trap™ Q HP (GE Healthcare). The stabilized two-chain (scFv α CD20)-Fc-MBM-1x2 DOTA bundle was eluted using a step elution of 300 mM NaCl for 18 min and a linear gradient from 300 mM to 1000 mM NaCl for 80 min at a flow rate of 1.0 ml / min.

[0168] The stabilized two-chain (scFv α CD20)-Fc-MBM-1x2 DOTA bundle was then separated from free two-chain fusion protein, two-chain fusion protein bound to three or four DOTA bundles, and aggregated material using an anion exchange column, Q HP.

[0169] Figure 22 shows the results of SDS-PAGE analysis of fractions collected from the anion exchange column Q HP. Lanes A, B, and C correspond to the unbound molecular construct, the molecular construct bound to a DOTA bundle, and the stabilized molecular construct bound to two DOTA bundles before being loaded onto the column, respectively.

[0170] The pure product, stabilized two-chain (scFv α CD20)-Fc-MBM-1x2 DOTA bundle, was collected from fractions #40 and #41 shown in Figure 22. [Example]

[0171] MALDI-TOF analysis of stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundles The stabilized two-chain (scFv α CD19)-Fc-MBM-1×2DOTA bundle from Example 19 was analyzed using mass spectrometry MALDI-TOF. The MALDI-TOF results in Figure 23 show that the stabilized two-chain (scFv α CD19)-Fc-MBM-1×2DOTA bundle sample had m / z (z=1): [M+H] + and m / z(z=2):[M+2H] 2+The results show that the nucleotides have molecular weights of 114,435 and 57,306 daltons, which correspond to the nucleotides 114,435 and 57,306 daltons, respectively. [Example]

[0172] 89 Preparation of Y-labeled two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle In this example, a Y(NO3)3 solution was added to a solution of purified stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle from Example 19 at a ratio of 6:1 [Y 3+ The reaction mixture was incubated at room temperature for 6 hours. 3+ Ions were removed from the solution using a NAP-10 Sephadex G-25 column. 89 Y] 3+ The stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle chelated with α was analyzed using mass spectrometry MALDI-TOF. The MALDI-TOF results in Figure 24 show that the molecular construct has a molecular weight of 114,391 daltons. [Example]

[0173] 175 Preparation of Lu-labeled two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle In this example, a LuCl solution was added to a solution of purified stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle at a ratio of 60:1 [Lu 3+ The reaction mixture was then incubated at 45°C for 1.5 hours. 175 The resulting Lu-DOTA protein chelates were free Lu 3+ The ions were removed from the solution by spinning filtration (Amicon centrifugal filter, 10 kDa). 175The stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle chelated with Lu was analyzed using MALDI-TOF mass spectrometry. The MALDI-TOF results in Figure 25 show that the molecular construct has a molecular weight of 114,928 daltons. [Example]

[0174] 111 Preparation of In-labeled two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle 111 For the preparation of In-labeled fusion proteins, carrier-free 111 An aliquot of In was transferred to a tube, and 20 volumes of metal-free 0.1 M HEPES buffer was added to adjust the pH of the solution to 4.5. Then, a solution of the two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle (30-50 μg) in 0.1 M HEPES buffer (pH 4.5) was added to a final protein concentration of 0.3-0.6 mg / mL. The resulting solution was gently mixed and incubated at 40-45°C for 60 min. Diethylenetriaminepentaacetic acid (DTPA) was added in an amount 1000 times the amount of protein to release the ATP. 111 In 3+ The reaction was stopped by trapping the ions. After incubation at 37°C for 30 minutes, 111 In-DTPA chelate 111 The In-labeled product was removed and the solvent replaced with 0.9% saline by spinning filtration (Amicon centrifugal filter, 10 kDa).

[0175] collected 111 The specific activity of the In-labeled product was determined by measuring the radioactivity of an appropriate aliquot of the sample using a gamma counter. [Example]

[0176] 111 Radioincorporation assay of In-labeled two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle Radiochemical purity assessment was performed by instant thin layer chromatography (iTLC). The solution of the radiolabeled product from Example 24 was diluted 1:10 or 1:20 with the original solvent, and then 1 μL of the sample was spotted on one edge of a 1 × 10 cm strip of TLC SG paper. The paper was developed using ascending chromatography with 0.5 M sodium citrate (pH 4.5). Radioactivity was determined using a radio-TLC scanner. Protein-associated radioactivity was expressed as a percentage of total radioactivity. As shown in Figure 26, 111 The radiochemical purity of the In-labeled two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle is greater than 98%. [Example]

[0177] in serum 111 Stability study of In-labeled two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle 111 The in vitro stability of the In-labeled two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle was evaluated in mouse serum at 37°C for 96 hours. 111 The In-labeled molecular constructs were suspended in 0.2 M ammonium acetate (pH 5). 111 In-labeled molecular constructs were diluted 1:10 in mouse serum and then incubated at 37° C. for periods up to 96 hours. At selected time points (0, 12, 24, 48, and 96 hours), 111 The In-labeled molecules were removed and analyzed using radio-TLC as described in Example 25.

[0178] As shown in Figure 27, 111 The In-labeled two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle showed sufficient stability during 96 h incubation in mouse serum at 37°C. [Example]

[0179] Site-specific binding of recombinant two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle To identify the cysteine ​​residues of the two-chain (scFv α CD19)-Fc-MBM-1 linked to the DOTA bundle, the sample was digested and analyzed using LC-MS. Briefly, 5 μl of the sample (0.4 μg / mL) from Example 11 was diluted with 15 μl of 25 mM tetraethylammonium tetrahydroborate (TEAB) and 2 μl of 200 mM TCEP and incubated at 55°C for 1 hour. Then, 2 μl of 375 mM iodoacetic acid (IAA) was added to the reaction mixture for 30 minutes at room temperature. After alkalinization with IAA, the solution was digested overnight with trypsin and Glu-C at 37°C.

[0180] The results of mass spectrometry (data not shown) showed that the m / z value of the fragment in the MS spectrum corresponded to 4530.99 daltons, which is consistent with the molecular weight of the molecular construct and the fragment containing the amino acid sequence of one DOTA bundle, SLSLSPGGGGACPGHA (amino acid residues 468 to 483 of SEQ ID NO: 13). [Example]

[0181] Binding activity of stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundles to human B lymphoma cell lines Two stabilized molecular constructs, (scFv α CD19)-Fc-MBM-1x2DOTA bundle and (scFv α CD19)-Fc-MBM-3x2DOTA bundle, were analyzed for their ability to bind to human CD19 in the human B lymphoma cell line Raji. Assays were performed using 1x10 antibodies (RB4v1.2), (scFv α CD19)-Fc-MBM-1x2DOTA bundle, and (scFv α CD19)-Fc-MBM-3x2DOTA bundle as positive controls, with 0.001 μg / ml of each construct in 1% BSA in PBS. 6The staining was performed by incubating CD19-expressing Raji cells on ice for 30 minutes. An anti-HER2 antibody (trastuzumab) was used as a negative control. Cell staining was analyzed by FACS (FACSCanto II; BD Biosciences) using FITC-conjugated goat anti-human IgG.Fc (diluted 1:200 in PBS / BSA) (Caltag, Buckingham, UK) for 20 minutes in the dark at 4°C. Figures 27A and 27B show the results of cell staining analysis of two molecular constructs containing MBM-1 and MBM-3, respectively, on CD19-expressing Raji cells, which demonstrate that the two constructs actively bound to Raji cells.

[0182] These molecular constructs were also analyzed for their ability to bind to human CD19 in the human B lymphoma cell line Ramos. The assay was performed using a protocol similar to that used in the cell binding assay with Raji cells. Staining of Ramos cells was analyzed by FACS (FACSCanto II; BD Biosciences) using FITC-conjugated goat anti-human IgG.Fc, with anti-CD19 IgG used as a positive control. An anti-HER2 antibody (trastuzumab) was used as a negative control. Figures 27C and 27D show the results of cell staining analysis of two molecular constructs containing MBM-1 and MBM-3, respectively, in CD19-expressing Ramos cells. These two constructs significantly and positively bound to Ramos cells. [Example]

[0183] Stability of stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle To assess the stability of the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2DOTA bundle, the molecular construct was placed in Tris buffer (100 mM Tris buffer, 50 mM Bis-Tris buffer, and 290 mM NaCl at pH 7.3) with glutathione or human serum albumin and then incubated at 37°C for 30 days.

[0184] As shown in Figure 29, the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle exhibited sufficient stability during incubation with glutathione or human serum albumin (HSA) at 37°C for 30 days. As can be seen in Figure 28, in both glutathione- and HSA-treated groups, approximately 90% of the intact molecular construct remained in solution without any retro-Michael addition products. Lanes U and C correspond to the unbound molecular construct and the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle, respectively, stored at 4°C as controls. [Example]

[0185] Examination of retro-Michael adducts of stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundles As a further confirmation of the stability of the two-chain (scFv α CD19)-Fc-MBM-1×2DOTA bundle, the stabilized molecular constructs were incubated with glutathione or HSA and then analyzed by Western blot.

[0186] For Western blot analysis of stabilized samples, briefly, samples were separated on an 8% SDS-PAGE gel and transferred to a poly(vinylidene fluoride) (PVDF) membrane (Millipore). The membrane blot was blocked for 1 hour at room temperature in PBS containing 5% BSA and 0.05% Tween 20. After washing three times with phosphate-buffered saline Tween-20 (PBST) containing PBS and 0.05% Tween-20, the blot was incubated with a goat anti-human IgG.Fc antibody conjugated with horseradish peroxidase (Millipore). The membrane was then washed three times with PBST, and immunoreactive bands were detected using ECL™ Western blotting detection reagents (Millipore) and exposed to a Fujifilm (Tokyo, Japan).

[0187] Figure 30 shows the results of Western blot analysis of the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle. As shown in Figure 29, the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle exhibits sufficient stability during incubation with glutathione or HSA at 37°C for 30 days compared to the stabilized molecular construct stored at 4°C (lane C). Lane U is the unbound molecular construct as a control. [Example]

[0188] Half-life of stabilized two-chain (scFv α CD19)-Fc-MBM-1×2 DOTA bundle The half-life of the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2DOTA bundle was measured in mice after intravenous administration. The (scFv α CD19)-Fc-MBM-1x2DOTA bundle in serum samples was monitored using ELISA. Eight- to ten-week-old BALB / c mice were purchased from BioLasco, Taipei, Taiwan. Mice were divided into groups of three and intravenously injected with approximately 200 μL of 3.33 μM protein.

[0189] The pharmacokinetic profiles of the anti-CD19 antibody (RB4v1.2), the two-chain (scFv αCD19)-Fc-MBM-1 fusion protein, as shown in Figure 31, indicate that the half-lives of the anti-CD19 antibody (RB4v1.2), the (scFv αCD19)-Fc-MBM-1 fusion protein according to Example 3, the (scFv αCD19)-Fc-MBM-1x2DOTA bundle molecular construct according to Example 18, and the stabilized (scFv αCD19)-Fc-MBM-1x2DOTA bundle molecular construct according to Example 19 are approximately 129, 179.6, 153.6, and 193.5 hours, respectively. In conclusion, the stabilization treatment results in an extended half-life of the stabilized molecular constructs. [Example]

[0190] Targeting Efficacy of a Stabilized Two-Chain (scFv αCD19)-Fc-MBM-1×2DOTA Bundle against CD19-Expressing Xenograft Tumors In this example, we investigated the targeting effect of a stabilized two-chain (scFv αCD19)-Fc-MBM-1×2DOTA bundle against CD19-expressing tumors in a mouse xenograft model using an in vivo imaging system (IVIS). Prior to IVIS imaging, the stabilized two-chain (scFv αCD19)-Fc-MBM-1×2DOTA bundle from Example 19 and an anti-CD19 antibody (RB4v1.2) were conjugated using the Dylight680 Antibody Labeling Kit (Thermo Scientific) according to the manufacturer's instructions.

[0191] 8-10 week old NOD-SCID (NOD.CB17-Prkdc) scid / Jnarl) were purchased from the Laboratory Animal Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. Two weeks before treatment, 1 × 10 -7 B-cell lymphoma Raji cells were injected intraperitoneally. Mice were divided into groups of three and intravenously injected with approximately 200 μL of 6.65 μM labeled molecules. At various time points, mice were anesthetized with isoflurane in O and placed in a supine position on an IVIS Spectrum in vivo imaging system (PerkinElmer). Fluorescent images were captured at ex / em = 675 / 720 using Living Image Software V3.2. Images were captured at the designated time points using the IVIS Spectrum imager and analyzed using Living Image Software.

[0192] Fluorescence images from NOD-SCID mice were taken and analyzed at 0, 24, 48, and 72 hours after administration of the Dylight680 binding protein. Figure 32 shows the distribution of fluorescently labeled molecules in the mouse model. NOD-SCID mice were intravenously injected with anti-CD19 antibody (RB4v1.2) (1 and 2) and the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle (3 and 4).

[0193] As shown in Figure 32, at 24, 48, and 72 hours after intravenous injection, the penetration of the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle and anti-CD19 antibody (RB4v1.2) into tumors located on the right side was significantly greater than that observed in normal organs on the left side. Thus, the stabilized molecular construct exhibits a significant targeting effect on CD19-expressing tumors in a mouse xenograft model. [Example]

[0194] Biodistribution of stabilized two-chain (scFv alpha CD19)-Fc-MBM-1x2DOTA bundle in CD19-expressing xenograft tumors For analysis of the biodistribution of stabilized (scFv α CD19)-Fc-MBM-1×2DOTA bundles in a xenograft mouse model, the procedure described in Example 32 was used with some modifications.

[0195] Briefly, mice were treated with 1 × 10 IgG per mouse for 2–3 weeks prior to treatment. 7 Mice were subcutaneously injected with 1000 μL of Raji cells, a B-cell lymphoma. Mice were divided into groups of three and intravenously injected with approximately 200 μL of 6.65 μM of the labeled molecule.

[0196] The biodistribution in NOD-SCID mice was analyzed by tissue ELISA 7 days after administration of the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle and anti-CD19 antibody. Briefly, the animals were sacrificed 7 days after injection. Blood was exsanguinated from the heart, and tissue samples from 11 organs were collected. The tissue samples were homogenized using a homogenizer and further analyzed to measure the concentration of the injected molecules by ELISA.

[0197] Figure 33 shows the distribution of the stabilized two-chain (scFv α CD19)-Fc-MBM-1x2 DOTA bundle (TE-1122) in a mouse model. The y-axis of the biodistribution diagram represents the tissue-to-blood ratio. This result also demonstrates that the stabilized molecular construct can target CD19-expressing tumors in a mouse xenograft model. [Example]

[0198] Synthesis of Aib-GLP-1 agonists with terminal cysteine ​​residues In this example, an ε-aminobutyric acid (Aib)-substituted glucagon-like peptide-1 (GLP-1) agonist with a free cysteine ​​at the C-terminus was prepared. The amino acid sequence of this Aib-GLP-1 agonist-Cys molecular construct is set forth in SEQ ID NO: 37. Figure 34 is a schematic diagram showing the structure of this molecular construct. Similar to semaglutide, the second amino acid residue is substituted with an Aib (or U) residue to improve resistance to dipeptidyl peptidase IV (DPP4) degradation. Furthermore, in this molecular construct, a 15-amino acid flexible linker (amino acid residues 31-45 of SEQ ID NO: 37) is introduced before the last glycine residue of semaglutide (i.e., amino acid residue 46 of SEQ ID NO: 37), and a terminal cysteine ​​residue is added to the C-terminus.

[0199] The structure of Aib-GLP-1 agonist-Cys was designed by the present inventors, and its synthesis was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China).

[0200] The purified sample of Aib-GLP-1 agonist-Cys was analyzed by reversed-phase analytical HPLC on a Supelco C18 column (250 mm × 4.6 mm; 5 μm) using a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, a linear gradient of 0% to 100% acetonitrile over 30 min, a flow rate of 1.0 ml / min, and a column temperature of 25°C.

[0201] Identification of the Aib-GLP-1 agonist-Cys product was performed using mass spectrometry ESI-MS. A sample of the Aib-GLP-1 agonist-Cys product showed a strong molecular ion at 1483.4, which is [M+H] + , and indicates that the actual molecular weight of the present Aib-GLP-1 agonist-Cys agonist is 1482.4 daltons. [Example]

[0202] Synthesis of somatostatin analogues containing a terminal cysteine ​​residue. In this example, a somatostatin analog with a free cysteine ​​at the N-terminus was prepared. The amino acid sequence of this Cys-octreotide molecular construct is set forth in SEQ ID NO: 38. Figure 35 is a schematic diagram showing the structure of this molecular construct. This molecular construct has 14 amino acid residues, including an N-terminal cysteine ​​residue, followed by a flexible linker of five amino acid residues and the octreotide sequence. Like the original octreotide, the seventh amino acid residue (Phe) and the tenth amino acid residue (Trp) of this Cys-octreotide molecular construct are in the D-form, a disulfide bridge is formed between the eighth and thirteenth cysteine ​​residues, and the C-terminal threonine residue is in the threoninol form. Additionally, the N-terminus of the Cys-octreotide molecular construct is modified with an acetyl group.

[0203] The structure of this Cys-octreotide molecular construct was designed by the present inventors, and its synthesis, which is synthesized by standard solid-phase methods, was outsourced to Ontores Biotechnologies Co., Ltd. (Hangzhou, China), and the Cys-octreotide product has a purity of more than 95%.

[0204] The Cys-octreotide product was identified using mass spectrometry MALDI-TOF. Mass spectrometry analysis was performed at the Mass Core Facility of the Institute of Molecular Biology (IMB), Academia Sinica, Taipei, Taiwan. Measurements were performed on a Bruker Autoflex III MALDI-TOF / TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Figure 36 shows the results of mass spectrometry MALDI-TOF, which indicates that the molecular construct has a molecular weight of 1493.587 daltons. [Example]

[0205] Synthesis of PSMA ligands containing cysteine ​​residues In this example, a PSMA ligand containing a free cysteine ​​residue was prepared, and the structure of this molecular construct is given in FIG.

[0206] The structure of this Cys-PSMA ligand molecular construct was designed by the inventors, and its synthesis was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China). The Cys-PSMA ligand product has a purity of more than 95.0%.

[0207] The identification of the Cys-PSMA ligand was carried out using mass spectroscopy ESI-MS. Figure 38 shows the results of mass spectroscopy ESI-MS, which shows that the molecular construct has a strong molecular ion at 522.4, which is [M+H] + and indicates that the actual molecular weight of the Cys-PSMA ligand is 521.4 daltons. [Example]

[0208] Synthesis of calcitonin-MBM-1 In this example, a calcitonin molecular construct bearing a metal-binding motif at its C-terminus was prepared. The amino acid residues of this calcitonin-MBM-1 molecular construct are listed in SEQ ID NO: 39. Figure 39 is a schematic diagram showing the structure of this molecular construct. Similar to calcitonin, a disulfide bridge is formed between the first and seventh cysteine ​​residues. Furthermore, in this molecular construct, a flexible linker of eight amino acid residues (amino acid residues 33-40 of SEQ ID NO: 39) is introduced after the last proline residue of calcitonin (i.e., amino acid residue 32 of SEQ ID NO: 39), and an MBM-1 motif (ACPGHA, SEQ ID NO: 7) is added to the C-terminus of the flexible linker.

[0209] The structure of this calcitonin-MBM-1 molecular construct was designed by the present inventors, and its synthesis was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China). [Example]

[0210] Synthesis of teriparatide-MBM-1 In this example, a molecular construct of teriparatide bearing a metal-binding motif at its C-terminus was prepared. The amino acid sequence of this teriparatide-MBM-1 molecular construct is set forth in SEQ ID NO:40. Teriparatide is a form of parathyroid (PTH) hormone consisting of the first 34 amino acids, which are the active portion of the hormone. It is used in the treatment of certain forms of osteoporosis. In the teriparatide-MBM-1 molecular construct, a flexible linker of 15 amino acid residues (amino acid residues 35-49 of SEQ ID NO:40) is introduced after the last phenylalanine residue of teriparatide (i.e., amino acid residue 34 of SEQ ID NO:40), and the MBM-1 motif (ACPGHA, SEQ ID NO:7) is added to the C-terminus of the flexible linker.

[0211] The structure of this teriparatide-MBM-1 molecular construct was designed by the present inventors, and its synthesis was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China). [Example]

[0212] Synthesis of leuprolide-MBM-3 In this example, a molecular construct of leuprolide bearing a metal-binding motif at its C-terminus was prepared. The amino acid sequence of this leuprolide-MBM-3 molecular construct is set forth in SEQ ID NO: 41. Leuprolide (also known as leuprorelin) is a gonadotropin-releasing hormone (GnRH) analog that acts as an agonist at the pituitary GnRH receptor and is used in the treatment of prostate and breast cancer. Commercially available leuprolide is an oligopeptide having nine amino acid residues. In this leuprolide-MBM-3 molecular construct, a flexible linker of eight amino acid residues (amino acid residues 10-17 of SEQ ID NO: 41) is introduced after the last proline residue of calcitonin (i.e., amino acid residue 9 of SEQ ID NO: 41), and the MBM-3 motif (GCGGHA, SEQ ID NO: 6) is added to the C-terminus of the flexible linker.

[0213] The structure of this leuprolide-MBM-3 molecular construct was designed by the present inventors, and its synthesis was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China). The identification of the synthesized peptide was carried out using mass spectrometry ESI-MS. The results of mass spectrometry ESI-MS showed that this molecular construct has a strong molecular ion at 1091.97, which is [M+2H] 2+ , indicating that the actual molecular weight of the leuprolide-MBM-3 molecular construct is 2181.35 Daltons. [Example]

[0214] Synthesis of lenalidomide bundles In this example, the inventors designed two drug bundles with a maleimide-containing peptide having three lysine residues as a central core and three lenalidomide molecules attached to the central core. The lenalidomide bundles were synthesized using a hybrid methodology: standard Fmoc-based solid-phase synthesis was performed to synthesize the central core, followed by solution-phase synthesis to attach lenalidomide molecules modified with linking arms to the side chains of the lysine residues in the central core. Manufacturing was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China).

[0215] Figure 40 shows the structure of the Mal-peptide3-lenalidomide bundle, in which the central core has the sequence EGEGEAGGKGAGKGAGKG (SEQ ID NO: 42), in which the first amino acid residue is modified with a maleimide-ethyl group. Prior to being attached to the central core, each lenalidomide molecule is modified with a para-aminobenzylcarbamate (PABC)-alanine-valine-PEG linking arm. The lenalidomide molecule is linked to the ε-amino group of a lysine residue in the central core via the free end of the linking arm.

[0216] The resulting synthesized Mal-peptide 3-lenalidomide bundle was identified using mass spectroscopy ESI-MS. Figure 41 shows the mass spectroscopy ESI-MS results, which show that the drug bundle has a strong molecular ion at 1321.9, which is [M+3H] 3+ which indicates that the actual molecular weight of the drug bundle is 3962.02 Daltons.

[0217] Figure 42 shows the structure of the Mal-peptide4-lenalidomide bundle, in which the central core has the sequence EDEDEAGGKGAGKGAGKG (SEQ ID NO: 43), in which the first amino acid residue is modified with a maleimide-ethyl group. The lenalidomide molecule is modified with a linking arm as described above and linked to the ε-amino group of a lysine residue in the central core via the free end of the linking arm.

[0218] The resulting Mal-peptide 4-lenalidomide bundle was identified using mass spectrometry ESI-MS. The ESI-MS results showed that the drug bundle possesses a strong molecular ion at 1379.2, which corresponds to [M+3H] 3+ which indicates that the actual molecular weight of the drug bundle is 4135.15 Daltons. [Example]

[0219] Synthesis of Mal-fatty acid bundles In this example, a molecular construct of Mal-peptide 5 core and one stearoyl diacid chain and one palmitoyl acid chain was prepared.

[0220] Figure 43 shows the structure of this Mal-peptide 5 fatty acid bundle. The central core has the sequence maleimide-ethyl-EGEGE-X1-K-X2-OMe (SEQ ID NO: 44), where the first amino acid residue is modified with a maleimide-ethyl group, the last lysine residue is modified with a methoxy group (-OMe), and both X1 and X2 are PEGylated amino acids with four EG repeats. One stearoyl diacid chain and one palmitoyl acid chain were each linked to the K residue of the peptide central core by forming an amide bond between the CO2H group of the fatty acid and the amine group of the K residue. The structure of this Mal-peptide 5-fatty acid bundle was designed by the inventors, and its synthesis was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China). The Mal-peptide 5-fatty acid bundle product has a purity of more than 97.0%.

[0221] The identification of the synthesized fatty acid bundle was carried out using mass spectrometry ESI-MS. The ESI-MS results in Figure 44 show that this molecular construct has a strong molecular ion at 1143.6, which is [M+2H] 2+ and indicates that the actual molecular weight of the Mal-peptide 5-fatty acid bundle is 2285.66 daltons. [Example]

[0222] Synthesis of 3-DOTA arm linker unit In this example, a 3-DOTA arm linker unit was prepared to carry three peptides. Figure 45 shows the structure of this 3-arm linker. In particular, the linker unit comprises a central core having the sequence acetyl-KGAGGKGAGGKG (SEQ ID NO: 45, peptide core 6), with the first lysine residue modified with an acetyl group. The linker unit also comprises three peptide linking arms having the sequence maleimide-ethyl-EGEGEAGKGAG (SEQ ID NO: 46), with the first glutamic acid residue modified with a maleimide-ethyl group and the lysine residues modified with a DOTA molecule.

[0223] The structure of this 3-DOTA arm linker unit was designed by the present inventors, and its synthesis was outsourced to Shanghai WuXi AppTech Co., Ltd. (Shanghai, China). The identification of the 3-DOTA arm linker unit was carried out using mass spectrometry ESI-MS. The ESI-MS results in Figure 46 show that this molecular construct has a strong molecular ion at 1357.8, which is [M+4H] 4+ This corresponds to the actual molecular weight of the 3-DOTA arm linker unit being 5427.2 daltons. [Example]

[0224] Construction, expression, and purification of recombinant MBM-1-IL-2 In this example, a conjugated protein having the sequence set forth in SEQ ID NO: 47 was constructed, expressed, and purified using protocols similar to those described in the preceding working example. In particular, this MBM-1-IL-2 molecule construct contains the MBM-1 motif, followed by a short flexible linker and the IL-2 sequence of SEQ ID NO: 9.

[0225] The culture suspension was harvested, and the recombinant fusion protein expressed in the medium was purified using an anion exchange column. Prior to purification, the recombinant MBM-1-IL-2 fusion protein was adjusted to pH 8.5. The protein was then applied to a pre-equilibrated Q Sepharose anion exchange resin (GE Healthcare) with 50 mM Bis-Tris buffer at pH 8.5. The MBM-1-IL-2 fusion protein was eluted using a three-step elution with 200 mM, 500 mM, and 1 M NaCl, respectively, in a 4 M urea solution at pH 8.5.

[0226] The eluted samples were analyzed using 10% SDS-PAGE as shown in Figure 47. The MBM-1-IL-2 fusion protein appeared as a major band at approximately 16 kDa, which is consistent with the expected size (indicated by an arrow). [Example]

[0227] Synthesis of a two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle In this example, the (scFv α CD38)-Fc-MBM-1 fusion protein from Example 10 and the Mal-peptide 4-lenalidomide bundle from Example 40 were combined to generate a molecular construct with a two-chain (scFv α CD38)-Fc-MBM-1 fusion protein and two lenalidomide bundles linked to respective cysteine ​​residues in the metal-binding motif of the fusion protein. Briefly, the purified (scFv α CD38)-Fc-MBM-1 fusion protein was prepared in sodium succinate buffer (30 mM sodium succinate, pH 5.3, 0.02% Tween 20, and 100 mM sucrose) and reduced by incubation with 45 μM TCEP at room temperature for 30 minutes with gentle shaking. After the reduction reaction, excess TCEP was removed by dialysis against 10 mM sodium succinate buffer (pH 5.3, 0.02% Tween 20, and 100 mM sucrose) containing 60 μM Zn(II) ions. The reduced protein sample was then treated with 15 μM Mal-peptide 4-lenalidomide bundle and incubated at room temperature for 1 hour. Unreacted lenalidomide bundle was removed using a desalting column, and the product was analyzed by SDS-PAGE.

[0228] Figure 48 shows the results of SDS-PAGE analysis of this molecular construct. As shown in Figure 48, this molecular construct has a molecular weight of approximately 120 kDa (indicated by arrow #1 in lane 2), which is somewhat larger than expected. Unbound molecular construct was present in lane 3 (indicated by arrow #2). As shown in Figure 48, the binding yield of the two-chain (scFv α CD38)-Fc-MBM-1 with the lenalidomide bundle is approximately 85%. [Example]

[0229] Synthesis of octreotide x fatty acid bundle In this example, Mal-peptide 5-fatty acid bundle from Example 41 and Cys-octreotide from Example 35 were conjugated using a protocol similar to that described above, such that the maleimide group of the Mal-peptide 5-fatty acid bundle was attached to the -SH group of the terminal cysteine ​​residue of Cys-octreotide, thereby generating the molecular construct of octreotide x fatty acid bundle (see Figure 49). [Example]

[0230] Synthesis of Aib-GLP-1 agonist × fatty acid bundle In this example, Mal-peptide 5-fatty acid bundle according to Example 41 and Aib-GLP-1 agonist-Cys according to Example 34 were conjugated using a protocol similar to that described above, such that the maleimide group of Mal-peptide 5-fatty acid bundle was attached to the -SH group of the terminal cysteine ​​residue of Aib-GLP-1 agonist-Cys, thereby generating the molecular construct of Aib-GLP-1 agonist x fatty acid bundle (see Figure 50A). [Example]

[0231] Synthesis of teriparatide-MBM-1× fatty acid bundle In this example, the Mal-peptide 5-fatty acid bundle from Example 41 and teriparatide-MBM-1 from Example 38 were combined using a protocol similar to that described above to generate the molecular construct teriparatide-MBM-1 x fatty acid bundle (see Figure 50B). [Example]

[0232] Synthesis of leuprolide-MBM-3× fatty acid bundle In this example, the Mal-peptide 5-fatty acid bundle from Example 41 and the leuprolide-MBM-3 from Example 39 were combined using a protocol similar to that described above to generate the molecular construct of leuprolide-MBM-3 × fatty acid bundle (see Figure 51A). The synthesized leuprolide-MBM-3 × fatty acid bundle was identified using mass spectrometry MALDI-TOF, and the results in Figure 51B show that this molecular construct has a molecular weight of 4466.019 Daltons. The molecular weight of 2181.038 Daltons indicated an excess of leuprolide-MBM-3 molecules in the 1.5:1 [leuprolide-MBM-3:Mal-peptide 5-fatty acid] molar ratio of this reaction. [Example]

[0233] Synthesis of 3-DOTA arm linker units x 3 PSMA ligands In this example, the 3-DOTA arm linker unit according to Example 42 and three Cys-PSMA ligands according to Example 36 were combined using a protocol similar to that described above to generate a molecular construct of 3-DOTA arm linker unit x 3 PSMA ligand (Figure 52A). The identification of the synthesized 3-DOTA arm linker unit x 3 PSMA ligand was performed using mass spectrometry ESI-MS, and the results in Figure 52B show that this molecular construct has a strong molecular ion at 1749, which is [M+4H] 4+ This indicates that the actual molecular weight of the 3-DOTA arm linker unit x 3 PSMA ligand is 6992.18 daltons. [Example]

[0234] Purification of stabilized two-chain (scFv α CA19-9)-Fc-MBM-1x2DOTA bundle The procedure for stabilizing the recombinant two-chain (scFv α CA19-9)-Fc-MBM-1x2DOTA bundle was performed as described in the previous examples.

[0235] The two-chain fusion protein conjugated to two DOTA bundles from the previous example was adjusted to pH 5.0 and then applied to a pre-equilibrated (0.1 mM EDTA, 50 mM Bis-Tris at pH 5.0) anion exchange column Q Sepharose (GE Healthcare), and the sample was eluted at a flow rate of 1.0 ml / min using a three-step elution: a first elution of 320 mM NaCl for 70 min, followed by a second elution of 330 mM for 100 min, and a final elution of 1000 mM NaCl for 50 min.

[0236] The two-chain (scFv α CA19-9)-Fc-MBM-1 × 2 DOTA bundle was separated from free two-chain (scFv α CA19-9)-Fc-MBM-1 fusion protein, two-chain (scFv α CA19-9)-Fc-MBM-1 bound to three or four DOTA bundles, and aggregated material using an anion-exchange column, Q-Sepharose.

[0237] The purified product, two-chain (scFv α CA19-9)-Fc-MBM-1x2 DOTA bundle, was collected. Lanes 1 and 2 shown in Figure 53 correspond to the unbound molecular construct and the molecular construct bound to two DOTA bundles, respectively. [Example]

[0238] Purification of stabilized two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle The molecular construct of the two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle according to Example 44 was stabilized using a protocol similar to that described in the previous examples.

[0239] The stabilized molecular construct of the two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle was adjusted to pH 7.0 and then applied to a pre-equilibrated (50 mM NaHPO, 1 M NaCl at pH 7.0) hydrophobic interaction column (HIC) Phenyl HP (GE Healthcare). The stabilized molecular construct was eluted with a linear gradient of 1000 mM to 0 mM NaCl over 80 min at a flow rate of 1.0 ml / min.

[0240] The collected sample from the preceding HIC purification was applied to a size-exclusion chromatography column, ENrich™ SEC650 (Bio-Rad), to separate the stabilized molecular construct from aggregated material. The purified product was collected. Lanes 1 and 2 of the 10% non-reducing SDS-PAGE shown in Figure 54 correspond to the unbound molecular construct and the molecular construct bound to two lenalidomide bundles, respectively.

[0241] The purified stabilized molecular construct of the two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle was further analyzed using mass spectrometry MALDI-TOF. The mass spectrometry results in the lower panel of Figure 55 show that the sample has molecular weights of 58382 and 116705 daltons, which is m / z (z=1): [M+H] + , and m / z (z = 2): [M + 2H] 2+ correspond to the following:

[0242] Mass spectrometry analysis of the two-chain (scFv α CD38)-Fc-MBM-1 control shown in the upper panel of Figure 55 shows that the sample has molecular weights of 54,300 and 108,537 daltons, which corresponds to m / z (z=1): [M+H] + , and m / z (z = 2): [M + 2H] 2+ correspond to the following: [Example]

[0243] Half-life of a stabilized two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle in NOD-SCID mice The half-life of the stabilized two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle was measured in mice after intravenous administration. The molecular construct in serum samples was monitored using an ELISA method. Eight- to ten-week-old NOD-SCID mice were purchased from BioLasco, Taipei, Taiwan. Mice were divided into groups of three per group and intravenously injected with 100 μL of 7.6 μM of the molecule via an intravenous bolus.

[0244] The results show that the half-lives of the parent anti-CD38 hIgG1.Fc antibody (Figure 56A) and the two-chain(scFv αCD38)-Fc-MBM-1x2 lenalidomide bundle (Figure 56B) are approximately 13.1 and 23.9 hours, respectively. Using non-compartmental pharmacokinetic modeling, the results indicate that the two-chain(scFv αCD38)-Fc-MBM-1x2 lenalidomide bundle has a longer half-life than that of conventional anti-CD38 antibodies. [Example]

[0245] Cytotoxic activity of purified two-chain (scFv α CD38)-Fc-MBM-1×2 lenalidomide bundle against H929 and CD38-positive U266 cells H929 cells (5 x 10 3 (cells / well) were added to wells of a 96-well plate in RPMI 1640 medium containing 10% fetal bovine serum. After 2 hours, cells were treated with different concentrations (two-fold dilutions from 20 μM) of purified two-chain (scFv α CD38)-Fc-MBM-1 (without lenalidomide bundle) and two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle. After 2 hours of incubation, the culture medium was removed by centrifugation at 400 g for 5 minutes and replaced with fresh medium. The cells were further incubated for an additional 120 hours (5 days) and 168 hours (7 days). Cell viability was then determined using the alamarBlue Cell Viability Reagent Kit (Invitrogen) according to the manufacturer's instructions.

[0246] The protocol to determine the cytotoxic activity of purified two-chain (scFv α CD38)-Fc-MBM-1 with or without two lenalidomide bundles against CD38-positive U266 cells was similar to that described above for H929 cells.

[0247] Figure 57A shows the viability of H929 cells in the four treatment groups. The two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle (denoted "scFv-Fc-len") caused approximately 80% cytolysis of H929 cells at 20 μM after 5 days of incubation. The two-chain (scFv α CD38)-Fc-MBM-1 (denoted "scFv-Fc") used as a negative control showed no cytotoxic effect.

[0248] Figure 57B shows the viability of CD38-positive U266 cells in the four treatment groups. The two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle resulted in approximately 80% cytolysis of CD38-positive U266 cells at 20 μM after 5 days of incubation.

[0249] Figure 57C shows the viability of CD38-negative U266 cells in the four treatment groups. The two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle (denoted "scFv-Fc-len") is unable to induce cytolysis of CD38-negative U266 cells at each molar concentration.

[0250] Taken together, the results in Figures 57A-57C demonstrated that the two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle had a strong targeting effect. [Example]

[0251] Antibody-dependent cell-mediated cytotoxicity (ADCC) of purified two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle against CD38-expressing H929 and U266 cells In this example, an in vitro assay was performed to investigate the antibody-dependent cell-mediated cytotoxicity (ADCC) effect of the two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle on cytolysis of CD38-expressing H929 and CD38-positive U266 cells.

[0252] Human whole peripheral blood mononuclear cells (PBMCs) were isolated from donors and tested for ADCC function. H929 cells or CD38-positive U266 cells were incubated with the two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle at final concentrations of 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, or 0.0064 nM. Then, they were mixed with human PBMCs at an E:T ratio of 25 and incubated at 37°C for 5 hours. A parental anti-CD38 mAb was used as a positive control. Cell lysis was analyzed using the LDH cytotoxicity assay kit (Enzo Life Sciences).

[0253] The data presented here demonstrate that the purified two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle (designated "scFv-Fc-len") recruits effector cells and elicits cytolytic responses in a manner similar to its parental anti-CD38 mAb (Figure 58A). The parental anti-CD38 antibody was used as a positive control (designated "IgG"), and an isotype control antibody (designated "control IgG") was also used.

[0254] Figure 58B shows that the purified two-chain (scFv α CD38)-Fc-MBM-1x2 lenalidomide bundle (denoted "scFv-Fc-len") also displays a cytolytic response against CD38-positive U266 cells in a manner similar to its parental counterpart.

[0255] It should be understood that the above description of the embodiments is provided by way of example only, and that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. While various embodiments of the invention have been described above with a certain degree of particularity or with reference to one or more specific embodiments, those skilled in the art could make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. A linker unit, a central core that is a polypeptide having a length of 3 to 120 amino acid residues, 2 to 10 lysine (K) residues; an SH-reactive group linked to the N-terminus of the first K residue or the C-terminus of the last K residue of the central core by forming an amide bond with the alpha amino group of the first K residue or the carboxyl group of the last K residue, wherein the central core carries a localized negative charge present within the first 5 to 15 amino acid residues starting from the amino acid residue linked to the SH-reactive group; a central core comprising: 2 to 10 targeting elements, effector elements, or pharmacokinetic elements, each targeting element, effector element, or pharmacokinetic element is linked to a K residue of the core; or When the linker unit further comprises 2 to 10 linking arms, one end of each linking arm is linked to a K residue of the core via forming an amide bond with the ε-amino group of the K residue, and the other end of each linking arm is linked to a targeting element, an effector element, or a pharmacokinetic element, and the linking arm is a peptide comprising 2 to 12 non-K amino acid residues or a polyethylene glycol (PEG) chain having 2 to 24 repeats of EG units. 2 to 10 targeting, effector, or pharmacokinetic elements; A linker unit comprising:

2. the central core further comprises one or more fillers; each of the fillers comprises (1) 1 to 12 non-K amino acid residues or (2) a PEGylated amino acid having 1 to 12 repeats of ethylene glycol (EG) units; any two of the K residues are separated by the filler; The linker unit of claim 1 .

3. the central core further comprises terminal spacers; the terminal spacer is an N-terminal spacer linked to the N-terminus of the first K residue or a C-terminal spacer linked to the C-terminus of the last K residue, the terminal spacer comprising: (1) 1 to 12 non-K amino acid residues; or (2) a PEGylated amino acid having 1 to 12 repeats of ethylene glycol (EG) units; the SH-reactive group is linked to the terminal amino acid residue of the terminal spacer by forming an amide bond therewith; The linker unit according to claim 1 or 2.

4. 2. The linker unit of claim 1, wherein the localized negative charge is imparted by at least one glutamic acid (E) or aspartic acid (D) residue.

5. The linker unit of claim 1 , wherein the effector element is a negatively charged chemical moiety.

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