RU2814475C2 - Composite polypeptide having metal-binding motif and molecular structure containing thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to novel framework conjugates of therapeutic polypeptides. Invention discloses a composite polypeptide comprising a central nucleus of lysine amino acid residues, to which various functional elements are attached through short linker sequences, which are small molecules capable of causing a therapeutic effect in the human body. According to some embodiments, the present invention is aimed at increasing the half-life of a peptide hormone or cytokine.

EFFECT: according to some embodiments, the present invention relates to a composite polypeptide containing an antibody or a fragment thereof, targeting an antigen of the surface of cancer cells, which can be used in medical practice for the therapy of tumour diseases.

7 cl, 54 ex, 58 dwg

Description

BACKGROUND OF THE INVENTION

1. FIELD OF TECHNOLOGY

The present invention relates to composite polypeptides having a metal-binding motif, which is a short oligopeptide capable of complexing with a zinc cation.

2. DESCRIPTION OF RELATED ART

Bioconjugation is a technique used to form a covalent bond between a biomolecule and another molecule, which may or may not be a biomolecule. Biomolecules generally refer to molecules present in organisms that are essential for a biological process. Biomolecules include natural or synthetic polypeptides or proteins, carbohydrates, lipids, nucleic acids, and metabolites.

The concept of weaponizing 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”) originated in 1970. The synthesis of such bioconjugates often involves conjugation reactions such as binding of surface accessible lysine, cysteine or tyrosine residues, and modifications of surface accessible lysine or tryptophan or N- and C residues - ends. However, the above-mentioned amino acid residues are present in large quantities in the parent antibody, and the conjugation reaction is usually a random process. As a consequence, the conjugation reaction lacks chemoselectivity and efficiency and often results in heterogeneous products. For example, previous studies of huN901-DM1, a maytansinoid-monoclonal antibody immunoconjugate, showed that a sample of lysine-conjugated ADC contained ADCs with varying drug-to-antibody ratios (DAR) ranging from 0 to 6 and possibly containing more than 4.5 million unique molecules.

In view of the above, there is an increasing need for conjugation strategies that will effectively conjugate a functional element to a specific site on a biomolecule, in particular a peptide-based molecule. It is believed that the present invention is a response to this need.

SUMMARY OF THE INVENTION

The following presents a simplified summary of the invention to provide a basic understanding to the reader. The Summary of the Invention is not a broad overview of the disclosure of the invention, and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to introduce some of the concepts disclosed herein in a simplified form as a prelude to the more detailed description that will be presented later.

In one aspect, the present invention provides a metal-binding motif that is capable of complexing with a zinc ion under suitable conditions.

According to one embodiment of the present invention, the metal binding motif comprises the amino acid sequence CX ₁ X ₂ HA (SEQIDNo.1) in order from N-terminus to C-terminus, or in order from C-terminus to N-terminus, where X ₁ represents glycine or proline, and X ₂ represents glycine or alanine, and the metal binding motif is located at the N- or C-terminus of the parent polypeptide. Illustrative examples of metal-binding motifs include, but are not limited to, CGGHA (SEQIDNo. 2), CPGHA (SEQIDNo. 3), CGAHA (SEQIDNo. 4), CPAHA (SEQIDNo. 5), GCGGHA (SEQIDNo. 6), ACPGHA (SEQIDNo. 6). 7), and GCPGHA (SEQIDNo. 8).

In another aspect, the present invention is directed to a composite polypeptide that contains a metal binding motif in accordance with the above aspect/embodiment. Thus, the composite polypeptide is capable of forming a complex with a metal ion (eg, zinc ion) through a metal-binding motif under suitable conditions. Thus, the sulfhydryl group of a cysteine residue complexed with a metal ion is more reactive than the sulfhydryl group of a cysteine residue without complexation with the metal ion. As can be determined, the complex may undergo chemical conjugation with another chemical compound or biomolecule in a site-specific manner, thereby forming a bioconjugate.

In accordance with one embodiment of the present invention, the composite polypeptide includes a parent polypeptide and a metal binding motif located at the N- or C-terminus of the parent polypeptide. In some optional examples, there is a sequence of 1 to 10 glycine residues between the parent peptide and the metal binding motif, while in other examples, the actual metal binding motif precedes or follows the parent polypeptide entirely.

In yet another aspect, one or more functional elements can be covalently conjugated to a composite polypeptide in accordance with the above aspect/variants of the present invention by reacting with the sulfhydryl (SH) group of a cysteine residue in the metal-binding motif of the composite polypeptide, thereby forming a molecular construct (or bioconjugate ).

In optional embodiments of the present invention, the SH-reactive group is a maleimide, iodoacetyl, bromoacetyl, vinylsulfonyl, monosulfonyl, methylsulfonylbenzothiazole, or 2-pyridylthiol group.

In some optional embodiments, the composite polypeptide is in dimeric form; for example, the composite polypeptide may include part of an Fc region, F(ab') ₂ , or an antibody. In these cases, each constituent polypeptide chain has one functional element conjugated to it.

According to various embodiments of the present invention, the functional element is a small molecule entity capable of causing a therapeutic effect. For example, the small molecule entity may be a cytotoxic drug, a toll-like receptor agonist, or a chelator complexed with a radioactive nuclide. Alternatively, the functional element is a fatty acid chain capable of modifying the pharmacokinetic profile of the parent polypeptide or the molecular construct as a whole.

Alternatively, the functional element is in the form of a linker unit (also referred to as a “ligament”). In some embodiments of the present invention, the linker unit comprises a central core and a plurality of targeting, effector, or pharmacokinetic elements.

In particular, the central core contains from 2 to 10 lysine (K) residues. Any two K residues are 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 contains a terminal spacer, and the SH-reactive group is linked to the terminal amino acid residue of the terminal spacer, forming an amide bond with it. 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. Each of the fillers and terminal spacers contains, independently, (1) from 1 to 12 K amino acid residues other than K, or (2) a PEGylated amino acid having from 1 to 12 ethylene glycol (EG) unit repeats.

In some embodiments of the present invention, the central core carries a local negative charge at or adjacent to the amino acid residue associated with the SH-reactive group. For example, a local negative charge is present within the first 5-15 amino acid residues within an amino acid residue, starting with the amino acid residue associated with the SH-reactive group. In particular, a local negative charge is present in the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, starting with the amino acid residue associated with the SH-reactive group. Local negative charge can be imparted by the incorporation of one or more negatively charged amino acid residues at pH=7, such as aspartate (D) and glutamate (E) residues. Alternatively, the core may be conjugated to a variety of negatively charged effector elements.

In some examples, each targeting, effector, or pharmacokinetic element is linked to a K residue of the core by forming an amide bond with the ε-amino group of the K residue. In some other cases, the molecular construct further contains from 2 to 10 linking arms, with one end of each linking arm linked to a core K residue via amide bond formation with the ε-amino group of the K residue, and the other end of each linking arm linked to each targeting, effector or pharmacokinetic element. For example, the binding arm may be a peptide containing 2-12 amino acid residues other than K, or a polyethylene glycol (PEG) chain having 2-24 EG repeat units.

In some embodiments of the present invention, the composite polypeptide described above serves as a targeting element capable of directing a molecular construct to a site of interest or increasing the relative level of a molecular construct at a site of interest. In these cases, the linker unit (or bundle) may carry multiple effector elements, such as the small molecule described above.

In some embodiments of the present invention, the composite polypeptide described above serves as an effector element capable of producing a desired therapeutic effect in a subject. In these cases, the linker unit (or bundle) may carry a variety of pharmacokinetic elements, such as a C ₈ - ₂₈ fatty acid derivative or a C ₈ - ₂₈ dicarboxylic fatty acid derivative, to modify or optimize the pharmacokinetic profile of the molecular construct or composite polypeptide in a subject. In various embodiments of the present invention, the ε-amino group of the K residue is linked to a C _8-28 fatty acid derivative or a C _8-28 dicarboxylic fatty acid derivative. Fatty acid molecules can bind to serum albumin and therefore improve the kinetic properties of the modified parent polypeptide in vivo.

Also included within the scope of the present invention is the linker unit described above which is capable of forming a molecular construct with a given composite polypeptide.

In yet another aspect, the present invention is directed to a linker unit capable of carrying a plurality of constituent polypeptides in accordance with the above-mentioned aspect/embodiments of the present invention. In embodiments of the present invention, a linker unit having a plurality of constituent polypeptides covalently linked to its central core is sometimes referred to as a polypeptide linker.

According to some embodiments of the present invention, the present linker unit comprises a central core and a plurality of linking arms.

Specifically, the central core contains 2 to 10 lysine (K) residues and a conjugated group. Any two K residues are adjacent to each other or separated by filler. In some cases, the conjugated group is linked to the first or last K residue of the central core by forming an amide bond with it. Examples of the conjugate group include, but are not limited to, azide, alkyne, tetrazine, cyclooctene and cyclooctene groups. In other cases, the central core additionally contains a terminal spacer, and the conjugated group is linked to the terminal amino acid residue by the terminal spacer, forming an amide bond with it. 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. Each of the filler and terminal spacer contains, independently, (1) from 1 to 12 amino acid residues other than K, or (2) a PEGylated amino acid having from 1 to 12 ethylene glycol (EG) unit repeats.

In some embodiments, one end of each linking arm is linked to the K core residue by 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 binding arm may be a peptide containing 2-12 amino acid residues other than K, or a polyethylene glycol (PEG) chain having 2-24 EG repeat units.

According to some embodiments of the present invention, the linker unit further comprises from 2 to 10 constituent polypeptides in accordance with the above-mentioned aspect/embodiments of the invention and a functional element. Each constituent polypeptide is conjugated to the thiol group of a cysteine residue in the metal-binding motif via SH-reactive cross-linking chemistry.

According to some optional embodiments of the present invention, each linking arm carries a local negative charge on or adjacent to the amino acid residue associated with the SH-reactive group. In particular, a local negative charge is present within the first 5-15 amino acid residues within an amino acid residue, starting with the amino acid residue associated with the SH-reactive group. For example, a local negative charge is present in the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, starting with the amino acid residue associated with the SH-reactive group. Local negative charge can be imparted by the incorporation of one or more negatively charged amino acid residues at pH=7, such as aspartate (D) and glutamate (E) residues.

Many of the attendant features and advantages of the present invention will become better understood with reference to the following detailed description taken in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present description will be better understood from the following detailed description, read in light of the accompanying drawings, wherein:

Fig. 1 is a MALDI-TOF result for the central core of Mal-Peptide 1 according to one working example of the present invention;

Fig. 2 illustrates the structure of a Mal-Peptide 2-DOTA binder according to one working example of the present invention;

Fig. 3A illustrates the reverse phase analytical HPLC elution profile of the Mal-Peptide 2-DOTA binder;

Fig. 3B is a MALDI-TOF result for a Mal-Peptide 2-DOTA binder according to one working example of the present invention;

Fig. 4A illustrates the structure of recombinant 2-stranded (scFvαCD19)-Fc-MBM-1 according to one working example of the present invention;

Fig. 4B illustrates a non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCD19)-Fc-MBM-1 in accordance with one working example of the present invention;

Fig. 5 illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCD19)-Fc-MBM-2 according to one working example of the present invention;

Fig. 6 illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCD19)-Fc-MBM-3 in accordance with one working example of the present invention;

Fig. 7A illustrates a reducing SDS-PAGE analysis of anti-CD19 antibody MBM-1 or anti-CD19 antibody MBM-2 in accordance with one working example of the present invention;

Fig. 7B illustrates a retrieval SDS-PAGE analysis of anti-CD19 antibody MBM-1 or anti-CD19 antibody MBM-2 in accordance with one working example of the present invention;

Fig. 8 illustrates non-reducing SDS-PAGE analysis of four (scFvαCD33)-Fc-MBM molecular constructs in accordance with one working example of the present invention;

Fig. 9 illustrates non-reducing SDS-PAGE analysis of recombinant (scFvαCD20)-Fc-MBM-1 in both V _L -V _H and V _H -V _L configurations according to one working example of the present invention;

Fig. 10A illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _H -V _L scFvαCD20)-Fc-MBM-2 in accordance with one working example of the present invention;

Fig. 10B illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCD20)-Fc-MBM-3 according to one working example of the present invention;

Fig. 11A illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCA19-9)-Fc-MBM-1 in accordance with one working example of the present invention;

Fig. 11B illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCA19-9)-Fc-MBM-2 according to one working example of the present invention;

Fig. 12A illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCC38)-Fc-MBM-1 in accordance with one working example of the present invention;

Fig. 12B illustrates a non-reducing SDS-PAGE analysis of recombinant 2-stranded (V _L -V _H scFvαCC38)-Fc-MBM-2 in accordance with one working example of the present invention;

Figure 13A illustrates the structure of recombinant 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages according to one working example of the present invention;

Fig. 13B illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages according to one working example of the present invention;

Fig. 14 illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (scFvαCD19)-Fc-MBM-2 X 2 DOTA linkages according to one working example of the present invention;

Fig. 15 illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (scFvαCD19)-Fc-MBM-3 X 2 DOTA linkages;

Fig. 16 illustrates retrieval SDS-PAGE analysis of intact anti-CD19 antibody-MBM-1 X 2 DOTA bundles in accordance with one worked example of the present invention;

Fig. 17 illustrates retrieval SDS-PAGE analysis of intact anti-CD19 antibody-MBM-2 X2 DOTA bundles in accordance with one worked example of the present invention;

Fig. 18 illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (scFvαCD33)-Fc-MBM-1 X 2 DOTA linkages according to one working example of the present invention;

Fig. 19 illustrates non-reducing SDS-PAGE analysis of recombinant 2-stranded (scFvαCD20)-Fc-MBM-1 X 2 DOTA linkages in accordance with one working example of the present invention;

Fig. 20 illustrates the elution profile of an FPLC anion exchange column on stabilized 2-strand (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages in accordance with one working example of the present invention;

Fig. 21 illustrates SDS-PAGE analysis of fractions collected from an anion exchange column on stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages in accordance with one working example of the present invention;

Fig. 22 illustrates SDS-PAGE analysis of fractions collected from an anion exchange column on stabilized 2-chain (scFvαCD20)-Fc-MBM-1 X 2 DOTA linkages in accordance with one working example of the present invention;

Fig. 23 is a MALDI-TOF result for stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages according to one working example of the present invention;

Fig. 24 is a MALDI-TOF result for stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages chelated with the ⁸⁹ Y ³⁺ ion according to one working example of the present invention;

Fig. 25 is a MALDI-TOF result of stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages chelated with ¹⁷⁵ Lu ³⁺ ion according to one working example of the present invention;

Fig. 26 is the result of a TLC analysis of the radiochemical purity of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles labeled with ¹¹¹ In, in accordance with one working example of the present invention;

Fig. 27 illustrates the stability test result of ¹¹¹ In-labeled stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles analyzed by Western blotting according to one working example of the present invention;

From Fig. 28A of FIG. 28D illustrates the results of a staining assay for stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages according to one working example of the present invention;

Fig. 29 illustrates the result of a stability test of stabilized 2-strand (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages analyzed by SDS-PAGE according to one working example of the present invention;

Fig. 30 illustrates the result of a stability test of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages analyzed by TLC according to one working example of the present invention;

Fig. 31 illustrates the half-lives of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles in accordance with one working example of the present invention;

Fig. 32 illustrates the targeting effect of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles on a CD19-expressing xenograft tumor according to one working example of the present invention;

Fig. 33 illustrates the result of a biodistribution assay of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles on a CD19-expressing xenograft tumor according to one working example of the present invention;

Fig. 34 is a schematic diagram showing the structure of an Aib-GLP-1 Cys agonist according to one working example of the present invention;

Fig. 35 is a schematic diagram showing the structure of a Cys-octreotide according to one working example of the present invention;

Fig. 36 illustrates a MALDI-TOF/TOF result for Cys-octreotide in accordance with one working example of the present invention;

Fig. 37 is a schematic diagram showing the structure of a Cys-PSMA ligand according to one working example of the present invention;

Fig. 38 illustrates the result of an ESI-MSCys-PSMA ligand according to one working example of the present invention;

Fig. 39 is a schematic diagram showing the structure of calcitonin-MBM-1 according to one working example of the present invention;

Fig. 40 is a schematic diagram showing the structure of the Mal-peptide-3-lenalidomide bundle according to one working example of the present invention;

Fig. 41 illustrates an ESI-MS result for the Mal-peptide-3-lenalidomide linkage according to one working example of the present invention;

Fig. 42 is a schematic diagram showing the structure of the Mal-peptide-4-lenalidomide bundle according to one working example of the present invention;

Fig. 43 is a schematic diagram showing the structure of a Mal-peptide-5-fatty acid linkage according to one working example of the present invention;

Fig. 44 illustrates an ESI-MS result of a Mal-peptide 5-fatty acid linkage according to one working example of the present invention;

Fig. 45 is a schematic diagram showing the structure of a 3-DOTA linker arm according to one working example of the present invention;

Fig. 46 illustrates an ESI-MS result of a 3-DOTA linker arm according to one working example of the present invention;

Fig. 47 illustrates SDS-PAGE analysis of MBM-1-IL-2 according to one working example of the present invention;

Fig. 48 illustrates SDS-PAGE analysis of 2-stranded (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles in accordance with one working example of the present invention;

Fig. 49 is a schematic diagram showing the structure of an octreotide X fatty acid bundle according to one working example of the present invention;

Fig. 50A is a schematic diagram showing the structure of an Aib-GLP-1 agonist X fatty acid bundle in accordance with one working example of the present invention;

Fig. 50B is a schematic diagram showing the structure of a teriparatide-MBM-1 X fatty acid bundle according to one working example of the present invention;

Fig. 51A is a schematic diagram showing the structure of a leuprolide-MBM-3 X fatty acid bundle according to one working example of the present invention;

Fig. 51B illustrates a MALDI-TOF result of a leuprolide-MBM-3 X fatty acid coupling according to one worked example of the present invention;

Fig. 52A is a schematic diagram showing the structure of the 3-DOTA linker unit branch of the X 3 PSMA ligands according to one working example of the present invention;

Fig. 52B illustrates the ESI-MS result of 3-DOTA linker arm X 3 PSMA ligands according to one working example of the present invention;

Fig. 53 illustrates SDS-PAGE analysis of purified 2-stranded (scFvαCA19-9)-Fc-MBM-1 X 2 DOTA linkages in accordance with one working example of the present invention;

Fig. 54 illustrates SDS-PAGE analysis of purified 2-chain (scFvαCD38)-Fc-MBM-1 X 2 Lenalidomide bundles in accordance with one working example of the present invention;

Fig. 55 illustrates a MALDI-TOF result for purified 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles according to one working example of the present invention;

Fig. 56A and FIG. 56B illustrates the half-lives of the parental anti-CD38 hIgG1.Fc antibody and 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles according to one working example of the present invention;

From Fig. 57A of FIG. 57C illustrates the targeting effect of 2-chain (scFvαCD38)-Fc-MBM-1 X 2lenalidomide linkages in accordance with one working example of the present invention; And

Fig. 58A and FIG. 58B illustrates the ADCC effect of 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles in accordance with one working example of the present invention.

DESCRIPTION OF THE INVENTION

The detailed description provided below in connection with the accompanying drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or used. The description outlines the functions of the example and the sequence of steps for building and working with the example. However, the same or equivalent functions and sequences can be performed using different examples.

For convenience, certain terms used in the description, examples, and appended claims are collected herein. Unless otherwise specified herein, scientific and technical terminology used in the present invention should have the meanings commonly understood and used by one of ordinary skill in the art.

Unless the context otherwise requires, it is understood that singular terms shall include the plural forms of the same and plural terms shall include the singular. Additionally, 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. In addition, 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 also used herein and The appended claims are intended to cover one A, one B, one C, A and B together, B and C together, A and C together, and A, B and C together.

Although the numerical ranges and parameters defining the broad scope of the invention are approximate, the numerical values set forth in the specific examples are reported to the greatest accuracy possible. However, any numerical value inherently contains certain errors, necessarily resulting from the standard deviation found in the corresponding test measurements. In addition, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a predetermined value or range. Alternatively, the term "about" means within the acceptable standard error of the mean as considered by one skilled in the art. Except for operating/demonstration examples, or unless otherwise expressly stated, all numerical ranges, quantities, values and percentages, such as quantities of materials, length of time, temperatures, operating conditions, ratios of quantities, etc., disclosed herein are should be understood as modified in all cases by the term “about.” Accordingly, unless otherwise indicated, the numerical parameters set forth in the present invention and the accompanying claims are approximate and may vary as desired. At a minimum, each numerical parameter must be interpreted in light of at least the number of significant figures reported and using normal rounding techniques. As used herein, ranges may be expressed as from one endpoint to another endpoint or between two endpoints. All ranges disclosed herein include endpoints unless otherwise noted.

As used herein, the term “targeting element” refers to a portion of a molecular construct that directly or indirectly binds to a target of interest (eg, a receptor on the surface of a cell or a protein in a tissue), thereby facilitating the transport of the molecular construct to the target of interest. In some examples, the targeting element may direct the molecular construct closer to the 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 surface of the cell. In some cases, the targeting element may be internalized along with the actual molecular construct once it binds to the target of interest, hence translocating into the cytosol of the target cell. The targeting element may be an antibody or ligand for a cell surface receptor, or it may be a molecule that binds such an antibody or ligand, thereby indirectly targeting the present molecular construct to a target site (eg, the surface of a selected cell). Localization of the effector (therapeutic agent) to the affected site will be enhanced or favored using the present molecular constructs compared to a therapeutic agent without targeting functionality. Localization depends on degree or relative proportion; it is not intended to completely or completely localize the effector to the affected area.

According to the present invention, the term "effector element" refers to a portion of a molecular construct that causes biological activity (eg, induces or suppresses immune activity, has a cytotoxic effect, inhibits enzymes, etc.) or other functional activity (eg, recruitment of immunocytes or other therapeutic molecules) after the molecular construct is directed to its target site. The "effect" may be therapeutic or diagnostic. Effector elements include those that bind to cells and/or extracellular immunoregulatory factors. The effector element includes agents such as proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties (both small molecule drugs and biologics), compounds, elements and isotopes, as well as fragments thereof.

According to the present invention, "pharmacokinetic element" means an element capable of changing at least one of the following characteristics of a molecular construct: solubility, clearance, half-life and bioavailability. For example, the pharmacokinetic element may include a long chain PEG 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 a C _8-28 dicarboxylic fatty acid chain.

The term "bioconjugate" in accordance with the present invention refers to a molecular construct containing a composite polypeptide of the present invention and one or more functional elements.

Although the terms first, second, third, etc. may be used herein to describe various elements, components, regions and/or sections, these elements (and components, regions and/or sections) are not limited to these terms. Moreover, the use of such serial numbers does not imply sequence or order unless the context clearly indicates so. Rather, these terms are simply used to distinguish one element from another. Thus, the first element discussed below may be referred to as the second element without departing from the teachings of the exemplary embodiments of the invention.

As used herein, the terms “bond,” “pair,” and “conjugate” are used interchangeably to refer to any means of connecting two components, either through a direct connection or through an indirect connection between two components.

As used herein, the term “polypeptide” refers to a polymer having at least two amino acid residues. Typically, a polypeptide contains amino acid residues ranging in length from 2 to about 200 residues; however, it also includes macromolecules containing more than 200 amino acid residues. Where an amino acid sequence is presented herein, L-, D- or beta-amino acid variants of that sequence are also contemplated. Polypeptides also include amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. In addition, the term applies to amino acids linked by a peptide bond or other "modified bonds", for example, when the peptide bond is replaced by an α-ester, β-ester, thioamide, phosphoramide, carbamate, hydroxylate, and the like.

In some embodiments, the present invention provides conservative substitutions of amino acids containing any of the sequences described in the present invention. In various embodiments of the invention, one, two, three, four or five different residues are replaced. The term "conservative substitution" is used to designate amino acid substitutions that do not significantly change the activity (eg, biological or functional activity and/or specificity) of the molecule. Typically, conservative amino acid substitutions involve replacing one amino acid with another amino acid with similar chemical properties (such as charge or hydrophobicity). Some conservative substitutions include "analogue substitutions", where a standard amino acid is replaced by a non-standard (eg, rare, synthetic, etc.) amino acid that differs minimally from the parent residue. Amino acid analogues are obtained synthetically from standard amino acids without significantly changing the structure of the parent amino acids; they are isomers or precursors of metabolites. In this application, the amino acid residues are (1) lysine, which contains an amino group in its side chain, (2) cysteine, which contains a thiol group in its side chain, (3) serine and threonine, which contain a hydroxyl group in its side chain, and (4 ) aspartic acid and glutamic acid, which contain a carboxyl group in their side chain, are considered the four distinctive groups of amino acids. Each of these four groups of amino acids contains a unique functional group on its side chains that can be used for conjugation with various chemical moieties. Unnatural amino acids that contain the same functional groups in the side chains can be substituted for similar purposes.

In some embodiments, polypeptides containing at least 80%, preferably at least 85 or 90%, and more preferably at least 95 or 98% sequence identity with any of the sequences described in the present invention are also contemplated.

"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 polypeptide sequence, after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, 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 the purposes of this description, sequence comparisons of two polypeptide sequences were performed using the Blastp (protein-proteinBLAST) computer program provided online by the National Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given polypeptide sequence A to a given polypeptide sequence B (which may alternatively be stated as a given polypeptide sequence A that has a certain % amino acid sequence identity with a given polypeptide sequence B) is calculated using the formula as follows:

%

where X is the number of amino acid residues counted as identical matches by the sequence alignment program BLAST when aligning sequences A and B in that program, and Y is the total number of amino acid residues in sequence A or sequence B, whichever Briefly speaking.

The term "PEGylated amino acid" as used herein refers to a polyethylene glycol (PEG) chain with one amino group and one carboxyl group. Typically, the PEGylated amino acid has the formula NH ₂ -(CH ₂ CH ₂ O) _n -CO ₂ H. As used herein, the value of n ranges from 1 to 20; preferably from 2 to 12.

The term "conjugating moiety" as used herein refers to a molecule having one or more functional groups (also called a "conjugating group") that is chemically reactive and capable of covalently bonding to other chemical entities. Non-limiting examples of the functional group include hydroxyl, carbonyl, carboxyl, thiol, amine, tert-butyldimethylsilyl (TBDMS), N-hydroxysuccinimidyl (NHS), SH-reactive group (e.g. maleimide, haloacetyl, sulfone or pyridyl disulfide), iodine, iodoacetamide azide, alkyne, tetrazine, cyclooctene and cyclooctene groups. According to embodiments of the present invention, the conjugating moiety of a given molecular construct has two functional groups, one of which is a carboxyl or amino group for binding to the alpha-amino or carboxyl group of the terminal amino acid residue of the core through the formation of an amide bond between them, so that the conjugating moiety is bound to N - or the C-terminal amino acid residue of the core; and the other is an SH-reactive group for bonding to a second element via SH-reactive cross-linking chemistry.

As used herein, the term “end” in relation to a polypeptide refers to the amino acid residue at the N- or C-terminus of the polypeptide. With respect to a polymer, the term "end" refers to a structural unit of the polymer (eg, polyethylene glycol of the present invention) that is located at the end of the polymer backbone. In the present specification and claims, the term “free end” is used to mean a terminal amino acid residue or structural unit that is not chemically linked to any other molecule.

The term "antigen" or "Ag" is used interchangeably and refers to a molecule that causes an immune response. This immune response may include a secretory, humoral and/or cellular antigen-specific response. As used herein, the term "antigen" may be any of a protein, a polypeptide (including mutants or biologically active fragments thereof), a polysaccharide, a glycoprotein, a glycolipid, a nucleic acid, or a combination thereof.

In the present description and claims, the term "antibody" or "antibody fragment" is used in the broadest sense to cover fully assembled antibodies, antibody fragments that bind antigens, such as antigen binding fragment (Fab/Fab'), F(ab' fragment )2 (having two antigen-binding Fab moieties linked together by disulfide bonds), variable fragment (Fv), single chain variable fragment (scFv), bispecific single chain variable fragment (bi-scFv), nanoantibodies (also called single domain antibodies, sdAb), monoantibodies and Diabodies. "Antibody fragments" include a portion of an intact antibody, preferably an antigen binding region or variable region of an intact antibody. The antibody fragment may contain a pair of scFvs fused to the N- or C-terminus of a pair of CH2-CH3 segments derived from human immunoglobulin γ4 or γ1. Generally, "antibody" refers to a protein consisting of one or more polypeptides, generally encoded by immunoglobulin genes or fragments of immunoglobulin genes. Well-known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as many immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define the classes of immunoglobulins: IgG, IgM, IgA, IgD and IgE, respectively. It is known that the typical structural unit of an immunoglobulin (antibody) forms a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 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, an antibody fragment can be obtained by modifying the nature of the antibody or by denovo synthesis using recombinant DNA techniques. In some embodiments of the present disclosure, the antibody and/or antibody fragment may be bispecific and may have various configurations. For example, bispecific antibodies may contain two different antigen binding sites (variable regions). In various embodiments, bispecific antibodies can be produced by a hybridoma method or a recombinant DNA method. In some embodiments, the bispecific antibodies have binding specificities for at least two different epitopes. In many molecular configurations that use antibody fragments, the antibody fragments can be replaced by antibody mimetics that bind to the same antigenic components as the antibody fragments. Antibody mimetics include anticalins, DARPs, affibodies, flomers, ankyrins, avimers, and others.

The term “specifically binds” as used herein refers to the ability of an antibody or antigen-binding fragment thereof to bind an antigen with a dissociation constant (Kd) of no more than about 1 x 10 ⁻⁶ M, 1 x 10 ⁻⁷ M, 1 x 10 ⁻⁸ M , 1×10 ⁻⁹ M, 1×10 ⁻¹⁰ M, 1×10 ⁻¹¹ M, 1×10 ⁻¹² M and/or the ability to bind an antigen with an affinity that is at least twice its affinity for a nonspecific antigen.

As used herein, the term “treatment” includes preventative (eg, prophylactic), curative, or palliative treatment; and “treatment” as used herein also includes preventive (eg, prophylactic), curative or palliative treatment. In particular, the term "treatment" as used herein refers to the use or administration of the present molecular construct, or a pharmaceutical composition containing it, to a subject who has a medical condition or a symptom associated with a medical condition, disease, or disorder secondary to to a medical condition or predisposition to a disease, for the purpose of partially or completely alleviating, improving, mitigating, delaying the onset, suppressing the progression, reducing the severity and/or reducing the frequency of one or more symptoms or features of said specific disease, disorder and/or condition. Treatment may be administered to a subject who exhibits no signs of a disease, disorder and/or condition, and/or to a subject who exhibits only early signs of a disease, disorder and/or condition, in order to reduce the risk of developing pathology associated with the disease, disorder and/or condition. /or condition.

The term "effective amount" as used herein refers to an amount of a given molecular construct that is sufficient to produce the desired therapeutic response. An effective amount of the agent is not required to treat the disease or condition, but provides treatment for the disease or condition such that the onset of the disease or condition is delayed, delayed, or prevented, or the symptoms of the disease or condition are improved. An effective amount may be divided into one, two or more doses in a suitable form for administration one, two or more times over a designated period of time. The specific effective or sufficient amount will vary depending on factors such as the specific condition being treated, the physical condition of the patient (for example, the patient's weight, age, or gender), the type of patient being treated, the duration of treatment, the nature of concomitant therapy (if any). available), as well as the specific drugs used and the structure of the compounds or their derivatives. The effective amount may be expressed, for example, as the total weight of the active ingredient (eg, in grams, milligrams or micrograms) or as a ratio of the weight of the active ingredient to body weight, for example, as milligrams per kilogram (mg/kg).

The terms "use" and "administration" are used interchangeably herein to refer to the application of a molecular construct or pharmaceutical composition of the present invention to a subject in need of treatment thereof.

The terms “subject” and “patient” are used interchangeably herein and are intended to refer to an animal, including the human species, that is amenable to treatment with the molecular construct, pharmaceutical composition and/or method of the present invention. The term "subject" or "patient" is intended to include both male and female genders unless one gender is specifically indicated. Accordingly, the term “subject” or “patient” includes any mammal that may benefit from a treatment method of the present invention. Examples of “subject” or “patient” include, but are not limited to, human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird, and poultry. In an exemplary embodiment, the patient is a human. The term "mammal" refers to all members of the class Mammalia, including humans, primates, domestic and farm animals such as rabbits, pigs, sheep and cattle; as well as zoo, sports or pet animals; and rodents such as mouse and rat. The term "non-human mammal" refers to all members of the class mammals other than humans.

In various embodiments of the present invention, the term “metal binding motif” refers to a short oligopeptide capable of binding a metal ion, for example Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ²⁺ , Mn ²⁺ , orCu ²⁺ . In some embodiments, metal-binding motifs capable of binding a zinc ion are also referred to as “zinc-binding motifs”; however, as those skilled in the art will appreciate, such labels can also bind to other metal ions with physical and/or chemical properties similar to the zinc ion.

The present invention is based at least on the design of several new metal-binding motifs that can be fused to the N- or C-terminus of the parent polypeptide. The present metal-binding motifs are advantageous for the design of bioconjugates in several aspects. First, true metal-binding motifs are sequences of human origin and thus are less immunogenic compared to traditional metal-binding motifs that are not part of the human genome. Second, the expression yield of composite polypeptides containing metal-binding motifs is desirable, and the composite polypeptides thus expressed are reasonably stable during storage, conjugation, and purification processes. Third, the present metal-binding motifs provide a facile site-specific conjugation reaction between the SH group of the cysteine residue in the metal binding motif and the SH-reactive group of the functional element, provided that the cysteine residues of the parent polypeptide are substantially inactive. Accordingly, the present invention provides a versatile means for the design of multifunctional bioconjugates. The following are aspects and embodiments of the present invention.

(I) Metal-binding motif

The first aspect of the present invention is directed to a metal-binding motif that, under suitable conditions, can form a complex with a metal ion such as Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ²⁺ , Mn ²⁺ , orCu ²⁺ .

According to one embodiment of the present invention, a metal binding motif comprising the amino acid sequence CX ₁ X ₂ HA (SEQIDNo.1) in order from N-terminus to C-terminus or in order from C-terminus to N-terminus, where X ₁ is glycine or proline, and X ₂ represents glycine or alanine. Illustrative examples of a metal-binding motif include, but are not limited to, CGGHA (SEQIDNo. 2), CPGHA (SEQIDNo. 3), CGAHA (SEQIDNo. 4), CPAHA (SEQIDNo. 5), GCGGHA (SEQIDNo. 6), ACPGHA (SEQIDNo. 6). 7), or GCPGHA (SEQIDNo. 8).

(II) Composite polypeptide containing a metal-binding motif

In another aspect, the present invention is directed to a composite polypeptide that contains a metal-binding motif according to the above-mentioned aspect/embodiments of the invention. Thus, the composite polypeptide is capable of forming a complex with a metal ion through a metal-binding motif under suitable conditions.

According to one embodiment of the present invention, the composite polypeptide comprises a parent polypeptide and a metal binding motif in accordance with the above aspect/embodiments of the present invention. In various embodiments, the metal binding motif is fused to the N- or C-terminus of the parent polypeptide, directly or to one or more intervening amino acid residues. In optional embodiments of the invention, the intermediate sequence may contain from two to ten glycine residues.

According to various embodiments of the present invention, when the metal binding motif is located at the N terminus of the parent polypeptide, the metal binding motif may have the sequence CX ₁ X ₂ HA (SEQIDNo.1) in order from N terminus to C terminus or in order from C terminus to the N-terminus, where X ₁ represents glycine or proline and X ₂ represents glycine or alanine. Similarly, when the metal-binding motif is located at the C-terminus of the parent polypeptide, the metal-binding motif may have the sequence CX ₁ X ₂ HA (SEQIDNo.1) in N-terminal to C-terminal order or C-terminal to N-terminal order. end, where X ₁ represents glycine or proline, and X ₂ represents glycine or alanine.

In various embodiments of the present invention, the parent polypeptide may be a peptide hormone or equivalents thereof. Peptide hormone equivalents include functional fragments, precursors, analogs or derivatives that are 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, insulin equivalents include, but are not limited to, lispro, aspart, glulisine, detemir, degludec and glargine. Calcitonin equivalents include, but are not limited to, procalcitonin and adrenomedullin. Somatostatin equivalents include, but are not limited to, octreotide and lanreotide.

In some embodiments, the parent polypeptide may be a peptidomimetic ligand that binds to cell surface receptors. Peptidomimetics are small protein-like chains that mimic a peptide. For example, a series of glutamate-urea-lysine peptidomimetics with the binding affinity of PSMA have been developed.

In optional embodiments of the present invention, 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 a whole antibody are also within the scope of the present invention, and examples of which include, but are not limited to, bispecific antibodies, chimeric antibodies, human antibodies, humanized antibodies, single chain antibodies (scAbs), single chain variable fragments (scFvs), tandem di -scFvs, tandem tri-scFvs, diantibodies, triantibodies, tetrantibodies, Fab fragments, F(ab')2 fragments, Fds, domain antibodies and mini-antibodies.

Alternatively, the parent polypeptide may be a cytokine or equivalents thereof. Cytokine equivalents include functional fragments, precursors, analogs or derivatives that are known to those skilled in the art. Illustrative examples of 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 alpha (TNF-α).

In some embodiments of the present invention, the parent polypeptide may be a single chain antibody fragment, such as a single domain antibody (sdAb), single chain antibody (scAb), single chain variable fragment (scFv), bispecific T cell invader (BiTE), tandem di-scFv and tandem tri-scFv. In some other embodiments of the present invention, the parent polypeptide may be an antibody fragment derived from the heavy chain or light chain of immunoglobulins, and the present composite polypeptide includes a portion of a full-length antibody, a fragment crystallizable region (Fc region), a fragment of an antigen binding fragment (Fab), a Fab' , F(ab') ₂ , Fab', Fv, (scFv) ₂ , sc(Fv) ₂ , diantibody, Fd, Fd' or mini-antibodies. Immunoglobulins can be derived from immunoglobulin D (IgD), IgE or IgG. The antibody may be a bispecific antibody, a chimeric antibody, a human antibody, or a humanized antibody.

In some embodiments, the antibody (as well as a functional fragment thereof) is specific for a cell surface antigen that is associated with and/or overexpressed on a diffuse tumor. Illustrative 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, CD319. Antibodies (or fragments or derivatives thereof) against these cell surface antigens can be used as targeting elements capable of directing a molecular construct containing them to the site of disease. In other optional embodiments, the antibody (as well as a functional fragment or derivative thereof) is specific for other cell surface antigens, and such antibody can cause a therapeutic effect in a 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 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 (CA 19 -9), CA 125, carcinoembryonic antigen (CEA), mucin 1 (MUC 1), ganglioside GD2, melanoma-associated antigen (MAGE), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, mucin-related Tn, sialylated Tn, GloboH, 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 an antibody against such TAA can be used as a targeting element as part of the molecular construct provided herein. In some other optional embodiments, the antibody (or fragment thereof) is specific for a growth factor selected from the group consisting of epidermal growth factor (EGF), mutant EGF, epiregulin, heparin-binding epidermal growth factor (HB-EGF), endothelial growth factor vascular A (VEGF-A), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). In some embodiments, the antibody (or fragment thereof) is specific for a cytokine described above.

(III) Molecular design of the functional element and the composite polypeptide containing the metal-binding motif.

In yet another aspect, one or more functional elements can be covalently conjugated to a composite polypeptide in accordance with the above-mentioned aspect/embodiments of the present invention by reacting with the sulfhydryl (SH) group of a cysteine residue in the metal-binding motif of the composite polypeptide, thereby forming a molecular construct (or bioconjugate ) according to the present invention. In particular, the metal-binding activity of the composite polypeptide allows site-specific conjugation, resulting in homogeneous molecular constructs.

In optional embodiments of the present invention, the SH-reactive group is a maleimide, iodoacetyl, bromoacetyl, vinylsulfonic, monosulfonic, methylsulfonylbenzothiazole, or 2-pyridyldithiol group.

As can be appreciated, the functional element may be an effector molecule capable of producing a desired therapeutic effect in a subject. Alternatively, the functional element may be a targeting element capable of directing the molecular construct to a target site in the body of a subject. Alternatively, the functional element is a pharmacokinetic element capable of altering the pharmacokinetic profile of a molecular construct in a subject. Still alternatively, the functional element is in the form of a linker unit or bundle. In certain embodiments of the present invention, the linker unit comprises a central core and a plurality of targeting, effector, or pharmacokinetic elements; the structure of said linker unit is discussed in section (IV) below.

Examples of effector molecules that can be used in the present invention include, but are not limited to, cytotoxic drugs, toll-like receptor (TLR) agonists, or chelators (alone or in combination with a radioactive nuclide).

Obviously, a cytotoxic drug that exhibits cytotoxic effects on certain cells can be antiestrogens (for example, tamoxifen, raloxifene and megestrol), LHRH agonists (for example, gosclin and leuprolide), antiandrogens (for example, flutamide and bicalutamide), a photodynamic therapy agent ( e.g. vertoporphin, phthalocyanine, photosensitizer Pc4 and dimethoxyhypocrellin A), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trophosfamide, chlorambucil, estramustine and melphalan), nitrosoureas (e.g. carmustine and lomustine), alkyl sulfonates (e.g. busulfan and treosulfan), triazines (eg dacarbazine, temozolomide), platinum containing compounds (eg cisplatin, carboplatin, oxaliplatin), vinca alkaloids (eg vincristine, vinblastine, vindesine and vinorelbine), taxoids (eg paclitaxel, docetaxal and taxol), epipodophyllines (eg , etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mitomycin C), antimetabolites, DHFR inhibitors (eg, methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (eg, mycophenolic acid, tiazofurin , ribavirin and EICAR), ribonucleotide reductase inhibitors (eg, hydroxyurea and deferoxamine), uracil analogs (eg, 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogues (eg, cytarabine ( araC), cytosine arabinoside and fludarabine), purine analogues (eg, mercaptopurine and thioguanane), vitamin D3 analogues (eg, EB 1089, CB 1093 and KH 1060), isoprenylation inhibitors (eg, lovastatin), dopaminergic neurotoxins (eg, 1-methyl -4-phenylpyridinium ion), cell cycle inhibitors (eg, staurosporine), actinomycin (eg, actinomycin D, dactinomycin), bleomycin (eg, bleomycin A2, bleomycin B2, peplomycin), anthracycline (eg, daunorubicin, doxorubicin, idarubicin, epirubicin , pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (for example, verapamil), Ca2+ ATPase inhibitors (for example, thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (for example, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestavrtinib, neratinib, nilotinib, semaxanib, sunitinib, toceranib, vandetanib, vatalanib, rituximab, nilotinib, sorafenib, everolimus, temsirolimus, proteasome inhibitors (eg, bortezomib), mTOR inhibitors (eg, rapamycin, temsirolimus, everolimus, and Rida forolimus) , oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campatecin, plicamycin, asparaginase, aminopterin, methopterin, porphyromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectin, procarbazine, discodermolide, carminomycin, aminopterin or hexamethylmelamine. In one specific embodiment of the present invention, the cytotoxic drug is mertansine, auristatin, maytansine, doxorubicin, calicheamicin, or camptothecin. In one specific embodiment of the present invention, the cytotoxic drug is mertansine, auristatin, maytansine, doxorubicin, calicheamicin, or camptothecin.

Illustrative examples of toll-like receptor agonists include lipoteichoic acid, glucan, motolimod, imiquimod, resiquimod, gardiquimod, CpG oligodeoxynucleotide (CpGDON), lipopolysaccharide (LPS), monophosphoryl liposide A, and zymosan.

In various embodiments, the chelator is selected from the group consisting of 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-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7- bis(carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl )pentadioic acid (DOTAGA), 1,4,7-triazacyclononane phosphinic 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 (PTA) , N'-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO) and 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 some embodiments, the radioactive nuclide is ¹¹¹ In, ¹³¹ I, or ¹⁷⁷ Lu; in other embodiments, the radioactive nuclide may be ⁹⁰ Y, ⁶⁸ Ga, ^99m Tc, ⁶⁴ Cu, ¹⁵³ Gd, ¹⁵⁵ Gd, ¹⁵⁷ Gd, ²¹³ Bi, ²²⁵ Ac or Fe.

In other embodiments, the selection of effector elements for the molecular constructs of the present invention also covers 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 for 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) immunoenhancing cytokines (IFN-α, IFN-γ, IL-2, TNF-α).

Targeting elements for use in embodiments of the present invention may be selected depending on the disease being treated. For example, targeting elements for the treatment of various diseases include (1) antibody fragments specific for extracellular matrix components in joints, skin or bones, such as collagen I, collagen II, collagen III, collagen V, collagen VII, collagen IX, collagen. XI, α-aggrecan and osteonectin; (2) antibody fragments specific for a cluster of differentiation markers, such as CD19, CD20, CD22, CD30, CD52, CD79a, CD79b, CD38, CD56, CD74, CD78, CD138; or , CD319, CD5, CD4, CD7, CD8, CD30; or CD13, CD14, CD15, CD33, CD34, CD36, CD37, CD41, CD61, CD64, CD65, and CD11c and other surface antigens of cells of lymphoid and myeloid origin and plasma cells, or (3) antibody fragments specific for receptors or antigens that are overexpressed on the surface of solid tumor cells, such as human epidermal growth factor receptor (HER1), HER2/Neu, HER3, Tn, GloboH, ganglioside GD-2, CA125, CA19-9 and carcinoembryonic antigen (CEA).

Targeting elements can also be antibodies to hormones, growth factors or cytokines, in which receptors for hormones, growth factors or cytokines are expressed on tumor cells or other diseased cells. In some other optional embodiments, the targeting element of the present molecular construct is a growth factor.

In some embodiments of the present invention, at least one of the parent polypeptide and the functional element of the present invention is a growth factor-specific antibody fragment. In some 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). In a similar concept to that described above, when the targeting element is a growth factor (eg, EGF), the present molecular construct is capable of specifically targeting a receptor-expressing cell/tissue/organ (eg, a tumor cell with an EGF receptor expressed on it). If the effector element is an antibody fragment specific for a growth factor (eg, VEGF-A), it can hijack and neutralize a growth factor-associated signal transduction pathway (eg, VEGF-A-induced angiogenesis). According to one working example, the present molecular construct is useful in the treatment of solid tumors in which the effector element is an antibody fragment specific for VEGF-A.

Examples of pharmacokinetic elements are C _8-28 fatty acid derivatives or C _8-28 dicarboxylic fatty acid derivatives. Each pharmacokinetic element is associated with one of the K residues through the ε-amino acid group of the K residue. In various embodiments of the present invention, the functional element is a fatty acid derivative that is a derivative of octanoic acid, pelargonic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid 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), docosahexaenoic acid (DHA). In some embodiments of the present invention, the functional element is a derivative of a dicarboxylic fatty acid, which is a derivative of suberic acid, azelaic acid, sebacic acid, undecanedicarboxylic acid, dodecanedicarboxylic acid, brassilic acid, tetradecanedicarboxylic acid, pentadecanedicarboxylic acid, thapsic acid, heptadecanedicarboxylic acid, or octadecanedicarboxylic acid . In some embodiments, the present functional element is a myristic acid or palmitic acid derivative. In other embodiments, the present functional element is a tetradecanedicarboxylic acid or thapsic acid derivative.

(IV) Linker unit for conjugation to a composite polypeptide containing a metal binding motif

In some optional embodiments of the invention, the functional element conjugated to the above-mentioned composite polypeptide is in the form of a ligament or linker unit. In some embodiments of the present invention, the linker unit comprises a central core and a plurality of targeting, effector, or pharmacokinetic elements. As can be understood, such linker units also fall within the scope of protection of the present invention.

In some embodiments of the present invention, the linker unit comprises a central core and a plurality of targeting, effector, or pharmacokinetic elements in accordance with the aforementioned aspect/embodiments of the invention.

Specifically, the central core is a polypeptide that ranges from 3 to 120 amino acid residues in length and contains from 2 to 10 lysine (K) residues; for example, a true core may contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 K residues. Any two K residues are adjacent to each other or separated by 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 additionally contains a terminal spacer, and the SH-reactive group is associated with the terminal amino acid residue of the terminal spacer, forming an amide bond with it. Thus, it is possible to conjugate the terminal SH-reactive group of a given linker unit to the SH-group of a cysteine residue of the metal-binding motif of composite polypeptides, provided that the cysteine residues of the parent polypeptide are substantially inactive, thereby achieving site-specific conjugation that results in homogeneous molecular constructs.

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. Each of the filler and terminal spacer contains, independently, (1) from 1 to 12 amino acid residues other than K, or (2) a PEGylated amino acid having from 1 to 12 ethylene glycol (EG) unit repeats.

In cases where the linker unit does not have binding arms, each targeting, effector or pharmacokinetic element is associated with 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 structure additionally contains from 2 to 10 linkers branches, one end of each linking arm is linked to the core K residue by forming 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, effector or pharmacokinetic element. For example, the binding arm may be a peptide containing 2-12 amino acid residues other than K, or a polyethylene glycol (PEG) chain having 2-24 EG repeat units.

According to optional embodiments of the present invention, prior to conjugation to a targeting, effector, or pharmacokinetic element, the free end of the binding arm (i.e., the end that is not bound to the central core lysine residue) may bear a linking group selected from the group consisting of amine, carboxyl , N-hydroxysuccinimidyl (NHS), azide, alkyne, cyclooctyne, tetrazine and cyclooctene groups. Depending on the linking group (e.g., amine, carboxyl, NHS, azide, alkyne, cyclooctyne, tetrazine, or cyclooctene group), modification of the amino group of the side chain of the lysine residue (in cases where there is no linking arm) or the true linking group present at the free end of the linker branches, a functional element (such as a targeting element, a therapeutic effector element, or a pharmacokinetic element) can be designed with an appropriate functional group such that the functional element can be linked to the free end of the linking branch through any of the following chemical reactions,

(1) forming 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 azide-alkyne cycloaddition click reactions (CuAAC reaction): one of the linking group and functional element has an azide or picol azide group, while the other has an alkyne group;

(3) inverse electron demand Diels-Alder reactions (iEDDA): one of the linking group and functional element has a tetrazine group, while the other has a cyclooctene group (eg, TCO or norbornene group); or

(4) strain-facilitated azide-alkyne cycloaddition (SPAAC) reactions: one of the coloring group and functional element has an azide group, and the other has a cyclooctyne group.

In various embodiments of the present invention, the tetrazine group is 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, 1,2,4,5-tetrazine, or derivatives thereof; the cyclooctene group is a norbornene or trans-cyclooctene (TCO) group; and the cyclooctyne group is selected from the group consisting of dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononine (BCN) and dibenzoazacyclooctine (DIBAC or DBCO). According to one embodiment of the present invention, the tetrazine group is 6-methyltetrazine.

According to some optional embodiments of the present invention, the central core carries a local negative charge on or adjacent to the amino acid residue associated with the SH-reactive group. In particular, a local negative charge is present within the first 5-15 amino acid residues within an amino acid residue, starting with the amino acid residue associated with the SH-reactive group. For example, a local negative charge is present in the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, starting with the amino acid residue associated with the SH-reactive group. A local negative charge can be imparted by the inclusion of one or more negatively charged amino acid residues at pH=7, such as an aspartate (D) and glutamate (E) residue.

Alternatively, the core may be coupled to multiple effector elements that are negatively charged such that the central core carries a local negative charge on or adjacent to the SH-reactive group. For example, the effector element may contain a chelator (eg, DOTA, DOTAGA, etc.), which can increase the net negative charge of the linker unit as a whole.

The targeting, effector, and pharmacokinetic elements described in section (III) above are applicable in embodiments of section (IV), and their detailed description is omitted here for brevity. In various embodiments, linker units carrying a plurality of effector elements are referred to as “effector bundles” or “drug bundles”, a linker unit carrying a plurality of chelators is referred to as “chelator bundles”, a linker unit carrying a plurality of targeting elements is referred to as “targeting elements”. bundles", a linker unit carrying a plurality of scFvs is referred to as "scFv bundles", a linker unit carrying a plurality of pharmacokinetic elements is referred to as "pharmacokinetic (PK) bundles", and a linker unit carrying a plurality of fatty acid chains and/or dicarboxylic fatty acids is referred to as "bundles". fatty acids (FA)".

In some embodiments, the fatty acid (or dicarboxylic fatty acid) derivative is a chemically modified fatty acid (or dicarboxylic fatty acid) molecule. For example, the carboxyl group of a fatty acid molecule (or one of the carboxyl groups of a dicarboxylic fatty acid molecule) reacts with a chemical moiety with two functional groups in which one functional group is carboxyl-reactive (thus forming a covalent bond with the (dicarboxylic) fatty acid molecule ), while the other is a functional group that reacts with the amino group of the side chain of a lysine residue. According to optional embodiments of the present invention, the chemical compound modifying the (dicarboxylic) fatty acid molecule is a glutamate residue, an aspartate residue, an amino-EG2 acid, a gamma-aminobutyric acid, or the like; however, the present invention is not limited to this.

As can be understood, in cases where the parent polypeptide does not contain a cysteine residue or other amino acid residues containing a sulfhydryl group, a terminal cysteine residue can be introduced in place of the terminal metal-binding motif to form a composite polypeptide, and the present linker unit can form a conjugate with such a composite polypeptide through terminal cysteine residue.

(V) Linker unit carrying multiple composite polypeptides containing a metal-binding motif

In yet another aspect, the present invention is directed to a linker unit capable of carrying a plurality of constituent polypeptides in accordance with the above-mentioned aspect/embodiments of the present invention. In some embodiments, such linker units are referred to as “peptide bonds.”

In some embodiments of the present invention, the linker unit comprises a central core, 2 to 10 linking arms, and 2 to 10 component polypeptides according to the above-mentioned aspect/embodiments of the invention. The binding arms of such a linker unit have an SH-reactive group at their free end, and since each constituent polypeptide has a true metal-binding motif, it is possible to conjugate the constituent polypeptides to the binding arm in a site-specific manner, in a state in which the cysteine residues of the parent polypeptide are substantially inactive.

In particular, the central core is a polypeptide that ranges from 3 to 120 amino acid residues in length, with the core containing from 2 to 10 lysine (K) residues; for example, the present core may contain 2, 3, 4, 5, 6, 7, 8, 9 or 10 K residues and a conjugated group. Any two K residues are adjacent to each other or separated by filler.

In some cases, the central core additionally contains a conjugated group associated with the first or last K residue of the central core, forming an amide bond with it. Examples of the conjugate group include, but are not limited to, azide, alkyne, tetrazine, cyclooctene and cyclooctene groups.

In other cases, the central core additionally contains a terminal spacer, and the conjugating group is linked to the terminal amino acid residue of the terminal spacer, forming an amide bond with it. 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.

Each of the filler and terminal spacer contains, independently, (1) from 1 to 12 amino acid residues other than K, or (2) a PEGylated amino acid having from 1 to 12 ethylene glycol (EG) unit repeats. In general, the above-mentioned terminal spacer or filler may be (1) an oligopeptide of 1-12 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues, other than amino acid residue K, or (2) a PEGylated amino acid with 1 to 12 EG units (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 units EG). In particular, in the case where the present core contains multiple K residues, the terminal spacer or filler may contain either a PEGylated amino acid having 2-12 EG units or 1-12 non-K amino acid residues, wherein each of the amino acid residues other than K is suitably 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), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W) leftovers; preferably each of the amino acid residues other than K is suitably selected from the group consisting of G, S, R, H, T, N, Q, P, A, V, I, L, M, F, Y, and W residues ; more preferably, each of the amino acid residues other than K is G and/or S residues, respectively.

According to some embodiments of the invention, one end of each linking arm is linked to the K residue of the core by forming an amide bond with the ε-amino group of the K residue, while the other end of each linking arm has a sulfhydryl group (SH)-reactive group. For example, the binding arm may be a peptide containing 2-12 amino acid residues other than K, or a polyethylene glycol (PEG) chain having 2-24 EG repeat units.

Exemplary SH-reactive groups include maleimide, haloacetyl (eg, iodoacetyl or bromoacetyl), sulfonic (eg, vinyl sulfone, monosulfone, methylsulfonylbenzothiazole) and pyridyl disulfide (eg, 2-pyridyl dithiol) groups.

In some embodiments of the present invention, the binding arm is a peptide containing 2-12 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues (each of which may be a natural or non-natural amino acid residue) other than amino acid residue K. For example, a binding arm is a peptide containing 2-12 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 working example, the binding arm is a peptide containing 5-10 amino acid residues that are independently selected from the group consisting of residues G, S, E and R. Alternatively, the binding arm may be a PEG chain having 2-24 (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 5-15 repeats of EG units; more preferably 6-12 repeats of EG units. It is understood that the peptide or PEG chain of the linking arm can be replaced by a polymer of approximately the same length. A polymer containing carbohydrate or other hydrophilic moieties is suitable for use as linking arms.

In some embodiments of the present invention, each linking arm carries a local negative charge on or adjacent to the amino acid residue associated with the SH-reactive group. In particular, a local negative charge is present within the first 5-15 amino acid residues within an amino acid residue, starting with the amino acid residue associated with the SH-reactive group. For example, a local negative charge is present in the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, starting with the amino acid residue associated with the SH-reactive group. A local negative charge can be provided by the inclusion of one or more negatively charged amino acid residues at pH=7, such as an aspartate (D) and glutamate (E) residue.

As can be understood, when the present core contains at least two terminal spacers or fillers, each of the terminal spacers or fillers may be the same or different; that is, each terminal spacer or filler may contain the same or different amino acid sequences and/or EG units. According to one embodiment of the present invention, the present core contains three fillers, in which one of the spacers is a PEGylated amino acid having 8 EG unit repeats, and the other two spacers are, respectively, one S residue and one G residue. According to another embodiment of the present invention, the present the core contains one terminal spacer and four fillers, in which one of the spacers consists of four G residues and two S residues, while the other four spacers respectively consist of one G residue and one S residue. According to another embodiment of the present invention, the present the core contains two terminal spacers and three fillers, each of which is a PEGylated amino acid having 4 EG unit repeats.

In optional embodiments of the present invention, the aforementioned linker unit comprising a plurality of constituent polypeptides (or peptide bundle) may be conjugated to an additional linker unit by reacting with a central core conjugating group to form a molecular construct. For example, said additional linker unit may be an effector link, a targeting link, or a PK link. For example, the additional linker unit may have a structure similar to that described in the applicant's previously filed patent applications. Briefly, the additional linker unit may have a structure similar to that described in section (III) above, except that the terminal SH-reactive group is replaced by a conjugating group that corresponds to the conjugating group of the peptide linkage.

In embodiments of the invention where the targeting or effector elements carried by said additional linker unit are peptide-based elements, said peptide-based element may have a zinc-binding motif described above, and the additional linker unit may adopt the structure of the present peptide bundle, thereby forming a molecular construct consisting of two peptide bundles carrying different types of functional elements.

In some optional embodiments, the conjugation group is part of a conjugation moiety that has a functional group capable of forming a covalent bond with the α-amino group (-NH2) of a terminal amino acid residue (i.e., the first linking amino acid residue or the N-terminal amino acid residue N- terminal spacer) or the carboxyl group (-COOH) of the terminal amino acid residue (ie, the last binding amino acid residue or the C-terminal amino acid residue of the C-terminal spacer), such that the conjugating moiety is bound to it. In a certain embodiment, the core may have only one of the N- and C-terminal spacers and has both first and second conjugated moieties that are respectively linked to two terminal amino acid residues (which may be a terminal binding amino acid residue or a terminal amino acid residue of a terminal spacer ). There are also embodiments of the invention in which the core contains both N- and C-terminal spacers, and two conjugated fragments are respectively linked to the terminal amino acid residues of the two terminal spacers. In preferred embodiments, the covalent bond formed between the conjugating moiety and the terminal amino acid residue is an amide bond. As can be understood, to ensure the homogeneity of the resulting linkage, it is important that one conjugated moiety has only one functional group, which can react with either the α-amino group or the carboxyl group.

Optionally, the conjugating moiety further includes a PEG chain having 2-10 (ie, 2, 3, 4, 5, 6, 7, 8, 9 or 10) EG unit repeats connecting the carboxyl or amino group and the conjugating group; for example, a PEG chain may have 4, 6, 7 or 8 EG unit repeats.

According to various embodiments of the present invention, two conjugated moieties may have the same or different reactivity. Preferably, when the second conjugating group of the conjugating moiety is an azide, alkyne or cyclooctyne group, then the linking group of the linking arm cannot be any of an azide, alkyne or cyclooctyne group to avoid reaction between the second conjugating group and the linking group; rather, the linking group may be a tetrazine, cyclooctene, amine, carboxyl or N-hydroxysuccinimidyl (NHS) group. Alternatively, when the second conjugating group is a tetrazine or cyclooctene group, the linking group may not be a tetrazine or cyclooctene group; instead, the linking group may be an azide, alkyne, cyclooctyne, amine, carboxyl or N-hydroxysuccinimidyl (NHS) group.

As can be understood, the number of optional binding branches associated with the core is mainly determined by the number of binding amino acid residues (ie K residues) contained in the core. Since the present core includes at least two binding amino acid residues, the present targeting or effector bundle may include multiple binding branches.

As can be understood, the description regarding effector, targeting or pharmacokinetic elements applies to all aspects/embodiments of the invention including such elements, unless the context clearly indicates otherwise.

As can be appreciated, during solid phase synthesis of a core peptide center, instead of attaching functional elements to the core after the core has been synthesized, it is also possible to include a K amino acid residue modified by a specific functional element in the peptide chain. Therefore, in various embodiments, K residues modified with different functional elements can be added sequentially during solid-phase synthesis to produce a batch of homogeneous pharmacokinetic couplings, where each pharmacokinetic coupling can have two or more different functional elements associated with it.

The following examples are provided to explain certain aspects of the present invention and to assist those skilled in the art in practicing the invention. These examples should in no way 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 given herein, use the present invention to its fullest extent.

Example 1

Synthesis of the central core Mal-peptide 1

In this example, a peptide having five lysine residues was developed by the present inventor, and production was outsourced to KareBayCo., Ltd. (Monmouth Junction, USA). This peptide can be used as a central core to construct a linker unit or functional bundle. Mal-peptide 1 (maleimido-ethyl-GGSGGSKGSKGSKGSKGSK; SEQIDNo. 11) was synthesized using a standard Fmoc-based solid phase method. Mal-peptide 1 had a purity of 95.67%.

Identification of the peptide synthesized in this way was carried out using MALDI-TOF mass spectrometry. Mass spectrometry analyzes were performed at the Mass Core Facility of 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 1 illustrates the result of MALDI-TOF mass spectrometry, which shows that Mal-peptide 1 has a molecular weight of 1,790.916 daltons.

Example 2

Synthesis of the Mal-peptide 2-DOTA linkage

In this example, a chelator linkage (see Figure 2) containing a maleimide-containing peptide with three lysine residues as the central core and three DOTA molecules conjugated to the central core (Mal-peptide 2-DOTA linkage) was constructed by the present inventors, and production was outsourced to KareBayCo., Ltd. (Monmouth Junction, USA)

Mal-peptide 2 (maleimido-ethyl-GGSGGSGKGGSGGSGKGGSGGSGK, SEQIDNo. 12) for use as the central core was synthesized using a standard Fmoc-based solid-phase method, and then DOTA molecules were conjugated to the central core using liquid-phase synthesis.

The purified chelator linkage sample thus synthesized was analyzed using reversed-phase analytical high-performance liquid chromatography (HPLC) on a SupelcoC18 column (250 mm × 4.6 mm; 5 μm) using a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, linear gradient from 0% to 100% acetonitrile over 30 minutes, at a flow rate of 1.0 ml/min and a column temperature of 35°C. Figure 3A illustrates the reversed-phase HPLC profile of the chelator bundle from Example 2, which shows that the chelator bundle peak has a retention time of 7.06 minutes.

Identification of the Mal-peptide 2-DOTA binding was performed by MALDI-TOF mass spectrometry. In FIG. 3B shows a MALDI-TOF mass spectrometry result showing that the present chelator linkage has a molecular weight of 3,093,454 daltons.

Example 3

Construction, expression and purification of recombinant 2-stranded (scFvαCD19)-Fc-MBM-1

V _L and V _H scFv specific for human CD19 were derived from the monoclonal antibody RB4v1.2. The gene sequence encoding the recombinant scFv-CH2-CH3 (human γ1) chain was configured by fusing the gene sequence encoding the human CD19-specific scFv with the upstream gene sequence encoding the flexible hinge region and CH2 domain of IgG1.Fc, and the gene sequence , encoding metal binding motif (MBM)-1, ACPGHA(SEQIDNo. 7), was fused to the gene sequence encoding the CH3 domain of IgG1.Fc and the short flexible linker GGGG (SEQIDNo. 9).

The ScFvs were configured in two orientations, i.e., V _L -linker-V _H and V _H -linker-V _L , where V _L and V _H were linked by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQIDNo. 10). The amino acid sequences of the recombinant chain of the present (scFvαCD19)-Fc-MBM-1 in the configurations V _L -linker-V _H and V _H -linker-V _L are shown in SEQIDNO: 13 and 14, respectively, and the general structure of these 2-strand (scFvαCD19 )-Fc-MBM-1 molecular constructs are presented in Fig. 4A.

To produce the above recombinant proteins, a mammalian overexpression system based on the Expi293F™ cell line was used. This system used an ExpiFectamine™ 293 transfection kit (LifeTechnologies, Carlsbad, USA) consisting of the Expi293F™ cell line, ExpiFectamine™ 293 cationic lipid-based reagent and ExpiFectamine™ 293 transfection enhancers 1 and 2, and media that was part of the system expression (Gibco, New York, USA).

The gene sequence constructed as described above was placed into the pcDNA3 expression cassette. Expi293F cells were seeded at a density of 2.0 x ¹⁰⁶ viable cells/ml in Expi293F expression medium and maintained for 18-24 hours before transfection to ensure that cells were actively dividing during transfection. For transfection in a 2-liter Erlenmeyer shake flask, 7.5 x 10 ⁸ cells in 255 ml of medium were transfected using ExpiFectamine™ 293 transfection reagent according to the manufacturer's instructions. Transfected cells were incubated at 37°C for 16-18 hours after transfection in an orbital shaker (125 rpm), and then the incubated cells were added with ExpiFectamine™ 293 transfection enhancer 1 and enhancer 2 in a shaking flask and incubated for another 5- 6 days. Culture supernatants were collected and the expressed recombinant proteins fused to hIgG1.Fc in the medium were purified using protein A affinity chromatography.

After buffer exchange to phosphate-buffered saline (PBS), the concentration of (scFvαCD19)-Fc-MBM-1 present was determined and analyzed using 10% SDS-PAGE. The results show that the real (scFvαCD19)-Fc-MBM-1 had a molecular mass of about 120 kDa on non-reducing SDS-PAGE, slightly larger than the expected size of 110 kDa. In fig. 4B shows an example of a non-reducing SDS-PAGE result for 2-stranded (V _L -V _H scFvαCD19)-Fc-MBM-1 (see major band indicated by arrow). As can be understood, the SDS-PAGE results here are intended to quickly confirm the approximate molecular weight of the molecular construct thus synthesized, and a more precise molecular weight analysis has been determined using mass spectrometric analysis. For example, the MALDI-TOF result of the present molecular construct is presented in Figure 23.

Example 4

Construction, expression and purification of recombinant 2-stranded (scFvαCD19)-Fc-MBM-2

In this example, recombinant 2-stranded (scFvαCD19)-Fc-MBM-2 in the V _L -linker-V _H configuration was constructed, expressed, and purified using protocols similar to those outlined in the worked example above, except that used another metal-binding motif, GCPGHA (SEQIDNo. 8, hereinafter MBM-2). The amino acid sequences of the recombinant chain of the present (V _L -V _H scFvαCD19)-Fc-MBM-2 are shown in SEQIDNo. 15.

The SDA-PAGE results in Figure 5 show that the recombinant chain of this molecular construct is approximately 120 kDa in size (indicated by the arrow), which is slightly larger than the expected size.

Example 5

Construction, expression and purification of recombinant 2-stranded (scFvαCD19)-Fc-MBM-3

In this example, recombinant 2-stranded (scFvαCD19)-Fc-MBM-3 in the V _L -linker-V _H configuration was constructed, expressed, and purified using protocols similar to those outlined in the worked example above, except that used another metal-binding motif, GCGGHA (SEQIDNo. 6, hereafter MBM-3). The amino acid sequences of the recombinant chain of the present (scFvαCD19)-Fc-MBM-3 in the V _L -linker-V _H configuration are shown in SEQIDNo. 16.

Characterization of the molecular construct (V _L -V _H scFvαCD19)-Fc-MBM-3 was performed using SDS-PAGE. The SDA-PAGE results in Figure 6 show that the recombinant chain of the new design is approximately 110 kDa (indicated by arrow), which is the expected size.

Example 6

Construction, expression and purification of recombinant anti-CD19 antibody-MBM

Recombinant IgG (human γ1) molecular constructs were constructed by fusing a short flexible linker, GGGG (SEQIDNo. 9) and an MBM sequence (in this example, MBM-1 or MBM-2) to the C-terminus of the heavy chain of an intact CD19-specific antibody . Two gene sequences were inserted into the pG1K expression cassette with a multiple cloning site. Expression and purification of the present recombinant anti-CD19 antibody-MBM was carried out using protocols similar to those described in the working example above.

The amino acid sequences of the heavy chain of the antibody specific for human CD19 antibody-MBM-1 or MBM-2, respectively, are indicated in SEQIDNO: 17 and 18, and the amino acid sequence of the light chain of the intact anti-CD19 antibody is indicated in SEQIDNo. 19.

Characterization of molecular constructs was performed using SDS-PAGE. The reducing SDS-PAGE results in Figure 7A show that control protein (lane 1), human CD19 antibody-MBM-1 (lane 2), and human CD19 antibody-MBM-2 (lane 3) have a higher molecular weight band of approximately 55 kDa and a lower molecular weight band of approximately 25 kDa, which corresponds to the expected sizes of the heavy chain and light chain of the construct, respectively.

The general structure of the anti-CD19 antibody-MBM-1 molecular construct is shown in FIG. 7B.

Example 7

Construction, expression and purification of recombinant 2-stranded (scFvαCD33)-Fc-MBM

V _L and V _H scFv specific for human CD33 were derived from the monoclonal antibody huMy9-6. The gene sequence encoding the recombinant chain (scFvαCD33)-CH2-CH3 (human γ1) was configured by fusing the gene sequence encoding the scFv specific for human CD33 with the upstream gene sequence encoding the flexible hinge region and CH2 domain of IgG1.Fc, and the gene sequence encoding MBM-1 or MBM-2 was fused to a subsequent gene sequence encoding the CH3 domain of IgG1.Fc and a short flexible linker, GGGG (SEQIDNo. 9).

The ScFvs were configured in two orientations, i.e., V _L -linker-V _H and V _H -linker-V _L , where V _L and V _H were linked by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQIDNo. 10). The amino acid sequences of the recombinant chain present (scFvαCD33)-Fc-MBM-1 in the configurations V _L -linker-V _H and V _H -linker-V _L are shown in SEQIDNO: 20 and 21, respectively, while the amino acid sequences of the recombinant chain present (scFvαCD33 )-Fc-MBM-2 in the V _L -linker-V _H and V _H -linker-V _L configurations are shown in SEQIDNO: 22 and 23, respectively.

Expression and purification of the present recombinant 2-stranded (scFvαCD33)-Fc-MBM was carried out using protocols similar to those described in the working example above.

Characterization of molecular constructs was performed using nonreducing SDS-PAGE. The SDS-PAGE results in Figure 8 show that each of (V _L -V _H scFvαCD33)-Fc-MBM-1 (lane 1), (V _H -V _L scFvαCD33)-Fc-MBM-1 (lane 2), ( V _L -V _H scFvαCD33)-Fc-MBM-2 (lane 3), (V _H -V _L scFvαCD33)-Fc-MBM-2 (lane 4) have a size of about 120 kDa, which is slightly larger than the expected size of 110 kDa .

Example 8

Construction, expression and purification of recombinant 2-stranded (scFvαCD20)-Fc-MBM

V _L and V _H scFv specific for human CD20 were derived from the monoclonal antibody ocrelizumab. The metal binding motifs MBM-1, MBM-2 and MBM-3 were respectively fused to the C-terminal CH3 domain of the recombinant chain (scFvαCD20)-CH2-CH3 (human γ1) through a short flexible linker, GGGG (SEQIDNo. 9). ScFv had a V _L -linker-V _H or V _H -linker-V _L orientation. V _L and V _H were linked by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQIDNo. 10).

Expression and purification of the present recombinant 2-stranded (scFvαCD20)-Fc-MBM-1, -MBM-2 or -MBM-3 were performed using protocols similar to those described in the working example above. Sequences of the recombinant chain of the present (V _L -V _H scFvαCD20)-Fc-MBM-1, (V _H -V _L scFvαCD20) -Fc-MBM-1, (V _H -V _L scFvαCD20)-Fc-MBM-2, ( V _L -V _H scFvαCD20)-Fc-MBM-3 and (V _H -V _L scFvαCD20)-Fc-MBM-3 are given in SEQIDNO: 24, 25, 26, 27 and 28, respectively.

Expression and purification of the present recombinant 2-stranded (scFvαCD20)-Fc-MBM was carried out using protocols similar to those described in the working example above.

Characterization of the molecular constructs was performed using non-reducing SDS-PAGE. The SDS-PAGE results in Figure 9 show that both (V _L -V _H scFvαCD20)-Fc-MBM-1 (lane 1) and (V _H -V _L scFvαCD20)-Fc-MBM-1 (lane 2) molecular constructs are approximately 120 kDa in size, slightly larger than the expected size of 110 kDa. The SDS-PAGE results in Figures 10A and 10B, respectively, indicate that the molecular constructs V _H -V _L scFvαCD20)-Fc-MBM-2 and (V _L -V _H scFvαCD20)-Fc-MBM-3 are approximately 120 kDa in size , which is slightly larger than the expected size of 110 kDa.

Example 9

Construction, expression and purification of recombinant 2-stranded (scFvαCA19-9)-Fc-MBM

Anti-CA19-9 antibody-producing mouse B-cell hybridoma HB8059 was purchased from Developmental Studies Hybridoma Bankat the University of Iowa. Poly(A)+ RNA was reverse transcribed using the SuperScriptIIIRT-PCR system (Invitrogen, Waltham, USA), and first-strand cDNA was synthesized. V _H and V _L nucleotide and amino acid sequences of HB8059 have not been published. To determine the variable region sequences of HB8059, V _H and V _L cDNAs were amplified by PCR using the DNA primer set provided by Ig-primerSets (Novagen, Madison, USA) according to the manufacturer's instructions. The amino acid sequence V _H and V _L of the human CA19-9-specific monoclonal antibody HB8059 is described in SEQIDNO: 29 and 30.

MBM-1 or MBM-3 was fused to the C-terminus of the CH3 domain (scFvαCA19-9)-CH2-CH3 (human γ1) of the recombinant chain through a short flexible linker GGGG (SEQIDNo. 9). The ScFvs had a V _L -linker-V _H orientation. V _L and V _H were linked by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQIDNo. 10). The recombinant chain sequences of the present scFvαCA19-9)-Fc-MBM-1 and (scFvαCA19-9)-Fc-MBM-3 are shown as SEQIDNO: 31 and 32, respectively.

Expression and purification of the present recombinant 2-stranded (scFvαCA19-9)-Fc-MBM was carried out using protocols similar to those described in the working example above.

Characterization of the molecular construct was performed using SDS-PAGE. The SDS-PAGE results in Figure 11A and Figure 11B show that the recombinant chain of both the (scFvαCA19-9)-Fc-MBM-1 molecular and (scFvαCA19-9)-Fc-MBM-3 constructs is approximately 120 kDa in size (band 1), which is slightly larger than the expected size of 110 kDa.

Example 10

Construction, expression and purification of recombinant 2-stranded (scFvαCD38)-Fc-MBM

V _L and V _H scFv-specific for human CD38 were derived from the monoclonal antibody daratumumab. The metal binding motifs MBM-1 and MBM-2 were respectively fused to the C-terminal CH3 domain of the recombinant chain (scFvαCD38)-CH2-CH3 (human γ1) via a short flexible linker GGGG (SEQIDNo. 9). ScFv had a V _L -linker-V _H or V _H -linker-V _L orientation. V _L and V _H were linked by the hydrophilic linker GSTSGSGKPGSGEGSTKG (SEQIDNo. 10). Expression and purification of the present recombinant 2-stranded (scFvαCD38)-Fc-MBM-1 or -MBM-2 was carried out using protocols similar to those described in the working example above. Sequences of the recombinant chain present (V _L -VHscFvαCD38)-Fc-MBM-1, (VH-V _L scFvαCD38)-Fc-MBM-1, (V _L -VHscFvαCD38)-Fc-MBM-2 and (VH-V _L scFvαCD38 )-Fc-MBM-2 are listed in SEQIDNO: 33, 34, 35 and 36, respectively.

Characterization of the molecular construct was performed using SDS-PAGE. The SDS-PAGE result in Figure 12A shows that the recombinant chain (V _L -V _H scFvαCD38)-Fc-MBM-1 (lane 1) has a size of about 120 kDa, which is slightly larger than the expected size of 110 kDa. The SDS-PAGE result in Figure 12B shows that the recombinant chain of (V _L -V _H scFvαCD38)-Fc-MBM-2 has a size of about 120 kDa, which is slightly larger than the expected size of 110 kDa.

Example 11

Preparation of recombinant 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles

In this example, a molecular construct was prepared having two DOTA linkages respectively conjugated to two cysteine residues of the 2-strand metal binding motif (scFvαCD19)-Fc-MBM-1; a schematic diagram illustrating the structure of this molecular construct is shown in Figure 13A. To reduce the cysteine residue at the C-terminus of the purified recombinant 2-stranded (V _L -V _H scFvαCD19)-Fc-MBM-1 from Example 3, recombinant proteins in sodium succinate buffer (10 mM sodium succinate, pH6.0 and 30 mM sucrose) reconstituted by incubating with 45 μM tris(2-carboxyethyl)phosphine (TCEP) at room temperature for 30 min 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. Reconstituted protein samples were then treated with 15 μM Mal-peptide 2-DOTA coupling from Example 2 and then incubated for 1 hour at room temperature. The unreacted DOTA binder was removed using a desalting column and the product was analyzed by SDS-PAGE.

Figure 13B shows the result of SDS-PAGE analysis of this molecular construct (illustrated below), which shows that the present molecular construct has a molecular mass of about 120 kDa (lane 2, indicated by arrow), which is slightly larger than expected. The unconjugated fusion protein was in lane 1. As can be seen in Figure 13B, the conjugation yield of DOTA bundles to recombinant 2-stranded (scFvαCD19)-Fc-MBM-1 is approximately 90%. These protein bands on SDS-PAGE were quantified by ImageJ.

Example 12

Preparation of recombinant 2-stranded (scFvαCD19)-Fc-MBM-2 X 2 DOTA bundles

In this example, conjugation of two DOTA linkages to the recombinant 2-stranded (V _L -V _H scFvαCD19)-Fc-MBM-2 from Example 4 was performed as described in the previous Example.

In FIG. 14 shows the result of SDS-PAGE analysis of this molecular construct, which shows that the present molecular construct has a molecular mass of about 120 kDa (lane 2, indicated by arrow), which is consistent with or slightly larger than the expected size. The unconjugated fusion protein was in lane 1. As can be seen in Figure 14, the conjugation yield of DOTA bundles to recombinant 2-stranded (scFvαCD19)-Fc-MBM-2 is approximately 90%.

Example 13

Preparation of recombinant 2-stranded (scFvαCD19)-Fc-MBM-3 X 2 DOTA bundles

In this example, conjugation of two DOTA linkages to the recombinant 2-stranded (V _L -V _H scFvαCD19)-Fc-MBM-3 from Example 5 was performed as described in the previous Example.

In FIG. 15 shows the result of SDS-PAGE analysis of this molecular construct, which shows that the present molecular construct has a molecular mass of about 120 kDa (lane 2, indicated by arrow), which is consistent with or slightly larger than the expected size. The unconjugated fusion protein was in lane 1. As can be seen in FIG. 15, the yield of conjugation of DOTA bundles to recombinant 2-stranded (scFvαCD19)-Fc-MBM-3 is approximately 90%.

Example 14

Preparation of anti-CD19 antibody-MBM-1 X 2 DOTA bundles

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

In fig. 16 shows SDS-PAGE analysis of the present molecular construct of anti-CD19 antibody-MBM-1 X 2 DOTA binders. As shown in Figure 16, the heavy chain molecular weight is approximately 54 kDa (indicated by arrow #1 in lane 2), which is the expected size. Arrow #2 in lane 2 represents the unconjugated anti-CD19 antibody-MBM-1 heavy chain, and arrow #3 represents the anti-CD19 antibody-MBM-1 light chain.

Example 15

Preparation of anti-CD19 antibody-MBM-2 X 2 DOTA bundles

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

In FIG. 17 shows SDS-PAGE analysis of the present molecular construct. As shown in FIG. 17, the molecular weight of the heavy chain is approximately 54 kDa (indicated by arrow #1 in lane 2), which is the expected size. Arrow #2 in lane 2 represents the unconjugated anti-CD19 antibody-MBM-2 heavy chain, and arrow #3 represents the anti-CD19 antibody-MBM-2 light chain.

Example 16

Preparation of recombinant 2-stranded (scFvαCD33)-Fc-MBMX 2 DOTA bundles

In this example, conjugation of two DOTA linkages to recombinant 2-stranded (V _L -V _H scFvαCD33)-Fc-MBM-1 or -MBM-2 from Example 7 was performed as described in the previous example.

In FIG. 18 shows the result of conjugation analysis of DOTA bundles to recombinant 2-stranded (scFvαCD33)-Fc-MBM-1 by SDS-PAGE. As shown in Figure 18, this molecular construct has a molecular mass of approximately 120 kDa (indicated by arrow #1 in lane 2), which is at or slightly larger than the expected size. Arrow #2 in lane 1 is the unconjugated fusion protein. SDS-PAGE results show that the conjugation yield of DOTA tethers to recombinant 2-stranded (scFvαCD33)-hIgG1.Fc-MBM-1 is approximately 65%.

Example 17

Preparation of recombinant 2-stranded (scFvαCD20)-Fc-MBM-1 X 2 DOTA bundles

In this example, conjugation of two DOTA linkages to the recombinant 2-stranded (V _L -V _H scFvαCD20)-Fc-MBM-1 from Example 8 was performed as described in the previous Example.

In FIG. 19 shows the result of conjugation analysis of DOTA bundles to recombinant 2-stranded (scFvαCD20)-Fc-MBM-1 by SDS-PAGE. As shown in Figure 19, this molecular construct has a molecular mass of approximately 120 kDa (indicated by arrow #1 in lane 2), which is at or slightly larger than the expected size. Arrow #2 in lane 2 is the unconjugated fusion protein. The SDS-PAGE result in Figure 19 also shows that the conjugation yield of DOTA tethers to recombinant 2-stranded (scFvαCD20)-Fc-MBM-1 is approximately 90%.

Example 18

Preparation of recombinant 2-chain (scFvαCA19-9)-Fc-MBMX 2 DOTA

In this example, conjugation of two DOTA linkages to recombinant 2-stranded (scFvαCA19-9)-Fc-MBM-1 or -MBM-3 from Example 9 was performed as described in the previous Example. SDS-PAGE results show that the conjugation yields of DOTA tethers to recombinant 2-stranded(scFvαCA19-9)-Fc-MBM-1 and -MBM-3 are approximately 65% and 70%, respectively (data not shown).

Example 19

Stabilization of 2-stranded (scFv α CD19)-Fc-MBM-1 X 2 DOTA bundles

To stabilize the 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles, a ring opening reaction was performed using the following protocol.

The reaction product of the molecular construct obtained in Example 14 was a buffer exchange to Tris buffer (100 mM Tris at pH 9.0, 100 mM L-arginine, and 100 mM sodium chloride) using a NAP-10 SephadexG-25 column ( GEHealthcare). The resulting solution was then heated to 37°C for 5 hours. The solution was cooled and the buffer was replaced by centrifugation with 50 mM Bis-Tris buffer at pH 5.5. The final samples were concentrated to ~1-3 mg/mL protein.

For purification, the stabilized product was adjusted to pH 5.5 and then applied to a pre-equilibrated (50 mM Bis-Tris buffer at pH 5.5) Hi-Trap™QHP anion exchange column (GEHealthcare). Stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles were eluted using a step elution of 250 mM NaCl over 15 minutes and a linear gradient from 250 mM to 324 mM NaCl at a flow rate of 1.0 ml/min over 50 minutes .

A QHP anion exchange column was used to separate stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages from free 2-stranded fusion protein, 2-stranded fusion protein conjugated to three or four DOTA linkages, and aggregated material. The purified product, 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkage, was concentrated and buffer exchanged with Tris buffer (50 mM Tris buffer at pH 7.0 and 290 mM NaCl).

Fig. 20 is an FPLC elution profile of a QHP anion exchange column on stabilized 2-strand (scFvαCD19)-Fc-MBM-1 X 2 DOTA binders. As shown in Figure 20, the stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles eluted at a peak of 69.91 ml. Figure 21 shows the results of SDS-PAGE analysis of fractions collected from the QHP anion exchange column. Lanes A, B, C and D respectively correspond to an unconjugated molecular construct, a molecular construct conjugated to DOTA linkages, a stabilized molecular construct conjugated to two DOTA linkages in the feed solution, and a stabilized molecular construct conjugated to two DOTA linkages before loading onto the column.

The pure product, stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages, was collected from fractions #53, #54 and #55 shown in Figure 21.

Example 20

Stabilization of 2-stranded (scFv α CD20)-Fc-MBM-1 X 2 DOTA bundles

Purification of Stabilized 2-Chain (scFvαCD20)-Fc-MBM-1 X 2 DOTA Linkages

The procedure for stabilizing the 2-strand (scFvαCD20)-Fc-MBM-1 X 2 DOTA linkages was similar to that described in Example 19.

For purification, the stabilized product was adjusted to pH 5.1 and then applied to a pre-equilibrated (50 mM acetic acid at pH 5.1) Hi-Trap ^TM QHP anion exchange column (GEHealthcare). Stabilized 2-stranded (scFvαCD20)-Fc-MBM-1 X 2 DOTA bundles were eluted using a step elution of 300 mM NaCl over 18 minutes and a linear gradient from 300 mM to 1000 mM NaCl at a flow rate of 1.0 ml/min over 80 minutes .

A QHP anion exchange column was used to separate stabilized 2-stranded (scFvαCD20)-Fc-MBM-1 X 2 DOTA linkages from free 2-stranded fusion protein, 2-stranded fusion protein conjugated to three or four DOTA linkages, and aggregated materials.

In FIG. Figure 22 shows the results of SDS-PAGE analysis of fractions collected from a QHP anion exchange column. Lanes A, B, and C, respectively, correspond to an unconjugated molecular construct, a molecular construct conjugated to DOTA tethers, and a stabilized molecular construct conjugated to two DOTA tethers before loading onto the column.

The pure product, stabilized 2-strand(scFvαCD20)-Fc-MBM-1 X 2 DOTA linkages, was collected from fractions #40 and #41 shown in Figure 22.

Example 21

MALDI-TOF analysis of stabilized 2-stranded (scFv α CD19)-Fc-MBM-1 X 2 DOTA bundles

The stabilized 2-strand (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages from Example 19 were analyzed using MALDI-TOF mass spectroscopy. The MALDI-TOF result in Figure 23 shows that the sample of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles has a molecular weight of 114.435 and 57.306 Daltons, respectively, corresponding to m/z (z=1): [M +H] ⁺ andm/z (z=2): [M+2H] ²⁺ .

Example 22

Preparation of ⁸⁹ Y-labeled 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles

In this example, a solution of Y(NO ₃ ) ₃ was added to a solution of purified stabilized 2-chain(scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages from Example 19 in a molar ratio of 6:1 [Y ³⁺ ion:protein]; the reaction mixture was incubated for 6 hours at room temperature. Free Y ³⁺ ions were removed from the solution using a NAP-10 SephadexG-25 column. Stabilized 2-strand(scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages chelated with [ ⁸⁹ Y] ³⁺ were analyzed by MALDI-TOF mass spectroscopy. The MALDI-TOF result in Figure 24 shows that the present molecular construct has a molecular mass of 114.391 daltons.

Example 23

Preparation of ¹⁷⁵ Lu labeled 2-stranded (scFv α CD19)-Fc-MBM-1 X 2 DOTA bundles

In this example, a solution of LuCl ₃ was added to a solution of purified stabilized 2-chain (scFvαCD19)-Fc-MBM-1×2 DOTA linkages at a molar ratio of 60:1 [Lu ³⁺ ion:protein]; the reaction mixture was then incubated at 45°C for 1.5 hours to obtain the ¹⁷⁵ Lu-DOTA protein chelate. Free Lu ³⁺ ions were removed from the solution by centrifugal filtration (Amiconcentrifugalfilter, 10 kDa). Stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles chelated with two ¹⁷⁵ Lu were analyzed by MALDI-TOF mass spectroscopy. The MALDI-TOF result in Figure 25 shows that the present molecular construct has a molecular mass of 114.928 daltons.

Example 24

Preparation of ¹¹¹ ln-labeled 2-stranded (scFv α CD19)-Fc-MBM-1 X 2 DOTA bundles

To prepare ¹¹¹ In labeled fusion protein, an aliquot of carrier-free ¹¹¹ In in 50 mM HCl was transferred to a tube and twenty volumes of metal-free 0.1 M HEPES buffer were added to adjust the pH of the solution to 4.5. A solution of 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages (30-50 μg) in 0.1 M HEPES buffer (pH 4.5) was then 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 minutes. Diethylene triamine pentaacetic acid (DTPA) was added in an amount 1000 times the amount of protein to capture free ¹¹¹ In ³⁺ ions and quench the reaction. After incubation at 37°C for 30 minutes, ^{the 111} In-DTPA chelates were removed from ^{the 111} In labeled product and the solvent was replaced with 0.9% saline by centrifugal filtration (Amiconcentrifugalfilter, 10 kDa).

The specific activities of the collected ¹¹¹ In-labeled product were determined by measuring the radioactivity of a corresponding aliquot of the sample using a γ-counter.

Example 25

Radio-incorporation analysis ^{of 111} In labeled 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles

Radiochemical purity was assessed using flash thin layer chromatography (iTLC). A solution of the radiolabeled product from Example 24 was diluted 1:10 or 1:20 with the original solvent; 1 μL of sample was then applied to one end of a 1 × 10 cm strip of SG TLC paper. The paper was developed using ascending chromatography using 0.5 M sodium citrate (pH 4.5). Radioactivity was determined using a radio-TLC scanner. Protein-bound radioactivity was expressed as a percentage of total radioactivity. As shown in Figure 26, the radiochemical purity ^{of 111} In-labeled 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles exceeds 98%.

Example 26

Stability Test of ¹¹¹ In Labeled 2-Chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA Bundles in Serum

The in vitro stability of ¹¹¹ In labeled 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA binders was assessed in mouse serum at 37°C for 96 hours. ^{The 111} In labeled molecular construct from Example 24 was suspended in 0.2 M ammonia acetate (pH 5). ^{The 111} In labeled molecular construct was diluted 1:10 with mouse serum and then incubated at 37°C for up to 96 hours. At selected time points (0, 12, 24, 48 and 96 hours), ^{the 111} In-labeled molecule was removed and analyzed using radio-TLC as described in Example 25.

As shown in Figure 27, ¹¹¹ In-labeled 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles exhibit satisfactory stability when incubated in mouse serum at 37°C for 96 hours.

Example 27

Site-specific conjugation of recombinant 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles

To identify the cysteine residue in the 2-stranded (scFvαCD19)-Fc-MBM-1 that is conjugated to a DOTA linkage, the sample was digested and analyzed by LC-MS. Briefly, 5 μl of sample (0.4 μg/ml) from Example 11 was diluted in a solution containing 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 at room temperature for 30 minutes. After alkylation with IAA, the enzymes trypsin and Glu-C were added to the solution at 37°C overnight for enzymatic digestion.

The result of mass spectrometric analysis (data not shown) shows that the m/z value of the fragment in the MS spectrum corresponds to 4530.99 daltons, which corresponds to the molecular mass of the fragment containing the amino acid sequence SLSLSPGGGGACPGHA (amino acid residues 468-483 SEQIDNo. 13) of the molecular construct and one DOTA bundle.

Example 28

Binding activity of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles to human lymphoma B cells

Two stabilized molecular constructs, (scFvαCD19)-Fc-MBM-1 X 2 DOTA and (scFvαCD19)-Fc-MBM-3 X 2 DOTA bundles, were analyzed for their ability to bind to human CD19 on the Raji human B lymphoma cell line Assay was performed by incubating 1 x 10 ⁶ CD19 expressing Raji cells with 0.001 μg/ml of each construct in PBS, 1% BSA on ice for 30 minutes using anti-CD19 antibody (RB4v1.2), (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles and (scFvαCD19)-Fc-MBM-3 X 2 DOTA bundles as positive controls. Anti-HER2 antibody (trastuzumab) was used as a negative control. Cell staining was analyzed by FACS (FACSCantoII; BDBiosciences) using FITC-conjugated goat anti-human IgG.Fc antibody (diluted 1:200 in PBS/BSA) (Caltag, Buckingham, UK) at 4°C for 20 minutes in the dark . Figures 27A and 27B show the results of staining assays for two molecular constructs containing MBM-1 and MBM-3 on CD19-expressing Raji cells, respectively, which show that the two constructs bound essentially positively to Raji cells.

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

Example 29

Stability of Stabilized 2-Chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA Linkages

To assess the stability of stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages, molecular constructs were placed in Tris buffer (100 mM Tris buffer at pH 7.3, 50 mM Bis-Tris buffer and 290 mM NaCl) with glutathione or human serum albumin and then incubated at 37°C for 30 days.

As shown in FIG. 29, stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages exhibit satisfactory stability when incubated with glutathione or human serum albumin (HSA) at 37°C for 30 days. As can be seen in Figure 28, in both glutathione and HAS treated groups, approximately 90% of the intact molecular construct was still in solution without retro-Michael product attachment. Bands U and C, respectively, correspond to the unconjugated molecular construct and stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles stored at 4°C as controls.

Example 30

Study of the addition of a retro-Michael product stabilized by 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA binders.

As further confirmation of the stability of the 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages, the stabilized molecular construct was incubated with glutathione or HSA and then analyzed by Western blotting.

Briefly, for Western blot analysis of stabilized samples, samples were separated on 8% SDS-PAGE gels and transferred to a poly(vinylidene fluoride) (PVDF) membrane (Millipore). Membrane blots were blocked with PBS containing 5% BSA and 0.05% Tween 20 for 1 hour at room temperature. After washing three times with phosphate-buffered saline tween-20 (PBST) containing PBS and 0.05% Tween-20, the blots were incubated with goat anti-human IgG.Fc antibody conjugated to horseradish peroxidase (Millipore). The membranes were then washed 3 times with PBST and immunoreacted bands were detected using ECL™ Western Blot Detection Reagents (Millipore) and exposed to Fujifilm (Tokyo, Japan).

In FIG. 30 shows the result of stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages by Western blotting. As shown in Figure 29, stabilized 2-strand (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages show satisfactory stability when incubated with glutathione or HSA at 37°C for 30 days compared to the stabilized molecular construct, which is stored at 4°C (band C). Band U represents an unconjugated molecular construct as a control.

Example 31

Half-life of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA binders

Measurement of half-lives of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles was carried out in mice after intravenous administration. (scFvαCD19)-Fc-MBM-1 X 2 DOTA Bundles in the serum sample were observed using ELISA. 8–10 week old BALB/c mice were purchased from BioLasco, Taipei, Taiwan. The mice were divided into three mice in each group and approximately 200 μL of 3.33 μM protein were intravenously injected.

The pharmacokinetic profiles of the anti-CD19 antibody (RB4v1.2), 2 chained (scFvαCD19)-Fc-MBM-1 fusion protein, as shown in Figure 31, indicate that the half-lives of the anti-CD19 antibody (RB4v1.2), (scFvαCD19) -Fc-MBM-1 fusion protein from Example 3, the molecular construct (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles from Example 18, and the duration of the stabilized molecular construct (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles from Example 19 is approximately 129, 179.6, 153.6, and 193.5 hours, respectively. In conclusion, stabilization treatment results in prolongation of the half-life of the stabilized molecular construct.

Example 32

Targeting effect of stabilized 2-strand (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles on CD19-expressing xenograft tumor

In this example, an in vivo imaging system (IVIS) was used to investigate the effect of targeting stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles to CD19-expressing tumors in a mouse xenograft model. Prior to IVIS imaging, the Dylight 680 Antibody Labeling Kit (ThermoScientific) was used to conjugate the stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA linkages from Example 19 and the anti-CD19 antibody (RB4v1.2) according to the manufacturer's instructions .

NOD-SCID (NOD.CB17-Prkdc ^scid /JNarl) 8–10 weeks old were purchased from the Laboratory Animal Facility of Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. Mice were injected intraperitoneally with 1 x 10 ⁷ Raji B-cell lymphoma cells per mouse 2 weeks before treatment. The mice were divided into three mice in each group and were intravenously injected with approximately 200 μL of the 6.65 μM labeled molecule. At various time points, mice were anesthetized with isoflurane in _O2 and placed in the IVISSpectrumInvivo imaging system (PerkinElmer) in the supine position. Fluorescence images were acquired at ex/em=675/720 using LivingImageSoftwareV3.2. Images were acquired at the indicated time points using an IVISSpectrum thermal imager and analyzed using Living Image software.

Fluorescence images of NOD-SCID mice were recorded and analyzed at 0, 24, 48, and 72 hours after administration of DyLight 680 conjugated proteins. In FIG. 32 shows the distribution of fluorescently labeled molecules in a mouse model. NOD-SCID mice were intravenously injected with anti-CD19 antibody (RB4v1.2) (1 and 2) and stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA binders (3 and 4).

As shown in Figure 32, at 24, 48 and 72 hours after intravenous injection, the penetration of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles and anti-CD19 antibodies (RB4v1.2) into tumors located on the right side, significantly more than that observed with a normal organ on the left. In summary, the stabilized molecular construct demonstrates a satisfactory targeting effect on CD19-expressing tumors in a mouse xenograft model.

Example 33

Biodistribution of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles in CD19-expressing xenograft tumor.

To analyze the biodistribution of stabilized (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles in a mouse xenograft model, the procedure outlined in Example 32 was used, with some modifications.

Briefly, mice were injected subcutaneously with 1 x 10 ⁷ Raji B-cell lymphoma cells per mouse 2-3 weeks before treatment. The mice were divided into three mice in each group and were intravenously injected with approximately 200 μL of the 6.65 μM labeled molecule.

The biodistribution of NOD-SCID mice was analyzed by tissue ELISA 7 days after administration of stabilized 2-chain (scFvαCD19)-Fc-MBM-1 X 2 DOTA binders and anti-CD19 antibodies. Briefly, animals were sacrificed on day 7 post-injection. Blood was hemostasis from the heart, and tissue samples were taken from 11 organs. Tissue samples were homogenized using a homogenizer and further analyzed to measure the concentration of introduced molecules using ELISA.

In FIG. 33 shows the distribution of stabilized 2-stranded (scFvαCD19)-Fc-MBM-1 X 2 DOTA bundles (TE-1122) in a mouse model. The Y-axis of the biodistribution diagram represents the ratio of tissue to blood. The result also shows that the stabilized molecular construct can target CD19-expressing tumors in a mouse xenograft model.

Example 34

Synthesis of the Aib-GLP-1 agonist having a terminal cysteine residue

In this example, an ε-aminobutyric acid (Aib) substituted glucagon-like peptide-1 (GLP-1) agonist having a free cysteine at the C-terminus was prepared; the amino acid sequence of this molecular construct Aib-GLP-1-Cys is described in SEQIDNo. 37. Fig. 34 is a schematic diagram illustrating the structure of this molecular construct. Like semaglutide, the second amino acid residue is replaced with an Aib (or U) residue to increase resistance to degradation by dipeptidyl peptidase IV (DPP 4); however, in this molecular design, a flexible linker of 15 amino acid residues (amino acid residues 31 to 45 of SEQID No. 37) is introduced before the last glycine residue of semaglutide (i.e., amino acid residue 46 of SEQID No. 37), and is added to the C-terminus terminal cysteine residue.

The structure of Aib-GLP-1 agonist-Cys was developed by the present inventors, and the synthesis was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China).

The purified Aib-GLP-1 agonist-Cys sample was analyzed using analytical reverse phase HPLC on a SupelcoC18 column (250 mm × 4.6 mm; 5 μm) with a mobile phase of acetonitrile and 0.1% trifluoroacetic acid, linear gradient from 0% to 100% acetonitrile over 30 minutes at a flow rate of 1.0 ml/min and a column temperature of 25°C.

Identification of the Aib-GLP-1 agonist-Cys product was performed using ESI-MS mass spectrometry. The Aib-GLP-1 agonist-Cys product sample shows a strong molecular ion at 1,483.4, which corresponds to [M+H] ⁺ , indicating that the actual molecular weight of the actual Aib-GLP-1 agonist-Cys agonist is 1,482.4 daltons.

Example 35

Synthesis of a somatostatin analog having a terminal cysteine residue

In this example, a somatostatin analog having a free cysteine at the N-terminus was prepared; the amino acid sequence of this Cys-octreotide molecular construct is described in SEQIDNo. 38. Fig. 35 is a schematic diagram illustrating the structure of this molecular construct. This molecular construct has 14 amino acid residues, into which an N-terminal cysteine residue is introduced, followed by a flexible linker of 5 amino acid residues and an octreotide sequence. Like the parent octreotide, the seventh amino acid residue (Phe) and tenth amino acid residue (Trp) of the present Cys-octreotide molecular construct are in the D-form, a disulfide bridge is formed between the eighth and thirteenth cysteine residues, and the threonine residue at the C-terminus is in the threoninol form . In addition, the N-terminus of the Cys-octreotide molecular construct is modified with an acetyl group.

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

Identification of the Cys-octreotide product was performed using MALDI-TOF mass spectrometry. Mass spectrometry analyzes were performed at the Mass Core Facility at 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 result of MALDI-TOF mass spectrometry, which shows that the molecular construct of the present invention has a molecular weight of 1,493,587 daltons.

Example 36

Synthesis of PSMA ligand containing a cysteine residue

In this example, a PSMA ligand containing a free cysteine residue was prepared, the structure of this molecular construct is presented in Figure 37.

The structure of this Cys-PSMA ligand molecular construct was developed by the present inventors, and the synthesis was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China). The Cys-PSMA ligand product has a purity of greater than 95.0%.

Cys-PSMA ligand identification was performed using ESI-MS mass spectrometry. Figure 38 shows the ESI-MS mass spectrometry result, which shows that the present molecular construct has a strong molecular ion at 522.4, which corresponds to [M+H] ⁺ , indicating that the actual molecular weight of the Cys-PSMA ligand is 521.4 Da .

Example 37

Synthesis of calcitonin-MBM-1

In this example, a calcitonin molecular construct was prepared having a metal-binding motif at its C-terminus; the amino acid sequence of this calcitonin-MBM-1 molecular construct is described in SEQIDNo. 39. Fig. 39 is a schematic diagram illustrating the structure of this molecular construct. Like calcitonin, a disulfide bridge is formed between the first and seventh cysteine residues; however, in this molecular design, a flexible linker of 8 amino acid residues (amino acid residues 33 to 40 of SEQID No. 39) is introduced after the last proline residue of calcitonin (i.e., amino acid residue 32 of SEQID No. 39), and the MBM-1 motif ( ACPGHA, SEQIDN. 7) added to the C-terminus of the flexible linker.

The structure of the present molecular construct calcitonin-MBM-1 was developed by the present inventors, and the synthesis was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China).

Example 38

Synthesis of teriparatide-MBM-1

In this example, a teriparatide molecular construct was prepared having a metal-binding motif at its C-terminus; the amino acid sequence of this teriparatide-MBM-1 molecular construct is described in SEQIDNo. 40. Teriparatide is a form of parathyroid hormone (PTH) consisting of the first 34 amino acids, which is the active part of the hormone. It is used in the treatment of some forms of osteoporosis. In the teriparatide-MBM-1 molecular construct, a flexible linker of 15 amino acid residues (amino acid residues 35 to 49 of SEQIDNo. 40) is introduced after the last phenylalanine residue of teriparatide (i.e., amino acid residue 34SEQIDNo. 40), and the MBM-1 motif (ACPGHA , SEQID No. 7) is added to the C-terminus of the flexible linker.

The structure of this teriparatide-MBM-1 molecular construct was developed by the present inventors, and the synthesis was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China).

Example 39

Synthesis of leuprolide-MBM-3

In this example, a leuprolide molecular construct was prepared having a metal-binding motif at its C-terminus; the amino acid sequence of this leuprolide-MBM-3 molecular construct is described in SEQIDNo. 41. Leuprolide (also known as leuprorelin) is a gonadotropin-releasing hormone (GnRH) analogue that acts as a pituitary GnRH receptor agonist that is used in the treatment of prostate and breast cancer. Commercially available leuprolide is an oligopeptide containing nine amino acid residues. In the present leuprolide-MBM-3 molecular construct, a flexible linker of 8 amino acid residues (amino acid residues 10 to 17 of SEQID No. 41) is introduced after the last proline residue of calcitonin (i.e., amino acid residue 9 of SEQID No. 41), and the MBM-3 motif (GCGGHA, SEQIDNo. 6) is added to the C-terminus of the flexible linker.

The structure of this leuprolide-MBM-3 molecular construct was developed by the present inventors, and the synthesis was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China). Identification of the synthesized peptide was carried out using ESI-MS mass spectrometry. The ESI-MS mass spectrometry result shows that the actual molecular construct has a strong molecular ion at 1.091.97, which corresponds to [M+2H] ²⁺ , indicating that the actual molecular weight of the leuprolide-MBM-3 molecular construct is 2.181.35 daltons.

Example 40

Syntheses of lenalidomide bundles

In this example, the present inventors have developed two sets of drugs containing a maleimide-containing peptide with three lysine residues as the central core and three lenalidomide molecules conjugated to the central core. The present lenalidomide linkage was synthesized using a combination method in which standard Fmoc-based solid-phase synthesis was carried out to synthesize the central core, and then liquid-phase synthesis was carried out to conjugate binding arm-modified lenalidomide molecules to the side chain of lysine residues in the central core. Manufacturing was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China).

Fig. 40 illustrates the structure of a Mal-peptide 3-lenalidomide linkage where the central core has the sequence EGEGEAGGKGAGKGAGKG (SEQIDNo. 42) where the first amino acid residue is modified with a maleimidoethyl group. On the other hand, before conjugation 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 through the free end of the linking arm.

Identification of the Mal-peptide 3-lenalidomide bundle thus synthesized was carried out using ESI-MS mass spectrometry. Figure 41 shows the ESI-MS mass spectrometry result, which shows that the drug set has a strong molecular ion at 1.321.9, which corresponds to [M+3H] ³⁺ , indicating that the actual molecular weight of the drug set is 3.962.02 daltons.

Fig. 42 illustrates the structure of a Mal-peptide 4-lenalidomide linkage where the central core has the sequence EDEDEAGGKGAGKGAGKG (SEQIDNo. 43) where the first amino acid residue is modified with a maleimido-ethyl group. The lenalidomide molecule is modified with a linking arm as described above and is linked to the ε-amino group of a lysine residue in the central core through the free end of the linking arm.

Identification of the Mal-peptide 4-lenalidomide linkage synthesized in this way was carried out using ESI-MS mass spectrometry. The ESI-MS result shows that the actual drug set has a strong molecular ion at 1.379.2, which corresponds to [M+3H] ³⁺ , indicating that the actual molecular weight of the drug set is 4.135.15 daltons.

Example 41

Synthesis of Mal-fatty acid bundle

In this example, a molecular construct of the Mal-Peptide 5 core and one chain of stearoyl diacid and one chain of palmitoic acid was prepared.

Fig. 43 illustrates the structure of this Mal-peptide 5-fatty acid bundle, where the central core has the sequence maleimido-ethyl-EGEGE-X ₁ -KX ₂ -K-OMe (SEQIDNo. 44), where the first amino acid residue is modified with a maleimido-ethyl group, the last the lysine residue is modified with a methoxy group (-OMe), and both X ₁ and X ₂ are PEGylated amino acids with 4 EG repeats. One stearoyl diacid chain and one palmitoyl acid chain were respectively linked to the K residues of the core peptide center by forming an amide bond between the CO ₂ H group of the fatty acid and the amino group of the K residue. The structure of the present Mal-peptide 5-fatty acid bundle was developed by the present inventors, and the synthesis was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China). The related product Mal-peptide 5-fatty acid has a purity of greater than 97.0%.

Identification of the synthesized fatty acid bundle was performed using ESI-MS mass spectrometry. The ESI-MS result in Figure 44 shows that the present molecular construct has a strong molecular ion of 1.143.6, which corresponds to [M+2H] ²⁺ , indicating that the actual molecular weight of the Mal-peptide 5-fatty acid bundle is 2.285.66 Daltons .

Example 42

Synthesis of 3-DOTA branch linker unit

In this example, a 3-DOTA linker arm was prepared to carry three peptides. Fig. 45 illustrates the structure of this 3-arm linker. In particular, the linker unit contains a central core having the sequence acetyl-KGAGGKGAGGKG (SEQIDNo. 45, peptide core 6), where the first lysine residue is modified with an acetyl group. The linker unit also includes three peptide linking arms having the sequence maleimido-ethyl-EGEGEAGKGAG (SEQIDNO: 46), where the first glutamate residue is modified with a maleimido-ethyl group and the lysine residue is modified with a DOTA molecule.

The structure of the presented 3-DOTA linker branch was developed by the present inventors, and the synthesis was outsourced to ShanghaiWuXiAppTechCo., Ltd. (Shanghai, China). Identification of the 3-DOTA branch of the linker unit was performed using ESI-MS mass spectrometry. ESI-MS result in Fig. 46 shows that the present molecular construct has a strong molecular ion at 1.357.8, which corresponds to [M+4H] ⁴⁺ , indicating that the actual molecular weight of the 3-DOTA linker unit was 5.427.2 Daltons.

Example 43

Construction, expression and purification of recombinant MBM-1-IL-2

In this example, a fusion protein having the sequence described in SEQIDNo. 47 was constructed, expressed and purified using protocols similar to those outlined in the previous working examples. Specifically, the present molecular construct MBM-1-IL-2 has the MBM-1 motif followed by a short flexible linker SEQIDNo. 9 and IL-2 sequence.

Culture supernatants were collected and the expressed recombinant fusion proteins in the medium were purified using an anion exchange column. Before purification, the recombinant MBM-1-IL-2 fusion protein was adjusted to pH 8.5. The protein was then applied to pre-equilibrated (50 mM Bis-Tris buffer at pH 8.5) Q Sepharose anion exchange resin (GEHealthcare). The MBM-1-IL-2 fusion protein was eluted using a 3-step elution with 200 mM, 500 mM, and 1 M NaCl in 4 M urea at pH 8.5, respectively.

Eluted samples were analyzed using 10% SDS-PAGE shown in FIG. 47. The MBM-1-IL-2 fusion protein was detected as a major band at approximately 16 kDa, which is the expected size (indicated by the arrow).

Example 44

Synthesis of 2-chain (scFv α CD38)-Fc-MBM-1 X 2 lenalidomide bundles

In this example, the (scFvαCD38)-Fc-MBM-1 fusion protein from Example 10 and the Mal-peptide 4-lenalidomide linkage from Example 40 were conjugated to produce a molecular construct having a 2-stranded (scFvα CD38 fusion protein)-Fc-MBM- 1 and 2 lenalidomide linkages conjugated to the corresponding cysteine residue of the metal-binding motifs of the fusion protein. Briefly, purified fusion protein (scFvαCD38)-Fc-MBM-1 was prepared in sodium succinate buffer (30 mM sodium succinate, pH 5.3, 0.02% tween 20 and 100 mM sucrose) and reconstituted 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. Reduced protein samples were then treated with 15 μM Mal-peptide coupling 4-lenalidomide and then incubated for 1 hour at room temperature. Unreacted lenalidomide binders were removed using a desalting column and the product was analyzed by SDS-PAGE.

In FIG. 48 shows the result of analysis of the molecular construct of the present invention by SDS-PAGE. As shown in Figure 48, this molecular construct has a molecular mass of approximately 120 kDa (indicated by arrow #1 in lane 2), which is slightly larger than the expected size. The unconjugated molecular construct was in lane 3 (indicated by arrow #2). As shown in Figure 48, the conjugation yield of the 2-strand(scFv α CD38)-Fc-MBM-1 linkage to lenalidomide is approximately 85%.

Example 45

Synthesis of the fatty acid octreotide X bundle

In this example, the Mal-peptide 5-fatty acid linkage from Example 41 and the cis-octreotide from Example 35 were conjugated using protocols similar to those outlined above, such that the maleimide group of the Mal-peptide 5-fatty acid linkage is conjugated to the -SH group of the terminal cysteine Cys-octreotide residue, resulting in the formation of the molecular structure of the octreotide X fatty acid bundle (see Fig. 49).

Example 46

Synthesis of the Aib-GLP-1 agonist X fatty acid bundle

In this example, the Mal-peptide 5-fatty acid linkage from Example 41 and the Aib-GLP-1 agonist -Cys from Example 34 were conjugated using protocols similar to those outlined above, such that the maleimido group of the Mal-Peptide 5-Fatty acid linkage is conjugated to - SH group of the terminal cysteine residue of the Aib-GLP-1 agonist Cys, thereby forming the molecular construct of the Aib-GLP-1 agonist X fatty acid bundle (see Figure 50A).

Example 47

Synthesis of teriparatide-MBM-1 X fatty acid bundle

In this example, the Mal-peptide 5-fatty acid linkage from Example 41 and teriparatide-MBM-1 from Example 38 were conjugated using protocols similar to those outlined above to produce the teriparatide-MBM-1 X fatty acid linkage molecular construct (see Figure 50B).

Example 48

Synthesis of leuprolide-MBM-3 X fatty acid bundle

In this example, the Mal-peptide 5-fatty acid linkage from Example 41 and leuprolide-MBM-3 from Example 39 were conjugated using protocols similar to those outlined above to produce the leuprolide-MBM-3 X fatty acid linkage molecular construct (see Figure 51A). Identification of the synthesized leuprolide-MBM-3 X fatty acid coupling was carried out using MALDI-TOF mass spectrometry, and the result in Figure 51B shows that this molecular construct has a molecular weight of 4,466.019 daltons. The molecular weight of 2,181.038 daltons indicates an excess of the leuprolide-MBM-3 molecule at a molar ratio of 1.5:1 [leuprolide-MBM-3: Mal-peptide-5-fatty acid] in this reaction.

Example 49

Synthesis of 3-DOTA branch linker unit X 3 PSMA ligands

In this example, the 3-DOTA linker arm from Example 42 and the three Cys-PSMA ligands from Example 36 were conjugated using protocols similar to those outlined above to generate the molecular construct of the 3-DOTA linker arm X 3 PSMA ligands (Figure 52A). Identification of the synthesized 3-DOTA arms of the X 3 PSMA linker ligands was carried out using ESI-MS mass spectrometry, and the result in Figure 52B shows that this molecular construct has a strong molecular ion at 1.749, corresponding to [M+4H] ⁴⁺ , indicating that the actual molecular weight of the 3-DOTA branch of the linker unit X 3 PSMA ligands is 6,992.18 daltons.

Example 50

Purification of Stabilized 2-Chain (scFvαCA19-9)-Fc-MBM-1 X 2 DOTA Linkages

The stabilization procedure for recombinant 2-stranded (scFvαCA19-9)-Fc-MBM-1 X 2 DOTA linkages was performed as described in the previous examples.

The 2-strand fusion protein conjugated to two DOTA linkages from the previous examples 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) Q Sepharose anion exchange column ( GEHealthcare). The sample was then eluted using a 3-step elution; a first elution of 320 mM NaCl for 70 minutes, then a second elution of 330 mM for 100 minutes, and then a final elution of 1000 mM NaCl for 50 minutes at a flow rate of 1.0 ml/min.

The 2-stranded (scFvαCA19-9)-Fc-MBM-1 X 2 DOTA bundles were separated from the free 2-stranded (scFvαCA19-9)-Fc-MBM-1 fusion protein, the 2-stranded (scFvαCA19-9)-Fc- MBM-1 conjugated to three or four DOTA linkages and aggregated material using a Q Sepharose anion exchange column.

The purified product, 2-stranded (scFvαCA19-9)-Fc-MBM-1 X 2 DOTA linkages, was collected. Lane 1 and lane 2, shown in Figure 53, respectively, correspond to the unconjugated molecular construct and the molecular construct conjugated to two DOTA linkages.

Example 51

Purification of Stabilized 2-Chain (scFvαCD38)-Fc-MBM-1 X 2 Lenalidomide Linkages

The 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide linkage molecular construct from Example 44 was stabilized using protocols similar to those outlined in the previous examples.

The stabilized molecular construct of 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide linkages was brought to pH 7.0 and then applied to a pre-equilibrated (50 mM Na ₂ HPO ₄ at pH 7.0, 1 M NaCl) hydrophobic interaction column (HIC) PhenylHP (GEHealthcare). The stabilized molecular construct was eluted using a linear gradient from 1000 mM to 0 mM NaCl at a flow rate of 1.0 ml/min over 80 minutes.

Collected samples from the previous HIC purification were applied to an ENrich™SEC650 size exclusion chromatography column (Bio-Rad) to separate the stabilized molecular construct from the aggregated material. The purified product was collected. Lane 1 and lane 2 of the 10% non-reducing SDS-PAGE shown in Figure 54, respectively, correspond to the unconjugated molecular construct and the molecular construct conjugated to two lenalidomide linkages.

The purified stabilized molecular construct of 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide linkages was further analyzed using MALDI-TOF mass spectroscopy. The result of the mass spectrometric analysis in the bottom diagram of Figure 55 shows that the sample has a molecular weight of 58.382 and 116.705 daltons, which corresponds to m/z (z=1): [M+H] ⁺ and m/z (z=2): [ M+2H] ²⁺ , respectively.

The result of mass spectrometric analysis of 2-chain (scFvαCD38)-Fc-MBM-1 as a control shown in the top diagram of Figure 55 shows that the sample has a molecular weight of 54.300 and 108.537 daltons, which corresponds to m/z (z=1 ): [M+H] ⁺ and m/z (z=2): [M+2H] ²⁺ , respectively.

Example 52

Half-life of stabilized 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles in NOD-SCID mice

Measurement of the half-lives of stabilized 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles was carried out in mice after intravenous administration. The molecular construct in the serum sample was observed using the ELISA method. NOD-SCID mice, 8–10 weeks old, were purchased from BioLasco, Taipei, Taiwan. The mice were divided into three mice in each group and given an intravenous bolus of 100 μL of 7.6 μM molecules.

The results show that the half-lives of the parental anti-CD38 hIgG1.Fc antibody (Figure 56A) and the 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles (Figure 56B) are approximately 13.1 and 23. 9 hours respectively. Results using a non-compartmental pharmacokinetic model indicate that the present 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles have a longer half-life than the conventional anti-CD38 antibody.

Example 53

Cytotoxic activity of purified 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles towards H929 and CD38-positive U266 cells

H929 cells (5×10 ³ /well) were added to the wells of 96-well plates in RPMI1640 medium containing 10% fecal bovine serum. After 2 hours, cells were treated with various concentrations (2-fold dilutions from 20 μM) of purified 2-chain (scFvαCD38)-Fc-MBM-1 (without lenalidomide linkage) and 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide ligaments After incubation for 2 hours, the culture medium was removed by centrifugation at 400 g for 5 minutes and replaced with fresh medium, and 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.

Protocols for determining the cytotoxic activity of purified 2-chain (scFvαCD38)-Fc-MBM-1 with or without two lenalidomide linkages on CD38-positive U266 were similar to those described above for H929 cells.

In FIG. 57A shows the viability of H929 cells in four treatment groups. 2-stranded (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles (designated “scFv-Fc-len”) caused approximately 80% cytolysis of H929 cells at 20 μM after incubation for 5 days. 2-stranded (scFvαCD38)-Fc-MBM-1 (designated “scFv-Fc”), which was used as a negative control, showed no cytotoxic effect.

In FIG. 57B shows the viability of CD38-positive U266 cells in four treatment groups. 2-stranded (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles caused approximately 80% cytolysis of CD38-positive U266 cells at 20 μM after incubation for 5 days.

In FIG. 57C shows the viability of CD38-negative U266 cells in the four treatment groups. 2-stranded (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles (designated “scFv-Fc-len”) failed to induce any cytolysis of CD38-negative U266 cells at each molar concentration.

Overall, the results in Figures 57A-57C showed that the 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles have a strong targeting effect.

Example 54

Antibody-dependent cell-mediated cytotoxicity (ADCC) of purified 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles on CD38-expressing H929 and U266 cells.

In this example, an in vitro assay was performed to examine the antibody-dependent cell-mediated cytotoxicity (ADCC) of 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles on the lysis of CD38-expressing H929 and CD38-positive U266 cells.

Human peripheral blood mononuclear cells (PBMCs) were isolated from donors to study ADCC function. H929 cells or CD38-positive U266 cells were incubated with 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles at a final concentration of 200, 40, 8, 1.6, 0.32 or 0.064 nM, and then mixed with human PBMC at the ratio E:T equal to 25 and incubated at 37°C for 5 hours. Parental anti-CD38mAb was used as a positive control. Cytolysis was analyzed using the LDH Cytotoxicity Assay Kit (EnzoLifeSciences).

Data presented here show that purified 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles (designated “scFv-Fc-len”) recruited effector cells in a manner similar to its parental anti-CD38 mAb to exhibit cytolytic activity (Fig. 58A). Parental anti-CD38 antibody was used as a positive control (designated “IgG”), and an isotype control antibody (designated “IgG control”) was also used.

In FIG. 58B shows that purified 2-chain (scFvαCD38)-Fc-MBM-1 X 2 lenalidomide bundles (designated “scFv-Fc-len”) also exhibit cytolytic activity on CD38-positive U266 cells similar to the parent counterpart.

It should be understood that the above description of embodiments of the invention is given by way of example only and that various modifications may be made by those skilled in the art. The above description, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of specificity or with reference to one or more individual embodiments, those skilled in the art can make numerous changes to the disclosed embodiments without departing from the spirit or scope of this invention.

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LIST OF SEQUENCES

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Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr

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Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

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Pro GlyThr Ser Pro ArgArgTrp Ile Tyr Asp Thr Ser Lys Leu Ala

180 185 190

Ser Gly Val Pro Ala ArgPhe Ser Gly Ser Gly Ser GlyThr Asp Tyr

195 200 205

Ser Leu Thr Ile Ser Ser Met Glu Pro Glu Asp Ala AlaThr Tyr Tyr

210 215 220

Cys His GlnArgGly Ser Tyr ThrPheGlyGlyGlyThr Lys Leu Glu

225 230 235 240

Ile Lys ArgGlyGlyGly Ala Ser Asp Lys Thr His ThrCys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr

275 280 285

Cys Val ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

370 375 380

Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu Val Lys GlyPhe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGlyGln Pro Glu

405 410 415

AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrpGlnGlnGly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGlyGly Ala Cys Pro

465 470 475 480

GlyHisAla

<210> 15

<211> 483

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 15

Glu Ile Val Leu ThrGln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met ThrCys Ser Ala Ser SerGly Val Asn Tyr Met

20 25 30

His Trp Tyr GlnGln Lys Pro GlyThr Ser Pro ArgArgTrp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala ArgPhe Ser Gly Ser

50 55 60

Gly Ser GlyThr Asp Tyr Ser Leu Thr Ile Ser Ser Met Glu Pro Glu

65 70 75 80

Asp Ala AlaThr Tyr TyrCys His GlnArgGly Ser Tyr ThrPheGly

85 90 95

GlyGlyThr Lys Leu Glu Ile Lys ArgGly Ser Thr Ser Gly Ser Gly

100 105 110

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys GlyGln Val Gln Leu Val

115 120 125

Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Leu Ser

130 135 140

Cys Lys Thr Ser Gly Tyr ThrPheThr Ser AsnTrp Met His Trp Val

145 150 155 160

Lys Gln Ala Pro GlyGlnGly Leu Glu Trp Ile Gly Glu Ile Asp Pro

165 170 175

Ser Asp Ser Tyr ThrAsn Tyr AsnGlnAsnPheGlnGly Lys Ala Lys

180 185 190

Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Val Ser Ser

195 200 205

Leu Arg Ser Asp AspThr Ala Val Tyr TyrCys Ala ArgGly Ser Asn

210 215 220

Pro Tyr TyrTyr Ala Met Asp Tyr TrpGlyGlnGlyThr Ser Val Thr

225 230 235 240

Val Ser SerGlyGlyGly Ala Ser Asp Lys Thr His ThrCys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr

275 280 285

Cys Val ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

370 375 380

Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu Val Lys GlyPhe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGlyGln Pro Glu

405 410 415

AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrpGlnGlnGly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGlyGlyGlyCys Pro

465 470 475 480

GlyHisAla

<210> 16

<211> 483

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 16

Glu Ile Val Leu ThrGln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met ThrCys Ser Ala Ser SerGly Val Asn Tyr Met

20 25 30

His Trp Tyr GlnGln Lys Pro GlyThr Ser Pro ArgArgTrp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala ArgPhe Ser Gly Ser

50 55 60

Gly Ser GlyThr Asp Tyr Ser Leu Thr Ile Ser Ser Met Glu Pro Glu

65 70 75 80

Asp Ala AlaThr Tyr TyrCys His GlnArgGly Ser Tyr ThrPheGly

85 90 95

GlyGlyThr Lys Leu Glu Ile Lys ArgGly Ser Thr Ser Gly Ser Gly

100 105 110

Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys GlyGln Val Gln Leu Val

115 120 125

Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Leu Ser

130 135 140

Cys Lys Thr Ser Gly Tyr ThrPheThr Ser AsnTrp Met His Trp Val

145 150 155 160

Lys Gln Ala Pro GlyGlnGly Leu Glu Trp Ile Gly Glu Ile Asp Pro

165 170 175

Ser Asp Ser Tyr ThrAsn Tyr AsnGlnAsnPheGlnGly Lys Ala Lys

180 185 190

Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Val Ser Ser

195 200 205

Leu Arg Ser Asp AspThr Ala Val Tyr TyrCys Ala ArgGly Ser Asn

210 215 220

Pro Tyr TyrTyr Ala Met Asp Tyr TrpGlyGlnGlyThr Ser Val Thr

225 230 235 240

Val Ser SerGlyGlyGly Ala Ser Asp Lys Thr His ThrCys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr

275 280 285

Cys Val ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

370 375 380

Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu Val Lys GlyPhe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGlyGln Pro Glu

405 410 415

AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrpGlnGlnGly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGlyGlyGlyCysGly

465 470 475 480

GlyHisAla

<210> 17

<211> 458

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 17

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr ThrPheThr Ser Asn

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro GlyGlnGly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Tyr ThrAsn Tyr AsnGlnAsnPhe

50 55 60

GlnGly Lys Ala Lys Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp AspThr Ala Val Tyr TyrCys

85 90 95

Ala ArgGly Ser Asn Pro Tyr TyrTyr Ala Met Asp Tyr TrpGlyGln

100 105 110

GlyThr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser GlyGlyThr Ala Ala

130 135 140

Leu GlyCys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

TrpAsn Ser Gly Ala Leu Thr Ser Gly Val His ThrPhe Pro Ala Val

165 170 175

Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val ValThr Val Pro

180 185 190

Ser SerSer Leu GlyThrGlnThr Tyr Ile CysAsn Val Asn His Lys

195 200 205

Pro Ser AsnThr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser ArgThr Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys

305 310 315 320

Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu

355 360 365

ThrCys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser AsnGlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser ArgTrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro

435 440 445

GlyGlyGlyGly Ala Cys Pro Gly His Ala

450 455

<210> 18

<211> 458

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 18

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr ThrPheThr Ser Asn

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro GlyGlnGly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Tyr ThrAsn Tyr AsnGlnAsnPhe

50 55 60

GlnGly Lys Ala Lys Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp AspThr Ala Val Tyr TyrCys

85 90 95

Ala ArgGly Ser Asn Pro Tyr TyrTyr Ala Met Asp Tyr TrpGlyGln

100 105 110

GlyThr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser GlyGlyThr Ala Ala

130 135 140

Leu GlyCys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

TrpAsn Ser Gly Ala Leu Thr Ser Gly Val His ThrPhe Pro Ala Val

165 170 175

Leu Gln Ser SerGly Leu Tyr Ser Leu Ser Ser Val ValThr Val Pro

180 185 190

Ser SerSer Leu GlyThrGlnThr Tyr Ile CysAsn Val Asn His Lys

195 200 205

Pro Ser AsnThr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser ArgThr Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys

305 310 315 320

Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu

355 360 365

ThrCys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser AsnGlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser ArgTrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro

435 440 445

GlyGlyGlyGlyGlyCys Pro Gly His Ala

450 455

<210> 19

<211> 211

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 19

Glu Ile Val Leu ThrGln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met ThrCys Ser Ala Ser SerGly Val Asn Tyr Met

20 25 30

His Trp Tyr GlnGln Lys Pro GlyThr Ser Pro ArgArgTrp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala ArgPhe Ser Gly Ser

50 55 60

Gly Ser GlyThr Asp Tyr Ser Leu Thr Ile Ser Ser Met Glu Pro Glu

65 70 75 80

Asp Ala AlaThr Tyr TyrCys His GlnArgGly Ser Tyr ThrPheGly

85 90 95

GlyGlyThr Lys Leu Glu Ile Lys ArgThr Val Ala Ala Pro Ser Val

100 105 110

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser GlyThr Ala Ser

115 120 125

Val ValCys Leu LeuAsnAsnPhe Tyr Pro Arg Glu Ala Lys Val Gln

130 135 140

Trp Lys Val Asp Asn Ala Leu Gln Ser GlyAsn Ser Gln Glu Ser Val

145 150 155 160

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser SerThr Leu

165 170 175

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

180 185 190

Val Thr His GlnGly Leu Ser Ser Pro Val Thr Lys Ser PheAsnArg

195 200 205

GlyGluCys

210

<210> 20

<211> 489

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 20

Glu Ile Val Leu ThrGln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met Ser Cys Lys Ser SerGln Ser Val PhePhe Ser

20 25 30

Ser SerGln Lys Asn Tyr Leu Ala Trp Tyr GlnGln Ile Pro GlyGln

35 40 45

Ser Pro Arg Leu LeuThr Tyr Trp Ala Ser ThrArg Glu Ser Gly Val

50 55 60

Pro Asp ArgPheThrGly Ser Gly Ser GlyThr Asp PheThr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr TyrCys His Gln

85 90 95

Tyr Leu Ser SerArgThrPheGlyGlnGlyThr Lys Leu Glu Ile Lys

100 105 110

ArgGly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

115 120 125

Thr Lys GlyGln Val Gln Leu GlnGln Pro Gly Ala Glu Val Val Lys

130 135 140

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr ThrPhe

145 150 155 160

Thr Ser Tyr Tyr Ile His Trp Ile Lys GlnThr Pro GlyGlnGly Leu

165 170 175

Glu Trp Val Gly Val Ile Tyr Pro GlyAsn Asp Asp Ile Ser Tyr Asn

180 185 190

Gln Lys PheGlnGly Lys Ala Thr Leu Thr Ala Asp Lys Ser SerThr

195 200 205

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

210 215 220

Tyr TyrCys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val TrpGly

225 230 235 240

GlnGlyThrThr Val Thr Val Ser SerGlyGlyGly Ala Ser Asp Lys

245 250 255

Thr His ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

ArgThr Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn

305 310 315 320

Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr

370 375 380

Leu Pro Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu Thr

385 390 395 400

Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

405 410 415

Ser AsnGlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser ArgTrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

GlyGlyGly Ala Cys Pro Gly His Ala

485

<210> 21

<211> 489

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 21

Gln Val Gln Leu GlnGln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr ThrPheThr Ser Tyr

20 25 30

Tyr Ile His Trp Ile Lys GlnThr Pro GlyGlnGly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro GlyAsn Asp Asp Ile Ser Tyr AsnGln Lys Phe

50 55 60

GlnGly Lys Ala Thr Leu Thr Ala Asp Lys Ser SerThrThr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr TyrCys

85 90 95

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val TrpGlyGlnGlyThr

100 105 110

Thr Val Thr Val Ser SerGly Ser Thr Ser Gly Ser Gly Lys Pro Gly

115 120 125

Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val Leu ThrGln Ser Pro

130 135 140

Gly Ser Leu Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys

145 150 155 160

Ser SerGln Ser Val PhePhe Ser SerSerGln Lys Asn Tyr Leu Ala

165 170 175

Trp Tyr GlnGln Ile Pro GlyGln Ser Pro Arg Leu LeuThr Tyr Trp

180 185 190

Ala Ser ThrArg Glu Ser Gly Val Pro Asp ArgPheThrGly Ser Gly

195 200 205

Ser GlyThr Asp PheThr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp

210 215 220

Leu Ala Ile Tyr TyrCys His Gln Tyr Leu Ser SerArgThrPheGly

225 230 235 240

GlnGlyThr Lys Leu Glu Ile Lys ArgGlyGlyGly Ala Ser Asp Lys

245 250 255

Thr His ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

ArgThr Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn

305 310 315 320

Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr

370 375 380

Leu Pro Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu Thr

385 390 395 400

Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

405 410 415

Ser AsnGlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser ArgTrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

GlyGlyGly Ala Cys Pro Gly His Ala

485

<210> 22

<211> 489

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 22

Glu Ile Val Leu ThrGln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met Ser Cys Lys Ser SerGln Ser Val PhePhe Ser

20 25 30

Ser SerGln Lys Asn Tyr Leu Ala Trp Tyr GlnGln Ile Pro GlyGln

35 40 45

Ser Pro Arg Leu LeuThr Tyr Trp Ala Ser ThrArg Glu Ser Gly Val

50 55 60

Pro Asp ArgPheThrGly Ser Gly Ser GlyThr Asp PheThr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr TyrCys His Gln

85 90 95

Tyr Leu Ser SerArgThrPheGlyGlnGlyThr Lys Leu Glu Ile Lys

100 105 110

ArgGly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

115 120 125

Thr Lys GlyGln Val Gln Leu GlnGln Pro Gly Ala Glu Val Val Lys

130 135 140

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr ThrPhe

145 150 155 160

Thr Ser Tyr Tyr Ile His Trp Ile Lys GlnThr Pro GlyGlnGly Leu

165 170 175

Glu Trp Val Gly Val Ile Tyr Pro GlyAsn Asp Asp Ile Ser Tyr Asn

180 185 190

Gln Lys PheGlnGly Lys Ala Thr Leu Thr Ala Asp Lys Ser SerThr

195 200 205

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

210 215 220

Tyr TyrCys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val TrpGly

225 230 235 240

GlnGlyThrThr Val Thr Val Ser SerGlyGlyGly Ala Ser Asp Lys

245 250 255

Thr His ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

ArgThr Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn

305 310 315 320

Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr

370 375 380

Leu Pro Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu Thr

385 390 395 400

Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

405 410 415

Ser AsnGlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser ArgTrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

GlyGlyGlyGlyCys Pro Gly His Ala

485

<210> 23

<211> 489

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 23

Gln Val Gln Leu GlnGln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr ThrPheThr Ser Tyr

20 25 30

Tyr Ile His Trp Ile Lys GlnThr Pro GlyGlnGly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro GlyAsn Asp Asp Ile Ser Tyr AsnGln Lys Phe

50 55 60

GlnGly Lys Ala Thr Leu Thr Ala Asp Lys Ser SerThrThr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr TyrCys

85 90 95

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val TrpGlyGlnGlyThr

100 105 110

Thr Val Thr Val Ser SerGly Ser Thr Ser Gly Ser Gly Lys Pro Gly

115 120 125

Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val Leu ThrGln Ser Pro

130 135 140

Gly Ser Leu Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys

145 150 155 160

Ser SerGln Ser Val PhePhe Ser SerSerGln Lys Asn Tyr Leu Ala

165 170 175

Trp Tyr GlnGln Ile Pro GlyGln Ser Pro Arg Leu LeuThr Tyr Trp

180 185 190

Ala Ser ThrArg Glu Ser Gly Val Pro Asp ArgPheThrGly Ser Gly

195 200 205

Ser GlyThr Asp PheThr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp

210 215 220

Leu Ala Ile Tyr TyrCys His Gln Tyr Leu Ser SerArgThrPheGly

225 230 235 240

GlnGlyThr Lys Leu Glu Ile Lys ArgGlyGlyGly Ala Ser Asp Lys

245 250 255

Thr His ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

ArgThr Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn

305 310 315 320

Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr

370 375 380

Leu Pro Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu Thr

385 390 395 400

Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

405 410 415

Ser AsnGlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser ArgTrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

GlyGlyGlyGlyCys Pro Gly His Ala

485

<210> 24

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 24

Asp Ile Gln Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile ThrCysArg Ala Ser SerSer Val Ser Tyr Met

20 25 30

His Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser ArgPhe Ser Gly Ser

50 55 60

Gly Ser GlyThr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr TyrCysGlnGlnTrp Ser PheAsn Pro ProThr

85 90 95

PheGlyGlnGlyThr Lys Val Glu Ile Lys ArgGly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln

115 120 125

Leu Val Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Tyr ThrPheThr Ser Tyr Asn Met His

145 150 155 160

Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Ala Ile

165 170 175

Tyr Pro GlyAsnGly Asp Thr Ser Tyr AsnGln Lys Phe Lys GlyArg

180 185 190

PheThr Ile Ser Val Asp Lys Ser Lys AsnThr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys Ala Arg Val

210 215 220

Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val TrpGlyGlnGly

225 230 235 240

Thr Leu Val Thr Val Ser SerGlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

Gly Ala Cys Pro Gly His Ala

485

<210> 25

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 25

Glu Val Gln Leu Val Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr ThrPheThr Ser Tyr

20 25 30

Asn Met His Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro GlyAsnGly Asp Thr Ser Tyr AsnGln Lys Phe

50 55 60

Lys GlyArgPheThr Ile Ser Val Asp Lys Ser Lys AsnThr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

GlyGlnGlyThr Leu Val Thr Val Ser SerGly Ser Thr Ser Gly Ser

115 120 125

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met

130 135 140

ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile ThrCysArg Ala Ser SerSer Val Ser Tyr Met His Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr Ala Pro Ser Asn

180 185 190

Leu Ala Ser Gly Val Pro Ser ArgPhe Ser Gly Ser Gly Ser GlyThr

195 200 205

Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

210 215 220

Tyr TyrCysGlnGlnTrp Ser PheAsn Pro ProThrPheGlyGlnGly

225 230 235 240

Thr Lys Val Glu Ile Lys ArgGlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

Gly Ala Cys Pro Gly His Ala

485

<210> 26

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 26

Glu Val Gln Leu Val Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr ThrPheThr Ser Tyr

20 25 30

Asn Met His Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro GlyAsnGly Asp Thr Ser Tyr AsnGln Lys Phe

50 55 60

Lys GlyArgPheThr Ile Ser Val Asp Lys Ser Lys AsnThr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

GlyGlnGlyThr Leu Val Thr Val Ser SerGly Ser Thr Ser Gly Ser

115 120 125

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met

130 135 140

ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile ThrCysArg Ala Ser SerSer Val Ser Tyr Met His Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr Ala Pro Ser Asn

180 185 190

Leu Ala Ser Gly Val Pro Ser ArgPhe Ser Gly Ser Gly Ser GlyThr

195 200 205

Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

210 215 220

Tyr TyrCysGlnGlnTrp Ser PheAsn Pro ProThrPheGlyGlnGly

225 230 235 240

Thr Lys Val Glu Ile Lys ArgGlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

GlyGlyCys Pro Gly His Ala

485

<210> 27

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 27

Asp Ile Gln Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile ThrCysArg Ala Ser SerSer Val Ser Tyr Met

20 25 30

His Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser ArgPhe Ser Gly Ser

50 55 60

Gly Ser GlyThr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr TyrCysGlnGlnTrp Ser PheAsn Pro ProThr

85 90 95

PheGlyGlnGlyThr Lys Val Glu Ile Lys ArgGly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln

115 120 125

Leu Val Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Tyr ThrPheThr Ser Tyr Asn Met His

145 150 155 160

Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Ala Ile

165 170 175

Tyr Pro GlyAsnGly Asp Thr Ser Tyr AsnGln Lys Phe Lys GlyArg

180 185 190

PheThr Ile Ser Val Asp Lys Ser Lys AsnThr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys Ala Arg Val

210 215 220

Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val TrpGlyGlnGly

225 230 235 240

Thr Leu Val Thr Val Ser SerGlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

GlyGlyCysGlyGlyHisAla

485

<210> 28

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 28

Glu Val Gln Leu Val Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr ThrPheThr Ser Tyr

20 25 30

Asn Met His Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro GlyAsnGly Asp Thr Ser Tyr AsnGln Lys Phe

50 55 60

Lys GlyArgPheThr Ile Ser Val Asp Lys Ser Lys AsnThr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

GlyGlnGlyThr Leu Val Thr Val Ser SerGly Ser Thr Ser Gly Ser

115 120 125

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met

130 135 140

ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile ThrCysArg Ala Ser SerSer Val Ser Tyr Met His Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr Ala Pro Ser Asn

180 185 190

Leu Ala Ser Gly Val Pro Ser ArgPhe Ser Gly Ser Gly Ser GlyThr

195 200 205

Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

210 215 220

Tyr TyrCysGlnGlnTrp Ser PheAsn Pro ProThrPheGlyGlnGly

225 230 235 240

Thr Lys Val Glu Ile Lys ArgGlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

GlyGlyCysGlyGlyHisAla

485

<210> 29

<211> 108

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 29

Asp Ile Gln Met ThrGln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile ThrCys Lys Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser TrpPheGlnGln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala AsnArg Leu Val Asp Gly Val Pro Ser ArgPhe Ser Gly

50 55 60

Ser Gly Ser GlyGln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Gly Ile Tyr TyrCys Leu Gln Tyr Asp Glu Phe Pro Arg

85 90 95

ThrPheGlyGlyGlyThr Lys Leu Glu Ile Lys Arg

100 105

<210> 30

<211> 115

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 30

Glu Val Lys Leu GlnGln Ser GlyGlyGly Leu Val Gln Pro GlyGly

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser GlyPheThrPhe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val ArgGln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile GlyAsn Lys GlyAsnAsn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys GlyArgPheThr Val Ser Arg Asp Asp Ser Lys Ser Arg

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp ThrGlyThr Tyr

85 90 95

Tyr CysThrThrArgPhe Ala Tyr TrpGlyGlnGlyThrThr Val Thr

100 105 110

ValSerSer

115

<210> 31

<211> 481

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 31

Asp Ile Gln Met ThrGln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile ThrCys Lys Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser TrpPheGlnGln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala AsnArg Leu Val Asp Gly Val Pro Ser ArgPhe Ser Gly

50 55 60

Ser Gly Ser GlyGln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Gly Ile Tyr TyrCys Leu Gln Tyr Asp Glu Phe Pro Arg

85 90 95

ThrPheGlyGlyGlyThr Lys Leu Glu Ile Lys ArgGly Ser Thr Ser

100 105 110

Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val

115 120 125

Lys Leu GlnGln Ser GlyGlyGly Leu Val Gln Pro GlyGly Ser Met

130 135 140

Lys Leu Ser Cys Ala Ala Ser GlyPheThrPhe Ser Asp Ala Trp Met

145 150 155 160

Asp Trp Val ArgGln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu

165 170 175

Ile GlyAsn Lys GlyAsnAsn His Ala Thr Tyr Tyr Ala Glu Ser Val

180 185 190

Lys GlyArgPheThr Val Ser Arg Asp Asp Ser Lys Ser Arg Val Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Val Glu Asp ThrGlyThr Tyr TyrCys

210 215 220

ThrThrArgPhe Ala Tyr TrpGlyGlnGlyThrThr Val Thr Val Ser

225 230 235 240

Ser GlyGlyGly Ala Ser Asp Lys Thr His ThrCys Pro ProCys Pro

245 250 255

Ala Pro Glu Leu LeuGlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys

260 265 270

Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val ThrCys Val

275 280 285

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsnTrp Tyr

290 295 300

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

305 310 315 320

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

325 330 335

Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys

340 345 350

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln

355 360 365

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

370 375 380

ThrArgAsnGln Val Ser Leu ThrCys Leu Val Lys GlyPhe Tyr Pro

385 390 395 400

Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGlyGln Pro Glu AsnAsn

405 410 415

Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu

420 425 430

Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrpGlnGlnGlyAsn Val

435 440 445

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr ThrGln

450 455 460

Lys Ser Leu Ser Leu Ser Pro GlyGlyGlyGly Ala Cys Pro Gly His

465 470 475 480

Ala

<210> 32

<211> 481

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 32

Asp Ile Gln Met ThrGln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile ThrCys Lys Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

Leu Ser TrpPheGlnGln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala AsnArg Leu Val Asp Gly Val Pro Ser ArgPhe Ser Gly

50 55 60

Ser Gly Ser GlyGln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Gly Ile Tyr TyrCys Leu Gln Tyr Asp Glu Phe Pro Arg

85 90 95

ThrPheGlyGlyGlyThr Lys Leu Glu Ile Lys ArgGly Ser Thr Ser

100 105 110

Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val

115 120 125

Lys Leu GlnGln Ser GlyGlyGly Leu Val Gln Pro GlyGly Ser Met

130 135 140

Lys Leu Ser Cys Ala Ala Ser GlyPheThrPhe Ser Asp Ala Trp Met

145 150 155 160

Asp Trp Val ArgGln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu

165 170 175

Ile GlyAsn Lys GlyAsnAsn His Ala Thr Tyr Tyr Ala Glu Ser Val

180 185 190

Lys GlyArgPheThr Val Ser Arg Asp Asp Ser Lys Ser Arg Val Tyr

195 200 205

Leu Gln Met Asn Ser Leu Arg Val Glu Asp ThrGlyThr Tyr TyrCys

210 215 220

ThrThrArgPhe Ala Tyr TrpGlyGlnGlyThrThr Val Thr Val Ser

225 230 235 240

Ser GlyGlyGly Ala Ser Asp Lys Thr His ThrCys Pro ProCys Pro

245 250 255

Ala Pro Glu Leu LeuGlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys

260 265 270

Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val ThrCys Val

275 280 285

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsnTrp Tyr

290 295 300

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

305 310 315 320

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

325 330 335

Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys

340 345 350

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln

355 360 365

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

370 375 380

ThrArgAsnGln Val Ser Leu ThrCys Leu Val Lys GlyPhe Tyr Pro

385 390 395 400

Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGlyGln Pro Glu AsnAsn

405 410 415

Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu

420 425 430

Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrpGlnGlnGlyAsn Val

435 440 445

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr ThrGln

450 455 460

Lys Ser Leu Ser Leu Ser Pro GlyGlyGlyGlyGlyCysGlyGly His

465 470 475 480

Ala

<210> 33

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 33

Glu Ile Val Leu ThrGln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr GlnGln Lys Pro GlyGln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser AsnArg Ala ThrGly Ile Pro Ala ArgPhe Ser Gly

50 55 60

Ser Gly Ser GlyThr Asp PheThr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr TyrCysGlnGlnArg Ser AsnTrp Pro Pro

85 90 95

ThrPheGlyGlnGlyThr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln

115 120 125

Leu Leu Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Val Ser GlyPheThrPheAsn Ser Phe Ala Met Ser

145 150 155 160

Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile

165 170 175

Ser Gly Ser GlyGlyGlyThr Tyr Tyr Ala Asp Ser Val Lys GlyArg

180 185 190

PheThr Ile Ser Arg Asp Asn Ser Lys AsnThr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr PheCys Ala Lys Asp

210 215 220

Lys Ile Leu TrpPheGly Glu Pro Val Phe Asp Tyr TrpGlyGlnGly

225 230 235 240

Thr Leu Val Thr Val Ser SerGlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

Gly Ala Cys Pro Gly His Ala

485

<210> 34

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 34

Glu Val Gln Leu Leu Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser GlyPheThrPheAsn Ser Phe

20 25 30

Ala Met Ser Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser GlyGlyGlyThr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys GlyArgPheThr Ile Ser Arg Asp Asn Ser Lys AsnThr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr PheCys

85 90 95

Ala Lys Asp Lys Ile Leu TrpPheGly Glu Pro Val Phe Asp Tyr Trp

100 105 110

GlyGlnGlyThr Leu Val Thr Val Ser SerGly Ser Thr Ser Gly Ser

115 120 125

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val Leu

130 135 140

ThrGln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr

165 170 175

GlnGln Lys Pro GlyGln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser

180 185 190

AsnArg Ala ThrGly Ile Pro Ala ArgPhe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp PheThr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala

210 215 220

Val Tyr TyrCysGlnGlnArg Ser AsnTrp Pro ProThrPheGlyGln

225 230 235 240

GlyThr Lys Val Glu Ile Lys GlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

Gly Ala Cys Pro Gly His Ala

485

<210> 35

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 35

Glu Ile Val Leu ThrGln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr GlnGln Lys Pro GlyGln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser AsnArg Ala ThrGly Ile Pro Ala ArgPhe Ser Gly

50 55 60

Ser Gly Ser GlyThr Asp PheThr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr TyrCysGlnGlnArg Ser AsnTrp Pro Pro

85 90 95

ThrPheGlyGlnGlyThr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln

115 120 125

Leu Leu Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Val Ser GlyPheThrPheAsn Ser Phe Ala Met Ser

145 150 155 160

Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile

165 170 175

Ser Gly Ser GlyGlyGlyThr Tyr Tyr Ala Asp Ser Val Lys GlyArg

180 185 190

PheThr Ile Ser Arg Asp Asn Ser Lys AsnThr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr PheCys Ala Lys Asp

210 215 220

Lys Ile Leu TrpPheGly Glu Pro Val Phe Asp Tyr TrpGlyGlnGly

225 230 235 240

Thr Leu Val Thr Val Ser SerGlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

GlyGlyCys Pro Gly His Ala

485

<210> 36

<211> 487

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 36

Glu Val Gln Leu Leu Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser GlyPheThrPheAsn Ser Phe

20 25 30

Ala Met Ser Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser GlyGlyGlyThr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys GlyArgPheThr Ile Ser Arg Asp Asn Ser Lys AsnThr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr PheCys

85 90 95

Ala Lys Asp Lys Ile Leu TrpPheGly Glu Pro Val Phe Asp Tyr Trp

100 105 110

GlyGlnGlyThr Leu Val Thr Val Ser SerGly Ser Thr Ser Gly Ser

115 120 125

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val Leu

130 135 140

ThrGln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr

165 170 175

GlnGln Lys Pro GlyGln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser

180 185 190

AsnArg Ala ThrGly Ile Pro Ala ArgPhe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp PheThr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala

210 215 220

Val Tyr TyrCysGlnGlnArg Ser AsnTrp Pro ProThrPheGlyGln

225 230 235 240

GlyThr Lys Val Glu Ile Lys GlyGlyGly Ala Ser Asp Lys Thr His

245 250 255

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr

275 280 285

Pro Glu Val ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Asp Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu ThrArgAsnGln Val Ser Leu ThrCys Leu

385 390 395 400

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

GlyGln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

TrpGlnGlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro GlyGlyGly

465 470 475 480

GlyGlyCys Pro Gly His Ala

485

<210> 37

<211> 47

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>VARIANT

<222> 2

<223>Xaa is ε-aminobutyric acid

<400> 37

His Xaa Glu GlyThrPheThr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val ArgGlyArgGlyGly

20 25 30

GlyGly Ser GlyGlyGlyGly Ser GlyGlyGlyGly Ser GlyCys

35 40 45

<210> 38

<211> 14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>VARIANT

<222> 7

<223> D-form

<220>

<221> VARIANT

<222> 10

<223> D-form

<400> 38

Cys Ser GlyGlyGlyGlyPheCysPheTrp Lys ThrCysThr

1 5 10

<210> 39

<211> 46

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 39

Cys Ser Asn Leu Ser ThrCys Val Leu Gly Lys Leu Ser Gln Glu Leu

1 5 10 15

His Lys Leu GlnThr Tyr Pro ArgThrAsnThrGly Ser GlyThr Pro

20 25 30

GlyGlyGly Ser GlyGlyGly Ser Ala Cys Pro Gly His Ala

35 40 45

<210> 40

<211> 55

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 40

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

AsnPheGlyGlyGlyGly Ser GlyGlyGlyGly Ser GlyGlyGlyGly

35 40 45

Ser Ala Cys Pro Gly His Ala

50 55

<210> 41

<211> 23

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>VARIANT

<222> 1

<223> Xaa is a pyroglutamate

<220>

<221>VARIANT

<222> 6

<223> D-form

<400> 41

Xaa His Trp Ser Trp Leu LeuArg Pro GlyGly Ser Gly Ser GlyGly

1 5 10 15

Ser GlyCysGlyGly His Ala

20

<210> 42

<211> 18

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 42

Glu Gly Glu Gly Glu Ala GlyGly Lys Gly Ala Gly Lys Gly Ala Gly

1 5 10 15

LysGly

<210> 43

<211> 18

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 43

Glu Asp Glu Asp Glu Ala GlyGly Lys Gly Ala Gly Lys Gly Ala Gly

1 5 10 15

LysGly

<210> 44

<211> 9

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<220>

<221> VARIANT

<222> 6.8

<223>Xaa is a PEGylated amino acid having 4

ethylene glycol repeat unit (EG)

<400> 44

Glu Gly Glu Gly Glu Xaa Lys Xaa Lys

15

<210> 45

<211> 12

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 45

Lys Gly Ala GlyGly Lys Gly Ala GlyGly Lys Gly

1 5 10

<210> 46

<211> 11

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 46

Glu Gly Glu Gly Glu Ala Gly Lys Gly Ala Gly

1 5 10

<210> 47

<211> 144

<212>PRT

<213> Artificial sequence

<220>

<223>Synthetic

<400> 47

Ala Cys Pro Gly His Ala GlyGlyGlyGly Ser Ala Pro Thr Ser Ser

1 5 10 15

Ser Thr Lys LysThrGln Leu Gln Leu Glu His Leu LeuLeu Asp Leu

20 25 30

Gln Met Ile Leu AsnGly Ile AsnAsn Tyr Lys Asn Pro Lys Leu Thr

35 40 45

Arg Met Leu ThrPhe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

50 55 60

Lys His Leu GlnCys Leu Glu GluGlu Leu Lys Pro Leu Glu Glu Val

65 70 75 80

Leu Asn Leu Ala Gln Ser Lys AsnPhe His Leu Arg Pro Arg Asp Leu

85 90 95

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

100 105 110

ThrPhe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

115 120 125

Leu AsnArgTrp Ile ThrPhe Ser Gln Ser Ile Ile Ser Thr Leu Thr

130 135 140

<---

Claims (32)

1. A molecular construct for the treatment of a tumor containing a composite polypeptide and a functional element, where a compound polypeptide contains a parent polypeptide, a linker and a short oligopeptide, where the short oligopeptide comprises the amino acid sequence GCGGHA (SEQ ID NO: 6), ACPGHA (SEQ ID NO: 7) or GCPGHA (SEQ ID NO: 8) in order from N-terminus to C-terminus and the short oligopeptide is linked to N- or C-terminal -the end of the parent polypeptide using a linker; the linker has a length of from 1 to 15 amino acid residues, independently selected from the group consisting of glycine (G) and serine (S) residues; And the parent polypeptide is an antibody or antibody fragment that is specific for a tumor-associated cell surface antigen; And the functional element has a sulfhydryl (SH)-reactive group, which is conjugated to the thiol group of the cysteine residue in the short oligopeptide, the functional element includes: central core, wherein the central core contains from 2 to 10 lysine residues (K), optionally, one or more fillers, in which any two K residues are adjacent to each other or separated by the filler, optionally, a terminal spacer, wherein the terminal spacers are 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 fillers and the terminal spacer comprises, independently, (1) 1 to 12 amino acid residues other than K, or (2) a PEGylated amino acid having 1 to 12 ethylene glycol (EG) unit repeats, and an SH-reactive group linked to the first or last K residue of the central core by forming an amide bond with it, or, when a terminal spacer is present, an SH-reactive group linked to the terminal amino acid residue of the terminal spacer, forming an amide bond with it; and from 2 to 10 effector elements, where each effector element is a lenalidomide molecule, an auristatin molecule or a 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) molecule complexed with radioactive nuclide; where each effector element is associated with a K core residue, or, when the molecular construct further contains from 2 to 10 binding arms, one end of each linking arm is associated with the K residue of the core by forming an amide bond with the ε-amino group of the K residue and the other end of each linking arm is associated with each effector element, where the binding arm is a peptide , containing 2-12 amino acid residues other than K; or a polyethylene glycol (PEG) chain having 2 to 24 EG repeat units. 2. The molecular structure according to claim 1, characterized in that the SH-reactive group is a maleimide, iodoacetyl, bromoacetyl, vinylsulfone, monosulfone, methylsulfonylbenzothiazole or 2-pyridyldithiol group. 3. The molecular construct according to claim 1, characterized in that the composite polypeptide is in dimeric form, each of which has one functional element conjugated to it. 4. The molecular structure according to claim 1, characterized in that the central core carries a local negative charge on or near the amino acid residue associated with the SH-reactive group. 5. The molecular construct of claim 4, wherein the central core has at least one glutamate residue (E) or aspartate residue (D) on or adjacent to the amino acid residue associated with the SH-reactive group. 6. The molecular design according to claim 4, characterized in that a local negative charge is present in the first 5-15 amino acid residues, starting with the amino acid residue associated with the SH-reactive group. 7. A molecular construct for extending the half-life of the parent polypeptide, containing a composite polypeptide and a functional element, in which the composite polypeptide contains a parent polypeptide, a linker and a short oligopeptide, and the short oligopeptide contains the amino acid sequence GCGGHA (SEQ ID NO: 6), ACPGHA (SEQ ID NO: 7) or GCPGHA (SEQ ID NO: 8) in order from N-terminus to C-terminus, and the short oligopeptide is linked to N- or C-terminus of the parent polypeptide using a linker; the linker has a length of from 1 to 15 amino acid residues, independently selected from the group consisting of glycine (G) and serine (S) residues; And the parent polypeptide is a peptide hormone or cytokine; And the functional element has a sulfhydryl (SH)-reactive group, which is conjugated to the thiol group of a cysteine residue in a short oligopeptide, where the functional element contains: a central core, wherein the central core contains from 2 to 10 lysine residues (K), optionally, one or more fillers, in which any two of the K residues are adjacent to each other or separated by the filler, optionally, a terminal spacer, wherein the terminal spacers are 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 fillers and the terminal spacer contains independently (1 ) 1 to 12 amino acid residues other than K, or (2) a PEGylated amino acid having 1 to 12 ethylene glycol (EG) unit repeats, and an SH-reactive group linked to the first or last K residue of the central core by forming an amide bond therewith, or, when a terminal spacer is present, an SH-reactive group linked to the terminal amino acid residue of the terminal spacer to form an amide bond therewith; and from 2 to 10 pharmacokinetic elements, where each pharmacokinetic element is a C8-28 fatty acid derivative or a C8-28 dicarboxylic fatty acid derivative; Where each pharmacokinetic element is associated with the ε-amino group of the K residue, or, when the molecular construct further contains from 2 to 10 linking arms, one end of each linking arm is associated with 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 associated with each pharmacokinetic element, where the linking arm is a peptide containing 2-12 amino acid residues other than K; or a polyethylene glycol (PEG) chain having 2 to 24 EG repeat units.