阅读说明：本技术 半衰期延长的药物及其文库、以及制备方法和应用 (Half-life-prolonged drug, library thereof, preparation method and application ) 是由 周界文 潘利强 闰长青 周柳娟 于 2019-10-29 设计创作，主要内容包括：本发明提供了半衰期延长的药物及其文库、以及制备方法和应用。具体地,本发明提供一种药物文库,所述药物文库包括：(a)药物单元；和(b)n个半衰期延长单元；其中,所述的药物单元包括药物元件部分以及与所述药物元件部分相连的第一核酸元件部分；所述的半衰期延长单元包括半衰期延长元件部分以及与所述的半衰期延长元件部分相连的第二核酸元件部分；并且所述药物单元的一个所述的第一核酸元件部分与至少一个半衰期延长单元的第二核酸元件部分可通过形成碱基互补的配对结构,从而构成“药物单元-半衰期延长单元”复合物；其中,n为≥1的正整数。可根据需要,快速、高效、低成本、高得率地组装半衰期延长的药物。(The invention provides a drug with prolonged half-life, a library thereof, a preparation method and application thereof. Specifically, the present invention provides a drug library comprising: (a) a drug unit; and (b) n half-life extending units; wherein the drug unit comprises a drug element portion and a first nucleic acid element portion attached to the drug element portion; said half-life extending unit comprising a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion; and one of said first nucleic acid element portions of said drug units and a second nucleic acid element portion of at least one half-life extender unit may be formed by base-complementary pair formation to form a "drug unit-half-life extender unit" complex; wherein n is a positive integer not less than 1. The drug with prolonged half-life can be assembled quickly, efficiently, at low cost and high yield according to the needs.)

1. A drug library, comprising: (a) a drug unit; and (b) n half-life extending units;

wherein the drug unit comprises a drug element portion and a first nucleic acid element portion attached to the drug element portion;

said half-life extending unit comprising a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion;

and one of said first nucleic acid element portions of said drug units and a second nucleic acid element portion of at least one half-life extender unit may be formed by base-complementary pair formation to form a "drug unit-half-life extender unit" complex;

wherein n is a positive integer not less than 1.

2. The drug library of claim 1, wherein the "drug unit-half-life extending unit" complex has the structure of formula I:

T₀-[L₀-A₀]n(I)

wherein the content of the first and second substances,

T₀is a drug element portion;

L₀is a nucleic acid linker (linker) for linking T₀And A₀And comprising a base-complementary pairing structure of said first nucleic acid element portion and said second nucleic acid element portion;

A₀is an FcRn binding element, or a binding element that binds to an FcRn binding protein (e.g., serum albumin);

n is a positive integer not less than 1.

3. The drug library of claim 1, wherein the drug unit has a structure according to formula II:

T─X1─L1─Y1─W1─Z1(II)

the half-life extending unit has a structure represented by formula III:

A─X2─L2─Y2─W2─Z2(III)

in the formula (I), the compound is shown in the specification,

t is a drug element moiety;

a is a half-life extending element moiety;

x1, X2 are each independently a non-or redundant peptide;

l1, L2 are each independently a linker molecule;

y1, Y2 and Z1, Z2 are each independently a non-or redundant nucleic acid;

w1, W2 are each independently a nucleic acid sequence selected from the group consisting of: l nucleic acid, peptide nucleic acid, locked nucleic acid, thio modified nucleic acid, 2' -fluoro modified nucleic acid, 5-hydroxymethyl cytosine nucleic acid, or a combination thereof;

"—" is a covalent bond;

wherein the nucleic acid W1 of the drug unit has at least one complementary pair of regions and the nucleic acid W2 of the half-life extending unit has at least one complementary pair of regions, which are partially or fully complementary.

4. The drug library of claim 3, wherein the drug element moiety is selected from the group consisting of: antibodies, ligands that activate or inhibit receptors, proteins, biologically active enzymes, nucleic acid drugs, small molecule drugs, or combinations thereof.

5. The drug library of claim 3, wherein the half-life extending element moiety is selected from the group consisting of: natural Albumin (Albumin), recombinant Albumin, anti-Albumin antibodies (including nanobodies, single chain antibodies, fabs, monoclonal antibodies), aptamers (aptamers) that specifically bind Albumin, proteins and aptamers (aptamers) that bind FcRn directly, proteins with any long half-life, or combinations thereof.

6. The drug library of claim 3, wherein the linker molecules L1, L2 each independently have a bifunctional linker conjugating a modified end of a nucleic acid W1, W2 or Y1, Y2 having a modifying group and a specific attachment site for T or A or X1, X2.

7. A method of assembling a drug having an extended half-life (e.g., a protein drug), comprising:

(a) selecting drug units and half-life extending units from the drug library of claim 1 based on pharmaceutical information; and

(b) mixing the drug units and at least one half-life extending unit to assemble the half-life extending drug.

8. A half-life-extending drug, wherein the half-life-extending drug is a half-life-extending drug formed by a drug unit and n half-life-extending units through nucleic acid complementation to form a base-complementary paired structure (e.g., a double-stranded paired structure), wherein n is a positive integer of 1 or more;

wherein said drug unit comprises a drug element portion and a first nucleic acid element portion linked to said drug element portion, said half-life extending unit comprises a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion, and said nucleic acid element portion of one of said drug units and the nucleic acid element portion of at least one half-life extending unit form base-complementary paired structures (e.g., double-stranded paired structures) by complementation.

9. The half-life extending pharmaceutical of claim 8, wherein said nucleic acid element portion is selected from the group consisting of: l nucleic acid, peptide nucleic acid, locked nucleic acid, thio modified nucleic acid, 2' -fluoro modified nucleic acid, 5-hydroxymethyl cytosine nucleic acid, or a combination thereof.

10. A pharmaceutical composition comprising

(i) The half-life extended pharmaceutical of claim 8 as an active ingredient; and

(ii) a pharmaceutically acceptable carrier.

Technical Field

The invention relates to the field of biotechnology medicine. In particular to a drug with prolonged half-life and a library thereof, and a preparation method and application thereof.

Background

For protein or protein-based drugs, it is advantageous to increase their half-life to improve therapeutic efficacy and to reduce drug dosage.

In order to prolong the half-life of protein or polypeptide drugs, the existing methods include preparing them as follows: forms of Fc-fusion proteins, HSA-fusion proteins; preparing Fc-protein/polypeptide conjugate and HSA-protein/polypeptide conjugate by chemical coupling; and replacing the HSA in the fusion protein or conjugate with a protein or polypeptide (such as an anti-HSA antibody), a small molecule, a nucleic acid (such as an aptamer), etc., which can bind to HSA; chemical modification methods such as PEG, glycosylation and sialic acid.

PEG modification is a method for improving the half-life of protein or polypeptide drugs by a main chemical method, has the advantages of strong biocompatibility of PEG and capability of increasing drug solubility and stability, and has the following defects: the PEG modification can greatly influence the activity of active proteins such as biological enzyme and the like, the PEG with large molecular weight has immunogenicity, the PEG molecule has poor uniformity, and the PEG chemical drug substance control difficulty is high. In addition, chemical modification methods such as PEG rely on increasing molecular weight to increase half-life of protein or polypeptide drugs, and thus have limited half-life enhancing effects.

Although Fc or HSA fusion proteins can extend half-life, methods for extending half-life of Fc or HSA fusion proteins will generate novel epitopes at the Fc/HSA fusion site of the protein or polypeptide of interest. Furthermore, Fc can only be fused to the C-terminus, while HSA can only be fused to the N-or C-terminus of the protein or polypeptide of interest. Furthermore, this method is not applicable to native proteins or polypeptides.

Therefore, there is a need in the art to develop a simple, flexible, efficient and modular connection method for specific heterocoupling of protein drugs and half-life prolonging factors (HSA, HSA binding module, etc.), so as to prolong the half-life of protein drugs and improve their pharmacokinetic properties.

Disclosure of Invention

The invention aims to provide a simple, flexible, efficient and modular method for specific heterologous coupling of a protein drug and a half-life prolonging factor and a coupled drug prepared by the method.

Another object of the present invention is to provide a low-cost, modular FcRn targeting unit, which can be used as a half-life enhancing module to flexibly link with a protein drug, thereby significantly extending the half-life of the protein drug, and a module library comprising the modular FcRn targeting unit.

In a first aspect of the invention, there is provided a drug library comprising: (a) a drug unit; and (b) n half-life extending units;

wherein the drug unit comprises a drug element portion and a first nucleic acid element portion attached to the drug element portion;

said half-life extending unit comprising a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion;

and one of said first nucleic acid element portions of said drug units and a second nucleic acid element portion of at least one half-life extender unit may be formed by base-complementary pair formation to form a "drug unit-half-life extender unit" complex;

wherein n is a positive integer not less than 1.

In another preferred embodiment, the drug unit and the half-life extending unit are directly or indirectly base complementary linked.

In another preferred embodiment, the "base-complementary" includes direct base-complementary and/or indirect base-complementary.

In another preferred embodiment, said "direct base complementarity" comprises direct base complementarity between said first nucleic acid element portion and said second nucleic acid element portion.

In another preferred embodiment, the term "indirect base-complementary" includes that the first nucleic acid element portion and the second nucleic acid element portion are base-complementary via one or more (e.g., 2, 3, 4, 5, 6) single-stranded complementary sequences of complementary nucleic acid molecules (i.e., nucleic acid F) to form a triple or more paired structure.

In another preferred embodiment, the complementary nucleic acid molecule is in single stranded form.

In another preferred embodiment, the helper nucleic acid molecule is selected from the group consisting of: a separate nucleic acid molecule, a second nucleic acid element portion of another half-life extending unit, a first nucleic acid element portion of another drug unit, or a combination thereof.

In another preferred embodiment, the complementary nucleic acid molecule is a nucleic acid not conjugated to a protein drug.

In another preferred embodiment, the complementary nucleic acid molecule is a first nucleic acid element portion of a different drug unit and/or a second nucleic acid element portion of a different half-life extending unit.

In another preferred embodiment, the first nucleic acid element portion, the second nucleic acid element portion, and/or the complementary nucleic acid molecule is a levorotatory nucleic acid, or a nucleic acid modified with a modifying group.

In another preferred embodiment, said first nucleic acid element is not linked to the N-terminus (amino terminus) of said drug element portion and/or is also not linked to the C-terminus (carboxy terminus) of said drug element portion.

In another preferred embodiment, said first nucleic acid element is linked to a non-terminal amino acid residue portion of said drug element portion.

In another preferred embodiment, said second nucleic acid element is not linked to the N-terminus (amino-terminus) of said half-life extending element portion and/or is not linked to the C-terminus (carboxy-terminus) of said half-life extending element portion.

In another preferred embodiment, said second nucleic acid element is linked to a non-terminal (non-terminal) amino acid residue portion of said half-life extending element portion.

In another preferred embodiment, the half-life extending element portion of the half-life extending unit is not Fc or a fragment thereof or modified Fc or a fragment thereof (e.g., glycosylated Fc, NH₂Modified Fc).

In another preferred embodiment, the half-life extending element moiety in the half-life extending unit is not PEG (polyethylene glycol).

In another preferred embodiment, the "drug unit-half-life extender" complex has 1 half-life extender.

In another preferred embodiment, the "drug unit-half-life extender" complex has 2 or more different half-life extender units.

In another preferred embodiment, the "drug unit-half-life extender unit" complex has a half-life extender unit to drug unit molar ratio (A/D) of 0.5-10, preferably 1-8, more preferably 2-5.

In another preferred embodiment, the "drug unit-half-life extending unit" complex has 1 drug unit.

In another preferred embodiment, the "drug unit-half-life extending unit" complex has 2 or more different drug units.

In another preferred embodiment, the "drug unit-half-life extender unit" complex has a half-life extender unit to drug unit molar ratio (A/D) of 0.5-10, preferably 1-8, more preferably 2-5.

In another preferred embodiment, the drug element portion is a protein drug.

In another preferred embodiment, the drug element moiety is a polypeptide drug.

In another preferred embodiment, the drug element moiety is a fusion protein drug.

In another preferred embodiment, the "drug unit-half-life extending unit" complex has the structure of formula I:

T₀-[L₀-A₀]n (I)

wherein the content of the first and second substances,

T₀is a drug element portion;

L₀is a nucleic acid linker (linker) for linking T₀And A₀And comprising a base-complementary pairing structure of said first nucleic acid element portion and said second nucleic acid element portion;

A₀is an FcRn binding element, or a binding element that binds to an FcRn binding protein (e.g., serum albumin);

n is a positive integer not less than 1.

In another preferred embodiment, the drug unit has the structure shown in formula II:

T─X1─L1─Y1─W1─Z1 (II)

the half-life extending unit has a structure represented by formula III:

A─X2─L2─Y2─W2─Z2 (III)

in the formula (I), the compound is shown in the specification,

t is a drug element moiety;

a is a half-life extending element moiety;

x1, X2 are each independently a non-or redundant peptide;

l1, L2 are each independently a linker molecule;

y1, Y2 and Z1, Z2 are each independently a non-or redundant nucleic acid;

w1, W2 are each independently a nucleic acid sequence selected from the group consisting of: l nucleic acid, peptide nucleic acid, locked nucleic acid, thio modified nucleic acid, 2' -fluoro modified nucleic acid, 5-hydroxymethyl cytosine nucleic acid, or a combination thereof;

"—" is a covalent bond;

wherein the nucleic acid W1 of the drug unit has at least one complementary pair of regions and the nucleic acid W2 of the half-life extending unit has at least one complementary pair of regions, which are partially or fully complementary.

In another preferred embodiment, the drug element moiety is selected from the group consisting of: antibodies, ligands that activate or inhibit receptors, proteins, biologically active enzymes, nucleic acid drugs, small molecule drugs, or combinations thereof.

In another preferred embodiment, the half-life extending element moiety is selected from the group consisting of: natural Albumin (Albumin), recombinant Albumin, anti-Albumin antibodies (including nanobodies, single chain antibodies, fabs, monoclonal antibodies), aptamers (aptamers) that specifically bind Albumin, proteins and aptamers (aptamers) that bind FcRn directly, proteins with any long half-life, or combinations thereof.

In another preferred embodiment, X1 and X2 are each independently 0 to 30 amino acids.

In another preferred embodiment, X1, X2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

In another preferred embodiment, the linker molecules L1, L2 each independently have a bifunctional linker, which can couple the modified end of the nucleic acids W1, W2 or Y1, Y2 with a modification group and a specific attachment site for T or a or X1, X2.

In another preferred embodiment, the reactive groups of the linker molecules L1, L2 are each independently selected from: maleimide, haloacetyl, thiopyridine.

In another preferred embodiment, the haloacetyl group is selected from: iodoacetyl and bromoacetyl.

In another preferred embodiment, the drug element moiety T is a proteinaceous drug element.

In another preferred embodiment, the protein drug element T is wild type or mutant.

In another preferred embodiment, the mutation does not affect the function of the drug.

In another preferred embodiment, the mutation comprises the introduction of one or more cysteine residues (Cys) in the proteinaceous pharmaceutical element.

In another preferred embodiment, the cysteine residue is located at any position (e.g., N-terminal, C-terminal, or any position in between) of the proteinaceous pharmaceutical element.

In another preferred embodiment, the mutation comprises the introduction of one or more cysteine residues at the carboxy-terminus (C-terminus) of the proteinaceous pharmaceutical element (e.g. antibody).

In another preferred embodiment, the cysteine residue is used for ligation of DNA.

In another preferred embodiment, the proteinaceous drug element is FVIII.

In another preferred embodiment, Y1, Y2 are each independently 0-30 nucleotides.

In another preferred embodiment, each of Y1 and Y2 is independently a l-nucleic acid.

In another preferred embodiment, Y1, Y2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

In another preferred embodiment, each of Y1 and Y2 is independently AAAA, AAA or AA.

In another preferred embodiment, Z1 and Z2 are each independently 0 to 30 nucleotides.

In another preferred embodiment, each of Z1, Z2 is independently a l-nucleic acid.

In another preferred embodiment, Z1, Z2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

In another preferred embodiment, each of Z1 and Z2 is independently AAAA, AAA or AA.

In another preferred embodiment, the nucleic acids W1, W2 are l-nucleic acids.

In another preferred embodiment, the nucleic acids W1, W2 are selected from: DNA, RNA.

In another preferred embodiment, the modifying group is selected from the group consisting of: NH (NH)₂Alkynyl, mercapto (SH), Carboxyl (COOH), or combinations thereof.

In another preferred embodiment, the modifying group is NH₂。

In another preferred embodiment, the position of the modifying group on the nucleic acid W1, W2 and/or Y1, Y2 is selected from: 5 'end, 3' end, and any intermediate site.

In another preferred embodiment, there is a transition region between any 2 complementary pairing regions of the nucleic acids W1, W2 with a length of 0-10 nt.

In another preferred embodiment, the transition region is AAAA, AAA or AA.

In another preferred embodiment, the length of the complementary pairing region is 5 to 100 nt; preferably 8 to 50 nt; more preferably 10 to 30 nt; and more preferably 12 to 25 nt; most preferably 16-20 nt.

In a second aspect of the invention, there is provided a method of assembling a half-life extended drug (e.g., a protein drug) comprising:

(a) selecting drug units and half-life extending units from a drug library according to the first aspect of the invention based on pharmaceutical information; and

(b) mixing the drug units and at least one half-life extending unit to assemble the half-life extending drug.

In another preferred embodiment, the assembly is by complementarity of the nucleic acid element portions to form base-complementary paired structures (e.g., double-stranded paired structures).

In another preferred embodiment, in the half-life extending drug, the nucleic acid element portion of each drug unit forms a base-complementary pair structure (e.g., a double-stranded pair structure) with the nucleic acid element portions of one or two or three half-life extending units.

In another preferred embodiment, the assembly is such that the nucleic acid element portion is complementary to the single-stranded complementary sequence of the complementary-assisting nucleic acid molecule (i.e., nucleic acid F) to form a base-complementary paired structure (e.g., a double-stranded paired structure).

In another preferred embodiment, the complementary nucleic acid molecule is in single stranded form.

In another preferred embodiment, the nucleic acid F is a nucleic acid to which a protein drug is not coupled.

In another preferred embodiment, the nucleic acid F is a levorotatory nucleic acid, or a nucleic acid modified with a modifying group.

In another preferred embodiment, the length of the nucleic acid F is 1 to 1.5 times the sum of the number of pairs of monomeric nucleic acids in all (b).

In another preferred example, the pharmaceutical information is protein drug information required for treating a disease of a subject to be treated, including the type, administration mode, and pharmacokinetics of the protein drug.

In another preferred example, the assembling conditions are: 5-50 degrees (preferably 25-40 degrees), and reacting for 1-15 minutes (preferably 5-10 minutes).

In another preferred example, the assembling conditions are: the pH value is 6-8.

In a third aspect of the present invention, there is provided a half-life-extended drug, which is a half-life-extended drug in which a drug unit and n half-life-extension units are formed by nucleic acid complementation to form a base-complementary paired structure (e.g., a double-stranded paired structure), wherein n is a positive integer of 1 or more;

wherein said drug unit comprises a drug element portion and a first nucleic acid element portion linked to said drug element portion, said half-life extending unit comprises a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion, and said nucleic acid element portion of one of said drug units and the nucleic acid element portion of at least one half-life extending unit form base-complementary paired structures (e.g., double-stranded paired structures) by complementation.

In another preferred embodiment, the nucleic acid element portion is resistant to nuclease degradation.

In another preferred embodiment, the drug is a protein drug having the structure of formula I:

T₀-[L₀-A₀]n (I)

wherein the content of the first and second substances,

T₀is a protein drug;

L₀is a nucleic acid linking element for linking T₀And A₀；

A₀Is an FcRn binding element; or a binding element that binds to an FcRn binding protein (e.g., serum albumin);

n is a positive integer not less than 1.

In another preferred embodiment, the nucleic acid element portion is selected from the group consisting of: l nucleic acid, peptide nucleic acid, locked nucleic acid, thio modified nucleic acid, 2' -fluoro modified nucleic acid, 5-hydroxymethyl cytosine nucleic acid, or a combination thereof.

In another preferred embodiment, the drug units and half-life extending units are from the drug library of the first aspect of the invention.

In another preferred embodiment, the drug with prolonged half-life is a multimeric protein drug, and the multimeric protein drug is a multimer formed by forming base-complementary pairing structures (such as double-stranded pairing structures) by nucleic acid complementation of D protein drug monomers, wherein D is a positive integer greater than or equal to 2.

In another preferred embodiment, D is a positive integer from 2 to 6; preferably D is 2, 3, 4 or 5.

In another preferred embodiment, the half-life extending drug is a half-life extending protein drug.

In another preferred embodiment, the half-life of the extended half-life protein drug is greater than the in vivo half-life of the protein drug element alone, H1, which degrades in vivo.

In another preferred embodiment, the ratio of H1/H2 is 1-100, preferably 10-50, more preferably 10-20.

In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising

(i) The half-life-extended drug according to the third aspect of the present invention as an active ingredient; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injection and lyophilized preparation.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a mechanism diagram of the present invention for prolonging the half-life of a drug by nucleic acid pairing.

Figure 2 shows the structure of a protein drug linked to an FcRn targeting unit in one embodiment of the invention.

FIG. 3 shows agarose gel electrophoresis before and after four different L-DNA single strands form a tetrameric framework.

Figure 4 shows the half-life of long acting factor viii in a hemophilia mouse model.

Detailed Description

After extensive and intensive research, the inventor firstly develops a drug with prolonged half-life period, a library thereof, a preparation method and application. The drug library and the preparation method can be used for assembling the drug with prolonged half-life period rapidly and efficiently at low cost and high yield according to the needs. The present invention has been completed based on this finding.

Specifically, the present invention provides a half-life extended drug comprising: (a) a drug unit; and (b) n half-life extending units coupled to the drug unit by nucleobase complementarity; wherein said drug unit comprises a drug element portion (motif) and a first nucleic acid element portion linked to said drug element portion, said half-life extending unit comprises a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion, and said nucleic acid element portion of one of said drug units and the nucleic acid element portion of at least one of said half-life extending units are complementary to form a base-complementary pair (e.g., a double-stranded pair) to form said drug; wherein n is a positive integer not less than 1. The half-life extended drugs of the present invention form stable nucleobase complementary paired structures (rather than complex peptide bonds or other chemical modifications, etc.) by rapid assembly (e.g., 1 minute) only. Experiments have shown that the agents of the invention can specifically bind to FcRn via the FcRn binding element, or a binding element that binds to an FcRn binding protein (e.g. serum albumin). Unexpectedly, when the drug of the invention is endocytosed by hematopoietic cells or vascular endothelial cells in blood through endocytosis, the drug can enter an inclusion body (endosome) and be combined with FcRn in the endosome, thereby entering a recycling way and being resistant to degradation under various environments such as intracellular and inclusion bodies. Thus, the drug of the present invention can effectively avoid entering a degradation pathway to be degraded by lysosome (lysosome). In addition, the drug of the present invention can be dissociated from FcRn under neutral physiological conditions (e.g., about pH 7.4) after being circulated to the cell surface, and released into the blood, thereby achieving recycling (see fig. 1).

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

As used herein, the terms "protein drug", "polypeptide drug" are used interchangeably and refer to a drug containing amino acids that form a polypeptide sequence by peptide bonds, which drug may consist of only the amino acid sequence or may consist essentially of the amino acid sequence. In addition, the term also includes unmodified or modified forms, e.g., glycosylated protein drugs, pegylated protein drugs, protein drugs in ADC form.

As used herein, the terms "half-life extending unit", "half-life extending module", "half-life enhancing module" are used interchangeably and refer to an element or module comprising a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion and configured to form a "drug unit-half-life extending unit" complex by base complementation, thereby extending the half-life of the protein drug complex.

As used herein, the term "protein drug complex" or "drug unit-half-life extender unit complex" are used interchangeably and refer to a drug complex formed by base-complementary drug units and half-life extender units.

Medicine T

In the present invention, the drug unit includes a drug element portion and a first nucleic acid element portion connected to the drug element portion.

Wherein, the drug element part is protein drug and polypeptide drug.

Typically, the proteinaceous drugs include, but are not limited to, antibodies, fusion proteins, growth factors, hormones (e.g., insulin, growth hormone, etc.).

In a preferred embodiment of the invention, the proteinaceous agent is a long-acting factor eight (FVIII) for the treatment of haemophilia a.

Half-life extending unit A

In the present invention, the half-life extending unit includes a half-life extending element portion and a second nucleic acid element portion linked to the half-life extending element portion.

Specifically, the invention utilizes a nucleic acid pairing-mediated multispecific antibody self-assembly technology (patent application No. 201710322583.3), a protein or polypeptide (half-life extender) targeting FcRn and a drug are respectively connected with a nucleic acid element to form an FcRn targeting unit, and then the protein or polypeptide and the drug are coupled together through complementary pairing of the nucleic acid elements to form a long-acting drug which can be recovered by cells.

The half-life extending element moieties (half-life extending factors) can be divided into the following broad categories:

2.1 HSA protein

The HSA protein may be HSA extracted from a blood source, or HSA recombinantly expressed in a low-cost expression system such as yeast. HSA has a free Cys34, and can be used for site-directed coupling of activated single-stranded degradation-resistant nucleic acid (such as reverse nucleic acid). Of courseNH on the HSA surface if site-directed coupling is not required₂It can also be used for random coupling of single-stranded nucleic acids.

2.2 HSA binding proteins

HSA binding protein types include nanobodies, single chain antibodies, fabs, and full-length IgG antibodies. According to the invention, the preferred HSA binding protein is a nano-antibody and a single-chain antibody which have low preparation cost.

2.3 HSA-binding polypeptides and domains

Polypeptides of specific sequence may also bind to HSA, such as the polypeptide SA21 (amino acid sequence: Ac-RLIEDICLPRWGCLWDED-NH)₂SEQ ID NO.6), wherein the core sequence is DICLPRWGCLW (SEQ ID NO.7), and the polypeptides containing the core sequence can be combined with HSA to different degrees to become the half-life promoting factors of the invention.

Other protein domains can also become HSA binding protein domains through directed engineering, evolution and other means, such as Affinibody, DART and the like, and small fragment antibodies are artificially engineered.

2.4 HSA binding nucleic acids

A portion of the aptamer (aptamer) can specifically bind to HSA, such as one of the RNA types with aptamer sequence 5'-GUGGACUAUACCGCGUAAUGCUGCCUCCAC-3' (SEQ ID NO. 8). The preferred HSA binding nucleic acids of the present invention are preferably of the same type as the nucleic acid framework, and are conveniently synthesized and prepared, e.g., both L-RNA and L-DNA may be selected.

Furthermore, nucleic acids modified with dendritic alkyl chains (dendritic alkyl chains) can also bind to HSA, and for example, j.am.chem.soc.2017.139.21.7355-7362 reports that a DNA cage (DNA cube) assembled from dendritic alkyl-modified DNA can bind to HSA with high affinity.

For protein or polypeptide half-life extending factors, to facilitate their conjugation to activated L-nucleic acids, a specific site (e.g., mutation site, Cys) is introduced into the half-life extending factor for conjugation to a linker.

For nucleic acids, the half-life extender can be synthesized simultaneously with the sequence of the same type of nucleic acid used for assembly.

Drug libraries

The present invention provides a drug library comprising: (a) a drug unit; and (b) n half-life extending units;

wherein the drug unit comprises a drug element portion and a first nucleic acid element portion attached to the drug element portion;

said half-life extending unit comprising a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion;

and one of said first nucleic acid element portions of said drug units and a second nucleic acid element portion of at least one half-life extender unit may be formed into a "drug unit-half-life extender unit" complex by forming base-complementary paired structures (e.g., double-stranded paired structures);

wherein n is a positive integer not less than 1.

As used herein, the term "complementary" includes direct complementary pairing or pairing (e.g., double-stranded pairing) that is complementary to form a base-complementary pairing structure (e.g., double-stranded pairing structure) via one or more (e.g., 2, 3, 4, 5, 6) single-stranded complementary sequences of the complementary nucleic acid molecule (i.e., nucleic acid F).

In a preferred embodiment of the invention, the complementary nucleic acid molecule (i.e.nucleic acid F) is a multimerization facilitator molecule (strand).

In a preferred embodiment of the invention, the drug unit and the half-life extending unit form a multimer in the presence of the multimerization-facilitating molecule (chain).

Typically, the "complementary" is a direct complementary pairing.

In a preferred embodiment of the present invention, the "complementary" is a paired structure formed by the complementary formation of base complementarity through the single-stranded complementary sequence of 1 auxiliary complementary nucleic acid molecule (i.e., nucleic acid F).

In another preferred embodiment of the present invention, the "complementary" is a paired structure formed by the complementary of single-stranded complementary sequences of 2 auxiliary complementary nucleic acid molecules (i.e., nucleic acid F) (as shown in FIG. 2).

The library of the invention contains at least 1 drug unit and at least 1 half-life extending unit, wherein the drug unit and the half-life extending unit are assembled to form a drug, and the preferred drug has the structure shown in the formula I.

In another preferred embodiment of the invention, the library of the invention comprises 1 drug unit and at least one (e.g. 2, 3, 4) half-life extending unit.

Typically, the drug unit has the structure of formula II above.

Typically, the half-life extending unit has the structure of formula III above.

Due to the specific structure of the drug units and half-life extending units of the invention, not only can the drug be rapidly assembled, but also the resulting drug has an unexpectedly extended half-life (significantly increased stability in vivo), can exist and maintain activity in vivo for a long time, without being rapidly degraded.

For example, in the treatment of cancer (or other metabolic diseases), multiple targets and multiple pathways are often involved, and not only the etiology, constitution, and age of each patient are different, but also tumor heterogeneity or disease subtypes are present in a single patient, and the pharmacokinetics and metabolic pathways are often different, and thus, it is often necessary to use drugs with different half-lives for multiple targets. With the development of personalized therapy or precise therapy technology, development of protein drugs (such as multispecific antibodies) which can be rapidly prepared, have low cost, good targeting property and good stability and have controllable (adjustable) half-life is urgently needed in the field. The libraries of the present invention meet such a need.

It will be appreciated that while the drug library of the present invention contains, or consists essentially of, or consists of, the above-described drug units and half-life extending units of the present invention, the drug library may also contain other therapeutic agents, particularly other protein drugs, representative examples of which include (but are not limited to): antibodies, compounds, fusion proteins. For example, the libraries of the invention may additionally contain one or more conventional antibodies with therapeutic effect, or conventional drugs with prolonged drug half-life, or conventional metabolic modulators.

It will be appreciated that the number of half-life extending units in the libraries of the invention is not limited and can be any positive integer n ≧ 2. For example, n is any positive integer from 3 to 100; n is 3 to 50; more preferably n is 5 to 20; most preferably n is from 5 to 8.

Further, in the present invention, the antibody element in the half-life extending unit is not particularly limited, and representative examples (selected from the group consisting of: single chain antibodies, nanobodies, fabs, monoclonal antibodies, or combinations thereof.

For the libraries of the invention, various sources of proteins, polypeptides can be used to prepare pharmaceutical units; various sources of antibodies can be used to make the half-life extending unit. One of the outstanding features of the drug library of the present invention is that antibody fragments expressed by prokaryotic systems (e.g., E.coli) or eukaryotic systems (e.g., yeast, CHO cells) can be used, thereby greatly reducing the production cost.

Typically, in use, the drug assembly is conveniently accomplished by nucleic acid complementation framework by selecting the corresponding drug units and half-life extension units (including species and number) according to the need (e.g., the condition and diagnostic result of a subject, the protein drug information required for treating the disease in the subject to be treated, including the species, administration mode, pharmacokinetics of the protein drug). For example, in use, the half-life extender units are assembled after metabolic conditions, and the number or proportion of half-life extender units (e.g., 2, 3, 4, or more than 4) are determined accordingly, depending on the target condition of the disease in the patient.

In the preparation of the drug, the corresponding drug units and half-life extending units, which can be coupled directly or indirectly to each other in pairs, are selected from the library and mixed in the desired antibody ratio, allowing the assembly process to be completed within 1 minute.

In the library of the present invention, the nucleic acid elements of the drug units and the nucleic acid elements of the half-life extending units can be designed into a high polymer framework such as dimers, trimers, tetramers, etc. through sequence design, thereby completing the preparation of a polymer with 3 or even 4 half-life extending units carried by one drug molecule, which cannot be easily achieved by the traditional drug half-life design and preparation technology.

Once assembled to form the multimeric protein medicament, it can be used in the corresponding individual depending on the therapeutic purpose.

L-nucleic acids (L-nucleic acids)

L-nucleic acid refers to a nucleic acid which is present in a mirror image relative to a naturally occurring D-nucleic acid (D-nucleic acid), and can be classified into L-DNA (L-DNA) and L-RNA (L-RNA). The left-hand (chiral center) is mainly present in the deoxyribose or ribose portion of nucleic acids, with mirror inversion. Therefore, L-nucleic acids cannot be degraded by nucleases (e.g., exonucleases, endonucleases) that are ubiquitous in plasma.

The invention relates to a medicament with prolonged half-life period

The half-life-extended drug of the present invention is a half-life-extended drug formed by a drug unit and n half-life-extended units forming a base-complementary paired structure (e.g., a double-stranded paired structure) by nucleic acid complementation, wherein n is a positive integer of 1 or more; wherein said drug unit comprises a drug element portion and a first nucleic acid element portion linked to said drug element portion, said half-life extending unit comprises a half-life extending element portion and a second nucleic acid element portion linked to said half-life extending element portion, and said nucleic acid element portion of one of said drug units and the nucleic acid element portion of at least one half-life extending unit form base-complementary paired structures (e.g., double-stranded paired structures) by complementation.

In another preferred embodiment, the nucleic acid element portion is resistant to nuclease degradation.

In another preferred embodiment, the nucleic acid element portion is selected from the group consisting of: l nucleic acid, peptide nucleic acid, locked nucleic acid, thio modified nucleic acid, 2' -fluoro modified nucleic acid, 5-hydroxymethyl cytosine nucleic acid, or a combination thereof.

The drug of the present invention can be formed by, for example, assembling a drug unit represented by formula II and a half-life extending unit represented by formula III.

Typically, a multimeric drug is one that is assembled from one drug unit and multiple half-life extending units, such as a di-, tri-, tetra-, penta-, or hexamer.

In a preferred embodiment, the drug of the present invention is linked using a levorotatory nucleic acid. The research of the invention shows that nucleic acid is a double-chain molecule which can be rapidly and specifically paired, so that if a half-life extension element part (such as HSA, a single-chain antibody, a nano antibody, Fab and the like) is coupled with a nucleic acid single chain, and a drug element part is coupled with the nucleic acid single chain, two or more than two nucleic acid single chains can be rapidly paired by designing a nucleic acid sequence, so that a drug unit and one or more half-life extension units can be rapidly assembled, and the preparation of the drug with the extended half-life can be completed.

In the present invention, in order to achieve the effect of extending the half-life of a drug, it is necessary to use a drug unit having a specific structure and a half-life extending unit. In a preferred embodiment, the drug half-life can be prolonged significantly by replacing dextro nucleic acids (e.g., D-DNA, D-RNA) with L-nucleic acids (e.g., L-DNA, L-RNA, etc.). One reason is that L-nucleic acid cannot be degraded by exonucleases, endonucleases, etc. present in the human body, so that the drug mediated by self-assembly of L-nucleic acid (L-nucleic acid) will be extremely stable in vivo.

Preparation method

Design and preparation of L-nucleic acid chain frameworks

According to the present invention, an L-nucleic acid strand framework is formed of two or more L-nucleic acid single strands by base pairing. The 5 'or 3' end of each L-nucleic acid single strand is activated to form a group (e.g., NH) for subsequent modification₂Etc.), and then coupled to an activating group on the L-nucleic acid single strand with one end of a linker (e.g., SMCC, SM (PEG), SPDP, etc.). The L-nucleic acid with the linker can be assembled into the desired L-nucleic acid strand framework. After it is determined that the linker-bearing L-nucleic acids can successfully self-assemble into a framework, the linker-bearing L-nucleic acid single chains can be coupled to antibodies for subsequent assembly, respectively.

The L-nucleic acid framework of the invention can be prepared essentially by the following steps.

1.1 designing an L-nucleic acid Single Strand capable of fast self-Assembly

Determining a required number N of half-life boosting modules (e.g., three half-life boosting modules); determining the number of required L-nucleic acid single strands M (M > -N +1) according to the half-life promoting module number N; designing a corresponding number of L-nucleic acid single-stranded sequences, adjusting the stability of a target nucleic acid framework by increasing or decreasing the number of base pairs, and reducing the possibility of non-specific pairing between nucleic acid chains.

According to a preferred embodiment of the invention, to design a tetrameric L-nucleic acid framework (M ═ 4), four L-nucleic acids are designed which can be paired according to a certain rule (as shown in fig. 2). Wherein any one of the L-nucleic acid single strands can be specifically complementarily paired with the other two L-nucleic acid single strands, but not with the fourth strand. And the absolute value of gibbs energy change Δ G of the specific complementary pair is much larger than that of the non-specific pair, for example, in the preferred embodiment, the absolute value of gibbs energy change Δ G of the specific complementary pair of each arm is about 26 kcal/mole (kcal/mole), and the absolute value of the gibbs energy change Δ G of the non-specific pair of each arm is less than 7 kcal/mole (kcal/mole), which means that the assembly of tetramer is easier than that of the non-specific pair-by-pair, and the tetramer form is most stable in the reaction system. The tetrameric L-nucleic acid framework may be linked to 1-3 half-life enhancing factors.

1.2 activation of L-DNA or L-RNA

Activation of the L-nucleic acid involves modification of the reactive group at its 5 'or 3' end and subsequent conjugate of the linker. The active group modification can be customized by a nucleic acid synthesis company; the linker typically possesses a bifunctional group, i.e., one end can be conjugated to an active group of the nucleic acid, and the other end can be attached to a specific site (e.g., SH) on the antibody.

According to a preferred embodiment of the invention, all L-nucleic acids constituting the framework are added NH at the 5' end₂Modification followed by coupling of NH on nucleic acids via amide bond using linker, i.e., dual heterofunctional crosslinker reagent SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylate succinimidyl ester sodium salt)₂. The maleimide group at the other end of the linker is now free and can be used for subsequent conjugation to the thiol (SH) group on the antibody, thus completing the activation of the L-nucleic acid.

2. Method for producing antibody-L-nucleic acid complex

First, the 5 'or 3' end of the L-nucleic acid is substituted with NH₂Modification, then according to the difference of the linker, can be following several main preparation methods, wherein one end functional group of the linker is NHS (N-hydroxysuccinimide) or Sulfo-NHS (N-hydroxysuccinimide sulfonic acid sodium salt), for fast coupling of NH at one end of L-nucleic acid₂A group. Linkers containing dual hetero-functional groups are all first bound to NH of an L-nucleic acid₂And secondly, after reducing the sulfhydryl on the antibody, reacting the other end of the sulfhydryl with the sulfhydryl to form a stable chemical bond.

2.1 Maleimide (Maleimide). The linker is used to conjugate the thiol group of the antibody with maleimide. Maleimide reacts rapidly with free thiols on the antibody to form thioether bonds. Common linkers are SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate), sm (peg) (polyethylene glycol-modified succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate), and the like.

2.2 Haloacetyl (Haloacetyl). The linker is used to conjugate to a thiol group on the antibody as a haloacetyl group, such as iodine, bromoacetyl. The halogen ion can replace the sulfhydryl on the antibody through nucleophilic action to form stable thioether bond. Common linkers are SBAP (N-maleimidomethyl [ 4-bromoacetyl ] aminobenzoate), SIAB (N-maleimidomethyl [ 4-iodoacetyl ] aminobenzoate), and the like.

2.3 Thiopyridine (Pyridyldithiol). The linker group used to conjugate to a thiol group on the antibody is thiopyridine. Thiopyridines can react with free thiols to form disulfide bonds. Common linkers are SPDP (N-hydroxysuccinimide 3- (2-pyridyldithio) propionate) and the like.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention can be used directly for therapy (e.g., anti-tumor therapy), and thus can be used to prolong the half-life of the drug, and in addition, other therapeutic agents can be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention are:

(1) the half-life period prolonging module can be used for finishing the assembly with target protein by utilizing the left-handed nucleic acid chain mediation within one minute;

(2) the half-life prolonging factor in the half-life promoting module has wide modification space, and HSA or HSA binding protein, small molecules and polypeptide (such as anti-HSA single-chain antibody, nano-antibody and Fab) in any form can be selected;

(3) the half-life period improving module can be independently stored for later use, and then various protein or polypeptide medicines are assembled and coupled through simple nucleic acid pairing;

(4) the half-life period prolonging module can flexibly prepare long half-life period medicines containing different numbers of half-life period prolonging modules according to requirements so as to flexibly adjust pharmacokinetic properties;

(5) according to the half-life extension module, the half-life extension factors are all low-cost and easily prepared proteins or polypeptides, such as an anti-HSA nano antibody capable of being expressed in escherichia coli, HSA protein with the concentration of 50g/L in human blood and the like, so that the long-acting drug prepared based on the module has the advantages of simplicity in preparation, strong universality, low cost and the like;

(6) the invention provides a flexible preparation method for connecting a drug with an FcRn targeting factor and application of the drug in prolonging the half-life period of the drug in an animal body through an FcRn-mediated drug recovery path.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The experimental materials referred to in the present invention are commercially available without specific reference.

Example 1 design of the structural framework of tetrameric DNA

Four L-nucleic acids were designed that could pair in a quadrilateral shape (as shown in FIG. 2). Wherein any one of the L-nucleic acid single strands can be specifically complementarily paired with the other two L-nucleic acid single strands, but not with the fourth strand. The absolute value of the gibbs energy variation Δ G of the specific complementary pair is much larger than that of the non-specific pair, and the absolute value of the gibbs energy variation Δ G of the specific complementary pair of each arm is larger than 25 kcal per mole (kcal/mole), while the absolute value of the gibbs energy variation Δ G of the non-specific complementary pair of each arm is smaller than 7 kcal per mole (kcal/mole), which means that the assembly mode of the tetramer is easier to occur than that of the non-specific pairwise pair, and the form of the tetramer is most stable in the reaction system.

The four L-DNA single-stranded sequences designed according to the above principle are as follows (from 5 'to 3'):

chain 1(L-DNA 1): SEQ ID NO.1

5'-AAGAGGACGCAATCCTGAGCACGAGGTCT-3'

Strand 2(L-DNA 2): SEQ ID NO.2

5'-AACTGCTGCCATAGTGGATTGCGTCCTCT-3'

Strand 3(L-DNA 3): SEQ ID NO.3

5'-AATGAGTGCATTCGGACTATGGCAGCAGT-3'

Chain 4(L-DNA 4): SEQ ID NO.4

5'-AAGACCTCGTGCTCACCGAATGCACTCAT-3'

Having NH at the 5' end₂Group modification for coupling to NHS of SMCC. The base sequence of either strand is complementary-paired with the other two strands, and the amount of change in paired Gibbs energy Δ G of each portion is about-34 kcal per mole (kcal/mole).

Example 2 Synthesis and validation of the tetrameric DNA framework

Synthesis of 5' NH by Biotechnology services (e.g., Chemmene Inc., Biosyn Inc., etc.)₂The sequence of the modified L-DNA single strand, four single strands, is shown in example 1.

With phosphate buffer (50mM NaH)₂PO₄150mM NaCl, pH7.0) was dissolved to prepare a mother liquor having a final concentration of 200 uM. The SMCC powder was dissolved in dimethyl sulfoxide (DMSO) and 250mM SMCC stock was freshly prepared. Adding 10-50 times molar mass of SMCC mother liquor into L-DNA single-stranded mother liquor, quickly mixing, and reacting at room temperature for 30 min-2 h. After completion of the reaction, 10% by volume of 1M Tris-HCl (pH 7.0) was added to the reaction solution, and after mixing, the reaction solution was incubated at room temperature for 20 minutes for terminating the reaction of excess SMCC. After the incubation is finished, 100% absolute ethyl alcohol with 2 times volume of the reaction solution is added, and the mixture is placed in a refrigerator with the temperature of-20 ℃ for 25 minutes after being uniformly mixedThe L-DNA was precipitated sufficiently. The pellet was centrifuged (12000rpm, 10min), washed with 1mL 70% ethanol, centrifuged at 12000rpm for 1min to remove the supernatant, and the washing was repeated 5 times to remove the excess SMCC sufficiently. And naturally drying the remaining white precipitate in the air for 5-10 min, and then re-suspending and dissolving by using a phosphate buffer solution to obtain the SMCC-L-DNA compound (namely the SMCC-L-DNA single strand).

The concentration of each SMCC-L-DNA single strand was determined. Taking a proper amount of four SMCC-L-DNA single strands to be reacted, preheating at 40 ℃ for 5min, then mixing four SMCC-L-DNA single strands with equimolar amount under the condition of 40 ℃, and incubating for 1 min. 0.25ul of SMCC-L-DNA single strand and the reaction product were analyzed by 3% agarose gel electrophoresis.

As shown in FIG. 3, the size of the single-stranded SMCC-L-DNA is about 25bp, while the main band formed by mixing is about 100bp, which indicates that four different single-stranded SMCC-L-DNA forms a tetrameric framework.

EXAMPLE 3 preparation of half-Life prolonging factor-anti-HSA Nanobody mutants

Cysteine mutation is introduced into the carboxyl terminal of the anti-HSA nano antibody. The nano antibody has disulfide bonds, but can be expressed in the cytoplasm of escherichia coli because of small molecular weight and abnormally stable structure.

The gene sequence of the nano antibody for resisting HSA is optimized to a codon preferred by Escherichia coli, and then the nano antibody is subcloned into pET21b plasmid.

The amino acid sequence of the nano antibody for resisting HSA is SEQ ID NO. 5. For purification, double Strep labels are added at the N ends of the nano antibodies.

SEQ ID NO.5, amino acid sequence of anti-HSA nanobody mutant:

SAWSHPQFEKGGGSGGGSGGSAWSHPQFEKENLYFQSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGSC

respectively transforming Escherichia coli BL21(DE3) with 1ul of the constructed expression vector, picking out single colony of transformed BL21(DE3) to LB culture medium (containing 100ug/mL ampicillin), culturing at 37 ℃ until OD600 is 0.7, adding IPTG with final concentration of 1mM for induction expression, and continuously culturing at 37 ℃ for 3-4 hours. The cells after completion of expression were collected by centrifugation and resuspended in phosphate buffer (50mM NaH)₂PO₄150mM NaCl, pH7.0), protease inhibitor cocktail (cocktail) (Sigma) (Add one tablet of SIGMAFAST to solution)^TMProtease inhibitor tablets were fully dissolved) and disrupted by sonication. DNaseI hydrolase was added and incubated on ice for 1 hour. After the incubation, the supernatant was collected by centrifugation at 17000rpm for 20 minutes. The nanobodies in the supernatant were purified using a Strep affinity column, after the supernatant was passed through the column at a rate of 0.25ml/min, the column was washed with a large amount of Tris buffer (50mM Tris-HCl, 150mM NaCl, pH 7.4) at a flow rate of 1ml/min until the hetero-proteins no longer flowed out (according to UV absorption on AKTA protein chromatography system), and the nanobodies bound to the column were gradient-eluted using a Strep-Tactin elution buffer (50mM Tris-HCl, 150mM NaCl, 2.5mM DethiobiotinpH 7.4).

As a result: obtaining the high-purity anti-HSA nano antibody mutant.

EXAMPLE 4 conjugation and purification of Nanobody-L-DNA

The purified single-chain antibody is incubated with reducing agent (such as TCEP, DTT, mercaptoethanol, etc.) in 10-50 times molar excess for 30min at room temperature. After the incubation, the reducing agent in the reaction system was rapidly removed by a PD-10 desalting column while replacing the buffer with a phosphate buffer (50mM NaH)₂PO₄150mM NaCl, pH 7.0). Immediately after the measurement of the concentration of the single-chain antibody, 1 to 4-fold molar excess of SMCC-L-DNA single chain (prepared in example 2) was added, mixed well and reacted at room temperature for 1 hour.

Removing unreacted and excessive single chains of SMCC-L-DNA by using a Strep affinity column, and collecting the nano-antibody and the nano-antibody-L-DNA mixture. The buffer was replaced with the loading buffer of the anion exchange column.

Since nucleic acids such as DNA are negatively charged, nanobody-L-DNA is further separated and purified by an anion exchange column (HiTrap Q HP column), and unreacted nanobody is removed. The separation process is realized by gradient elution, the loading buffer solution is 50mM Tris-HCl, pH 8.5, the elution buffer solution is 50mM Tris-HCl, 1M NaClpH 8.5, and 0-100% of elution buffer solution is subjected to gradient elution, and unreacted nano antibody and nano anti-antibody are subjected to gradient elutionThe body-L-DNA shows peaks successively. Collecting nano antibody-L-DNA, concentrating, and replacing buffer with 50mM NaH by PD-10 desalting column₂PO₄，150mM NaCl，pH 7.4。

Example 5 self-Assembly of half-Life enhancing Module with protein of interest drugs

The process of self-assembly of the half-life enhancing module with a protein drug of interest is described below, taking as an example a long-acting factor eight (FVIII) drug design for treatment of hemophilia a. In this embodiment, the octafactor is used as a target protein drug, and the octafactor may be a blood-derived octafactor extracted from human blood, or a recombinant octafactor obtained by genetic engineering.

Mixing factor VIII with 10-50 times molar excess of reducing agent (such as TCEP, DTT, mercaptoethanol, etc.), and incubating at room temperature for 30 min. After the incubation, the reducing agent in the reaction system was rapidly removed by a PD-10 desalting column while replacing the buffer with a phosphate buffer (50mM NaH2 PO)₄150mM NaCl, pH 7.0). Immediately after the concentration of the octamer was determined, 1 to 4-fold molar excess of SMCC-L-DNA single strand (prepared in example 2) was added, mixed well and reacted at room temperature for 1 hour. The product after reaction is the drug unit: octafactor-L-DNA.

The concentrations of the anti-HSA nanobody-L-DNA and the octafactor-L-DNA were measured, respectively. Preheating a proper amount of the components at 40 ℃ for 5min, mixing the HSA nanobody-L-DNA and the octafactor-L-DNA according to a molar ratio of 1:1 at 40 ℃, and incubating for 1 min. Thus, FVIII-HSAnb comprising the half-life enhancing module and the octafactor active moiety is assembled, i.e. long acting octafactor.

FVIII-deficient mice 6-8 weeks old were used to demonstrate the half-life extending effect of FVIII-HSAnb in vivo. 8 mice were randomly divided into experimental and control groups, and FVIII-HSAnb and FVIII were intravenously injected at a dose of 5 IU/mouse, respectively. The blood sampling time is one day before administration, 5min after administration, 30h,48h,60h, 72h and 96 h. After blood sampling, the plasma was rapidly separated and frozen in a-80 ℃ freezer. After all plasma samples were obtained, FVIII activity was assayed using the APTT method.

The half-lives of FVIII and FVIII-HSAnb were calculated to be 5.6h and 22.7h, respectively (as shown in FIG. 4).

Therefore, the FVIII-HSAnb designed by the invention can prolong the half-life by about 4 times compared with the common FVIII.

Discussion of the related Art

The IgG type monoclonal antibody (monoclonal antibody) has the longest half-life in the immunoglobulin type antibody, and can be as long as 21 days. Thus, IgG-type antibodies are the predominant type of therapeutic antibodies at present.

The long half-life and high serum concentration of IgG-type mabs is achieved by binding to the neonatal receptor (FcRn) and entering its mediated metabolic regulation. The Fc portion of IgG can undergo specific pH-dependent binding to FcRn. After the IgG type monoclonal antibody is endocytosed by hematopoietic cells or vascular endothelial cells in blood through endocytosis, the IgG type monoclonal antibody can enter an endosome (endosome) and be combined with FcRn in the blood, so as to enter a recycling path, avoid entering a degradation path, be degraded by lysosome (lysosome), circulate to the cell surface, be dissociated from the FcRn under neutral physiological conditions (pH 7.4) and be released into the blood.

Albumin is another protein that can extend half-life by binding to FcRn, which can be as long as 19 days. Human serum albumin is divided into four domains I-IV, in which domain III is the portion that binds FcRn and domain I contains a free Cysteine residue (Cysteine 34, Cys 34).

For protein or polypeptide drugs with long half-life requirements, such as cytokine drugs, coagulation factors, etc., FcRn binding proteins, such as Fc and HSA, are better candidates.

Methods for extending half-life of Fc or HSA fusion proteins will generate novel epitopes at the site where the protein or polypeptide of interest is fused to Fc/HSA. Furthermore, Fc can only be fused to the C-terminus, while HSA can only be fused to the N-or C-terminus of the protein or polypeptide of interest. Furthermore, this method is not applicable to native proteins or polypeptides.

The invention develops a chemical coupling method for the first time, which specifically realizes heterologous coupling of protein or polypeptide and an FcRn binding module (HSA or HSA binding module and the like) by utilizing a chemical molecule or a connector, thereby prolonging the half-life period of protein drugs and improving the pharmacokinetic properties of the protein drugs.

Compared with Fc or HSA fusion proteins, the method of the present invention is a more flexible scheme, and can be applied to various groups (such as SH, NH) of target protein or polypeptide₂Active groups on non-natural amino acids, etc.) can also be applied to the modification of natural proteins or polypeptides.

Furthermore, the methods of the invention do not or substantially do not introduce new epitopes.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Ansheng (Shanghai) pharmaceutical science & technology Co., Ltd

<120> half-life-prolonged drug, library thereof, preparation method and application

<130> P2019-1201

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

aagaggacgc aatcctgagc acgaggtct 29

<210> 2

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

aactgctgcc atagtggatt gcgtcctct 29

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

aatgagtgca ttcggactat ggcagcagt 29

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

aagacctcgt gctcaccgaa tgcactcat 29

<210> 5

<211> 155

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Asn

20 25 30

Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

35 40 45

Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

50 55 60

Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys

65 70 75 80

Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu

85 90 95

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

100 105 110

Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

115 120 125

Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln

130 135 140

Gly Thr Leu Val Thr Val Ser Ser Gly Ser Cys

145 150 155

<210> 6

<211> 18

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 6

Arg Leu Ile Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu

1 5 10 15

Asp Asp

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10

<210> 8

<211> 30

<212> RNA

<213> Artificial sequence (Artificial sequence)

<400> 8

guggacuaua ccgcguaaug cugccuccac 30