TWI719963B - Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same - Google Patents

Abstract

The invention is related to fusion proteins of human somatostatin (e.g., SST-14 or SST-28) and human serum albumin, comprising a region at least 85% homologous to human somatostatin and a region at least 85% homologous to human serum albumin or a region with a partial amino acid sequence of human serum albumin, wherein linker peptide sequences may be present between somatostatin and somatostatin moieties or somatostatin and albumin moieties. Also disclosed are constructs wherein the somatostatin moiety contains multiple tandem repeats of a somatostatin sequence. In selected embodiments, the orientation of the somatostatin and albumin moieties can be varied, and such sequences may impact the binding and efficacy of the disclosed fusion proteins. Also disclosed are methods of making and using the aforementioned constructs.

Description

Composition containing albumin and its analogue fusion protein, its preparation and use method Cross reference of related applications

This application claims the right of priority in the U.S. Provisional Patent Application No. US 62/121,487 filed on February 26, 2015, and the content of the case is incorporated herein by reference.

The present invention relates to a fusion protein comprising somatostatin (or an analog or derivative thereof), a linker or spacer, and albumin (or an analog or variant thereof).

The present invention also relates to a recombinant fusion protein containing a human serum albumin part and a somatostatin part separated by a spacer sequence and its analogs.

Somatostatin ("SST") is a secretion product of various endocrine and non-endocrine tissues and is widely distributed throughout the body. Sostatin inhibits the secretion and release of pituitary, pancreas and gastrointestinal hormones, as well as the production of cytokines, intestinal energy and absorption, vasoconstriction and cell proliferation. Recent studies have found that SST is useful for the treatment of cancer. It inhibits tumor growth, inhibits the proliferation of endocrine tumors and many other solid tumors (such as breast cancer, colorectal cancer, liver cancer, and lung cancer). Somatostatin molecules have two biologically active forms: somatostatin-14 (SST-14, cyclic myristin) and somatostatin-28 (SST-28, the N-terminal extended form of SST-14). SST-14 is a 14-residue cyclic peptide containing disulfide between the cysteine at position 3 and 14 key. SST-28 is the N-terminal extended form (28 residues) of the same precursor, which is proteolytically cleaved to produce SST-14. Although the two have similar activities, their respective efficacy and histological characteristics are different. For example, SST-14 exhibited more significant inhibition of glucagon and gastrin, while SST-28 exhibited more significant inhibition of growth hormone and insulin. Both forms of somatostatin exert their respective biological functions through receptors on target cells and intracellular pathways. Five somatostatin receptor subtypes (SSTR 1-5) have been identified. SSTR2 has two splice variants with different carboxyl termini: SSTR2A and SSTR2B.

The industry has fully realized the beneficial effect of sostatin on the treatment of certain hypersecretion endocrine disorders and its anti-proliferative effect on tumors. However, due to enzymatic degradation and endocytosis, the half-life of somatostatin in vivo is only 2-3 min, which limits the clinical utility of somatostatin. In the past decade, several stable somatostatin analogs have been developed. For example, octreotide and lanreotide are used to treat growth hormone (GH)-secreting adenomas and carcinoids. However, due to the change in binding affinity to SSTR, there are still treatment limitations. Therefore, in this technology, there is still a need for a somatostatin construct that achieves a high in vivo half-life while maintaining the desired binding affinity for SSTR.

Albumin, the most abundant protein in plasma, is produced in the liver as a monomer protein of 67kDa and it generates 80% of the colloidal osmotic pressure in plasma. Human granule ball community stimulating factor (G-CSF), human growth hormone (GH), human insulin, human interferon-a-2b (INF-2b) and interleukin-28B (IL-28B) fused with HSA are effective It is used to construct long-acting candidate therapeutic drugs. However, there are few reports of comparative studies on the biology and molecular mechanism between the HSA fusion protein and the parent molecule.

Chinese patent applications CN102391376A and CN102675467A (both of which are incorporated herein by reference) disclose somatostatin-albumin fusion proteins. However, there is still a need for further development of somatostatin-albumin fusion protein.

The present invention provides somatostatin-albumin fusion protein and its analogues, and its production and use Use method. The construct prepared according to the present invention includes an albumin (or its analogue) part, a somatostatin part (SST-14, SST-28) and a spacer (such as a spacer peptide or a linker peptide) separating the two parts .

In one embodiment, the present invention provides a nucleotide sequence encoding an albumin-sostatin fusion protein or polypeptide sequence, which comprises: (a) a region comprising a nucleotide sequence encoding a human somatostatin peptide (SST) (B) A region containing a nucleotide sequence encoding human serum albumin or a fragment thereof (ALB); (c) A spacer region (L) containing a nucleotide sequence encoding a polypeptide of 2 to 100 residues in length ); Wherein, the spacer sub-region (c) exists between the region (a) and the region (b), or between the region (a) and the region (a).

In a specific embodiment, the nucleotide sequence is selected as appropriate to encode an albumin-somastatin fusion protein consisting of one of the following formulas I-X.

SST-(L) _x1 -ALB (I); ALB-(L) _x1 -SST (II); [SST-(L) _x1 ] _y1 -ALB (III); ALB-[(L) _x1 -SST] _y1 (IV); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 (V); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 -(L) _x3 -ALB (VI); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 -(L) _x3 -ALB-[(L) _x4 -SST] _y3 ( _{VII); ALB- (L) x1} - [SST- (L) x2] y1 -ALB (VIII); ALB- (L) x1 - [SST- (L) x2] y1 -ALB - [(L) x3 - SST] _y2 -(L) _x1 -ALB (IX); and ALB-(L) _x1 -[SST-(L) _x2 ] _y1 -ALB-[(L) _x3 -SST] _y2 -(L) _x1 -ALB -[(L) _x4 -SST] _y3 (X); where x1, x2, x3, x4, y1, y2 or y3 are each independently 0 or an integer selected from 1 to 10, provided that at least one L exists in In the nucleotide sequence encoding the albumin-sostatin fusion protein.

The fusion protein according to the present invention is also described herein as a polypeptide. The polypeptide according to the present invention may optionally include one or more non-natural amino acid or amino acid residues in certain embodiments.

Somatostatin-albumin fusion protein and its analogs roughly include human SST peptide part, linker or spacer and human albumin part. The SST peptide portion may include its analogs and derivatives that actively inhibit the activity of human growth hormone. Depending on the circumstances, the SST peptide portion is obtained from natural or synthetic sources. The albumin portion is, for example, human albumin and/or active fragments or subdomains thereof. The linker or spacer is selected to enhance the stability of the somatostatin-albumin fusion protein. More specifically, somatostatin-albumin fusion protein and its analogs have the following structure.

In one embodiment, the present invention provides a fusion protein comprising: (a) a region containing human somatostatin peptide (SST); (b) a region containing human serum albumin or a fragment thereof (ALB); (c) A spacer region (L) containing a polypeptide with a length of 2 to 100 residues; wherein the spacer region (c) exists between the region (a) and the region (b), or the region (a) and the region ( a) Between.

In another embodiment, the present invention includes a fusion protein comprising a structure of formula IX selected from: SST-(L) _x1 -ALB (I); ALB-(L) _x1 -SST (II); [SST -(L) _x1 ] _y1 -ALB (III); ALB-[(L) _x1 -SST] _y1 (IV); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 ( V); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 -(L) _x3 -ALB (VI); [SST-(L) _x1 ] _y1 -ALB-[(L ) _x2 -SST] _y2 -(L) _x3 -ALB-[(L) _x4 -SST] _y3 (VII); ALB-(L) _x1 -[SST-(L) _x2 ] _y1 -ALB (VIII); ALB -(L) _x1 -[SST-(L) _x2 ] _y1 -ALB-[(L) _x3 -SST] _y2 -(L) _x1 -ALB (IX); and ALB-(L) _x1 -[SST-( L) _x2 ] _y1 -ALB-[(L) _x3 -SST] _y2 -(L) _x1 -ALB-[(L) _x4 -SST] _y3 (X); where x1, x2, x3, x4, y1 y2 or y3 are each independently 0 or an integer selected from 1 to 10, provided that at least one L is present in the fusion protein.

In another embodiment of the present invention, x1, x2, x3, and x4 are each independently selected from an integer of 1 to 5, or an integer of 1 to 4. In another embodiment of the present invention, y1, y2, and y3 are each independently selected from an integer from 1 to 5, or an integer from 1 to 4.

In a more specific embodiment of the present invention, the SST portion comprises a sequence encoding SST-14 or SST-28 (represented as SEQ ID NO: 17 or 18, respectively) or has at least 85% identity with any of these sequences One or more tandem repeats of the sequence.

The SST part is SST-14 or SST-28 depending on the situation.

In certain embodiments, the somatostatin-albumin fusion protein of the present invention includes variants of albumin, including mammalian serum albumin, such as human serum albumin, for example, having SEQ ID NO: 25, or having A sequence with at least 85% sequence identity.

The linker or spacer according to another embodiment of the present invention encompasses a peptide that is covalently linked to somatostatin on one end and to albumin on the other end. In another embodiment of the present invention, the spacer includes a peptide sequence having 2 to 100 amino acid residues.

The terms "linker" or "spacer" are used interchangeably herein and refer to a short amino acid sequence used to separate multiple domains in a single protein. Two in protein or The lack of linkers between more discrete domains can lead to reduced or improper functionality of protein domains, for example, due to steric hindrance, reduced catalytic activity or binding affinity to receptor/ligand. The use of linker domains of artificial linkers in chimeric proteins can increase the space between the domains. Preferably, the choice of linker or spacer has nothing to do with somatostatin and albumin.

Linker L is a flexible or alpha-helix structured polypeptide linker or spacer. In certain embodiments, L contains at least one GGGGS, A(EAAAK) ₄ A, (AP)n (wherein n is an integer selected from 10 to 34), (G)8, (G)5, or any combination thereof .

The albumin-sostatin fusion construct described herein may also include a signal peptide sequence ("SP"). The signal peptide is understood to refer to the short amino acid sequence present at the N-terminus of the polypeptide, which guides the cellular localization of the newly synthesized protein. For example, the signal peptide can cause the protein to be localized to a given intracellular region (e.g., nucleus), inserted into the membrane (e.g., cell membrane or endoplasmic reticulum), or secreted from the cell. In addition to guiding localization, signal peptides can also be incorporated into recombinant proteins to improve stability, change the degree of performance, and help the recombinant protein to fold properly. The signal peptide sequence of the precursor protein is usually removed by the signal peptidase in the host cell to produce the mature protein.

The albumin-sostatin fusion construct described herein may also include an affinity or purification tag as part of the polypeptide sequence to facilitate purification. These tags are used as part of affinity chromatography methods (eg, high performance liquid chromatography (HPLC)) to purify protein samples from crude biological sources. Suitable purification tags include (but are not limited to): polyhistidine (for example, His-6 or H6), glutathione-S-transferase (GST), maltose binding protein (MBP), chitin binding protein ( CBP), FLAG-tag (FLAG octapeptide). When it is necessary to remove the affinity tag from the fusion protein, a specific enzyme cleavage site can be introduced in the linker region. Enzymes commonly used to remove affinity tags include (but are not limited to): factor Xa, enterokinase, thrombin, TEV protease, and rhinovirus 3C protease.

The present invention also provides a pharmaceutical group comprising the fusion protein of the present invention by administering an effective amount A method for treating endocrine-releasing diseases or disorders in mammals (such as human subjects), wherein the endocrine-releasing diseases or disorders are conditions responsive to the administration of somatostatin.

For example, the disease or condition is a cancer selected from the group consisting of breast cancer, colorectal cancer, liver cancer, endocrine cancer, neuroendocrine cancer, pancreatic cancer, prostate cancer, and lung cancer. In certain embodiments, the cancer exhibits somatostatin receptor type 1, 2, 3, 4, or 5.

It should also be understood that the use of the singular form (such as "一(a)", "一(an)" and "the") throughout this application is for convenience, however, unless the context or clear statement otherwise There are instructions, otherwise these singular forms are intended to include plural meanings. In addition, it should be understood that every journal article, patent, patent application, publication, and the like mentioned in this article is incorporated herein by reference in its entirety and used for all purposes.

All numerical ranges should be understood to include every numerical point within this numerical range, and should be interpreted as enumerating each numerical point individually. The endpoints of all ranges for the same components or properties are included and are intended to be independently combinable.

As used herein, the term "about" means within 10% of the reported value, preferably within 5% of the reported value.

The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not substantially change the claimed composition or method The basic and novel characteristics of this.

The fusion protein of SST and albumin in this application provides advantages over natural SST: (a) higher in vivo stability, (b) higher binding affinity with SST receptor, (c) higher Protein performance yield, and (d) better pharmacokinetic/pharmacodynamic characteristics.

Before describing the present invention in detail below, it should be understood that since the specific methods, schemes and reagents described herein are variable, the present invention is not limited to these elements. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention. As a result, the scope of the present invention is only limited by the scope of the attached patent application. Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as commonly understood by ordinary technicians.

The present invention covers somatostatin-albumin fusion proteins and their analogs and methods of their production and use. The construct prepared according to the present invention includes an albumin (or its analogue) part, a somatostatin part (for example, SST-14, SST-28) and a spacer separating the two parts.

The somatostatin-albumin fusion protein of certain embodiments of the present invention includes a variant of albumin (including human serum albumin) and/or a derivative of somatostatin. The spacer of another embodiment of the present invention encompasses peptides that are covalently linked to somatostatin on one end and to albumin on the other end. The spacer in other embodiments of the present invention includes a peptide sequence having 2 to 100 amino acids.

In one embodiment, the present invention provides a fusion protein comprising: SST; L; and ALB, wherein SST is based on somatostatin or an analog or derivative thereof; L based spacer or linker; ALB based on albumin or Its analogs or variants.

In some embodiments, the fusion protein of the present invention is selected from the following formula I-X.

SST-(L) _x1 -ALB (I); ALB-(L) _x1 -SST (II); [SST-(L) _x1 ] _y1 -ALB (III); ALB-[(L) _x1 -SST] _y1 (IV); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 (V); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 -(L) _x3 -ALB (VI); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 -(L) _x3 -ALB-[(L) _x4 -SST] _y3 ( _{VII); ALB- (L) x1} - [SST- (L) x2] y1 -ALB (VIII); ALB- (L) x1 - [SST- (L) x2] y1 -ALB - [(L) x3 - SST] _y2 -(L) _x1 -ALB (IX); and ALB-(L) _x1 -[SST-(L) _x2 ] _y1 -ALB-[(L) _x3 -SST] _y2 -(L) _x1 -ALB -[(L) _x4 -SST] _y3 (X); where x1, x2, x3, x4, y1, y2 or y3 are each independently 0 or an integer selected from 1 to 10, provided that at least one L is present in Fusion protein.

In another embodiment, the present invention provides a nucleotide sequence encoding an albumin-sostatin fusion protein, the fusion protein comprising: SST; L; and ALB, wherein SST is a somatostatin or an analogue or derivative thereof物; L-based spacer or linker; ALB-based albumin or its analogs or variants.

In certain embodiments, the nucleotide sequence of the present invention is selected to encode the albumin- _{somastatin fusion protein from the following: SST-(L) x1} -ALB (I); ALB-(L) _x1 -SST (II); [SST-(L) _x1 ] _y1 -ALB (III); ALB-[(L) _x1 -SST] _y1 (IV); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 (V); [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 -(L) _x3 -ALB (VI); [SST-(L) _x1 ] _{y1 -ALB - [(L) x2 -SST} ] y2 - (L) x3 -ALB - [(L) x4 -SST] y3 (VII); ALB- (L) x1 - [SST- (L) x2] y1 - ALB (VIII); ALB-(L) _x1 -[SST-(L) _x2 ] _y1 -ALB-[(L) _x3 -SST] _y2 -(L) _x1 -ALB (IX); and ALB-(L) _x1 -[SST-(L) _x2 ] _y1 -ALB-[(L) _x3 -SST] _y2 -(L) _x1 -ALB-[(L) _x4 -SST] _y3 (X); where x1, x2 x3, x4, y1, y2, or y3 are each independently 0 or an integer selected from 1 to 10, provided that at least one L is present in the nucleotide sequence encoding the albumin-sostatin fusion protein.

Another embodiment of the present invention provides a nucleotide sequence encoding an albumin-sostatin fusion protein, wherein the spacer sequence is composed of a sequence encoding the amino acid sequence represented by SEQ ID NO: 31 or -GGGGS-.

Another embodiment of the present invention provides a nucleotide sequence encoding an albumin-sostatin fusion protein, wherein the second region (b) encodes at least 85% sequence with SEQ ID NO: 19 (albumin or fragments thereof) Consistent peptide.

An embodiment of the present invention provides a nucleotide sequence encoding an albumin-sostatin fusion protein, wherein the first region (a) encodes SEQ ID NO: 17 or 18 (SST-14, SST-28) or fragments thereof A polypeptide with at least 85% sequence identity.

The present invention also provides a nucleotide sequence encoding an albumin-sostatin fusion protein, which comprises: (a) a region containing one or more adjacently repeated nucleotide sequences of the sequence encoding human somatostatin peptide (B) A region containing a nucleotide sequence encoding human serum albumin or a fragment thereof; (c) A spacer region containing a nucleotide sequence encoding a polypeptide having a length of 2 to 100 residues; wherein, the spacer Region (c) exists between region (a) and region (b) or between region (a) and region (a); among them, one or more adjacent repeats of the sequence encoding human somatostatin peptide SST-14 or SST-28 (such as SEQ ID NOs: 17 and 18, respectively), or a sequence that has at least 85% identity with one of the two sequences; or wherein the spacer sequence is represented by the code as SEQ ID NO: 31 or GGGGS or the amino acid sequence of SEQ ID NO: 30A (EAAAK) ₄ A); or wherein, the region (a) is composed of SST-14 or SST-28 (as shown as SEQ ID NO: 23 and 24) one or more adjacent repeats or a sequence composition having at least 85% identity with either of the two sequences.

In addition, the present invention provides a polypeptide sequence of an albumin-sostatin fusion protein, which comprises: (a) a region comprising a polypeptide sequence of a somatostatin peptide (which may be a human somatostatin peptide); (b) a region comprising serum albumin A region of the polypeptide sequence of a protein (which may be human serum albumin) or a fragment thereof; (c) a spacer region comprising a polypeptide of 2 to 100 residues in length.

The spacer sub-region (c) may exist between the region (a) and the region (b) or between the region (a) and the region (a). In addition, region (a) may include one or more of the sequence encoding SST-14 or SST-28 (represented as SEQ ID NO: 17 or 18, respectively) or a sequence that has 85% identity with one of these sequences Tandem repeats.

Another embodiment of the present invention provides a plastid construct, which represents an albumin-somatostatin fusion protein having any fusion protein or polypeptide sequence described above.

Another embodiment of the present invention includes using the above-mentioned plastid construct to transform Bacterial cells.

Another embodiment of the present invention includes an albumin-body that has been isolated and purified and has the above-mentioned polypeptide sequence (for example, an albumin-somstatin fusion protein or a polypeptide sequence expressing a plastid construct of the protein) Inhibin fusion protein.

For fusion proteins (such as SEQ ID NOs: 1-5, 7-10, and 13-16), it should be noted that these fusion proteins are used as a signal peptide (SEQ ID NO: 20) with 22 residues. To code.

Somatostatin-albumin fusion protein

The present invention covers polypeptide constructs, wherein the somatostatin part is at least 85% sequence identity with the nucleotide sequence of endogenous human SST-14 or SST-28 (SEQ ID Nos: 23 and 24, respectively) The nucleotide code.

The present invention also covers polypeptide constructs in which the human serum albumin part is encoded by nucleotides having at least 85% sequence identity with the nucleotide sequence of endogenous human serum albumin (SEQ ID NO: 25). The present invention further encompasses polypeptide constructs in which the human serum albumin part is a fragment of endogenous human serum albumin protein, for example, in which it is encoded by a nucleotide consisting of a subsequence of SEQ ID NO:25. For example, the human serum albumin fragment optionally includes one or more of the three human serum albumin globular domains, each of which contains two subdomains, called subdomains IA, IB, IIA, IIB, IIIA and IIIB (Dockal, 1999, The Journal Of Biological Chemistry, 274 (41): 29303-29310).

The present invention also covers polypeptide constructs, wherein the somatostatin portion has a polypeptide sequence that is at least 85%, preferably at least 90%, and endogenous SST-14 or SST-28 polypeptide sequence (SEQ ID NOs: 17 and 18, respectively) Polypeptide sequence with sequence identity.

The present invention also covers polypeptide constructs, wherein the human serum albumin portion has a polypeptide sequence that has at least 85% sequence identity with the polypeptide sequence of mature human serum albumin (SEQ ID NO: 19).

The present invention also covers a fusion protein, which includes a signal peptide, a purification tag (His-6), a first linker, a human serum albumin part, a second linker and a somatostatin part. In one embodiment, the fusion protein is represented by the polypeptide of SEQ ID NO: 9 or a sequence with 85% sequence identity.

The present invention also covers a fusion protein comprising a somatostatin part, a first linker, a human serum albumin part, a second linker, a somatostatin part, and a purification tag (His-6). In one embodiment, the fusion protein is represented by the polypeptide of SEQ ID NO: 10 or a sequence with 85% sequence identity.

The present invention also covers a nucleotide sequence (SEQ ID NO: 11) which encodes a fusion protein comprising an N-terminal human serum albumin portion and a C-terminal somatostatin portion separated by a peptide spacer. The present invention further covers a nucleotide sequence that encodes an albumin-somatostatin fusion construct having 85% sequence identity with SEQ ID NO:11.

The present invention also covers the nucleotide sequence (SEQ ID NO: 12), the encoding of which includes the peptide A fusion protein of the N-terminal somatostatin part and the C-terminal human serum albumin part separated by a spacer. The present invention further covers a nucleotide sequence which encodes an albumin-somatostatin fusion construct having 85% sequence identity with SEQ ID NO:12.

The present invention also covers polypeptide constructs, wherein the somatostatin portion contains two or more copies of the SST-14 or SST-28 sequence arranged in tandem, namely "(SST-14) ₂ "or "(SST- 14) ₃ "or "(SST-28) ₂ " or "(SST-28) ₃ ". Optionally, the linker sequence is included between two or more tandem statin parts, and/or the signal peptide sequence is included at the N-terminus of the fusion protein.

The present invention also encompasses polypeptide constructs in which the somatostatin portion contains two or more copies of the SST-14 sequence, which are arranged in such a way that at least one copy of SST14 is attached to both sides of the albumin. Optionally, the linker sequence is included between two or more tandem sostatin parts and between somatostatin and albumin, and/or the signal peptide sequence is included at the N-terminus of the fusion protein. For example, the polypeptide construct may include a signal peptide, two SST-14 parts separated by a spacer, a second spacer and an HSA part as representatives. Optionally, the construct omits the N-terminal signal peptide.

The present invention also covers polypeptide constructs, wherein the somatostatin portion contains two or three copies of the SST-28 sequence arranged in tandem, namely "(SST-28) ₂ "or "(SST-28) ₃ ", respectively. Optionally, the linker sequence is included between two or more tandem somastatin moieties.

The present invention also covers polypeptide constructs comprising any albumin-sostatin fusion protein described in the preceding paragraph, wherein the in vivo half-life of the albumin-sostatin fusion protein is longer than that of endogenous SST-14 or SST- 28 peptides.

The present invention also covers polypeptide constructs, which comprise any albumin-sostatin fusion protein described in the preceding paragraph, wherein the albumin-sostatin fusion protein has a comparison with endogenous SST-14 or SST-28 Approximately equal or greater binding affinity to the somatostatin receptor.

The present invention also covers albumin-sostatin fusion protein, which comprises the expression SEQ ID The N-terminal albumin portion of NO: 15, SEQ ID NO: 16 and SEQ ID NO: 2, the internal SST portion, and the C-terminal albumin portion represented by SEQ ID NO: 7 and SEQ ID NO: 8. Optionally, the N-terminal may further include a signal peptide. Optionally, one or more albumin and SST domains can each be separated by an independently selected linker sequence (as represented by SEQ ID NO: 1).

In some embodiments, the SST portion may include a pair or a plurality of tandem SST sequences, for example, (SST-14) ₂ or (SST-28) ₃ , and contains or between two or more tandem SST repeats. Does not contain intermediate spacer sequences. Optionally, one or more purification tag sequences may be included between the two parts or in the sequence at the N-terminus or C-terminus to facilitate purification of the fusion protein. Alternative embodiments include a pair of SST-14 parts separated by a spacer, as represented by SEQ ID NO:4. In another embodiment, the purification tag (for example, His6) can be omitted, as shown in the polypeptide sequence represented by SEQ ID NO:5.

Somatostatin

The somatostatin used in the present invention can be any somatostatin, its analogue or derivative. It can be human somatostatin or any other isolated or natural somatostatin.

The present invention also encompasses polypeptide constructs, wherein the somatostatin portion includes somatostatin analogs. Preferably, this analog is suitable for expression as part of a fusion protein in a recombinant host cell. It should be understood that suitable somatostatin analog sequences can be used in place of the SST-14 or SST-28 sequences included in any of the examples disclosed herein.

The present invention also encompasses polypeptide constructs, wherein the somatostatin portion comprises two or more tandem repeats of the somatostatin polypeptide sequence (for example, SST-14 or SST-28, respectively; SEQ ID NOs: 17 and 18) . Each of the repeat somatostatin polypeptide sequences may be a polypeptide sequence that has at least 85% sequence identity with SST-14 or SST-28. The repeat variant sequences are independently selected, that is, in some embodiments the repeats are the same, while in other embodiments they are unique.

albumin

The albumin used in the present invention can be any albumin, its analogue or variant. White The protein may be human serum albumin, or any other isolated or natural albumin.

The present invention also encompasses polypeptide constructs, wherein the human serum albumin portion comprises a polypeptide sequence variant that has substitutions due to the presence of additional or fewer cysteine residues compared to the native form (SEQ ID NO: 25) The arrangement or number of sex disulfide bonds.

Spacer or linker

As mentioned earlier, spacers or linkers can be used in the present invention. The spacer or linker may not be related to somatostatin or albumin.

The present invention also encompasses polypeptide constructs in which a peptide spacer, which is alternatively called a linker, consists of a polypeptide sequence of about 2 to about 100 amino acid residues in length. The present invention further encompasses polypeptide constructs, wherein the peptide spacer is about 2 to about 50 amino acid residues in length, preferably about 2 to about 30, or more preferably about 3 to about 20 amino acid residues in length. Acid residues.

The present invention also encompasses polypeptide constructs in which the peptide spacer (alternatively referred to as a linker) has the polypeptide sequence "GGGGS" (SEQ ID NO: 31). Polypeptides rich in Gly, Ser and Thr provide special advantages: (i) the polypeptide backbone can rotate freely, allowing adjacent domains to move freely relative to each other; (ii) enhanced solubility; (iii) resistance to proteolysis. In addition, many natural linkers exhibit an α-helical structure. The α-helical structure is more rigid and more stable than the Gly-rich linker. An empirical rigid linker with the sequence of A (EAAAK) ₄ A (SEQ ID NO: 30) can be used to separate the functional domains. In addition to the role of linking protein domains together, artificial linkers can provide other advantages for the manufacture of fusion proteins, such as improved biological activity, increased protein performance, and desired pharmacokinetic properties.

Preparation of Sostatin-Albumin Fusion Protein

The embodiment of the present invention provides a method for preparing a somatostatin-albumin fusion protein. In the examples, the somatostatin-albumin fusion protein of the present invention is prepared by the expression of the recombinant fusion protein containing the coding gene and introducing the vector into the host. For example, the fusion protein is obtained by expression in a host such as yeast. For example, Pichia pastoris GS115 (Pichia pastoris GS115) can be used as a suitable expression host and used to construct a recombinant expression carrier system pPIC9K. In addition, mammalian strains such as CHO or HEK293 can be used as a better performance host.

The present invention also encompasses a plastid construct capable of expressing an albuminostatin fusion protein, which comprises a nucleotide sequence encoding the somatostatin albumin fusion protein as described in any of the preceding paragraphs. For example, suitable plastid constructs include (but are not limited to) the pcDNA3.1 vector represented by SEQ ID NO: 26, in which the DNA sequence encoding any albumin-sostatin fusion protein disclosed herein is linked thereto Multiple selection sites of the vector. Other suitable protein expression vectors known in the art can be selected based on the expression host (for example, expression vectors with a mammalian promoter system will be suitable for expression in human cell lines, and if desired in either yeast or bacteria In one of the expressions, the expression plastids of the organisms will be selected).

The present invention also encompasses a bacterial or yeast protein expression system comprising a bacterial or yeast cell transformed with a plastid construct comprising a somatostatin albumin fusion protein encoding as described in any of the preceding paragraphs Nucleotide sequence. Suitable bacterial strains include, for example, Escherichia coli . Suitable yeast strains include, for example, Pichia pastoris. Exemplary plastid constructs include pPIC9K (Invitrogen) as represented by SEQ ID NO: 27, wherein the nucleotide sequence encoding any albumin-sostatin fusion protein set forth herein is incorporated into multiple selections of the vector In the reproductive site.

The present invention also encompasses isolated and purified fusion statin fusion proteins, which have the polypeptide sequence as described in any of the preceding paragraphs.

The utility of somatostatin-albumin fusion protein

The fusion protein of the present invention can be used to treat conditions known in the industry that are treated with somatostatin. Therefore, the present invention also covers a method of treating cancer in a human individual by administering the isolated and purified albumin-sostatin fusion protein as described in any of the preceding paragraphs, wherein the cancer is selected from breast cancer, colorectal cancer , Liver cancer and lung cancer.

The present invention also encompasses a method of treating cancer in a human individual by administering a composition containing the fusion protein of the present invention (eg, an isolated and purified albumin-sostatin fusion protein as described in any of the preceding paragraphs). The composition may also include suitable carriers.

Design 11 SST14-albumin fusion protein constructs with different linker sequences. Eight of these constructs were made into plastid fusion genes and generated by instant expression of HEK293 on a 100mL scale. The protein is collected from the culture medium, purified by means of albumin-based affinity purification, and dialyzed against the storage buffer. The fusion proteins were evaluated for their binding affinity to the SSTR2 receptor, and for the cell-based activity that inhibits cAMP production in the SSTR2-overexpressing CHO-K1 cell line. The results of these studies indicate that the length and type of the linker significantly affect the SSTR2 receptor binding affinity, cell-based functional activity in vitro, and fusion protein yield.

Instance

The selected embodiments of the present invention will be explained in more detail with reference to the following experiments and comparative examples. These examples are for illustrative purposes only, and are not intended to limit the scope of the present invention.

Example 1: Performance in the mammalian system Example 1-1. Recombinant gene synthesis

Prepare 8 constructs (see Table 4). First, the gene is synthesized and then the target sequence is inserted into the pcDNA3.1 vector.

Example 1-2. Plastid generation

Use Maxi-prep or Mega-prep to generate approximately 20mg of each DNA

Examples 1-3. Transfection and protein production (A) Suspended cell method

FreeStyle ^TM 293-F cells were seeded in the flask ^{at 0.55-0.6×10 6 cells/mL.} After about 24 hours, the cells were seeded in the shake flask ^{at 1.1-1.2×10 6 cells/mL.} Prepare DNA at 500ug DNA/80mL in FreeStyle medium. PEI was prepared at 1.8 mL polyethyleneimine (PEI)/80 mL in FreeStyle medium. The DNA is mixed in FreeStyle medium, and an effective amount of PEI is added to the DNA solution, and the mixture is vortexed and incubated at room temperature for about 15 minutes to form a DNA-PEI complex. Add 80 mL of the incubated DNA-PEI complex to the cell culture. After about 3 hours, add TC Yeastolate feed (BD) to a final concentration of 4 g/L culture. After about 7 to 8 days, the medium is harvested by centrifugation.

(B) Adhesive cell method

About 24 hours before transfection, HEK293 cells were inoculated in the flask to a confluency of 50%-90%, and complete medium was added. After about 24 hours, the cells were washed, and then basal medium was added.

Prepare DNA and PEI solutions by adding DNA to serum-free medium. The PEI solution was added to the DNA solution and incubated at room temperature for 15 minutes to form a DNA-PEI complex.

The PEI/DNA mixture is added to the cells, and the mixture is incubated at 37°C for about 4 to 6 hours. The medium was removed and fresh medium containing glutamic acid and serum was added, followed by incubation at 37°C for 4 days.

After about 4 days, the medium was harvested, and the supernatant was collected by centrifugation. The sediment was supplemented with a fresh medium containing L-glutamic acid for an additional 3 days to repeat the harvesting process.

Example 1-4: Protein concentration, Ni-NTA purification and buffer exchange

In the end-view purification method (continuous chromatography or manual batch purification), the collected medium is concentrated to a certain volume by the TFF system (Millipore).

Using chromatography or batch system, the concentrated protein in the binding buffer at about 4 ℃ The solution was incubated with fresh Ni-NTA resin and washed with washing buffer. The protein is eluted with the elution buffer and the fractions are collected and concentrated to recover the purified protein. The protein can be further purified using particle size chromatography purification.

The buffer of the final eluate can be exchanged for the desired buffer by dialysis.

Example 2: The yield of several SST-albumin fusion proteins

SST-HSA fusion proteins are all expressed in soluble form with high yield. The length or nature of the linker can affect the protein yield and solubility of the fusion protein. The results indicate that as the fusion protein construct becomes longer and more complex, the yield decreases slightly. However, all structures exhibited yields for scale-up production.

Example 3: The binding affinity of several SST-albumin fusion proteins

This analysis measures the ^{binding of [125} I] somatostatin to the human somatostatin sst2 receptor. _{Using standard techniques, membranes were prepared using CHO-K 1} cells stably transfected with plastids encoding human somatostatin sst2 receptor in modified HEPES pH 7.4 buffer. Incubate 0.1 mg ^‡ aliquots of the membrane with 0.03 nM [ ¹²⁵ I] somatostatin and the tested fusion protein at 25°C for 240 minutes. Estimate non-specific binding in the presence of 1 μM sostatin. The membrane was filtered and washed 3 times and the filters were counted to determine the specifically bound [ ¹²⁵ I] somatostatin.

^{The competitive binding study of 125} I-Tyr-Sostatin relative to the fusion protein showed the following results. The inhibition efficiency differs depending on the structure of the fusion protein. The fusion protein construct (SEQ ID NO: 1) with two α-helical linkers A (EAAAK) ₄ A showed 100% inhibition of the interaction between somatostatin and its receptor. The SEQ ID NO:1 construct has two somatostatin parts on both the N-terminal side and the C-terminal side of human serum albumin. A smaller construct (SEQ ID NO: 2) with somatostatin linked by the same α-helical linker on the C-terminal side of human serum albumin showed 96% inhibition. The end-view length of the same construct with the more flexible GGGGS linker showed lower inhibition of 82% to 85%. The length of the GGGGS linker also affects inhibition. The construct of the GGGGS linker with 5 amino acids (SEQ ID NO: 9 and SEQ ID NO: 8) shows 57% to 59% inhibition, and 15 amino acids (SEQ ID NO: 15) or The 30 amino acid GGGGS linker (SEQ ID NO: 16) construct showed greater than 80% inhibition, indicating that GGGGS longer than 5 amino acids would be more advantageous for SST function. The more rigid A (EAAAK) ₄ A (a-helix) linker will be more effective than the flexible GGGGS linker in binding. Multiple SSTs can increase the effective concentration of ligand for SST receptor binding. The position of the histidine purification tag does not affect the binding. Changing the orientation or position of albumin in the fusion protein can further increase the efficiency of protein binding.

Example 4. Inhibition of CAMP accumulation by several SST-albumin fusion proteins in SSTR2-expressing cells

The human recombinant statin sst2a receptor expressed in CHO-K1 cells was used. Incubate the test compound and/or vehicle together with the cells (2 x 105/ml) in an incubation buffer at 37°C for 20 minutes. A 50% or more (50%) reduction in cAMP induced by the test compound relative to the 10 nM octreotide response is indicative of possible sst2a receptor agonist activity.

Inhibition of cAMP accumulation has been observed in CHO-K1 cells expressing SST receptor type 2. The EC ₅₀ value is 260 nM. Having a longer linker of construct (SEQ ID NO: 1,15 and 2) having a lower value ₅₀ EC, this data analysis is consistent with the binding. When comparing the EC ₅₀ values of albumin-(GGGGS) ₃ -SST14 and albumin-(GGGGS) ₃ -SST14, the α-helical linker seems to be more effective in inhibiting cAMP production.

<110> NAL PHARMACEUTICAL GROUP LIMITED, Mainland China

<120> Composition of fusion protein containing albumin and its analogues, its preparation and use method

<140> TW105106088

<140> 2016-02-26

<150> US 62/121,487

<151> 2015-02-26

<160> 48

<170> PatentIn version 3.5

<210> 1

<211> 657

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 1

<210> 2

<211> 621

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 2

<210> 3

<211> 1203

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 3

<210> 4

<211> 1222

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 4

<210> 5

<211> 1216

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 5

<210> 6

<211> 8

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 6

<210> 7

<211> 614

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 7

<210> 8

<211> 621

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 8

<210> 9

<211> 613

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 9

<210> 10

<211> 613

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 10

<210> 11

<211> 604

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 11

<210> 12

<211> 604

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 12

<210> 13

<211> 623

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 13

<210> 14

<211> 661

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 14

<210> 15

<211> 614

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 15

<210> 16

<211> 629

<212> PRT

<213> Labor

<220>

<223> Fusion protein, mature protein

<400> 16

<210> 17

<211> 14

<212> PRT

<213> Homo sapiens

<400> 17

<210> 18

<211> 28

<212> PRT

<213> Homo sapiens

<400> 18

<210> 19

<211> 585

<212> PRT

<213> Homo sapiens

<400> 19

<210> 20

<211> 22

<212> PRT

<213> Labor

<220>

<223> Signal peptide of fusion protein

<400> 20

<210> 21

<211> 20

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 21

<210> 22

<211> 40

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 22

<210> 23

<211> 42

<212> DNA

<213> Homo sapiens

<400> 23

<210> 24

<211> 84

<212> DNA

<213> Homo sapiens

<400> 24

<210> 25

<211> 1755

<212> DNA

<213> Homo sapiens

<400> 25

<210> 26

<211> 5428

<212> DNA

<213> Labor

<220>

<223> Mammalian expression vector

<400> 26

<210> 27

<211> 9276

<212> DNA

<213> Labor

<220>

<223> Yeast expression vector

<400> 27

<210> 28

<211> 45

<212> DNA

<213> Labor

<220>

<223> Flexible extended linker

<400> 28

<210> 29

<211> 66

<212> DNA

<213> Labor

<220>

<223> Alpha helix linker

<400> 29

<210> 30

<211> 22

<212> PRT

<213> Labor

<220>

<223> Alpha helix peptide linker

<400> 30

<210> 31

<211> 5

<212> PRT

<213> Labor

<220>

<223> Flexible extended peptide linker

<400> 31

<210> 32

<211> 16

<212> PRT

<213> Labor

<220>

<223> Linker with thrombin cleavage site

<400> 32

<210> 33

<211> 6

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 33

<210> 34

<211> 15

<212> PRT

<213> Labor

<220>

<223> Linker with thrombin cleavage site

<400> 34

<210> 35

<211> 16

<212> PRT

<213> Labor

<220>

<223> Linker with thrombin cleavage site

<400> 35

<210> 36

<211> 11

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 36

<210> 37

<211> 11

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 37

<210> 38

<211> 11

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 38

<210> 39

<211> 5

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 39

<210> 40

<211> 16

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 40

<210> 41

<211> 8

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 41

<210> 42

<211> 8

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 42

<210> 43

<211> 18

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 43

<210> 44

<211> 8

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 44

<210> 45

<211> 5

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 45

<210> 46

<211> 5

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 46

<210> 47

<211> 11

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 47

<210> 48

<211> 26

<212> PRT

<213> Labor

<220>

<223> Linker

<400> 48

Claims (17)

A fusion protein comprising the following formula (V): [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 (V), wherein SST is a somatostatin or its analogue; L A spacer or linker having the sequence of A (EAAAK) ₄ A (SEQ ID NO: 30); ALB is albumin; and x1, x2, y1 or y2 are each independently 0 or an integer selected from 1 to 6 , The premise is that at least one L is present in the fusion protein. The fusion protein of claim 1, wherein the SST is natural or synthetically produced. The fusion protein of claim 1, wherein the SST comprises a sequence encoding SST-14 or SST-28 (respectively represented as SEQ ID NO: 17 or 18) or a sequence having at least 85% identity with any of these sequences One or more tandem repeats of the sequence. The fusion protein of claim 1, wherein the SST is SST-14 or SST-28. The fusion protein of claim 1, wherein L is an α-helical structured polypeptide linker or spacer. The fusion protein of claim 1, wherein ALB is mammalian serum albumin. The fusion protein of claim 6, wherein the mammalian serum albumin is SEQ ID NO: 25 or a sequence with at least 85% sequence identity. The fusion protein of claim 1, wherein x1 and x2 are each independently an integer selected from 1 to 5. The fusion protein of claim 1, wherein y1 and y2 are each independently an integer selected from 1 to 5. Such as the fusion protein of claim 1, which has been isolated and purified. A polynucleotide encoding a polypeptide comprising the following formula (V): [SST-(L) _x1 ] _y1 -ALB-[(L) _x2 -SST] _y2 (V), wherein the SST series somatostatin or Its analogues; L is a _{spacer or linker with the sequence of A (EAAAK) 4} A (SEQ ID NO: 30); the ALB is albumin; and x1, x2, y1 or y2 are each independently 0 or selected An integer from 1 to 6, provided that at least one L is present in the fusion protein. Such as the polynucleotide of claim 11, which encodes the polypeptide sequence, wherein the SST comprises a sequence encoding SST-14 or SST-28 (respectively represented as SEQ ID NO: 17 or 18) or a sequence with SEQ ID NO: 17 or SEQ. ID NO: 18 One or more tandem repeats with at least 85% identity. A plastid construct expressing an albumin-somatostatin fusion protein comprising the fusion protein of any one of claims 1 to 10. A bacterial host cell transformed with the plastid construct of claim 13. A use of the fusion protein according to any one of claims 1 to 10 in the manufacture of a medicine for the treatment of endocrine-releasing diseases or disorders in human individuals, wherein the endocrine-releasing diseases or disorders are the administration of somatostatin Reactive symptoms. Such as the use of claim 15, wherein the condition is cancer selected from the group consisting of breast cancer, colorectal cancer, liver cancer, endocrine cancer, neuroendocrine cancer, pancreatic cancer, prostate cancer, and lung cancer. The use of claim 16, wherein the cancer exhibits somatostatin receptor type 1, 2, 3, 4, or 5.