TW201634482A - 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 fusion protein of albumin and its analogs, preparation thereof and use method thereof Cross-reference to related applications

The present application claims the benefit of priority to U.S. Provisional Patent Application Serial No. No. No. No. No. Ser.

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

The present invention also relates to a recombinant fusion protein comprising a human serum albumin portion and a somatostatin portion separated by a spacer sequence and the like.

Somatostatin ("SST") is a secretory product of various endocrine and non-endocrine tissues and is widely distributed throughout the body. Somatostatin inhibits the secretion of pituitary, pancreatic and gastrointestinal hormones, as well as interleukin production, intestinal motility and absorption, vasoconstriction and cell proliferation. Recent studies have found that SST has utility in the treatment of cancer, which 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. The somatostatin molecule has two biologically active forms: somatostatin-14 (SST-14, cyclic tetradecapeptide) and somatostatin-28 (SST-28, an N-terminally extended form of SST-14). SST-14 is a 14-residue cyclic peptide containing disulfide between the 3rd and 14th cysteine key. SST-28 is an N-terminal extended form (28 residues) of the same precursor that is proteolytically cleaved to generate SST-14. Although they have similar activities, their individual efficacy and histological characteristics are different. For example, SST-14 exhibits a more pronounced inhibition of glycemic and gastrin, while SST-28 exhibits a more pronounced inhibition of growth hormone and insulin action. Both forms of somatostatin exert their respective biological functions by means of receptors and intracellular pathways on target cells. Five somatostatin receptor subtypes (SSTR 1-5) have been identified, and SSTR2 has two splice variants with different carboxy termini: SSTR2A and SSTR2B.

The beneficial effects of somatostatin on the treatment of certain excessively secreted endocrine disorders and their anti-proliferative effects on tumors have been well recognized in the industry. However, due to enzymatic degradation and endocytosis, the in vivo half-life of somatostatin is only 2-3 min, which limits the clinical utility of somatostatin. Several stable somatostatin analogues have been developed over the past decade. For example, octreotide and lanreotide are used to treat growth hormone (GH)-secreting adenomas and carcinoids. However, there are still treatment limitations due to changes in binding affinity to SSTR. Thus, there remains a need in the art for somatostatin constructs that achieve high in vivo half-lives while maintaining the desired binding affinity for SSTR.

The most abundant protein albumin in plasma is produced in the liver as a 67 kDa monomeric protein and produces 80% of the colloid osmotic pressure in plasma. Human particle sphere stimulating factor (G-CSF), human growth hormone (GH), human insulin, human interferon-a-2b (INF-2b) and interleukin-28B (IL-28B) fused to HSA are effective It is used to construct long-acting candidate therapeutic drugs. However, there are few reports on comparative studies in biological and molecular mechanisms between HSA fusion proteins and parental molecules.

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

The present invention provides somatostatin-albumin fusion proteins and analogs thereof, and their production and Use the method. Constructs prepared according to the present invention include albumin (or an analogue thereof) moiety, a somatostatin moiety (SST-14, SST-28), and a spacer separating the two (eg, a spacer peptide or a linker peptide) .

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

In a particular embodiment, the nucleotide sequence is optionally selected to encode an albumin-sostatin fusion protein consisting of one of Formulas I-X below.

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); wherein 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 A nucleotide sequence encoding an albumin-sostatin fusion protein.

A fusion protein according to the invention is also described herein as a polypeptide. Polypeptides according to the invention may, in certain embodiments, include one or more unnatural amino acid or amino acid residues, as appropriate.

Somatostatin-albumin fusion proteins and analogs thereof generally include a human SST peptide moiety, a linker or spacer, and a human albumin moiety. The SST peptide moiety can include analogs and derivatives thereof that actively inhibit human growth hormone activity. The SST peptide moiety is obtained from natural or synthetic sources, as appropriate. The albumin moiety is, for example, human albumin and/or its active fragment or subdomain. A linker or spacer is selected to enhance the stability of the somatostatin-albumin fusion protein. More specifically, the somatostatin-albumin fusion protein and analogs thereof have the following structures.

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

In another embodiment, the invention includes a fusion protein comprising a structure selected from the group consisting of: 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 invention, x1, x2, x3, x4 are each independently selected from an integer from 1 to 5, or an integer from 1 to 4. In still another embodiment of the present invention, y1, y2, y3 are each independently selected from an integer of 1 to 5, or an integer from 1 to 4.

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

The SST part is SST-14 or SST-28 as appropriate.

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

A linker or spacer according to another embodiment of the invention encompasses a peptide covalently linked to vostatin at one end and covalently linked to albumin at the other end. In another embodiment of the invention, the spacer comprises a peptide sequence having from 2 to 100 amino acid residues.

The term "linker" or "spacer" is used interchangeably herein to refer to a short amino acid sequence used to separate multiple domains in a single protein. Two or two in the protein The lack of a linker between more discrete domains can result in reduced or inappropriate functionality of the protein domain, for example, due to steric hindrance, reduced catalytic activity or reduced binding affinity to the receptor/ligand. The use of a linker protein domain of an artificial linker in a chimeric protein increases the space between domains. Preferably, the choice of linker or spacer is independent of somatostatin and albumin.

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

The albumin-sostatin fusion constructs described herein may also include a signal peptide sequence ("SP"). A signal peptide is understood to mean a short amino acid sequence present at the N-terminus of a polypeptide that directs cellular localization of a newly synthesized protein. For example, a signal peptide can localize a protein to a given intracellular region (eg, a nucleus), to a membrane (eg, a cell membrane or endoplasmic reticulum), or to secretion from a cell. In addition to targeting, signal peptides can also be incorporated into recombinant proteins to improve stability, alter the degree of performance, and aid in proper folding of recombinant proteins. The signal peptide sequence of the precursor protein is typically removed by signal peptidase in the host cell to produce a mature protein.

The albumin-sostatin fusion constructs set forth herein may also include an affinity or purification tag as part of a polypeptide sequence to facilitate purification. These tags are used as part of an affinity chromatography method (eg, high performance liquid chromatography (HPLC)) to purify protein samples from crude biological sources. Suitable purification tags include, but are not limited to, polyhistamines (eg, His-6 or H6), glutathione-S-transferase (GST), maltose binding protein (MBP), chitin-binding proteins ( CBP), FLAG-tag (FLAG octapeptide). When an affinity tag is required to be removed from the fusion protein, a specific enzyme cleavage site can be introduced into 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 invention also provides a pharmaceutical group comprising the fusion protein of the invention by administering an effective amount thereof A method for treating an endocrinely released disease or condition in a mammal, such as a human subject, wherein the endocrinely released disease or condition is a condition responsive to administration of a somatostatin.

For example, the disease or condition is 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 a somatostatin receptor type 1, 2, 3, 4 or 5.

It is also understood that the singular forms (such as "a", "an" and "the" are used throughout the application for convenience purposes, however, unless the context or There are indications, otherwise the singular forms are intended to include the plural. In addition, it should be understood that each of the journal articles, patents, patent applications, publications, and the like referred to herein are hereby incorporated by reference in its entirety for all purposes.

All numerical ranges are to be understood as inclusive of the value of the End points for all ranges of the same component or property are included and are intended to be independently combined.

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 alter the claimed composition or method. This is the case with basic and novel features.

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

Before the present invention is explained in detail below, it is to be understood that the invention is not limited to such elements, as the specific methods, protocols, and reagents described herein are varied. It is also understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to The scope of the present invention is limited only by the scope of the accompanying claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning

The present invention encompasses somatostatin-albumin fusion proteins and analogs thereof, and methods of their production and use. Constructs made in accordance with the present invention include albumin (or analogs thereof) moieties, somatostatin moieties (e.g., SST-14, SST-28) and spacers separating the two moieties.

The somatostatin-albumin fusion proteins of certain embodiments of the invention include variants of albumin (including human serum albumin) and/or derivatives of somatostatin. A spacer of another embodiment of the invention encompasses a peptide covalently linked to vostatin at one end and covalently linked to albumin at the other end. The spacers in other embodiments of the invention include peptide sequences having from 2 to 100 amino acids.

In one embodiment, the invention provides a fusion protein comprising: SST; L; and ALB, wherein the SST is a somatostatin or an analogue or derivative thereof; an L-series spacer or linker; an ALB albumin or Its analogue or variant.

In certain embodiments, the fusion protein of the invention is selected from Formulas I-X below.

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); wherein 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 still another embodiment, the present invention provides a nucleotide sequence encoding an albumin-sostatin fusion protein, the fusion protein comprising: SST; L; and ALB, wherein the SST system is a somatostatin or an analog or derivative thereof L-series or linker; ALB-based albumin or an analog or variant thereof.

In certain embodiments, the nucleotide sequence of the invention is selected to encode an albumin-sostatin fusion protein 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 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 consists of a sequence encoding an amino acid sequence represented by SEQ ID NO: 31 or -GGGGS-.

A further embodiment of the 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 a fragment thereof) Consistent polypeptide.

An embodiment of the 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 a fragment thereof A polypeptide having at least 85% sequence identity.

The invention also provides a nucleotide sequence encoding an albumin-sostatin fusion protein comprising: (a) a region comprising a nucleotide sequence comprising one or more adjacent repeats of a sequence encoding a human somatostatin peptide (b) a region comprising a nucleotide sequence encoding human serum albumin or a fragment thereof; (c) a spacer region comprising a nucleotide sequence encoding a polypeptide of from 2 to 100 residues in length; wherein the spacer Region (c) is present between region (a) and region (b) or between region (a) and region (a); wherein one or more adjacent repeat encodings of the sequence encoding the human somatostatin peptide SST-14 or SST-28 (shown as SEQ ID NOS: 17 and 18, respectively), or a sequence that is at least 85% identical to one of the two sequences; or wherein the spacer sequence is represented by the code as SEQ ID NO: 31 or GGGGS or the sequence composition of the amino acid sequence indicated as SEQ ID NO: 30A (EAAAK) ₄ A); or wherein the region (a) is represented by SST-14 or SST-28 (as expressed as One or more contiguous repeats of SEQ ID NOS: 23 and 24) or a sequence composition that is at least 85% identical to either of the two sequences.

Furthermore, the present invention provides a polypeptide sequence of an albumin-sostatin fusion protein comprising: (a) a region comprising a polypeptide sequence of a somatostatin peptide (which may be a human somatostatin peptide); (b) comprising serum white a region of the polypeptide sequence of the protein (which may be human serum albumin) or a fragment thereof; (c) a spacer region comprising a polypeptide of from 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 comprise one or more of a sequence encoding SST-14 or SST-28 (represented as SEQ ID NO: 17 or 18, respectively) or a sequence that is 85% identical to one of the sequences. Repeat the segments in series.

Another embodiment of the invention provides a plastid construct that exhibits an albumin-sostatin fusion protein having any of the fusion protein or polypeptide sequences described above.

Yet another embodiment of the invention includes transformation using the plastid constructs described above Bacterial cells.

Another embodiment of the invention includes an albumin-body isolated and purified and having the polypeptide sequence described above (eg, an albumin-sostatin fusion protein or a polypeptide sequence representing a plastid construct of the protein) Inhibitin fusion protein.

For fusion proteins (eg, SEQ ID NOS: 1-5, 7-10, and 13-16), it should be noted that these fusion proteins serve as a protein prior to the signal peptide (SEQ ID NO: 20) having 22 residues. To code.

Somatostatin-albumin fusion protein

The invention encompasses polypeptide constructs wherein the somatostatin moiety has at least 85% sequence identity with the nucleotide sequence of endogenous human SST-14 or SST-28 (SEQ ID No: 23 and 24, respectively) Nucleotide coding.

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

The invention also encompasses polypeptide constructs wherein the somatostatin moiety has at least 85%, preferably at least 90% of the polypeptide sequence of endogenous SST-14 or SST-28 (SEQ ID NOS: 17 and 18, respectively) Sequence identity of the polypeptide sequence.

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

The invention also encompasses fusion proteins comprising a signal peptide, a purification tag (His-6), a first linker, a human serum albumin moiety, a second linker, and a somatostatin moiety. In one embodiment, the fusion protein is represented by the polypeptide of SEQ ID NO: 9 or a sequence having 85% sequence identity thereto.

The invention also encompasses fusion proteins comprising a somatostatin moiety, a first linker, a human serum albumin moiety, a second linker, a somatostatin moiety, 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 having 85% sequence identity thereto.

The present invention also encompasses 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 invention further encompasses a nucleotide sequence encoding an albumin-sostatin fusion construct having 85% sequence identity to SEQ ID NO:11.

The invention also encompasses a nucleotide sequence (SEQ ID NO: 12) encoding a peptide comprising a peptide A fusion protein of the N-terminal somatostatin portion and the C-terminal human serum albumin portion separated by a spacer. The invention further encompasses a nucleotide sequence encoding an albumin-sostatin fusion construct having 85% sequence identity to SEQ ID NO: 12.

The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or more copies of the SST-14 or SST-28 sequences arranged in tandem, namely "(SST-14) ₂ " or "(SST- 14) ₃ " or "(SST-28) ₂ " or "(SST-28) ₃ ". Optionally, the linker sequence is comprised between two or more tandem somatostatin moieties, and/or the signal peptide sequence is included at the N-terminus of the fusion protein.

The invention also encompasses polypeptide constructs wherein the voxatin moiety comprises two or more copies of the SST-14 sequence, which are arranged in such a manner that at least one SST14 copy is ligated to the sides of the albumin, respectively. Optionally, the linker sequence is comprised between two or more tandem somatostatin moieties and between somatostatin and albumin, and/or the signal peptide sequence is included at the N-terminus of the fusion protein. For example, a polypeptide construct can include a signal peptide, two SST-14 portions separated by a spacer, a second spacer, and an HSA portion as representatives. The construct omits the N-terminal signal peptide as appropriate.

The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or three copies of the SST-28 sequences arranged in tandem, namely "(SST-28) ₂ " or "(SST-28) ₃ ", respectively. Optionally, a linker sequence is included between two or more tandem somatostatin moieties.

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

The invention also encompasses a polypeptide construct comprising any of the albumin-sostatin fusion proteins described in the preceding paragraph, wherein the albumin-sostatin fusion protein has an endogenous SST-14 or SST-28 compared to endogenous SST-14 or SST-28 Approximately equal or greater binding affinity to a somatostatin receptor.

The invention also encompasses an albumin-sostatin fusion protein comprising the representation SEQ ID NO: 15, the N-terminal albumin portion of 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. The N-terminus may further comprise a signal peptide, as appropriate. Optionally, one or more albumin and SST domains can each be separated by an independently selected linker sequence (shown as SEQ ID NO: 1).

In some embodiments, the SST portion can comprise a pair or a plurality of tandem SST sequences, eg, (SST-14) ₂ or (SST-28) ₃ , and contain or between two or more tandem SST repeats No intermediate spacer sequence. Optionally, one or more purification tag sequences can be included in the sequence between the two portions or at the N-terminus or C-terminus to facilitate purification of the fusion protein. An alternate embodiment includes partitioning one of the SST-14 portions by a spacer, as represented by SEQ ID NO: 4. Another embodiment may omit the purification tag (e.g., His6) as shown by the polypeptide sequence represented as SEQ ID NO:5.

Somatostatin

The somatostatin used in the present invention may be any somatostatin, an analog or a derivative thereof. It can be a human somatostatin or any other isolated or natural somatostatin.

The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises a somatostatin analog. Preferably, such an analog is suitable for expression as part of a fusion protein in a recombinant host cell. It will be appreciated 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 invention also encompasses polypeptide constructs wherein the voxatin moiety comprises two or more tandem repeats of a somatostatin polypeptide sequence (eg, SST-14 or SST-28; SEQ ID NOS: 17 and 18, respectively) . Each of the repeating somatostatin polypeptide sequences can be a polypeptide sequence having at least 85% sequence identity to SST-14 or SST-28. The repeat variant sequences are independently selected, i.e., in some embodiments the repeats are identical, while in other embodiments they are unique.

albumin

The albumin used in the present invention may be any albumin, analog or variant thereof. White The protein may be human serum albumin, or any other isolated or native albumin.

The invention also encompasses polypeptide constructs wherein the human serum albumin portion comprises a polypeptide sequence variant having an alternative to the presence of additional or less cysteine residues as compared to the native form (SEQ ID NO: 25) The arrangement or number of disulfide bonds.

Spacer or linker

As mentioned previously, a spacer or linker can be used in the present invention. The spacer or linker can be independent of somatostatin or albumin.

Polypeptide constructs are also encompassed by the invention, wherein the peptide spacer, alternatively referred to as a linker, consists of a polypeptide sequence of from about 2 to about 100 amino acid residues in length. The invention further encompasses polypeptide constructs wherein the peptide spacer is from about 2 to about 50 amino acid residues in length, preferably from about 2 to about 30, or more preferably from about 3 to about 20 amine groups in length. Acid residue.

The 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 and Ser and Thr provide particular advantages: (i) the polypeptide backbone is free to rotate such that adjacent domains move freely relative to each other; (ii) enhanced solubility; (iii) resistance to protein breakdown. In addition, many natural linkers exhibit an alpha-helical structure. The alpha-helical structure is more rigid and more stable than the Gly-rich linker. The functional domain can be separated using an empirical rigid linker having the sequence of A(EAAAK) ₄ A (SEQ ID NO: 30). In addition to the role of linking protein domains together, artificial linkers can provide additional advantages for the manufacture of fusion proteins, such as improving biological activity, increasing protein performance, and achieving desired pharmacokinetic properties.

Preparation of somatostatin-albumin fusion protein

Embodiments of the invention provide methods of preparing somatostatin-albumin fusion proteins. In the embodiments, the somatostatin-albumin fusion protein of the present invention is produced by the expression of the recombinant fusion protein containing the gene and introduction of the vector into the host. For example, fusion proteins are obtained by expression in a host such as yeast. For example, P. pastoris GS115 ( Pichia pastoris GS115) can be used as a suitable expression host and used to construct a recombinant expression vector pPIC9K. In addition, mammalian lines such as CHO or HEK293 can be used as preferred expression hosts.

The invention also encompasses a plastid construct capable of expressing an albumin somatostatin fusion protein comprising a nucleotide sequence encoding a somatostatin albumin fusion protein as described in any of the preceding paragraphs. For example, a suitable plastid construct includes, but is not limited to, a pcDNA3.1 vector represented by SEQ ID NO: 26, wherein the DNA sequence encoding any of the albumin-sostatin fusion proteins disclosed herein is ligated thereto Multiple selection sites for the vector. Other suitable protein expression vectors known in the art can be selected based on the expression host (e.g., a expression vector having a mammalian promoter system would be suitable for expression in a human cell line, if desired in yeast or bacteria) In one of the performances, the plastids of the organisms will be selected).

The 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 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 of the albumin-sostatin fusion proteins set forth herein is incorporated into the vector. In the colony.

The invention also encompasses an isolated and purified fusion voxel fusion protein having a polypeptide sequence as described in any of the preceding paragraphs.

The effect of somatostatin-albumin fusion protein

The fusion proteins of the invention are useful in the treatment of conditions known in the art for treatment with somatostatin. Accordingly, the present invention also encompasses a method of treating cancer in a human subject by administering an isolated and purified albumin-sostatin fusion protein as described in any of the preceding paragraphs, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer , liver cancer and lung cancer.

The invention also encompasses a method of treating cancer in a human subject by administering a composition comprising a fusion protein of the invention (e.g., an isolated and purified albumin-sostatin fusion protein as described in any of the preceding paragraphs). The composition may also include a suitable carrier.

Eleven SST14-albumin fusion protein constructs with different linker sequences were designed. Eight of these constructs were made into a fusion gene in the plastid and produced by transient expression of HEK293 on a 100 mL scale. Proteins were collected from the culture medium, purified by albumin-based affinity purification, and dialyzed against storage buffer. The fusion proteins were evaluated for the binding affinity of the fusion proteins to the SSTR2 receptor and for the cell-based activity of inhibiting cAMP production in the SSTR2-overexpressing CHO-K1 cell line. The results of these studies indicate that the length and type of linker significantly affects SSTR2 receptor binding affinity, in vitro cell-based functional activity, and fusion protein yield.

Instance

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

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

Eight constructs were prepared (see Table 4). First, the gene was synthesized and then the target sequence was inserted into the pcDNA3.1 vector.

Example 1-2. plastid generation

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

Example 1-3. Transfection and protein production (A) Suspension cell method

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

(B) Adhesive cell method

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

DNA and PEI solutions were prepared 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 was added to the cells and the mixture was 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.

The medium was harvested after about 4 days, and the supernatant was collected by centrifugation. The precipitate was replenished with fresh medium containing L-glutamic acid for re-cultivation for 3 days to repeat the harvesting process.

Example 1-4: Protein Concentration, Ni-NTA Purification, and Buffer Exchange

The collected medium was concentrated to a volume by a TFF system (Millipore) by a side-by-side purification method (continuous chromatography or manual batch purification).

Concentrated protein in binding buffer at about 4 °C using a chromatography or batch system The solution was incubated with fresh Ni-NTA resin and washed with a washing buffer. The protein was eluted with an elution buffer and the fractions were collected and concentrated to recover the purified protein. The protein can be further purified using particle size screening chromatography purification.

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

Example 2: Yield of several SST-albumin fusion proteins

SST-HSA fusion proteins are expressed in a soluble form in 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 constructs exhibit yields for scale-up production.

Example 3: Binding affinity of several SST-albumin fusion proteins

This assay measures the binding of [ ¹²⁵ I] somatostatin to the human somatostatin sst2 receptor. Using standard techniques, using stably transfected plasmid sst2 somatostatin receptor encoded by the human body CHO-K ₁ cells 7.4 Preparation of membrane buffer was modified in HEPES pH. The aliquot of 0.1mg ^‡ film 0.03nM ^[125 I] compstatin and incubated with fusion proteins tested at 25 ℃ 240 minutes. Non-specific binding was estimated in the presence of 1 μM somatostatin. The membrane was filtered and washed 3 times and the filter was counted to determine the specifically bound [ ¹²⁵ I] somatostatin.

The competitive binding studies of ¹²⁵ I-Tyr-sostatin relative to the fusion protein show the following results. The inhibition efficiency varies depending on the structure of the fusion protein. A fusion protein construct (SEQ ID NO: 1) having two α-helix linker A (EAAAK) ₄ A showed 100% inhibition of the interaction of somatostatin with its receptor. The SEQ ID NO: 1 construct has two somatostatin portions on both the N-terminal side and the C-terminal side of human serum albumin. A smaller construct (SEQ ID NO: 2) having a somatostatin linked by the same α-helix linker on the C-terminal side of human serum albumin showed 96% inhibition. The same construct end-length length of the more flexible GGGGS linker showed a 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) showed 57% to 59% inhibition with 15 amino acids (SEQ ID NO: 15) or The construct of the 30 amino acid GGGGS linker (SEQ ID NO: 16) 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 efficient in binding than the flexible GGGGS linker. Multiple SSTs can increase the effective concentration of ligands for SST receptor binding. The location of the histidine purification tag does not affect binding. Altering 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 somatostatin sst2a receptor expressed in CHO-K1 cells was used. Test compounds and/or vehicles were incubated with cells (2 x 105/ml) in incubation buffer for 20 minutes at 37 °C. A 50% or more (50%) reduction in cAMP induced by the test compound relative to the 10 nM octreotide reaction indicates a possible sst2a receptor agonist activity.

Inhibition of accumulation of cAMP has been observed in CHO-K1 cells expressing SST receptor type 2. The EC ₅₀ value was 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 albumin - (GGGGS) _{3 -SST14} and albumin - (GGGGS) ₅₀ when the value of EC ₃ -SST14, α- helical linker seems to be more effective inhibition of cAMP generation.

<110> China Shangan Nengtai Pharmaceutical Co., Ltd.

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

<130> 4019-1015-W

<150> US 62/121,487

<151> 2015-02-26

<160> 48

<170> PatentIn version 3.5

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Claims (21)

A fusion protein comprising: SST; L; and ALB, wherein the SST is a somatostatin, an analog or derivative thereof; an L-series spacer or a linker; and an ALB-based albumin, an analog or variant thereof. The fusion protein of claim 1, which is selected from the group consisting of: 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 independently 0 or An integer selected from 1 to 10, provided that at least one L is present in the fusion protein. The fusion protein of claim 1, wherein the SST is naturally or synthetically produced. The fusion protein of claim 1, wherein the SST comprises or has at least 85% identity to a sequence encoding SST-14 or SST-28 (represented as SEQ ID NO: 17 or 18, respectively) One or more tandem repeats of the sequence. The fusion protein of claim 1, wherein the SST is SST-14 or SST-28. A fusion protein according to claim 1 wherein the L is a flexible or alpha helically structured polypeptide linker or spacer. The fusion protein of claim 1, wherein the L system has a peptide of 2 to 100 amino acids. The fusion protein of claim 6, wherein the peptide comprises at least one GGGGS, A(EAAAK) ₄ A, (AP) _n (where n is selected from an integer from 10 to 34), (G) ₈ , (G) ₅ or Any combination of them. The fusion protein of claim 1, wherein the ALB is mammalian serum albumin. The fusion protein of claim 1, wherein the mammalian serum albumin is SEQ ID NO: 25 or a sequence having at least 85% sequence identity thereto. The fusion protein of claim 2, wherein x1, x2, x3, x4 are each independently selected from an integer from 1 to 5. The fusion protein of claim 2, wherein y1, y2, y3 are each independently selected from an integer from 1 to 5. The fusion protein of claim 1 which is isolated and purified. A nucleotide sequence encoding a polypeptide comprising: SST; L; and ALB, wherein the SST is a somatostatin or an analogue or derivative thereof; an L-series spacer or a linker; The ALB is albumin or an analog or variant thereof. The nucleotide sequence of claim 14, which encodes a polypeptide selected from the group consisting of: 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 Each is independently 0 or an integer selected from 1 to 10, provided that at least one L is present in the polypeptide. The nucleotide sequence encoding the polypeptide sequence of claim 14, wherein the SST comprises a sequence encoding SST-14 or SST-28 (represented as SEQ ID NO: 17 or 18, respectively) or with SEQ ID NO: 17 or SEQ ID NO: 18 has one or more tandem repeats of at least 85% identity sequence. A plastid construct comprising an albumin-sostatin fusion protein comprising the fusion protein of claim 1. A bacterial host cell transformed with a plastid construct as claimed in claim 17. A fusion protein according to claim 1 for use in the manufacture of endocrine for the treatment of human subjects Use of the disease or condition to be released, wherein the endocrinely released disease or condition is a condition responsive to administration of a somatostatin. The use of claim 19, wherein the condition is 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 20, wherein the cancer exhibits a somatostatin receptor type 1, 2, 3, 4 or 5.