TECHNICAL FIELD

One or more embodiments of the present invention relate to a novel ribosome display complex and a production method therefor.

BACKGROUND

In recent years, a therapeutic agent for a specific disease and a molecule having a high affinity for a target molecule have been searched from a polypeptide library having various amino acid sequences. It is thought that the reason for this is that a polypeptide has fewer side effects than a low molecular weight compound due to high specificity and selectivity of a polypeptide. In addition, a polypeptide has an advantage that a library containing many polypeptides can be prepared more easily than a library of a low molecular weight compound, since DNA encoding a polypeptide in which the amino acid sequence in a specific part is randomized can be readily obtained by a PCR technique using a random primer (Non-patent documents 1 to 3).

It has attracted attention to develop a pharmaceutical product of which function is expanded by chemically modifying a polypeptide such as an antibody with a functional molecule. For example, it is described in Non-patent documents 4 and 5 that an antibody-drug conjugate (ADC) was applied as an anticancer drug. Such a compound formed by binding a polypeptide and a functional molecule has been a focus of attention in the drug discovery field, since a property of a polypeptide and a function of a functional molecule are combined, and each characteristic is exploited and each shortcoming is compensated (Non-patent documents 6 to 10).

For example, since a polypeptide has high specificity for a target molecule, a polypeptide is considered to have lower side effect than a low molecular weight drug. On the one hand, a polypeptide is not stable, since a polypeptide is degraded by an enzyme such as protease and peptidase in a living body. Accordingly, the stability of a peptide in a living body may be improved by introducing an unnatural amino acid or cyclizing the peptide with a modifying reagent. In addition, since the structure of a polypeptide becomes stable by cyclization, an affinity for and a selectivity to a target compound may be improved, and a resistance to a degrading enzyme and a cell membrane permeability may be obtained (Non-patent documents 6 to 10).

Thus, a technology to prepare a library containing polypeptides to which a functional molecule such as a modifying reagent for cyclization and an anticancer drug is bound is brought to attention.

As such a technology to prepare a library containing compounds formed by binding a functional molecule and a polypeptide, a complex in which a displayed polypeptide (phenotype) corresponds to the gene encoding the polypeptide (genotype) on a one-to-one basis has been known (Non-patent document 1).

As the above-described method, phage display method is often used. In phage display method, a method for preparing a library by displaying a polypeptide on a bacteriophage and modifying the polypeptide has been known. A library can be produced by such phage display method at low cost by using comparatively simple equipment. The prepared library can be used for screening to isolate a useful polypeptide. There is, however, only a few report of the examples thereof, since the procedure to specifically bind a polypeptide displayed on a phage and a functional molecule is difficult and the process is cumbersome due to many steps (Non-patent documents 6, 7 and 10).

As a library of polypeptides to which a functional molecule is bound, a library obtained by inserting a special amino acid into a specific position of a polypeptide chain in translation system in vitro and reacting the amino acid residue in the peptide with a reactive functional group to cyclize the peptide (Non-patent documents 8 and 9). In addition, a method for cyclizing a peptide by directly reacting an mRNA-peptide complex with a modifying reagent in mRNA display method is reported (Non-patent document 9).

The above-described method for preparing a library using in vitro translation system is very superior to phage display method in terms of variation. As the in vitro translation system method, ribosome display method (RD method), mRNA display method and cDNA display method are exemplified. Among the exemplified methods, ribosome display method (RD method) is excellent, since a library containing 1012or more kinds of polypeptides can be produced in several minutes by merely mixing in vitro translation system and mRNA. On the one hand, mRNA display method and cDNA display method are cumbersome, since the number of steps to produce a library is large, such as a step of annealing mRNA and puromycin DNA.

It has not been reported that a functional molecule is bound to a polypeptide in RD method. The reason for this is that a phenotype-genotype complex used in RD method is formed by non-covalently biding a displayed peptide and a nucleic acid containing a gene through a ribosome and a ribosome itself is vulnerable to a reaction to bind a functional molecule. Specifically, the reason why it has been not reported that a functional molecule is bound in RD method is that 1) a complex in RD method is more unstable than that of mRNA display method, since a phenotype-genotype complex is produced by a covalent bond in mRNA display method, and 2) the functions of a ribosome and a complex tend to become poor by binding a functional molecule, since cysteine and lysine contained in a ribosomal protein which constitutes a ribosome are reactive. In fact, for example, there are totally 36 cysteines and 686 lysines in 55 kinds of ribosomal proteins ofEscherichia coli.

For example, cysteine plays a structurally and functionally important role as a residue of a polypeptide. A SH group of a cysteine residue forms a disulfide bona with a SH group of a cysteine residue of the same polypeptide and other polypeptide. The disulfide bond often contributes to a higher order structure of a polypeptide. Since a SH group not only forms a disulfide bond but also acts as a substrate of various reactions, a SH group is a good candidate as a modification site in the case where a polypeptide chain is artificially modified. On the one hand, when a specific cysteine residue is specifically modified, another cysteine residue at other unintentional position may be often reacted due to the high reactivity thereof. As a result, a higher order structure of a polypeptide is often broken. The same applies to lysine, histidine and tryptophan. Thus, it is not easy to devise a method for modifying a target cysteine without impairing the function thereof in a polypeptide, a ribosome and a RD complex containing a polypeptide and a ribosome having a plurality of cysteine, lysine, histidine and tryptophan. Specifically, a complicated reaction step procedure is required, such as a method for removing an unintended cysteine from a ribosomal protein by a genetic engineering and a preliminary reaction by which an unintended cysteine becomes unreactive. If such a step is included in a method for producing a library, an advantage that a library can be produced with fewer steps as a characteristics of RD method is lost.

NON-PATENT DOCUMENT

Non-patent document 1: H. Leemhuis, other 3 persons, “New genotype-phenotype linkages for directed evolution of functional proteins”, Current Opinion in Structural Biology 2005, 15: 472-478Non-patent document 2: D. Lipovsek, other 1 person, “In-vitro protein evolution by ribosome display and mRNA display”, Journal of immunological methods, 290(2004), 51-67Non-patent document 3: H. M. E. Azzazy, other 1 person, “Phage display technology: clinical applications and recent innovations”, Clinical Biochemistry, 35(2002), 425-445Non-patent document 4: H. L. Perez, other 6 persons, “Antibody-drug conjugates: current status and future directions”, Drug Discovery Today, Volume 19, Number 7, July 2014Non-patent document 5: S. C. Alley, other 2 persons, “Antibody-drug conjugates: targeted drug delivery for cancer”, Current Opinion in Chemical Biology, 2010, 14: 529-537Non-patent document 6: C. Heinis, other 3 persons, “Phage-encoded combinatorial chemical libraries based on bicyclic peptides”, Nature Chemical Biology, 5, 502-507(2009)Non-patent document 7: I. R. Rebollo, other 1 person, “Phage selection of bicyclic peptides”, Methods 60(2013), 46-54Non-patent document 8: T. Kawakami, other 2 persons, “Messenger RNA-Programmed Incorporation of Multiple N-Methyl-Amino Acids into Linear and Cyclic Peptides”, Chemistry & Biology, vol. 15(1), p. 32-42, January 2008Non-patent document 9: K. Josephson, other 2 persons, “mRNA display: from basic principles to macrocycle drug discovery”, Drug Discovery Today, volume 19, Number 4, April 2014, pp. 388-399Non-patent document 10: K. Fukunaga, other 4 persons, “Construction of a crown ether-like supramolecular library by conjugation of genetically-encoded peptide linkers displayed on bacteriophage T7”, Chemical Communications, 2014, 50, 3921-3923

In fact, the present inventors experimentally confirmed that when the inventors tried to produce a RD complex after a ribosome is reacted with a cyclizing reagent, DBP: 1,3-dibromo-2-propanone, as a modifying reagent, a RD complex cannot be formed.

SUMMARY

Accordingly, one or more embodiments of the present invention provide a ribosome complex which can be produced without a complicated reaction procedure and of which displayed polypeptide is modified without impairing a function of the ribosome, particularly function to produce a polypeptide library, and a method for producing the ribosome complex.

The inventors intensively studied and found that when a library of modified polypeptides is prepared by ribosome display method and a polypeptide is modified by an artificial material, there is a risk of losing the function of the polypeptide, since an amino acid reside has a reactive side chain functional group, such as cysteine, in a ribosomal protein of a ribosome; but an adhesive function of a ribosome to bind RNA to the polypeptide translated from the RNA can be maintained without losing the inherent function and a polypeptide in a ribosome display complex can be easily modified by modifying a polypeptide with an artificial material at an appropriate timing.

Hereinafter, one or more embodiments of the present invention are described.[1] A method for producing a ribosome display complex,

wherein the ribosome display complex comprises a polypeptide chain, an mRNA molecule and a ribosome,

the polypeptide chain comprises 1 or more reactive amino acid residues selected from the group consisting of a cysteine residue, a lysine residue, a histidine residue and a tryptophan residue,

a side chain reactive functional group of the reactive amino acid residue is modified, and

the mRNA molecule comprises a base sequence encoding an amino acid sequence of the polypeptide chain,

comprising the steps of:

translating the mRNA molecule in a cell-free peptide synthesis system utilizing the ribosome to obtain a ribosome complex comprising an unmodified polypeptide chain, the mRNA molecule and the ribosome, and

modifying the unmodified polypeptide chain by reacting the side chain reactive functional group in the unmodified polypeptide chain with a modifying reagent.[2] The method for producing the ribosome display complex according to the above [1], wherein the modifying reagent is a compound represented by the following formula (1):


wherein A is a group capable of forming a linkage by reacting with a side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue, B is a linker group or a single bond, C is a functional group, a is an integer of 1 or more, c is 0 or an integer of 1 or more, provided that when the a is an integer of 2 or more, a plurality of A are the same as or different from each other.[3] The method for producing the ribosome display complex according to the above [2], wherein the A is a halogenated alkyl group, an activated carbonyl group, an unsaturated hydrocarbon group, an epoxy group, a sulfonyl-containing group, an isocyanate group, an isothiocyanate group, a carbene-precursor group, a carbene-containing group, a disulfide bond-containing group or a thiol group.[4] The method for producing the ribosome display complex according to the above [2] or [3], wherein

the linker group B is a group having one or more selected from a hetero atom-containing polar group, a chained or cyclic aliphatic hydrocarbon group optionally having a hetero atom-containing polar group between carbon atoms and optionally having a substituent group, and an aromatic ring optionally having a substituent group solely or in combination,

the hetero atom-containing polar group is —O—, —S—, —NR1— (wherein R1is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group), —CO—, —COO—, —CONR2— (wherein R2is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group), —N═N— or —SO2—,

the substituent group on the aliphatic hydrocarbon group is a halogeno group, an aryl group, a carboxy group, an alkoxycarbonyl group or a hydroxy group,

the substituent group on the aromatic ring is a halogeno group, an alkyl group, an aralkyl group, a carboxy group, an alkoxycarbonyl group, a hydroxyalkyl group or a carboxyalkyl group.

[5] The method for producing the ribosome display complex according to the above [4], wherein

the linker group B has -B1- unit, -B2- unit, -B2-B1- unit or - B2-B1-B3- unit,

the B1 is a group having one or more selected from a chained or cyclic aliphatic hydrocarbon group optionally having a hetero atom-containing polar group between carbon atoms and optionally having a substituent group, and an aromatic ring optionally having a substituent group solely or in combination,

B2 and B3 are independently hetero atom-containing polar groups,

B1 or B2 are bound to the A,

the hetero atom-containing polar group and the substituent group have the same meanings as the above.[6] The method for producing the ribosome display complex according to the above [5], wherein the modifying reagent is any one of compounds represented by the following formulae:


wherein A, B1, B2 and B3 have the same meanings as the above, n is an integer of 1 or more, one or more of B1, B2 and B3 may be bound by the one or more C.[7] The method for producing the ribosome display complex according to any one of the above [2] to [6], when the A is the halogenated alkyl group, a carbon atom, bound by the halogeno group in the halogenated alkyl group is a carbon atom at a position of the carbonyl group or a carbon atom directly binding to the aromatic ring.[8] The method for producing the ribosome display complex according to any one of the above [2] to [7], wherein

the polypeptide chain comprises 2 or more reactive amino acid residues selected from the group consisting of the cysteine residue, the lysine residue, the histidine residue and the tryptophan residue,

the a is 2 or more in the modifying reagent represented by the formula (1),

a ring is formed by the polypeptide chain and the modifying reagent in the step of reacting the side chain reactive functional group in the unmodified polypeptide chain with the modifying reagent.[9] The method for producing the ribosome display complex according to any one of the above [1] to [8], wherein the polypeptide chain has 100 to 5000 amino acid residues.[10] The method for producing the ribosome display complex according to any one of the above [1] to [9], wherein the reactive amino acid residue is included between a 2ndposition from an N-terminal and a 30thposition from a C-terminal inclusive in the polypeptide chain.[11] The method for producing the ribosome display complex according to any one of the above [1] to [10], wherein a random, sequence having 1 to 30 amino acid residues is included between a 2ndposition from, an N-terminal and a 30thposition from a C-terminal inclusive in the polypeptide chain.[12] The method for producing the ribosome display complex according to any one of the above [1] to [11], wherein the ribosome is source a fromEscherichia coli.[13] A ribosome display complex,

comprising a polypeptide chain, an mRNA molecule and a ribosome,

wherein the polypeptide chain comprises 1 or more reactive amino acid residues selected from the group consisting of a cysteine residue, a lysine residue, a histidine residue and a tryptophan residue,

a side chain reactive functional group of the 1 or more reactive amino acid residues is modified, and

the mRNA molecule comprises a base sequence encoding an amino acid sequence of the polypeptide chain.[14] The ribosome display complex according to the above [13], wherein a modification structure of the side chain reactive functional group is a chemical structure represented by the following formula (2):


wherein Ax is a binding group formed by reacting with a side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue, B is a linker group or a single bond, C is a functional group, a is an integer of 1 or more, c is 0 or an integer of 1 or more, provided that when the a is an integer of 2 or more, a plurality of Ax are the same as or different from each other.[15] The ribosome display complex according to the above [14], wherein the Ax is a chemical bond formed by reacting a halogenated alkyl group, an activated carbonyl group, an unsaturated hydrocarbon group, an epoxy group, a sulfonyl-containing group, an isocyanate group, an isothiocyanate group, a carbene-precursor group, a carbene-containing group, a disulfide bond-containing group or a thiol group with the side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue.[16] The ribosome display complex according to the above [15], wherein

the linker group B is a group having one or more selected from a hetero atom-containing polar group, a chained or cyclic aliphatic hydrocarbon group optionally having a hetero atom-containing polar group between carbon atoms and optionally having a substituent group, and an aromatic ring optionally having a substituent group solely or in combination,

the hetero atom-containing polar group is —O—, —S—, —NR1— (wherein R1is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group), —CO—, —COO—, —CONR2— (wherein R2is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group), —N═N— or —SO2—,

the substituent group on the aliphatic hydrocarbon group is a halogeno group, an aryl group, a carboxy group, an alkoxycarbonyl group or a hydroxy group,

the substituent group on the aromatic ring is a halogeno group, an alkyl group, an aralkyl group, a carboxy group, an alkoxycarbonyl group, a hydroxyalkyl group or a carboxyalkyl group.[17] The ribosome display complex according to the above [16], wherein

the linker group B has -B1- unit, -B2- unit, -B2-B1- unit or -B2-B1-B3- unit,

the B1 is a group having one or more selected from a chained or cyclic aliphatic hydrocarbon group optionally having a hetero atom-containing polar group between carbon atoms and optionally having a substituent group, and an aromatic ring optionally having a substituent group solely or in combination,

B2 and B3 are independently hetero atom-containing polar groups,

B1 or B2 is bound to the Ax,

the hetero atom-containing polar group and the substituent group have the same meanings as the above.[18] The ribosome display complex according to the above [17], wherein the modification structure is represented by the following formulae:


wherein Ax, B1, B2 and B3 have the same meanings as the above, n is an integer of 1 or more, one or more of B1, B2 and B3 may be bound by the one or more C.[19] The ribosome display complex according to any one of the above [14] to [18], when the Ax is a chemical bond formed between a halogenated alkyl group and the side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue, a carbon atom bound by the halogeno group in the halogenated alkyl group is a carbon atom at α position of the carbonyl group or a carbon atom directly binding to the aromatic ring.[20] The ribosome display complex according to any one of the above [14] to [19], wherein

the polypeptide chain comprises 2 or more reactive amino acid residues selected from the group consisting of the cysteine residue, the lysine residue, the histidine residue and the tryptophan residue,

the a is 2 or more in the modification structure represented by the formula (2),

a ring is formed by the polypeptide chain and the modification structure.[21] The ribosome display complex according to any one of the above [13] to [20], wherein the polypeptide chain consists of 100 to 5000 amino acid residues.[22] The ribosome display complex according to any one of the above [13] to [21], wherein the reactive amino acid residue is included between a 2ndposition from an N-terminal and a 30thposition from a C-terminal inclusive in the polypeptide chain,[23] The ribosome display complex according to any one of the above [13] to [22], wherein a random sequence having 1 to 30 amino acid residues is included between a 2ndposition from an N-terminal and a 30thposition from a C-terminal inclusive in the polypeptide chain.[24] The ribosome display complex according to any one of the above [13] to [23], wherein the ribosome is sourced fromEscherichia coli.

According to one or more embodiments of the present invention, a ribosome complex of which cysteine residue, lysine residue, histidine residue or tryptophan residue in a displayed polypeptide is modified with a functional molecule can be provided by very simple and easy steps. A ribosomal protein which constitutes a ribosome complex has a much larger number of amino acid reside having a reactive side chain functional group, such as a cysteine residue, than a displayed polypeptide. In consideration of this fact, the above-described effect is very surprising. Specifically, a ribosome is composed of 55 ribosomal proteins and 3 RNA, and a certain ribosome totally contains 36 cysteines, 686 lysines and 151 histidines. Nevertheless, according to one or more embodiments of the present invention, a displayed polypeptide can be chemically modified by the very simple step without inhibiting the adhesive function between the polypeptide and m RNA, in other words, with maintaining a RD complex. Such an effect according to one or more embodiments of the present invention is unpredictable and extremely useful.

