WO2021132379A1 - 変異型rsv fタンパク質及びその利用 - Google Patents
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Definitions
- the present invention relates to a mutant RSV F protein and its use.
- Respiratory syncytial virus (RSV) infection is a respiratory infection that is prevalent worldwide.
- RSV infection causes lower respiratory tract inflammation in more than 30 million people annually, and more than 3 million people require hospital treatment annually. Therefore, many efforts have been made so far toward the development of a vaccine to prevent RSV infection.
- clinical trials targeted for infants using formalin-inactivated virus as a vaccine antigen were conducted, but instead of reducing infectious diseases, the hospitalization rate due to RSV infection increased 16 times. The result was that.
- F antigen which is one of the surface antigens of the virus
- the F antigen is one of the surface antigens of the virus
- the F antigen is denatured in the process of inactivation treatment, and only the antibody having low RSV neutralizing activity is induced to prevent the immune reaction. ing.
- Non-Patent Document 1 Research on RSV F proteins has also been advanced, and mature F proteins are most stable with metastable pre-fusion conformations (sometimes referred to herein as "pre-F” or "pre-type F protein”). Pre-fusion conformation (sometimes referred to herein as "post-F” or “post-type F protein”), and pre-fusion conformation is common due to its instability.
- Non-Patent Document 2 the pre-type F protein forms a trimer, and pre It was found that some of the epitopes (epitope ⁇ , I, II, III, IV, V, etc.) present in the type F protein are lost due to the structural change to post-F.
- Non-Patent Document 3 Infants with insufficient immune maturation are at high risk of exacerbation of symptoms at the time of infection, which is due to the fact that infants at the time of initial infection or with a small number of infections can hardly induce immunity targeting epitopes ⁇ and V.
- Non-Patent Document 4 It is important to efficiently induce an antibody having a high ability to neutralize virus infection in a vaccine antigen, and an ideal vaccine antigen is an antigen capable of preferentially inducing an antibody against epitopes ⁇ and V having a neutralizing ability. It is believed that.
- An object of the present invention is to provide a technique for efficiently inducing an antibody having a high ability to neutralize RSV infection.
- the present inventors have found that a mutant RSVF protein containing a specific amino acid mutation efficiently induces an antibody having a high ability to neutralize RSV infection, and completed the present invention.
- the present invention is as follows.
- a mutant RSVF protein having a mutation wherein the mutation replaces leucine corresponding to leucine at position 141 or leucine corresponding to leucine at position 142 with cysteine in the amino acid sequence of SEQ ID NO: 1.
- the mutant RSV F protein according to [2] wherein the RSV subtype A is an RSV A2 strain or an RSV Long strain.
- a fusion protein comprising the mutant RSV F protein according to any one of [1] to [14] and a multimerization domain fused to the C-terminus of the mutant RSV F protein.
- the fusion protein according to [15] wherein the multimerization domain is a foldon domain.
- [17] The fusion protein according to [16], which comprises any of the amino acid sequences of SEQ ID NOs: 10-13.
- the multimer which is associated through.
- the multimer according to [18], wherein the multimer is a trimer.
- the F protein or the fusion protein contains a particleization domain, and two or more of the mutant RSV F protein or the multimer are aggregated via the particleization domain to form a particle, and the particleization domain is ,
- the particulateized product which is located closer to the epitope I than the epitope ⁇ on the three-dimensional structure of the mutant RSVF protein or the multimer.
- two or more fusion proteins for producing a particle product in which a particle product domain is bound to the C-terminal of the mutant RSVF protein or the fusion protein are assembled via the particle product domain.
- the particleized body according to [20].
- the particleization domain is an Fc domain consisting of the amino acid sequence of SEQ ID NO: 30, and the particleization domain binds to the Z domain of protein A immobilized on VLP derived from a modified HBs antigen.
- [25] The C-terminal of the mutant RSVF protein according to any one of [1] to [14], or the fusion protein according to any one of [15] to [17], and particles fused to the C-terminal thereof.
- a fusion protein for the production of particulates including a chemical domain.
- An expression unit comprising the polynucleotide according to [28].
- a host cell comprising the expression unit according to [29].
- An immunogen comprising the multimer of the above or the particleized product according to any one of [20] to [24].
- a pharmaceutical composition comprising the expression unit according to [29] or the immunogen according to [31].
- An RSV vaccine comprising the expression unit according to [29] or the immunogen according to [31].
- the present invention it is possible to provide a technique for efficiently inducing an antibody having a high ability to neutralize RSV infection. It is possible to provide a mutant RSV F protein whose stability as a pre-type F protein is significantly improved as compared with the conventional case.
- the mutant RSV F protein of the present invention has an effect of inducing an antibody group considered to have a high effect on virus neutralization, particularly in humans, preferentially over an antibody group considered to contribute less to virus neutralization ( Hereinafter, it is referred to as an effect of preferentially inducing an antibody group considered to have a high effect on virus neutralization).
- the graph which shows the trimerization in Example 3 which concerns on one aspect of this invention.
- a graph showing the storage stability of epitope ⁇ in Example 7 according to one aspect of the present invention (examination of an insertion linker).
- the graph which shows the trimerization in Example 7 which concerns on one aspect of this invention (examination of an insertion linker).
- the graph which shows the recognition of the epitope I in Example 7 which concerns on one aspect of this invention (examination of an insertion linker).
- the photograph which shows the polyacrylamide gel electrophoresis result in Example 9 which concerns on one aspect of this invention.
- One aspect of the present invention is a mutant RSV F protein having a mutation, and the mutation is directed to leucine corresponding to leucine at position 141 of the amino acid sequence of SEQ ID NO: 1 or cysteine of leucine corresponding to leucine at position 142.
- This is a mutant RSV F protein in which a disulfide bond is formed between the cysteines, which is a substitution of leucine corresponding to leucine at position 373 and a substitution of leucine with cysteine.
- Natural RSV F protein is first expressed as an F0 polypeptide (precursor).
- the F0 polypeptide is processed by an intracellular furin-like protease at two sites after the endoplasmic reticulum translocation signal is cleaved to produce the F1 polypeptide, the F2 polypeptide, and the Pep27 polypeptide.
- the Pep27 polypeptide is excised and therefore does not form part of the mature F protein.
- the F2 polypeptide is derived from the N-terminal portion of the F0 polypeptide and is linked to the F1 polypeptide via two disulfide bonds.
- the F1 polypeptide is derived from the C-terminal portion of the F0 polypeptide and anchors the mature F protein to the membrane via a transmembrane domain.
- the three protomers of the F2-F1 heterodimer form mature F proteins as trimers, which are pre-type F proteins that have the function of fusing the viral membrane and the target cell membrane.
- the mutant RSV F protein of the present invention contains an F2 polypeptide and an F1 polypeptide from the N-terminal side.
- the F1 polypeptide that does not contain a transmembrane region and an intracellular region (hereinafter, may be referred to as an F1 polypeptide (extracellular region)) is preferably used.
- the mutant RSV F protein of the present invention may contain a pep27 polypeptide on the N-terminal side of the F1 polypeptide by eliminating one of the furin-like protease recognition sites by a mutation described later.
- a signal sequence may be contained on the N-terminal side of the F2 polypeptide, a linker between the F1 polypeptide and the F2 polypeptide, and a tag used for purification or the like may be further contained on the C-terminal side thereof.
- the mutant RSV F protein of the present invention preferably forms a trimer.
- the mutant RSV F protein of the present invention is preferably derived from RSV subtype A or RSV subtype B.
- RSV subtype A include RSV A2 strain, RSV Long strain, RSV S2 strain, and RSV line 19 strain, and among them, RSV A2 strain or RSV Long strain is preferable.
- RSV subtype B include RSV 18537 strain, RSV B1 strain, and RSV 9320 strain, and among them, RSV 18537 strain is preferable.
- the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of the F0 polypeptide of the F protein of the RSV A2 strain, and each component of the F0 polypeptide will be described with reference to this.
- the amino acid sequence of the signal sequence is the amino acid sequence at positions 1 to 25.
- the amino acid sequence of the F2 polypeptide is the amino acid sequence at positions 26 to 109.
- the amino acid sequence of the pep27 region is the amino acid sequence of positions 110 to 136.
- the amino acid sequence of the F1 polypeptide (extracellular region) is the amino acid sequence at positions 137 to 513.
- the amino acid sequence of the F1 polypeptide is the amino acid sequence at positions 514 to 574.
- the amino acid sequence of the F1 polypeptide is a sequence having 85% or more, 90% or 95% or more identity with the amino acid sequence at positions 514 to 574 of SEQ ID NO: 1. Good.
- the RSV F protein can include an F2 polypeptide and an F1 polypeptide (extracellular region).
- the RSVF protein containing the F2 polypeptide and the F1 polypeptide (extracellular region) contains the amino acid sequence at positions 26 to 109 of SEQ ID NO: 1 and the amino acid sequence at positions 137 to 513 in this order, SEQ ID NO: The amino acid sequence of 2 is exemplified.
- the RSV F protein can include an F2 polypeptide and an F1 polypeptide (extracellular region) in which the pep27 region is linked.
- the RSV F protein containing the F2 polypeptide and the F1 polypeptide (extracellular region) in which the pep27 region is linked in this order has the amino acid sequence of SEQ ID NO: 3 including the amino acid sequence of positions 26 to 513 of SEQ ID NO: 1. Illustrated. It should be noted that these polypeptides can be separate polypeptide chains, preferably they are linked by disulfide bonds. That is, the RSVF protein can be a complex of such polypeptides.
- the mutant RSVF protein of the present invention comprises the substitution of leucine corresponding to leucine at position 141 or leucine corresponding to leucine at position 142 with cysteine in the amino acid sequence of SEQ ID NO: 1 and leucine corresponding to leucine at position 373. Includes mutations in substitution with cysteine.
- the mutant RSVF protein forms a new disulfide bond between the introduced cysteines in addition to the naturally occurring disulfide bond, whereby the preservability of epitope ⁇ (pre-type is maintained). ) Is improved.
- it is particularly preferable that leucine corresponding to leucine at position 141 of SEQ ID NO: 1 is replaced with cysteine and leucine corresponding to leucine at position 373 is replaced with cysteine.
- the mutation position of the mutant RSV F protein is indicated by the amino acid number of the reference sequence represented by SEQ ID NO: 1.
- Leucine corresponding to leucine at position 141, leucine corresponding to leucine at position 142, and leucine corresponding to leucine at position 373 of the amino acid sequence of SEQ ID NO: 1 are SEQ ID NOs: regardless of the origin of the mutant RSVF protein. It refers to leucine corresponding to leucine at position 141, leucine corresponding to leucine at position 142, and leucine corresponding to leucine at position 373 in the amino acid sequence of 1.
- leucine at position 141 when the amino acid sequence of the RSVF protein into which the mutation should be introduced is aligned with the amino acid sequence of SEQ ID NO: 1, leucine at position 141, leucine at position 142, and leucine at position 373 of the amino acid sequence of SEQ ID NO: 1 It refers to the leucine residue located in each of the above.
- leucine at position 141 in the amino acid sequence of SEQ ID NO: 1 is at position 140.
- Leucine can be said to be leucine corresponding to leucine at position 141 of the amino acid sequence of SEQ ID NO: 1.
- the amino acid sequence of the RSVF protein into which the mutation should be introduced When the amino acid sequence of the RSVF protein into which the mutation should be introduced is aligned with the amino acid sequence of SEQ ID NO: 1, it becomes leucine corresponding to leucine at position 141 and leucine at position 142 of the amino acid sequence of SEQ ID NO: 1.
- an amino acid other than leucine is present at the position of the corresponding leucine and / or the leucine corresponding to the leucine at position 373, an embodiment of substituting the amino acid with cysteine is also included in the mutant RSVF protein of the present invention.
- the amino acid other than leucine is cysteine
- the substitution with cysteine is not required and the amino acid may be cysteine as it is.
- F2 produced from a mutant F0 polypeptide comprising an amino acid sequence in which the amino acid sequence of SEQ ID NO: 1 is substituted with leucine at position 141 or leucine at position 142 and leucine at position 373 with cysteine.
- the amino acid sequence of SEQ ID NO: 2 includes an amino acid sequence in which leucine at position 89 or leucine at position 90 and leucine at position 321 are replaced with cysteine.
- One aspect of the present invention is the amino acid of SEQ ID NO: 1 in an amino acid sequence having 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 1.
- An F2 poly produced from a mutant F0 polypeptide containing an amino acid sequence in which leucine corresponding to leucine at position 141 or leucine at position 142 or leucine corresponding to leucine at position 142 and leucine corresponding to leucine at position 373 are substituted with cysteine.
- a disulfide bond is formed between the cysteines and has an ability to induce an antibody against the pre-type F protein of RSV.
- the amino acid sequence of SEQ ID NO: 1 Leucine corresponding to leucine at position 141 (89th position in SEQ ID NO: 2) or leucine corresponding to leucine at position 142 (90th position in SEQ ID NO: 2), and leucine corresponding to leucine at position 373 (321st in SEQ ID NO: 2).
- % (%) identity with respect to an amino acid sequence is defined as sequence identity, introducing gaps if necessary to obtain maximum% identity, and any conservative substitution of sequence identity. It is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular reference polypeptide sequence, if not considered part. Alignment for the purpose of measuring% identity can be done by various methods within the skill of one of ordinary skill in the art, such as publicly available computers such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. It can be achieved by using software. One of ordinary skill in the art can determine the appropriate parameters for aligning the sequences, including any algorithm required to achieve maximum alignment for the full length of the sequences being compared.
- % identity values are obtained by using the sequence comparison computer program BLAST in pairwise alignment.
- the% identity of a given amino acid sequence A with a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues with scores consistent to be identical by the program alignment of A and B of the sequence alignment program BLAST, and Y is the total number of amino acid residues of B. Is. It will be appreciated that if the length of amino acid sequence A differs from the length of amino acid sequence B, then the% identity of A to B is different from the% identity of B to A. Unless otherwise noted, all% identity values here are obtained using the BLAST computer program as shown in the paragraph immediately above.
- the amino acid sequence having a predetermined identity with the predetermined amino acid sequence is due to substitution, deletion, insertion, or addition in the predetermined amino acid sequence.
- the substitution is preferably a conservative substitution.
- Constant substitution is the replacement of an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the protein. For example, there is a case where one hydrophobic residue is replaced with another hydrophobic residue, a case where a certain polar residue is replaced with another polar residue having the same charge, and the like.
- Examples of functionally similar amino acids capable of such substitution include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like as non-polar (hydrophobic) amino acids.
- Examples of the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine and the like.
- Examples of positively charged (basic) amino acids include arginine, histidine, lysine and the like.
- Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- mutant RSV F protein of the present invention is a mutant RSV F protein having a mutation, wherein the mutation is to a non-acidic amino acid of glutamic acid corresponding to glutamic acid at position 60 of the amino acid sequence of SEQ ID NO: 1.
- mutant RSVF protein that is a replacement for.
- the non-acidic amino acid has the effect that the mutant RSVF protein preferentially induces an antibody group that is considered to have a high effect on improving the storage stability of epitope ⁇ , promoting trimerization, and neutralizing the virus.
- Specific examples thereof include neutral amino acids, basic amino acids, and non-polar (hydrophobic) amino acids. More specifically, the neutral amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine and cysteine, and the basic amino acids include arginine, histidine and lysine, which are non-polar (hydrophobic).
- amino acids examples include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
- the non-acidic amino acid is preferably a neutral amino acid and / or a non-polar (hydrophobic) amino acid.
- the neutral amino acids threonine and serine are preferable, and among the non-polar (hydrophobic) amino acids, it is preferable to be selected from the group consisting of methionine, phenylalanine and leucine. Therefore, the non-acidic amino acids are methionine and phenylalanine.
- Leucine, threonine, and serine are preferably selected from the group.
- One aspect of the present invention is an F2 polypeptide and an F1 polypeptide (extracellular region) produced from a mutant F0 polypeptide containing an amino acid sequence in which glutamic acid at position 60 of the amino acid sequence of SEQ ID NO: 1 is replaced with a non-acidic amino acid.
- the amino acid sequence of SEQ ID NO: 2 includes an amino acid sequence in which glutamic acid at position 35 is replaced with a non-acidic amino acid.
- One aspect of the present invention is the amino acid of SEQ ID NO: 1 in an amino acid sequence having 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 1.
- a mutant RSV F protein having the ability to induce an antibody against the F protein.
- the amino acid sequence of SEQ ID NO: 1 It contains an amino acid sequence in which glutamic acid (position 35 in SEQ ID NO: 2) corresponding to glutamic acid at position 60 is replaced with a non-acidic amino acid.
- the mutation of glutamic acid at position 60 may be introduced together with the above-mentioned mutation of replacing leucine with cysteine.
- the mutant RSVF protein of the present invention is further mutated in that the amino acid constituting the furin recognition site present on the C-terminal side of the pep27 region is replaced with an amino acid such that the furin recognition site is not recognized by furin. It may be a type RSVF protein.
- the mutant RSV F protein has an F2 polypeptide and an F1 polypeptide (extracellular region) in which the pep27 region is linked, whereby the epitope ⁇ remains conserved. , It has the effect of promoting trimerization.
- the pep27 region is a region corresponding to the 110th to 136th positions of the amino acid sequence of SEQ ID NO: 1, and is intracellularly composed of a first recognition site on the N-terminal side and a second recognition site on the C-terminal side by furin. Is disconnected.
- the N-terminal side of the pep27 region is the F2 polypeptide and the C-terminal side is the F1 polypeptide.
- an F2 polypeptide and an F1 polypeptide are produced, and these are cysteine residues originally possessed by each polypeptide. A disulfide bond is formed between them to form a complex.
- the RSV F protein does not contain the pep27 region, but the mutation does not cleave the second recognition site, and the first recognition that exists between the C-terminal side of the F2 polypeptide and the pep27 region. Only the site is cut by furin.
- a polypeptide containing an F1 polypeptide linking the pep27 region and an F2 polypeptide are produced, and these form a disulfide bond between the cysteine residues originally possessed by each polypeptide to form a complex.
- this aspect may be a complex containing a polypeptide fragment containing an F1 polypeptide linking the pep27 region and an F2 polypeptide.
- the substitution may be a substitution of one or more amino acids among the amino acids constituting the furin recognition site.
- the amino acid constituting the furin recognition site consists of arginine corresponding to arginine at position 133, arginine corresponding to arginine at position 135, and arginine corresponding to arginine at position 136 in the amino acid sequence of SEQ ID NO: 1. It is one or more amino acids selected from, and it is preferable that the amino acid is replaced with a non-basic amino acid.
- the arginine corresponding to arginine at position 133, the arginine corresponding to arginine at position 135, and the arginine corresponding to arginine at position 136 in the amino acid sequence of SEQ ID NO: 1 may be independently substituted with non-basic amino acids. ..
- the non-basic amino acid is not limited as long as it has the effect of promoting trimerization while maintaining the preservation of the epitope ⁇ , but specifically, it is a neutral amino acid, an acidic amino acid, or a non-polar (hydrophobic).
- Sex) Amino acids can be exemplified. More specifically, neutral amino acids include glycine, serine, threonine, tyrosine, glutamine, aspartic acid, and cysteine, and acidic amino acids include aspartic acid and glutamic acid, which are non-polar (hydrophobic) amino acids. Examples include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Among them, neutral amino acids are preferable, and asparagine is more preferable.
- mutant RSV F protein of the present invention is that in the amino acid sequence of SEQ ID NO: 3, leucine at position 116 or leucine 117, and leucine at position 348 are replaced with cysteine, and arginine at position 108, 110. It contains an amino acid sequence in which an amino acid selected from the group consisting of arginine at position and arginine at position 111 is replaced with a non-basic amino acid.
- mutant RSV F protein of the present invention is an amino acid sequence having 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 3. , Leucine corresponding to arginine at position 141 of the amino acid sequence of SEQ ID NO: 1 (leucine at position 116 in SEQ ID NO: 3) or arginine corresponding to arginine at position 142 (leucine at position 117 in SEQ ID NO: 3), and position 373.
- Leucine corresponding to leucine (leucine at position 348 in SEQ ID NO: 3) is replaced with cysteine, and arginine corresponding to arginine at position 133 in the amino acid sequence of SEQ ID NO: 1 (arginine at position 108 in SEQ ID NO: 3), 135 Amino acids selected from the group consisting of arginine corresponding to arginine at position 3 (arginine at position 110 in SEQ ID NO: 3) and arginine corresponding to arginine at position 136 (arginine at position 111 in SEQ ID NO: 3) are non-basic amino acids. Contains the substituted amino acid sequence.
- mutant RSV F protein of the present invention in the amino acid sequence of SEQ ID NO: 3, glutamic acid at position 35 is replaced with a non-acidic amino acid, and arginine at position 108, arginine at position 110, and position 111.
- glutamic acid at position 35 is replaced with a non-acidic amino acid
- arginine at position 108, arginine at position 110, and position 111 Contains an amino acid sequence in which an amino acid selected from the group consisting of arginine is replaced with a non-basic amino acid.
- mutant RSV F protein of the present invention is an amino acid having 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 3.
- glutamic acid corresponding to glutamic acid at position 60 of the amino acid sequence of SEQ ID NO: 1 is replaced with a non-acidic amino acid, and arginine at position 133 of the amino acid sequence of SEQ ID NO: 1 is used.
- Corresponding arginine (arginine at position 108 in SEQ ID NO: 3), arginine corresponding to arginine at position 135 (arginine at position 110 in SEQ ID NO: 3), and arginine corresponding to arginine at position 136 (arginine at position 111 in SEQ ID NO: 3). ) Includes an amino acid sequence in which the amino acid selected from the group consists of non-basic amino acids.
- the pep27 region may be replaced with an artificial linker in the amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more identity with SEQ ID NO: 3 or SEQ ID NO: 3.
- the sequence of the artificial linker is not particularly limited, but it is preferably an amino acid residue length of 6 to 27, preferably a sequence containing Gly and / or Ser, and more specifically, Gly-Ser-Gly.
- -Ser-Gly-Ser SEQ ID NO: 18
- (Gly-Gly-Gly-Gly-Ser) 2 SEQ ID NO: 19
- (Gly-Gly-Gly-Gly-Ser) 3 SEQ ID NO: 20
- SEQ ID NO: 20 can be exemplified. ..
- the mutant RSVF protein of the present invention replaces leucine corresponding to leucine at position 141 or leucine at position 142 and leucine corresponding to leucine at position 373 with cysteine in the amino acid sequence of SEQ ID NO: 1 above.
- the mutation that replaces leucine with cysteine the mutation of glutamate at position 60, and / or the mutation of the furin recognition site, it further corresponds to threonine at position 189 of the amino acid sequence of SEQ ID NO: 1.
- It may be a mutant RSVF protein in which the amino acid and / or the amino acid corresponding to serine at position 190 is replaced with a hydrophobic amino acid.
- the mutant RSVF protein according to this embodiment has excellent storage stability of epitope ⁇ , has an effect of promoting trimerization, and also has an effect of preferentially inducing an antibody group considered to have a high effect on virus neutralization. While maintaining the effect, the expression level is remarkably improved in the expression system.
- the amino acid corresponding to threonine at position 189 and / or the amino acid corresponding to serine at position 190 of the amino acid sequence of SEQ ID NO: 1 may be independently substituted with hydrophobic amino acids.
- the mutant RSVF protein containing the substitution has excellent storage stability of epitope ⁇ , and preferentially induces an antibody group considered to have an effect of promoting trimerization and a high effect on virus neutralization.
- the mutant RSVF protein according to this embodiment has a lower risk than conventional vaccines because it is difficult to be recognized by an antibody that is ineffective or poor in neutralizing viral infection.
- the mutant RSVF protein according to this embodiment is difficult to be recognized by an antibody that recognizes epitope I.
- Epitope I induces an antibody having a significantly low ability to neutralize viral infection
- the symptoms are worsened by activation of immune cells that hardly contribute to neutralization of viral infection.
- the mutant RSVF protein according to this embodiment which is difficult to be recognized by an antibody that recognizes epitope I, has a lower risk than the conventional vaccine.
- the hydrophobic amino acid preferentially induces an antibody group in which the mutant RSV F protein is considered to have excellent storage stability of epitope ⁇ , promotion of trimerization, and high effect on virus neutralization.
- the effect of significantly improving the expression level in the expression system while maintaining the effect of it is not limited as long as it has the effect of being ineffective in neutralizing viral infection or difficult to be recognized by a poor antibody.
- Examples include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Above all, it is preferable to select from the group consisting of valine, isoleucine, and leucine.
- the 189th position in SEQ ID NO: 1 is the amino acid at position 137 of SEQ ID NO: 2, and the position 190 of SEQ ID NO: 1 is the amino acid at position 138 of SEQ ID NO: 2.
- position 189 in SEQ ID NO: 1 is the amino acid at position 164 of SEQ ID NO: 3
- position 190 of SEQ ID NO: 1 is the amino acid at position 165 of SEQ ID NO: 3
- one or both of these mutations are introduced in addition to the above-mentioned mutation in which leucine is replaced with cysteine, the mutation in glutamate at position 60, and / or the mutation in the furin recognition site. May be done.
- one aspect of the present invention further corresponds to lysine at position 42 of the amino acid sequence of SEQ ID NO: 1.
- It may be a mutant RSVF protein in which the amino acid to be used is replaced with arginine and / or the amino acid corresponding to valine at position 384 is replaced with threonine.
- the mutant RSVF protein according to this embodiment has excellent storage stability of epitope ⁇ , and also maintains the effect of promoting trimerization and the effect of preferentially inducing an antibody group considered to have a high effect on virus neutralization. As it is, it has the effect of significantly improving the expression level in the expression system.
- the 42nd position in SEQ ID NO: 1 is the 17th amino acid of SEQ ID NO: 2
- the 384th position of SEQ ID NO: 1 is the 332th amino acid of SEQ ID NO: 2, which has 90% or more identity with SEQ ID NO: 2.
- one or both of these mutations are introduced in addition to the leucine-substituting mutation and / or the glutamic acid at position 60 mutation, as well as the mutations at positions 189 and / or 190. You can.
- position 42 in SEQ ID NO: 1 is the amino acid at position 17 of SEQ ID NO: 3
- position 384 of SEQ ID NO: 1 is the amino acid at position 359 of SEQ ID NO: 3
- one or both of these mutations replace leucine with cysteine, glutamate at position 60, and / or mutation at the furin recognition site, and mutations at positions 189 and / or 190. In addition, it may be introduced.
- the mutant RSV F protein may further contain a known mutation as long as it does not interfere with the effect of the mutant RSV F protein.
- the expression system may further contain mutations for improving the expression level.
- the amino acid corresponding to proline at position 102 of the amino acid sequence of SEQ ID NO: 1 is replaced with alanine
- the amino acid corresponding to isoleucine at position 379 is replaced with valine
- position 447 One or more substitutions selected from the group consisting of the amino acids corresponding to methionine substituted with valine can be exemplified.
- the 102nd position in SEQ ID NO: 1 is the amino acid at position 77 of SEQ ID NO: 2
- the position 379 of SEQ ID NO: 1 is the amino acid at position 327 of SEQ ID NO: 2
- the position 447 of SEQ ID NO: 1 is 395 of SEQ ID NO: 2.
- the amino acid sequence which is the amino acid at the position and has 85%, 90% or 95% or more identity with SEQ ID NO: 2 one or both of these mutations replaces leucine with cysteine, and / or 60.
- glutamic acid at position 189 and / or 190 and the mutation at position 42 and / or 384 it may be introduced.
- the 102nd position of SEQ ID NO: 1 is the amino acid at the 77th position of SEQ ID NO: 3, the 379th position of SEQ ID NO: 1 is the amino acid at the 354th position of SEQ ID NO: 3, and the 447th position of SEQ ID NO: 1 is the 422 of SEQ ID NO: 3.
- the amino acid sequence at position 3 or 85%, 90% or 95% or more identity with SEQ ID NO: 3 one or both of these mutations replace leucine with cysteine, glutamic acid at position 60.
- mutant RSV F protein of the present invention is a mutant RSV F protein containing any of the amino acid sequences of SEQ ID NOs: 6 to 9. Specifically, it contains the following amino acid mutations.
- SEQ ID NO: 6 In the amino acid sequence of SEQ ID NO: 6 (variant RSV_F protein contained in FH_50), in SEQ ID NO: 3, leucine at position 141 of SEQ ID NO: 1 is replaced with cysteine, and leucine at position 373 of SEQ ID NO: 1 is replaced with cysteine. , The 135th arginine of SEQ ID NO: 1 is replaced with asparagine, the 136th arginine of SEQ ID NO: 1 is replaced with asparagine, the 189th threonine of SEQ ID NO: 1 is replaced with valine, and the 190th position of SEQ ID NO: 1 is replaced.
- the amino acid sequence of SEQ ID NO: 6 is divided into the amino acid sequence of the F2 polypeptide and the amino acid sequence of the pep27-added F1 polypeptide (extracellular region), but here, they are represented as one bound amino acid sequence.
- the amino acid sequence of the F2 polypeptide of the target protein is compared with the amino acid sequence of the pep27-added F1 polypeptide (extracellular region) bound in this order. .. The same applies hereinafter.
- SEQ ID NO: 7 In the amino acid sequence of SEQ ID NO: 7 (variant RSV_F protein contained in FH_81), in SEQ ID NO: 3, leucine at position 141 of SEQ ID NO: 1 is replaced with cysteine, and leucine at position 373 of SEQ ID NO: 1 is replaced with cysteine. , The 135th arginine of SEQ ID NO: 1 is replaced with asparagine, the 136th arginine of SEQ ID NO: 1 is replaced with asparagine, the 189th threonine of SEQ ID NO: 1 is replaced with valine, and the 190th position of SEQ ID NO: 1 is replaced.
- Serin was replaced with valine, glutamate at position 60 of SEQ ID NO: 1 was replaced with methionine, proline at position 102 of SEQ ID NO: 1 was replaced with alanine, isoleucine at position 379 of SEQ ID NO: 1 was replaced with valine, and the sequence. It is an amino acid sequence in which methionine at position 447 of No. 1 is replaced with valine.
- SEQ ID NO: 8 variant RSV_F protein contained in FH_82
- SEQ ID NO: 3 leucine at position 141 of SEQ ID NO: 1 is replaced with cysteine
- leucine at position 373 of SEQ ID NO: 1 is replaced with cysteine.
- the 135th arginine of SEQ ID NO: 1 is replaced with asparagine
- the 136th arginine of SEQ ID NO: 1 is replaced with asparagine
- the 189th threonine of SEQ ID NO: 1 is replaced with valine
- the 190th position of SEQ ID NO: 1 is replaced.
- Serin was replaced with valine, lyucine at position 42 of SEQ ID NO: 1 was replaced with arginine, valine at position 384 of SEQ ID NO: 1 was replaced with sleonine, proline at position 102 of SEQ ID NO: 1 was replaced with alanine, and the sequence. It is an amino acid sequence in which isoleucine at position 379 of No. 1 is replaced with valine and methionine at position 447 of SEQ ID NO: 1 is replaced with valine.
- SEQ ID NO: 9 variant RSV_F protein contained in FH_85
- leucine at position 141 of SEQ ID NO: 1 is replaced with cysteine
- leucine at position 373 of SEQ ID NO: 1 is replaced with cysteine.
- the 135th arginine of SEQ ID NO: 1 is replaced with asparagine
- the 136th arginine of SEQ ID NO: 1 is replaced with asparagine
- the 189th threonine of SEQ ID NO: 1 is replaced with valine
- the 190th position of SEQ ID NO: 1 is replaced.
- Serine is replaced with valine, glutamate at position 60 of SEQ ID NO: 1 is replaced with methionine, lysine at position 42 of SEQ ID NO: 1 is replaced with arginine, valine at position 384 of SEQ ID NO: 1 is replaced with threonine, and the sequence. It is an amino acid sequence in which proline at position 102 of No. 1 is replaced with alanine, isoleucine at position 379 of SEQ ID NO: 1 is replaced with valine, and methionine at position 447 of SEQ ID NO: 1 is replaced with valine.
- One aspect of the present invention is an amino acid sequence having 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity with any of the amino acid sequences of SEQ ID NOs: 6-9.
- Each is a mutant RSV F protein containing the above amino acid substitutions, forming a disulfide bond between the cysteines, and having the ability to induce an antibody against the pre-type F protein of RSV.
- the definition of an amino acid sequence having a predetermined identity with a predetermined amino acid sequence is the same as that described above.
- Fusion protein Another aspect of the invention is a fusion protein comprising said mutant RSVF protein and at least a multimerized domain.
- the fusion protein may contain a polypeptide other than the mutant RSVF protein and the multimerization domain, and the mutant RSVF protein and the polypeptide other than the multimerization domain interfere with the effect of the mutant RSVF protein.
- the present invention is not particularly limited, and specific examples thereof include motifs such as a thrombin cutting motif and purification tags such as His-tag and StrepTagII.
- the multimerization domain is not particularly limited as long as it is a polypeptide required for multimerization of the mutant RSVF protein, but a foldon domain for trimerization can be exemplified (Yizhi Tao et. Al. 1997). . Structure 5 (6): 789) (Frank S et. Al. 2001. Journal of molecular biology 308 (5): 1081).
- Examples of the foldon domain include a foldon domain derived from bacteriophage T4 fibritin.
- the amino acid sequence the amino acid sequence of SEQ ID NO: 21 can be exemplified.
- the fusion protein examples include a fusion protein having the amino acid sequences of SEQ ID NOs: 6 to 9 in which the Foldon domain is linked to the C-terminal of the mutant RSVF protein.
- those having the amino acid sequences of SEQ ID NOs: 10 to 13 can be mentioned.
- the amino acid sequences of SEQ ID NOs: 10 to 13 are divided into the amino acid sequence of the F2 polypeptide and the amino acid sequence in which the folddon domain is linked to the C-terminal of the pep27-added F1 polypeptide (extracellular region). It is represented as one amino acid sequence.
- the multimer is a fusion protein associated with a multimerization domain such as a foldon domain.
- the multimer is preferably a triquantifier of the fusion protein.
- polynucleotide Another aspect of the invention is the mutant RSVF protein, or a polynucleotide encoding the fusion protein.
- the polynucleotide encoding the mutant RSV F protein was introduced with the desired mutation by standard genetic engineering techniques with reference to the polynucleotide sequence encoding the RSVF protein before the introduction of the mutation (SEQ ID NO: 74). It can be obtained as a polynucleotide.
- the polynucleotide encoding the fusion protein is also a standard genetic engineering technique using a polynucleotide encoding the mutant RSVF protein and a polynucleotide encoding a polypeptide other than the mutant RSVF protein. Can be obtained by
- polynucleotide sequences of SEQ ID NOs: 14 to 17 are exemplified as the polynucleotide sequences used for expressing the fusion protein of the present invention and producing it in a host.
- SEQ ID NO: 14 is a polynucleotide sequence for expressing a fusion protein having the amino acid sequence of SEQ ID NO: 10 and producing it in a host, and a sequence encoding a signal peptide on the 5'end side of the sequence encoding the fusion protein. Is added.
- SEQ ID NO: 15 is a polynucleotide sequence for expressing a fusion protein having the amino acid sequence of SEQ ID NO: 11 and producing it in a host, and is a sequence encoding a signal peptide on the 5'end side of the sequence encoding the fusion protein. Is added.
- SEQ ID NO: 16 is a polynucleotide sequence for expressing a fusion protein having the amino acid sequence of SEQ ID NO: 12 and producing it in a host, and a sequence encoding a signal peptide on the 5'end side of the sequence encoding the fusion protein. Is added.
- SEQ ID NO: 17 is a polynucleotide sequence for expressing a fusion protein having the amino acid sequence of SEQ ID NO: 13 and producing it in a host, and a sequence encoding a signal peptide on the 5'end side of the sequence encoding the fusion protein. Is added.
- each codon may use any codon as long as it encodes the intended amino acid.
- each codon may or may not be optimized, but it is preferably optimized.
- the polynucleotide sequence of these polynucleotide sequences It may be a polynucleotide that hybridizes with a polynucleotide having a complementary sequence under stringent conditions.
- the stringent condition includes, for example, a condition in which after Southern hybridization, washing is performed at 68 ° C., 0.1 ⁇ SSC, and a salt concentration corresponding to 0.1% SDS.
- the expression unit may include an element necessary for expression such as a promoter sequence and a transcription termination signal sequence, as long as it contains a mechanism for expressing the polynucleotide, and is used in standard genetic engineering techniques. It may be an expression unit.
- the expression unit may be included, for example, in a recombinant vector.
- the recombinant vector include a plasmid vector and a viral vector, and examples thereof include a vector that can be expressed in prokaryotic cells, a vector that can be expressed in eukaryotic cells, and a vector that can be expressed in cells derived from mammals.
- a host cell comprising said expression unit.
- it is a host cell transformed with a recombinant vector containing the expression unit.
- the host cell is not particularly limited as long as the protein encoded by the polynucleotide is expressed from the expression unit, and may be used in standard genetic engineering techniques. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells, and cells derived from mammals can be exemplified.
- the introduction of the polynucleotide into the host cell can be carried out by using a known means such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method.
- the mutant RSVF protein or fusion protein can be expressed to produce the mutant RSVF protein, fusion protein or a multimerizer thereof.
- the mutant RSVF protein, fusion protein or quantifier thereof produced can be purified by known means.
- an immunogen comprising said mutant RSVF protein, said fusion protein, said multiplier, or particle form below. That is, the mutant RSVF protein, the fusion protein, the multimer, or the particulate can be used as an immunogen.
- the immunogen may contain any one of the mutant RSVF protein, the fusion protein, the multimer, or the particleized form, as long as it does not interfere with the effect of the mutant RSVF protein. Or two types may be included, or all may be included. Of these, a multimer, particularly a trimer, is preferable, and a particle is preferable, and it is used as an immunogen.
- the production of the multimerized product is as described above, and the particleized product will be described in detail below.
- the immunogen of the present invention may contain preservatives, excipients and the like contained in a standard immunogen.
- the particle form is not particularly limited as long as it is a particle formed by aggregating two or more mutant RSVF proteins or multimerizers and can efficiently induce an antibody having a high neutralizing ability.
- the mutant RSVF protein or the multimer is a particle formed by assembling two or more of them via a quantification domain.
- the shapes of the particleized body and the particles are not necessarily limited to a spherical shape, and it is sufficient that the particles are aggregated in a certain direction.
- the mutant RSVF protein or the fusion protein was further fused with a particleized domain, and the mutant RSVF protein or a multimer of the fusion protein aggregated via the particleized domain to form particles. It is preferable that it is a thing. It is preferable to use the mutant RSVF protein or a fusion protein for producing a particulate product in which a particle-forming domain is bound to the C-terminal of the fusion protein to form particles.
- the fusion protein for producing particulates will be described later.
- the particleized domain is preferably located closer to epitope I than to epitope ⁇ in terms of the three-dimensional structure of the mutant RSVF protein or multimer. That is, it is preferable that the particle is a particle in which the core is formed by the aggregation of the particleization domains, the epitope I of the mutant RSVF protein or the multimer is present inside, and the epitope ⁇ is exposed to the outside. Therefore, in the particleized product according to one aspect of the present invention, the mutant RSVF protein or the multimer is exposed at least the epitope ⁇ of the RSVF protein via the particleization domain, and at least the epitope I of the RSVF protein is exposed.
- "to prevent the epitope I from being exposed” does not need to mean that the epitope I is not completely exposed, as long as the accessibility to the antibody to the epitope I and various immune cells is reduced.
- the epitope ⁇ and the epitope I of the mutant RSV F protein are at both poles in the three-dimensional structure of the protein, so that the particleization domain forming the core is the epitope I rather than the epitope ⁇ . Due to the closeness, epitope I, which is an unnecessary epitope, is masked as an antigen, and epitope ⁇ , which is a useful epitope, can easily function as an antigen.
- the unnecessary epitope is generally used in humans to neutralize RSV virus infection when the mutant RSV F protein, its fusion protein, their multimerizer, or their particleized product is used as an immunogen.
- a useful epitope is generally the ability to neutralize RSV virus infection in humans when the mutant RSV F protein, its fusion protein, their multimerizer, or their particleized product is used as an immunogen. It means an epitope that induces an antibody having a high protein content, and specific examples thereof include epitope ⁇ and epitope V.
- the particleization domain for particle formation is a polypeptide that pulverizes a mutant RSV F protein or multimer, which can bind to scaffold particles (eg, scaffold particle-binding peptide), and can be hydrophobically aggregated (eg, scaffold particle-binding peptide).
- scaffold particles eg, scaffold particle-binding peptide
- hydrophobically aggregated eg, scaffold particle-binding peptide
- Examples include hydrophobic peptide chains) or those having self-association (eg, self-associating peptides).
- the material of the scaffold particles is not particularly limited, and examples thereof include proteins having particle-forming ability, lipid bilayers, envelopes, liposomes, iosomes, virosomes, ferrosomes, gold nanoparticles, resins, silica, polymer micelles, and gels. Be done.
- proteins having a particle-forming ability include viral proteins and variants of viral proteins that form VLPs (Virus-like particles) and VPs (Virus Particles) (for example, JP-A-2004-2313).
- hepatocyte virus surface antigen protein modified to lack the original infectivity to hepatocytes and further modified to present a scaffold molecule, which protein is expressed in eukaryotic cells. Then, it is expressed and accumulated as a membrane protein on a membrane such as the endoplasmic reticulum membrane, and released as a VLP. More specifically, as the scaffold particle, a protein-derived VLP prepared by inserting the Fc binding domain (Z domain) of protein A into the PreS region of the hepatitis B virus surface antigen (HBs antigen) protein can be mentioned, for example. , Bionanocapsule-ZZ (Beacle's catalog number: BCL-DC-002) can be used.
- a molecule that binds to the scaffold particle-binding peptide as a particleization domain is immobilized on the surface of the scaffold particle.
- the Fc-binding domain of protein A can be mentioned, and the mutant RSVF protein or a multimer thereof can be bound via a scaffold particle-binding peptide.
- the bond between the scaffold molecule and the scaffold particle-binding peptide may be either a covalent bond or a non-covalent bond.
- covalent bonds include alkyne (acetylene, etc.)-azide bonds, cysteine-maleimide bonds, primary amine-NHS ester bonds, and charge-shift bonds (hydrazide), which are collectively referred to as click chemistry.
- -Aldehyde bond is exemplified), a method of forming a covalent bond by a polypeptide (a method using SpyTag-SpyCatcher, etc.) and the like.
- non-covalent bonds include ionic interactions, hydrophobic bonds, hydrogen bonds and the like.
- a combination of Protein tag and Protein tag binding molecule (biotin-avidin, Fc-Protein A, GST (Glutathione S-transferase) -glutathione, MBP (Maltose binding protein) -maltose, His Tag-nickel, SBP tag -Streptavidin is exemplified), a combination of an antibody and an antigen, and the like.
- the combination of the scaffold molecule and the scaffold particle-binding peptide is preferably a combination of the Fc domain of IgG1 (SEQ ID NO: 30) and the Fc-binding domain of protein A (Z domain).
- the Fc domain of IgG1 may be used for the scaffold molecule, the Fc binding domain of protein A may be used for the scaffold particle binding peptide, the Fc domain of IgG1 may be used for the scaffold particle binding peptide, and the Fc binding domain of protein A may be used for the scaffold molecule. You may. As long as the scaffolding molecule or the scaffolding particle-binding peptide can be covalently or non-covalently bound to each other, partial fragments thereof can be used or modified.
- a hydrophobic peptide chain is bound to the mutant RSV F protein or fusion protein, and the mutant RSV F protein or fusion protein forms particles via the hydrophobic peptide chain.
- the hydrophobic peptide chain means a peptide chain containing a hydrophobic amino acid and self-assembling to form the particleized product, having 5 to 50 amino acid residues, preferably 10 to 30 amino acid residues.
- examples of the highly hydrophobic amino acid include valine, leucine, phenylalanine, isoleucine and the like, with leucine or isoleucine being more preferable.
- the proportion of hydrophobic amino acids in the hydrophobic peptide chain is preferably 20-60%.
- the hydrophobic amino acid appears on the surface of the hydrophobic peptide chain.
- the ratio of hydrophobic amino acids on the surface is preferably 10% or more, but if the ratio is too high, the hydrophobic amino acids on the surface are 50% or less, 30% or less, and 20% because they are not aggregated and secreted in cells. The following is preferable.
- a higher ratio of hydrophobic amino acids on the inside is preferable because the structure of the hydrophobic peptide chain is stable.
- any amino acid other than the hydrophobic amino acid may be used, but it is particularly preferable to contain 20% or more of histidine in order to enhance the secretory effect.
- the hydrophobic peptide chain is preferably a repeating sequence of a peptide sequence consisting of 7 amino acids in which hydrophobic amino acids are arranged at the 1st, 3rd, and 5th positions. The number of repetitions is, for example, 2 to 5, preferably 2 to 3.
- Preferred examples of the hydrophobic peptide chain include CC02, CC03, CC07 and CC08 (SEQ ID NOS: 40, 41, 42 and 43 in that order), of which CC07 (SEQ ID NO: 42) is more preferred.
- the self-associating protein domain is bound to the mutant RSV F protein or fusion protein, and the mutant RSV F protein or fusion protein forms particles through the self-associating protein domain.
- self-assembling proteins include hepatitis B virus core antigen (HBcAg), human papillomavirus capsid protein (HPV L1), hepatitis E virus capsid protein (HEV capsid), and so on.
- the self-associating protein may be a self-associating partial fragment or a variant.
- RSV F when bound to the RSV F protein or quantifier of the present invention, RSV F is located at a site far from the association surface for self-association of HBcAg and outside the self-association. It is preferred to bind the protein or multimer. Furthermore, when such a bond is made, it is preferable to insert a linker between the helix of the ⁇ 3 domain and the helix of the ⁇ 4 domain in order to obtain the original three-dimensional structure of HBcAg.
- the linker is, for example, a polypeptide of 10 to 30 amino acids, for example, a GS linker.
- the sequence in which the FLAG tag is bound to such a variant of HBcAg includes SEQ ID NO: 54.
- the fusion protein for producing a particle product is a protein used for producing the particle product, and is a scaffold described above that is fused to the C-terminal of the mutant RSVF protein of the present invention or the C-terminal of the fusion protein. Includes pulverized domains such as particle-bound peptides, hydrophobic peptide chains, and self-associating protein domains.
- the fusion protein for producing the present particle product is aggregated via the particle domain to form a particle product. When the distance between the C-terminals of the fusion protein constituting the multimer of the present invention changes by binding to the particle-forming domain, the particle-forming domain and the C-terminal (multimerized domain) are used.
- the linker is not particularly limited, but is preferably a peptide having 5 to 20 amino acids, and more preferably a peptide having 5 to 10 amino acids.
- the sequence is not particularly limited as long as it does not inhibit the vaccine effect of the fusion protein, and is, for example, a GS linker consisting of glycine and serine.
- the position of the linker may be between the multimerization domain and the particleization domain of the mutant RSVF protein or fusion protein, and tag sequences may be included before and after the linker.
- the tag sequence is not particularly limited, but a tag sequence usually used for protein purification is preferably used, for example, FLAG tag (SEQ ID NO: 28), His-tag (SEQ ID NO: 25), Strep-tagII (SEQ ID NO: 26) and the like. Can be exemplified.
- the reading frame is aligned with the DNA encoding the mutant RSV F protein or the fusion protein, the DNA encoding the particleization domain, and, if necessary, the DNA encoding the linker or tag. It can be obtained by ligating and expressing in the host.
- the expression and purification method can be carried out in the same manner as the expression and purification of the mutant RSV F protein or fusion protein.
- a scaffold particle-binding peptide is bound to a mutant RSVF protein (sequences of their precursor proteins: SEQ ID NOs: 31 to 39, and a poly encoding the same.
- SEQ ID NOs: 57-65 an embodiment in which a hydrophobic peptide chain is bound to a mutant RSVF protein (sequences of their precursor proteins: SEQ ID NOs: 44-49, and a polynucleotide sequence encoding it: SEQ ID NO: 66-71)
- an autoassociative protein domain is bound to a mutant RSVF protein
- the fusion protein for producing a particle product may contain a tag sequence such as a FLAG tag (DYKDDDDK: SEQ ID NO: 28) and a tag in which 3 amino acids (GGS) are added to the FLAG tag (GGSDYKDDDDK: SEQ ID NO: 29).
- a tag sequence such as a FLAG tag (DYKDDDDK: SEQ ID NO: 28) and a tag in which 3 amino acids (GGS) are added to the FLAG tag (GGSDYKDDDDK: SEQ ID NO: 29).
- the particleized product can be obtained by atomizing the particle product using the fusion protein for producing the particle product obtained above, and then purifying the particle product if necessary.
- the fusion protein for producing particulates is mixed with the scaffold particles to form particles, and then the non-particleified mutant RSV F protein or its product is removed to obtain particles. This can be done by purifying the chemicals.
- the scaffolding particles for example, HBsAg VLP (Beacle catalog number: BCL-DC-002) can be used.
- the particleization step involves associating the fusion protein for producing a particulate product through the hydrophobic peptide chain to form particles, and then atomizing the fusion protein. This can be done by purifying the particulates, except for the unmodified RSV F protein or its products.
- the particle formation step involves associating the fusion protein for the production of the particulate product through the self-associative protein domain to form particles. This can be done by purifying the granulated product, except for the unparticleted variant RSV F protein or its products.
- the particleized product of the present invention it is desirable that, for example, 2 or more, preferably 10 or more, and more preferably 20 or more as a trimer are bonded per particleized material. Further, in the particleized product of the present invention, it is desirable that 0.0001 or more, preferably 0.0005 or more, and more preferably 0.001 or more as a trimer are bonded per 1 nm 2 surface area of the particleized product. ..
- the surface area of the particleized body is the surface area of the particleized body when it is assumed that the particleized body is a sphere having a theoretical diameter measured by a dynamic light scattering method.
- the number per particle body surface area can be calculated, for example, by dividing the number per particle body surface area by the particle body surface area.
- particulates By producing particulates, selecting those having a low binding ability to epitope I and a high binding ability to epitope ⁇ , those skilled in the art can optimally count the number of particles per particle or per 1 nm 2 particle surface area. You can select the number of.
- the RSV F protein or multimer used for granulation is not limited to the mutant RSV F protein or multimer of the present invention, and is not particularly limited as long as it can be used as an RSV vaccine.
- a mutant RSV F protein other than the mutant RSV F protein of the present invention or a multimer thereof can also be used.
- those described in International Publication No. 2017/172890 and International Publication No. 2017/109629 may be used. That is, the particle formation technique of the present invention can be generally used in the particle formation of RSV F protein or its multimer, and the particle form of the present invention includes the particle form of RSV F protein or its multimer.
- the RSV F protein or its multiplier includes a particle form in which two or more particles are assembled via a particleization domain to form particles, and the particleization domain and the method of particleization include the above-mentioned mutant RSV F.
- the particleization domains and methods described in Particleization of proteins or multipliers can be applied.
- composition Another aspect of the present invention is a pharmaceutical composition comprising the expression unit or the immunogen as an active ingredient.
- the expression unit or immunogen can be a pharmaceutical composition for the prevention or treatment of respiratory syncytial virus infection.
- RSV vaccine Another aspect of the invention is an RSV vaccine comprising said expression unit or said immunogen. This is also an embodiment in which the pharmaceutical composition according to the above aspect is an RSV vaccine.
- the route of administration of the pharmaceutical composition is not particularly limited, and either parenteral administration (eg, intradermal, intramuscular, subcutaneous, transdermal, mucosal) or oral administration is administered. It can be administered to mammals by route. Examples of mammals include humans, mice, rats, rabbits, dogs, cats, cows, pigs, monkeys, and chimpanzees, but humans are preferred.
- the dosage form of the pharmaceutical composition and the method for preparing the pharmaceutical composition are well known to those skilled in the art, and the pharmaceutical composition can be produced by blending the expression unit or the immunogen with a pharmaceutically acceptable carrier or the like. it can.
- Pharmaceutically acceptable carriers are, for example, excipients, lubricants, binders and disintegrants in solid formulations, or solvents, lysis aids, suspending agents, isotonic agents, buffers in liquid formulations. Examples thereof include agents and pain-relieving agents. Further, if necessary, an appropriate amount of additives such as ordinary preservatives, antioxidants, colorants, sweeteners, adsorbents, and wetting agents can be used.
- the pharmaceutical composition may include an adjuvant.
- the adjuvant include a gel type, a bacterial cell type, an oil emulsion type, a polymer nanoparticle type, a synthetic type, and a cytokine type.
- the gel type include aluminum hydroxide, aluminum phosphate, calcium phosphate and the like.
- the bacterial cell type include CpG, cholera toxin, Escherichia coli hyperthermic toxin, pertussis toxin, and Muramyl dipeptide (MDP).
- MDP Muramyl dipeptide
- the oil emulsion type include Freund's incomplete adjuvant, MF59, and SAF.
- Examples of the polymer nanoparticle type include immunostimulatory complexes, liposomes, biodegradable microspheres, and saponin-derived QS-21.
- Examples of the synthetic type include nonionic block copolymers, muramyl peptide analogs, polyphosphazene, and synthetic polynucleotides.
- Examples of the cytokine type include IFN- ⁇ , IL-2, IL-12 and the like.
- Dosage forms for parenteral administration include injectable preparations (eg, drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral preparations, intraspinal administration preparations), external preparations.
- injectable preparations eg, drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral preparations, intraspinal administration preparations
- external preparations eg, ointment, poultice, lotion
- suppository inhalant eye agent, eye ointment, nasal drop, ear drop, liposome agent and the like can be exemplified.
- Dosage forms for oral administration are solid or liquid dosage forms, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, troches. Examples include agents.
- the pharmaceutical composition of the present invention is preferably in
- the effective dose of the pharmaceutical composition is, for example, pharmacological findings such as administration route, type of disease, degree of symptom, age, sex, weight of patient, severity of disease, pharmacokinetics and toxicological characteristics. It is decided by the doctor based on various factors such as whether or not a drug delivery system is used and whether it is administered as a part of a combination of other drugs.
- Example 1 The formation of covalent bonds (disulfide bonds) using cysteine is useful for stabilizing the structure of proteins.
- F protein RSV F protein
- International Publication No. 2017/172890, International Publication No. 17 International Publication No. 17
- 2017/109629 replaces amino acids that are members of secondary structure with cysteine and forms disulfides in distant parts, so the resulting mutants have a natural structure (natural F protein is a protein).
- natural F protein is a protein
- the precursor protein of the F_native antigen is a polypeptide (amino acid numbers 1 to 513 of SEQ ID NO: 1) obtained by removing the transmembrane region and the intracellular region of the F1 polypeptide from the natural F0 protein of the A2 strain. , P102A, I379V and M447V, a polypeptide in which the C-terminal side of the polypeptide contains the Foldon domain (SEQ ID NO: 21), Thrombin recognition sequence, His tag and StrepTagII (SEQ ID NO: 22) are fused in order. Is.
- mutants have mutations of P102A, I379V and M447V because they were prepared based on the precursor protein of F_native antigen.
- the F_native precursor protein undergoes processing during the expression process, and the signal peptide and pep27 region are shed.
- the F_native antigen is produced by expressing a polynucleotide (SEQ ID NO: 5) encoding a precursor protein (SEQ ID NO: 4) of the F_native antigen using mammalian cells as a host.
- the F_native antigen is a precursor protein of the F_native antigen (SEQ ID NO: 4) that forms a quaternary structure after being processed during the expression process.
- the signal peptide and pep27 region were shed by processing, and the remaining F2 polypeptide sequence and F1 polypeptide (extracellular region), Foldon domain, Thrombin recognition sequence, His tag, and Strep-Tag II-containing sequence were bound by disulfide bonds.
- the homotrimer is formed mainly by the binding force generated by the foldone domain.
- the specific method for producing the F native antigen is shown below.
- the above polynucleotide (SEQ ID NO: 5) was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) by standard genetic engineering techniques. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for mammalian cells expressing the precursor protein of the F_native antigen was constructed.
- the vector for introduction into cells was purified by standard plasmid vector purification methods.
- F_native antigen expression was performed by mammalian cell secretion expression using Expi293 Expression System (Thermo Fisher Scientific).
- the purified vector is introduced into Expi293 cells using ExpiFectamin293Reagent, which is a gene transfer reagent of the same kit, and cultured for about 5 days to secrete and express the encoded F_native antigen, centrifuge and pore size 0.22 ⁇ m. Only the supernatant component was obtained using the filter of.
- F_native antigen Purification of the F_native antigen was performed by affinity chromatography using Ni Sepharose High Performance (GE Healthcare). After reacting the Ni carrier with a substance obtained by diluting the above culture supernatant component with a binding buffer (20 mmol / L Tris-HCl pH 7.4, 500 mmol / L NaCl, 20 mmol / L Imidazole) about 3 times for a whole day and night. , Elution buffer (20 mmol / L Tris-HCl pH 7.4, 500 mmol / L NaCl, 500 mmol / L Imidazole) to elute each antigen. Then, the solvent was replaced with PBS (pH 7.4) using an ultrafiltration membrane, and the antigen was used as an F_native antigen.
- a binding buffer (20 mmol / L Tris-HCl pH 7.4, 500 mmol / L NaCl, 20 mmol / L Imidazole
- the binding property of each antigen to each antibody was measured using Octet QK384 (ForteBio) under the conditions of PBS (pH 7.4), 25 ° C., and 1000 rpm.
- Each antibody solution was applied to the Protein A sensor chip (ForteBio) equilibrated with PBS (pH 7.4) for 180 sec to bind each antibody to the sensor chip, and then each antigen solution was applied for 180 sec.
- the amount of increase in the detected value at time was taken as the value of the binding signal of each antigen to each antibody.
- As the antibody to be used for the sensor chip a D25 antibody capable of specifically detecting epitope ⁇ was used.
- the value of the binding signal was normalized by determining the ratio to the value of the binding signal when Palivizumab (Synagis intramuscular injection, AbbVie), which can bind regardless of the conformational change of the F antigen, was used.
- the antigens FH_08 and FH_09 containing the mutant protein in which the cysteine point mutation was introduced at two places for the formation of the disulfide bond have significantly preserved epitope ⁇ as compared with the F_native antigen containing the F protein before the mutation introduction. Results were obtained indicating that the sex was improved (Fig. 1).
- Example 2 The native F protein first undergoes cleavage of the first furin recognition site to form a monomeric structure, and then undergoes cleavage of the second furin recognition site to eliminate the pep27 region, thereby promoting trimerization. There is a report that it will be done (Anders Krarup et al. 2015. Nature communications 6: 8143). However, since the pep27 region is highly conserved in sequence length and amino acid type among virus strains isolated from nature, it is considered that it may have an indispensable effect on the formation of the three-dimensional structure of the antigen. Therefore, we decided to introduce a mutation into the second furin recognition site to inhibit cleavage of the pep27 region and to investigate the effect of the mutation on trimer formation.
- FH_08 + R135N, R136N is a precursor protein of the antigen FH_08, in which arginine at position 135 in the amino acid sequence of SEQ ID NO: 1 is mutated to asparagine and arginine at position 136 is mutated to asparagine. Indicates that you are doing.
- FH_09 + R135N, R136N means that in the precursor protein of antigen FH_09, arginine at position 135 in the amino acid sequence of SEQ ID NO: 1 is mutated to asparagine, and arginine at position 136 is mutated to asparagine. Represents.
- GSGSGS SEQ ID NO: 18
- GGGGS GGGGS 2
- GGGGS GGGGS 3
- a polynucleotide encoding the precursor protein of each of the above antigens was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for each mammalian cell expressing the precursor protein of each antigen was constructed. The vector for introduction into cells was purified by standard plasmid vector purification methods.
- Example 2 Expression and purification of each antigen were carried out in the same manner as in Example 1.
- the binding property of each antigen to each antibody was also measured by the same operation as in Example 1, but AM14 antibody capable of detecting pre-F trimer formation was added as an antibody to be used for the sensor chip.
- AM14 antibody capable of detecting pre-F trimer formation was added as an antibody to be used for the sensor chip.
- For the binding signal values of D25 antibody and AM14 antibody determine the ratio to the binding signal values when using Synagis antibody (AbbVie, Synagis intramuscular injection) that can bind regardless of the conformal change of F antigen. Normalized with.
- the antigens FH_21, FH_24, and FH_25 containing the mutant protein to which the mutation was added to the second furin recognition site are trimeric with the preservation of epitope ⁇ as compared with the antigen FH_08 before the addition of the mutation. Results were obtained indicating that the formation was improved. Among them, the improvement in trimer formation was remarkable in the antigen FH_21, which is closer to the pep27 region of the F_native antigen, than in the case where the pep27 region was replaced with an artificial linker (FH_24, FH_25) (Figs. 2 and 3).
- the amino acids at positions 189 and 190 of the precursor protein of F_native antigen are threonine and serine, which are hydrophilic amino acids in that they have a hydroxyl group in the side chain.
- the amino acids located proximal to the three-dimensional structure of the amino acids at positions 189 to 190 are isoleucine at position 57, isoleucine at position 167, leucine at position 171, valine at position 179, and leucine at position 260. Since all of these are hydrophobic amino acids, it is possible that their structural stability is impaired.
- Table 3 summarizes the mutation details of the precursor protein of each antigen prepared below.
- “FH_21 + T189I” in Table 3 indicates that the threonine at position 189 in the amino acid sequence of SEQ ID NO: 1 is mutated to isoleucine in the precursor protein of the antigen FH_21.
- “FH_21 + T189V” indicates that in the precursor protein of antigen FH_21, threonine at position 189 in the amino acid sequence of SEQ ID NO: 1 is mutated to valine.
- FH_21 + T189I, S190V indicates that threonine at position 189 in the amino acid sequence of SEQ ID NO: 1 is mutated to isoleucine and serine at position 190 is mutated to valine in the precursor protein of antigen FH_21.
- FH_21 + T189L, S190V indicates that threonine at position 189 in the amino acid sequence of SEQ ID NO: 1 is mutated to leucine and serine at position 190 is mutated to valine in the precursor protein of antigen FH_21.
- FH_21 + T189V, S190V indicates that threonine at position 189 and serine at position 190 in the amino acid sequence of SEQ ID NO: 1 are mutated to valine in the precursor protein of antigen FH_21.
- FH_50 + K42R, V384T indicates that in the precursor protein of the antigen FH_50, the lysine at position 42 in the amino acid sequence of SEQ ID NO: 1 is mutated to arginine and the valine at position 384 is mutated to threonine.
- a polynucleotide encoding the precursor protein of each of the above antigens was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for each mammalian cell expressing the precursor protein of each antigen was constructed. The vector for introduction into cells was purified by standard plasmid vector purification methods.
- each antigen FH_50, FH_51, FH_52, FH_53, FH_55 to which the mutation to the hydrophobic amino acid (V, I, L) at positions 189 to 190 was added has an epitope as compared with the antigen FH_21 before the mutation was added. Results were obtained showing that the expression level was significantly improved while maintaining the storage stability of ⁇ and the formation of trimers.
- the antigen FH_82 to which two point mutations (K42R, V384T) extracted from natural mutations that can occur in natural viruses have been added, has a significantly higher expression level while maintaining the conservation of epitope ⁇ and the formation of trimers. Results showing improvement were obtained (FIGS. 4, 5, and 6).
- the amino acid at position 60 of F_native precursor protein is glutamic acid, and since it belongs to acidic amino acids, it is generally known that the solvent is negatively charged under neutral conditions. This amino acid is adjacent to aspartic acid at position 194, which is also negatively charged under neutral conditions in terms of three-dimensional structure, and it was considered that the structure may be destabilized by the proximity of the negative charge. Therefore, we decided to carry out a mutation that changes the amino acid at position 60 to an amino acid other than the acidic amino acid in the amino acid sequence of F_native precursor protein.
- a polynucleotide encoding the precursor protein of each of the above antigens was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for each mammalian cell expressing the precursor protein of each antigen was constructed. The vector for introduction into cells was purified by standard plasmid vector purification methods.
- the antigen ⁇ FP furin wt F ecto (Kurt A. Swanson et al. 2014. Journal of Virology. 88 (20): 11802), which is known to have a post-F trimer structure, was also prepared. It was decided to use it for the evaluation of the antigen.
- Table 5 summarizes the mutations of precursor protein of the antigen prepared below.
- the precursor protein of antigen FH_81-85 is the precursor protein of antigen FH_50 introduced with mutations of E60M, K42R + V384T or E60M + K42R + V384T, respectively.
- a polynucleotide encoding the precursor protein of each of the above antigens was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques.
- the precursor protein of the antigen F_DS-Cav1 has an amino acid sequence consisting of SEQ ID NO: 23, and the precursor protein of ⁇ FP furin wt F ecto has an amino acid sequence consisting of SEQ ID NO: 24. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for each mammalian cell expressing the precursor protein of each antigen was constructed.
- the vector for introduction into cells was purified by standard plasmid vector purification methods.
- the antigens FH_50, FH_81, FH_82, and FH_85 have the same ability to neutralize virus infection, while the conservation of epitope ⁇ and the formation of trimers are not so different from those of the antigen F_DS-Cav1 in the prior literature.
- Results indicate that Epitope I, which is known to induce significantly lower antibodies, is rarely detected (FIGS. 9, 10, 11). This is because vaccines carry the risk of exacerbating symptoms by activating immune cells that contribute little to virus neutralization, but the introduction of newly found mutations is higher than the introduction of DS-Cav1 mutations. It suggests that the risk is low.
- Antigen adsorption operation was performed on 3 healthy adult sera using magnetic beads Dynabeads His-Tag Isolation and Pulldown (Thermo Fisher Scientific, 10103D).
- 20 ⁇ g of antigens F_DS-Cav1, FH_50, FH_81, FH_82 or FH_85 were added to and bound to 45 ⁇ L of beads in a bead solution washed twice with PBS (pH 7.4), and then bovine serum albumin was added. Blocked at.
- 100 ⁇ L of each healthy adult serum was diluted 10-fold with PBS (pH 7.4), beads were added and incubated (4 ° C, 1 hour, rotated), and then the beads were removed to obtain an antigen. Removal of the binding antibody was performed.
- a 4-fold dilution series was prepared by appropriately adding buffer to healthy adult serum or healthy adult serum after antigen adsorption operation, and the mixture was added to a plate and incubated (room temperature, 1 hour, standing). After that, the diluted serum is removed, a peroxidase-binding anti-human IgG antibody (Jackson Immuno Research, 709-035-149) is bound, and the color is further developed with a standard peroxidase detection reagent, and then the color is developed by absorbance measurement using a standard plate reader. The strength of was calculated.
- the relationship between the serum dilution rate and the absorbance was obtained by segmented linear interpolation between the measurement points, and the inverse number of the dilution rate at which the absorbance was 0.2 was used as the antibody titer of the antibody that binds to post-F, and the serum before the adsorption operation was used.
- the relative value of the decrease in serum antibody titer after the adsorption operation when the antibody titer was set to 100% was taken as the adsorption rate (Fig. 12).
- a 5-fold dilution series was prepared by appropriately adding PBS (pH 7.4) to healthy adult serum or healthy adult serum after antigen adsorption operation, and RS virus was mixed and incubated (room temperature, 30 minutes, static). After placement), 4 ⁇ 10 4 cells were seeded in each well of a 96-well plate, added to Vero cells cultured for 1 day, and incubated (37 ° C., humidity, 2 hours, standing).
- 1% (v) of Sodium Pyruvate Solution (Nacalai, 06977-34) and Antibiotic-Antimycotic Mixed Stock Solution (Nacalai, 09366-44) were added to DMEM, High Glucose (Thermo Fisher Scientific, 11965). / v) 100 ⁇ L of the added medium was added per well, and the mixture was further incubated (37 ° C., humidity, 16 hours, standing).
- the cells were fixed with paraformaldehyde and permeabilized with Triton X-100 (Nakarai, 35501-15), and the cells were subjected to Anti-RSV Antibody, fusion protein, all type A, B strains, clone 133-. Stain the nuclei of infected cells and whole cells with 1H, Alexa Fluor 488 (Merck, MAB8262X) and Heochst 33258 (Dojindo, 343-07961) and in the infected cell region with Imager IN Cell Analyzer (GE Healthcare). The number of virus-infected cells was counted by counting the number of nuclei.
- the reciprocal of IC 50 was used as the virus infection neutralizing titer.
- the virus infection neutralizing titer of serum before the adsorption operation was set to 100%, the relative value of the decrease in the virus infection neutralizing titer after the adsorption operation was taken as the adsorption rate (Fig. 13).
- the antigens FH_50, FH_81, FH_82, and FH_85 have lower binding properties of the post-F recognition antibody present in healthy adult serum than the antigen F_DS-Cav1 of the prior literature, and the antibody is halved in the adsorption operation. It was shown that only the degree of binding was possible. Nevertheless, almost all the antibodies contributing to the virus infection neutralizing ability were bound like F_DS-Cav1, and it was also shown that the antibody remaining by the adsorption operation had almost no virus infection neutralizing ability.
- Mouse immune serum was obtained by immunizing BALB / cAnNCrlCrlj (Japan Charles River) (female, 7 weeks old) with each mutant protein.
- Antigens FH_50, FH_81, FH_82, FH_85 or F_DS-Cav1 (antigen concentration: 0.80 mg / mL) dissolved in PBS (pH 7.4) and aluminum hydroxide (InvivoGen, vac-alu-250) were mixed well in equal amounts.
- the solution was intramuscularly administered to the hind limbs and thighs of the mice on the 0th day of breeding and the 21st day of breeding (5 animals per group).
- 50 ⁇ L of each mouse immune serum was diluted 20-fold with PBS (pH 7.4), beads were added and incubated (4 ° C, 1 hour, rotated), and then the beads were removed to obtain an antigen. Removal of the binding antibody was performed.
- a 5-fold dilution series was prepared by appropriately adding a buffer to the mouse immune serum or the mouse immune serum after the antigen adsorption operation, and the mixture was added to a plate and incubated (room temperature, 2 hours). Then, the diluted serum was removed, a peroxidase-binding anti-mouse antibody antibody (DaKo, P0161) was bound, and the color was further developed with a standard peroxidase detection reagent, and then the intensity of color development was determined by absorbance measurement with a standard plate reader.
- a peroxidase-binding anti-mouse antibody antibody DaKo, P0161
- the relationship between the serum dilution rate and the absorbance was obtained by segmental linear interpolation between the measurement points, and the inverse number of the dilution rate at which the absorbance was 0.2 was used as the antibody titer of the detected anti-F antigen antibody, and the serum before the adsorption operation was used.
- the relative value of the decrease in serum antibody titer after the adsorption operation when the antibody titer was 100% was taken as the adsorption rate (Fig. 14).
- the antigens FH_50, FH_81, FH_82, and FH_85 had a smaller induction rate of the post-F recognition antibody than the antigen F_DS-Cav1 in the prior literature.
- the results suggest that when the antigens FH_50, FH_81, FH_82, and FH_85 are used as vaccine antigens, the risk of inducing antibodies that do not contribute to virus infection protection is low.
- Example 6 Comparison with the preceding product 2
- the F protein moieties of the antigens FH_50, FH_81, FH_82, FH_85 carried out in Example 5 all contain mutations in L141C and L373C.
- the mutation was introduced in Example 1 to introduce a disulfide bond, but in the previous patent (International Publication No. 2017/109629), the introduction of a disulfide bond into a relatively close site (Mutant ID: pXCS524. L142C, N371C) have been investigated, and further mutated optimized antigens (Mutant ID: pXCS881.
- the antigens FH_08, FH_82, and FH85 used were those described in Example 1, Example 3, and Example 5, respectively.
- the polynucleotide encoding these precursor proteins is inserted into the pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques, and the nucleotide sequence indicating that the target DNA sequence is inserted is inserted. Confirmed by analysis, an expression vector for each mammalian cell expressing the precursor protein of each antigen was constructed. The vector for introduction into cells was purified by standard plasmid vector purification methods.
- the antigen FH_08 induces an antibody having a significantly lower ability to neutralize virus infection, while having better storage of epitope ⁇ and formation of trimers than the antigen Pf524 into which the mutation of the previous patent has been introduced.
- the optimized antigens FH_82 and FH_85 are superior to the antigens Pf881 into which the mutation of the previous patent, which is also optimized, have been introduced, while the preservation of epitope ⁇ and the formation of trimers are superior, while they are infected with virus. Results indicate that epitope I, which is known to induce antibodies with significantly lower harmony, is rarely detected.
- the antigens FH_82 and FH_85 had a smaller induction rate of the post-F recognition antibody than the antigen Pf881 into which the mutation of the previous patent was introduced.
- the results suggest that they preferentially induce antibody groups that are considered to have a higher effect on virus neutralization than Pf881.
- Example 7 Particleing of antigen using scaffold particles
- an antigen site recognized by a post-F recognition antibody such as an epitope I recognition antibody. It is considered effective to reduce the accessibility of antibodies against the virus and various immune cells by the steric disorder effect. Therefore, we considered the possibility of obtaining the steric hindrance effect by immobilizing the antigen on appropriate scaffold particles so that the recognition site of the post-F recognition antibody is on the inside.
- the density is sparse because the interval between the antigens is longer than a certain length, or a flexible linker having a length longer than a certain length is provided between the scaffold particle and the antigen. If it is introduced, it is possible that the above-mentioned steric hindrance effect cannot be obtained. Therefore, we decided to investigate GS linkers of multiple lengths using HBsAg VLP, which is considered to be feasible for relatively high-density antigen presentation.
- Bionanocapsule-ZZ (Beacle, BCL-DC-002) capable of binding an Fc fusion protein was used.
- the precursor protein of the antigen F_DS-Cav1-Fc (SEQ ID NO: 31), the precursor protein of FH_82-Fc (SEQ ID NO: 32), and the precursor protein of FH_85-Fc (SEQ ID NO: 33) were prepared.
- the GS linker (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGS (SEQ ID NO: 19) or GGGGS (SEQ ID NO: 27) immediately before the Fc sequence ) was inserted, and it was prepared by the following method based on the corresponding precursor protein (SEQ ID NOs: 34 to 39).
- the polynucleotide encoding each precursor protein was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for each mammalian cell expressing the precursor protein of each antigen was constructed.
- the vector for introduction into cells was purified by standard plasmid vector purification methods.
- Expi293 Expression System (Thermo Fisher Scientific). Each purified vector is introduced into Expi 293 cells using Expi Fectamin 293 Reagent, which is a gene transfer reagent of the same kit, and cultured for about 4 to 5 days to secrete and express each antigen encoded by centrifugation and pore size. Only the supernatant component was obtained using a 0.22 ⁇ m filter. Purification of each antigen was performed by a standard affinity purification method using a Protein A purification column. After purification, the solvent was replaced with PBS (pH 7.4) by using an ultrafiltration membrane.
- PBS pH 7.4
- Each prepared antigen is mixed with HBsAg VLP at a ratio of about 135 molecules (about 45 molecules in trimer) on average per HBsAg VLP particle, and allowed to stand at room temperature for 1 hour to form scaffold particles. Fixed. At this time, it was confirmed by performing Blue Native PAGE that almost no unimmobilized antigen was present.
- Table 7 summarizes the antigen used for the evaluation in this example and the particleized product in which the antigen is immobilized on the scaffold particles (hereinafter, may be referred to as “particleted antigen”).
- any of the GS linker insertions carried out improved the formation of the trimer while maintaining the binding property of the antibody to the epitope ⁇ , and had the effect of diminishing the detected value of the epitope I.
- the attenuating effect of epitope I detection was shown to be reduced when too long a GS linker was inserted between the scaffold particles and the antigen.
- a length of about GGGGSGGGGS does not have a significant effect, but a longer length of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75) reduces the effect of attenuating epitope I detection. It was seen.
- the theoretical diameter of the particleized antigen FH_85-VLP was estimated to be 89.28 nm by a dynamic light scattering method using Zetasizer ⁇ V (Malvern) (Fig. 25). Since an average of about 135 molecules (about 45 molecules of the trimer) is presented per particle, the average molecular density per 1 nm 2 theoretical surface area is calculated to be about 0.005 molecules (about 0.0018 molecules as a trimer). it can.
- the desired steric hindrance effect can be achieved if the density is such that at least about 0.005 molecules or more of antigen molecules are present per 1 nm 2 theoretical surface area estimated by the dynamic light scattering method. Similar results were obtained with DS-Cav1-VLP and FH_82-VLP (Fig. 25).
- each particleized antigen use a well-mixed solution of PBS or PBS and aluminum hydroxide (InvivoGen, vac-alu-250) in equal amounts.
- the antigen is intramuscularly administered to the hind limbs and thighs of mice twice at intervals of about 3 weeks, and blood is obtained about 3 weeks after the second administration.
- the obtained blood is incubated at room temperature and then centrifuged to collect serum.
- An ELISA test in which the antigens of each conformation are stratified is performed to confirm the degree of induction of antibodies that bind to pre-F or post-F in serum.
- the antigen adsorption operation and the reduction rate of the antigen recognition antibody by the antigen adsorption operation are measured.
- the method conforms to the method shown in Example 5.
- this particle formation confirms the effect of preferentially inducing antibody groups that are considered to have a high effect on virus neutralization.
- Example 8 Particleification of antigen using hydrophobic peptide chain
- the effect of reducing the accessibility of antibodies and various immune cells to the antigen site recognized by the post-F recognition antibody shown in Example 7 by the steric hindrance effect is , I thought that it could be realized by a method that does not use scaffold particles. Therefore, in this example, it was decided to investigate particle formation by a hydrophobic assembly using a hydrophobic peptide chain (a polypeptide chain containing a hydrophobic amino acid).
- hydrophobic peptide chain used needs to have the property of hydrophobically assembling under the conditions of a vaccine preparation or in vivo, but it is difficult to put it into practical use with a sequence that significantly reduces the efficiency of antigen secretion and expression by cells.
- hydrophobic polypeptide chain should be investigated. And said.
- the sequence containing the Thrombin recognition sequence, His tag, and Strep-Tag II (SEQ ID NO: 22) in the precursor protein of the antigen F_DS-Cav1 is subjected to various hydrophobic peptide chains (SEQ ID NOs: 40 to 43) and the FLAG tag which is a tag for purification.
- An antigen was prepared based on the precursor protein substituted with the sequence consisting of (SEQ ID NOs: 44 to 47).
- the hydrophobic peptide sequences examined are summarized in Table 8-1.
- the polynucleotide encoding each precursor protein was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for each mammalian cell expressing each precursor protein was constructed.
- the vector for introduction into cells was purified by standard plasmid vector purification methods.
- each antigen was carried out in the same manner as in Example 7. Purification of each antigen was performed by affinity purification using ANTI-FLAG M2 Affinity Gel (Sigma, A2220). Although FLAG peptide was used for elution of affinity purification, FLAG peptide was removed from each antigen sample by using an ultrafiltration membrane. PBS (pH 7.4) was used as the solvent. Whether or not the antigen was secreted and expressed was determined by performing general SDS-PAGE after purification and confirming whether or not the target protein could be separated and purified with high purity. In addition, the feasibility of particle formation was confirmed by performing a general Blue Native PAGE after purification.
- the difference between the hydrophobic peptide chains of F_DS-Cav1-CC02 and F_DS-Cav1-CC03 is that the non-hydrophilic amino acid alanine is replaced by the hydrophilic amino acid lysine or glutamic acid. Therefore, we first considered changing the alanine of F_DS-Cav1-CC02 to histidine, which has moderate hydrophilicity and a pK of about pH6. Since it is known that the pH of the solvent at the time of preparation is near neutral, while the pH in the secretory pathway of cells is lower, the peptide chain containing histidine is relatively high at the time of secretory expression. It was expected to have hydrophilic properties and relatively hydrophobic properties at the time of formulation.
- the sequence containing the Thrombin recognition sequence, His tag, and StrepTagII (SEQ ID NO: 22) in the precursor proteins of the antigens FH_82 and FH_85 is arranged into a sequence consisting of the optimal hydrophobic peptide chain (SEQ ID NO: 42) and the FLAG tag which is a purification tag.
- Antigens were prepared based on the substituted precursor proteins (SEQ ID NOs: 48 and 49).
- the polynucleotide encoding the precursor protein was inserted into a pcDNA3.4 vector (Thermo Fisher Scientific) using standard genetic engineering techniques. It was confirmed by nucleotide sequence analysis that the target DNA sequence was inserted, and an expression vector for each mammalian cell expressing each precursor protein was constructed.
- the vector for introduction into cells was purified by standard plasmid vector purification methods.
- each antigen was carried out in the same manner as in the examination of the hydrophobic peptide chain. Purification of each antigen was carried out in the same manner as in the examination of hydrophobic peptide chains.
- Table 8-2 summarizes the antigens (sometimes referred to as "particulate antigens") used for evaluation in this example.
- the theoretical diameters of the antigen FH_85 and the particleized antigen FH_85-CC07 were estimated by dynamic light scattering using a DynaPro Plate Reader III (Wyatt) and found to be 13.8 nm and 60.0 nm (Fig. 31).
- the molecular weight ratio of two particles is proportional to the 2nd to 3rd power of the theoretical diameter ratio, so the molecular weight of FH_85-CC07 is about 20 to 80 times the molecular weight of FH_85. Is estimated.
- FH85-CC07 is considered to present about 60 to 240 molecules per particle (about 20 to 80 molecules as a trimer), and the average molecular density per 1 nm 2 theoretical surface area is 0.005 to 0.022. It can be calculated to be about a molecule (about 0.0017 to 0.0073 molecules as a trimer). Similar results were obtained with FH_82-CC07 (Fig. 31). Therefore, it was estimated that the pulverized antigen using the hydrophobic peptide chain had an antigen density equal to or higher than that of the pulverized antigen using the scaffold particles shown in Example 6.
- Mouse immune serum was obtained by immunizing BALB / cAnNCrlCrlj (Japan Charles River) (female, 6 weeks old) with each antigen and particleized antigen.
- Antigen or particulate antigen dissolved in PBS (pH 7.4) F_DS-Cav1, F_DS-Cav1-CC07, FH_82-CC07 or FH_85-CC07 (antigen concentration: 0.40 mg / mL) on the 0th and 21st days of breeding
- 50 ⁇ L was intramuscularly administered (5 animals per group).
- Example 9 Particleification of antigen using self-associating protein domain
- the accessibility of antibodies and various immune cells to the antigen site recognized by the post-F recognition antibody shown in Examples 7 and 8 is reduced by the steric disorder effect. It was considered that the effect of causing the antigen could be realized by fusion of the self-associating protein domain to the C-terminal side of the antigen. In particular, it was considered that by regularly aligning the antigens using the self-associating protein domain, a higher steric hindrance effect than the particle formation by the hydrophobic assembly shown in Example 8 could be obtained. Therefore, in this example, it was decided to investigate particle formation utilizing the self-association of the core antigen (HBcAg) of hepatitis B virus.
- HBcAg core antigen
- sequence containing the Thrombin recognition sequence, His tag, and Strep-Tag II in the antigen F_DS-Cav1 is GS linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 75), the self-association domain of HBcAg (SEQ ID NO: 50), In the one replaced with the peptide sequence consisting of the and His tags (SEQ ID NO: 51), self-association was not observed although expression was observed (verification result by BlueNativePAGE).
- the N-terminal of the self-association domain of HBcAg exists near the binding surface when HBcAgs self-associate with each other, and it is considered that self-association may be hindered by fusing a large protein to the N-terminal. .. Therefore, in order to place the RSVF antigen moiety at a position far from the binding surface at the time of self-association, a site (between the helix existing in the ⁇ 3 domain and the ⁇ 4 domain) in which the particles protrude outward when HBcAg self-associates. It was decided to divide it into the C-terminal side and the N-terminal side rather than the partial).
- the sequence containing the Thrombin recognition sequence, His tag, and StrepTagII (SEQ ID NO: 22) in the precursor proteins of the antigens F_DS-Cav1 and FH_85 is GS linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 75), and the self-association domain of HBcAg.
- a 10-fold dilution series was prepared by adding appropriate buffer to each antigen, added to the plate and incubated (room temperature, 2 hours, standing). After that, the added antigen solution is removed, peroxidase-binding anti-FLAG M2 antibody (Sigma-Aldrich, A8592) is bound, and the color is further developed with a standard peroxidase detection reagent, and then the intensity of color development is measured by absorbance measurement with a standard plate reader. Asked. The relationship between the dilution rate of the antigen and the absorbance was obtained by segmental linear interpolation between the measurement points, and the reciprocal of the dilution rate at which the absorbance was 0.2 was used as the binding force of the added antigen to the solidified antibody.
- the value obtained by dividing the binding force to the D25 antibody by the binding force to Palivizumab was defined as the epitope ⁇ detection signal, and the value obtained by dividing the binding force to the 131-2A antibody by the binding force to Palivizumab was defined as the epitope I detection signal (FIGS. 34 and 35). ..
- the pulverized antigen aggregated by the self-associating protein domain in which pre-F is added to the C-terminal of the amino acid sequence has further the binding property of the antibody to epitope ⁇ than the pulverization by the hydrophobic peptide chain. Results have been shown to improve and attenuate the detection of epitope I, which is known to induce antibodies with significantly lower ability to neutralize viral infections. The above results suggest that this particle formation improves the effect of preferentially inducing antibody groups that are considered to have a high effect on virus neutralization.
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Abstract
Description
また、安定的に取得することが困難であったpre-Fの構造が明らかとなった(非特許文献2)ことにより、pre型Fタンパク質は3量体を形成していること、また、pre型Fタンパク質に存在するエピトープ(エピトープΦ、I、II、III、IV、V等)のうち、いくつかのエピトープはpost-Fへの構造変化によって失われることが分かった。
本発明は以下の通りである。
[2]前記変異型RSV Fタンパク質が、RSVサブタイプA又はRSVサブタイプBに由来する、[1]に記載の変異型RSV Fタンパク質。
[3]前記RSVサブタイプAがRSV A2株又はRSV Long株である、[2]に記載の変異型RSV Fタンパク質。
[4]前記RSVサブタイプBがRSV 18537株である、[2]に記載の変異型RSV Fタンパク質。
[5]配列番号2と85%以上の同一性を有するアミノ酸配列において、配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン又は142位のロイシンに相当するロイシン、及び373位のロイシンに相当するロイシンがシステインに置換されたアミノ酸配列を含み、前記システイン間でジスルフィド結合が形成され、RSVのpre型Fタンパク質に対する抗体を誘導する能力を有する、[1]~[4]のいずれかに記載の変異型RSV Fタンパク質。
[6]さらに、pep27領域のC末端側に存在する、フーリン認識部位を構成するアミノ酸が、前記フーリン認識部位がフーリンによって認識されないように置換されている、[1]~[4]のいずれかに記載の変異型RSV Fタンパク質。
[7]前記フーリン認識部位を構成するアミノ酸が、配列番号1のアミノ酸配列の133位のアルギニンに相当するアルギニン、135位のアルギニン相当するアルギニン、及び136位のアルギニンに相当するアルギニンからなる群から選択されるアミノ酸であり、
当該アミノ酸が、非塩基性アミノ酸に置換されている、[6]に記載の変異型RSV Fタンパク質。
[8]配列番号3と85%以上の同一性を有するアミノ酸配列において、配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン又は142位のロイシンに相当するロイシン、及び373位のロイシンに相当するロイシンがシステインに置換され、さらに、配列番号1のアミノ酸配列の133位のアルギニンに相当するアルギニン、135位のアルギニン相当するアルギニン、及び136位のアルギニンに相当するアルギニンからなる群から選択されるアミノ酸が、非塩基性アミノ酸に置換されたアミノ酸配列を含み、前記システイン間でジスルフィド結合が形成され、RSVのpre型Fタンパク質に対する抗体を誘導する能力を有する、[6]または[7]に記載の変異型RSV Fタンパク質。
[9]前記非塩基性アミノ酸がアスパラギンである、[7]または[8]に記載の変異型RSV Fタンパク質。
[10]さらに、配列番号1のアミノ酸配列の189位のスレオニンに相当するスレオニン、及び/又は190位のセリンに相当するセリンが疎水性アミノ酸に置換されている、[1]~[9]のいずれかに記載の変異型RSV Fタンパク質。
[11]前記疎水性アミノ酸が、それぞれ独立して、バリン、イソロイシン、及びロイシンからなる群から選択される、[10]に記載の変異型RSV Fタンパク質。
[12]さらに、配列番号1のアミノ酸配列の42位のリジンに相当するリジンがアルギニンに置換されており、及び/又は、384位のバリンに相当するバリンがスレオニンに置換されている、[1]~[11]のいずれかに記載の変異型RSV Fタンパク質。
[13]変異を有する変異型RSV Fタンパク質であって、前記変異は、配列番号1のアミノ酸配列の60位のグルタミン酸に相当するグルタミン酸の非酸性アミノ酸への置換である、変異型RSV Fタンパク質。
[14]前記非酸性アミノ酸が、メチオニン、フェニルアラニン、ロイシン、スレオニン、及びセリンからなる群から選択される、[13]に記載の変異型RSV Fタンパク質。
[15][1]~[14]のいずれかに記載の変異型RSV Fタンパク質と、前記変異型RSV Fタンパク質のC末端に融合された多量体化ドメインを含む、融合タンパク質。
[16]前記多量体化ドメインがフォルドンドメインである、[15]に記載の融合タンパク質。
[17]配列番号10~13のいずれかのアミノ酸配列を含む、[16]に記載の融合タンパク質。
[18][1]~[14]のいずれかに記載の変異型RSV Fタンパク質の多量化体であって、[15]~[17]のいずれかに記載の融合タンパク質が前記多量体化ドメインを介して会合したものである、前記多量化体。
[19]前記多量化体が3量体である、[18]に記載の多量化体。
[20][1]~[14]のいずれかに記載の変異型RSV Fタンパク質または[15]~[17]のいずれかに記載の多量化体の粒子化体であって、前記変異型RSV Fタンパク質または前記融合タンパク質は粒子化ドメインを含み、前記変異型RSV Fタンパク質または前記多量化体が該粒子化ドメインを介して2個以上集合して粒子を形成しており、前記粒子化ドメインは、変異型RSV Fタンパク質または多量化体の立体構造上、エピトープΦよりもエピトープIに近い位置に存在する、前記粒子化体。
[21]前記粒子化体は、前記変異型RSV Fタンパク質または前記融合タンパク質のC末端に粒子化ドメインが結合した粒子化体製造用融合タンパク質が、前記粒子化ドメインを介して2個以上集合したものである、[20]に記載の粒子化体。
[22]前記多量化体が[16]に記載の融合タンパク質からなる3量体である、[20]または[21]に記載の粒子化体。
[23]前記粒子化ドメインが配列番号30のアミノ酸配列配列からなるFcドメインであって、当該粒子化ドメインが改変型HBs抗原由来のVLPに固定化されているプロテインAのZドメインと結合することにより集合したものである、[22]に記載の粒子化体。
[24]前記粒子化ドメインが配列番号42のアミノ酸配列からなるペプチドである、[22]に記載の粒子化体。
[25][1]~[14]のいずれかに記載の変異型RSV Fタンパク質のC末端、または[15]~[17]のいずれかに記載の融合タンパク質と、そのC末端に融合した粒子化ドメインを含む、粒子化体製造用融合タンパク質。
[26]前記粒子化ドメインが配列番号30のアミノ酸配列配列からなるFcドメインである、[25]に記載の粒子化体製造用融合タンパク質。
[27]前記粒子化ドメインが配列番号42のアミノ酸配列からなるペプチドである、[25]に記載の粒子化体製造用融合タンパク質。
[28][1]~[14]のいずれかに記載の変異型RSV Fタンパク質、[15]~[17]のいずれかに記載の融合タンパク質、または[25]~[27]のいずれかに記載の粒子化体製造用融合タンパク質をコードするポリヌクレオチド。
[29][28]に記載のポリヌクレオチドを含む、発現ユニット。
[30][29]に記載の発現ユニットを含む、宿主細胞。
[31][1]~[14]のいずれかに記載の変異型RSV Fタンパク質、[15]~[17]のいずれかに記載の融合タンパク質、[18]~[19]のいずれかに記載の多量化体又は[20]~[24]のいずれかに記載の粒子化体を含む、免疫原。
[32][29]に記載の発現ユニット又は[31]に記載の免疫原を含む、医薬組成物。
[33][29]に記載の発現ユニット又は[31]に記載の免疫原を含む、RSVワクチン。
[34]ヒト用である、[32]に記載の医薬組成物または[33]に記載のRSVワクチン。
前記RSVサブタイプAとしては、具体的にはRSV A2株、RSV Long株、RSV S2株、RSV line 19株が例示でき、中でも、RSV A2株又はRSV Long株であることが好ましい。
前記RSVサブタイプBとしては、具体的にはRSV 18537株、RSV B1株、RSV 9320株が例示でき、中でも、RSV 18537株であることが好ましい。
配列番号1において、シグナル配列のアミノ酸配列は、1位~25位のアミノ酸配列である。
配列番号1において、F2ポリペプチドのアミノ酸配列は、26位~109位のアミノ酸配列である。
配列番号1において、pep27領域のアミノ酸配列は、110位~136位のアミノ酸配列である。
配列番号1において、F1ポリペプチド(細胞外領域)のアミノ酸配列は、137位~513位のアミノ酸配列である。
配列番号1において、F1ポリペプチド(膜貫通領域と細胞内領域)のアミノ酸配列は、514位~574位のアミノ酸配列である。なお、F1ポリペプチド(膜貫通領域と細胞内領域)のアミノ酸配列は、配列番号1の514位~574位のアミノ酸配列と85%以上、90%または95%以上同一性を有する配列であってよい。
RSV Fタンパク質は、F2ポリペプチドと、pep27領域が連結したF1ポリペプチド(細胞外領域)を含むことができる。F2ポリペプチドと、pep27領域が連結したF1ポリペプチド(細胞外領域)をこの順で含むRSV Fタンパク質は、配列番号1の26位~513位のアミノ酸配列を含む、配列番号3のアミノ酸配列が例示される。
なお、これらのポリペプチドは別々のポリペプチド鎖であることができ、好ましくは、それらはジスルフィド結合で連結されている。すなわち、RSV Fタンパク質は、このようなポリペプチドの複合体であることができる。
本発明の変異型RSV Fタンパク質は、配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン又は142位のロイシンに相当するロイシンのシステインへの置換、及び373位のロイシンに相当するロイシンのシステインへの置換の変異を含む。前記変異型RSV Fタンパク質は上記アミノ酸変異を有することにより、天然に生じるジスルフィド結合に加え、導入されたシステイン間で新たなジスルフィド結合が形成され、これにより、エピトープΦの保存性(pre型を維持)の向上という効果を奏する。3量体の形成のためには、特に配列番号1の141位のロイシンに相当するロイシンがシステインに、373位のロイシンに相当するロイシンがシステインへ置換されているものが好ましい。
例えば、N末端側が配列番号1のアミノ酸配列と比較して1アミノ酸短い変異型RSV Fタンパク質のアミノ酸配列の場合、配列番号1のアミノ酸配列における141位のロイシンは140位となるが、そのようなロイシンは配列番号1のアミノ酸配列の141位のロイシンに相当するロイシンということができる。
尚、変異を導入すべきRSV Fタンパク質のアミノ酸配列について、配列番号1のアミノ酸配列とアラインメントを行ったときに、配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン、142位のロイシンに相当するロイシン、及び/または373位のロイシンに相当するロイシンの位置にロイシン以外のアミノ酸が存在する場合、当該アミノ酸をシステインに置換する態様も本発明の変異型RSV Fタンパク質に含まれる。また、当該ロイシン以外のアミノ酸がシステインである場合には、システインへの前記置換を要さず、そのままシステインであってよい。
分率X/Yの100倍
ここで、Xは配列アラインメントプログラムBLASTのA及びBのプログラムアラインメントによって同一であると一致したスコアのアミノ酸残基の数であり、YはBの全アミノ酸残基数である。アミノ酸配列Aの長さがアミノ酸配列Bの長さと異なる場合、AのBに対する%同一性は、BのAに対する%同一性とは異なることは理解されるであろう。特に断らない限りは、ここでの全ての%同一性値は、直ぐ上のパラグラフに示したようにBLASTコンピュータプログラムを用いて得られる。
本発明の変異型RSV Fタンパク質の他の態様は、変異を有する変異型RSV Fタンパク質であって、前記変異は、配列番号1のアミノ酸配列の60位のグルタミン酸に相当するグルタミン酸の非酸性アミノ酸への置換である、変異型RSV Fタンパク質である。
前記非酸性アミノ酸は、中性アミノ酸、及び/又は非極性(疎水性)アミノ酸であることが好ましい。
また、中性アミノ酸の中でも、スレオニン、セリンが好ましく、非極性(疎水性)アミノ酸の中でも、メチオニン、フェニルアラニン、ロイシンからなる群から選択されることが好ましいため、前記非酸性アミノ酸は、メチオニン、フェニルアラニン、ロイシン、スレオニン、及びセリンからなる群から選択されることが好ましい。
本発明の変異型RSV Fタンパク質は、さらに、pep27領域のC末端側に存在する、フーリン認識部位を構成するアミノ酸が、前記フーリン認識部位がフーリンによって認識されないようなアミノ酸に置換されている、変異型RSV Fタンパク質であってもよい。このようなアミノ酸置換を有することにより、変異型RSV Fタンパク質は、F2ポリペプチドと、pep27領域が連結したF1ポリペプチド(細胞外領域)を有し、それによりエピトープΦの保存性を保ったまま、三量体化が促進されるという効果を奏する。
配列番号1のアミノ酸配列の133位のアルギニンに相当するアルギニン、135位のアルギニン相当するアルギニン、及び136位のアルギニンに相当するアルギニンは、それぞれ独立に、非塩基性アミノ酸に置換されていてもよい。
本発明の変異型RSV Fタンパク質は、上記配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン又は142位のロイシンに相当するロイシン、及び373位のロイシンに相当するロイシンをシステインへ置換する変異(以下、ロイシンをシステインに置換する変異と称する)、60位のグルタミン酸の変異、および/またはフーリン認識部位の変異に加えて、さらに、配列番号1のアミノ酸配列の189位のスレオニンに相当するアミノ酸、及び/又は190位のセリンに相当するアミノ酸が疎水性アミノ酸に置換されている、変異型RSV Fタンパク質であってもよい。
本態様に係る変異型RSV Fタンパク質は、エピトープΦの保存性に優れ、三量体化が促進するという効果およびウイルス中和に高い効果を持つと考えられる抗体群を優先的に誘導する効果も保ったまま、発現系において発現量が著しく向上するという効果を奏する。
配列番号1のアミノ酸配列の189位のスレオニンに相当するアミノ酸、及び/又は190位のセリンに相当するアミノ酸は、それぞれ独立に、疎水性アミノ酸に置換されていてもよい。
また、当該置換を含む変異型RSV Fタンパク質は、エピトープΦの保存性に優れ、三量体化が促進するという効果およびウイルス中和に高い効果を持つと考えられる抗体群を優先的に誘導する効果も保ったまま、発現系において発現量が著しく向上するという効果に加えて、ウイルス感染の中和に効果のない又は乏しい抗体によって認識されにくい。逆に、ウイルス感染の中和に効果のない又は乏しい抗体が誘導されると、ウイルス感染の中和に寄与しない又はほとんど寄与しない免疫細胞が活性化し、かえって症状を悪化するリスクが想定されるところ、後述する実施例から分かるように、本態様に係る変異型RSV Fタンパク質は、ウイルス感染の中和に効果のない又は乏しい抗体によって認識されにくいため、従来のワクチンよりもそのリスクが低い。
具体例を挙げれば、本態様に係る変異型RSV Fタンパク質は、エピトープIを認識する抗体によって認識されにくい。エピトープIは、ウイルス感染の中和能の著しく低い抗体を誘導することから、抗体によってエピトープIが認識されると、ウイルス感染の中和にほとんど寄与しない免疫細胞の活性化によって、かえって症状を悪化するリスクが想定されるところ、後述する実施例から分かるように、エピトープIを認識する抗体によって認識されにくい本態様に係る変異型RSV Fタンパク質は、従来のワクチンよりもそのリスクが低い。
また、配列番号1における189位は配列番号3の164位のアミノ酸であり、配列番号1の190位は配列番号3の165位のアミノ酸であり、配列番号3またはそれと85%以上、90%以上または95%以上同一性を有するアミノ酸配列において、これらの一方または両方の変異が、上記ロイシンをシステインに置換する変異、60位のグルタミン酸の変異、および/またはフーリン認識部位の変異に加えて、導入されてよい。
本態様に係る変異型RSV Fタンパク質は、エピトープΦの保存性に優れ、三量体化の促進という効果およびウイルス中和に高い効果を持つと考えられる抗体群を優先的に誘導する効果も保ったまま、発現系において発現量が著しく向上するという効果を奏する。
また、配列番号1における42位は配列番号3の17位のアミノ酸であり、配列番号1の384位は配列番号3の359位のアミノ酸であり、配列番号3またはそれと90%以上同一性を有するアミノ酸配列において、これらの一方または両方の変異が、上記ロイシンをシステインに置換する変異、60位のグルタミン酸の変異、および/またはフーリン認識部位の変異、さらには上記189位および/または190位の変異に加えて、導入されてよい。
また、配列番号1における102位は配列番号3の77位のアミノ酸であり、配列番号1の379位は配列番号3の354位のアミノ酸であり、配列番号1の447位は配列番号3の422位のアミノ酸であり、配列番号3またはそれと85%、90%または95%以上同一性を有するアミノ酸配列において、これらの一方または両方の変異が、上記ロイシンをシステインに置換する変異、60位のグルタミン酸の変異、および/またはフーリン認識部位の変異、さらには上記189位および/または190位の変異や42位および/または384位の変異に加えて、導入されてよい。
尚、所定のアミノ酸配列と所定の同一性を有するアミノ酸配列の定義は、既述の内容と同一である。
本発明の他の態様は、前記変異型RSV Fタンパク質と、少なくとも多量体化ドメインとを含む、融合タンパク質である。
融合タンパク質は変異型RSV Fタンパク質および多量体化ドメイン以外のポリペプチドを含んでもよく、当該変異型RSV Fタンパク質および多量体化ドメイン以外のポリペプチドは、前記変異型RSV Fタンパク質の効果を妨げるものでなければ特に限定されないが、具体的には、トロンビン切断モチーフなどのモチーフ、His-tagやStrepTagIIなどの精製用タグが例示できる。
フォルドンドメインとしては、バクテリオファージT4フィブリチン由来のフォルドンドメインが例示できる。そのアミノ酸配列としては、配列番号21のアミノ酸配列が例示できる。
具体的には、配列番号10~13のアミノ酸配列を有するものが挙げられる。
実際は、配列番号10~13のアミノ酸配列はF2ポリペプチドのアミノ酸配列と、pep27付加F1ポリペプチド(細胞外領域)のC末端にフォルドンドメインが連結したアミノ酸配列に分かれるが、ここではそれらを結合した一つのアミノ酸配列として表す。
本発明の他の態様は、前記融合タンパク質の多量化体である。前記多量化体は、前記融合タンパク質がフォルドンドメインなどの多量体化ドメインを介して会合したものである。多量化体は、前記融合タンパク質の3量化体であることが好ましい。
本発明の他の態様は、前記変異型RSV Fタンパク質、又は前記融合タンパク質をコードするポリヌクレオチドである。
前記変異型RSV Fタンパク質をコードするポリヌクレオチドは、変異導入前のRSV Fタンパク質をコードするポリヌクレオチド配列(配列番号74)を参考に、標準的な遺伝子工学技術により、所望の変異が導入されたポリヌクレオチドとして得ることができる。
また、前記融合タンパク質をコードするポリヌクレオチドも、前記変異型RSV Fタンパク質をコードするポリヌクレオチド、及び当該変異型RSV Fタンパク質以外のポリペプチドをコードするポリヌクレオチドを用いて、標準的な遺伝子工学技術により得ることができる。
配列番号14は配列番号10のアミノ酸配列を有する融合タンパク質を発現させて宿主で産生させるためのポリヌクレオチド配列であるが、当該融合タンパク質をコードする配列の5’末端側にシグナルペプチドをコードする配列が付加されている。
配列番号15は配列番号11のアミノ酸配列を有する融合タンパク質を発現させて宿主で産生させるためのポリヌクレオチド配列であるが、当該融合タンパク質をコードする配列の5’末端側にシグナルペプチドをコードする配列が付加されている。
配列番号16は配列番号12のアミノ酸配列を有する融合タンパク質を発現させて宿主で産生させるためのポリヌクレオチド配列であるが、当該融合タンパク質をコードする配列の5’末端側にシグナルペプチドをコードする配列が付加されている。
配列番号17は配列番号13のアミノ酸配列を有する融合タンパク質を発現させて宿主で産生させるためのポリヌクレオチド配列であるが、当該融合タンパク質をコードする配列の5’末端側にシグナルペプチドをコードする配列が付加されている。
本発明の他の態様は、前記ポリヌクレオチドを含む、発現ユニットである。
前記発現ユニットは、前記ポリヌクレオチドを発現させる機構を含むものであればよく、例えば、プロモーター配列や転写終結シグナル配列等の発現に必要な要素を含んでよく、標準的な遺伝子工学技術に用いられる発現ユニットでよい。発現ユニットは、例えば、組換えベクターに含まれてよい。組換えベクターとしては、プラスミドベクターやウイルスベクターが例示でき、原核細胞において発現可能なベクターや、真核細胞において発現可能なベクター、哺乳動物由来の細胞において発現可能なベクターが挙げられる。
本発明の他の態様は、前記発現ユニットを含む、宿主細胞である。好ましくは、前記発現ユニットを含む組換えベクターで形質転換された宿主細胞である。
前記宿主細胞としては、前記発現ユニットから前記ポリヌクレオチドがコードするタンパク質が発現されれば特に限定されず、標準的な遺伝子工学技術に用いられるものでよい。具体的には、大腸菌、枯草菌のような原核細胞や、真核細胞、哺乳動物由来の細胞が例示できる。
宿主細胞へのポリヌクレオチド導入は、リン酸カルシウム法、DEAEデキストラン法、エレクトロポレーション法、リポフェクション法などの公知の手段を用いて行うことができる。
宿主細胞を適当な条件で培養することで、前記変異型RSV Fタンパク質または融合タンパク質を発現させ、前記変異型RSV Fタンパク質、融合タンパク質またはそれらの多量化体を生産することができる。生産された前記変異型RSV Fタンパク質、融合タンパク質またはそれらの多量化体は公知の手段により、精製することができる。
本発明の他の態様は、前記変異型RSV Fタンパク質、前記融合タンパク質、前記多量化体、又は下記粒子化体を含む、免疫原である。
すなわち、前記変異型RSV Fタンパク質、前記融合タンパク質、前記多量化体、又は前記粒子化体は、免疫原として使用することができる。当該免疫原は、前記変異型RSV Fタンパク質の効果を妨げない限り、前記変異型RSV Fタンパク質、前記融合タンパク質、前記多量化体、又は前記粒子化体のいずれか1種を含んでもよく、いずれか2種を含んでもよく、すべてを含んでもよい。このうち多量化体、特に三量体が好ましく、また粒子化体が好ましく、免疫原として用いられる。多量化体の製造については、上記の通りであり、粒子化体については、以下に詳細に説明する。なお、本発明の免疫原は標準的な免疫原が含む保存剤や賦形剤等を含んでもよい。
本発明の他の態様は、前記変異型RSV Fタンパク質または前記多量化体の粒子化体である。
粒子化体は、変異型RSV Fタンパク質または多量化体が2個以上集合して粒子を形成したものであって、中和能の高い抗体を効率よく誘導できるものであれば特に制限されないが、例えば、前記変異型RSV Fタンパク質または前記多量化体が粒子化ドメインを介して2個以上集合して粒子を形成したものである。ここで、粒子化体および粒子の形状は必ずしも球状に限られず、一定方向を向いて集合していれば足りる。
すなわち、前記変異型RSV Fタンパク質または前記融合タンパク質にさらに粒子化ドメインを融合させ、前記変異型RSV Fタンパク質または前記融合タンパク質の多量化体が該粒子化ドメインを介して集合して粒子を形成したものであることが好ましい。
前記変異型RSV Fタンパク質または前記融合タンパク質のC末端に粒子化ドメインが結合した粒子化体製造用融合タンパク質を用いて粒子化させることが好ましい。粒子化体製造用融合タンパク質については後述する。
したがって、本発明の一態様にかかる粒子化体は、変異型RSV Fタンパク質または多量化体が、粒子化ドメインを介して少なくともRSV Fタンパク質のエピトープΦが露出し、少なくともRSV Fタンパク質のエピトープIが露出しないように2個以上集合したものである粒子化体である。ここで、「エピトープIが露出しないように」とは、エピトープIが完全に露出しないことまでは要さず、エピトープIへの抗体や各種免疫細胞のアクセシビリティが低下する程度であればよい。
足場粒子の材料としては、特に制限されないが、粒子形成能を有するタンパク質、脂質二重膜、エンベロープ、リポソーム、ニオソーム、virosome、フェロソーム、金ナノ粒子、樹脂、シリカ、高分子ミセル、ゲルなどが挙げられる。
粒子形成能を有するタンパク質の例として、VLP (Virus-like particle)やVP (Virus Particle)を形成する、ウイルスタンパク質やウイルスタンパク質の改変体が挙げられる(例えば、特開2004-2313号公報)。例えば、本来の肝細胞に対する感染能を欠失するように改変され、さらに足場分子を提示するように改変されたB型肝炎ウイルス表面抗原タンパク質が挙げられ、当該タンパク質は、真核細胞で発現させると、小胞体膜等の膜上に膜タンパク質として発現、蓄積され、VLPとして放出される。
足場粒子としてより具体的には、B型肝炎ウイルス表面抗原(HBs抗原)タンパク質のPreS領域に、プロテインAのFc結合ドメイン(Zドメイン)を挿入して作製したタンパク質由来のVLPが挙げられ、例えば、Bionanocapsule-ZZ(Beacle社カタログ番号:BCL-DC-002)が使用できる。
足場粒子の表面には、粒子化ドメインとしての足場粒子結合ペプチドと結合する分子(足場分子)が固定化されていることが好ましい。
例えば、プロテインAのFc結合ドメインを挙げることができ、足場粒子結合ペプチドを介して変異型RSV Fタンパク質またはその多量化体を結合させることができる。
具体的には、Protein tagとProtein tag結合分子の組み合わせ(ビオチン-アビジン、Fc-Protein A、GST(Glutathione S-transferase)-グルタチオン、MBP(Maltose binding protein)-マルトース、His Tag-ニッケル、SBPタグ-ストレプトアビジンが例示される)、抗体と抗原の組み合わせなどが挙げられる。
足場分子と足場粒子結合ペプチドの組み合わせとしては好ましくは、IgG1 のFcドメイン(配列番号30)とプロテインAのFc結合ドメイン(Zドメイン)の組み合わせである。IgG1 のFcドメインを足場分子に、プロテインAのFc結合ドメインを足場粒子結合ペプチドに使用してもよいし、IgG1 のFcドメインを足場粒子結合ペプチドに、プロテインAのFc結合ドメインを足場分子に使用してもよい。
なお、上記足場分子または足場粒子結合ペプチドは互いに共有結合または非共有結合できる限り、それらの部分断片を使用したり改変したりすることができる。
この態様では、変異型RSV Fタンパク質または融合タンパク質に疎水性ペプチド鎖が結合しており、該疎水性ペプチド鎖を介して変異型RSV Fタンパク質または融合タンパク質が粒子を形成する。
疎水性ペプチド鎖は、疎水性アミノ酸を含み、自己集合して当該粒子化体を形成する、アミノ酸残基数5~50、好ましくは10~30のペプチド鎖を意味する。ここで、疎水性の強いアミノ酸としては、バリン、ロイシン、フェニルアラニン、イソロイシン等が挙げられ、ロイシンまたはイソロイシンがより好ましい。疎水性ペプチド鎖における疎水性アミノ酸の割合は好ましくは20~60%である。
疎水性ペプチド鎖においては、疎水性アミノ酸が疎水性ペプチド鎖の表面に現れることが好ましい。表面の疎水性アミノ酸の割合は10%以上であることが好ましいが、比率が高すぎると、細胞内で凝集して分泌されないため、表面の疎水性アミノ酸が50%以下、30%以下、20%以下が好ましい。一方、内側の疎水性アミノ酸の比率は高いほうが、疎水性ペプチド鎖の構造が安定するため好ましい。
疎水性アミノ酸以外のアミノ酸は、いずれのものでもよいが、特に分泌効果を高めるためにヒスチジンを20%以上含むことが好ましい。
疎水性ペプチド鎖を7アミノ酸からなるペプチドの繰り返し配列とすることにより、ヘリックス構造を形成し、かつ上記設計が行いやすいため、好ましく用いられる。例えば、疎水性ペプチド鎖は、1、3、5番目に疎水性アミノ酸が配置される7アミノ酸からなるペプチド配列の繰り返し配列が好ましい。繰り返し数は例えば2~5であり、2~3であることが好ましい。
疎水性ペプチド鎖の好ましい例としては、CC02、CC03、CC07、CC08(順に、配列番号40、41、42、43)が挙げられ、この中ではCC07(配列番号42)がより好ましい。
この態様では、変異型RSV Fタンパク質または融合タンパク質に自己会合性タンパク質ドメインが結合しており、該自己会合性タンパク質ドメインを介して変異型RSV Fタンパク質または融合タンパク質が粒子を形成する。
自己会合性を有するタンパク質(Self-assembly protein)の例としては、B型肝炎ウイルスのコア抗原(HBcAg)、ヒトパピローマウイルスのカプシドタンパク(HPV L1)、E型肝炎ウイルスのカプシドタンパク(HEV capsid)、Bacteriophage Qβ coat protein、ノーウォークウイルス(ノロウイルス、NoV) Capsid、Parvovirus B19 capsid、Alfalfa mosaic virus、Feritin、encapsulinなどが挙げられる。自己会合性を有するタンパク質は自己会合性を有する部分断片でもよいし、改変体でもよい。
HBcAgを使用する場合には、本発明のRSV Fタンパク質または多量化体と結合する際には、HBcAgの自己会合のための会合面から遠い部位でかつ、自己会合したものの外側の部位とRSV Fタンパク質または多量化体とを結合させることが好ましい。さらに、このような結合を行った場合、HBcAgの本来の立体構造を取らせるために、α3ドメインのヘリックスとα4ドメインのヘリックスの間にリンカーを挿入することが好ましい。リンカーは例えば10~30アミノ酸のポリペプチドであり、例えば、GSリンカーである。このようなHBcAgの改変体にFLAGタグが結合している配列としては、配列番号54が挙げられる。
粒子化体製造用融合タンパク質は、上記の粒子化体の作製に使用されるタンパク質であって、本発明の変異型RSV Fタンパク質のC末端、または融合タンパク質のC末端に融合する、上述した足場粒子結合ペプチド、疎水性ペプチド鎖、自己会合性タンパク質ドメインなどの粒子化ドメインを含む。
本粒子化体製造用融合タンパク質が粒子化ドメインを介して、集合することにより粒子化体となる。
なお、本発明の多量化体を構成する融合タンパク質のC末端間の距離が、粒子化ドメインと結合することによって変化してしまう場合には、粒子化ドメインと当該C末端(多量体化ドメイン)との間にリンカーを挿入することによって、上記C末端間の距離への影響を小さくすることができる。リンカーは特に制限されないが、5~20アミノ酸のペプチドであることが好ましく、5~10アミノ酸のペプチドであることがより好ましい。配列は融合タンパク質のワクチン効果を阻害しない限り特に制限されないが、例えば、グリシンとセリンからなるGSリンカーである。
リンカーの位置は、変異型RSV Fタンパク質または融合タンパク質の多量体化ドメインと粒子化ドメインの間であればよく、リンカーの前後にタグ配列を含んでもよい。タグ配列は特に制限されないが、タンパク質の精製に通常用いられるタグ配列が好ましく用いられ、例えば、FLAGタグ(配列番号28)、His-tag(配列番号25)、Strep-tagII(配列番号26)などが例示できる。
なお、粒子化体製造用融合タンパク質は、FLAGタグ(DYKDDDDK:配列番号28)、FLAGタグに3アミノ酸(GGS)が付加したタグ(GGSDYKDDDDK:配列番号29)などのタグ配列を含んでもよい。
粒子化体は、上記で得られた粒子化体製造用融合タンパク質を用いて粒子化させたのちに、必要に応じて粒子化体を精製することで得ることができる。
粒子化工程は、足場粒子を用いる場合は、粒子化体製造用融合タンパク質を足場粒子と混合して粒子化させたのちに、粒子化されていない変異型RSV Fタンパク質またはその産物を除き、粒子化体を精製することにより行うことができる。足場粒子としては、例えば、HBsAg VLP(Beacle社カタログ番号:BCL-DC-002)が使用できる。
疎水性ペプチド鎖を含む粒子化体製造用融合タンパク質を用いる場合、粒子化工程は、粒子化体製造用融合タンパク質を、疎水性ペプチド鎖を介して会合させて粒子化させたのちに、粒子化されていない変異型RSV Fタンパク質またはその産物を除き、粒子化体を精製することにより行うことができる。
自己会合性タンパクドメインを含む粒子化体製造用融合タンパク質を用いる場合、粒子化工程は、粒子化体製造用融合タンパク質を、自己会合性タンパクドメインを介して会合させて粒子化させたのちに、粒子化されていない変異型RSV Fタンパク質またはその産物を除き、粒子化体を精製することにより行うことができる。
また、本発明の粒子化体においては、粒子化体表面積1 nm2あたり、例えば三量体としては0.0001個以上、好ましくは0.0005個以上、より好ましくは0.001個以上が結合していることが望ましい。ここで、粒子化体表面積とは、粒子化体を、動的光散乱法により測定された理論直径を直径とする球体と仮定したときの表面積のことである。粒子化体表面積あたりの個数は、例えば1粒子化体あたりの個数を粒子化体表面積で除する方法で算出できる。
粒子化体を生成し、エピトープIに対する結合能が低く、かつ、エピトープΦに対する結合能が高いものを選択することにより当業者は最適な1粒子化体当たりの個数や粒子化体表面積1nm2あたりの個数を選択できる。
本発明の他の態様は、前記発現ユニット又は前記免疫原を有効成分として含む、医薬組成物である。
前記発現ユニット又は前記免疫原は、RSウイルス感染症の予防又は治療のための医薬組成物とすることができる。
また、本発明の他の態様は、前記発現ユニット又は前記免疫原を含む、RSVワクチンである。これは、前記態様に係る医薬組成物がRSVワクチンである態様でもある。
ゲルタイプとしては、水酸化アルミニウム、リン酸アルミニウム、リン酸カルシウムなどが例示できる。
菌体タイプとしては、CpG、コレラ毒素、大腸菌易熱性毒素、百日咳毒素、ムラミルジペプチド(Muramyl dipeptide;MDP)などが例示できる。
油乳濁液タイプとしては、フロイント不完全アジュバント、MF59、SAFなどが例示できる。
高分子ナノ粒子タイプとしては、免疫刺激複合体、リポソーム、生分解性マイクロスフェア、サポニン由来QS-21などが例示できる。
合成タイプとしては、非イオン性ブロックコポリマー、ムラミルペプチドアナログ、ポリホスファゼン、合成ポリヌクレオチドなどが例示できる。
サイトカインタイプとしては、IFN-γ、IL-2、IL-12などが例示できる。
経口投与のための剤型は、固体または液体の剤型、具体的には錠剤、被覆錠剤、丸剤、細粒剤、顆粒剤、散剤、カプセル剤、シロップ剤、乳剤、懸濁剤、トローチ剤等が例示できる。
本発明の医薬組成物は好ましくは液体の剤型であり、注射剤が好ましい。
タンパク質の構造の安定化には、システインを用いた共有結合(ジスルフィド結合)の形成が有用である。先行研究では、RSV Fタンパク質(以下、「Fタンパク質」と称することがある)においてpre-F構造を安定化する様々なジスルフィド変異が報告されている(国際公開第2017/172890号、国際公開第2017/109629号)が、2次構造のメンバーであるアミノ酸をシステインに置き換えていたり、距離が遠い部分にジスルフィドを形成させていたりするため、得られる変異体は天然構造(天然型Fタンパク質がタンパク質発現時に取ると考えられる融合前構造)と異なる構造となるリスクが大きい。そこで、2次構造を取っておらず、かつ、システインに置き換えたときのS原子間の距離が5Åよりも近いアミノ酸ペアをシステインに置き換えることで構造の安定化を実施することとした。距離の計算においては、公開されている構造モデル(PDB登録番号:4MMS)を用いて、任意の2アミノ酸をシステインに置き換えて二面角N-Ca-Cb-Sがエネルギー的に安定な配座を発生させ、最短となるS原子間の距離を算出した(表1)。その結果、後述する抗原FH_08およびFH_09に含まれる変異型RSV Fタンパク質(本明細書では、「変異タンパク質」と称することがある。)の変異箇所が候補として挙げられた。この2候補は、変異箇所における変異前のアミノ酸の側鎖に極性のある官能基が無いため、天然型Fタンパク質に存在する極性基間相互作用(水素結合など)を失わせることに起因する構造不安定化のリスクが無い点でも優れていることが考えられた。尚、表1中の「L141C」は、配列番号1のアミノ酸配列における141位のロイシンがシステインに変異していることを表す。以下、表中では同様の表記を用いている。なお、F_native抗原の前駆体タンパク質(配列番号4)はA2株の天然型F0タンパク質からF1ポリペプチドの膜貫通領域と細胞内領域を除いたポリペプチド(配列番号1のアミノ酸番号1~513)において、P102A、I379VおよびM447Vの変異を有するポリペプチドのC末端側に、順に、フォルドンドメイン(配列番号21)、Thrombin認識配列、HisタグおよびStrepTagIIを含む配列(配列番号22)を融合したポリペプチドである。本実施例ではいずれの変異体もF_native抗原の前駆体タンパク質を元に作成されたため、P102A、I379VおよびM447Vの変異を有する。なお、F_native前駆体タンパク質は発現過程でプロセシングを受け、シグナルペプチドとpep27領域が脱落する。
F native抗原の具体的な製造方法を以下に示す。上記ポリヌクレオチド(配列番号5)をpcDNA3.4ベクター(Thermo Fisher Scientific社)に標準的な遺伝子工学技術により挿入した。目的のDNA配列が挿入されていることを塩基配列解析によって確認し、F_native抗原の前駆体タンパク質を発現する哺乳類細胞用発現ベクターを構築した。細胞に導入するためのベクターを標準的なプラスミドベクター精製方法により精製した。
天然型Fタンパク質は、まず第1フーリン認識部位の切断を受けて単量体構造を取った後に、第2フーリン認識部位の切断を受けてpep27領域が消失することによって、三量体化が促進されるとする報告がある(Anders Krarup et al. 2015. Nature communications 6:8143)。ところが、pep27領域は天然から単離されたウイルス株間で配列の長さ及びアミノ酸の種類が高度に保存されているため、寧ろ、抗原の立体構造形成に不可欠な効果がある可能性を考えた。そこで、第2フーリン認識部位に変異を導入してpep27領域の切断を阻害するとともに、該変異の三量体形成に対する効果を調べることとした。下記で作製した各抗原の前駆体タンパク質の変異内容は、表2にまとめた。表2中の「FH_08 + R135N, R136N」は、抗原FH_08の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における135位のアルギニンがアスパラギンに変異し、かつ、136位のアルギニンがアスパラギンに変異していることを表す。「FH_09 + R135N, R136N」は、抗原FH_09の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における135位のアルギニンがアスパラギンに変異し、かつ、136位のアルギニンがアスパラギンに変異していることを表す。なお、抗原FH23~25では抗原FH08の前駆体タンパク質におけるpep27(p27)配列を各種GSリンカーに置換した。GSリンカーはGSGSGS(配列番号18)、(GGGGS)2(配列番号19)、(GGGGS)3(配列番号20)を用いた。
各抗原の各抗体への結合性も実施例1と同様の操作で測定したが、センサーチップに供する抗体として、pre-Fの三量体形成を検出可能なAM14抗体を追加した。D25抗体およびAM14抗体の結合シグナルの値は、F抗原のコンホメーション変化に依らず結合可能なシナジス抗体(アッヴィ、シナジス筋注液)を用いた際の結合シグナルの値との比を求めることで正規化した。
F_native抗原の前駆体タンパク質の189位と190位のアミノ酸はスレオニンとセリンであり、側鎖にヒドロキシル基を持つという点で親水性アミノ酸である。ところが、189~190位のアミノ酸の立体構造上の近位に存在するアミノ酸は57位のイソロイシン、167位のイソロイシン、171位のロイシン、179位のバリン、260位のロイシン等であるが、それらはいずれも疎水性アミノ酸であるため、構造の安定性を損ねている可能性が考えられた。そこで、189~190位のアミノ酸を疎水性アミノ酸(バリン、ロイシン、イソロイシン)に変異させることで構造を安定化させ、発現量向上に寄与する可能性を考えた。下記で作製した各抗原の前駆体タンパク質の変異内容は表3にまとめた。表3中の「FH_21 + T189I」は、抗原FH_21の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における189位のトレオニンがイソロイシンに変異していることを表す。「FH_21 + T189V」は、抗原FH_21の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における189位のトレオニンがバリンに変異していることを表す。「FH_21 + T189I, S190V」は、抗原FH_21の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における189位のトレオニンがイソロイシンに、190位のセリンがバリンに、変異していることを表す。「FH_21 + T189L, S190V」は、抗原FH_21の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における189位のトレオニンがロイシンに、190位のセリンがバリンに、変異していることを表す。「FH_21 + T189V, S190V」は、抗原FH_21の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における189位のトレオニンがバリンに、190位のセリンがバリンに、変異していることを表す。「FH_50 + K42R, V384T」は、抗原FH_50の前駆体タンパク質において、さらに、配列番号1のアミノ酸配列における42位のリジンがアルギニン、384位のバリンがトレオニンに変異していることを表す。さらに、天然のウイルス単離株にて生じている点変異を抽出し、それぞれ単独変異の検討を行ったところ、2つの点変異(K42R,V384T)で発現量を向上させる効果が認められたため、この変異も併せて導入して検討を行うこととした。
各抗原の発現量は、標準的な分光光度計を用いた吸光度測定によって測定した。精製後の抗原溶液の吸光度(280 nm)A280、アミノ酸一次配列情報から推定されるモル吸光度係数εと分子量M、精製後の抗原溶液量Vp、発現培養液量Vcを用い発現培養体積当たりの発現量y(y=A280xM/εxVp /Vc)を算出した。
以上の操作により、189~190位の疎水性アミノ酸(V、I、L)への変異を追加した各抗原FH_50、FH_51、FH_52、FH_53、FH_55は、変異追加前の抗原FH_21に比べて、エピトープΦの保存性と三量体の形成を保ったまま、著しく発現量が向上していることを示す結果を得た。更に自然界のウイルスで起こりうる天然変異から抽出された2つの点変異(K42R、V384T)を追加した抗原FH_82は、エピトープΦの保存性と三量体の形成を保ったまま、更に著しく発現量が向上していることを示す結果を得た(図4、図5、図6)。
F_nativeの前駆体タンパク質の60位のアミノ酸はグルタミン酸であり、酸性アミノ酸に属するため溶媒が中性の条件下で負電荷を帯びることが一般的に知られている。このアミノ酸は、立体構造上、同じく中性条件下で負電荷を帯びる194位のアスパラギン酸と隣接しており、負電荷の近接により構造が不安定化する可能性を考えた。そこで、F_nativeの前駆体タンパク質のアミノ酸配列において、60位のアミノ酸を酸性アミノ酸以外のアミノ酸に変更する変異を実施することとした。ただし、60位のアミノ酸をアスパラギンに変更する際は、60~62位のアミノ酸が一般的に知られるN型糖鎖結合モチーフ(Asn-X-Ser/Thr)となることを避けるため、同時に62位のアミノ酸をセリンからアラニンに変更することとした。下記で作製した抗原の前駆体タンパク質の変異内容は、表4にまとめた。
各抗原のD25抗体およびAM14抗体への結合性は、実施例2と同様の操作で測定した。
以上の操作により、60位をM、F、L、S、Tに変更した際にエピトープΦの保存性と三量体の形成が向上していることを示す結果を得た(図7、図8)。
F抗原のpre-Fを安定化させる技術として最もよく報告されている変異の一つにDS-Cav1変異(S155C、S290C、S190F、V207L)がある(Jason S. McLellan et al. 2013. Science 342: 592)。そこで、先述の実施例で見出された有用な抗原FH_50及び抗原FH_50にさらに有用な変異を導入した表5に記載の抗原をDS-Cav1変異を導入した抗原F_DS-Cav1と比較することとした。また、post-F三量体構造を取ることが知られている抗原ΔFP furinwtFecto(Kurt A. Swanson et al. 2014. Journal of Virology. 88(20): 11802)も併せて作製し、抗原の評価に用いることとした。下記で作製した抗原の前駆タンパク質の変異内容を、表5にまとめた。抗原FH_81~85の前駆体タンパク質は抗原FH_50の前駆体タンパク質においてそれぞれE60M、K42R+V384TまたはE60M+K42R+V384Tの変異を導入したものである。
各抗原の発現量は、実施例3と同様の操作で実施した。
各抗原の各抗体への結合性は実施例2と同様の操作で測定したが、センサーチップに供する抗体として、エピトープIを認識する131-2A抗体を追加した。D25抗体、AM14抗体および131-2A抗体の結合シグナルの値は、F抗原のコンホメーション変化に依らず結合可能なシナジス抗体(アッヴィ、シナジス筋注液)を用いた際の結合シグナルの値との比を求めることで正規化した。
先の操作によって、抗原FH_50、FH_81、FH_82、FH_85は、ウイルス中和に寄与しない免疫の誘導を抑える効果がある可能性が示された。その効果をより詳細に検討するため、健常成人血清を用いて、抗原FH_50、FH_81、FH_82、FH_85およびF_DS-Cav1について、健常成人血清中のどのような抗体に結合するのか検証することとした。
先の操作によって、変異タンパク質はウイルス中和に寄与しない免疫の誘導を抑える効果があることが示唆された。その効果をさらに詳細に検討するため、抗原FH_50、FH_81、FH_82、FH_85又はF_DS-Cav1をマウスに免疫することで誘導される抗体の分析を実施した。
実施例5で実施した抗原FH_50、FH_81、FH_82、FH_85のFタンパク質部分は、いずれもL141CおよびL373Cの変異を含んでいる。当該変異は実施例1においてジスルフィド結合を導入するために導入した変異であるが、先行特許(国際公開第2017/109629号)にて比較的近い部位へのジスルフィド結合の導入(Mutant ID: pXCS524.L142C、N371C)が検討され、さらに変異を加えた最適化抗原(Mutant ID: pXCS881.L142C、N371C、S55C、L188C、T54H、V296I、D486S、E487Q、D489S)が検討されている。そこで、先述の実施例で見出された有用な抗原を、pXCS524変異またはpXCS881変異を導入した抗原Pf524またはPf881と比較することとした。下記で作製した抗原の前駆体タンパク質の変異内容を、表6にまとめた。
各抗原の発現量は、実施例3と同様の操作で測定した。
各抗原の各抗体への結合性は実施例5と同様の操作で測定したが、結合シグナルの値は、各抗原溶液を180 sec供した時の検出値から、PBS(pH7.4)を180 sec供した時の検出値を引いた値とした(図15、図16、図17)。
上記の実験によって、抗原FH_82、FH_85は、ウイルス中和に寄与しない免疫の誘導が先行特許の変異を導入した変異体Pf881に比べて低いという効果があることが示唆された。その効果をさらに詳細に検討するため、抗原FH_82、FH_85又はPf881をマウスに免疫することで誘導される抗体の分析を実施した。
ウイルス感染防御に寄与しない抗体を誘導するリスクをさらに減少させるためには、エピトープI認識抗体等のpost-F認識抗体によって認識される抗原部位に対する抗体や各種免疫細胞のアクセシビリティを立体障害効果によって低下させることが有効であると考えらえれる。そこで、post-F認識抗体の認識部位が内側になるように抗原を適当な足場粒子上に固定させることで、当該立体障害効果を得ることができる可能性を考えた。
ところが、抗原を適当な足場粒子上に提示させる際に、抗原の間隔がある一定以上長いために密度が疎であったり、足場粒子と抗原との間に一定以上の長さのフレキシブルなリンカーを導入したりすると、先述の立体障害効果が得られない可能性が考えられる。そこで、比較的高密度な抗原提示が実現可能であると考えられるHBsAg VLPを用い、複数の長さのGSリンカーを検討することとした。
足場粒子として、Fc融合タンパクを結合可能なBionanocapsule-ZZ(Beacle社、BCL-DC-002)を用いた。
抗原の前駆体タンパク質として、前述のF_DS-Cav1、FH_82、FH_85の前駆体タンパク質におけるThrombin認識配列、Hisタグ、Strep-Tag IIを含む配列(配列番号22)をFc配列(配列番号30)に置換した抗原F_DS-Cav1-Fcの前駆体タンパク質(配列番号31)、FH_82-Fcの前駆体タンパク質(配列番号32)、FH_85-Fcの前駆体タンパク質(配列番号33)を作製した。さらに、抗原F_DS-Cav1-Fcの前駆体タンパク質、FH_85-Fcの前駆体タンパク質について、Fc配列の直前にGSリンカー(GGGGSGGGGSGGGGSGGGGS(配列番号75)、GGGGSGGGGS(配列番号19)あるいはGGGGS(配列番号27))を挿入したものについては、対応する前駆体タンパク質(配列番号34~39)を元に以下の方法で作製した。
各抗原の精製は、Protein A精製カラムを用いた標準的なアフィニティー精製法にて実施した。精製後は限外濾過膜を用いることでPBS(pH7.4)に溶媒置換した。
作製した各抗原は、HBsAg VLP 1粒子当たり、平均して約135分子(3量体にして約45分子)となる比率でHBsAg VLPと混合し、室温で1時間静置することで足場粒子に固定化した。この時、固定化していない抗原がほとんど存在していないことは、BlueNativePAGEを行うことで確認した。
抗原または粒子化抗原であるF_DS-Cav1、F_DS-Cav1-VLP、FH_82、FH_82-VLP、FH_85およびFH_85-VLPの各抗体への結合性は、実施例6と同様の操作で測定した(図19、図20、図21)。
以上の操作により、F抗原領域のpre安定化方法(pre安定化のためのアミノ酸変異の内容)に依らず、pre-Fをそのアミノ酸配列のC末端に付加したドメインを介して足場粒子に固定化すると、エピトープΦに対する抗体の結合性を保ったまま三量体の形成が向上することが示された。さらに、ウイルス感染中和能の著しく低い抗体を誘導することで知られているエピトープIの検出値は減弱することが示された。以上の結果は、本粒子化抗原をワクチン抗原として用いた際に、ウイルス中和に高い効果を持つと考えられる抗体群を優先的に誘導することを示唆している。
抗原または粒子化抗原であるF_DS-Cav1、F_DS-Cav1-4GS-VLP、F_DS-Cav1-2GS-VLP、F_DS-Cav1-1GS-VLP、F_DS-Cav1-VLP、FH_85、FH_85-4GS-VLP、FH_85-2GS-VLP、FH_85-1GS-VLPおよびFH_85-VLPの各抗体への結合性を実施例6と同様の操作で測定した(図22、図23、図24)。
以上の操作により、実施した何れのGSリンカーの挿入によっても、エピトープΦに対する抗体の結合性を保ったまま三量体の形成を向上し、エピトープIの検出値は減弱する効果があることが示された。ただし、エピトープI検出の減弱の効果は、足場粒子と抗原との間に長すぎるGSリンカーが挿入されると、低下することが示された。具体的には、GGGGSGGGGS(配列番号19)程度の長さであれば顕著な影響は見られないが、より長いGGGGSGGGGSGGGGSGGGGS(配列番号75)の長さになるとエピトープI検出を減弱させる効果の低下が見られた。
以上の結果は、足場粒子と抗原との間に一定以上の長さのフレキシブルなリンカーを挿入すると、立体障害効果によってpost-F認識抗体の認識部位へのアクセシビリティを低下させる効果が減弱することを示唆している。
粒子化抗原FH_85-VLPの理論直径をZetasizer μV(Malvern)を用いた動的光散乱法によって見積もったところ、89.28 nmであった(図25)。1粒子当たり平均約135分子(3量体約45分子)を提示してることから、理論表面積1 nm2当たりの平均分子密度は、約0.005 分子(3量体としては、約0.0018 分子)と計算できる。なお、ここでは球体の表面積Sはその半径rを用いて以下の式によって求められることとした。
S=4πr2
DS-Cav1-VLPおよびFH_82-VLPにおいても同様の結果であった(図25)。
上記実験によって、足場粒子HBsAg VLPに結合したpre-F抗原はウイルス中和に寄与しない免疫の誘導を抑える効果があることが示唆された。その効果をさらに詳細に検討するため、F_DS-Cav1、F_DS-Cav1-VLP、FH_82、FH_82-VLP、FH_85またはFH_85-VLPをマウスに免疫することで誘導される抗体の分析を実施する。
マウス免疫血清は、BALB/c(雌、5~8週齢)に各粒子化抗原を免疫することで取得する。各粒子化抗原の溶媒組成としてPBSまたはPBSと水酸化アルミニウム(InvivoGen、 vac-alu-250)を等量ずつよく混合した液を使用する。抗原は約3週間間隔で2回マウスの後肢大腿部に筋肉内投与し、2回目の投与から更に約3週間後に血液を取得する。取得した血液は室温でインキュベートした後に遠心分離して血清を回収する。
血清中にpre-Fあるいはpost-Fに結合する抗体をどの程度誘導したか確認するため、各コンホメーションの抗原を固層化したELISA試験を実施する。
また、post-F認識抗体の誘導割合を算出するために抗原吸着操作および抗原吸着操作による抗原認識抗体の減少率の測定を実施する。方法は実施例5で示した方法に準拠する。
結論として、本粒子化によって、ウイルス中和に高い効果を持つと考えられる抗体群を優先的に誘導する効果が確認される。
実施例7で示したpost-F認識抗体によって認識される抗原部位に対する抗体や各種免疫細胞のアクセシビリティを立体障害効果によって低下させる効果は、足場粒子を用いない方法によっても実現できる可能性を考えた。そこで、本実施例では、疎水性ペプチド鎖(疎水性のアミノ酸を含むポリペプチド鎖)を用いた疎水集合による粒子化を検討することとした。
用いる疎水性ペプチド鎖は、ワクチン製剤あるいは生体内での条件下で疎水集合する性質を有する必要があるが、細胞による抗原の分泌発現効率を著しく低下する配列では実用が困難である。一般的に疎水性のアミノ酸を多く含むポリペプチド鎖をタンパクに融合した場合、そのタンパクの細胞における生産効率は低下することが知られているため、適切な疎水性ポリペプチド鎖の検討をすることとした。
前記抗原F_DS-Cav1の前駆体タンパク質におけるThrombin認識配列、Hisタグ、Strep-Tag IIを含む配列(配列番号22)を各種疎水性ペプチド鎖(配列番号40~43)および精製用タグであるFLAGタグから成る配列(配列番号44~47)に置換した前駆体タンパク質を元に抗原を作製した。検討した疎水性ペプチド配列を表8-1にまとめた。
各抗原の精製は、ANTI-FLAG M2 Affinity Gel(Sigma,A2220)を用いたアフィニティー精製によって実施した。アフィニティー精製の溶出にはFLAGペプチドを使用したが、限外濾過膜を用いることで各抗原サンプル中のFLAGペプチドの除去を行った。溶媒にはPBS(pH7.4)を使用した。
抗原の分泌発現の可否は、精製後に一般的なSDS-PAGEを行い、目的タンパクを高純度に分離精製できているかどうかを確認することで行った。また、粒子化の可否は、精製後に一般的なBlueNativePAGEを行うことで確認した。
F_DS-Cav1-CC02とF_DS-Cav1-CC03の疎水性ペプチド鎖の違いは、親水性ではないアミノ酸であるアラニンが、親水性アミノ酸であるリジンあるいはグルタミン酸に置き換わっている点である。そこでまず、F_DS-Cav1-CC02のアラニンを、中程度の親水性を持ち、pKがpH6程度であるヒスチジンに変更することを考えた。製剤時の溶媒のpHは中性付近であるのに対し、細胞の分泌経路中のpHはより低いpHであることが知られているため、ヒスチジンを含むペプチド鎖は、分泌発現時は比較的親水的な性質となり、製剤時には比較的疎水的な性質となることが期待された。
また、設計した疎水性ペプチド鎖がコイルドコイル構造を形成すると仮定すると、フォルドンドメインの直下に接続された疎水性ペプチド鎖の21個のアミノ酸の位相は、順にdefgabcdefgabcdefgabcとなることが予想される。3量体で疎水相互作用するa、d位にロイシンとイソロイシンを配置する場合、天然のタンパクのアミノ酸配列を検討した先行研究(Joel P Schneider et al. 1998. Folding & Design. 3:R29)によると、イソロイシンをa位、ロイシンをd位とする方がより高頻度であるため、F_DS-Cav1-CC02のa位とd位のロイシン、イソロイシンを入れ替えることを考えた。
以上から、新たにF_DS-Cav1-CC07を設計し、分泌発現の可否および粒子化の可否を検討したところ、分泌発現と粒子化の両方が達成された(図27)。ヒスチジン、リジン、グルタミン酸の位置を変更したF_DS-Cav1-CC08では分泌発現が困難になることから、単なるアミノ酸の組成のみの問題ではなく、立体構造上の配置が重要であることも示唆された。
前記抗原FH_82およびFH_85の前駆体タンパク質におけるThrombin認識配列、Hisタグ、StrepTagIIを含む配列(配列番号22)を最適な疎水性ペプチド鎖(配列番号42)および精製用タグであるFLAGタグから成る配列に置換した前駆体タンパク質(配列番号48、49)を元に、抗原を作製した。
各抗原の精製は、疎水性ペプチド鎖の検討と同様の操作で実施した。
作製した各粒子化抗原の各抗体への結合性は、実施例6と同様の操作で測定した(図28、図29、図30)。
以上の操作により、F抗原領域のpre安定化方法(pre安定化のためのアミノ酸変異の内容)に依らず、pre-Fをそのアミノ酸配列のC末端に付加した疎水性ペプチド鎖によって集合させた粒子化抗原は、エピトープΦに対する抗体の結合性を保ったまま三量体の形成が向上していることを示す結果を得た。さらに、ウイルス感染中和能の著しく低い抗体を誘導することで知られているエピトープIの検出は減弱することを示す結果を得た。以上の結果は、本抗原をワクチン抗原として用いた際に、本粒子化によって、ウイルス中和に高い効果を持つと考えられる抗体群を優先的に誘導する効果が向上することを示唆している。
抗原FH_85および粒子化抗原FH_85-CC07の理論直径をDynaPro Plate Reader III(Wyatt)を用いた動的光散乱法によって見積もったところ、13.8 nmおよび60.0 nmであった(図31)。2つの粒子の分子量の比は、一般的なタンパク質においては、その理論直径の比の2~3乗に比例するため、FH_85-CC07の分子量はFH_85の分子量の20~80倍程度になっていると見積もられる。つまり、FH85-CC07は1粒子当たり60~240分子程度(3量体としては20~80分子程度)を提示していると考えられ、理論表面積1 nm2当たりの平均分子密度は、0.005~0.022分子程度(3量体としては、0.0017~0.0073 分子程度)と計算できる。
FH_82-CC07においても、同様の結果であった(図31)。
従って、疎水性ペプチド鎖を用いた粒子化抗原は、実施例6で示した足場粒子を用いた粒子化抗原と比べて、同等程度かそれ以上の抗原密度であることが見積もられた。
先の操作によって、疎水性ペプチド鎖が結合した抗原の粒子化は、ウイルス中和に寄与しない免疫の誘導を抑える効果があることが示唆された。その効果をさらに詳細に検討するため、前記抗原または粒子化抗原F_DS-Cav1F_DS-Cav1-CC07、FH_82-CC07又はFH_85-CC07をマウスに免疫することで誘導される抗体の分析を実施した。
実施例7、8で示したpost-F認識抗体によって認識される抗原部位に対する抗体や各種免疫細胞のアクセシビリティを立体障害効果によって低下させる効果は、抗原のC末端側への自己会合性タンパクドメインの融合によっても実現できる可能性を考えた。特に、自己会合性タンパクドメインを用いて規則正しく抗原を整列させることで、実施例8で示した疎水集合による粒子化よりも高い立体障害効果が得られる可能性を考えた。
そこで、本実施例では、B型肝炎ウイルスのコア抗原(HBcAg)の自己会合性を利用した粒子化を検討することとした。
ただし、前記抗原F_DS-Cav1におけるThrombin認識配列、Hisタグ、Strep-Tag IIを含む配列(配列番号22)をGSリンカー(GGGGSGGGGSGGGGSGGGGS)(配列番号75)、HBcAgの自己会合ドメイン(配列番号50)、およびHisタグからなるペプチド配列(配列番号51)に置き換えたものでは、発現はするものの自己会合が見られなかった(BlueNativePAGEによる検証結果)。HBcAgの自己会合ドメインのN末端は、HBcAg同士が自己会合する際の結合面に近い位置に存在しており、N末端に大きなタンパク質を融合することで自己会合を妨げている可能性を考えた。
そこで、自己会合する際の結合面から遠い位置にRSV F抗原部分を配置するため、HBcAgが自己会合する際に粒子の外向きに突出する部位(α3ドメインとα4ドメインに存在するヘリックスの間の部分)よりもC末側とN末側に分割することとした。また、想定しないジスルフィド結合の生成リスクを減らすため、自己会合に影響が無いと考えられている2つの点変異(C48SおよびC107A)を導入することとした。具体的には、前記抗原F_DS-Cav1およびFH_85の前駆体タンパク質におけるThrombin認識配列、Hisタグ、StrepTagIIを含む配列(配列番号22)をGSリンカー(GGGGSGGGGSGGGGSGGGGS)(配列番号75)、HBcAgの自己会合ドメインのC末側(配列番号52)並びに、GSリンカー(GGGGSGGGGSGGGGSGGGGS)(配列番号75)、HBcAgの自己会合ドメインのN末側(配列番号53)及び、FLAGタグがその順番に結合した配列(配列番号54)に置き換えたF_DS-Cav1-HBcCNの前駆体タンパク質(配列番号55)及びFH_85-HBcCNの前駆体タンパク質(配列番号56)を元に抗原(「粒子化抗原」と称することがある)を作製した。
F_DS-Cav1-HBcCNについて、実施例8と同様の操作で発現、精製して分泌発現の可否および粒子化の可否を検討したところ、分泌発現と粒子化の両方が達成された(図33)。ただし、比較的発現量が低かったため、発現方法の改良が望まれた。そこで、DS-Cav1-HBcCNあるいはFH_85-HBcCNの前駆体をコードするポリヌクレオチドについて、GS Xceed Gene Expression System(Lonza社)を用いて安定発現CHO株を樹立した。樹立された細胞株を培養し、各抗原を分泌発現させた後に培養上清成分を取得した。
各粒子化抗原の精製は、実施例8と同様の操作で実施した。
各粒子化抗原の各抗体への結合性を確認するため、Palivizumab(シナジス筋注液、アッヴィ)、131-2A抗体、D25抗体をそれぞれ固層化したELISA試験を実施した。抗体結合バッファーには炭酸重炭酸バッファー(Sigma-Aldrich, C3041-50CAP)、サンプルの希釈及びプレートの洗浄にはTBS(TaKaRa, T9142)を用いた。96ハーフウェルプレートに対して10 μg/mLのPalivizumabまたは131-2A抗体またはD25抗体を添加して直接固層化し、ウシ血清アルブミンにてブロッキングした。各抗原に対して適当にバッファーを添加することで10倍希釈系列を作製し、プレートに添加してインキュベート(室温、2時間、静置)した。その後添加した抗原溶液を除き、ペルオキシダーゼ結合抗FLAG M2抗体(Sigma-Aldrich, A8592)を結合させ、更に標準的なペルオキシダーゼ検出試薬で発色させた後に標準的なプレートリーダーによる吸光度測定にて発色の強度を求めた。測定点間は区分線形補間することで抗原の希釈率と吸光度の関係を求め、吸光度が0.2となる希釈率の逆数を、固層化した抗体に対する添加した抗原の結合力とした。D25抗体に対する結合力をPalivizumabに対する結合力で割り算した値をエピトープΦ検出シグナル、131-2A抗体に対する結合力をPalivizumabに対する結合力で割り算した値をエピトープI検出シグナルとした(図34、図35)。
以上の操作により、pre-Fをそのアミノ酸配列のC末端に付加した自己会合性タンパクドメインによって集合させた粒子化抗原は、疎水性ペプチド鎖による粒子化よりも更にエピトープΦに対する抗体の結合性を向上させ、ウイルス感染中和能の著しく低い抗体を誘導することで知られているエピトープIの検出を減弱することを示す結果を得た。以上の結果は、本粒子化によって、ウイルス中和に高い効果を持つと考えられる抗体群を優先的に誘導する効果が向上することを示唆している。
Claims (34)
- 変異を有する変異型RSV Fタンパク質であって、前記変異は、配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン又は142位のロイシンに相当するロイシンのシステインへの置換、及び373位のロイシンに相当するロイシンのシステインへの置換であり、前記システイン間でジスルフィド結合が形成される、変異型RSV Fタンパク質。
- 前記変異型RSV Fタンパク質が、RSVサブタイプA又はRSVサブタイプBに由来する、請求項1に記載の変異型RSV Fタンパク質。
- 前記RSVサブタイプAがRSV A2株又はRSV Long株である、請求項2に記載の変異型RSV Fタンパク質。
- 前記RSVサブタイプBがRSV 18537株である、請求項2に記載の変異型RSV Fタンパク質。
- 配列番号2と85%以上の同一性を有するアミノ酸配列において、配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン又は142位のロイシンに相当するロイシン、及び373位のロイシンに相当するロイシンがシステインに置換されたアミノ酸配列を含み、前記システイン間でジスルフィド結合が形成され、RSVのpre型Fタンパク質に対する抗体を誘導する能力を有する、請求項1~4のいずれか一項に記載の変異型RSV Fタンパク質。
- さらに、pep27領域のC末端側に存在する、フーリン認識部位を構成するアミノ酸が、前記フーリン認識部位がフーリンによって認識されないように置換されている、請求項1~4のいずれか一項に記載の変異型RSV Fタンパク質。
- 前記フーリン認識部位を構成するアミノ酸が、配列番号1のアミノ酸配列の133位のアルギニンに相当するアルギニン、135位のアルギニン相当するアルギニン、及び136位のアルギニンに相当するアルギニンからなる群から選択されるアミノ酸であり、
当該アミノ酸が、非塩基性アミノ酸に置換されている、
請求項6に記載の変異型RSV Fタンパク質。 - 配列番号3と85%以上の同一性を有するアミノ酸配列において、配列番号1のアミノ酸配列の141位のロイシンに相当するロイシン又は142位のロイシンに相当するロイシン、及び373位のロイシンに相当するロイシンがシステインに置換され、さらに、配列番号1のアミノ酸配列の133位のアルギニンに相当するアルギニン、135位のアルギニン相当するアルギニン、及び136位のアルギニンに相当するアルギニンからなる群から選択されるアミノ酸が、非塩基性アミノ酸に置換されたアミノ酸配列を含み、前記システイン間でジスルフィド結合が形成され、RSVのpre型Fタンパク質に対する抗体を誘導する能力を有する、請求項6または7に記載の変異型RSV Fタンパク質。
- 前記非塩基性アミノ酸がアスパラギンである、請求項7または8に記載の変異型RSV Fタンパク質。
- さらに、配列番号1のアミノ酸配列の189位のスレオニンに相当するスレオニン、及び/又は190位のセリンに相当するセリンが疎水性アミノ酸に置換されている、請求項1~9のいずれか一項に記載の変異型RSV Fタンパク質。
- 前記疎水性アミノ酸が、それぞれ独立して、バリン、イソロイシン、及びロイシンからなる群から選択される、請求項10に記載の変異型RSV Fタンパク質。
- さらに、配列番号1のアミノ酸配列の42位のリジンに相当するリジンがアルギニンに置換されており、及び/又は、384位のバリンに相当するバリンがスレオニンに置換されている、請求項1~11のいずれか一項に記載の変異型RSV Fタンパク質。
- 変異を有する変異型RSV Fタンパク質であって、前記変異は、配列番号1のアミノ酸配列の60位のグルタミン酸に相当するグルタミン酸の非酸性アミノ酸への置換である、変異型RSV Fタンパク質。
- 前記非酸性アミノ酸が、メチオニン、フェニルアラニン、ロイシン、スレオニン、及びセリンからなる群から選択される、請求項13に記載の変異型RSV Fタンパク質。
- 請求項1~14のいずれか一項に記載の変異型RSV Fタンパク質と、前記変異型RSV Fタンパク質のC末端に融合された多量体化ドメインを含む、融合タンパク質。
- 前記多量体化ドメインがフォルドンドメインである、請求項15に記載の融合タンパク質。
- 配列番号10~13のいずれかのアミノ酸配列を含む、請求項16に記載の融合タンパク質。
- 請求項1~14のいずれか一項に記載の変異型RSV Fタンパク質の多量化体であって、前記請求項15~17のいずれか一項に記載の融合タンパク質が前記多量体化ドメインを介して会合したものである、前記多量化体。
- 前記多量化体が3量体である、請求項18に記載の多量化体。
- 請求項1~14のいずれか一項に記載の変異型RSV Fタンパク質または請求項15~17のいずれか一項に記載の多量化体の粒子化体であって、前記変異型RSV Fタンパク質または前記融合タンパク質は粒子化ドメインを含み、前記変異型RSV Fタンパク質または前記多量化体が該粒子化ドメインを介して2個以上集合して粒子を形成しており、前記粒子化ドメインは、変異型RSV Fタンパク質または多量化体の立体構造上、エピトープΦよりもエピトープIに近い位置に存在する、前記粒子化体。
- 前記粒子化体は、前記変異型RSV Fタンパク質または前記融合タンパク質のC末端に粒子化ドメインが結合した粒子化体製造用融合タンパク質が、前記粒子化ドメインを介して2個以上集合したものである、請求項20に記載の粒子化体。
- 前記多量化体が請求項16記載の融合タンパク質からなる3量体である、請求項20または21に記載の粒子化体。
- 前記粒子化ドメインが配列番号30のアミノ酸配列からなるFcドメインであって、当該粒子化ドメインが改変型HBs抗原由来のVLPに固定化されているプロテインAのZドメインと結合することにより集合したものである、請求項22に記載の粒子化体。
- 前記粒子化ドメインが配列番号42のアミノ酸配列からなるペプチドである、請求項22に記載の粒子化体。
- 請求項1~14のいずれか一項に記載の変異型RSV Fタンパク質のC末端、または請求項15~17のいずれか一項に記載の融合タンパク質と、そのC末端に融合した粒子化ドメインを含む、粒子化体製造用融合タンパク質。
- 前記粒子化ドメインが配列番号30のアミノ酸配列配列からなるFcドメインである、請求項25に記載の粒子化体製造用融合タンパク質。
- 前記粒子化ドメインが配列番号42のアミノ酸配列からなるペプチドである、請求項25に記載の粒子化体製造用融合タンパク質。
- 請求項1~14のいずれか一項に記載の変異型RSV Fタンパク質、請求項15~17のいずれか一項に記載の融合タンパク質、または請求項25~27のいずれか一項に記載の粒子化体製造用融合タンパク質をコードするポリヌクレオチド。
- 請求項28に記載のポリヌクレオチドを含む、発現ユニット。
- 請求項29に記載の発現ユニットを含む、宿主細胞。
- 請求項1~14のいずれか一項に記載の変異型RSV Fタンパク質、請求項15~17のいずれか一項に記載の融合タンパク質、請求項18~19のいずれか一項に記載の多量化体、又は請求項20~24のいずれか一項に記載の粒子化体を含む、免疫原。
- 請求項29に記載の発現ユニット、又は請求項31に記載の免疫原を含む、医薬組成物。
- 請求項29に記載の発現ユニット、又は請求項31に記載の免疫原を含む、RSVワクチン。
- ヒト用である、請求項32に記載の医薬組成物または請求項33に記載のRSVワクチン。
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CA3165855A CA3165855A1 (en) | 2019-12-23 | 2020-12-23 | Mutant rsv f protein and use thereof |
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CN202080089393.1A CN114929877A (zh) | 2019-12-23 | 2020-12-23 | 突变型rsv f蛋白及其利用 |
AU2020411873A AU2020411873B2 (en) | 2019-12-23 | 2020-12-23 | Mutant RSV F protein and use thereof |
KR1020227024647A KR20220116516A (ko) | 2019-12-23 | 2020-12-23 | 변이형 rsv f 단백질 및 그 이용 |
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US17/788,717 US20230086093A1 (en) | 2019-12-23 | 2020-12-23 | Mutant rsv f protein and use thereof |
EP20908021.7A EP4083212A4 (en) | 2019-12-23 | 2020-12-23 | MUTANT RSV-F PROTEIN AND ITS USE |
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CN117487823A (zh) * | 2023-09-28 | 2024-02-02 | 怡道生物科技(苏州)有限公司 | 呼吸道合胞体病毒mRNA疫苗及其制备方法和应用 |
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CN117586358B (zh) * | 2024-01-19 | 2024-08-13 | 北京安百胜生物科技有限公司 | 一种具有免疫原性的呼吸道合胞病毒(rsv)多肽 |
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