Patent Application
20190016760
This invention relates generally to the production of multimeric recombinant proteins.
Proteins are responsible for a majority of the cellular functions such as molecular recognition (for example in the immune system), signaling pathways (hormones), the transport of metabolites and nutrients and the catalysis of biochemical reactions (enzymes).
The function of proteins results from their three-dimensional structure, that is to say how the amino acids of the polypeptide chain are arranged relative to each other in space. It is usually only in its folded state (native state) that a protein can exert its biological activity.
Whereas most proteins have a primary structure (amino acid sequence), a secondary structure (alpha-helices and beta-sheets), and a tertiary structure (three-dimensional), protein oligomers have an additional level called the quaternary structure that is part of the three-dimensional structure. Oligomers are complexes of several polypeptides. They can contain several copies of an identical protein referred to as a sub-unit and are referred to as homo-oligomers, or they may consist of more than one type of protein sub-unit, in which case they are referred to as hetero-oligomers. Hemoglobin, the oxygen carrier in blood, is an example of a protein containing identical subunits. Nitrogenase, the microbial enzyme responsible for the reduction of nitrogen gas to ammonia, is an example of a protein containing non-identical sub-units.
Numerous recombinant proteins of interest are oligomeric in nature, for example antibodies, many transmembrane proteins such as transmembrane receptors, porins, viral surface antigens, heat shock proteins, viral capsid proteins, ferritin, insulin, many enzymes such as glutathione peroxidase, catalase or superoxide dismutase, collagen and many others.
For instance, influenza virus haemagglutinin (HA) is a homotrimeric glycoprotein on the surface of the virus which is responsible for interaction of the virus with host cell receptors. The three-dimensional structure of HA is described in detail in Nature, 289, 366-373 (1981). Protective immune responses induced by vaccination against influenza virus are primarily directed to the viral HA protein. Recombinant HA protein (rHA) represents therefore an interesting antigen for the development of influenza vaccines.
Another oligomeric antigen of interest is the Invasion Plasmid Antigen D (IpaD) protein of Shigella that was found to form either pentamers, or in the presence of IpaB, tetramers, at the needle tip of the bacteria (Cheung et al., Molecular Microbiology, 95(1), 31-50 (2015)).
A further oligomeric antigen of interest is the Membrane expression of Ipa H (MxiH) protein of Shigella that was found to form a helical assembly of subunits that produces the Shigella needle (Cordes et al., The Journal of Biological Chemistry, 278(19), 17103-17107 (2003)).
One of the challenges in the recombinant protein field is that recombinant proteins do not always have the same three-dimensional conformation as the native protein. Yet the function of proteins often results from their three-dimensional structure.
Similarly, in respect of oligomers, if the recombinant protein does not keep the quaternary structure of the native protein, the function of the recombinant protein may be altered or suppressed.
For instance, William C. Weldon et al., in Plos One, 5(9), e12466 (2010), showed that poor trimerization of a recombinant influenza haemagglutinin could play a role in its low immunogenicity.
There is therefore a need to produce recombinant proteins which better retain the oligomeric structure and desired biological function of the native protein.
Chih-Jen Wei et al., in Journal of Virology, 82(13), 6200-6208 (2008), describe the trimerization of influenza rHA using the foldon sequence of the T4 phage.
The inventors have surprisingly determined that a fragment of the sequence of the lamprey variable lymphocyte receptor B (VLR-B) antibody may be used to multimerize a heterologous fusion protein.
Lamprey is a jawless vertebrate with an adaptive immune system comprised of clonally diverse lymphocytes that express variable lymphocyte receptors (VLRs) created by combinatorial assembly of leucine-rich repeat gene segments. The VLR-B can be secreted and can function analogously to antibodies in jawed vertebrates.
Surprisingly we found that fusion of a nucleic acid sequence encoding a protein of interest and a nucleic acid sequence encoding a peptide found at the extreme C-terminus of lamprey VLR-B antibodies, i.e. C-terminal to the Stalk region (the domain named “C-TERM” in FIG. 11C of WO 2008/016,854), encodes a recombinant protein which is capable of oligomerization with several degrees of oligomerization.
More surprisingly we found that the multimeric recombinant proteins obtained are stable.
And even more surprisingly we found that the stable multimeric recombinant proteins obtained have several degrees of oligomerization while retaining the biological activity of their native form.
According to an embodiment, a molecule is obtained which comprises a first amino acid sequence which has at least 80% identity to SEQ ID NO: 1 and a second amino acid sequence which is heterologous to said first sequence.
According to another embodiment, a recombinant protein is obtained which comprises a first amino acid sequence which has at least 80% identity to SEQ ID NO: 1 and a second amino acid sequence which is heterologous to said first sequence.
According to another embodiment a recombinant nucleic acid is constructed which comprises a first nucleic acid sequence with at least 80% identity to SEQ ID NO: 3 and a second nucleic acid sequence which is heterologous to said first sequence.
Another aspect is directed to an expression cassette comprising a recombinant nucleic acid as described above wherein the recombinant nucleic acid is operably linked to a promoter.
Another aspect is directed to a host cell transformed with the expression cassette.
The invention is also directed to a stable homo-multimeric recombinant protein which comprises a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein, which is fused to a protein having an amino acid sequence with at least 80% identity to SEQ ID NO: 1.
Another embodiment is directed to a pharmaceutical composition comprising a molecule or a recombinant protein of the invention and a pharmaceutically acceptable carrier or diluent.
In another aspect the invention provides an immunogenic composition comprising a molecule or a recombinant protein of the invention.
In another embodiment, the molecule or the recombinant protein of the invention is for use as a medicament.
In a further aspect of the invention, the molecule or the recombinant protein of the invention is for use in inducing an immune response to an antigen in a subject.
The invention is also directed to a method for multimerizing a recombinant protein comprising:
a) fusing a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3 to the nucleic acid sequence coding for said recombinant protein, with the proviso that said recombinant protein is not a lamprey VLR-B antibody protein,
b) expressing the fusion protein encoded by said nucleic acid sequence, under conditions which lead to the multimerization of said recombinant protein.
In the context of the invention, protein “oligomers” or “polymers” or “multimers” have the same meaning, i.e. proteins having a quaternary structure, being complexes of at least two polypeptides, said polypeptides may be identical or different. Accordingly, in the context of the invention, “multimerization”, “oligomerization” and “polymerization” have the same meaning, as do “multimerized”, “oligomerized” and “polymerized” or “multimerizing”, “oligomerizing” and “polymerizing”.
“Recombinant proteins” are proteins encoded by recombinant nucleic acids. They are expressed from recombinant nucleic acids in a host cell. “Recombinant nucleic acid” is used herein to describe a nucleic acid molecule which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature and/or is linked to a polynucleotide other than that to which it is linked in nature. The recombinant proteins of the invention comprise a protein fragment from the VLR-B antibody of lamprey and a protein of interest which is heterologous to the protein fragment from the VLR-B antibody of lamprey. As described herein, the recombinant proteins of the invention comprise a protein fragment from the extreme C-terminus of VLR-B antibodies of Lamprey.
In the context of the invention, a “molecule” is the junction by any means between a protein fragment from the VLR-B antibody of lamprey and a protein of interest which is heterologous to the protein fragment from the VLR-B antibody of lamprey. For example, a molecule of the present invention may be created by joining the VLR-B protein and the heterologous protein of interest via a covalent linkage. Examples of such covalent linkages include a peptide bond, an ester linkage, an amide linkage and a disulfide bond. As described herein, the protein fragment from the VLR-B antibody of lamprey comes from the extreme C-terminus of VLR-B antibodies of Lamprey.
By “first amino acid sequence” and “second amino acid sequence” in the description of the molecule or the recombinant protein of the invention, it is not meant that a specific order of the sequences is contemplated. It is just for clarity of the embodiment to better distinguish the two sequences comprised in the molecule or recombinant protein of the invention.
By “first nucleic acid sequence” and “second nucleic acid sequence” in the description of the recombinant nucleic acid of the invention, it is not meant that a specific order of the sequences is contemplated. It is just for clarity of the embodiment to better distinguish the two sequences comprised in the recombinant nucleic acid of the invention.
In the context of the invention, the first sequence, either amino acid or nucleic acid sequence, designates respectively, an amino acid or a nucleic acid sequence, derived from the C-terminus of the VLR-B antibody of lamprey. According to the invention, the size of the first polypeptide sequence is typically between 24 and 43 amino acids long, particularly between 30 and 43 amino acids long, the bounds being included. Accordingly the size of the first polypeptide sequence may preferably be about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43 amino acids long. According to the invention, the size of the first nucleic acid sequence is typically between 72 and 129 base pairs long, particularly between 90 and 129 base pairs long, the bounds being included. Accordingly the size of the first nucleic acid sequence may preferably be about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 129 base pairs long.
In the context of the invention, the second sequence, either amino acid or nucleic acid sequence, designates respectively the amino acid sequence of a protein of interest or a fragment thereof or the nucleic acid sequence encoding a protein of interest or a fragment thereof. In the context of the present invention, a “fragment” of a protein as referred to herein retains the biological function of the full-length protein from which it is derived. Thus a fragment according to the present invention may be at least 20, at least 50, at least 75, at least 100 or at least 150 amino acids long.
Two sequences which are contained within a single recombinant molecule are “heterologous” relative to each other when they are not normally associated with each other in nature. In the context of the invention, a second sequence that is heterologous to a first sequence, either amino acid or nucleic acid sequence, means that the second heterologous sequence is not or does not comprise a sequence from the VLR-B antibody of lamprey. In the context of the invention, the heterologous sequence is not an amino acid sequence of, or a nucleic acid sequence coding for a polyhistidine-tag (His-tag). Furthermore, it is preferred that the heterologous sequence according to the present invention is at least 5, at least 10 or at least 15 amino acids long (or is a nucleotide sequence encoding such an amino acid sequence).
“Fusion proteins” are proteins created through the joining of two or more genes that originally coded for separate proteins. This typically involves removing the stop codon from a DNA sequence coding for the first protein, then appending the DNA sequence of the second protein in frame through ligation or overlap extension PCR. If more than two genes are fused, the other genes are added in frame in the same manner. The resulting DNA sequence will then be expressed by a cell as a single protein. The fusion proteins of the invention are obtained from a nucleic acid coding for a protein fragment from the VLR-B antibody of lamprey fused to a nucleic acid coding for any or all of proteins of interest or fragments thereof. In the context of the invention, the protein can be engineered to include the full sequence of a protein of interest, or only a portion of a protein of interest. The joining of the two or more genes may be made in any order, i.e. the sequences coding for proteins of interest, or fragments thereof, are located either 3′ or 5′ from the sequence coding for a fragment of the lamprey VLR-B antibodies. Preferably, the sequences coding for the proteins of interest, or fragments thereof, are located 5′ from the sequence coding for a fragment of the lamprey VLR-B antibodies. As described elsewhere herein, in the context of the present invention, the protein fragment from the VLR-B antibody of lamprey comes from the extreme C-terminus of the lamprey VLR-B antibody.
As used herein, a first sequence having at least x% identity to a second sequence means that x% represents the number of amino acids in the first sequence which are identical to their matched amino acids of the second sequence when both sequences are optimally aligned via a global alignment, relative to the total length of the second amino acid sequence. Both sequences are optimally aligned when x is maximum. The alignment and the determination of the percentage of identity may be carried out manually or automatically using a global alignment algorithm, for instance the Needleman and Wunsch algorithm, described in Needleman and Wunsch, J. Mol Biol., 48, 443-453 (1970), with for example the following parameters for polypeptide sequence comparison: comparison matrix: BLOSUM62 from Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89, 10915-10919 (1992), gap penalty: 8 and gap length penalty: 2; and the following parameters for polynucleotide sequence comparison: comparison matrix: matches=+10, mismatch=0; gap penalty: 50 and gap length penalty: 3.
A program which may be used with the above parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis. The aforementioned parameters are the default parameters respectively for peptide comparisons (along with no penalty for end gaps) and for nucleic acid comparisons.
An “antigen” refers to any agent, preferably a macromolecule, which can elicit an immunological response in an individual. The term may be used to refer to an individual macromolecule or to a homogeneous or heterogeneous population of antigenic macromolecules. As used herein, “antigen” is preferably used to refer to a protein molecule or portion thereof which contains one or more epitopes. An epitope is the part of the antigen that is recognized by antibodies or T cell receptors. Some epitopes are referred to as discontinuous conformational epitope. This means that the amino acids comprising these epitopes are proximal to each other in the three-dimensional structure of the protein, but appear distant from each other when one looks strictly at the one-dimensional linear amino acid sequence. Consequently, it is clear that the three-dimensional structure of the protein is extremely important in terms of what the immune system actually sees.
The “ectodomain” is the portion of a transmembrane anchored protein that extends beyond the membrane into the extracellular space.
“Scaffolds” are specific ligand-binding artificial structures usually generated from a combinatorial library of a chosen protein scaffold, by selective random mutagenesis of appropriate exposed surface residues followed by selection of variants with the desired binding activity. Kaspar Binz et al. reviewed numerous alternative protein scaffolds, in Nature Biotechnology, 86 (10), 1257-1268 (2005), and the well-established techniques to design the combinatorial library from them and to select the relevant variant, most predominantly phage display and related methods.
Various features of the embodiments can be more fully appreciated, with reference to the following detailed description of the embodiments and accompanying figures, in which:
According to an embodiment, a molecule is obtained which comprises a first amino acid sequence which has at least 80% identity to SEQ ID NO: 1 and a second amino acid sequence which is heterologous to said first sequence. In particular, the molecule according to the invention comprises a first amino acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 1.
According to an embodiment, a molecule is obtained which comprises a first amino acid sequence which has at least 80% identity to SEQ ID NO: 2 and a second amino acid sequence which is heterologous to said first sequence. In particular, the molecule according to the invention comprises a first amino acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 2.
In a preferred embodiment the 7 cysteines that correspond to positions 2, 7, 13, 19, 21, 24 and 27 of SEQ ID NO: 1 are conserved in the first io amino acid sequence. The molecule of the invention does not comprise a lamprey VLR-B antibody protein.
In a preferred embodiment the 8 cysteines that correspond to positions 2, 15, 20, 26, 32, 34, 37 and 40 of SEQ ID NO: 2 are conserved in the first amino acid sequence. The molecule of the invention does not comprise a lamprey VLR-B antibody protein.
According to an embodiment, a recombinant protein is obtained which comprises a first amino acid sequence which has at least 80% identity to SEQ ID NO: 1 and a second amino acid sequence which is heterologous to said first sequence. In particular, the recombinant protein according to the invention comprises a first amino acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 1.
According to an embodiment, a recombinant protein is obtained which comprises a first amino acid sequence which has at least 80% identity to SEQ ID NO: 2 and a second amino acid sequence which is heterologous to said first sequence. In particular, the molecule according to the invention comprises a first amino acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 2.
In a preferred embodiment the 7 cysteines that correspond to positions 2, 7, 13, 19, 21, 24 and 27 of SEQ ID NO: 1 are conserved in the first amino acid sequence. The recombinant protein of the invention does not comprise a lamprey VLR-B antibody protein.
Preferably, a molecule or recombinant protein of the invention does not comprise a leucine-rich repeat (LRR) module from a lamprey VLR-B antibody. A consensus sequence for an LRR module from a lamprey VLR-B antibody is LXXLXXLXLXXNXLXXXPXGXFDX, where X may be any amino acid (SEQ ID NO: 29). Preferably, a molecule or recombinant protein of the invention does not comprise a sequence falling within the scope of the group of sequences defined by SEQ ID NO: 29, i.e. a molecule or recombinant protein of the invention does not comprise SEQ ID NO: 29. Specific examples of LRR modules (see FIG. 11C of WO 2008/016854) include an N-terminal cap LRR (referred to as LRRNT), LRR1, variable LRR modules (referred to as LRRV), an end LRRV (known as LRRVe) and a C-terminal cap LRR (referred to as LRRCT). Preferably, a molecule or recombinant protein of the invention does not comprise one or more of an LRRNT, an LRR1, an LRRV and an LRRCT module from a lamprey VLR-B antibody. Lamprey VLR-B antibodies also comprise a connecting peptide (CP) and a Stalk region in addition to the LRR modules. Preferably, a molecule or recombinant protein of the invention does not comprise a CP or a Stalk region from a lamprey VLR-B antibody. Preferably, a molecule or recombinant protein of the invention does not comprise an LRR module, a CP or a Stalk region from a lamprey VLR-B antibody. Preferably, the only lamprey-derived amino acid sequence in a molecule or recombinant protein of the present invention is derived from the extreme C-terminus of a lamprey VLR-B antibody (i.e. the section of the protein C-terminal to the Stalk region, see FIG. 11C of WO 2008/016854). Preferably, the only lamprey-derived amino acid sequence in a molecule or recombinant protein of the present invention is a sequence having at least 80% identity to SEQ ID NO: 1 or io SEQ ID NO: 2, for example at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2.
Another embodiment is directed to a recombinant nucleic acid which comprises a first nucleic acid sequence with at least 80% identity to SEQ ID NO: 3 and a second nucleic acid sequence which is heterologous to said first sequence. In particular, the recombinant nucleic acid according to the invention comprises a first nucleic acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 3.
Another embodiment is directed to a recombinant nucleic acid which comprises a first nucleic acid sequence with at least 80% identity to SEQ ID NO: 4 and a second nucleic acid sequence which is heterologous to said first sequence. In particular, the recombinant nucleic acid according to the invention comprises a first nucleic acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 4.
In a preferred embodiment the first nucleic acid sequence encodes an amino acid sequence which comprises cysteine residues at positions within said amino acid sequence that correspond to positions 2, 7, 13, 19, 21, 24 and 27 of SEQ ID NO:1. The recombinant nucleic acid of the invention does not encode a lamprey VLR-B antibody. In a preferred embodiment the first nucleic acid sequence encodes an amino acid sequence which comprises cysteine residues at positions within said amino acid sequence that correspond to positions 2, 15, 20, 26, 32, 34, 37 and 40 of SEQ ID NO: 2.
Preferably, a recombinant nucleic acid of the invention does not encode a leucine-rich repeat (LRR) module from a lamprey VLR-B antibody. In particular, a recombinant nucleic acid as described herein does not encode an amino acid sequence having the sequence of SEQ ID NO: 29. Preferably, a recombinant nucleic acid of the invention does not encode one or more of an LRRNT module, an LRR1 module, an LRRV module, an LRRCT module, a CP and a Stalk region from a lamprey VLR-B antibody. Preferably, the only lamprey-derived amino acid sequence which is encoded by a recombinant nucleic acid of the present invention is derived from the extreme C-terminus of a lamprey VLR-B antibody (i.e. the section of the protein C-terminal to the Stalk region, see FIG. 11C of WO 2008/016854). Preferably, the only lamprey-derived nucleic acid sequence in a recombinant nucleic acid of the present invention is a sequence having at least 80% identity to SEQ ID NO: 3 or SEQ ID NO: 4, for example at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 3 or SEQ ID NO: 4.
A linker may be inserted between the first amino acid sequence and the second heterologous amino acid sequence. Linkers may be a short peptide sequence or another suitable covalent link between protein domains. Preferably, the linker is a short peptide sequence. Preferably said peptide linkers are composed of flexible residues like glycine (G) and serine (S) so that the adjacent protein domains are free to move relative to one another. Preferably said linker is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or at least 15 amino acid residues long. Any possible linker known by the person skilled in the art may be used for the purpose of the invention. For instance the linker may be G6S9 (which means 6 glycines followed by 9 serines) as used by William C. Weldon et al., in Plos One, 5(9), e12466 (2010); G8 as used by Ludmilla Sissoëff et al., in Journal of General Virology, 86, 2543-2552 (2005), or G4S3.
A spacer nucleic acid sequence coding for a peptide linker as described above may be inserted between the first nucleic acid sequence and the second heterologous nucleic acid sequence.
In a preferred embodiment the heterologous protein of interest is an antigen or fragment thereof. In this embodiment, the heterologous amino acid sequence is from an antigen amino acid sequence or the heterologous nucleic acid sequence is from an antigen nucleic acid sequence. For the purpose of the present invention, antigens can be obtained or derived from any appropriate source. Preferably, the source of the antigen is selected from the group consisting of influenza virus, HIV, cytomegalovirus, dengue virus, yellow fever virus, tick-borne encephalitis virus, hepatitis virus, japanese encephalitis virus, human papillomavirus, coxsackievirus, herpes simplex virus, rubella virus, mumps virus, measles virus, rabies virus, polio virus, rotavirus, respiratory syncytial virus, Ebola virus, Chikungunya virus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Clostridium difficile, Bordetella pertussis, Clostridium tetani, Haemophilus influenzae type b, Chlamydia pneumoniae, Chlamydia trachomatis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Mycobacterium diphtheriae, Shigella, Neisseria meningitidis, Streptococcus pneumoniae and Plasmodium falciparum. Preferably, the antigen has a molecular weight of less than 150 kDa, less than 125 kDa or less than 100 kDa. Most preferably, the antigen has a molecular weight of less than 100 kDa.
Preferably, the source of the antigen is selected from the group consisting of influenza virus, cytomegalovirus, dengue virus, yellow fever virus, hepatitis virus, japanese encephalitis virus, human papillomavirus, herpes simplex virus, rabies virus, polio virus, rotavirus, respiratory syncytial virus, Ebola virus, Chikungunya virus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Clostridium difficile, Bordetella pertussis, Clostridium tetani, Haemophilus influenzae type b, Mycobacterium diphtheriae, Shigella, Neisseria meningitidis and Streptococcus pneumoniae. Preferably, the source of the antigen is selected from influenza virus and Shigella.
In some embodiments a molecule or a recombinant protein of the invention may comprise more than one antigen which is heterologous to the lamprey VLR-B sequence as described herein. When the molecule or the recombinant protein comprises several antigens, these antigens are independently a complete protein of interest or a fragment of a protein of interest, and may be from the same organism or from different organisms. The antigen may be a fusion antigen from different proteins, or fragments thereof, of the same organism or from different organisms.
Preferably, the antigen for use in a molecule or a recombinant protein of the present invention is from an influenza virus. The influenza virus may be a seasonal or a pandemic influenza virus. The influenza virus may be any subtype of A strains, B strains, or C strains. In particular, the influenza A virus is selected from the group consisting of the H1N1, H2N2, H3N1, H3N2, H3N8, H5N1, H7N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7 viruses.
Preferably, the influenza antigen is selected from a haemagglutinin (HA), or fragment thereof, a matrix 2 protein (M2) (Holsinger et al., Virology, 183, 32-43 (1991)), or fragment thereof, and an HAM2 fusion protein. In the HAM2 fusion protein, HA and M2 are independently the complete protein or a fragment of the protein. In a more preferred embodiment, the antigen is an influenza haemagglutinin or fragment thereof.
Furthermore, for the purposes of the present invention, an antigen includes a protein having modifications, such as deletions, additions and substitutions to the native sequence, as long as the protein maintains sufficient immunogenicity. These modifications may be deliberate, for example through site-directed mutagenesis, or may be accidental, such as mutations which occur during expression of the antigens in a host cell. The antigen may also be a protein or a fragment thereof encoded by a consensus sequence.
Preferably, the antigen is the ectodomain of a transmembrane anchored protein. The ectodomain corresponds to the native protein wherein the transmembrane domain and cytoplasmic tail, if any, have been deleted in order to allow its secretion in the host which produces the antigen and its easy downstream purification.
Preferably, the antigen is the ectodomain of influenza virus HA.
In another preferred embodiment the protein of interest (i.e. the antigen for use in an antigen or recombinant protein of the present invention) is selected from cytomegalovirus (CMV) glycoprotein B (gB) (Scheffczick et al., FEBS Letters, 506, 113-116 (2001)), or a fragment thereof, cytomegalovirus UL130 protein (Patrone et al., J. Virol. 79(13), 8361-8373 (2005)) or a fragment thereof, ora gB-UL130 fusion protein, and the HIV glycoprotein 41 (Gp41) (Pancera et al., Nature, 514(7523), 455-461 (2014)), or a fragment thereof. In the gB-UL130 fusion protein, gB and UL130 are independently the complete protein or a fragment thereof.
In a more preferred embodiment, the antigen is the ectodomain of the CMV gB protein or of the HIV Gp41 protein. In the gB-UL130 fusion protein, gB is the complete protein or the ectodomain of the gB protein. In another preferred embodiment, the antigen is selected from the group consisting of the HIV Gp41 protein and the cytomegalovirus UL130 protein.
In another preferred embodiment, the antigen is a bacterial protein, for example a protein from Shigella sp. Preferably the antigen is from Shigella sonnei or Shigella flexneri. Preferably the antigen is IpaD or MxiH from Shigella sonnei or Shigella flexneri. In certain embodiments, the antigen is preferably not the CMV gB protein or the ectodomain of the CMV gB protein.
In another preferred embodiment, the protein of interest is an antibody or a scaffold. In this embodiment, the heterologous amino acid sequence is from an antibody or scaffold amino acid sequence or the heterologous nucleic acid sequence is from an antibody or scaffold nucleic acid sequence.
In a preferred embodiment the antibody or scaffold is specific for an antigen, i.e. specifically binds to an antigen. For the purpose of the present invention, antigens for which the antibody or scaffold is specific for can be obtained or derived from any appropriate source. Preferably, the source of the antigen is selected from the group consisting of influenza virus, HIV, cytomegalovirus, dengue virus, yellow fever virus, tick-borne encephalitis virus, hepatitis virus, japanese encephalitis virus, human papillomavirus, coxsackievirus, herpes simplex virus, rubella virus, mumps virus, measles virus, rabies virus, polio virus, rotavirus, respiratory syncytial virus, Ebola virus, Chikungunya virus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Clostridium difficile, Bordetella pertussis, Clostridium tetani, Haemophilus influenzae type b, Chlamydia pneumoniae, Chlamydia trachomatis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Mycobacterium diphtheriae, Shigella, Neisseria meningitidis, Streptococcus pneumoniae and Plasmodium falciparum.
Preferably, the source of the antigen is selected from the group consisting of influenza virus, cytomegalovirus, dengue virus, yellow fever virus, hepatitis virus, japanese encephalitis virus, human papillomavirus, herpes simplex virus, rabies virus, polio virus, rotavirus, respiratory syncytial virus, Ebola virus, Chikungunya virus, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Clostridium difficile, Bordetella pertussis, Clostridium tetani, Haemophilus influenzae type b, Mycobacterium diphtheriae, Shigella, Neisseria meningitidis and Streptococcus pneumoniae.
In a preferred embodiment the antibody is one of the alternative formats described by Roland Kontermann in Current Opinion in Molecular Therapeutics, 12(2), 176-183 (2010). In particular, the antibody is selected from the group consisting of a monoclonal antibody, a single domain antibody (dAb), a single-chain variable fragment (scFv), a Fab, a F(ab′)2 and a diabody (Db). In this embodiment, the heterologous amino acid sequence or the heterologous nucleic acid sequence is respectively from a monoclonal antibody, a dAb, a scFv, a Fab, a F(ab′)2 or a Db amino acid sequence, or from a monoclonal antibody, a dAb, a scFv, a Fab, a F(ab′)2 or a Db nucleic acid sequence.
Roland Kontermann also described bi-specific antibody formats in Current Opinion in Molecular Therapeutics, 12(2), 176-183 (2010). In some embodiments, the molecule, e.g. a recombinant protein, of the invention is a bi-specific antibody or a bi-specific scaffold, i.e. an antibody or a scaffold specific for two different antigens, or is a multi-specific antibody or a multi-specific scaffold, i.e. an antibody or a scaffold specific for more than two different antigens. In these embodiments, the heterologous amino acid sequence comprises at least two different antibody, monoclonal antibody, dAb, scFv, Fab, F(ab′)2, Db or scaffold amino acid sequences, or the heterologous nucleic acid sequence comprises at least two different antibody, monoclonal antibody, dAb, scFv, Fab, F(ab′)2, Db or scaffold nucleic acid sequences. The joining of the two or more genes may be made in any order, i.e. the sequences coding for the two or more proteins of interest, or fragments thereof, are located either 3′ or 5′ of the sequence coding for the fragment of the lamprey VLR-B antibody according to the present invention, or one of the sequences coding for a protein of interest, or fragment thereof, is located 5′ of the sequence coding for the fragment of the lamprey VLR-B antibody according to the present invention and the other sequence coding for a protein of interest, or fragment thereof, is located 3′. Preferably, the sequences coding for the two or more proteins of interest, or fragments thereof, are located 5′ from the io sequence coding for the fragment of the lamprey VLR-B antibody according to the present invention.
The molecule or the recombinant protein of the invention may be synthesized by any method well-known to the skilled person. Such methods include conventional chemical synthesis, in solid phase (R. B. Merrifield, J. Am. Chem. Soc., 85 (14), 2149-2154 (1963)), or in liquid phase, enzymatic synthesis (K. Morihara, Trends in Biotechnology, 5(6), 164-170 (1987)) from constitutive amino acids or derivatives thereof, cell-free protein synthesis (Katzen et al., Trends in Biotechnology, 23(3), 150-156 (2005)), as well as biological production methods by recombinant technology.
Any method known to the skilled person may be used for the chemical conjugation between the first amino acid sequence and the second amino acid sequence. Such methods include conventional chemical conjugation via a peptide bond (e.g. expression of the first and second amino acid sequences as a fusion protein from a recombinant nucleic acid), optionally with a peptide linker, or conjugation via any covalent link, e.g. a peptide bond, an ester linkage, an amide linkage or a disulfide bond. Preferably the first and second amino acid sequences are expressed together as a fusion protein.
Chemical synthesis of the molecule or recombinant protein of the invention can be particularly advantageous because it allows high purity, the absence of undesired by-products and ease of production.
The molecule or protein of the invention obtained by such methods can then optionally be purified using any method known to the skilled person.
Preferably, the recombinant protein of the invention is obtained using a biological production process with a recombinant host cell. In such a process, an expression cassette, containing a nucleic acid encoding the protein or fusion protein of the invention, is transferred into a host cell, which is cultured in conditions enabling expression of the corresponding protein or fusion protein. The protein or fusion protein thereby produced can then be recovered and purified.
The present invention is also directed to an expression cassette comprising a recombinant nucleic acid of the invention, wherein the recombinant nucleic acid is operably linked to a promoter. A number of expression cassettes have been described in the art, each of which typically comprises all of the elements which allow the transcription of a DNA or DNA fragment into mRNA and the translation of the latter into protein, inside a host cell. Typically, the elements necessary for the expression of a nucleic acid in a host cell include a promoter that is functional in the selected host cell and which can be constitutive or inducible; a ribosome binding site; a start codon (ATG); a region encoding a signal peptide, necessary for the recombinant protein to be secreted; a stop codon; and a 3′ terminal region (translation and/or transcription terminator). Other transcription control elements, such as enhancers, operators, and repressors can be also operatively associated with the polynucleotide to direct transcription and/or translation in the cell. The signal peptide-encoding region is preferably adjacent to the nucleic acid coding for the recombinant protein of the invention and placed in proper reading frame. The signal peptide-encoding region can be homologous or heterologous to the DNA molecule encoding the protein of interest or fusion protein of the invention and can be specific to the secretion apparatus of the host used for expression.
The open reading frame constituted by the recombinant nucleic acid of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host cell. Promoters and other elements necessary for the expression of a nucleic acid in a host cell are widely known and available to those skilled in the art.
Lastly, the nucleic acid sequences of the present invention may be codon optimized such that the transcription of the DNA encoding the proteins and/or the fusion proteins of the invention is enhanced and/or the translation of the mRNA encoding the proteins and/or the fusion proteins is prolonged.
A “codon-optimized DNA or mRNA sequence” means a nucleic acid sequence that has been adapted for a better expression into the host cell, by replacing one or more codons with one or more codons that are more frequently used in the genes of said host cell as described in US 2004/0209241 in the case of codon-optimized DNA sequences or to maximize the G/C content of the mRNA sequence according to the host cell used as described in US 2011/0269950 in the case of codon-optimized mRNA sequences. The codon optimization of the nucleic acid sequences is properly managed such that it does not change the amino acid sequence of the proteins and/or the fusion proteins, which are expressed in the host cells.
In another embodiment a host cell is transformed with an expression cassette of the invention. A host cell can be any cell, i.e., any eukaryotic or prokaryotic cell, into which an expression cassette can be inserted. According to the present invention, preferred host cells are eukaryotic or prokaryotic cells, including, but not limited to, animal cells (e.g., mammalian, bird, insect and fish host cells), plant cells (including eukaryotic algal cells), fungal cells, yeast cells, bacterial cells, and protist cells. Preferred prokaryote host cells useful in the invention include Escherichia coli, bacteria of Bacillus genus, Lactococcus lactis, Pseudomonas fluorescens, bacteria of Caulobacter genus, Corynebacterium glutamicum and Ralstonia eutropha. A particularly preferred prokaryote host cell for use in the present invention is Escherichia coli. Preferred eukaryote host cells useful in the invention include Leishmania tarentolae, Tetrahymena thermophila, Willaertia magna, Vero cell, CHO cell, 293 cell, 293T cell, SF9 cell, S2 cell, EB66 duck cell, Pichia pastoris, S. cerevisiae, Hansenula polymorpha, Nicotiana benthamiana cell, Physcomitrella patens cell, Oryza sativa cell, Oryza glaberrima cell, Medicago truncatula cell, Zea mays cell, Schizochytrium sp., Phaeodactylum tricornutum and Myceliophthora thermophila. A particularly preferred eukaryote host cell for use in the present invention is Leishmania tarentolae or CHO.
As glycosylation in eukaryote cells is different from and more complex than glycosylation in prokaryote cells, a protein of interest which is naturally expressed in an eukaryote cell is preferably expressed, as a fusion protein with the fragment of the lamprey VLR-B antibody according to the present invention, in an eukaryote host cell. Similarly, a protein of interest which is naturally expressed in a prokaryote cell is preferably expressed, as a fusion protein with the fragment of the lamprey VLR-B antibody according to the present invention, in a prokaryote host cell.
There are a variety of means and protocols for inserting expression cassettes into host cells including, but not limited to, transformation, transfection, cell or protoplast fusion, use of a chemical treatment (e.g., polyethylene glycol treatment of protoplasts, calcium treatment, transfecting agents such as LIPOFECTIN™ and LIPOFECTAMINE™ transfection reagents available from Invitrogen (Carlsbad, Calif.)), use of various types of liposomes, use of a mechanical device (e.g., nucleic acid coated microbeads), use of electrical charge (e.g., electroporation), and combinations thereof. It is within the skill of a practitioner in the art to determine the particular protocol and/or means to use to insert a particular vector molecule described herein into a desired host cell.
Recombinant host cells may be grown under a variety of specified conditions as determined by the requirements of the cells. For example, a host cell may possess certain nutritional requirements or a particular resistance or sensitivity to physical (e.g. temperature) and/or chemical (e.g. antibiotic) conditions. In addition, specific culture conditions may be necessary to regulate the expression of a desired gene (e.g. the use of inducible promoters). These varied conditions and the requirements to satisfy such conditions are understood and appreciated by practitioners in the art.
Methods for the purification of proteins are well-known to the skilled person. The obtained recombinant protein or fusion protein can be purified from lysates and cell extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatographic methods, immunoaffinity methods using specific mono- or polyclonal antibodies, etc. Preferably the obtained recombinant protein or fusion protein is purified from the culture medium supernatant.
Another embodiment is directed to a molecule or a recombinant protein of the invention which is capable of forming a stable multimer. In a preferred embodiment, the stable multimer of the present invention is a stable homo-multimeric recombinant protein comprising a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a protein having an amino acid sequence which has at least 80% identity to SEQ ID NO: 1. In particular, the stable homo-multimeric recombinant protein comprises a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a protein having an amino acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 1. Preferably the protein is influenza HA protein.
According to another preferred embodiment, the stable multimer of the present invention is a stable homo-multimeric recombinant protein comprising a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a protein having an amino acid sequence which has at least 80% identity to SEQ ID NO: 2. In particular, the stable homo-multimeric recombinant protein comprises a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a protein having an amino acid sequence which has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 2. Preferably the protein is influenza HA protein.
In a preferred aspect of these embodiments of the invention (i.e. the stable multimers), the 7 cysteines which correspond to positions 2, 7, 13, 19, 21, 24 and 27 of SEQ ID NO: 1 (or the 8 cysteines which correspond to positions 2, 15, 20, 26, 32, 34, 37 and 40 of SEQ ID NO: 2) are conserved in the amino acid sequence of the protein which is derived from the C-terminus of a Lamprey VLR-B and which is fused to a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein. In some embodiments a linker may be inserted between the amino acid sequence of the protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein and the fused amino acid sequence.
Preferably, the stable multimers of the invention do not comprise a leucine-rich repeat (LRR) module from a lamprey VLR-B antibody. In particular, a stable multimer as described herein does not comprise an amino acid sequence having the sequence of SEQ ID NO: 29. Preferably, a stable multimer of the invention does not comprise one or more of an LRRNT module, an LRR1 module, an LRRV module, an LRRCT module, a CP and a Stalk region from a lamprey VLR-B antibody. Preferably, the only lamprey-derived amino acid sequence which is present within a stable multimer of the present invention is derived from the extreme C-terminus of a lamprey VLR-B antibody (i.e. the section of the protein C-terminal to the Stalk region, see FIG. 11C of WO 2008/016854). Preferably, the only lamprey-derived amino acid sequence which is present in a stable multimer of the present invention is a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 2, for example at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2.
The invention also provides a stable homo-multimeric recombinant protein produced by an expression system from a nucleic acid molecule comprising a nucleic acid sequence encoding a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3. In particular, the stable homo-multimeric recombinant protein is produced by an expression system from a nucleic acid molecule comprising a nucleic acid sequence encoding a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a nucleic acid sequence having has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 3. Preferably the nucleic acid sequence encodes an influenza HA protein.
In some embodiments, the stable homo-multimeric recombinant protein is produced by an expression system from a nucleic acid molecule comprising a nucleic acid sequence encoding a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a nucleic acid sequence with at least 80% identity to SEQ ID NO: 4. In particular, the stable homo-multimeric recombinant protein is produced by an expression system from a nucleic acid molecule comprising a nucleic acid sequence encoding a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein fused to a nucleic acid sequence having has at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity or even 100% identity to SEQ ID NO: 4. Preferably the nucleic acid sequence encodes an influenza HA protein.
In a preferred aspect of these embodiments of the invention, the nucleic acid sequence which encodes the amino acid sequence derived from the C-terminus of a Lamprey VLR-B antibody (and which is fused to a nucleic acid sequence coding for a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein) encodes an amino acid sequence which comprises cysteine residues at positions within said amino acid sequence that correspond to positions 2, 7, 13, 19, 21, 24 and 27 of SEQ ID NO: 1 (or comprises cysteine residues at positions within said amino acid sequence that correspond to positions 2, 15, 20, 26, 32, 34, 37 and 40 of SEQ ID NO: 2). In some embodiments a spacer nucleic acid sequence coding for a peptide linker may be inserted between nucleic acid sequence coding for a protein selected from the group consisting of the ectodomain of an influenza HA protein, a Shigella IpaD protein and a Shigella MxiH protein and the fused nucleic acid sequence.
The invention also provides a pharmaceutical composition comprising a molecule or a recombinant protein of the invention and a pharmaceutically acceptable carrier or diluent. In a preferred embodiment, an immunogenic composition comprises a molecule or a recombinant protein of the invention. The molecule or the recombinant protein of the invention may also be for use as a medicament. In a preferred embodiment the molecule or the recombinant protein of the invention is for use in inducing an immune response to an antigen in a subject. In another preferred embodiment, a molecule or a recombinant protein, comprising an influenza antigen according to the invention, is for use in inducing an immune response against influenza virus. In a more preferred embodiment, the recombinant influenza HA protein according to the invention is for use in inducing an immune response against influenza virus. In another preferred embodiment, the immunogenic composition of the invention is a vaccine composition.
The pharmaceutical composition and the immunogenic composition of the invention may be formulated as conventional pharmaceutical or vaccine preparations. This can be done using standard pharmaceutical or vaccine formulation chemistries and methodologies, which are available to those skilled in the art. Any solvent, dispersing medium, charge, adjuvant, etc., commonly used in the formulation of pharmaceuticals and vaccines to enhance stability, sterility, potency or deliverability of the active agent, which does not produce any secondary reaction, for example an allergic reaction, especially in humans, may be used. The excipient is selected on the basis of the pharmaceutical or vaccine form chosen, the method and the route of administration. Appropriate excipients, and requirements in relation to pharmaceutical formulation, are described in “Remington's Pharmaceutical Sciences” (19th Edition, A. R. Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1995)), which represents a reference work in the field. Examples of pharmaceutically acceptable excipients are water, phosphate-buffered saline solutions and 0.3% glycine solution.
The pharmaceutical compositions and the immunogenic compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged and stored in liquid form or lyophilized, the lyophilized preparation being reconstituted with a sterile aqueous carrier prior to administration. In a preferred embodiment the pharmaceutical compositions and the immunogenic compositions are packaged and stored as micropellets via a prilling process as described in WO2009109550. The pH of the preparations typically will be between 3 and 11, e.g., between 5 and 9, 6 and 8, or 7 and 8, such as 7 to 7.5.
Once formulated or reconstituted, the pharmaceutical compositions and the immunogenic compositions can be delivered to a subject in vivo using a variety of known routes and techniques. For example, the liquid preparations can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, intradermal, intramuscular, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system. Liquid preparations can also be administered topically to skin or mucosal tissue, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques.
For oral administration, the pharmaceutical compositions and the immunogenic compositions may be formulated as, for example, a capsule, a tablet, a suspension, or a liquid.
The pharmaceutical compositions and the immunogenic compositions may also be prepared in a solid form (including granules, micropellets, powders or suppositories).
Another embodiment is directed to method for treating a patient, said method comprising administering to said patient a pharmaceutical composition of the invention. A preferred embodiment contemplates a method for inducing an immune response to an antigen in a patient, said method comprising administering to said patient an immunogenic composition or a vaccine composition, of the invention.
Another embodiment is directed to a method for multimerizing a recombinant protein comprising:
These conditions are known by the skilled person and essentially consist of avoiding extreme conditions, e.g. high concentration of solutes, extremes of pH, mechanical forces and the presence of chemical denaturants.
Another embodiment is directed to a method for multimerizing a recombinant protein comprising:
In a preferred embodiment the method is for multimerizing an antigen, an antibody or a scaffold. In a most preferred embodiment the method is for multimerizing a recombinant influenza HA or HA ectodomain protein.
Two sequences derived from the C-terminus of VLR-B antibodies of lamprey were evaluated through fusion to the C-terminus of the HA protein. The first tested sequence was SEQ ID NO: 1 and the second tested sequence was SEQ ID NO: 2. SEQ ID NO: 1 is a shortened version of SEQ ID NO: 2. SEQ ID NO: 1 corresponds to the 30 amino acids at the extreme C-terminus of VLR-B antibodies of Lamprey and SEQ ID NO: 2 corresponds to the 43 amino acids at the extreme C-terminus of VLR-B antibodies of Lamprey (see FIG. 11C of WO 2008/016,854). By extreme C-terminus it is meant the portion of the VLR-B C-terminal to the Stalk region.
A third sequence tested was the foldon sequence of the T4 phage (SEQ ID NO: 5).
The nucleic acid sequence coding for the HA ectodomain from influenza strain A/California/07/09 (H1N1), (which comprised its own signal sequence, but which did not comprise the sequences of the transmembrane and cytoplasmic tail regions of HA), was optimized for codon usage in Leishmania tarentolae by Geneart (Regensburg, Germany). This sequence is referred to herein as SEQ ID NO: 10.
The nucleic acid sequences coding for the three tested multimerization sequences (i.e. the two sequences derived from the C-terminus of the VLR-B antibody and the T4 phage foldon sequence) were individually fused to the nucleic acid sequence SEQ ID NO: 10 (which encodes the ectodomain of the HA protein from influenza strain A/California/07/2009) by Geneart (Regensburg, Germany). Accordingly, SEQ ID NO: 7 is the nucleic acid sequence SEQ ID NO: 3 (which is the nucleic acid sequence encoding the amino acid sequence SEQ ID NO: 1, i.e. the shortened fragment of the lamprey VLR-B antibody according to the present invention) fused to the nucleic acid sequence SEQ ID NO: 10. SEQ ID NO: 8 is the nucleic acid sequence SEQ ID NO: 4 (which is the nucleic acid sequence encoding the amino acid sequence SEQ ID NO: 2, i.e. the “long” (not shortened) fragment of the lamprey VLR-B antibody according to the present invention) fused to the nucleic acid sequence SEQ ID NO: 10 and SEQ ID NO: 9 is the nucleic acid sequence SEQ ID NO: 6 (which is the nucleic acid sequence encoding the amino acid sequence SEQ ID NO: 5, i.e. the foldon sequence of the T4 phage) fused to the nucleic acid sequence SEQ ID NO: 10.
SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 were each separately inserted into the SalI/NotI restriction site of the pLexsy-I-bleo2 expression cassette as shown in
The expression cassettes containing the HA sequence with or without one of the polymerization sequences were then digested by SwaI, and 1 μg of each purified linear SwaI fragment was, in separate experiments, transfected into the L. tarentolae T7-TR host strain via nucleoporation using the Nucleofector II device (Amaxa Biosystems, Cologne, Germany) and following the instructions of the Basic Parasite Nucleofector™ Kit 1 (Lonza, Bale, Switzerland). The transfected cells were transferred into 10 ml of BHI (Brain-Heart Infusion) medium (Jena Bioscience) containing 5 μg/ml Hemin, 50 units/ml penicillin, 50 μg/ml streptomycin (Pen/Strep to avoid bacterial contamination), 100 μg/ml nourseothricin (NTC) and 100 μg/ml hygromycin (NTC/Hygro: for maintaining T7 polymerase and TET repressor genes respectively in the T7-TR host) and incubated overnight at 26° C. in the dark. Twenty-four hours post transfection, a 2 ml aliquot of the suspension was centrifuged for 5 min at 2000 g, the pellet was resuspended in 50-100 μl of BHI medium and the cells were gently plated on fresh BHI-agar plates containing antibiotics plus 100 μg/ml of bleomycin (selective growth medium) for the selection of recombinant parasites. Approximately 7-9 days after plating, small colonies were visible and transferred to 0.2 ml of selective growth medium. Each recombinant clone of parasites was expanded into 10 ml of selective medium in a shake flask at 26° C.
Confirmation of the integration of the expression cassette containing HA sequences into the genome was performed by diagnostic PCR following the Jena Bioscience recommendation.
The confirmed recombinant parasites were cultivated in 100 ml BHI medium supplemented as described above with Hemin and antibiotics at 26° C., and agitated at 100 rpm in the dark. In order to induce the production of the rHA protein, the T7 driven transcription was induced by addition of 10 μg/ml of Tetracycline into the supplemented medium at the time of inoculation of the parasites.
For fermentation, 1 liter Biostat Qplus 12 fermenters (Sartorius AG, Aubagne, France), were used. Briefly 700 ml of supplemented BHI medium was inoculated with 1/10 of a recombinant parasite starter culture in exponential growth (0.4 OD600) and cultivated in the dark at 26° C., 100 rpm, 40% pO2, pH 7.4±0.1. Culture parameters were recorded using the MFCS/WIN software (Sartorius AG). Induction using 10 μg/ml of Tetracycline was performed in parallel with inoculation of the recombinant parasites (as was done for the shake flask cultures). Regulation of the pH with HCl 1N/NaOH 1N, and infusion of a 100 g/L solution of glucose at 1.5 ml/h began 43 h after induction while P1860 anti protease cocktail (1/800, Sigma, Saint Quentin Fallavier, France) was added at the same time.
Samples of the culture were taken every day in order to determine the optical density (OD600) of the culture (one OD600 is equivalent to approximately 1.5×107 parasites/ml), the concentration of various metabolites (Gln, Glu, Gluc, Lac, NH4+), and the cell mobility by microscopy.
After 48 h, the supernatants of the transformed Leishmania tarentolae cultures were collected and filtered on a 0.2 μm filter. Proteins were quantified in the samples by optical density measurement at 595 nm and samples were normalized.
20 μl of each sample was loaded and run on a SDS-PAGE gel (NuPAGE® Novex Bis Tris 4-12%, Life Technologies, Carlsbad, USA). The supernatant from a transformed Leishmania tarentolae culture cultivated over 48 h in the absence of the transcription inductor tetracycline served as a negative control.
To test the thermal stability of the different recombinant HA proteins obtained using the different expression plasmids, the three test samples and the negative control sample were divided in two, with one half of the sample being heated to 99° C. for 15 minutes using a heating block before migration on the SDS-PAGE gel, and the other half not being heated before migration on the SDS-PAGE gel. A further control sample on the gel contained a heated culture supernatant of Leishmania tarentolae (15 minutes at a temperature of 99° C.) transformed with a plasmid expressing another protein (i.e. an antibody against influenza).
A Western Blot of the SDS-PAGE gel was made using a nitrocellulose membrane (BioRad Laboratories, Hercules, USA), followed by a treatment with PBS, Tween 20 0.1% and milk 5% (DIFCO-BD, Sparks, USA) in order to block non-specific fixation sites.
The blot was probed using a rabbit polyclonal antibody against influenza A/California/07/09 HA, with a titer of 8000 (inhibition of haemagglutination) and a titer of 32 000 (seroneutralization), followed by an anti-rabbit IRDdye800CW antibody (Li-Cor BioSciences, Lincoln, USA) and the OPTI-4CN™ (BioRad Laboratories) substrate. The Western Blot was analyzed with an ODYSSEY (Li-Cor BioSciences) imaging system.
The results of the Western Blot are shown in
Recombinant HA ectodomain protein polymerized by fusion to the lamprey VLR-B antibody C-term domain SEQ ID NO: 2 (rHA poly) was produced as described in example 1.
After 72 h of induction with tetracycline in the medium of the L. tarentolae culture, shake flask harvests were performed and centrifuged for 30 min at 5,000 g. After concentration and diafiltration on a Sartorius sartocon slice 200 cassette, supernatants were placed on a Con A Sepharose 4B column of 1 ml. The recombinant HA was eluted using a 0.5M alpha-D-Methylmannoside in PBS-MM buffer. The eluate was dialysed against PBS/tween, concentrated on Ultracell 10K and filtered with a 0.22 μm filter. The recombinant HA was titrated by the microbradford technique. Each sample was resuspended in PBS+Tween 0.005%.
Two groups of 10 female Balb/C ByJ mice aged 8 weeks received two immunizations, one on day 0 and one on day 28, via the intramuscular (IM) route, of either 10 μg of influenza A/California/07/2009 rHA ectodomain protein polymerized by fusion to the lamprey VLR-B antibody C-term domain SEQ ID NO: 2 (rHA poly) (produced as described in example 1), or 10 μg of influenza A/California/07/2009 rHA ectodomain monomeric protein (rHA mono) produced in Leishmania tarentolae transformed with a plasmid expressing only the rHA ectodomain, i.e. not fused to a polymerization sequence (SEQ ID NO: 11). The 10 μg rHA proteins were resuspended in a Buffer (PBS+Tween 0.005%) and the volume injected was 2×50 μl (100 μl in total).
Finally, 5 female Balb/C ByJ mice aged 8 weeks received 100 μl of Buffer (2×50 μl).
Three weeks after the booster injection, blood samples were taken under anesthesia at D49 from all the animals. The anesthesia was performed by Imalgene® (1.6 mg of Ketamine) and Rompun (0.32 mg of Xylazine) administered in a volume of 200 μl via the intraperitoneal route. 1 ml of blood was collected in vials containing clot activator and serum separator (BD Vacutainer SST ref 367783). After a single night at +4° C. or one hour at 37° C., the blood was centrifuged at 10,000 rpm for 5 minutes or 3,000 rpm for 20 minutes and the serum was stored at −20° C. until analysis.
The presence of haemagglutination inhibitory antibodies against the influenza A/California/07/09 (H1N1) strain was assessed using chicken red blood cells (cRBCs). Assays were performed on individual Receptor Destroying Enzyme (RDE) treated serum samples and titers were expressed as the reciprocal of the highest dilution showing no haemagglutination, as described by Kendal et al., Haemagglutination inhibition, in Concepts and procedures for laboratory-based influenza surveillance, US Department of Health and Human Services and Pan-American Health Organization, Atlanta, Ga., 1982, pp. B17-B35.9.
The results of the inhibition of haemagglutination assay are shown in
The polymerization of recombinant influenza HA ectodomain protein via fusion with the lamprey sequences was also tested in another host cell.
The nucleic acid sequence coding for the HA ectodomain from influenza strain A/California/04/09 (H1N1) (Genbank Accession Number FJ966082), which comprised its own signal sequence, but which did not comprise the sequences of the transmembrane and cytoplasmic tail regions of HA, was optimized for codon usage in CHO by Geneart (Regensburg, Germany). This sequence is referred to herein as SEQ ID NO: 12.
The nucleic acid sequences coding for the three tested multimerization sequences (i.e. the two sequences derived from the C-term of the VLR-B antibody and the T4 phage foldon sequence), optimized for codon usage in CHO, were individually fused to the nucleic acid sequence SEQ ID NO: 12. Accordingly, SEQ ID NO: 13 is the nucleic acid sequence SEQ ID NO: 3 fused to the nucleic acid sequence SEQ ID NO: 12. SEQ ID NO: 14 is the nucleic acid sequence SEQ ID NO: 4 fused to the nucleic acid sequence SEQ ID NO: 12 and SEQ ID NO: 15 is the nucleic acid sequence SEQ ID NO: 6 fused to the nucleic acid sequence SEQ ID NO: 12. SEQ ID NO: 26 is the protein sequence encoded by SEQ ID NO: 13. SEQ ID NO: 27 is the protein sequence encoded by SEQ ID NO: 14. SEQ ID NO: 28 is the protein sequence encoded by SEQ ID NO: 15.
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 were each separately inserted into the HindIII/EcoRI restriction site of the pEE14.4 expression cassette shown in
The expression cassettes containing the HA sequence with or without one of the polymerization sequences were transfected into a CHO host cell (CHOK169 ATCC Number CB-CCL-61pUnK). 10 μg of each plasmid was separately introduced into 10×106 CHO cells via nucleoporation using the Nucleofector II device (Amaxa Biosystems, Cologne, Germany). The CHO cells were then plated on 2 ml of Ex-Cell® CHO fusion animal component free medium (SAFC Biosciences Sigma-Aldrich) containing 4 mM of L-glutamine at 37° C. The cultures were statically maintained at 37° C. under 5% CO2 for 24 h and then with agitation (100 rpm) for 48 h.
72 h after nucleoporation, the supernatants of the transformed CHO cultures were collected by centrifugation for 10 seconds at 10,000 rpm.
15 μl of each sample mixed with 5 μl NuPAGE® LDS Sample Buffer (4×) (Life Technologies) was loaded and run on a SDS-PAGE gel (NuPAGE® Novex 3-8% Tris-Acetate, Life Technologies, Carlsbad, USA). The supernatant from a CHO culture that was electroporated in the absence of any expression cassette served as a negative control. 20 μl of HiMark™ Pres stained High molecular Weight Protein Standard (LC5699 Life technlologies) was used as a molecular weight marker.
Sample separation was performed at 150V in Tris-acetate Buffer for 40 minutes (Life Technologies).
A Western Blot of the SDS-PAGE gel was made using a nitrocellulose membrane (BioRad Laboratories, Hercules, USA), followed by an overnight treatment with PBS and milk 5% (DIFCO-BD, Sparks, USA) in order to block non-specific fixation sites.
The blot was probed using a rabbit polyclonal antibody against influenza A/California HA diluted at 1/1000 in PBS, for 1 h at room temperature. The blot was then washed three times with PBS and Tween 20 0.05% before incubation with an anti-rabbit IRDdye800 sheep antibody (Rockland, Limerick, USA) diluted at 1/5000 in PBS. The Western Blot was analyzed with an ODYSSEY (Li-Cor BioSciences) imaging system.
The results of the Western Blot are shown in
The nucleic acid sequence coding for the IpaD protein from Shigella flexneri Serotype 2a Strain 301 (Q. Jin et al., Nucleic Acids Research, 30 (20), 4432-4441 (2002), Genbank Accession Number AF386526), was optimized for codon usage in E. coli by Geneart (Regensburg, Germany). This sequence is referred to herein as SEQ ID NO: 16.
SEQ ID NO: 16 was fused to the nucleic acid sequence SEQ ID NO: 4 also codon optimized for E. coli by Geneart (Regensburg, Germany) to generate SEQ ID NO: 17. The corresponding protein sequence is SEQ ID NO: 18. SEQ ID NO: 16 and SEQ ID NO: 17 were also fused to a sequence coding for a polyhistidine-tag (6×His) via a GGSLE linker, thus generating SEQ ID NO: 19 (IpaD-His, the GGSLE linker is between the IpaD sequence and the His-tag) and SEQ ID NO: 20 (IpaD-lamprey-His, the GGSLE linker is between the IpaD-lamprey sequence and the His-tag) respectively.
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20 were each separately inserted into the NcoI/XhoI restriction site of the pM1800 expression cassette as shown in
5 μg of the plasm ids containing the IpaD sequence with or without the polymerization sequence and with or without the linker and His-tag sequence were suspended in 10 μl of water. 0.5 μl of the suspension corresponding to the IpaD sequence with or without the polymerization sequence and without the linker and His-tag sequence was added to cultures of either E. coli BL21 DE3 C6000-03 (Life Technologies) or E. coli Shuffle (B) ref C3029H (New England Biolabs, i.e. E. coli engineered to promote the formation of disulfide bonds within proteins). 0.5 μl of the suspensions corresponding to the IpaD sequence with or without the polymerization sequence but with the linker and His-tag sequence were added to E. coli Shuffle (B). After mixing, the samples were placed on ice for 15 minutes. Then the samples were heat shocked at 42° C. for 30 seconds. The samples were then placed on ice for 2 minutes before dilution with 500 μl of room temperature S.O.C. Medium (Thermofisher). The samples were then incubated at 37° C. for 60 minutes before vigorous shaking (250 rpm).
100 μl of each sample was diluted and spread onto a LB medium containing Kanamycin (25 μg/ml) plate and incubated overnight at 37° C. A colony from each transformation plate was picked using a sterile inoculation loop and added to 2 ml LB broth/kanamycin 25 μg/ml. The cultures were then diluted in 25 ml of LB+Kanamycin (25 μg/ml) medium in order to obtain an optical density for seeding of OD600=0.05.
After 2 h of growth at 37° C. with agitation (200 rpm), when the cultures reached a OD600 of 0.4-0.8, the production of the recombinant protein was induced by IPTG 1 mM (i.e. addition of 25 μl of IPTG 1M).
The bacteria were maintained at 37° C. for about 4 h with agitation. One OD600 unit is taken from each Erlen flask and centrifuged. After removal of the supernatants, the pellet was stored at −20° C.
The pellets were resuspended in 75 μl of Tris EDTA (10 mM Tris, 1 mM EDTA, pH 8.0, Novagen)+1 μl of Ready lyse 35 KU/μl (Epicentre) diluted at 1/50+1 μl of Benzonase 25 U/μl (Novagen). The samples were then agitated for 20 minutes at 37° C. before adding 25 μl of NuPAGE® LDS Sample Buffer (4×) (Invitrogen). 20 μl of each sample was loaded and run on SDS-PAGE gels (NuPAGE® Novex® 3-8% Tris-Acetate, Life Technologies, Carlsbad, USA). 15 μl of HiMark™ Pres stained High molecular Weight Protein Standard (LC5699 Life technlologies) was used as a molecular weight marker.
pM1800 containing no IpaD sequence, inserted in the E. coli induced by IPTG, served as a negative control. Sample separation was performed at 150V in Tris-acetate Buffer for 1 hour (Life Technologies).
Western Blots of the SDS-PAGE gels were made using nitrocellulose membranes (BioRad Laboratories, Hercules, USA), followed by a treatment for 1 h with PBS and milk 5% (DIFCO-BD, Sparks, USA).
The blots were probed using a mouse monoclonal antibody against IpaD, followed by an Alexa fluor Goat anti-mouse antibody (Invitrogen) or an anti-mouse IRDye 800 antibody (Rockland) diluted at 1/5000 in PBS. The Western Blots were analyzed with an ODYSSEY (Li-Cor BioSciences) imaging system.
The results of the Western Blots are shown in
The results in
To test the thermal stability of the different recombinant IpaD proteins obtained, a further SDS-PAGE and Western Blot was conducted as described above, except that the test samples and the negative control sample were heated to 95° C. for 10 minutes using a heating block before migration on the SDS-PAGE gel.
The results of this Western Blot are shown in
The thermal stability of the polymers obtained from the IpaD protein fused to the lamprey VLR-B antibody C-terminal domain SEQ ID NO: 2 is of great interest, since increased stability should increase the shelf-life of an immunogenic composition containing such an antigen. Furthermore, a thermostable recombinant protein antigen is also expected to have a longer in vivo stability when injected into a patient.
The nucleic acid sequence coding for the MxiH protein from Shigella flexneri Serotype 2a Strain 301 was optimized for codon usage in E. coli by Geneart. This sequence is referred to herein as SEQ ID NO: 21.
SEQ ID NO: 21 was fused to the nucleic acid sequence SEQ ID NO: 4 also codon optimized for E. coli by Geneart to generate SEQ ID NO: 22. The corresponding protein sequence is SEQ ID NO: 23. SEQ ID NO: 21 and SEQ ID NO: 22 were also fused to a sequence coding for a polyhistidine-tag (6× His) via a GGSLE linker, thus generating SEQ ID NO: 24 (MxiH-His, the GGSLE linker is between the MxiH sequence and the His-tag) and SEQ ID NO: 25 (MxiH-lamprey-His, the GGSLE linker is between the MxiH-lamprey sequence and the His-tag) respectively.
SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 25 were each separately inserted into the NcoI/XhoI restriction site of the pM1800 expression cassette. Induction of the expression of the protein of interest is carried out via addition of IPTG
5 μg of the plasm ids containing the MxiH sequence with or without the polymerization sequence and with or without the linker and His-tag sequence were suspended in 10 μl of water. 0.5 μl of each suspension was added to either E. coli BL21 DE3 C6000-03 or E. coli Shuffle (B) ref C3029H and the bacteria were heat shocked as explained in example 4.
The samples were then cultured on LB medium, induced with IPTG, centrifuged and the cell pellets stored at −20° C. as described in Example 4.
The pellets were resuspended in 63 μl of Tris EDTA (10 mM Tris, 1 mM EDTA, pH 8.0, Novagen)+1 μl of Ready lyse 20 KU/μl (Epicentre) diluted at 1/20+1 μl of Benzonase 25 U/μl (Novagen). The samples were then agitated for 10 minutes at 37° C. before centrifugation at 13,000 rpm for 10 minutes.
60 μl of the supernatant was mixed with 20 μl of NuPAGE® LDS Sample Buffer (4×) (Invitrogen), while the pellet was suspended in 60 μl of Tris EDTA and 20 μl of NuPAGE® LDS Sample Buffer (4×) (Invitrogen).
15 μl of each sample was loaded and run on an SDS-PAGE gel (NuPAGE® 4-12% Bis-Tris gel, Life Technologies, Carlsbad, USA). 15 μl of SeeBlue® Plus2 Pre-Stained Standard (Life Technlologies) was used as a molecular weight marker.
pM1800 containing no MxiH sequence, inserted in IPTG-induced E. coli, served as a negative control. Sample separation was performed at 200V in MES buffer for 30 minutes (Life Technologies).
Western Blots of the SDS-PAGE gels were made as described in Example 4.
The blots were probed using a mouse polyclonal antibody against MxiH, diluted at 1/1000 in PBS, followed by Rabbit anti mouse IRDye 800 antibody (Rockland) diluted at 1/5000 in PBS. Another Western Blot was probed using a mouse monoclonal antibody against His (Sigma) diluted at 1/1000 in PBS, followed by Rabbit anti mouse IRDye 800 antibody (Rockland) diluted at 1/5000 in PBS. The blots were analyzed with an ODYSSEY (Li-Cor BioSciences) imaging system.
The results of the Western blots are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 14307096.9 | Dec 2014 | EP | regional |
This application is a Continuation of U.S. patent application Ser. No. 15/536,726, which was filed on Jun. 16, 2017, which is a 371 application of International Application No. PCT/EP2015/080653, which was filed on Dec. 18, 2015, and claims priority to European Patent Application No. EP 14307096.9, which was filed on Dec. 19, 2014, all of which are incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15536726 | Jun 2017 | US |
| Child | 16143079 | US |
