Patent Application
20230101439
The present invention relates to an engineered polypeptide derived from adenovirus pentane base protein. The polypeptide of the invention is based on the “upper” alpha-helical domain of the adenovirus pentane base as shown in the pentane base atomic structure, but it lacks essentially completely any amino acids of the beta-barrel sheet domain showing a jellyroll fold structure (the jellyroll fold domain). The polypeptide contains at least the large fragment (also referred to herein as the “big fragment”) of said alpha-helical domain of the pentane base, which fragment includes the RGD loop(s) and the VLP loop, and may contain also the second, short fragment of the alpha-helical domain of the adenovirus pentane base. The polypeptide of the invention provides a new scaffold for optimized presentation of peptidic entities such as oligopeptides, polypeptide sequences, protein domains, proteins and protein complexes made up of two, several or many subunits, preferably as high affinity agents to target molecules.
This application contains a Sequence Listing. The application thus incorporates by reference the material in the ASCII text file 34U6691.txt, created on Aug. 1, 2022, and having a size of 125,422 bytes.
A prerequisite for successful protein scaffold design for presentation of peptidic entities such as oligopeptides, polypeptide sequences, protein domains, proteins and protein complexes, is a compact, stable protein domain which can accommodate modalities representing exposed and flexible loop structures that can accommodate said entities. In a preferred embodiment of the invention these displayed entities can represent any peptidic sequence which may be recognized by any binding partner for peptidic structures, e.g. a binder sequence and/or paratope sequence and/or sequences, for example, from a randomized library that can be presented to any chemical or biochemical, respectively, structure capable to be recognized by said binder sequence (such as those exemplified above), e.g. an antigen and/or a toxin and/or a venom and/or a chemical (
Penton base proteins (protomers) from a number of Adenovirus (Ad) serotypes contain highly variable loop regions which can be functionalized for inserting foreign sequences encoding for oligopeptides, polypeptide sequences, protein domains, proteins and protein complexes as disclosed in WO2017/167988 A1. Adenovirus is one of the most commonly used gene therapy vector in humans. The adenovirus shell is predominantly built by two distinct proteins: the hexon protein, and the penton base protein, with the latter forming pentameric assemblies to which attaches the fiber protein characteristic for this virus. Penton base proteins of certain adenovirus serotypes were shown to spontaneously self-assemble into a multimeric superstructure when expressed recombinantly in absence of other adenoviral components. This superstructure represents a dodecahedron, formed by a total of 60 adenovirus base proteins arranged in twelve identical copies of the pentamer.
The technical problem underlying the invention is the provision of protein scaffolds for presentation of peptidic binding partners for target molecules.
The solution to the above technical problem is provided by the embodiments of the present invention as defined and disclosed in the claims, the present description and the accompanying drawings.
A close inspection of the high-resolution structure of the penton base protein (PDB ID 6HCR) evidenced that the penton base protein itself adopts a distinct two-domain architecture with one domain representing a beta-barrel jellyroll fold conjoined to a second domain stabilized by alpha-helices which in the present invention is referred to the “crown domain” (
Co-pending International Patent Application PCT/EP2019/070722 describes multimerizing polypeptides derived from the jellyroll fold domain of the penton base protein. While discovering that the penton base protein of adenovirus could be split into two domains, the inventor recognized that the alpha-helical crown domain itself is of great interest for adopting various non-adenoviral sequences as disclosed in WO 2017/167988 A1, and could be produced on its own.
The present invention provides engineered polypeptides consisting of or derived from, respectively, the adenovirus base-protein head domain (i.e. the penton base protein minus the multimerization domain), or specific fragments thereof, as an autonomous scaffold to form a separate, stable, highly versatile protein entity on its own. Because the highly variable loops in the crown domain are reminiscent of antibody complementarity determining regions (CDRs), the crown domain polypeptides according to the invention are also referred to as the “ADDobody” hereafter. The ADDobody polypeptide of the invention is capable of displaying multiple copies of any peptidic structure, in particular peptides, oligopeptides, protein domains, proteins or protein complexes. The ADDobody contains the large and small fragments of an adenovirus penton base alpha-helical domain. The invention also is directed to minimal ADDobodies (or miniADDobodies) containing only the large fragment of the alpha-helical domain.
These oligopeptides, polypeptide sequences, protein domains and proteins can include: (i) naturally occurring binder sequences or paratopes, (ii) binder sequences or paratopes obtained from random library and selection evolution (phage/ribosome display), (iii) antigenic entities that stimulate the immune system to trigger an immune response, for example for vaccination purposes, or for preparing antibodies or other binder molecules in cell culture, or in vitro in a test tube. Ideally such protein presenting such entities will be safe, non-immunogenic, efficient, and tunable. Moreover, they will be produced easily at industrial scale. In certain embodiments the polypeptide contains insertion sites which are within the VL-loop (also called V loop) and/or RGD loops as disclosed in WO 2017/167988 A1. According to the present invention, two more sites of flexibility for heterologous modification of the naturally sequences of existing adenovirus penton base proteins are disclosed which further adds to, e.g. flexible modification of the crown domain and the adenovirus penton base for including a multitude of possible heterologous peptidic structures.
In addition, the polypeptide of the invention can be engineered as a multivalent Virus Like Particle (VLP). The present disclosure describes the creation, design and production of the engineered polypeptide and its embodiment as a novel type of protein for presenting peptidic structures for presentation to target binding partners.
More particular, the present invention provides the following embodiments:
The invention provides an isolated engineered polypeptide comprising the amino acid stretches essentially corresponding to a first and a second fragment of the penton base wherein the first fragment of the polypeptide is present between the first and second amino acid stretches forming the jellyroll fold domain in the full length penton base and wherein the second fragment of the polypeptide is present between the second and third fragments forming the jellyroll fold domain in the full length penton base, respectively, wherein the isolated engineered domain lacks the amino acid stretches forming the jellyroll fold domain of the adenovirus penton base, wherein optionally the first and/or second fragments of the polypeptide contain(s) one or more heterologous modification(s).
Preferably, there is provided a polypeptide having the structure of the following general formula (I):
N-A-L-B-C (I)
A preferred fragment or group, respectively, N comprises an amino acid sequence facilitating the purification of the polypeptide, e.g. a His tag. The same applies also to fragment or group, respectively, C.
More preferably, fragment A of the polypeptides as defined herein comprises an amino acid sequence selected from the group consisting of the amino acid sequences according to the following Table 1:
wherein, optionally, fragment A contains one or more heterologous modifications.
Preferably, fragment B of the polypeptides as defined herein comprises an amino acid sequence selected from the group consisting of the amino acid sequences according to the following Table 2:
wherein, optionally, fragment B contains one or more heterologous modifications.
In preferred embodiments of the polypeptides according to the invention fragment A and/or B contain(s) one or more heterologous modifications wherein said one or more heterologous modifications is/are contained in the following sites:
According to the invention, it is surprisingly found that the floor region (also denoted as “floor site”) and the B loop show a flexible conformation as evidenced by X-ray chrystallography of an exemplary ADDobody of the invention.
It is to be understood that, with respect to the floor region (also referred to as “floor site”) and the B loop, which are both (more particularly the B loop) considerably conserved sites amongst the adenovirus penton base sequences of various adenovirus species, the one or more heterologous modification includes any insertion, deletion, replacement at any and of any, respectively, position of the residues outlined above, whereby the insertion or replacement may comprise one or more or all of the respective amino acids of the floor region and the B loop, respectively.
As regards the B loop, a preferred heterologous modification is a replacement of amino acid residues 1 to 6 of SEQ ID NO: 22 as defined above, preferably by a heterologous oligonucleotide, polypeptide, protein or protein complex.
In preferred embodiments of the invention, the polypeptide comprises one or more heterologous modifications at least in the RGD loop (i.e. the RGD loop region as defined above), the V loop and the floor site, wherein in certain embodiments of this type, the one or more heterologous modifications are located only in said RDG loop region, said V loop and said floor site. In other embodiments, the polypeptide of the invention comprises one or more heterologous modifications at least in the RGD loop region and the V loop, wherein in certain embodiments of this type, the one or more heterologous modifications are located only in said RDG loop region and said V loop. In other embodiments of the invention the polypeptide comprises one or more heterologous modifications at least in the floor region and the B loop, wherein in certain embodiments of this type, the one or more heterologous modifications are located only in said floor site and said B loop. It is understood that the one or more modifications may present in any combination of the sites for heterologous modification of fragment A and/or fragment B as defined above, including one or more heterologous modification in all of the above-defined sites.
More preferably, the N-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal):
More preferably, the C-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal):
Referring to WO 2017/167988 A1, it is to be understood that, according to the invention, the RGD loop region as disclosed and defined herein can be sub-divided into a first and a second RDG loop, more particularly as defined on pages 31 to 33 of WO 2017/167988.
Preferably, the N-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal):
Preferably, the C-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal):
According to another aspect, the present invention is also directed to a further isolated engineered polypeptide comprising the large fragment of the alpha-helical domain of an adenovirus penton base protein which polypeptide lacks the small fragment of the alpha-helical domain and the jellyroll fold domain of the adenovirus penton base protein, wherein said large fragment optionally contains one or more heterologous modifications. This further engineered polypeptide is referred to herein as “minimal crown domain” or “minimal ADDobody” or “miniADDobody”.
The minimal crown domain (or minimal ADDobody) of the invention has preferably a general structure as defined according to following formula (II):
N-A-C (II)
wherein N, A and —C are as defined above (formula (I).
The present invention also relates to a nucleic acid encoding a minimal ADDobody of the invention. The present invention further provides a vector comprising said nucleic acid encoding a minimal ADDobody, preferably said nucleic acid is contained in an expression cassette. There is further provided a recombinant host cell containing said minimal ADDobody vector. The invention further provides a method for producing a minimal ADDobody of the invention comprising the step of culturing the vector containing the minimal ADDobody coding sequence in an expression cassette under conditions allowing the expression of the minimal ADDobody, and optionally purifying the minimal ADDobody from the host cells.
A “heterologous modification” as defined herein may be any modification of the respective site (RGD loop region, V loop, floor site, B loop) compared to the respective naturally occurring sequence, preferably as found in the adenovirus penton base proteins as further described in more detail below. Preferably, the heterologous modification is selected from the group consisting of one or more single amino acid mutations in comparison to the wildtype sequence of fragment A and/or B, one or more replacements of wildtype amino acid stretches by one or more heterologous amino acids and/or amino acid stretches one or more insertions of heterologous amino acid stretches, one or more deletions of one or more amino acids and one or more amino acid modifications as well as any combination(s) thereof. Generally preferred modifications according to the invention are insertions in and/or replacements of amino acid residues of the wildtype adenoviral sequences of the sites as defined herein by non-adenoviral amino acid sequences, preferably non-adenoviral oligonucleotides, polypeptides, proteins and/or protein complexes.
A point mutation (there can be one or more, preferably in the respective sites of fragments A and/or B as defined herein) may, for example, the replacement of an amino acid by a coupling residue, i.e. a naturally or non-naturally occurring amino acid having a side chain capable of forming a covalent bond with a binding partner, for example another coupling residue present either on another polypeptide to be coupled to the coupling residue or in fragment A and/or B of the present invention and/or in the linker L and/or in fragment N and/or in fragment C of the polypeptide according to the invention. The latter may serve for stabilizing the structure of the ADDobody or minimal ADDobody, respectively, or by stabilizing dimers, decamers, pentamers and/or dodecahedrons of the polypeptides of the invention (for example, via head-to-tail, head-to-head or head-to-tail arrangement). Preferred coupling residues are amino acids D, E, K and C, with C being particularly preferred, since it may readily form a disulfide bond with another C under the appropriate redox conditions known in the art.
According to preferred embodiments the heterologous modification provides a target specific binding entity.
In preferred embodiments of the invention the target specific binding entity is selected from the group consisting of antigens, epitopes, CDRs, antibodies, antibody fragments such as an antigen binding (Fab) fragment, a Fab′ fragment, a F(ab′)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, a paratope, an alternative scaffold protein, and a fusion protein thereof.
According to the invention, a paratope, also called an antigen-binding site of an antibody, is a part of an antibody which recognizes and binds to an antigen, more particularly an epitope of an antigen. It is typically a short amino acid stretch, usually of 5 to 10 amino acids which are part of the Fab region of an antibody.
A specific polypeptide according to the invention has the following amino acid sequence (from N-terminal to C-terminal):
A variant of the above polypeptide of the invention according to SEQ ID NO: 27 has the following amino acid sequence (from N- to C-terminal):
A further specific polypeptide according to the invention has the following amino acid sequence (from N-terminal to C-terminal):
A variant of the above polypeptide of the invention according to SEQ ID NO: 27 has the following amino acid sequence (from N- to C-terminal):
The invention is further directed to a nucleic acid encoding a polypeptide according to the invention.
There is also provided a vector comprising the nucleic acid of the invention (which is meant synonymous to a nucleotide sequence encoding a polypeptide of the invention). The vector may contain the nucleic acid (or the nucleotide sequence) within an expression cassette.
The invention also provides a recombinant host cell comprising the nucleic acid or the vector.
The invention furthermore is directed to a method for the production of a polypeptide according to the invention comprising the step of culturing the host cell containing the vector comprising the nucleic acid with an expression cassette under conditions allowing the expression of said polypeptide. The production method preferably comprises the step of purifying the polypeptide from the cultured host cells.
The invention also provides an engineered adenovirus penton base protein comprising a polypeptide of the invention (i.e. an ADDobody or a minimal ADDobody) comprising one or more heterologous modifications, preferably one or more heterologous modifications at least in the floor region and/or the B loop (with respect to engineered penton base proteins comprising an ADDobody) or preferably one or more heterologous modifications at least in the floor region (with respect to engineered penton base proteins comprising a minimal ADDobody), fused to the multimerization domain (jellyroll fold domain) of an adenovirus penton base protein. Preferably, a multimerization domain is selected from an adenovirus selected from of human adenovirus serotype 2 (hAd2), human adenovirus serotype 3 (hAd3), human adenovirus serotype 4 (hAd4), human adenovirus serotype 5 (hAd5), human adenovirus serotype 7 (hAd7), human adenovirus serotype 11 (hAd11), human adenovirus serotype 12 (hAd12), human adenovirus serotype 17 (hAd17), human adenovirus serotype 25 (hAd25), human adenovirus serotype 35 (hAd35), human adenovirus serotype 37 (hAd37), human adenovirus serotype 41 (hAd41), gorilla adenovirus (gorAd), chimpanzee adenovirus (ChimpAd), simian adenovirus serotype 18 (sAd18), simian adenovirus serotype 20 (sAd20), simian adenovirus serotype 49 (sAd49), rhesus adenovirus serotype 51 (rhAd51), rhesus adenovirus serotype 52 (rhAd52), and rhesus adenovirus serotype 53 (rhAd53).
Engineered penton base proteins of the invention comprising an ADDobody polypeptide of the invention have typically a structure according to the following formula (III) (from N- to C-terminal):
D-A-E-B-F (III)
wherein A and B are the fragments of the alpha-helical crown domain as defined above, and D, E and F are the amino acid sequences of an adenovirus penton base forming the multimerization (jellyroll fold) domain, wherein one or more heterologous modifications is/are present in the floor region of fragment A and/or in the B loop of fragment B.
Engineered penton base proteins of the invention comprising a minimal ADDobody polypeptide of the invention have typically a structure according to the following formula (IV) (from N- to C-terminal):
D-A-E-Li-F (IV)
wherein A is the large fragment of the alpha-helical crown domain as defined above and D, E and F are the amino acid sequences of an adenovirus penton base forming the multimerization (jellyroll fold) domain, wherein, optionally and preferably, one or more heterologous modifications is/are present in the floor region of fragment A, and wherein Li is a linker selected from peptides, oligopeptides, polypeptides, proteins and protein complexes. Preferred linkers as Li are selected from oligopeptide linkers such as oligopeptides having 4 to 10 amino acids, i.e. 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably having amino acids G and S. A preferred example is GGGS (SEQ ID NO: 37). Another example is a linker composed of G and S and having multiple GGS repeats such as 2, 3, 4, 5 or more GGS repeats. A particularly preferred linker of this type is GGSGGS (SEQ ID NO: 38).
Preferred amino acid sequences forming the multimerization domain are taken from the adenovirus penton base sequences of SEQ ID Nos: 1 to 20.
More preferred amino acid sequences for fragments D, A and F are characterized as follows:
According to a preferred embodiment of the invention, amino acid stretch D of general formulae (III) and/or (IV) has the following consensus sequence (SEQ ID NO: 34):
U)1-47 PTJ1GRNSIRY SJ2J3x4PJ5J6DTT J7J8YLVDNKSA DIASLNYQND HSNFJ5TTVJ9Q NNDJ10J11PJ12EAJ13 TQTINJ14DJ15RS RWGJ16J17LKTIJ18 J19TZ1Z2Z3Z4Z5Z6Z7Z8 Z9Z10Z11Z12Z13Z14Z15
More preferred amino acid sequences of fragment D are outlined in the following Table 3:
According to a further preferred embodiment of the invention, amino acid stretch E of above general formulae (III) and/or (IV) has the following sequence (SEQ ID NO: 35):
Z17Z18Z19Z20Z21Z22Z23Z24Z25Z28 Z27QVYWSLPDJ20 MJ21DPVTFRST J22QJ23J24NJ26PVVGJ26 ELZ28Z28Z30
wherein: amino acid stretch E begins on the N-terminal side at an amino acid from Z17 to Z27 or at amino acid Q after Z27,
amino acid stretch B ends on the C-terminal side before Z28 at amino acid L or at an amino acid from Z28 to Z30;
More preferred amino acid sequences of fragment E are outlined in the following Table 4:
According to a further preferred embodiment of the invention, fragment F of above general formulae (Ill) and/or (IV) has the following sequence (SEQ ID NO: 36):
Z31Z32Z33ALTDHGT LPLRSSIJ27GV QRVTJ28TDARR RTCPYVYKA LGIVJ30PJ31VLS SRTF
wherein: amino acid stretch F begins on the N-terminal side at an amino acid from Z31 to Z33 or at amino acid A after Z33;
More preferred amino acid sequences of fragment F are outlined in the following Table 5:
It is to be understood that the start and end amino acids of each of fragments A, B, D, E and F of the engineered adenovirus penton base of the invention according to general formula (III) are preferably selected such that there is preferably no overlap of the residues forming the transition from one fragment to the following fragment when compared to the sequences as shown in SEQ ID NOs: 1 to 20.
It is further to be understood that the start and end amino acids of each of fragments A, D, E and F of the engineered adenovirus penton base of the invention according to general formula (IV) are preferably selected such that there is preferably no overlap of the residues forming the transition from one fragment to the following fragment when compared to the sequences as shown in SEQ ID NOs: 1 to 20.
The fragments A, B, D, E and F of engineered penton base proteins of the invention according to formula (III) are preferably comprised of amino acid sequences of the same adenovirus serotype, but chimeras are contemplated as well.
The fragments A, D, E and F of engineered penton base proteins of the invention according to formula (IV) are preferably comprised of amino acid sequences of the same adenovirus serotype, but chimeras are contemplated as well.
The invention also provides pentameric complexes of an engineered adenovirus penton base protein of the invention, preferably a penton base protein of formula (III) or (IV), most preferably a penton base protein of formula (III).
The invention is further directed to virus-like particles (VLP) comprising 12 pentameric complexes of an engineered adenovirus penton base protein of the invention.
The invention also provides the use of polypeptides as defined herein having one or more heterologous modifications as outlined herein and/or of VLPs containing such polypeptides having one or more heterologous modifications as defined herein as a medicament.
The invention also provides pharmaceutical compositions comprising a polypeptide as defined herein having one or more heterologous modifications as outlined herein and/or a VLP containing such polypeptides having one or more heterologous modifications as defined herein, optionally together with at least one pharmaceutically acceptable carrier, excipient and/or diluent.
The invention further provides a method for producing a VLP as disclosed herein, preferably a VLP composed of polypeptides containing one or more heterologous modifications as defined herein, comprising the step of incubating a solution of a polypeptide according the invention, preferably a polypeptide comprising one or more heterologous modifications as defined herein under conditions allowing the assembly of the polypeptide into a VLP.
The invention also provides the use of polypeptides as defined herein having one or more heterologous modifications as outlined herein and/or of VLPs containing such polypeptides having one or more heterologous modifications as defined herein in the treatment and/or prevention of an infectious disease, an immune disease, tumor or cancer.
The invention is also directed to a method of identifying a binding sequence to a target molecule comprising the steps of:
(i) preparing a library of vectors each containing a nucleotide sequence encoding a polypeptide according to the invention having a candidate binding sequence in an expression cassette, each polypeptide encoded by said nucleotide sequence comprising a candidate binding sequence as a heterologous modification in one or more of the sites (RGD loop and/or V loop and/or floor region and/or B loop) as defined herein, wherein the candidate binding sequence encoded by the nucleotide sequence in each vector is different (i.e. the vectors preferably contain a randomized library of nucleotide sequences encoding randomized candidate binding sequences);
(ii) expressing the polypeptides encoded by the nucleotide sequences in a host cell or a cell-free system;
(iii) contacting the polypeptides expressed in step (ii), optionally after purification from the host cells or the cell-free system, with the target molecule; and (iv) detecting which polypeptide(s) have/has bound to the target molecule.
More particularly, above step (i) may be one of the following steps (ia) and (ib):
(ia) preparing a library of vectors each containing a nucleotide sequence encoding a polypeptide having a candidate binding sequence in an expression cassette, each polypeptide encoded by said nucleotide sequence comprising a candidate binding sequence as a heterologous modification in one or more of RGD loop region and/or V loop and/or floor region and/or B loop as defined in above, i.e. the nucleotide sequences in said library encode an ADDobody as defined herein, wherein the candidate binding sequence encoded by the nucleotide sequence in each vector is different such that the vectors contain a randomized library of nucleotide sequences encoding randomized candidate binding sequences; or
(ib) preparing a library of vectors each containing a nucleotide sequence encoding a polypeptide having a candidate binding sequence in an expression cassette, each polypeptide encoded by said nucleotide sequence comprising a candidate binding sequence as a heterologous modification in one or more of RGD loop region and/or V loop and/or floor region as defined above, i.e. the nucleotide sequences in said library encode a minimal ADDobody (also denoted as “miniADDobody”) as defined herein, wherein the candidate binding sequence encoded by the nucleotide sequence in each vector is different such that the vectors contain a randomized library of nucleotide sequences encoding randomized candidate binding sequences.
The method preferably further comprises the step of determining the dissociation constant(s) (Kd) of the polypeptide(s) bound to the target molecule.
The term “specific binding” as used in the context of the present invention to mean that a binding moiety (e.g. an antibody) binds stronger to a target, such as an epitope, for which it is specific compared to the binding to another target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Targets can be recognized by their ligands which bind with a certain affinity to their targets and thus, the ligand binding to its respective target results in a biological effect. Preferably, the binding is both specific and occurs with a high affinity, preferably with a Kd of less than 10−7, 10−8, 10−9, 10−10 M or less. Such affinity is preferably measured at 37° C. Suitable assays include surface plasmon resonance measurements (e.g. Biacore), quartz crystal microbalance measurements (e.g. Attana), and competition assays.
As used herein, the term “Kd” (usually measured in “mol/L”, sometimes abbreviated as “M”) is intended to refer to the dissociation equilibrium constant of the particular interaction between a binding moiety (e.g. an antibody or fragment thereof) and a target molecule (e.g. an antigen or epitope thereof). Methods for determining Kd include, without limitation, ELISA and surface plasmon resonance assays.
The above identification method can also be provided as an evolutionary process wherein the identified sequences binding to the target are further optimized in subsequent rounds of library preparation (wherein the candidate sequences of each previous rounds are modified so as to provide improved binding to the target molecule), expression, and contacting with the target, optionally followed by determination of the dissociation constants so that improved binding candidates are selected in each subsequent rounds until a predetermined minimal dissociation constant is achieved, e.g. preferably a dissociation constant indicating a specific binding of the candidate sequence to the target, or until the dissociation constants are not further improved, typically not further lowered, in comparison to the previous round.
The nucleotide sequence encoding a thus selected and/or optimized, respectively, ADDobody (or minimal ADDobody) containing a, preferably the finally optimized, binding sequence can then genetically fused to the nucleic acid encoding a multimerization domain of the same or different, preferably the same, adenovirus penton base, for example by using appropriate restriction enzyme sites in the fragment encoding the optimized ADDobody or minimal ADDobody, respectively, and a vector containing the nucleotide sequence in an expression cassette. The nucleic acid encoding the, preferably optimized, ADDobody or minimal ADDobody, respectively, is than inserted in line with the nucleotide sequence encoding a multimerization domain so that a complete ADDobody-multimerization domain (or minimal ADDobody-multimerization domain) construct (i.e. an engineered penton base protein according to the invention) is generated within the expression cassette. The vector can then be introduced into appropriate host cells and the construct, also named an engineered adenovirus penton base, can be mass expressed and purified. VLPs can then be prepared from the purified engineered adenovirus penton bases which include multiple copies (up to 60 copies, if the binding sequence is present as a single copy per penton base in the VLP) of the selected/optimized binding sequence which leads, in terms of, for example, binding sequences directed to an antigen (or epitope thereof) to improved recognition of the target molecule. The system could be used, of course, for any binding partners, e.g. antigens or antibodies or fragments of such entities as further detailed herein.
The present invention also provides pentamers of the ADDobody of the invention. The present furthermore provides decamers of the ADDobody of the invention composed of two ADDobody pentamers.
It is to be understood that all engineered proteins and polypeptides disclosed herein can also be expressed from a corresponding expression vector in a cell-free expression system known in the art.
According to another aspect of the invention, there is provided an engineered penton base protein wherein said protein comprises a heterologous modification in the following sequence: (from N- to C-terminal)
of fragment A (also referred to as “floor site” or “floor region”), wherein
X1 is I or L, and is preferably I;
X2 is selected from the group consisting of K, Q and E, and is preferably Q;
X3 is P or A, and is preferably P,
X4 is selected from the group consisting of L, V and I, and is preferably L
X5 is selected from the group consisting of T, E, A, K and L, and is preferably E;
X6 is selected from the group consisting of E, K, T and Q, and is preferably K;
X7 is selected from the group consisting of S, P and D, and is preferably S;
X8 is selected from the group consisting of K, T and S, and is preferably K;
X9 is selected from the group consisting of K, S, N, G and D, and is preferably S;
X10 is L or V, and is preferably V;
X11 is I or L, and is preferably I;
X12 is selected from the group consisting of S, E and P, and is preferably E;
X13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid;
X14 is D or G, and is preferably ID,
X15 is selected from the group consisting of S, K, Q and T, and is preferably K; and
X16 is selected from the group consisting of T, N, I, K and M, and is preferably I;
and/or
in the following sequence (from N- to C-terminal) T-H-V-F-X17-R-F-P (SEQ ID NO: 22) of fragment B (also referred to as “B loop) wherein X17 is D or N, and is preferably N.
The linker L (formula (I)) according to the invention may be selected from oligopeptide linkers such as oligopeptides having 4 to 10 amino acids, i.e. 4, 5, 6, 7, 8, 9, or 10 amino acids. Larger oligopeptides of more than 10 amino acids, typically from 11 to 50 amino acids, are also contemplated. The linker L may also be a polypeptide, protein or protein complex provided that the linker L does not interfere with the proper folding and stability of the ADDobody. The same holds true for fragments N and C as defined herein. According to preferred embodiments of the invention, the linker L as defined herein may be selected from the amino acid sequences (from N- to C-terminal) GAMGSGIQ (SEQ ID NO: 29) and GANGDSGN (SEQ ID NO: 20).
As already outlined in the above embodiments of the invention (see Tables 1 and 2, the fragments A and/or B as well as the engineered adenonvirus penton base proteins of the invention are preferably based on an amino acid sequence which is each independently derived from penton base sequences selected from the group consisting of penton bases of human adenovirus serotype 2 (hAd2), human adenovirus serotype 3 (hAd3), human adenovirus serotype 4 (hAd4), human adenovirus serotype 5 (hAd5), human adenovirus serotype 7 (hAd7), human adenovirus serotype 11 (hAd11), human adenovirus serotype 12 (hAd12), human adenovirus serotype 17 (hAd17), human adenovirus serotype 25 (hAd25), human adenovirus serotype 35 (hAd35), human adenovirus serotype 37 (hAd37), human adenovirus serotype 41 (hAd41), gorilla adenovirus (gorAd), chimpanzee adenovirus (ChimpAd), simian adenovirus serotype 18 (sAd18), simian adenovirus serotype 20 (sAd20), simian adenovirus serotype 49 (sAd49), rhesus adenovirus serotype 51 (rhAd51), rhesus adenovirus serotype 52 (rhAd52), and rhesus adenovirus serotype 53 (rhAd53).
Preferred amino acid sequences of the above-indicated adenovirus penton bases are laid down in generally accessible databases such as UniProt and UniProtE, and especially preferred sequences referred to herein for the above-mentioned adenovirus subtypes are laid down in UniProt Acc. No. Q2YOH9 (human adenovirus serotype 3; SEQ ID NO: 1), UniProt Acc. No. P03276 (human adenovirus serotype 2; SEQ ID NO: 2), UniProt Acc. No. Q2KSF3 (human adenovirus serotype 4; SEQ ID NO: 3), UniProt Acc. No. P12538 (human adenovirus serotype 5; SEQ ID NO: 4), UniProt Acc. No. Q9JFT6 (human adenovirus serotype 7; SEQ ID NO: 5), UniProt Acc. No. D2DM93 (human adenovirus serotype 11; SEQ ID NO: 6), UniProt Acc. No. P36716 (human adenovirus serotype 12; SEQ ID NO: 7), UniProt Acc. No. F1DT65 (human adenovirus serotype 17; SEQ ID NO: 8), UniProt Acc. No. MOQUKO (human adenovirus serotype 25; SEQ ID NO: 9), UniProt Acc. No. Q7T941 (human adenovirus serotype 35; SEQ ID NO: 10), UniProt Acc. No. Q912J1 (human adenovirus serotype 37; SEQ ID NO: 11), UniProt Acc. No. F8WQN4 (human adenovirus serotype 41; SEQ ID NO: 12), UniProt Acc. No. E5L3Q9 (gorilla adenovirus; SEQ ID NO: 13), UniProt Acc. No. G9G849 (chimpanzee adenovirus; SEQ ID NO: 14), UniProt Acc. No. H8PFZ9 (simian adenovirus serotype 18; SEQ ID NO: 15), UniProt Acc. No. F6KSU4 (simian adenovirus serotype 20; SEQ ID NO: 16), UniProt Acc. No. F2VVTK5 (simian adenovirus serotype 49; SEQ ID NO: 17), UniProt Acc. No. A0A0A1EVWV1 (rhesus adenovirus serotype 51; SEQ ID NO: 18), UniProt Acc. No. A0A0A1EWX7 (rhesus adenovirus serotype 52; SEQ ID NO: 19), and UniProt Acc. No. A0A0A1 EWZ7 (rhesus adenovirus serotype 53; SEQ ID NO: 20).
The amino acid sequences of the above penton bases are as follows (the respective UniProt Acc. No. is indicated in brackets):
The polypeptides of the present invention are not confined to those known specific sequences for amino acid stretches A (minimal ADDobody) or A and B (ADDobody), respectively, forming the alpha-helical domain of the above-referenced adenovirus sub- and serotypes, respectively. Amino acid fragments A and B can also have similar amino acid sequences to the sequences of known adenovirus penton base protomers as long as the sequences of A and B are such that the resulting polypeptide adopts a conformationally stable crown or minimal crown domain under appropriate conditions as further outlined below. Typically, such similar sequences of fragments A and B share an amino acid sequence identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence of a known adenovirus penton base, preferably those of SEQ ID NOs: 1 to 20, more preferably amino acid stretches A and B as provided in Tables 1 and 2, with the proviso that, in embodiments of the invention where one or more heterologous modifications are present in the RGD region and/or the V loop and/or the floor segment and/or the B loop, said sequence identities as outlined above are to be understood as referring to the adenovirus penton base fragments A and B, preferably of the sequences as outlined above, excluding said RGD loop region and/or said V loop and/or said floor segment and/or said B loop. Also with respect to fragments D, E and F, it is to be understood that these fragments can each have an amino acid sequence similar to the respective parts of the known adenovirus penton base sequences, and preferred sequence identity values given above for fragment A and B also apply to fragments D, E and F.
As used herein, amino acid sequences are stated from N to C terminal using the single letter code of IUPAC, if not otherwise specifically indicated.
A particularly preferred ADDobody of the invention is based on the penton base protein of human adenovirus serotype 3 (hAd3). For preferred sequences as regards the amino acid positions of SEQ ID NO: 1 it is referred to Table 1 (large fragment or fragment A, respectively) and Table 2 (small fragment or fragment B, respectively). The same holds for the minimal ADDobody with respect to the large fragment or fragment A, respectively.
A particularly preferred ADDobody of the invention is based on the penton base protein of chimpanzee adenovirus (ChimpAd). For preferred sequences as regards the amino acid positions of SEQ ID NO: 14 it is referred to Table 1 (large fragment or fragment A, respectively) and Table 2 (small fragment or fragment B, respectively). The same holds for the minimal ADDobody with respect to the large fragment or fragment A, respectively.
As already outlined above, it is one premier embodiment of the invention to include one or more antigens or one or more epitopes thereof, more particularly one or more antigens or one or more epitopes thereof of an infectious agent such as a virus, bacterium or other pathogen, or a tumor or cancer antigen or one or more epitopes thereof, respectfully, into one or more of the sites preferably present in the fragment A and/or B of the ADDobody or minimal ADDobody of the invention as defined above.) As used herein, the term “antigen” or “epitope of an antigen” refers to a structure recognized by molecules of the immune response, e.g. antibodies, T cell receptors (TCRs) etc. In this context, it is also expressis verbis referred to the definitions of “antigen” and “epitope” disclosed in WO 2017/167988 A1 (antigen: page 16, epitope: pages 15 and 16). The heterologous modification(s) present in the polypeptides or engineered adeonovirus base protomers may also be mimotopes of corresponding naturally occurring epitopes.
Antigens or epitopes thereof, respectively, of infectious agents include, but are not limited to, e.g. viral infectious agents, such as HIV, hepatitis viruses such as hepatitis A virus, hepatitis B virus or hepatitis C virus, herpes virus, varicella zoster virus, rubella virus, yellow fever virus, dengue fever virus, flaviviruses (e.g. Zika virus), influenza viruses, Marburg disease virus, Ebola viruses and arboviruses such as Chikungunya virus. Antigens of bacterial infectious agents include, but are not limited to, antigens of e.g. Legionella, Helicobacter, Vibrio, infectious E. coli strains, Staphylococci, Salmonella and Streptococci. Antigens of infectious protozoan pathogens include, but are not limited to, antigens of Plasmodium, Trypanosome, Leishmania and Toxoplasma. Further examples of antigens of pathogenic agents include antigens of fungal pathogens such as antigens of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis and Candida albicans.
Specific examples of tumor antigens or epitopes thereof which can be used according to the invention include, but not limited to 707-AP, AFP, ART-4, BAGE, beta-catenin/m, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/Melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1, p190 minor bcr-abl, Pml/RAR.alpha., PRAME, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2 and VVT1.
A further embodiment of polypeptides of the invention relates to polypeptides wherein at last one of the sites of fragments A and/or B for heterologous modification as defined herein contains antibody sequences or parts of antibodies such as antibody fragments. In this context of the invention the term “antibody” is an immunoglobulin specifically binding to an antigen.
The term “antibody fragment” refers to a part of an antibody which retains the ability of the complete antibody to specifically bind to an antigen. Examples of antibody fragments include, but are not limited to, paratopes, Fab fragments, Fab′ fragments, F(ab′)2 fragments, heavy chain antibodys, single-domain antibodies (sdAb), scFv fragments, fragment variables (Fv), VH domains, VL domains, nanobodies, IgNARs (immunoglobulin new antigen receptors), di-scFv, bispecific T-cell engagers (BITEs), dual affinity re-targeting (DART) molecules, triple bodies, diabodies, a single-chain diabody and the like.
A “diabody” is a fusion protein or a bivalent antibody which can bind different antigens. A diabody is composed of two single protein chains (typically two scFv fragments) each comprising variable fragments of an antibody. Diabodies therefore comprise two antigen-binding sites and can, thus, target the same (monospecific diabody) or different antigens (bispecific diabody).
The term “single domain antibody” as used in the context of the present invention refers to antibody fragments consisting of a single, monomeric variable domain of an antibody. Simply, they only comprise the monomeric heavy chain variable regions of heavy chain antibodies produced by camelids or cartilaginous fish. Due to their different origins they are also referred to VHH or VNAR (variable new antigen receptor)-fragments. Alternatively, single-domain antibodies can be obtained by monomerization of variable domains of conventional mouse or human antibodies by the use of genetic engineering. They show a molecular mass of approximately 12-15 kDa and thus, are the smallest antibody fragments capable of antigen recognition. Further examples include nanobodies or nanoantibodies.
Antigen-binding entities useful in the context of the invention also include “antibody mimetic” which expression as used herein refers to compounds which specifically bind antigens similar to an antibody, but which compounds are structurally unrelated to antibodies. Usually, antibody mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa which comprise one, two or more exposed domains specifically binding to an antigen. Examples include inter alia the LACI-D1 (lipoprotein-associated coagulation inhibitor); affilins, e.g. human-y B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10th type III domain of fibronectin; adnectins: knottins (cysteine knot miniproteins); atrimers; evibodies, e.g. CTLA4-based binders, affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus; Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II, affilins; armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody mimetics as well (aptamers), but not artificial antibodies, antibody fragments and fusion proteins composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs.
Especially in the context of antigens or epitopes thereof as well as antibodies or antibody fragments, but also with respect to any protein-protein interaction such as receptor-ligand binding, included into the inventive polypeptides in one or more of the sites for heterologous modification in fragment A and/or fragment B of the ADDobody or minimal ADDobody, respectively, but also with respect to any protein-protein interaction such as receptor-ligand binding, it is possible to include a selection and/or evolutionary process for providing target binding-optimized sequences such as optimized antigens or epitopes thereof to exert an improved immune response thereto (cf.
A preferred selection/optimization process in the context of the invention is ribosome display as outlined in detail in Schaffitzel et al. (2001) in: Protein-Protein Interactions, A Molecular Cloning Manual: In vitro selection and evolution of protein-ligand interaction by ribosome display (Golemis E., ed.), pages 535-567, Cold Spring Harbor Laboratory Press, New York. The ribosome display protocol has the advantage of being carried out completely in vitro at all steps of the selection process. Further possible selection processes are also known in the art and include phage display (Smith (1985) Science 228, 1315-1317; Winter et al. (1994) Annu. Rev. Immunol. 12, 433-455), yeast two-hybrid systems (Fields and Song (19899 Nature 340, 245-246; Chien et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 88, 9578-9582), and cell surface display methods (Georgiu et al. (1993) Trends Biotechnol. 11, 6-10; Boder and Wittrup (1997) Nat. Biotechnol. 15, 553-557) as well as mRNA display, yeast display, and/or baculovirus capsid display.
The ribosome display process can basically be used in two ways for optimization of antigens or other amino acid sequences involved in targeting a specific molecule by use of the polypeptides of the invention. Either, the amino acid sequence for selection and/or optimization to bind to a target molecule can be selected first from an initial library of polypeptides sequences that can be as large as 1014 individual sequences, more typically 109 to 1010 sequences, optionally employing evolutionary procedures as described in detail in Schaffitzel et al. (2001), supra. After selection of the optimized sequences binding to the target molecule, the nucleotide sequence encoding it/them is/are cloned into an appropriate vector of the invention such that a polypeptide is expressed where the optimized amino acid is included in one or more sites as defined above (RDG region and/or V loop and/or floor region and/or B loop).
According to an alternative embodiment of this aspect of the invention, a library of potential candidate binding sequences is directly cloned into a nucleic acid of the invention such that each sequence encodes a polypeptide containing a candidate binding sequence which included in one or more sites as defined above (RDG region and/or V loop and/or floor region and/or B loop). The inventive polypeptides comprising an initial library of candidate binding sequences are than typically expressed in vitro and selection of optimized binding sequences is carried out according to the ribosome display methodology as outlined in detail in Schaffitzel et al. (2001), supra, or any other suitable selection/evolution methodology for candidate amino acid sequence binding to the targeted molecule known in the art such as phage display, mRNA display, yeast display and/or baculovirus capsid display.
As native penton base proteins do, the engineered adenovirus penton base proteins of the invention assemble into pentameric complexes, 12 of which in turn assemble into virus-like particles (VLPs) in a buffer solution of preferably pH about 5.0 to about 8.0. Preferred examples are buffer conditions at or near physiological conditions such as PBS, pH 7.4, or TBS or TBS-T pH 7.2 to 7.6. Under such conditions, the polypeptides of the invention form VLPs at a temperature of about from about 20 to about 42° C. The present invention is also directed to such pentameric complexes and VLPs.
Further subject matter of the invention is a nucleic acid coding for an ADDobody, minimal ADDobody or engineered adenovirus protein as defined herein.
According to the present invention, the terms “nucleic acid” and “polynucleotide” are used interchangeably and refer to DNA, RNA or species containing one or more nucleotide analogues. Preferred nucleic acids or polynucleotides according to the present invention are DNA, most preferred double-stranded (ds) DNA. Nucleotide sequences of the present disclosure are shown from 5′ to 3′, and the IUPAC single letter code for bases is used, if not otherwise used as indicated.
Embodiments of nucleic acids and vectors, respectively of the invention are also provided for insertion of the versatile heterologous modifications (as, for example, embodied by oligopeptides, polypeptides, proteins etc. as defined herein).
An insertion site in the context of this embodiment of the invention is preferably a recognition sequence of a restriction enzyme or of a homing endonuclease.
Restriction enzyme sites are generally well-known to the skilled person. Preferred examples are as defined above, but restriction sites can be selected from a wide variety and guidance can be found at the various manufacturers of restriction enzymes such as New England Biolabs, Inc., Ipswich, Mass., USA.
Examples of homing endonuclease (HE) sites include, but are not limited to, recognition sequences of PI-SceI, I-CeuI, I-PpoI, I-HmuI I-CreI, I-DmoI, PI-PfuI and I-MsoI, PI-PspI, I-SceI, other LAGLIDAG group members and variants thereof, SegH and Hef or other GIY-YIG homing endonucleases, I-ApeII, I-AniI, Cytochrome b mRNA maturase bl3, PI-Till and PI-TfuII, PI-ThyI and others; see Stoddard B. L. (2005) Q. Rev. Biophys. 38, 49-95. Corresponding enzymes are commercially available, e. g. from New England Biolabs Inc., Ipswich, Mass., USA.
In preferred embodiments of the present invention, the above-defined nucleic acid additionally comprises at least one site for integration of the nucleic acid into a vector or host cell. The integration site may allow for a transient or genomic incorporation.
With respect to the integration into a vector, in particular into a plasmid or virus, the integration site is preferably compatible for integration of the nucleic acid into an adenovirus, adeno-associated virus (AAV), autonomous parvovirus, herpes simplex virus (HSV), retrovirus, rhadinovirus, Epstein-Barr virus, lentivirus, semliki forest virus or baculovirus.
Particularly preferred integration sites that may be incorporated into the nucleic acid of the present invention can be selected from the transposon element of Tn7, λ-integrase specific attachment sites and site-specific recombinases (SSRs), in particular LoxP site or FLP recombinase specific recombination (FRT) site. Further preferred mechanisms for integration of the nucleic acid according to the invention are specific homologous recombination sequences such as lef2-603/Orf1629.
In further preferred embodiments of the present invention, the nucleic acid as described herein additionally contains one or more resistance markers for selecting against otherwise toxic substances. Preferred examples of resistance markers useful in the context of the present invention include, but are not limited to, antibiotics such as ampicillin, chloramphenicol, gentamycin, spectinomycin, and kanamycin resistance markers.
The nucleic acid of the present invention may also contain one or more ribosome binding site(s) (RBS)
Further subject-matter of the present invention relates to a vector comprising a nucleic acid as defined above.
Preferred vectors of the present invention are plasmids, expression vectors, transfer vectors, more preferred eukaryotic gene transfer vectors, transient or viral vector-mediated gene transfer vectors. Other vectors according to the invention are viruses such as adenovirus vectors, adeno-associated virus (AAV) vectors, autonomous parvovirus vectors, herpes simplex virus (HSV) vectors, retrovirus vectors, rhadinovirus vectors, Epstein-Barr virus vectors, lentivirus vectors, semliki forest virus vectors and baculovirus vectors.
Baculovirus vectors suitable for integrating a nucleic acid according to the invention (e.g. present on a suitable plasmid such as a transfer vector) are also subject matter of the present invention and preferably contain site-specific integration sites such as a Tn7 attachment site (which may be embedded in a lacZ gene for blue/white screening of productive integration) and/or a LoxP site. Further preferred baculovirus according to the invention contain (alternative to or in addition to the above-described integration sites) a gene for expressing a substance toxic for host flanked by sequences for homologous recombination. An example for a gene for expressing a toxic substance is the diphtheria toxin A gene. A preferred pair of sequences for homologous recombination is e.g. Isf2-603/Orf1629. The baculovirus can also contain further marker gene(s) as described above, including also fluorescent markers such as GFP, YFP and so on. Specific examples of corresponding baculovirus are, for example disclosed in WO 2010/100278 A1.
Further applicable vectors for use in the invention are disclosed in WO 2005/085456 A1.
Vectors useful in prokaryotic host cells comprise, preferably besides the above-exemplified marker genes (one or more thereof), an origin of replication (ori). Examples are BR322, ColE1, and conditional origins of replication such as OriV and R6Ky, the latter being a preferred conditional origin of replication which makes the propagation of the vector of the present application dependent on the pir gene in a prokaryotic host. OriV makes the propagation of the vector of the present application dependent on the trfA gene in a prokaryotic host.
Furthermore, the present invention is directed to a (recombinant) host cell containing a nucleic acid of the invention and/or a vector of the present invention.
The host cells may be prokaryotic or eukaryotic. Eukaryotic host cells may for example be mammalian cells, preferably human cells. Examples of human host cells include, but are not limited to, HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32,
MT-2, pancreatic β-cells, keratinocytes, bone-marrow fibroblasts, CHP212, primary neural cells, W12, SK-N-MC, Saos-2, WI38, primary hepatocytes, FLC4, 143TK, DLD-1, embryonic lung fibroblasts, primery foreskin fibroblasts, MRCS, and MG63 cells. Further preferred host cells of the present invention are porcine cells, preferably CPK, FS-13, PK-15 cells, bovine cells, preferably MDB, BT cells, bovine cells, such as FLL-YFT cells. Other eukaryotic cells useful in the context of the present invention are C. elegans cells. Further eukaryotic cells include yeast cells such as S. cerevisiae, S. pombe, C. albicans and P. pastoris. Furthermore, the present invention is directed to insect cells as host cells which include cells from S. frugiperda, more preferably Sf9, Sf21, Express Sf+, High Five H5 cells, and cells from D. melanogaster, particularly S2 Schneider cells. Further host cells include Dictyostelium discoideum cells and cells from parasites such as Leishmania spec.
Prokaryotic hosts according to the present invention include bacteria, in particular E. coli such as commercially available strains like TOP10, DH5α, HB101. BL21(DE3) etc.
The person skilled in the art is readily able to select appropriate vector construct/host cell pairs for appropriate propagation and/or transfer of the nucleic acid elements according to the present invention into a suitable host. Specific methods for introducing appropriate vector elements and vectors into appropriate host cells are equally known to the art and methods can be found in the latest edition of Ausubel et al. (ed.) Current Protocols In Molecular Biology, John Wiley & Sons, New York, USA.
In preferred embodiments of the present invention, the vector as defined above additionally comprises a site for site specific recombinases (SSRs), preferably one or more LoxP sites for Cre-lox specific recombination. In further preferred embodiments, the vector according to the present invention comprises a transposon element, preferably a Tn7 attachment site.
It is further preferred that the attachment site as defined above is located within a marker gene. This arrangement makes it feasible to select for successfully integrated sequences into the attachment site by transposition. According to preferred embodiments, such a marker gene is selected from luciferase, β-GAL, CAT, fluorescent encoding protein genes, preferably GFP, BFP, YFP, CFP and their variants, and the lacZa gene.
The present invention also provides the polypeptide, the nucleic acid encoding such a polypeptide, the vector containing a polypeptide-encoding nucleic acid, the host cell comprising such a vector as well as the VLP as defined above for use as a medicament, in particular for use in the treatment and/or prevention of an infectious disease, an immune disease, tumor or cancer.
Therefore, the present invention is also directed to pharmaceutical compositions comprising a polypeptide as defined herein, a nucleic acid encoding such a polypeptide, a vector containing a polypeptide-encoding nucleic, a host cell comprising such a vector or a VLP as described above, optionally together with at least one pharmaceutically acceptable carrier, excipient and/or diluent.
Generally, the preparation of pharmaceutical compositions in the context of the present invention, their dosages and their routes of administration are known to the skilled person, and guidance can be found in the latest edition of Remington's Pharmaceutical Sciences (Mack publishing Co., Eastern, Pa., USA).
The pharmaceutical compositions of the invention contain a therapeutically effective amount of the active ingredient as outlined above. The therapeutically effective amount depends on the active ingredient and in particular on the route of administration. The pharmaceutical composition according to the invention will preferably be applied by parenteral administration, in particular by infusion such as intravenous, intraarterial or intraosseous infusion, or by injection, e.g. intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, subcutaneous or intrathecal injection. In the case of anti-tumor therapy, the pharmaceutical composition such as a pharmaceutical composition containing VLPs according to the invention, can also be administered by intra-tumoral injection.
Inventive solutions for injection or infusion typically contain VLPs of the invention in water or an aqueous buffer solution, preferably an isotonic buffer at physiological pH. Liquid pharmaceutical compositions of the invention may contain further ingredients such as pharmaceutically acceptable stabilizers, suspending aids, emulsifyers and the likes. Further ingredients of the pharmaceutical composition of the invention are adjuvants, in particular in the context of application of the constructs of the invention for vaccination purposes.
Further subject matter of the invention are methods of treatment making use of the beneficial properties of the polypeptides, nucleic acids, host cells, vectors and/or VLPs of the invention. In a preferred embodiment, the invention provides a method for the prevention and/or treatment of an infectious disease comprising the step of administering to a subject, preferably a human, a therapeutically effective amount of the pharmaceutical composition as defined above, wherein the pharmaceutical composition comprises VLPs of the invention containing antigens or epitopes thereof, of the infective agent causing the infectious disease. Another embodiment is a method for preventing and/or treating a tumor or cancer disease the step of administering a therapeutically effective amount of the pharmaceutical composition as defined above to a subject, preferably a human, wherein the pharmaceutical composition comprises VLPs of the invention containing one or more tumor antigens or epitopes thereof.
According to another preferred embodiment, the invention provides a method for the prevention and/or treatment of an infectious disease comprising the step of administering to a subject, preferably a human, a therapeutically effective amount of the pharmaceutical composition as defined above, wherein the pharmaceutical composition comprises VLPs of the invention containing one or more antibodies or antibody fragments such as a paratope thereof, recognizing an antigen, in particular an epitope, of the infective agent causing the infectious disease. Another embodiment is a method for preventing and/or treating a tumor or cancer disease the step of administering a therapeutically effective amount of the pharmaceutical composition as defined above to a subject, preferably a human, wherein the pharmaceutical composition comprises VLPs of the invention one or more antibodies or antibody fragments such as a paratope thereof, recognizing a tumor or cancer antigen, in particular an epitope thereof.
The present invention is further directed to a method for producing the polypeptide as described herein comprising the step of cultivating the recombinant host cell in a suitable medium, wherein the host cell comprises a vector which comprises a nucleic encoding the polypeptide, under conditions allowing the expression of said polypeptide.
Preferably, the method for producing the polypeptide of the invention further comprises the step of recovering the expressed polypeptide from the host cells and/or the medium. Even more preferred, the method also comprises the step of purifying the recovered polypeptide by purification means known in the art such as centrifugation, gel chromatography, ion exchange chromatography, affinity chromatography etc.
The invention also provides a method for producing a VLP as defined herein comprising the step of incubating a solution of the polypeptide under conditions allowing the assembly of the polypeptide into a VLP as outlined before. The proper formation of VLPs can be tested by inspecting a sample solution with an electron microscope.
The present invention Is further illustrated by the following non-limiting examples:
Cloning, Expression and Purification of Adenovirus Penton Base Crown Domain of Human Adenovirus Serotype 3 (hAd3)
The nucleotide sequence coding for an ADDobody of the invention (named ADDomer2_Head) equipped with a His-Tag sequence
was cloned in expression vector pProEx using the restriction sites shown in
The resulting vector has the following sequence (ORF start and stop shown in bold, coding sequence in lower characters):
The polypeptide was produced and purified as follows:
[In the following text, the expression AD3Head is used for ADDobody]
The expression and purification protocol is described per 1 L culture. It should be upscaled accordingly if more expression cultures are required. Normally, 3-5 L expression culture for one big prep should be made, and the purified protein stored frozen.
pProEX_HTB_AD3Head plasmid encoding for the ADDomer head or ‘crown’ domain (the ADDobody) is transformed into BL21(DE3) competent cells and plated onto LB agarose plate containing ampicillin as selection marker
(MWCO 10 kDa). Centrifugation is performed at 3000 G at 4° C. to 500 ul.
An exemplary chromatogram is shown in
Protein is flash-frozen in 100 μl aliquots in low protein binding tubes and stored at −20° C.
A SDS polyacrylamide gel analysis of the pooled fractions before and after freezing is shown in
Crystallization and X-Ray Structure Determination of ADDobody Construct ADDomer2_Head
The truncated (i.e. maturated) polypeptide ADDomer2_Head (the amino acid sequence according to SEQ ID NO: 27 lacking the His tag-containing sequence
was crystalized and subjected to X-ray diffraction.
Crystallization conditions: sitting drop, PEG3350 20% (w/v), 0.1 M citrate pH 5.5, protein concentration: 5 mg/ml, drop size: 0.5 ul protein and 0.5 ul reservoir.
A representative single crystal is shown in
The X-ray diffraction gave the following results:
Spacegroup P1
a=102.124 b=103.837 c=177.578 α=92.308 β=94.314 γ=112.047
The solution structure is shown in
The unit cell contains 20 ADDobodies present in 2 decamers.
Importantly, the floor region of fragment A and the B loop of fragment B of the ADDobody forms a flexible structure in the isolated ADDobody
Cloning, Expression and Purification of Adenovirus Penton Base Crown Domain of Chimpanzee Adenovirus
The nucleotide sequence coding for a further ADDobody of the invention (named ChimpCrown) equipped with a His-Tag sequence
was cloned in expression vector pProEx as outlined in Example 1
Expression and purification was carried out according to the protocol of Example 1.
Cloning, Expression and Purification of Adenovirus Penton Base Crown Domain of Human Adenovirus Serotype 3 (hAd3) Containing a Heterologous Insertion
The nucleotide sequence coding for a modified ADDobody of the invention containing the major neutralizing epitope from Chikungunya virus STKDNFNVYKATRPYLAH (SEQ ID NO: 40) in the B loop of fragment B and replacing the sequence HVFNRF (SEQ ID NO: 41) in the wild-type fragment B of hAd3 penton base (equipped with a His-Tag sequence)
was cloned in expression vector pProEx as outlined in Example 1 Expression and purification was carried out according to the protocol of Example 1.
In summary, the present invention is particularly directed to the following items:
N-A-L-B-C (I)
wherein, optionally, fragment A contains one or more heterologous modifications.
wherein, optionally, fragment B contains one or more heterologous modifications.
5. The polypeptide according to any one of items 2 to 4 characterized in that fragment A and/or B contain(s) one or more heterologous modifications wherein said one or more heterologous modifications is/are contained in the following sites:
wherein
N-A-C (II)
wherein, optionally, fragment A contains one or more heterologous modifications.
wherein)
D-A-E-B-F (III)
D-A-E-Li-F (IV)
(U)1-47 PTJ1GRNSIRY SJ2J3x4PJ5J6DTT J7JBYLVDNKSA DIASLNYQND HSNFJ5TTVJ9Q NNDJ10J11PJ12EAJ13 TQT INJ14DJ15RS RWGJ16L-T17LKTIJ18 J19TZ1Z2Z3Z4Z5Z6Z7Z8 Z9Z10Z11Z12Z13Z14Z15
Z17Z18Z19Z20Z21Z22Z23Z24Z25Z26 Z27QVYWSLPDJ20 MJ21DPVTFRST J22QJ23J24NJ25PVVGJ26 ELZ28Z29Z30
Z31Z32Z33ALTDHGT LPLRSSIJ27GV QRVTJ28TDARR RTCPYVYKA LGIVJ30PJ31VLS SRTF
| Number | Date | Country | Kind |
|---|---|---|---|
| 20155742.8 | Feb 2020 | EP | regional |
| 20155982.0 | Feb 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/052884 | 2/5/2021 | WO |
