Patent Application
20140348826
The invention relates generally to a new trimerization domain useful for developing multifunctional and multivalent complexes. Particularly, the invention relates to the design of trimeric polypeptide complexes using polypeptide structural elements derived from the collagen XV protein, and their use in diagnostic and therapeutic systems in vivo and in vitro. The invention also relates to nucleic acids and vectors useful for producing said trimeric complexes.
Conversion of monovalent recombinant antibodies, such as scFv (single-chain variable fragments), Fabs (fragment antigen binding) or single-domains, into multivalent formats increases functional affinity, decreases dissociation rates when bound to cell-surface antigens, and enhances biodistribution (for a comprehensive review see Cuesta A M et al. Trends Biotech. 2010, 28:355-62).
The most common strategy to create multivalent antibodies has been the engineering of fusion proteins in which the antibody fragment makes a complex with homodimerization proteins. The most relevant recombinant dimeric antibodies are: the Fab2 antibody and its single-chain counterpart, sc(Fab′)2 (Fab2/sc(Fab)2); the ZIP miniantibody; the scFv-Fc antibody; and the minibody. The miniantibody is a 64 kDa molecule generated by the association of two scFv fragments through dimerization domains. Leucine zippers (Fos or Jun) are employed to mediate dimerization of scFv in a miniantibody molecule. Fos-Fos and Jun-Jun homodimer formation allow efficient production of stable, secreted, monospecific miniantibodies, and Fos-Jun heterodimer formation allows production of bispecific miniantibodies. The scFv-Fc antibody is a small IgG-like recombinant antibody (100-105 kDa) generated by fusion of a scFv with human IgG1 hinge and Fc regions (CH2, and CH3 domains). The 80 kDa minibody results from the fusion of a scFv to the human IgG1 CH3 domain. Other minibody-like molecules have been generated using the small immunoprotein technique, or by fusion to bacterial alkaline phosphatase.
Multimerization can be forced by shortening the peptide linker between the V domains of the scFv to self-associate into a dimer (diabody; 55 kDa). Further diabody derivatives are the triabody and the tetrabody, which fold into trimeric and tetrameric fragments by shortening the linker to less than 5 or to 0-2 residues, respectively. A different design for a (scFv)2 is the ‘bispecific T cell engager’ (BITE). BITEs are bispecific single-chain antibodies consisting of two scFv antibody fragments, joined via a flexible linker, that are directed against a surface antigen on target cells and CD3 on T cells.
Oligomerization domains from non-Ig proteins have been used to make multivalent antibodies (i.e. molecular constructs based on the bacterial barnase-barstar module). The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which the N- and C-termini are accessible for fusion. This module, which has a dissociation constant of about 10−14 M, has been exploited to generate stable, trimeric scFv (about 130 kDa). Similar formats have been generated by fusion of a scFv to a cytokine that naturally exists in trimeric form, the tumor necrosis factor (TNF). TNFα genetically fused to the C-terminus of a scFv antibody forms a homotrimeric structure that retains both TNFα activity and the ability to bind antigen recognized by the scFv. Also, fusion proteins of scFv fragments with the C-terminal multimerization domain of the tumor-suppressor protein p53 or with a streptavidin subdomain results in tetrameric antibodies (scFv-SA)4.
Another successful approach for the generation of multimeric antibodies has been the use of extracellular matrix-derived oligomerization domains. Trimerization of heterologous fusion proteins containing collagenous domain(s) has been accomplished by employing either a homogeneous or heterologous trimerization domain fused to the collagenous domain to drive the collagen triplex formation. Examples of a trimer-oligomerizing domain include a C-propeptide of procollagens, a coiled-coil neck domain of collectin family proteins, a C-terminal portion of FasL and a bacteriophage T4 fibritin foldon domain [Frank et al., (2001) J Mol Biol 308: 1081-1089; Holler et al., (2003) Mol Cell Biol 23: 1428-1440; Hoppe et al., (1994) FEBS Lett 344: 191-195]. A trimeric antibody, collabody (125 kDa), is based on the use of triplex-forming collagen-like peptides [(GPP)10].
Fusion of the trimerization region of the C-terminal non-collagenous domain (NC1) of collagen XVIII to the C-terminus of a scFv confers a natural trimeric state to the fused antibody (trimerbody; 110 kDa). WO 2006/048252 discloses fusion proteins comprising the scFv fragment of an antibody and the NC1 domain of collagen XVIII, said domain comprising a trimerization domain and an endostatin (ES) domain bound by a hinge peptide containing proteinase-sensitive sites, as well as trimers of said fusion proteins, wherein all the three fusion proteins contain an ES domain.
However, the stability of recombinant antibody-based molecules containing the trimerization region from collagen XVIII NC1 domain in serum is not very high; further, the stability of said recombinant antibody-based molecules in the presence of proteases decreases drastically, consequently, the eventual use of said molecules for uses in vivo is seriously limited, because the stability of engineered antibody fragments in serum and in protease-rich environment (e.g., tumors, inflammatory sites, etc.) are the critical parameters to determine their potential application in vivo.
Therefore, there is a need in the prior art to provide recombinant molecules containing a trimerization domain having a high stability in serum and in the presence of proteases.
The inventors have found that recombinant molecules containing the trimerization region from collagen XV NC1 domain are trimeric in solution, and significantly more stable in serum and in the presence of proteases than recombinant molecules containing the trimerization region from collagen XVIII NC1 domain. Hurskainen et al., in The Journal of Biological Chemistry, 285: 5258-5265, Feb. 19, 2010, disclose fusion proteins comprising a histidine tag and the NC1 domain of mouse collagen XV, said domain comprising a trimerization domain and a restin domain, as well as trimers of said fusion proteins for producing monoclonal N-terminal antibodies to mouse collagen XV; however, said document is silent about the stability of recombinant molecules containing the trimerization region from collagen XV NC1 domain in serum and in the presence of proteases.
The inventors have found that a collagen XV trimerizing structural element (XVTSE) forms, surprisingly, highly stable trimeric molecules. Stability of proteins in serum is a critical parameter to determine its potential application in vivo; therefore, said trimeric molecules may find a lot of applications in the field of therapy and diagnosis.
Thus, in an aspect, the invention relates to a trimeric polypeptide complex, hereinafter referred to as the “trimeric complex of the invention”, comprising three monomer polypeptides, wherein:
As used herein, the term “collagen XV trimerizing structural element” or “XVTSE”, refers to a polypeptide which comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 1:
or to a polypeptide having at least 60% amino acid sequence identity with the sequence shown in SEQ ID NO: 1, and maintains the ability to form trimers. SEQ ID NO: 1 comprises the amino acid sequence of the trimerization domain of human collagen XV (i.e., the human collagen XV NC1 domain without the ES domain—sometimes referred to in this description as “NC1ES-” or “trimerization domain”); said trimerization domain has the ability (capacity) to form trimers with other peptides having the same sequence at physiological conditions, i.e., conditions that permit in vivo protein expression in the cytosol of an eukaryotic or a prokaryotic organism. The ability of a polypeptide to form trimers can be determined by conventional methods known by the skilled person in the art. For example, by way of a simple illustration, the ability of a polypeptide to form a trimer can be determined by using standard chromatographic techniques. Thus, the polypeptide to be assessed is put under suitable trimerization conditions and the complex is subjected to a standard chromatographic assay under non denaturing conditions so that the eventually formed complex (trimer) is not altered. If the variant trimerizes properly, the molecular size of the complex would be three times heavier than the molecular size of a single molecule of the variant. The molecular size of the complex can be revealed by using standard methods such as analytical centrifugation, mass spectrometry, size-exclusion chromatography, etc.
In a particular embodiment, the XVTSE comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 1.
In another particular embodiment, the XVTSE comprises, or consists of, a polypeptide having at least 60% amino acid sequence identity with the sequence shown in SEQ ID NO: 1, normally at least 70%, usually at least 80%, advantageously at least 90%, more advantageously at least 95%, preferably at least 96%, more preferably at least 97%, still more preferably at least 98% or even more preferably at least 99%, and has the ability to form trimers. These variants result from the modifications of the polypeptide as defined in SEQ ID NO: 0.1 and include by the conservative (or non-conservative) substitution of one or more amino acids for other amino acids, the insertion and/or the deletion of one or more amino acids. The degree of identity between two amino acid sequences can be determined by any conventional method, for example, by means of standard sequence alignment algorithms known in the state of the art, such as, for example BLAST [Altschul S. F. et al., (1990) J Mol Biol. 215:403-10]. In a more particular embodiment, the XVTSE is a polypeptide having at least 80%, advantageously at least 90%, preferably at least 95%, more preferably 99% amino acid sequence identity with the sequence shown in SEQ ID NO: 1.
The trimerization region from collagen XVIII NC1 domain has also the ability to form trimers; nevertheless, the degree of identity of the trimerization region from collagen XV NC1 domain is about 32% with respect to the trimerization region from collagen XVIII NC1 domain.
The person skilled in the art will understand that the amino acid sequences referred to in this description can be chemically modified, for example, by means of chemical modifications which are physiologically relevant, such as, phosphorylations, acetylations, etc.
It has been found that the XVTSE forms surprisingly stable trimeric molecules which are even more stable than trimeric molecules comprising the N-terminal trimerization region of the collagen XVIII NC1 domain (Example 1). As it is well-known, the stability of proteins in serum and in protease-rich environments is a critical parameter to determine its potential application in vivo. As it is shown in
The trimeric complex of the invention is characterized in that at least one of the three constituent monomer polypeptides does not contain an endostatin domain or a restin domain. In a preferred embodiment, only one of the monomer polypeptides forming the trimeric complex of the invention does not contain an endostatin domain or a restin domain. In another embodiment, two of the monomer polypeptides forming the trimeric complex of the invention do not contain an endostatin domain or a restin domain. In yet another embodiment, none of the monomer polypeptides forming the trimeric complex of the invention contain an endostatin domain or a restin domain.
By the term “heterologous moiety” is herein meant any chemical entity that can be fused or linked covalently to a XVTSE and to which the XVTSE is not natively covalently bound. Hence, the heterologous moiety can be any covalent partner moiety known in the art for providing desired binding, detection, or effector properties. Illustrative, non-limitative, examples of heterologous moieties include:
In a particular embodiment, said heterologous moiety is an antibody, for example a monoclonal antibody (mAb), or a recombinant fragment thereof, e.g., a single chain Fv fragment (scFv), a single domain antibody, etc. In a particular embodiment, said heterologous moiety comprises the scFv L36 (Example 1) containing the variable heavy chain region (VH) fused with a (Gly4Ser)3 linker to the variable light chain (VL), whose sequence has been described by Sanz L et al. [Cancer Immunology and Immunotherapy, 2001 December; 50(10)557-65], wherein the C-terminus of said L36 scFv is linked to the N-terminus of said XVTSE, namely to the N-terminus of the human collagen XV NC1 trimerization region [L36-hXVNC1ES-], through a flexible peptide spacer. The scFv L36 recognizes laminins of different animal species, for example, from mice, rats, humans, etc., since it interacts with a region that is very preserved among different animal species [Sanz L et al. EMBO J 2003, Vol. 22(7):1508-1517].
In another particular embodiment, said heterologous moiety comprises the recombinant scFv MFE23, which specifically recognizes human carcinoembryonic antigen (CEA) (Example 1), containing the variable heavy chain region (VH) fused to the variable light chain (VL) through a peptide linker [(Gly4Ser)3] (Chester K A et al., (1994) Lancet 343: 455-456), wherein the C-terminus of said MFE23 scFv is linked to the N-terminus of said XVTSE, through a flexible peptide spacer.
In another particular embodiment, said heterologous moiety comprises a recombinant scFv derived from the mAb B1.8, which specifically recognizes the hapten NIP (Example 1), containing the variable heavy chain region (VH) fused to the variable light chain (VL) through a peptide linker [(Gly4Ser)3] (Chester K A et al., (1994) Lancet 343: 455-456), wherein the C-terminus of said B1.8 scFv is linked to the N-terminus of said XVTSE, through a flexible peptide spacer.
In another particular embodiment, the heterologous moiety comprises a specific affinity purification tag in order to obtain a substantially pure trimeric complex of the invention, particularly if each one of the three monomer polypeptides comprises one affinity purification tag, said tags being different each other (e.g., affinity purification tags “a”, “b” and “c”, wherein tag “a” is recognized by binding substance A, tag “b” is recognized by binding substance B, and tag “c” is recognized by binding substance C), and it is subjected to a three-step affinity purification procedure designed to allow selective recovery of only such trimeric complexes of the invention that exhibit affinity for the corresponding substances (A, B and C in the illustration). Said affinity purification tag can be fused directly in-line or, alternatively, fused to the monomer polypeptide via a cleavable linker, i.e., a peptide segment containing an amino acid sequence that is specifically cleavable by enzymatic or chemical means (i.e., a recognition/cleavage site). In a particular embodiment, said cleavable linker comprises an amino acid sequence which is cleavable by a protease such as an enterokinase, Arg-C endoprotease, Glu-C endoprotease, Lys-C endoprotease, factor Xa, etc.; alternatively, in another particular embodiment, said cleavable linker comprises an amino acid sequence which is cleavable by a chemical reagent, such as, for example, cyanogen bromide which cleaves methionine residues, or any other suitable chemical reagent. The cleavable linker is useful if subsequent removal of the affinity purification tags is desirable.
In a particular embodiment, when the heterologous moiety is a peptide, said heterologous moiety is preferably covalently linked to the XVTSE by via a peptide bond to the N- or C-terminus of the XVTSE peptide chain.
In a particular embodiment, said monomer polypeptide is directly covalently linked to said heterologous moiety. However, in another particular embodiment, said monomer polypeptide is not directly covalently linked to said heterologous moiety but it is linked through a spacer, i.e., an inert connecting or linking peptide of suitable length and character, between the monomer polypeptide and the heterologous moiety [i.e., forming a “monomer polypeptide-spacer-heterologous moiety” structure], or, alternatively, between the heterologous moiety and the monomer polypeptide [i.e., forming a “heterologous moiety-spacer-monomer polypeptide” structure]. In general, said spacer acts as a hinge region between said domains, allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains. In this sense, a preferred spacer would be a hinge region characterized by a structural ductility or flexibility allowing this movement. The length of the spacer can vary; typically, the number of amino acids in the spacer is 100 or less amino acids, preferably 50 or less amino acids, more preferably 40 or less amino acids, still more preferably, 30 or less amino acids, or even more preferably 20 or less amino acids.
Illustrative, non-limitative examples of spacers include a polyglycine linker; amino acid sequences such as: SGGTSGSTSGTGST (SEQ ID NO: 3), AGSSTGSSTGPGSTT (SEQ ID NO: 4), GGSGGAP (SEQ ID NO: 5), GGGVEGGG (SEQ ID NO: 6), etc. In a particular embodiment, said spacer is a peptide having structural flexibility (i.e., a flexible linking peptide or “flexible linker”) and comprises 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. In another particular embodiment, the spacer is a peptide containing repeats of amino acid residues, particularly Gly and Ser, or any other suitable repeats of amino acid residues. Virtually any flexible linker can be used as spacer according to this invention. Illustrative examples of flexible linkers include amino acid sequences such as Gly-Ser-Pro-Gly (GSPG; SEQ ID NO: 7), Ala-Ala-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Arg (AAAGGSGGSSGSR; SEQ ID NO: 8) or Gly-Ser-Gly-Ser-Gly-Ser-Gly-Ser (Gly-Ser)4 (GSGSGSGS; SEQ ID NO: 9). Nevertheless, in a particular embodiment said spacer is a flexible linker comprising the amino acid sequence Ala-Asn-Ser-Gly-Ala-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Gly-Ser-Asp-Gly-Ala-Ser-Gly-Ser-Arg (ANSGAGGSGGSSGSDGASGSR; SEQ ID NO: 10).
Alternatively, a suitable spacer can be based on the sequence of 10 amino acid residues of the upper hinge region of murine IgG3; said peptide (PKPSTPPGSS, SEQ ID NO: 11) has been used for the production of dimerized antibodies by means of a coiled coil (Pack P. and Pluckthun, A., 1992, Biochemistry 31:1579-1584) and can be useful as a spacer peptide according to the present invention. Even more preferably, it can be a corresponding sequence of the upper hinge region of human IgG3 or other human Ig subclasses (IgG1, IgG2, IgG4, IgM and IgA). The sequences of human Igs are not expected to be immunogenic in human beings. Additional spacers that can be used in the instant invention include the peptides of sequences: APAETKAEPMT (SEQ ID NO: 12), GAP, AAA or AAALE (SEQ ID NO: 13).
In a particular embodiment, the monomer polypeptide is covalently linked to only one heterologous moiety, preferably, said heterologous moiety is covalently linked to only one end of said monomer polypeptide. In this case, the heterologous moiety can be linked by any of the above mentioned means, e.g., via a peptide bond to the amino-terminal end (N-terminus) or, alternatively, to the carboxyl-terminal end (C-terminus) of said monomer polypeptide comprising the XVTES peptide chain. Thus, in a more particular embodiment, the monomer polypeptide is linked to only one end of the heterologous moiety and said heterologous moiety is linked to the N-terminus of said monomer polypeptide (this type of monomer polypeptide can be designated as “N-terminal monomer polypeptide” and represented as “HM-XVTSE”, wherein HM means heterologous moiety and XVTSE is that previously defined). In another particular embodiment, the monomer polypeptide is linked to only one end of the heterologous moiety and said heterologous moiety is linked to the C-terminus of said monomer polypeptide (this type of monomer polypeptide can be designated as “C-terminal monomer polypeptide” and represented as “XVTSE-HM”, wherein HM means heterologous moiety and XVTSE is that previously defined).
In another particular embodiment, the monomer polypeptide is covalently linked to two heterologous moieties, preferably each heterologous moiety is covalently linked to just one of the ends of the monomer polypeptide by any of the previously mentioned methods (i.e., via a peptide bond to the N- or C-terminus of the XVTSE peptide chain, etc.). Thus, according to this particular embodiment, one of the heterologous moieties is linked to the N-terminus and the other one is linked to the C-terminus of said monomer polypeptide comprising the XVTES peptide chain, whereas in another specific embodiment, both heterologous moieties are linked each other and linked to just one terminus (N-terminus or C-terminus) of said monomer polypeptide comprising the XVTES peptide chain.
Thus, in said specific embodiment, a first heterologous moiety is linked to the N-terminus of said monomer polypeptide and a second heterologous moiety is linked to the C-terminus of said monomer polypeptide (this type of monomer polypeptide can be designated as N-/C-terminal monomer polypeptide and represented as “HM1-XVTSE-HM2”, wherein HM1 means a first heterologous moiety, HM2 means a second heterologous moiety and XVTSE is that previously defined). Said first and second heterologous moieties can be equal or different. This particular embodiment, related to a monomer polypeptide comprising two heterologous moieties which are linked via peptide bonds to the N- and C-terminus, respectively, (i.e., N-/C-terminal monomer polypeptide) constitutes an interesting embodiment of the invention. This approach introduces a number of possibilities in terms of, for example, linking larger entities with oligomers of the invention by having specific activities coupled to each end of the monomers.
In another specific embodiment, a first heterologous moiety (HM1) is linked to a second heterologous moiety (HM2), in any order (HM1-HM2 or HM2-HM1), and also linked to only one terminus (i.e., the N-terminus or the C-terminus) of the monomer polypeptide thus forming, e.g., an “in-line” or “tandem” structure, linked to just one of the terminus of the monomer polypeptide, e.g., to the N-terminus, i.e., “HM1-HM2-XVTSE”, “HM2-HM1-XVTSE”, or to the C-terminus “XVTSE-HM1-HM2”, or “XVTSE-HM2-HM1” of the monomer polypeptide. Said first and second heterologous moieties can be equal or different. Preferably, in said “in-line” or “tandem” structures the heterologous moieties are linked each other by means of a spacer; in a particular embodiment, said spacer is flexible linking peptide or a “flexible linker” as defined above.
In a specific embodiment, at least one of the heterologous moieties is linked to the N-terminus and at least one of the heterologous moieties is linked to the C-terminus of said monomer polypeptide comprising the XVTES peptide chain, whereas in another specific embodiment, all the heterologous moieties are linked each other and linked to just one terminus (N-terminus or C-terminus) of said monomer polypeptide comprising the XVTES peptide chain.
Thus, in a specific embodiment, at least one heterologous moiety is linked to the N-terminus of said monomer polypeptide and at least one heterologous moiety is linked to the C-terminus of said monomer polypeptide to render a monomer polypeptide; this type of monomer polypeptide can be represented as “(HM1)n-XVTES-(HM2)m”, wherein the total number of heterologous moieties (n+m) is at least 3, each HM1 is, independently, equal or different to other HM1, and each HM2 is, independently, equal or different to other HM2. Further, each HM1 can be equal or different to each HM2. Illustrative, non-limitative, examples of this type of monomer polypeptide include monomer polypeptides in which one or more heterologous moieties are linked to the N-terminus of said monomer polypeptide and one or more heterologous moieties are linked to the C-terminus of said monomer polypeptide wherein the number of heterologous moieties is equal or higher than three.
As discussed above, the heterologous moieties can be directly linked each other, or, alternatively, in a particular embodiment, they can be linked though a spacer forming a “HM-spacer-HM” structure. The particulars or said spacer have been previously mentioned.
According to the instant invention, the trimeric complex of the invention comprises three monomer polypeptides, wherein:
The particulars of XVTSE and HM have been previously mentioned. As used herein, the term “endostatin domain” or “ES domain” refers to the fragment derived from the NC1 domain of collagen XVIII; whereas the term “restin domain” refers to the 22 kDa fragment derived from the NC1 domain of collagen XV.
Thus, in a particular embodiment, the trimeric complex of the invention comprises three monomer polypeptides, wherein (i) each of said monomer polypeptides comprises a XVTSE, wherein said XVTSE has the particulars mentioned above, and (ii) only one of said monomer polypeptides is covalently linked to at least one heterologous moiety. According to this particular embodiment, only one of the monomer polypeptides which constitute the trimeric complex of the invention is covalently linked to at least one heterologous moiety. In a particular embodiment, said monomer polypeptide is covalently linked to one heterologous moiety. In another particular embodiment, said monomer polypeptide is covalently linked to two or more, e.g., 2, 3, 4 or even more, heterologous moieties; said heterologous moieties can be equal or different each other. The heterologous moiety o moieties can be linked to the N-terminus, or to the C-terminus, of the monomer polypeptide which comprises the XVTSE peptide chain. If the monomer polypeptide is covalently linked to two or more heterologous moieties, said heterologous moieties can be linked to the N-terminus, or to the C-terminus, of the monomer polypeptide which comprises the XVTSE peptide chain, or alternatively, at least one heterologous moiety can be linked to the N-terminus of the monomer polypeptide and at least one heterologous moiety can be linked to the C-terminus of the monomer polypeptide.
Illustrative, non-limitative, examples of this embodiment of the trimeric complex of the invention wherein only one monomer polypeptide is linked to at least one heterologous moiety include trimeric complexes of the invention in which:
In another particular embodiment, the trimeric complex of the invention comprises three monomer polypeptides, wherein (i) each of said monomer polypeptides comprises a XVTSE, wherein said XVTSE has the particulars mentioned above, and (ii) each one of two of said monomer polypeptides is covalently linked to at least one heterologous moiety. According to this particular embodiment, each one of two of the three monomer polypeptides which constitute the trimeric complex of the invention, independently, is covalently linked to at least one heterologous moiety. Thus, according to this particular embodiment, the trimeric complex of the invention comprises a first monomer polypeptide covalently linked to at least one heterologous moiety and a second monomer polypeptide covalently linked to at least one heterologous moiety, wherein the heterologous moieties linked to each of said first and second monomer polypeptide can be equal or different. Further, as discussed above, said heterologous moiety or moieties can be linked to the N-terminus, or to the C-terminus, of the monomer polypeptide which comprises the XVTSE peptide chain, or alternatively, if any of the monomer polypeptides is covalently linked to two or more heterologous moieties, said heterologous moieties can be linked to the N-terminus, or to the C-terminus, of the monomer polypeptide which comprises the XVTSE peptide chain, or alternatively, at least one heterologous moiety can be linked to the N-terminus of the monomer polypeptide and at least one heterologous moiety can be linked to the C-terminus of the monomer polypeptide. The skilled person in the art will recognize that any combination of heterologous moieties can be present in this particular embodiment; i.e., in a specific embodiment, for example, a first monomer polypeptide can be linked to one or more heterologous moieties and a second monomer polypeptide can be linked to one or more heterologous moieties. In any case, said heterologous moiety or moieties can be linked to just one of the terminus (N- or C-terminus) of the monomer polypeptide or, alternatively, if any of the monomer polypeptide is covalently linked to two or more heterologous moieties, said heterologous moieties can be linked to the N-terminus, or to the C-terminus, of the monomer polypeptide which comprises the XVTSE peptide chain, or alternatively, at least one heterologous moiety can be linked to the N-terminus of the monomer polypeptide and at least one heterologous moiety can be linked to the C-terminus of the monomer polypeptide.
Illustrative, non-limitative, examples of this embodiment of the trimeric complex of the invention wherein each one of only two monomer polypeptides are linked to at least one heterologous moiety include trimeric complexes of the invention in which:
In another particular embodiment, the trimeric complex of the invention comprises three monomer polypeptides, wherein (i) each of said monomer polypeptides comprises a XVTSE, wherein said XVTSE has the particulars mentioned above, and (ii) each one of the three monomer polypeptides is covalently linked to at least one heterologous moiety. According to this particular embodiment, each one of the three monomer polypeptides which constitute the trimeric complex of the invention, independently, is covalently linked to at least one heterologous moiety. Thus, according to this particular embodiment, the trimeric complex of the invention comprises a first monomer polypeptide covalently linked to at least one heterologous moiety, a second monomer polypeptide covalently linked to at least one heterologous moiety, and a third monomer polypeptide covalently linked to at least one heterologous moiety, wherein the heterologous moieties linked to each of said first, second and third monomer polypeptide can be equal, with the proviso that at least one of said three monomer polypeptides does not contain an endostatin domain or a restin domain, or different each other, e.g., all the three heterologous moieties can be equal (with the above exception), or two of the three heterologous moieties can be equal each other and different to the other one, or, alternatively, all the three heterologous moieties can be different each other. The skilled person in the art will recognize that any combination of heterologous moieties can be present in this particular embodiment provided that the above mentioned provision is met; i.e., in a specific embodiment, for example, a first monomer polypeptide can be linked to one or more heterologous moieties, the second monomer polypeptide can be linked to one or more heterologous moieties and the third monomer polypeptide can be linked to one or more heterologous moieties. In any case, said heterologous moiety or moieties can be linked to just one of the terminus (N- or C-terminus) of the monomer polypeptide or, alternatively, if any of the monomer polypeptide is covalently linked to two or more heterologous moieties, said heterologous moieties can be linked to the N-terminus, or to the C-terminus, of the monomer polypeptide which comprises the XVTSE peptide chain, or alternatively, at least one heterologous moiety can be linked to the N-terminus of the monomer polypeptide and at least one heterologous moiety can be linked to the C-terminus of the monomer polypeptide.
Illustrative, non-limitative, examples of this embodiment of the trimeric complex of the invention wherein each one of the monomer polypeptides are linked to at least one heterologous moiety include trimeric complexes of the invention in which:
Thus, as disclosed above, the trimeric complex of the invention may be a homotrimer (i.e., all the monomer polypeptides are equal each other), with the proviso that at least one of said three monomer polypeptides does not contain an endostatin domain or a restin domain, or a heterotrimer (i.e., at least one of the monomer polypeptides is different from the other two monomer polypeptides).
As used herein, the term “homotrimer” refers to trimers wherein the heterologous moieties connected to each of the monomer polypeptides are the same, regardless of whether the monomer polypeptides contain or not an endostatin domain or a restin domain. As used herein, the term “heterotrimer” refers to trimers wherein at least two of the heterologous moieties connected to different monomer polypeptides are not same, regardless of whether the monomer polypeptides contain or not an endostatin domain or a restin domain.
In a particular embodiment, when the heterologous moiety is an antibody or a fragment thereof, the trimeric complex of the invention may be monospecific (e.g., when the heterologous moiety (e.g., antibody or fragment thereof) in each one of the three monomer polypeptides which constitute the trimeric complex of the invention is the same, with the proviso that at least one of said three monomer polypeptides does not contain an endostatin domain or a restin domain, i.e., it is specific for an antigen), bispecific (e.g., when one of the heterologous moieties (e.g., an antibody or fragment thereof recognizes a first antigen) in one of the monomer polypeptides which constitute the trimeric complex of the invention is different from another heterologous moiety (e.g., an antibody or fragment thereof which recognizes a second antigen—wherein said second antigen is different from said first antigen) in the same or different monomer polypeptide), or even, the specificity of the trimeric complex of the invention may be higher, (e.g., when one of the heterologous moieties (e.g., an antibody or fragment thereof recognizes a first antigen) in one of the monomer polypeptides which constitute the trimeric complex of the invention is different from another heterologous moiety (e.g., an antibody or fragment thereof which recognizes a second (different) antigen) in the same or different monomer polypeptide) and different from another heterologous moiety (e.g., an antibody or fragment thereof which recognizes a third (different) antigen) in the same or different monomer polypeptide).
Therefore, the trimeric complex of the invention constitutes an enormously flexible platform for different applications, including protein engineering.
Effectively, a particularly interesting embodiment of the invention is the possibility of designing oriented molecular assemblies, where one or more functional entities are located N-terminally to the XVTSE and one or more functional entities are located C-terminally to said trimerizing element (XVTSE). Such types of design may be particularly advantageous where a certain relative ratio is desired among the different functional entities included in a specific molecular unit. Such type of design may in addition be used if one or more functional entities for either structural or functional reasons appear incompatible within the same construct. Such may be the case, e.g., if one or more of the functional entities are expressed by large or bulky protein domains which for steric reasons might prevent formation of the trimeric molecular unit due to sterical constraints.
Further, the possibility of constructing bi-polar three-way fusion proteins in which one functionality is placed at the N-terminus of the XVTSE and a different functionality is placed at the C-terminus of said XVTSE is additionally advantageous in applications where large spatial separation between the two functionalities are desirable for optimal function. Examples of such application are, e.g., the deployment of binding domains (e.g. antibody-derived binding modules) for recognition and binding to binding sites located at or close to large structures like cell membranes in cases where it is advantageous to allow for binding of the other end of the trimerized molecule to a different, but also bulky target.
Hence, as discussed above, the trimeric complex of the invention may be used to join, e.g., bulky surfaces by said complex comprising at least one heterologous moiety which is positioned at the N-terminus of the XVTSE and at least one heterologous moiety which is positioned at the C-terminus of said XVTSE. The two heterologous moieties can be either part of the same monomer polypeptide or part of two separate monomer polypeptides.
The XVTSE will, essentially indiscriminately, form homo- and hetero-trimers with any molecule that also contains said trimerization module. For some applications, it may be advantageous to have available specially engineered derivatives of the XVTSE, which have been reengineered to disallow homotrimer formation and hence only allow hetero-trimerization. Thus, an important embodiment of the monomer polypeptide which constitutes the trimeric complex of the invention is constructed/reengineered so as to disfavour formation of complexes between identical XVTSEs; this also has the implication that said monomer polypeptides can advantageously be designed so as to disfavour formation of trimers including two monomer polypeptides having identical XVTSEs. One way of disfavouring the formation of homo-trimerization would be by “knobs into holes” mutagenesis.
The design/reengineering may be accomplished by introduction of amino acid substitution at sites in the monomer polypeptide intimately involved in the formation and stability of the trimer and, simultaneously, in a different construct introduce a compensatory amino acid substitution, all in all removing symmetry between individual monomer components of the triple helical structure so that the structural complementarity profile only allows the formation of hetero-trimers, but is incompatible with some or each of the homotrimer species.
The trimeric complex of the invention may be prepared by methods generally known in the art, based, for example, techniques of recombinant protein production. Hence the invention also relates to a method of preparing the trimeric complex of the invention, the method comprising isolating the trimeric complex of the invention from a culture comprising a host cell which carries and expresses a nucleic acid fragment which encodes at least one of the monomer polypeptides of the trimeric complex of the invention, and, optionally, subjecting the trimeric complex of the invention to further processing.
The nucleic acid fragment which is mentioned above, hereinafter referred to as the nucleic acid of the invention, is also a part of the invention and is defined as a nucleic acid fragment in isolated form which encodes a XVTSE as defined herein, i.e., a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or a polypeptide having at least 60% amino acid sequence identity with the sequence shown in SEQ ID NO: 1, said polypeptide having the ability to form trimers, or which encodes the polypeptide part of a monomer polypeptide according to the invention. In a particular, embodiment, the sequence of the nucleic acid of the invention comprises, or consists of, the nucleotide sequence shown in SEQ ID NO: 2:
which corresponds to the N-terminus region of the human collagen XV NC1 domain.
In a particular embodiment, the nucleic acid of the invention comprises the nucleotide sequence encoding a XVTSE as defined herein.
In another embodiment, the nucleic acid of the invention encodes a XVTSE as defined herein but does not encode the endostatin or restin domain.
In another particular embodiment, the nucleic acid of the invention comprises the nucleotide sequence encoding the polypeptide part of a monomer polypeptide which is present in the trimeric complex of the invention. The polypeptide part of said monomer polypeptide comprises the XVTSE and the heterologous moiety when the heterologous moiety is a peptide. As it has been previously mentioned, the monomer polypeptide may be fused to one or more heterologous moieties said one or more heterologous moieties being fused to one or both ends of the monomer polypeptide; thus, when said heterologous moieties are peptides, the nucleic acid of the invention comprises the nucleotide sequence encoding the XVTSE and the nucleotide sequence or sequences encoding said heterologous moiety or moieties. In that case, the 5′ end of the nucleotide sequence encoding said heterologous moiety can be fused to the 3′ end of the nucleotide sequence encoding the XVTSE, or, alternatively, the 3′ end of the nucleotide sequence encoding said heterologous moiety can be fused to the 5′ end of the nucleotide sequence encoding the XVTSE. Said nucleotide sequences can be operatively linked so that each sequence is correctly expressed by just one promoter or, alternatively, each nucleotide sequence is under the control of independent promoters. Said promoter(s) can be inducible or constitutive. The nucleic acid of the invention may include, if desired, operatively linked, the nucleotide sequence encoding the spacer between the monomer polypeptide and the heterologous moiety and/or, the nucleotide sequence encoding the cleavable linker between the monomer polypeptide and the tag (heterologous moiety).
The nucleic acid of the invention can be prepared by traditional genetic engineering techniques.
The above mentioned host cell, hereinafter referred to as the host cell of the invention, which is also a part of the invention, can be prepared by traditional genetic engineering techniques which comprise inserting the nucleic acid of the invention into a suitable expression vector, transforming a suitable host cell with the vector, and culturing the host cell under conditions allowing expression of the XVTSE or the polypeptide part of the monomer polypeptide of the invention. Said vector comprising the nucleic acid of the invention, hereinafter referred to as the vector of the invention, is also a part of the invention. The nucleic acid of the invention may be placed under the control of a suitable promoter which may be inducible or a constitutive promoter. Depending on the expression system, the polypeptide may be recovered from the extracellular phase, the periplasm or from the cytoplasm of the host cell.
Suitable vector systems and host cells are well-known in the art as evidenced by the vast amount of literature and materials available to the skilled person. Since the present invention also relates to the use of the nucleic acid of the invention in the construction of vectors and in host cells, the following provides a general discussion relating to such use and the particular considerations in practising this aspect of the invention.
In general, of course, prokaryotes are preferred for the initial cloning of the nucleic acid of the invention and constructing the vector of the invention. For example, in addition to the particular strains mentioned in the more specific disclosure below, one may mention by way of example, strains such as E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative rather than limiting.
Prokaryotes can be also utilized for expression, since efficient purification and protein refolding strategies are available. The aforementioned strains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may be used.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microorganism for expression.
Those promoters most commonly used in recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems and a tryptophan (trp) promoter system (EP 36776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors. Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means.
In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used. This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics, 85:23-33). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.
In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, Human Embryonic Kidney (HEK) 293 and MDCK cell lines.
Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
For use in mammalian cells, the control functions on the expression vectors are often provided by viral material; for example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus (CMV) and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., polyoma, adeno, etc.) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
Upon production of the monomer polypeptide which constitutes the trimeric complex of the invention, it may be necessary to process the polypeptides further, e.g. by introducing non-proteinaceous functions in the polypeptide, by subjecting the material to suitable refolding conditions (e.g. by using the generally applicable strategies suggested in WO 94/18227), or by cleaving off undesired peptide moieties of the monomer (e.g. expression enhancing peptide fragments which are undesired in the end product).
In the light of the above discussion, the methods for recombinantly producing said trimeric complex of the invention or monomer polypeptide according to the invention are also a part of the invention, as are the vectors carrying and/or being capable of replicating the nucleic acid of the invention in a host cell or in a cell-line. According to the invention the expression vector can be, e.g., a virus, a plasmid, a cosmid, a minichromosome, or a phage. Especially interesting are vectors which are integrated in the host cell/cell line genome after introduction in the host.
Another aspect of the invention are transformed cells (i.e., the host cell of the invention), useful in the above-described methods, carrying and capable of replicating the nucleic acid of the invention; the host cell can be a microorganism such as a bacterium, a yeast, or a protozoan, or a cell derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. Especially interesting are cells from the bacterial species Escherichia, Bacillus and Salmonella, and a preferred bacterium is E. coli.
Yet another aspect of the invention relates to a stable cell line producing the monomer polypeptide according to the invention or the polypeptide part thereof, and preferably the cell line carries and expresses a nucleic acid of the invention. Especially interesting are cells derived from the mammalian cell lines HEK and CHO.
As it is evident for the skilled person in the art, the trimer complex of the invention may be a homotrimer (i.e., all the three monomer polypeptides are identical, with the proviso that at least one of said three monomer polypeptides does not contain an endostatin domain or a restin domain) or a heterotrimer (i.e., at least one of the three monomer polypeptides is different from the others).
When the trimer complex of the invention is a homotrimer, with the above mentioned exception, the method for recombinantly producing said homotrimer comprises inserting the nucleic acid of the invention into a suitable expression vector, transforming a suitable host cell with the vector, and culturing the host cell under conditions allowing expression of the monomer polypeptide according to the invention and trimerization thereof. In this case, only one nucleic acid of the invention has to be prepared, said nucleic acid of the invention comprising the nucleotide sequence encoding the XVTSE and the nucleotide sequence encoding the polypeptide part of the heterologous moiety. The considerations applying to further processing mentioned above apply to this method also.
When the trimer complex of the invention is a heterotrimer, said heterotrimer may comprise (i) only one monomer polypeptide different from the other two monomer polypeptides, these two monomer polypeptides being identical each other, or, alternatively, (ii) three different monomer polypeptides.
In the first case, when the trimer complex of the invention is a heterotrimer wherein only one monomer polypeptide (MP1) is different from the other two monomer polypeptides (MP2), the method for recombinantly producing said heterotrimer comprises inserting a first nucleic acid of the invention comprising the nucleotide sequence encoding said monomer polypeptide (MP1) into a suitable expression vector, and a second nucleic acid of the invention comprising the nucleotide sequence encoding the other monomer polypeptide (MP2) into a suitable expression vector, transforming (co-transfecting) a suitable host cell with said vectors, and culturing the host cell under conditions allowing expression of the monomer polypeptides according to the invention and trimerization thereof to render the heterotrimer. In this case, two nucleic acids of the invention have to be prepared, one of them comprising the nucleotide sequence encoding a monomer polypeptide (e.g., MP1) and the other one encoding the other monomer polypeptide (MP2). The considerations applying to further processing mentioned above apply to this method also.
In the second case, when the trimer complex of the invention is a heterotrimer wherein all the three monomer polypeptides are different each other (e.g., MP1, MP2 and MP3), the method for recombinantly producing said heterotrimer comprises inserting a first nucleic acid of the invention comprising the nucleotide sequence encoding monomer polypeptide 1 (MP1) into a suitable expression vector, inserting a second nucleic acid of the invention comprising the nucleotide sequence encoding monomer polypeptide 2 (MP2) into a suitable expression vector, and inserting a third nucleic acid of the invention comprising the nucleotide sequence encoding monomer polypeptide 3 (MP3) into a suitable expression vector, transforming (co-transfecting) a suitable host cell with said vectors, and culturing the host cell under conditions allowing expression of the monomer polypeptides according to the invention and trimerization thereof to render the heterotrimer. In this case, three nucleic acids of the invention have to be prepared, one of them comprising the nucleotide sequence encoding one monomer polypeptide (e.g., MP1), another encoding other monomer polypeptide (MP2), and another comprising the nucleotide sequence encoding another monomer polypeptide (MP3). The considerations applying to further processing mentioned above apply to this method also.
A very important aspect of the invention is the possibility of generating a system designed especially for the individual circumstances. The basic idea is that the artificial selection of heterologous moieties and optionally active components, and functional entities result in a unique system as will be further disclosed in the following.
Using the XVTSE as a vehicle for assembling monovalent scFv or Fab antibody fragments into oligomeric and multivalent entities offer design advantages also in terms of generating chimaeric artificial antibodies having desirable pharmacokinetic and pharmacodynamic properties. Small derivatives like monomeric scFv fragments or bivalent “minibodies” are rapidly cleared from the circulatory system, whereas native Igs have longer half-life. Conversely, small derivatives like scFv and minibodies exhibit better extravasation properties. It is therefore expected that antibodies of a desired specificity may be optimized for particular diagnostic or therapeutic needs by engineering the pharmacological properties, using the XVTSE as a vehicle for controlled oligomerization of e.g. scFv fragments.
One example of such engineering would be the requirements for delivering a high dose of an imaging or toxin-conjugated antibody to a tumor, while ensuring as low a systemic exposure or imaging background as possible. In such case a XVTSE-fused or conjugated scFv fragment could be designed to exhibit strong multivalent binding to the tumor and rapid clearance of excess fusion protein or conjugate from circulation.
Accordingly, in a further aspect, the present invention also relates to the use of the trimeric complex of the invention as a vehicle for assembled antibody fragments thus generating chimeric artificial antibodies having preselected pharmacokinetic and/or pharmacodynamic properties.
The use of specific delivery systems also play an important role in connection with the present invention in that such systems may be utilized with respect to different use of the present invention both with respect to the a more general therapeutic application and with respect to gene therapy. Examples of suitable drug delivery and targeting systems are disclosed in Nature 392 supp. (30 Apr. 1998).
Furthermore, a review of the imaging and therapeutic potential of a range of known antibody derivatives has been published [Holliger & Hudson, Nature Biotechnology, 2005, vol 23, 1126-1136; and Wu & Senter, Nature Biotechnology, 2005, vol 23, 1137-1146] In direct continuation of their conclusions it will be apparent that oligomerisation of antibody derivatives like scFv derivatives may extend current technology in the designer-antibody field in many important aspects, some of which will be elaborated below.
One of the well-known problems inherent to mouse monoclonal antibodies that have been “humanized” by grafting of the murine antigen combining site onto a human Ig framework is that antigenicity of the chimaeric product in human patients is often difficult to suppress entirely, resulting in sometimes life threatening immune reactions to the diagnostic or therapeutic humanized antibody product. The risks of such side-effects are expected to be much reduced if the designed antibody is assembled from purely human proteins or protein fragments. Since, in a particular embodiment, the XVTSE trimerization unit described herein is identical to a portion of human collagen XV that is already present in human plasma and tissue, there is good reason to expect that the XVTSE will not elicit an antigenic response in a human subject if it is introduced as a component of a chimaeric product that is not otherwise antigenic in humans.
Accordingly, in another aspect, the present invention relates to the use of a trimeric complex of the invention or a monomer polypeptide according to the present invention as a component of a chimaeric product having low antigenicity in humans relative to formulations comprising on or more components of non-human origin.
In connection with the technology for radiolabelling of antibody derivatives, again, oligomerisation using XVTSE offer more elegant solutions to problems associated with labelling, as the XVTSE offers the possibility to construct one or two of the XVTSE monomer units in a heterotrimeric complex to harbour the site carrying the label. Thus, in this format labelling may also be confined to the non-antibody part of the complex, leaving the antigen-binding module entirely unmodified, and the complex may furthermore be formulated “in the field” as and when needed.
In molecular trapping approaches, it is important to increase the antibody size above the renal threshold to retard clearance rates. Trivalent antibodies (a particular embodiment of the trimeric complex of the invention), or trivalent soluble truncated versions of membrane receptors (triple trap) would have an increased binding stoichiometry to soluble cytokines and growth factors. Coupled with slowed clearance rates, the greater avidity of trivalent antibodies might translate into a greater ability than their monovalent or bivalent counterparts to bind and sequester soluble molecules. This can also be applied to other pathogenic particles against which antibody antidotes have been generated. These include toxins, such as botulinum neurotoxin A or anthrax toxin, and viruses, such as hepatitis viruses or varicella-zoster virus, in which multimerization has been demonstrated to be needed for effective neutralization.
In many receptor-mediated signal transduction pathways signals are triggered by the clustering of receptor molecules on the cell membrane. The XVTSEs therefore have important applications in the study and exploitation of receptor signalling, especially for receptors that trimerize upon ligand binding such as those of the TNF family of receptors (e.g. CD137, OX40) as ligands may be presented as trimers by genetic fusion or conjugation to a XVTSE unit. This also has important application in phage display technologies for discovering new ligands and new receptors as the engineering of a XVTSE unit fused in-line to a candidate ligand molecule will allow the display of a hetero-trimeric phage coat protein, in which only one of the monomer units is linked to the phage coat protein. This may be accomplished by appropriate insertion of amber codons at the site of fusion of phage coat protein to the XVTSE-ligand segment of the three-way fusion protein encoded by the recombinant phage. In appropriate E. coli cells the presence of this amber codon will result in translation termination in the majority of read-throughs, and hence most of the fusion protein product secreted to the periplasmic compartment in the phage-infected bacterium will be soluble XVTSE-ligand fusion protein, whereas a minority of the fusion protein will also contain a phage protein module. The majority of trimers that will be generated will therefore contain, at most, one monomeric unit that will ensure integration (display) in the mature recombinant phage particle.
A further advantage of the display technology described above relates to the fact that it is specially useful for selection on the basis of a relatively low affinity because of the entropic benefit contribution obtained by the proximity of the three binding moieties in confined spatial arrangement. Accordingly, the present invention in an important aspect, also relates to protein library technology wherein the XVTSE's described above are utilized.
The trimerization of candidate recombinant ligands is especially important as, for many receptors, the intracellular signal is induced by receptor clustering, which is only brought about if the external ligand exhibits multivalent binding to the receptor, so as to bridge two or more receptor molecules.
As mentioned above, it is an important aspect of the invention that the trimeric complex of the invention or a monomer polypeptide according to the invention may be used as a component of a chimaeric product having low antigenicity in humans. As the monomer polypeptide can be of human origin it is believed that the antigenicity in humans is low relative to formulations comprising on or more components of non-human origin.
One primary use of a trimeric complex of the invention or a monomer polypeptide according to the invention is for delivering an imaging or toxin-conjugated or genetically fused antibody to a target such as a tumor, or use as a vehicle delivering a substance to a target cell or tissue, as a vehicle for assembling antibody fragments into oligomeric or multivalent entities for generating chimeric artificial antibodies having pre-selected pharmacokinetic and/or pharmacodynamic properties.
The substance in question being one or more selected from the group of heterologous moieties as well as a pharmaceutical. Also a labelled construct wherein the label is coupled to one or more of the XVTSE monomer units is within the scope of the invention.
As explained in detail previously, an important and surprising use of the trimeric complex of the invention or a monomer polypeptide according to the present invention is for protein library technology, such as phage display technology.
A further use according to the invention includes the preparation and use of a pharmaceutical composition comprising the trimeric complex of the invention or a monomer polypeptide according to the invention and optionally a pharmaceutically acceptable excipient. The composition may be administered by a route selected from the group consisting of the intravenous route, the intra-arterial route, the intravitreal route, the transmembraneus route of the buccal, anal, vaginal or conjunctival tissue, the intranasal route, the pulmonary route, the transdermal route, the intramuscular route, the subcutaneous route, the intratechal route, the oral route, inoculation into tissue such as a tumor, or by an implant.
It is obvious from the disclosure of the present invention that the treating or preventing of a disease may be a further aspect comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition referred to above.
In a further use according to the invention, the trimeric complex of the invention comprises at least a heterologous moiety, wherein said heterologous moiety is an anti-angiogenesis compound for use in the prevention and/or treatment of an angiogenesis related disease.
In the context of the present invention, “angiogenesis” is understood to mean the physiological process that consists of the formation of new blood vessels from existing blood vessels. Angiogenesis is also known as neovascularization.
The expression “angiogenesis related disease” relates to all those diseases where pathogenic angiogenesis occur i.e. when said process is harmful or undesirable, whether cancerous or not. The scope of the present invention thus excludes the treatment of angiogenesis in situations where it is necessary, such as wound healing. Diseases associated to an undesired angiogenesis which may be treated with the compounds in accordance with the present invention, without limitation, are inflammatory diseases, especially chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, sarcoidosis and such like; autoimmune diseases; viral diseases; genetic diseases; allergic diseases; bacterial diseases; ophthalmological diseases such as diabetic retinopathy, premature retinopathy, proliferative atrial retinopathy, retinal vein occlusion, macular degeneration, senile discoid macular degeneration, neovascular ocular glaucoma, choroidal neovascularization diseases, retinal neovascularization diseases, rubeosis (angle neovascularization), corneal graft rejection, retrolental fibroplasia, epidermal keratoconjunctivitis, vitamin A deficiency, contact lens exhaustion, atopical keratitis, superior limbic keratitis, pterygium dry eye, Sjögrens syndrome, acne rosacea, phlyctenulosis, syphilis, mycobacterial infections, lipid degeneration, burns with corrosive substances, bacterial ulcers, mycotic ulcers, protozoan infections, Kaposi sarcoma, Mooren's ulcer, Terrien marginal degeneration, marginal keratolysis, scleritis, chronic retinal detachment and such like; atherosclerosis; endometriosis; obesity; cardiac insufficiency; advanced renal insufficiency; endotoxemia; toxic shock syndrome; meningitis; silicon-induced fibrosis; asbestos-induced fibrosis; apoplexia; periodontitis; gingivitis; macrocytic anaemia; refractory anaemia; 5q deletion syndrome; conditions where the vascularization is altered as infection by HIV, hepatitis, hemorrhagic telangiectasia or Rendu-Osler-Weber's disease.
In a preferred embodiment, the disease associated to an undesired angiogenesis is a disease selected from cancer, rheumatoid arthritis, psoriasis, sarcoidosis, diabetic retinopathy, premature retinopathy, retinal vein occlusion, senile discoid macular degeneration, atherosclerosis, endometriosis and obesity, preferably cancer.
In a particular embodiment the diseases associated to an undesired angiogenesis are inflammatory diseases. “Inflammatory disease” is understood to be any disease where there is an excessive or altered inflammatory response that leads to inflammatory symptoms. Said inflammatory diseases which may be treated by compounds of the invention include, without limitation, Addison's disease, acne vulgaris, alopecia areata, amyloidosis, ankylosing spondylitis, ulcerations, aphthous stomatitis, arthritis, arteriosclerosis, osteoarthritis, rheumatoid arthritis, bronchial asthma, Bechet's disease, Boeck's disease, intestinal inflammatory disease, Crohn's disease, choroiditis, ulcerative colitis, celiac's disease, cryoglobulinemia, macular degeneration, dermatitis, dermatitis herpetiformis, dermatomyositis, insulin dependent diabetes, juvenile diabetes, inflammatory demyelinating disease, Dupuytren contracture, encephalomyelitis, allergic encephalomyelitis, endophthalmia, allergic enteritis, autoimmune enteropathy syndrome, erythema nodosum leprosum, ankylosing spondylitis, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, cystic fibrosis, gingivitis, glomerulonephritis, Goodpasture syndrome, Graves syndrome, Hashimoto's disease, chronic hepatitis, histiocytosis, regional ileitis, iritis, disseminated lupus erythematous, systemic lupus erythematous, cutaneous lupus erythematous, lymphogranuloma, infectious mononucleosis, miastenia gravis, transverse myelitis, primary idiopathic myxedema, nephrosis, obesity, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, panniculitis, pemphigus vulgaris, periodontitis, polyarteritis nodosa, chronic polyarthritis, polymyositis, acute polyradiculitis, psoriasis, chronic obstructive pulmonary disease, purpura, gangrenous pioderma, Reiter's syndrome, diabetic retinopathy, rosacea, sarcoidosis, ataxic sclerosis, progressive systemic sclerosis, scleritis, sclerodermia, multiple sclerosis, disseminated sclerosis, acute anterior uveitis, vitiligo, Whipple's disease, diseases associated to AIDS, severe combined immunodeficiency and Epstein Barr's virus such as Sjögren's syndrome, osteoarticular tuberculosis and parasitic diseases such as leishmaniasis. Preferred inflammatory diseases are rheumatoid arthritis, psoriasis, sarcoidosis, diabetic retinopathy, macular degeneration, arteriosclerosis and obesity.
In another preferred embodiment the disease is cancer.
The terms “cancer” and “tumor” relate to the physiological condition in mammals characterized by unregulated cell growth. The trimeric polypeptide complexes according to the invention are useful for the treatment of any cancer or tumor, such as, without limitation, breast, heart, lung, small intestine, colon, splenic, kidney, bladder, head, neck, ovarian, prostate, brain, pancreatic, skin, bone, bone marrow, blood, thymic, uterine, testicular and liver tumors. Particularly, tumors which can be treated with said antibodies include but are not limited to adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and teratoma. Particularly, the tumor/cancer is selected from the group of acral lentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, germ-line tumors, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large-cell carcinoma, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small-cell carcinoma, soft tissue carcinoma, somatostatin secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm's tumor. In one embodiment of the present invention, the tumor is selected from the group consisting of: breast carcinoma, prostate carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, renal carcinoma, gastric carcinoma, ovarian carcinoma, papillary thyroid carcinoma, melanoma, hepatocellular carcinoma, bladder carcinoma, liposarcoma invasive carcinoma, neuroblastoma, esophageal squamous carcinoma, osteosarcoma, gallbladder carcinoma, oral squamous carcinoma, endometrial carcinoma, and medulloblastoma.
As used in the conventional pharmaceutical field the present invention includes a method wherein the trimeric complex of the invention or a monomer polypeptide according to the invention is administered by a route selected from the group consisting of the intravenous route, the intra-arterial route, the transmembraneus route of the buccal, anal or vaginal tissue, intranasal route, intravitreal route, the pulmonary route, the transdermal route, intramuscular route, subcutaneous route, intratechal route, the oral route, inoculation into tissue such as a tumor, or by an implant.
A further use according to the invention includes the use of the trimeric complex of the invention for delivering an imaging agent to a target cell or tissue. The trimeric complex of the invention may comprise at least one heterologous moiety that enables targeting to a cell or tissue, such an antibody fragment as described above, and an imaging agent.
The term “imaging agent” and “contrast agent”, are used herein interchangeably and refer to a biocompatible compound, the use of which facilitates the differentiation of different parts of the image, by increasing the “contrast” between those different regions of the image. The term “contrast agents” thus encompasses agents that are used to enhance the quality of an image that may nonetheless be generated in the absence of such an agent (as is the case, for instance, in MRI), as well as agents that are prerequisites for the generation of an image (as is the case, for instance, in nuclear imaging). Suitable contrast agent include, without limitation, contrast agents for Radionuclide imaging, for computerized tomography, for Raman spectroscopy, for Magnetic resonance imaging (MRI) and for optical imaging.
Contrast agents for radionuclide imaging include iodine 123, technicium 99, indium 111, rhenium 188, rhenium 186, copper 67, iodine 131, yttrium 90, iodine 125, astatine 211, gallium 67, iridium 192, cobalt 60, radium 226, gold 198, cesium 137 and phosphorus 32 ions. Examples of fluorogenic agents include gadolinium and renographin. Examples of paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (H)3 copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holraium (III) and erbium (III) ions.
Contrast agents for optical imaging include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green derivative, rhodamine green, a derivative of rhodamine green, an eosin, an erythrosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye and the various other fluorescent compounds disclosed herein.
Contrast agent for magnetic resonance imaging apparatus gadolinium chelates, manganese chelates, chromium chelates, 19F and iron particles.
MRI contrast agents include complexes of metals selected from the group consisting of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).
Finally, in another aspect, the invention relates to a method for the imaging of a target cell comprising contacting said cell with a trimeric polypeptide complex according to the invention wherein said trimeric polypeptide complex comprises a first heterologous moiety which is an imaging agent and a second heterologous moiety which is a molecule for which specific binding sites are present in the target cell.
The invention is hereby described by way of the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention.
The monoclonal antibody (mAb) specific for human c-myc used was 9E10 (Abcam, Cambridge, UK). The IRDye 800 (fluorochrome)-conjugated goat anti-mouse IgG polyclonal antibody (Fc specific) was from Rockland Immunochemicals Inc (Gilbertsville, Pa., USA). Laminin 111 extracted from the Engelbreth-Holm-Swarm (EHS) mouse tumor was from Becton Dickinson Labware (Bedford, Mass., USA). Human carcinoembryonic antigen (CEA), human and mouse sera, as well as Cathepsin L, and Elastase were from Sigma-Aldrich Inc. (St. Louis, Mo., USA).
HEK-293 cells (human embryo kidney epithelia; CRL-1573) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated Fetal Calf Serum (FCS) (all from Invitrogen, Carlsbad, Calif.) referred as to DMEM complete medium (DCM).
To construct the pCR3.1-L36-hXVNC1ES- expression vector, the polynucleotide encoding the N-terminal trimerization region from human collagen XV NC1 domain (hXVNC1ES-) [from nucleotide 3609 to nucleotide 3800] was synthesized by Geneart AG (Regensburg, Germany) and subcloned as NotIXbaI into the vector pCR3.1-L36 [Sanz, L. and Álvarez-Vallina, L. Antibody-based antiangiogenic cancer therapy (2005) Expert. Opin. Ther. Targets 9:1235-1245] containing the anti-laminin L36 single chain Fv (scFv) gene. The MFE23 (anti-human CEA) and the B1.8 (anti-hapten NIP) scFv expression cassettes were subcloned as HindIII-NotI from plasmid pVOM1.C23 and pVOM1.aNIP (kindly provided by Dr. R. E. Hawkins, from University of Manchester), into the vector pCR3.1-L36-hXVNC1ES-, resulting in pCR3.1-MFE23-hXVNC1ES- and pCR3.1-B1.8-hXVNC1ES-, respectively.
The vector pCR3.1-L36-mXVIIINC1ES- containing the anti-laminin L36 scFv and the N-terminal trimerization region from murine collagen XVIII NC1 domain (mXVIIINC1ES-) was constructed as described [Sanchez-Arevalo, L. et al., Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events (2006) Int. J. Cancer 119:455-462].
HEK-293 cells were transfected with pCR3.1-L36-mXVIIINC1ES- or pCR3.1-L36-hXVNC1ES-, pCR3.1-MFE-23-hXVNC1ES- or pCR3.1-B1.8-hXVNC1ES-expression vectors using Superfect (QIAGEN GmbH, Hilden, Germany), and selected in DCM supplemented with G418 (Geneticin) [500 μg/ml]. Supernatants from transient and stably transfected cell populations were analyzed for protein expression by ELISA, SDS-PAGE and Western blotting using anti-c-myc mAb. Stably transfected HEK-293 cells were used to collect serum-free conditioned medium (about 1 liter), and loaded onto a HisTrap HP 1 ml column using an ÄKTA Prime plus system (GE Healthcare, Uppsala, Sweden). The purified antibodies were dialyzed against PBS, analyzed by SDS-PAGE under reducing conditions, and stored at −20° C.
The ability of recombinant antibody constructs to bind laminin, CEA or NIP was studied by ELISA as described [Cuesta Á M, et al. (2009). In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences. PLoS ONE 4(4): e5381].
Analytical centrifugation and sedimentation equilibrium gradient experiments were performed at 20° C. in an Optima XL-A (Beckman Coulter, Miami, Fla.) analytical ultracentrifuge equipped with UV-visible optics, using an An50Ti rotor, with 3 mm double sector centerpieces of Epon charcoal. Short column (23 μl), low-speed sedimentation equilibrium was performed at 9,000 rpm, and the equilibrium scans were taken (after 20 h) at a wavelength of 297 nm. The baseline signal was measured after high-speed centrifugation (5 h at 42,000 rpm). Whole-cell apparent average molecular weight of recombinant antibodies was obtained using the program EQASSOC [Minton A Conservation of signal: a new algorithm for the elimination of the reference concentration as an independently variable parameter in the analysis of sedimentation equilibrium. In: Schuster T, Laue T, eds. Modern analytical ultracentrifugation (1994) Ed. Cambridge: Birkhauser Boston Inc., p. 81-93]. The sedimentation coefficients were calculated by continuous distribution c(s) Lamm equation model [Schuck P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling (2000). Biophys. J. 78:1606-1619) as implemented in the SEDFIT 11.8 program. These experimental sedimentation values were analyzed using the HETEROANALYSIS 1.1.33 program and corrected to standard conditions to get the corresponding s20,w values using the SEDNTERP program [Laue T. Computer-aided interpretation of analytical sedimentation data for proteins. In: Harding S E, Rowe A J, Horton J C, eds. Ultracentrifugation in biochemistry and polymer science (1992). Ed. Cambridge: Royal Society of Chemistry, p. 90-125]. Further hydrodynamic analysis (i.e., calculation of frictional coefficient ratio) was performed with the SEDFIT program to obtain the c(M) distribution.
To determine whether recombinant antibody constructs remained functional in serum, 1 microgram (μg) of each purified antibody (L36-hXVNC1ES- and L36-mXVIIINC1ES-) was incubated in 62% human or mouse serum at 37° C. for up to 72 h. Samples were removed for analysis at 3 h, 24 h, 48 h, and 72 h following the start of incubation and frozen until the entire study was completed. As a control, a second set of serum-exposed samples was frozen immediately to represent a zero time point. Aliquots were then subjected to Western blot, using an anti-c-myc mAb, and tested for their capability to bind laminin by ELISA.
To determine whether recombinant antibody constructs remained functional when incubated with different proteases, 1 microgram (μg) of each purified antibody (L36-hXVNC1ES- and L36-mXVIIINC1ES-) was incubated at 37° C. for 10 min with 200, 300, and 400 ng cathepsin L in 50 mM sodium acetate [pH 5.5], 2 mM dithiothreitol, 5 mM EDTA; and 50, 100, and 200 ng elastase in 100 mM Tris-HCl [pH 8.0]. Aliquots were then subjected to Western blot, using an anti-c-myc mAb, and tested for their capability to bind laminin by ELISA.
The NC1 domains of collagens XVIII and XV have similar primary structures [Sergei P. Boudko, “Crystal Structure of Human Collagen XVIII Trimerization Domain, A Novel Collagen Trimerization Fold”, JMB, Volume 392, Issue 3, 25 Sep. 2009, pages 787-802], which are organized into three different subdomains: an amino-terminal region potentially responsible for homotrimerization is followed by a protease-labile segment and the endostatin domain. The inventors have previously shown that fusion of the N-terminal trimerization region of the murine collagen XVIII NC1 domain (mXVIIINC1ES-) to the C-terminus of the L36 scFv fragment (L36-mXVIIINC1ES-) confers their natural oligomeric state to the fused antibody. The homo-trimeric molecules were isolated in functional active form from the cell culture supernatant of gene-modified 293 cells [Sanchez-Arevalo et al. (2006), ad supra].
In this study, inventors have extended the concept by designing trimeric antibodies using the N-terminal trimerization region of the human collagen XV NC1 domain (hXVNC1ES-). Starting from the L36 scFv gene a new recombinant antibody was generated. The L36 scFv-hXVNC1ES- fusion protein was secreted as soluble functional protein in transfected human 293 cells (
The functionality of purified recombinant L36-hXVNC1ES- antibody was demonstrated by ELISA against plastic immobilized laminin (
The oligomerization state of the different constructs was assessed by analytical centrifugation and sedimentation equilibrium gradient. Both L36-hXVNC1ES- and L36-hXVNC1ES- recombinant antibodies eluted from the column as a single peak with and estimated masses of 111.4 kDa for the L36-hXVIIINC1ES- recombinant antibody, and 117.6 kDa for the L36-hXVNC1ES- recombinant antibody (
To further evaluate type XV trimerization domain as a new platform for engineering multivalent antibodies, the inventors designed new constructs with specificity for the hapten NIP or the tumor-associated antigen CEA. The scFv B1.8 (anti-NIP) and the scFv MFE-23 (anti-CEA) were similarly assembled and expressed as soluble secreted antibody constructs in human HEK-293 cells. The expression and functionality of B1.8-hXVNC1ES- and MFE23-hXVNC1ES- antibodies was demonstrated by Western blot (
The stability of engineered antibody fragments in serum is the critical parameter to determine their potential application in vivo. Therefore, inventors compared the functionality of both NC1 collagen derived constructs (L36-mXVIIINC1ES- and L36-hXVNC1ES-) after incubation in mouse or human serum at 37° C., for prolonged periods of time. As shown in
Furthermore, their structural integrity and functional stability were compared by incubation, for 10 minutes at 37° C., with different amounts of proteases (cathepsin L and elastase). As shown in
1. Recombinant antibody molecules containing the trimerization region from collagen XV NC1 domain are trimeric in solution and recognize the cognate antigen as efficiently as recombinant antibody molecules, with the trimerization region from collagen XVIII NC1 domain.
2. Recombinant antibody molecules containing the trimerization region from collagen XV NC1 domain were significantly more stable in serum and in the presence of proteases than recombinant antibody molecules, with the trimerization region from collagen XVIII NC1 domain.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10382234.2 | Aug 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/064363 | 8/22/2011 | WO | 00 | 7/7/2014 |
