Patent Application
20110159018
The present invention relates to a complement activating construct comprising a complement factor H-derived short consensus repeat (fH-derived SCR) and a binding molecule which specifically recognizes a pathogen. More specifically, the fH-derived SCR is selected from the group consisting of SCR7, SCR9, SCR13SCR18-20 and artificial SCR (aSCR). Furthermore, an in vivo method for screening complement-based approaches for the treatment of the prevention, treatment or amelioration of an infection with a pathogen or a pathological condition associated with an infection with a pathogen is described.
Animal viruses such as animal RNA viruses can be inactivated and lysed by human serum (see: Takeuchi (1994) J Virol. 68: 8001-7; Cooper (1976) J Exp Med. 144: 970-84; Bartholomew (1978) J Immunol. 121: 1748-51; Bartholomew (1978) J Exp Med. 147: 844-53; Sherwin (1978) Int J Cancer. 21(1): 6-11; Jensen (1979) Hamatol Bluttransfus. 23: 501-3; Kobilinsky (1980) Infect Immun. 29(1): 165-70); Dierich (1996) in: Immunology of HIV infection, editor: S. Gupta, New York, Plenum Press, 365-376). This neutralising property is mediated by human complement. Also human retroviruses such as human T-lymphotropic virus (HTLV) or human immunodeficiency virus (HIV) activate the complement system. These viruses trigger the classical pathway already during the acute phase of infection, resulting in a deposition of C3-fragments on the viral surface (Ebenbichler (1991) J Exp Med. 174: 1417-24; Stoiber (2001) Immunol Reviews 180:168-76; Saifuddin (1995) AIDS Res Hum Retroviruses 11: 1115-22; Stoiber (1997) Annu Rev Immunol. 15: 649-674).
Although inactivated by animal sera, activation of the complement cascade by HTLV or HIV seems to result only in partial virolysis when incubated with human serum (Stoiber (1997) loc. cit.; Sullivan (1996) J Immunol 157: 1791-1798; Dierich (1996) Nature Med 2: 153-155). Responsible for this intrinsic resistance against human complement are host cell-derived proteins, which are acquired by HIV during the budding process (Frank (1996) AIDS 10: 1611-20). Among them are regulators of complement activation (RCA) such as CD46 (MCP), CD55 (DAF) or CD59 which down-regulate the complement system (Montefiori (1994) Virology 205: 82-92; Saifuddin (1995) J Exp Med. 182: 501-509; Schmitz (1995) J Clin Invest. 96: 1520-6; Marschang (1995) Eur J. Immunol. 25: 285-290; Takefman (1998) Virology 46: 370-378).
The intrinsic resistance of retroviruses against complement of their natural host seems to represent a general phenomenon. A mouse retrovirus is resistant to mouse serum, but is efficiently destroyed by complement of other species, such as human, feline or sheep (Spear (1991) Immunology 73: 377-82 and own unpublished observations). Similarly, as discussed above, HIV is not affected by human complement, but is lysed by animal sera within minutes. Thus, retroviruses have adapted similar but species specific protection mechanisms to keep complement activation in their natural host under the threshold necessary to induce virolysis. Therefore, opsonised virions accumulate in retrovirus-infected hosts.
Previously, it has been shown that viruses bind complement factor H (fH) in fluid phase. Since fH is a negative regulator of complement activation (RCA), said fH binding promotes significant protection against complement-induced lysis of virus particles such as HIV (Stoiber (1996) J Exp Med. 183: 307-310). The crucial role of fH for protection of the virus is evident, since incubation of HIV with fH-depleted sera results in up to 80% of complement-dependent virolysis in the presence of HIV-specific antibodies (Stoiber (1996) loc. cit.).
A common RCA motif is a repeat of about 60 amino acids (aa), i.e. short consensus repeats (SCRs) or complement consensus repeats (CCR) (Takeuchi (1994) loc. cit.). fH is organised in 20 SCR units (Prodinger (2004) in: Fundamental Immunology, editor: WE. Paul, Lippincott-Raven Publishers). The first 5 SCRs of fH have “decay-accelerating activity” serving as a cofactor for C3b inactivation (Prodinger (2004) loc. cit.). SCR 7, 9, 18-20 and probably SCR 13 mediate the binding of fH to negative charged surface elements such as heparin (Prodinger (2004) loc. cit.; Cheng (2006) Mol Immunol. 43: 972-9). Binding of fH to negatively charged host cells contributes to the protection of host cells against damage induced by the host's own complement.
EP-A1 0 854 150 describes reagents for the treatment of pathogen-induced disorders, whereby said reagents are peptide chains of less than 100 amino acids and are able to bind to a CFH (fH)-binding region. However, it could be shown that these peptides have still certain disadvantages and better treatment options are desired.
The technical problem underlying the present invention is to provide efficacious means and methods for prevention, treatment or amelioration of an infection with a pathogen or of a pathological condition associated with an infection with a pathogen or of a condition associated with a proliferative disease, like cancer. The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, this invention relates to a short consensus repeat-antibody construct (SCR-Ab) comprising:
In one embodiment, said fH-derived SCR is selected from the group consisting of SCR7, SCR9, SCR13, SCR18-20 and artificial SCR (aSCR) or a functional fragment of said fH-derived SCR7, SCR9, SCR13, SCR18-20 and aSCR.
The SCR-Ab constructs to be employed in accordance with this invention comprise a binding molecule that specifically recognizes a pathogen additionally to the herein defined complement factor H-derived short consensus repeat. As documented herein below and in the appended examples it was, in accordance with this invention, surprisingly found that the inventive SCR-Ab constructs are capable of lysing pathogens in vitro in a highly unexpected manner. In contrast thereto, SCR-derived polypeptides that do not comprise a pathogen-specific binding molecule failed to induce lysis of the pathogen under the same conditions. Accordingly, the embodiments of the present invention overcome the disadvantages of the prior art, like EP-A1 0 854 150. It was also found that the herein described SCR-Ab constructs are useful in the treatment of an infection with a pathogen in vivo. As documented in the examples SCR constructs that lacked pathogen-specific binding molecule or SCR constructs that are not linked to a pathogen-specific binding molecule are much less efficacious and partially non-functional. These inventive constructs are also useful in the medical intervention of cancer and/or of proliferative disorders, whereby in these embodiments binding molecules are to be employed that specifically bind to or recognize a cancer cell, tumour cell or a malignant cell; see also items below. Therefore, it is also envisaged that the inventive pharmaceutical concept described herein may be employed in a medical setting where it is desired to inhibit and/or eliminate pathogen infected host cells or malignant cells, like cancer cells, malignant cells or tumour cells.
Therefore, the present invention also relates to short consensus repeat-antibody construct (SCR-Ab) comprising:
In one embodiment, said fH-derived SCR is selected from the group consisting of SCR7, SCR9, SCR13 and SCR18-20 or a functional fragment of said fH-derived SCR7, SCR13, SCR18-20 and artificial SCR (aSCR).
Monoclonal Antibodies (mAbs) offer a promising perspective for treatment of certain types of tumours (Reichert (2007) Nat Rev Drug Discov 6:349-356). mAbs targeting angiogenesis-regulating growth factors, such as VEGF or tumour antigens, enable a more potent therapeutic efficacy when combined with conventional drugs, thereby unifying classical chemotherapeutic schemes with new immunotherapeutic concepts (i.e. chemoimmunotherapy). Targeting Her2/Neu in breast cancer, CD20 in lymphoma as well as EGFR and VEGF in gastrointestinal cancer has already been proven to prolong survival as compared to chemotherapy alone and therefore represents an important additional treatment option for these cancer types (Reichert (2007) loc.cit.). Moreover, data from clinical studies testing an antibody directed against an extracellular epitope of Her2/Neu (Trastuzumab) in the adjuvant setting on Her2/neu overexpressing breast cancers after surgical resection of the primary tumour demonstrated a ˜30 percent reduction in the risk of death, which was highly significant when compared to adjuvant chemotherapy alone.
These data support the concept that mAbs are most promising therapeutics when applied in patients with minimal tumour load (i.e. with micro-metastases or residual tumour cells after resection of the solid primary tumour). Despite the outstanding potency of mAb, their clinical efficacy in human cancer is far from optimal (Holz (1998) Recent Results Cancer Res 146:214-218). Therefore, several strategies including the application of radioimmuno-conjugates or bispecific antibodies are currently tested in pre-clinical models as well as in clinical trials to enhance their clinical efficacy (Reichert (2007) loc.cit.).
The binding of mAbs to tumour cells is thought to induce complement activation which may result in the destruction of tumour cells by complement-dependent cellular cytotoxicity (CDCC) and complement-mediated lysis (Durrant (2001). Curr Opin Investig Drugs 2:959-966). However these complement-mediated effector functions are limited, as tumours, similar to normal cells, are protected from complement-induced damage by regulators of complement activation (RCAs), which are over-expressed by certain tumours (Durrant (2001) loc.cit.). Among these RCAs are membrane anchored regulators such as CD46 (MCP), CD55 (DAF) or CD59 and in addition, RCAs in fluid phase, like factor H (1H) (Bjørge (2005) Br J Cancer 9:895-905; Prodinger (2004) Complement. In: Fundamental Immunology. Ed.: Paul W E, Lippincott-Raven Publishers).
The crucial role of fH for protection of several tumours is shown for ovarian, lung and colonic cancer cells (Prodinger (2004) loc.cit.; Ajona (2007) J Immunol 178:5991-5997; Fedarko (2000) J Biol Chem 275(22):16666-16672; Kinders (1998) Clin Cancer Res 4:2511-2520).
As described, a common motif of RCAs is a repeat of about 60 amino acids (aa), called short consensus repeats (SCRs) or complement consensus repeats (Prodinger (2004) loc.cit.). fH is organised in 20 SCR units (Prodinger (2004) loc.cit.). The first 5 SCRs of fH exhibit “decay-accelerating activity” and promote C3b inactivation (Prodinger (2004) loc.cit.). SCR 7, 9, 18-20 and probably SCR 13 mediate the binding of fH to negative charged surface elements such as heparin sulfates (Prodinger (2004) loc.cit.). Binding of fH to negatively charged host cells contributes to the protection against damage induced by the host's own complement.
As not only RCAs but also negatively charged surface structures are up-regulated in certain tumours (Fedarko (2000) loc.cit.), fH binds with high preference to the tumour surface and therefore contributes to the protection against complement-induce lysis. As exemplified herein, blocking of RCAs such as fH increases the efficacy of mAb therapy.
As used herein, the term “short consensus repeat”, “SCR” “complement consensus repeat” or “CCR” relates to the consensus repeat motifs which can be deduced from polynucleotide or amino acid sequences encoding regulators of complement activity (RCA). Particularly, SCR which mediate the binding of the RCA to negatively charged elements such as heparin may be of relevance in the context of the current invention. In one embodiment, the SCR comprised in the SCR-Ab construct of the present invention may be a fH-derived SCR selected from the group consisting of SCR7, SCR9, SCR13, SCR18-20 and artificial SCR (aSCR) or a functional fragment thereof. Yet, it is of note that SCRs also may have “decay-accelerating activity” which is associated with the down-regulating of complement activation.
As used herein, the term “artificial short consensus repeat”, “artificial SCR” or “aSCR” relates to amino acid sequences (or polynucleotide sequences encoding such amino acid sequences) that are non-naturally occurring regulators of complement activity (RCA). “Artificial SCRs” as used herein mediate the binding of a non-naturally occurring RCA to negatively charged elements such as heparin. Accordingly, “aSCR” represents, inter alia, an artificial derivate of fH or a related protein of the complement regulator protein family, which is capable of binding to heparin. Amino acid motifs which function as heparin binding sites and which contribute to the binding an “artificial SCR” to negatively charged surface elements are known in the art; see inter alia Smith (2000) J Virol 74:5659-5666 and Ghebremariam (2005) Ann N Y Acad Sci 1056:113-122. Accordingly, “artificial SCR”/“aSCR” may comprise a repetitive sequence which contains clusters of basic amino acid residues such as R or K resulting in the sequence motif “R/K-X-R/K”, wherein “X” is any naturally occurring amino acid. Furthermore, it is described in the art that fH-derived proteins capable of binding to heparin are made up of more than 9% positively charged amino acids and have an overall isoelectric point (pI) of greater than 7.0 (Smith (2000) J Virol 74:5659-5666). The capability of heparin-binding artificial SCRs or fragments thereof to induce lysis of a pathogen can be tested by using the in vitro lysis assays as described herein below and in particular in the appended examples.
Taken together, artificial SCRs (or fragments thereof) according to the present invention and comprised in the inventive short consensus repeat-antibody construct (SCR-Ab) may comprise (i) repetitive surface-exposed K/R-X-K/R motifs (wherein “X” is any naturally occurring amino acid); (ii) comprise more than 9% positively charged amino acids; and/or (iii) have an overall isoelectric point (pI) of greater than 7.0. One example of an artificial SCR according to the present invention may comprise the amino acid sequence as shown herein below in SEQ ID NO: 32 or may be encoded by a polynucleotide comprised in SEQ ID NO: 31. Yet, the person skilled in the art is readily in the position to use and to obtain further artificial SCR useful in the SCR-Ab constructs as provided herein, for instance by employing the herein described in vitro assays for testing heparin binding and/or the herein described in vitro lysis assays.
As used herein, the term “binding molecule” as comprised in the inventive short consensus repeat-antibody construct (SCR-Ab) of this invention relates to a molecule that is able to specifically interact with (a) potential binding partner(s) so that is able to discriminate between said potential binding partner(s) and a plurality of different molecules as said potential binding partner(s) to such an extent that, from a pool of said plurality of different molecules as potential binding partner(s), only said potential binding partner(s) is/are bound, or is/are significantly bound. Methods for the measurement of binding of a binding molecule to a potential binding partner are known in the art and can be routinely performed e.g. by using a Biacore apparatus.
In EP-A1 0 854 150 it is taught that fH-derived pathogen-binding peptide chains preferably comprise a SCR13 region-derived sequence, whereas the SCR7 region of fH is to be deleted. EP-A1 0 854 150 does not provide for or hint to SCR-Ab constructs as described in the present invention. Surprisingly (and in contrast to this prior teaching), the SCR-Ab constructs of this invention may also comprise other fH-derived SCR sequences, including, inter alia, SCR7, SCR9, SCR18-20 and artificial SCR (aSCR).
In one embodiment, the fH-derived SCR as comprised in the SCR-Ab construct of the present invention may comprise the amino acid sequence as shown herein below in SEQ ID NOs: 4, 22, 24, 6, 26, 8, 30 or 32 or a functional fragment thereof:
Said amino acid sequences SEQ ID NOs: 4, 22, 24, 6, 26, 8, 30 and 32 may be encoded by a polynucleotide comprised in SEQ ID NOs: 3, 21, 23, 5, 25, 7, 29 and 31, respectively:
It is evident for the skilled artisan that the present invention is not limited to the specific short consensus repeat-antibody construct (SCR-Ab) sequences as provided herein.
In one embodiment, the fH-derived SCR as comprised in the SCR-Ab construct of the present invention may comprise a polypeptide encoded by the complementary sequence of a polynucleotide that is able to hybridize, preferably under stringent conditions with the polynucleotide as comprised in the above described SEQ ID NOs: 3, 21, 23, 5, 25, 7, 29 and 31 and wherein said SCR is capable of binding a fH binding site.
In another embodiment, the fH-derived SCR as comprised in the SCR-Ab construct of the present invention may comprise a polypeptide encoded by the amino acid sequence that is at least 60% identical to the amino acid sequence as comprised in SEQ ID NO: 4, 22, 24, 6, 26, 8, 30 or 32 or is encoded by a nucleic acid molecule which is at least 60% identical to the nucleic acid sequence as comprised in SEQ ID NO: 3, 21, 23, 5, 25, 7, 29 or 31 and wherein said polypeptide is capable of binding a complement factor H binding site on said pathogen.
In yet another embodiment, the fH-derived SCR as comprised in the SCR-Ab construct of the present invention may comprise an ortholog of the polypeptide encoded by the amino acid as comprised in SEQ ID NO: 4, 22, 24, 6, 26, 8 or 30 or is encoded by a nucleic acid molecule which is an ortholog of the nucleic acid sequence as comprised in SEQ ID NO: 3, 21, 23, 5, 25, 7 or 29 and wherein said polypeptide is capable of binding a complement factor H binding site on said pathogen. Methods for identifying orthologs of a given polypeptide are well known in the art including the herein described hybridization methods.
In yet another embodiment, the fH-derived SCR as comprised in the SCR-Ab construct of the present invention may comprise a polypeptide encoded by the amino acid sequence comprised in SEQ ID NO: 4 or SEQ ID NO: 22 or a functional fragment thereof.
In a most preferred embodiment, the basic amino acid residues as comprised in the amino acid sequence encoding the fH-derived SCR as comprised in the SCR-Ab construct of the present invention are not exchanged. Basic amino acid residues are lysine, arginine and histidine.
Methods which are suitable for testing whether a SCR or a fragment thereof is capable of binding a fH binding site are known in the art. Binding of fH is mediated, inter alia, through binding of the SCRs to negative surface elements such as heparin (Prodinger (2004) loc. cit.; Cheng (2006) loc. cit.). The identification of fH-derived SCRs or functional fragments that are useful in the context of the present invention, therefore, may be achieved by using the heparin binding assay as described herein below in the Examples. An example of a further fH derived SCR that is capable of binding heparin is SCR9. The capability of heparin-binding SCRs or fragments thereof to induce lysis of a pathogen can be tested by using the in vitro lysis assays as described herein below in the examples. Yet, the person skilled in the art is aware that other SCR binding assays or in vitro lysis assays may also be useful for testing fH-derived SCR polypeptide sequences for their use in the inventive SCR-Ab constructs as described herein.
The term “hybridization” or “hybridizes” as used herein may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington D.C., (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as 0.1×SSC, 0.1% SDS at 65° C. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6×SSC, 1% SDS at 65° C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may represent nucleic acid sequences which code for a fH-derived SCR or a functional fragment thereof which have a length of at least 12 nucleotides, preferably at least 15, more preferably at least 18, more preferably of at least 21 nucleotides, more preferably at least 30 nucleotides, even more preferably at least 40 nucleotides and most preferably at least 60 nucleotides. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an anti-parallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A”. Complementarity between two single-stranded molecules may be “partial”, in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.
The term “hybridizing sequences” preferably refers to sequences which display a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97% identity with a nucleic acid sequence as described above encoding an antibody molecule. Moreover, the term “hybridizing sequences” preferably refers to sequences encoding an fH-derived SCR or a functional fragment thereof having a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97% identity with an amino acid sequence of the fH-derived SCR sequences as described herein above.
In accordance with the present invention, the term “identical” or “percent identity” in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.
Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
Moreover, the present invention also relates to nucleic acid molecules whose sequence is being degenerate in comparison with the sequence of an above-described hybridizing molecule. When used in accordance with the present invention the term “being degenerate as a result of the genetic code” means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid. In order to determine whether an amino acid residue or nucleotide residue in a given fH-derived SCR sequence corresponds to a certain position in the amino acid sequence or nucleotide sequence of any of e.g. SEQ ID NOs: 4, 22, 24, 6, 26, 8, 30 and 32, the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned further down below in connection with the definition of the term “hybridization” and degrees of homology.
For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.
Analogous computer techniques using BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:
and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson, Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.
The binding molecule as comprised in the SCR-Ab construct of the present invention may be selected from the group consisting of antibody molecules, receptor molecules, aptamers, DARPins and the like. The person skilled in the art is readily in the position to use and to obtain specific binding molecules, useful in the SCR-Ab constructs as provided herein.
In the context of the present invention, the term “antibody molecule(s)” or “antibody(ies)” comprises antibody molecule(s) like full immunoglobulin molecules, e.g. IgM, IgD, IgE, IgA or IgG, like IgG1, IgG2, IgG2b, IgG3 or IgG4 as well as to parts of such immunoglobulin molecules, like Fab-fragments, Fab′-fragments, F(ab)2-fragments, chimeric F(ab)2 or chimeric Fab′ fragments, chimeric Fab-fragments or isolated VH- or CDR-regions (said isolated VH- or CDR-regions being, e.g. to be integrated or engineered in corresponding “framework(s)”). Accordingly, the term “antibody molecule” also comprises known isoforms and modifications of immunoglobulins, like single-chain antibodies or single chain Fv fragments (scAB/scFv) or bispecific antibody constructs, said isoforms and modifications being characterized as comprising at least one antigen binding site which specifically recognizes an antigen on the surface of a virus particle. A specific example of the above described isoform or modification may be a sc (single chain) antibody in the format VH-VL or VL-VH. Also bispecific scFvs are envisaged, e.g. in the format VH-VL-VH-VL, VL-VH-VH-VL, VH-VL-VL-VH. Also comprised in the term “antibody molecule(s)” are diabodies and molecules that comprise an antibody Fc domain as a vehicle attached to at least one antigen binding moiety/peptide, e.g. peptibodies as described in WO 00/24782. The term “Antibody fragments” also comprises such fragments which are engineered to provide modified antibody effector functions such as antibody dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).
In one embodiment, the SCR-Ab of the invention comprises an antigen binding domain that is derived from the antibody molecules as comprised in the following Table 1.
Yet, the person skilled in the art is readily in the position to use and to obtain other suitable antibody molecules. For instance, antibody molecules known to specifically bind surface antigens of pathogens may be used as binding molecule comprised in the SCR-Ab construct of the present invention. Alternatively, suitable antibody molecules may be raised using standard methods known in the art, see, inter alia, Harlow and Lane “Antibodies: a laboratory manual” Cold Spring Harbor Laboratory Press (1988).
In another embodiment, the SCR-Ab of the invention comprises an antigen-binding domain as comprised in the antibody molecule selected from the group consisting of 2F5, 2G12, 3D6, 4E10 IgG1 and 4E10 IgG3 as defined in Table 1 that is provided herein above.
The term “receptor molecule” as used herein relates to proteins or fragments thereof which are capable of binding specific ligands. Binding of a ligand to a receptor molecule in its normal cellular context initiates a cellular response to the ligand. In the present invention, however, useful receptor molecules may also only comprise the ligand-binding portion of a receptor which is not capable of initiating a cellular response. The skilled person is aware that receptor molecules capable of binding pathogen-associated ligands are specifically useful in the context of the present invention. In a non-limiting example, a receptor molecule capable of binding a pathogen-associated ligand may be the CD4 receptor, preferably the human CD4 receptor. Other receptor molecules which are capable of binding pathogen-associated ligands include chemokine receptors such as the (human) CXCR4 receptor or the (human) β-chemokine receptor CCR5. Yet, the person skilled in the art is readily capable of identifying other examples of receptor molecules which are useful in the SCR-Ab construct as described herein. The term “aptamer” as used herein relates to nucleic acid molecules that are capable of specifically binding target molecules. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold (1995), Ann. Rev. Biochem 64, 763-797).
The term “DARPin” as used herein relates to designed binding proteins comprising ankyrin repeats as known in the art (Kohl (2003), PNAS 100, 1700-1705, Forrer (2003), FEBS letters 539, 2-6). Highly specific DARPins may be generated by screening DARPin libraries as described in WO 02/20565, e.g. by using ribosome display technology (Hanes (1997), PNAS 94, 4937-4942). Yet, the person skilled in the art is aware that other designed repeat protein libraries (DRP libraries), such as Leucine-rich repeat (LRR) libraries, may be used for screening binding molecules which are useful in the context of the present invention.
As used herein, “specifically recognizing” or “specifically binding” refers to the binding of a binding molecule to a target molecule, such molecules may be antibody molecules binding to a given antigen or receptor molecule binding to a given ligand and the like. According the law of mass action, the binding-equilibrium is dependent on the concentration of target molecule [t], the concentration of binding molecule [b] and the concentration of the binding molecule-target molecule complex [bt]. The equilibrium constant Kd, therefore, is defined as [t]×[b]/[bt]. Ligand binding of the binding molecule comprised in the SCR-Ab constructs as described herein may occur e.g. with an affinity (Kd) of about 10−13 to 10−6 M, e.g. with an affinity of about 10−13 to 10−7 M, e.g. with an affinity of about 10−13 to 10−8 M, e.g. with an affinity of about 10−13 to 10−9 M. As discussed before, the term “specifically recognizing/binding” in the context of the binding molecules for a given pathogen comprises in particular corresponding antibodies (or fragments or derivatives thereof).
In contrast thereto, the Kd for endogenous fH on cells is relatively high. Thus, only small amounts of endogenous fH are bound on the cell surface and the exchange from fH on the surface is fast. The Kd-value of fH-derived SCRs is expected to be in the same range as for the whole fH molecule. Previously described SCR constructs such as those described in EP-A1 0 854 150 do not comprise the pathogen-specific binding molecule as defined herein and, accordingly; show a substantial lower affinity (Kd) for pathogens or pathogen infected cells. Consequently, relatively high amounts of the respective SCR constructs are required in order to compete with the endogenous fH for binding on the cells and to sufficiently block fH-pathogen interactions; see, inter alia, the appended
The SCR-Ab constructs as described herein selectively bind pathogen-associated proteins exposed on pathogens and/or pathogen-infected cells with a high affinity via the comprised binding molecule. Accordingly, the fH-derived SCR constructs will only dissociate with the Kd of the binding molecule and are thought be “arrested” at the surface of pathogens and/or pathogen-infected cells. Without being bound by theory, the fH-derived SCR region comprised in the SCR-Ab constructs of the invention are thought to displace the host-derived fH from the surface of the pathogen and/or pathogen infected cells. This is thought to lead to the termination of the fH mediated inhibition of complement activation which finally will result in the complement-mediated lysis of the pathogens and/or pathogen infected cells.
The fH-derived SCR and the binding molecule as comprised in the SCR-Ab construct of the present invention may be covalently linked. The covalent linking of a fH-derived SCR with a binding molecule results in a molecule in which said fH-derived SCR and said binding Molecule are connected by (a) covalent bond(s). A covalent bond is a chemical bonding that is characterized by the sharing of pairs of electrons between atoms, as, inter alia, obtained by the herein exemplified cross-binding via chemical compounds. However, also the recombinant production of constructs as disclosed herein, i.e. SCR-Ab comprising a complement factor derived short consensus repeat and a covalently bound binding molecule specifically recognizing/binding to a pathogen is envisaged.
Alternatively, the fH-derived SCR and the binding molecule as comprised in the SCR-Ab construct of the present invention may be non-covalently linked. Non-covalent bonds are known in the art and include, but are not limited to the association of protein molecules as a result of protein-protein interaction. Non-limiting examples of non-covalent bonds that may be useful for linking a fH-derived SCR and a binding molecule in the context of the present invention include the biotin/streptavidin complex, lectin/glycoprotein complexes and antibody-antigen complexes. Yet, the person skilled in the art is readily capable of identifying other non-covalent bonds/complexes which are useful for non-covalently linking a fH-derived SCR with a binding molecule for generating SCR-Ab constructs as described herein.
In one embodiment, the fH-derived SCR and the binding molecule are comprised in a single-chain multi-functional polypeptide. A single-chain SCR-Ab construct e.g. may consist of (a) polypeptide(s) comprising (a) SCR-derived domain(s) and (a) binding-molecule domain(s). Said domains are connected by a polypeptide linker, wherein said linker is disposed between said SCR-derived domain(s) and said binding-molecule domain(s).
The SCR-Ab construct as described herein specifically recognizes a pathogen, wherein said pathogen may be a virus or a bacterium. Said virus may be selected from the group consisting of a double-stranded DNA virus, single-stranded DNA virus, double-stranded RNA virus, positive-sense single-stranded RNA virus, negative-sense single-stranded RNA virus, reverse transcribing RNA virus and reverse transcribing DNA virus. Said double-stranded DNA virus may include, but is not limited to herpes simplex virus, cytomegalo virus, varicella zoster virus, Epstein-Barr virus, roseolo virus, human herpesvirus-7 or Kaposi's sarcoma-associated virus. The positive-sense single-stranded RNA viruses as defined above include, but are not limited to corona virus, hepatitis C virus, dengue fever virus, polio virus, rubella virus, yellow fever virus or tick-borne encephalitis virus. The negative-sense single-stranded RNA viruses as defined above include, but are not limited to influenza virus, Ebola virus, Marburg virus, measles virus, mumps virus, rabies virus, parainfluenza virus, Lassa virus or lymphocytic choriomeningitis virus. Said reverse transcribing RNA virus may be a retrovirus, wherein said retrovirus e.g. may be selected from the group consisting of Rous sarcoma virus; (RSV) mouse mammary tumour virus (MMTV); Friend murine leukaemia virus (FV); feline leukaemia virus; feline sarcoma virus; bovine leukaemia virus; human T-lymphotropic virus (HTLV); bovine immunodeficiency virus; equine infectious anaemia virus; feline immunodeficiency virus; human immunodeficiency virus (HIV); simian immunodeficiency virus (SIV) and spumavirus. The reverse transcribing DNA virus as defined herein above includes, but is not limited to hepatitis B virus.
In another embodiment, the present invention relates to polynucleotides encoding the SCR-Ab constructs as described herein. Said SCR-Ab encoding polynucleotide e.g. may comprise, but is not limited to, a polynucleotide encoding a fH-derived SCR and a binding molecule that are comprised in a single chain multi-functional polypeptide. The term “polynucleotide”, as used herein, is intended to include nucleic acid molecules such as DNA molecules and RNA molecules. Said nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. Preferably, said polynucleotide may be comprised in a vector.
Furthermore, it is envisaged to transfect cells with the polynucleotides or vectors as described herein. Yet, in a further embodiment, the present invention relates to polynucleotides which upon expression encode the above-described polypeptides. Said polynucleotides may be fused to suitable expression control sequences known in the art to ensure proper transcription and translation of the polypeptide. Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
Preferably, the polynucleotide of the invention is comprised in a recombinant vector in which a polynucleotide encoding the herein described fH-SCR-constructs is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory N.Y. and Ausubel (1989), Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3, pPICZalpha A (Invitrogen), or pSPORT1 (GIBCO BRL). Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention.
In accordance with the above, the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide encoding a polypeptide of the invention. Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell populations. The vectors containing the polynucleotides of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. Such methods, for example, include the techniques described in Sambrook (1989), loc. cit. and Ausubel (1989), loc. cit. Accordingly, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, supra. As a further alternative, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells. The polynucleotide or vector of the invention which is present in host cell may either be integrated into the genome of the host cell or it may be maintained extra-chromosomally.
In a further aspect, the present invention comprises methods for the preparation of the SCR-Ab construct as described herein. The inventive SCR-Ab construct may be recombinantly produced, e.g. by cultivating a cell comprising the described polynucleotides or vectors which encode the inventive SCR-Ab constructs and isolating said constructs from the culture. The inventive SCR-Ab construct may be produced in any suitable cell-culture system including, but not limited to eukaryotic cells, e.g. pichia pastoris yeast strain X-33 or CHO cells. Further suitable cell lines known in the art are obtainable from cell line depositories, like the American Type Culture Collection (ATCC). The term “eukaryotic” is meant to include yeast, higher plant, insect and mammalian cells. The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. In a further embodiment, the present invention thus relates to a process for the preparation of a polypeptide described above comprising cultivating a cell of the invention under conditions suitable for the expression of the polypeptide and isolating the polypeptide from the cell or the culture medium.
The polypeptides of the invention, accordingly, can be isolated from the growth medium, cellular lysates or cellular membrane fractions. The isolation and purification of the expressed polypeptides of the invention may be by any conventional means, including ammonium sulphate precipitation, affinity columns, column chromatography, gel electrophoresis and the like and may involve the use of monoclonal or polyclonal antibodies directed, e.g., against a tag of e.g. the polypeptides of the invention; see, Scopes (1982), “Protein Purification”, Springer-Verlag, N.Y. The protein e.g. can be purified via its His-tag by using a Ni-NTA-column (Mack (1995), PNAS 92, 7021-7025) as described in the appended examples. Substantially pure polypeptides of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
The method for the preparation of the short consensus repeat-antibody construct (SCR-Ab) as described herein may also comprise coupling of the herein described al-derived SCR with a binding molecule. For instance, the fH-derived SCR may be coupled with a binding molecule by using sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB) chemical cross-linking as described in the appended examples. Yet, the person skilled in the art is readily capable of identifying other chemical cross-linkers which are useful in generating the SCR-Ab construct as described herein. Non-limiting examples of such cross-linking reagents are listed in the following Table 2.
Accordingly, any conventional cross-linking procedure may be applied for preparing the herein described SCR-Ab constructs, including, but not limited to, by forming protein-protein interactions such as biotin-streptavidin complexes or the antibody-antigen complexes.
In a further embodiment, the short consensus repeat-antibody construct (SCR-Ab) of the invention is comprised in a composition. Said composition may comprise one or more SCR-Ab constructs as provided herein. Said composition may be a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier and/or diluent. The use of the herein described SCR-Ab constructs for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of an infection with a pathogen or a pathological condition associated with an infection with a pathogen is also envisaged. Said pathogen may be a virus or a bacterium as defined herein above.
It is evident for the skilled artisan that for short consensus repeat-antibody construct (SCR-Ab) in which the comprised complement factor H-derived short consensus repeat (fH-derived SCR) and the binding molecule are non-covalently bound, said fH-derived SCR and said binding molecule may be administered concomitantly or sequentially. As exemplified herein below, e.g., the binding molecule may be administered first followed by the administration of the fH-derived SCR. Accordingly, the binding molecule that, e.g. specifically binds to a pathogen specifically associates to the fH-SCR forming the herein described non-covalently linked short consensus repeat-antibody construct (SCR-Ab). Therefore, the compositions of the present invention also comprise compositions in which both the complement factor H-derived short consensus repeat (fH-derived SCR) and the binding molecule are present independently.
The present invention also relates to the use of the nucleic acid molecules (polynucleotides), vectors, as well as transfected cells comprising said nucleic acid molecules (polynucleotides), vectors in medical approaches, like, e.g. cell based gene therapy approaches or nucleic acid based gene therapy approaches.
Said viral vectors are particularly suitable for gene therapy. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy, as well as vector systems, are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,466; U.S. Pat. No. 4,394,448, US2007/086985, US2007/082388, US2007/071770, US2007/003522 US2007/048285 (or corresponding EP1757703) or WO2004/111248 and references cited therein. Suitable vehicles/delivery vehicles, are inter alia, disclosed in WO2007/022030, WO2007/018562. Further suitable gene therapy constructs for use especially in lymphatic cells and/or tissues are known in the art; see Li, Ann NY Acad Sci 1082 (2006) 172-9.
In yet another embodiment, the present invention relates to method for the prevention, treatment or amelioration of an infection with a pathogen or a pathological condition associated with an infection with a pathogen can, in further embodiments using different binding molecules recognizing or binding to tumour cells, cancer cells and/or malignant cells for the treatment of an proliferative disorder like cancer, comprising administering the short consensus repeat-antibody construct (SCR-Ab) as described herein above or in the items below to a mammal in need of such prevention or treatment, wherein said mammal is a human. In a preferred embodiment, said virus may be a human immunodeficiency virus (HIV). Examples of a pathological condition associated with an infection with a pathogen may include, but are not limited to, acquired immune deficiency syndrome (AIDS) which is associated with a HIV infection, severe acute respiratory syndrome (SARS) which is associated with a corona virus infection, acute/chronic hepatitis C which is associated with hepatitis C virus infection or influenza which is associated with influenza virus infection.
As described, it is also envisaged that the inventive pharmaceutical concept described herein may be employed in a medical setting where it is desired to inhibit and/or eliminate pathogen infected host cells or malignant cells, like cancer cells. In said embodiment, the binding molecule comprised in the SCR-Abs or this invention specifically recognizes or binds to a cancer cell, malignant cell and/or tumour cell.
The SCR-Ab constructs to be employed in accordance with this invention may therefore (and alternatively) comprise a binding molecule that specifically recognizes or binds to a cancer cell, a malignant cell or a tumour cell additionally to the herein defined complement factor H-derived short consensus repeat. As documented herein below and in the appended examples it was, in accordance with this invention, surprisingly found that certain inventive SCR-Ab constructs are capable of lysing cancer cells in vitro in a highly unexpected manner.
Said cancer cell, malignant cell or tumour cell may be derived from any cancer or tumour type. In one embodiment the cancer cell or malignant cell is selected from the group consisting of but not limited to breast cancer cells, Burkitt's lymphoma cells, multiple myeloma cells, colorectal cancer cells, metastatic colorectal cancer cells, Non-Hodgkin's Lymphoma cells, lung cancer cells, chronic lymphocytic leukaemia cells, micro-metastases or residual tumour cells. Preferably, the cancer cell or malignant cell the tumour cells may be derived from micro-metastases or residual tumours.
Short consensus repeat-antibody construct (SCR-Ab) comprising an antibody molecule that specifically recognizes a cancer cell as described herein is useful for the prevention, treatment or amelioration of a cancerous disease in a subject. Preferably, said subject is a human.
As used herein, the cancerous disease may be selected from but not limited to breast cancer, Burkitt's lymphoma, multiple myeloma, colorectal cancer, metastatic colorectal cancer, Non-Hodgkin'Lymphoma, lung cancer, chronic lymphocytic leukaemia. Preferably, the cancerous disease originates from micro-metastases or residual tumours.
The antibody molecule comprised in the herein described short consensus repeat-antibody construct (SCR-Ab) useful for the treatment of a cancerous disease may be selected from the group consisting of but not limited to monoclonal antibodies recognizing an epitope selected from the group consisting of: CD9, CD19, CD20, CD22, CD30, CD33, CD40, CD46, CD55, CD56, CD138, erbB1, HER2/neu, IGFR, MUC-1, TAG-72, TAL-6, TRAILR and VEGFR. Antibody molecules that specifically recognize the above-described antigens are well known in the art. The compositions of the invention may be in solid or liquid form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). Furthermore, it is envisaged that the pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.
Such agents might be antibiotics, antiviral drugs or drugs acting on the gastro-intestinal system.
Administration of the suitable (pharmaceutical) compositions may be effected by different ways, e.g., by parenteral, subcutaneous, intraperitoneal, topical, intrabronchial, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. Parenteral administrations include intraperitoneal, intramuscular, intradermal, subcutaneous intravenous or intraarterial, administration. It is particularly preferred that said administration is carried out by injection and/or delivery, e.g., to a site in a brain artery or directly into brain tissue. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like a specific organ which is infected with a pathogen.
Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. Suitable carriers may comprise any material which, when combined with the SCR-Ab constructs of the invention, retains the biological activity of the comprised SCR-Ab construct; see Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed. Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present including, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumine or immunoglobuline, preferably of human origin.
These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Pharmaceutically active matter may be present in amounts between 1 μg and 20 mg/kg body weight per dose, e.g. between 0.1 mg to 10 mg/kg body weight, e.g. between 0.5 mg to 5 mg/kg body weight. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg per kilogram of body weight per minute. Yet, doses below or above the indicated exemplary ranges also are envisioned, especially considering the aforementioned factors.
The pharmaceutical compositions as described herein may be formulated to be short-acting, fast-releasing, long-acting, or sustained-releasing. Hence, the pharmaceutical compositions may also be suitable for slow release or for controlled release. Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody in which the matrices are in the form of shaped articles, e.g. films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers and poly-D-(−)-3-hydroxybutyric acid. Possible loss of biological activity and possible changes in the binding properties of SCR-Ab constructs comprised in sustained-release preparations may be prevented by using appropriate additives, by controlling moisture content and by developing specific polymer matrix compositions.
Furthermore, it is envisaged that the pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition. For example in patients suffering from an HIV-infection, such agents might be drugs acting on the immunological system, drugs used in anti-viral treatment, in particular in HIV-treatment (for example, HAART) and AIDS management and/or anti-inflammatory drugs. HAART therapy consists of a cocktail of three classes anti-viral drugs. The classes are nucleosidal reverse transcriptase inhibitors (NRTI), non-nucleosidal reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Usually 2 to 4 drugs from preferentially more than one class are combined to reduce viral load to almost non-detectable levels. Early treatment of infected patients with HAART prevents the transition of viral strains from usage of CCR5 to other chemokine receptors, like CXCR4 (Connor (1997) J. Exp. Med. 185, 621-628). Constructs as disclosed in the present invention can be administered in addition to HAART intravenously, subcutaneously, and/or into the cerebral-spinal fluid. Other agents for combination with the inventive constructs could comprise, inter alia, or integrase inhibitors such as raltegravir.
In a further embodiment, the composition as comprised herein may be a diagnostic composition, optionally further comprising suitable means for detection.
In yet another embodiment, the present invention provides for a kit comprising at least one SCR-Ab construct as defined. Advantageously, the kit of the present invention further comprises, optionally (a) buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of medical, scientific or diagnostic assays and purposes. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. In one embodiment, said kit may comprise the short consensus repeat-antibody construct (SCR-Ab) according to the present invention, wherein both the complement factor H-derived short consensus repeat (fH-derived SCR) and the binding molecule are present independently in one container. In another embodiment, said complement factor H-derived short consensus repeat (fH-derived SCR) and the binding molecule are present independently in more than one container and wherein a covalently bound SCR-Ab or a non-covalently bound SCR-Ab is formed after the contacting the comprised fH-derived SCR with the comprised binding molecule.
The kit of the present invention may be advantageously used, inter alia, for carrying out the method of the invention and could be employed in a variety of applications referred herein, e.g., as diagnostic kits, as research tools or medical tools. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
In addition to the above description, the present invention also relates to the following items which are particularly useful in the medical intervention of cancerous diseases, tumour diseases and/or hyperplastic diseases:
Item 12. A cell transfected with the polynucleotide of item 10 or the vector of item 11.
The embodiments provided herein above in relation to binding molecule directed against a pathogen and the corresponding short consensus repeat-antibody construct apply, mutatis mutandis, to the above-provided items relating to short consensus repeat-antibody constructs (SCR-Abs) comprising a binding molecule that specifically recognizes and/or binds to a cancer cell/malignant cell/tumour cell.
In all embodiments, it is envisaged that the SCR-Abs of this invention are particular useful in medical/pharmaceutical intervention. Accordingly, subjects in need of such a medical/pharmaceutical intervention may be treated with the constructs/compounds of this invention. The subject to be treated may be mammalian, in particular a human.
The present invention is additionally described by way of the following illustrative non-limiting examples that provide a better understanding of the present invention and of its many advantages.
The following examples illustrate the invention:
Codon-optimized plasmids comprising the genetic sequences encoding for the complement factor H-derived short consensus repeats (fH-SCRs) SCR2, SCR7, SCR9, SCR13 and SCR18-20 were purchased from GeneArt.
The SCR polynucleotide sequences encoding SCR2, SCR7, SCR9, SCR13 and SCR18-20 were amplified using the following primer sets (primers manufactured by Metabion):
The synthetic SCRs mSCR2, mSCR7, hSCR7, hSCR9, mSCR13, hSCR13, mSCR18-20 and hSCR18-20 (all GeneArt) were amplified by Real-Time PCR (BioRad) with the following protocol (Brillant SYBR-Green Q-PCR, BioRad):
The PCR products were analysed by agarose gel electrophoresis (2% TAE) and the specific bands were extracted and purified (Gel extraction Kit, Qiagen). Samples were digested with specific enzymes (Fermentas) and ligated into their destined vector (also digested with suitable enzymes). The list below gives an overview of cloning details:
mSCR2 in pPICZalphaA, restriction sites: EcoRI (5-prime) und XbaI (3-prime)
mSCR7 in pPICZalphaA, restriction sites: EcoRI (5-prime) und XbaI (3-prime)
hSCR7 in pPICZalphaA, restriction sites: EcoRI (5-prime) und XbaI (3-prime)
hSCR9 in pPICZalphaA, restriction sites: EcoRI (5-prime) und XbaI (3-prime)
mSCR13 in pPICZalphaA, restriction sites: EcoRI (5-prime) und XbaI (3-prime)
hSCR13 in pPICZalphaA, restriction sites: KpnI (5-prime) und XbaI (3-prime)
mSCR18-20 in pPICZalphaC, restriction sites: ClaI (5-prime) und XbaI (3-prime)
hSCR18-20 in pPICZalphaA, restriction sites: EcoRI (5-prime) und XbaI (3-prime)
The PCR resulted in the synthesis of DNA molecules consisting of the following polynucleotide sequences, comprising an EcoRI restriction site at the 5′ end (underlined) and a XbaI (SCR2, 7, 9, 13 and human SCR18-20) or a ClaI restriction site (mouse SCR18-20) at the 3′ end (double underlined):
gaattctcgaccaaaaaaccgtgtggtcatccgggtgataccccgatg
gaattctcgaccaaagtgcgcaaatgtgtgttccactacgtggaaaac
gaattcctcagaaaatgttattttccttatttggaaaatggatataat
gaattcaaatcttgtgatatcccagtatttatgaatgccagaactaaa
gaattctcgaccaaagcgaccgatcagctggaaaaatgccgcgttctg
ggtaccgataaacttaagaagtgcaaatcatcaaatttaattatactt
atcgatgaaagataacagctgcgttgatccgccgcatgttccgaatgc
gaattcgacacctcctgtgtgaatccgcccacagtacaaaatgatata
The above described polynucleotides encoding SCR2, SCR7, SCR9, SCR13 and human SCR18-20 were cloned into pPICZalphaA (Invitrogen) via the EcoRI (5-prime) and XbaI (3-prime) cloning sites. The mouse SCR18-20 was cloned into pPICZaIphaC (Invitrogen) via the EcoRI (5-prime) and the ClaI (3-prime) cloning site. The correct reading frame was confirmed by sequencing. The vectors were transfected into pichia pastoris yeast strain X-33 according to the protocol of the manufacturer (Invitrogen) and positively selected using Zeocin. Positive yeast clones were cultured at 30° C. for 96 h and expression was induced by adding repeatedly 1% Methanol (each 24 h). Expressing clones were analysed by dot blotting.
The resulting complement factor H-derived short consensus repeat (fH-SCR) polypeptides are defined by the following amino acid sequences:
The SCRs were purified from the yeast supernatant via NiNTA-columns as recommended by the manufacturer (Qiagen). Fractions positive in slot blots (see
Monoclonal antibody clone #48; (Ab #48; Chesebro et al. (1981) Virology 112(1): 131-44) that specifically recognizes the envelope protein of Friend Murine Leukaemia Virus (FV) was purified from the supernatant of hybridoma cells by G-Sepharose (Amersham) according to the protocol of the manufacturer.
Equimolar amounts of fH-SCR and Ab#48 antibody molecules were cross-linked using Sulfo-SMPB as recommended by the manufacturer (Pierce). The resulting short consensus repeat-antibody constructs (SCR-Ab) were purified by spin filters (Zeba). The successful cross-linking of the fH-SCR with the Ab#48 antibody molecules was analyzed by western blotting (see
To show that the generated SCRs bind to negatively charges surfaces, supernatants of the transfected and induced yeast strains was applied to a heparin column. For this, 20 ml hSCR18-20 were centrifuged, the resulting supernatant (SN) diluted 1:2 with dH2O and sterile filtered (0.2 μm sterile filter). The treated SN was applied to heparin affinity chromatography using a 1 ml HiTrap heparin column (GE healthcare) and a Pharmacia FPLC-System according to the following protocol:
Buffer A: ⅓ PBS
flow-rate: 1 ml/min
load: 3×40 ml prepared supernatant (1:2 H2O)
wash: buffer A
Gradient: linear up to 100% buffer B→hold for 10 min
equilibration: buffer A
The flow-through was collected and 2× reloaded. The column was washed extensively with low salt buffer (⅓ PBS) and bound proteins were eluted using a linear salt gradient, ranging from 50 mM to 1 M NaCl (high salt buffer: PBS with 1 M NaCl) in a total volume of 100 ml. Individual fractions of 2 ml were collected, and the presence of hSCR18-20 was assayed by SlotBlotting (polyclonal serum goat-anti-humanFH 1:1.000 (Quidel); rabbit-anti-goat HRP conjugated 1:2.000 (Dako) (
Isolated FV virus was incubated with normal mouse serum (NMS), in RPMI 1640 without any supplement as input virus control or in NMS containing less than 5% fH, designated as fH-depleted serum (Δ-fH-serum). All sera were diluted 1:10 in RPMI 1640. The RNA of remaining FV (which was not lysed by mouse complement) was isolated and amplified by reverse transcriptase real time-PCR. Virus was isolated by centrifugation (Hermle Z382K, Rotor 220.87V01, 1 h/25.000 g/4° C.) and quantified by Real-time RT-PCR. For this, viral RNA was isolated with the Viral RNA Mini Kit (Qiagen). 5 μl of eluted RNA either from in vitro assays or revealed from serum was taken as template for Real-time RT-PCR according to the following protocol: The PCR-Mix contained RT-PCR Reaction Mix, iScript Reverse Transcriptase, nuclease-free water (iScript One-Step RT-PCR Kit, BioRad), F-MuLV env-specific fluorogenic PCR probe (5′-FAM ACT CCC ACA TTG ATT TCC CCG, Metabion), upstream primer 5′-AAGTCTCCCCCCGCCTCTA-3′ (SEQ ID NO: 17) and downstream primer 5′-AGTGCCTGGTAAGCTCCCTGT-3′ (SEQ ID NO: 18). The viral RNA was transcribed to cDNA using the following PCR cycle profile: 30 min 45° C., 15 sec 95° C., 30 sec 60° C. (iCycler, BioRad). Real-time PCR and RT-PCR amplifications were performed in 25 μl reaction mixture with BioRad iScript One-Step RT-PCR Reaction Mix (BioRad), using an iCycler (BioRad). Comparisons between groups were made using differences in critical threshold values (Ct value). Experiments were performed in duplicates. An increase of about 3.3 in the Ct value corresponds to a reduction of about 1 log in viral titer; see following Table 3.
Thus fH depleted serum reduces the amount of FV at around 3 logs, since the Ct value of the control sera increased from Ct 24.5 to 33.0.
Similarly, Mouse Mammary Tumour Virus (MMTV), a further murine retrovirus, is efficiently lysed by complement and the viral titre is reduced for about five logs; see
The Ct values were determined by RT-PCR according to the following protocol: MMTV was incubated with NMS or fH-depleted serum (1:10 final dilution) in RPMI 1640 without any supplement at 37° C. for 15 min in the presence of RNAse. Lysis was stopped by the addition of the AVL buffer from the viral RNA isolation kit (Qiagen) to inactivate the RNAse. RNA isolation was performed as described in the kit by the manufacturer. To quantify the amount of MMTV after lysis, real time RT-PCR was performed using the QRT-PCR kit from Stratagene at 45° C. for 30 min followed by PCR of the cDNA in the presence of SybrGreen.
The following primers were used:
The cDNA was amplified using the following PCR cycle profile: 15 sec 95° C., 30 sec 60° C. for 50 cycles (iCycler, BioRad). Experiments were performed in duplicate.
The capacity of the herein described SCR-Ab constructs to enhance complement dependent lysis was first demonstrated using an in vitro lysis assay. Therefore, 103 spleen focus forming units (SFFU) of Friend virus were mixed with the construct containing SCR7, SCR13, SCR18-20 or SCR 2, the uncoupled mixture of Ab#48 antibody and SCR7 (Ab#48 single/SCR single), the antibody alone (Ab#48 single) or SCR alone (SCR single) in a ten-fold excess and incubated with normal mouse serum (NMS) in a 1:10 dilution for 30 min. As a control, fH-depleted mouse serum (fH depleted NMS) was used. Virus was isolated by centrifugation (Hermle Z382K, Rotor 220.87V01, 1 h/25.000 g/4° C.) and quantified by Real-time RT-PCR. For this, viral RNA was isolated with the Viral RNA Mini Kit (Qiagen). 5 μl of eluted RNA either from in vitro assays or revealed from serum was taken as template for Real-time RT-PCR according to the following protocol: The PCR-Mix contained RT-PCR Reaction Mix, iScript Reverse Transcriptase, nuclease-free water (iScript One-Step RT-PCR Kit, BioRad), F-MuLV env-specific fluorogenic PCR probe (5′-FAM ACT CCC ACA TTG ATT TCC CCG, Metabion), upstream primer 5′-AAGTCTCCCCCCGCCTCTA-3′ (SEQ ID NO: 17) and, downstream primer 5′-AGTGCCTGGTAAGCTCCCTGT-3′ (SEQ ID NO: 18). The viral RNA was transcribed to cDNA using the following PCR cycle profile: 30 min 45° C., 15 sec 95° C., 30 sec 60° C. (iCycler, BioRad). Real-time PCR and RT-PCR amplifications were performed in 25 μl reaction mixture with BioRad iScript One-Step RT-PCR Reaction Mix (BioRad), using an iCycler (BioRad). Experiments were performed in duplicates.
The results shown in the following Table 5 clearly indicate that FV is lysed only when the SCRs are coupled to an FV specific antibody molecule. As an example the cross-linked Ab#48 to SCR7 (SCR7-Ab#48) molecule is given in
In contrast, the mixture of uncoupled SCR(SCR single) and Ab#48 (Ab#48 single) was unable to induce complement-mediated lysis (CoML; Table 5). Constructs in which SCR13 or SCR18-20 were cross-linked to the Ab#48 behaved similar and induced CoML (data not shown).
Furthermore, the herein described SCR-Ab constructs were tested in vivo using BALB/c mice as this mouse strain is highly susceptible to FV infection.
For that reason, 5 μg of the respective SCR-Ab constructs was mixed with FV (500 SFFU/animal) for 30 min on ice in a total volume of 500 μl and applied to BALB/c mice (purchased from Charles River, Germany) via the tail vein. Control animals obtained 500 SFFU FV in 500 μl PBS without any supplement.
After one week, animals were sacrificed and the following parameters were determined: infectious centres in the spleen (infectious centre assay) and infected cells in the spleen (FACS analysis).
Serial dilutions of spleen cells from infected mice were plated onto susceptible Mus dunni. cells, co-cultivated for 3 days, fixed with ethanol, stained with F-MuLV envelope-specific Mab 720 (Dittmer (1998) J Virol 72: 6554-8), and developed with peroxidase-conjugated goat anti-mouse IgG and substrate to detect foci of infected cells.
The determination of infectious centres in the spleen revealed that the constructs which contained SCRs which bind to negatively charged surfaces were able to reduce the infection. While control constructs (SCR2-Ab#48) or infection in the absence of any Ab and SCR (positive control) induced no protection against FV infection, the SCR-Ab constructs containing SCR7, 13, or 18-20 or a mixture of SCR7, 13, 18-20 (mix) reduced the infectious centres up to 3 logs; see Table 6 as listed herein below:
Thus, only when SCRs 7, 13 or 18-20 were coupled to the virus-specific Ab, the amount of infectious centres was drastically reduced (about 3 logs) when compared to the control SCR, the absence of any constructs; see Table 6. As expected, uninfected control mice (negative control) gave no FV-specific signal in this assay.
In a second set of experiments, a mixture of uncoupled SCRs and Ab#48 (Ab#48 single/SCR7 single; equimolar amount of Ab and SCR, total 5 μg) was applied to the animals via the tail vein. After 6 h, BALB/c mice were infected with FV (500 SFFU) again via the tail vein. After one week, animals were sacrificed and the infection of the mice was determined as described above.
Titration of infectious centres in the spleen on a Reporter cell line (IC-Assay) and detection of infectious centres by FACS analysis of infected animals treated with a mixture of uncoupled SCRs and virus-specific Ab is shown in Table 7 as listed herein below:
As expected, the mixture of uncoupled SCRs and Ab#48 had no effect. Neither the amount of infected cells in the spleen, nor the infectious centres in the IC assay were significantly reduced when compared to the control settings in the absence of any construct, the Ab#48 alone, Ab#48 together with control SCR2 or the uncoupled mixtures of SCRs 7, 13 or 18-20 and Ab #48; see Table 7.
To determine the amount of FV infected cells in the spleen FACS analyses was performed. Suspensions of spleen cells were incubated with biotinylated monoclonal antibody clone #34; (Ab #34; Chesebro et al. (1981) Virology 112(1): 131-44) recognizing F-MuLV glyco-Gag on the surface of infected cells. After washing step, spleen cells were incubated with FITC-conjugated streptavidine (DAKO). The fluorescence signal was analyzed with a Becton Dickinson FACScan flow cytometer using CellQuest software.
The viral load in the spleen is measured by measuring the percent of infected cells with FACS analysis and by titration of spleen cells using a reporter cell line. The FACS analysis showed that the SCR-Ab constructs reduced the amount of FV infected cells (0.5-2.5 of infected cells in the spleen) compared to the absence of the constructs (FV, 15% infected cells) or a control SCR coupled to the Ab#48 (7% infected cells, see
To determine the induction of CoML by the cross-linked SCR-3D6 constructs, HIV-1 was pre-incubated with the SCR-3D6 in RPMI- for about 10 min on ice. NHS was added in a 1:10 dilution. Quickly 1 μg/μl RNase A was added and the samples were incubated at 37° C. for an hour. To compare the effect of the SCR-3D6 (constr.-SCR7 in
To determine the induction of CoML by SCR9, HIV-1 virus was incubated with the in RPMI-medium for about 10 min on ice together with normal human serum (NHS) in a 1:10 dilution. Quickly 1 μg/μl RNase A was added and the samples were incubated at 37° C. for an hour. To compare the effect of the SCR9 (SCR9 in
After the lysis experiment as described above (initial viral concentration contained 40 ng HIV-1 NL4-3 p24 equivalents) the pelleted samples (Hermle Z382K, Rotor 220.87V01, 1 h/25.000 g/4° C.) were resuspended and transferred to a u-bottom 96-well plate containing 100000 PBMCs in RPMI supplemented with IL-2 and 10% FCS. The plate was then incubated over night at 37° C. The next day the cells were washed and resuspended in fresh medium. 10 μl of each sample were taken and lysed 1:10 in 1% Igepal and stored at −80° C. for the p24 ELISA experiment. These samples were supposed to be day 0 of infection. Further samples were taken on day 5 and day 10 of infection. The amount of produced virus was determined by a p24-ELISA as described (Stoiber (1996) loc. cit.). The results of the HIV-1 infection assay are described in
Mouse Hepatitis Virus A59 (MHV; 20 μl of a stock with 3×108 plaque forming Units), a member of the coronavirus family, was incubated with SCR13 or SCR2 (control-SCR, both in a 100-fold excess) before NMS was added in a 1:5 dilution. Samples were incubated for 30 min at 37° C. The RNA of the lysed viruses was digested by RNAse. After centrifugation for 1 h at 13000 rpm at 4° C., the supernatant was discarded and the pellet of the remaining non-lysed MHV was resuspended in RPMI without any supplement before the viral RNA was isolated with the QIAGEN kit as recommended by the manufacturer. Isolated RNA was amplified by RT-PCR (upstream primer 5′-TCCTGGTTTTCTGGCATTACCCAG-3′ (SEQ ID NO:45), downstream primer 5′-CTGAGGCAATACCGTGCCGGGCGC-3′ (SEQ ID NO:46)) using the following profile:
Amplified viral cDNA was applied to an agarose gel electrophoresis and visualized by ethidium-bromide. The expected band at 380 bp is highlighted by an arrow in
SOK3-cells (1×106/ml) were incubated with an antibody specific for an extracellular domain of HER2/neu, a mAb against the tumour cell (1:100 dilution in PBS) and, after washing, exposed to normal human serum (NHS, 1:10 in RPM; 130 min at 37° C.) as a source of complement. The samples contained in addition SCR7 (data 12), or as control SCR2 (data 11). Samples contained a 60-fold molar excess considering that the serum concentration of fH is 500 μg/ml. After washing again, a FITC-labelled antibody against C3c was added (1:500) for 30 min on ice followed by a further washing step. The samples were incubated with propidium-iodine (PI), washed and fixed with PBS supplemented with 3% formaldehyde. The deposition of C3 (FL-1-H) and the PI-staining (FL2H) were determined by FACS analysis and analysed by FACs Diva software (BD Bioscience). Considering Examples 4, 5 and 6, the Coupling of SCR7 with a tumour cell-specific binding molecule is considered to lead, in accordance with this invention, to an even higher activity.
Raji cells (5×105) were incubated with the SCR7 or SCR9, respectively, at 37° C. for 1 h (in a 50-fold excess compared to fH (shown as 7-50 and 9-50 in
The codon-optimized plasmid comprising the genetic sequences encoding for an illustrative artificial short consensus repeats (aSCRi; SEQ ID NO: 32) was purchased from GeneArt. The aSCRi was cloned into pPICZalphaC (Invitrogen) via the ClaI (5-prime) and the NotI (3-prime) cloning site. The correct reading frame was confirmed by sequencing. The vectors were transfected into pichia pastoris yeast strain X-33 according to the protocol of the manufacturer (Invitrogen) and positively selected using Zeocin. Positive yeast clones were cultured at 30° C. for 96 h and expression was induced by adding repeatedly 0.5% Methanol (each 12 h). Expressing clones were analysed by Western blotting.
The binding of the illustrative artificial SCR (aSCRi) to heparin was analyzed with the following modification: The bound protein was eluted by a high salt buffer (PBS containing 500 mM NaCl) not by a gradient but by direct application of this buffer. The fraction was dialyzed against PBS and after concentration analyzed by Western blot. As the aSCRi does not represent a fH sequence, an polyclonal serum goat-anti-humanFH was substituted by a direct PDX-labelled anti-HIS antibody (1:2000, Sigma) which recognizes the his-tag at the C-terminus of the aSCRi. The blot in
To determine the induction of complement-mediated lysis (CoML) by artificial SCR, HIV-1 was incubated in RPMI-medium for about 10 min on ice together with normal human serum (NHS) in a 1:10 dilution and the monoclonal HIV-specific Antibody 2G12 (Polymun, Vienna) in a 1:500 dilution. Samples were then incubated at 37° C. for an hour. The aSCR having SEQ ID NO: 31 was applied in an equimolar amount and in a 120 molar excess calculated to the content of fH in the system. As positive control 1% Igepal in RPMI-medium was added to determine 100% lysis. As negative controls one sample contained the input virus with heat inactivated serum (hiNHS in
| Number | Date | Country | Kind |
|---|---|---|---|
| 07008954.5 | May 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/003554 | 5/2/2008 | WO | 00 | 6/25/2010 |
