BINDING MOLECULES TARGETING PATHOGENS

Abstract

A first aspect of the disclosure relates to the field of binding molecules targeted at pathogens. The disclosure further relates to proteinaceous binding molecules targeting cells displaying pathogen-associated molecular patterns, in particular targeting cell surface molecules associated with or derived from pathogens, more in particular cell surface proteins displaying peptides from intracellular (pathogen associated) proteins.

Claims

1.-19. (canceled)

20. A proteinaceous molecule, comprising: a single polypeptide chain comprising a first and a second specific binding domain separated by at least one linker, and an Fc monomer, and an effector moiety, wherein the first and second specific binding domains are each Vh domains, and wherein each specific binding domain specifically recognizes a different binding site present on or associated with a pathogen or on a cell infected with a pathogen, but which binding site is not present on a cell not infected with the pathogen.

21. The proteinaceous molecule of claim 20, wherein the single polypeptide chain further comprises: a third specific binding domain separated from the first and second binding domains by at least one linker.

22. A dimeric proteinaceous molecule, comprising two proteinaceous molecules of claim 1 dimerized to one another through two Fc monomers.

23. The dimeric molecule of claim 22, wherein the two proteinaceous molecules are different from one another.

24. The dimeric molecule of claim 20, wherein the effector moiety is apoptin.

25. A method of treating a subject suffering from an infectious disease, the method comprising: administering the proteinaceous molecule of claim 20 to the subject so as to treat the infectious disease.

26. A pharmaceutical formulation comprising: the proteinaceous molecule of claim 20, and suitable excipients.

27. A nucleic acid molecule encoding the proteinaceous molecule claim 20.

28. A vector comprising the nucleic acid molecule of claim 27.

29. A cell comprising the nucleic acid molecule of claim 27.

30. A method for producing proteinaceous molecule, the method comprising: culturing the cell of claim 29, allowing for expression of the proteinaceous molecule, and separating the proteinaceous molecule from the culture.

31. The cell of claim 29, wherein the nucleic acid molecule is integrated into the cell's genome.

32. A proteinaceous molecule of FIG. 1 or FIG. 3.

33. A cell comprising the vector of claim 28.

34. A method of treating a subject suffering from a cancer relating to an infection, the method comprising: administering the proteinaceous molecule of claim 20 to the subject so as to treat the infectious disease.

35. A method of treating a cell of the type wherein a binding site on a pathogen or on a cell infected with the pathogen is targeted with a binding molecule comprising a specific binding domain that specifically binds the binding site, and wherein the binding molecule further optionally comprises an effector moiety, the method comprising: utilizing in said method, a binding molecule that comprises at least four binding domains specific for said binding sites, said at least four binding domains connected to one another with peptide linkers, wherein at least two of the at least four binding domains specifically bind to different binding sites on the pathogen or cell infected with the antigen, and wherein the binding sites are not present on a cell not infected with the pathogen.

36. The method according to claim 35, wherein the binding molecule that comprises at least four binding domains comprises an Fc monomer.

37. The method according to claim 35, wherein the at least four binding domains are each Vh domains.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0037] FIG. 1. Exemplified topologies of binding molecules comprising one or more copies each of two or more different binding domains each binding to a different binding site and in one embodiment comprising effector moieties as part of the disclosure.

[0038] 1. Topologies of binding molecule comprising two different binding domains D1 and D2, and divalent for a binding site 1 and monovalent for a binding site 2.

[0039] 2. Binding molecule comprising two different binding domains and monovalent for a binding site 1 and multivalent for a binding site 2 (multi-valency is, for example, 3-6). Shown are two examples of many possible single-chain polypeptides, according to the disclosure. All possible permutations regarding the position of the single binding domain and the multiple copies of the second binding domain are also part of the disclosure, and are visualized by the ensemble of different domains and number of domains between accolades.

[0040] 3. Binding molecule comprising two different binding domains each binding to a different binding site and with two to six copies of a first binding domain and with two to six copies of a second binding domain, providing multi-valency for both binding sites. As an example, a binding molecule is shown in which binding domains binding to the same binding site are linked in consecutive order. All possible domain topologies obtained by permutations regarding domain positions in the single chain binding molecule of all binding domains of both kinds, are also part of the disclosure.

[0041] 4. Binding molecule comprising three, four, five or six different binding domains, thus, binding to three, four, five or six different binding sites, respectively, and monovalent or multivalent for a binding site 1, monovalent or multivalent for a binding site 2, etc., (the valencies for the three to six different binding sites are, for example, one to six). As an example, four binding molecules are shown in which one to six clustered identical binding domains are linked in consecutive order, with three, four, five and six different binding domains in the binding molecules, respectively. All possible domain topologies obtainable by permutations regarding domain positions in the single chain binding molecule, of all one to six copies of the three to six different binding domains, are also part of the disclosure.

[0042] 5. Binding molecule comprising two different binding domains each binding to a separate binding site and with one binding domain monovalent or multivalent for a binding site 1 and the second binding domain monovalent or multivalent for a binding site 2 (both valencies are, for example, 1-6), and with one or more effector moieties (covalently) bound to the binding molecule. As an example, a binding molecule is shown in which the two sets of one to six binding domains are linked in consecutive order, with the effector moiety covalently linked to the C-terminus of the binding molecule. All possible domain topologies obtainable by permutations regarding each domain position in the single chain binding molecule are also part of the disclosure.

[0043] 6. Similar to 5, now with three to six different binding domains, for each of which one to six copies of the unique binding domains are part of the binding molecule.

[0044] FIG. 2. Exemplified topologies of multi-specific binding molecules comprising two or more different binding domains each binding to a different binding site, and linked to an Fc monomer, which is in one embodiment provided as a single chain molecule and in a second embodiment as a two-chain Fc fragment comprising two Fc monomers with arms each comprising two or multiple binding domains, according to the disclosure.

[0045] 1. Binding molecule comprising one or multiple, preferably one to six copies each of two different binding domains, thus, mono-specific or multi-specific for a binding site a and mono-specific or multi-specific for a binding site b, and comprising an Fc monomer. Shown is an example of a single-chain molecule with n binding domains specific for binding site a and m binding domains specific for binding site b. All possible domain topologies regarding the position of the individual binding domains specific for binding site a or b are also part of the disclosure. It is also part of the disclosure that a third, fourth, etc., type of binding domain (preferably one to six copies of each different binding domain) are incorporated in binding molecules with specificity for a third, fourth, etc., unique binding site c, d, etc.

[0046] 2. Two-chain binding molecule formed upon dimerization through binding interactions between two Fc monomers of a binding molecule comprising multiple, preferably two to six identical binding domains, thus, mono-specific for a binding site a, and comprising an Fc monomer, forming an Fc fragment with two identical arms. Alternatively, two Fc monomers comprising arms with different binding domains and/or with different numbers of copies of identical binding domains are dimerized, resulting in two-chain hetero-dimeric binding molecules of the disclosure comprising Fc fragments with bi-specific arms.

[0047] 3. Two-chain binding molecule formed upon dimerization through binding interactions between Fc monomers as outlined in 1, forming an Fc fragment with two identical arms. Alternatively, two Fc monomers comprising different arms with different binding domains and/or with different domain topologies are dimerized, resulting in two-chain hetero-dimeric binding molecules of the disclosure comprising Fc fragments with bi-specific arms, with the number of copies n, m, o and p of binding domains each being two to six.

[0048] Examples of multiple different binding sites targeted in a monovalent or multivalent manner by different binding domains are given in the specification and in example 1. An effector moiety can be part of any of the outlined proteinaceous molecules, as detailed in the specification.

[0049] FIG. 3. Cartoon displaying examples of preferred domain topologies.

[0050] Examples are provided of possible combinations of V.sub.H domains and distinct linker sequences for the construction of multi-domain proteins that are mono-specific or multi-specific. In a-h various examples are provided of proteinaceous molecules of the disclosure, comprising two or three different binding domains, and comprising one, two, three or four copies of the various binding domains, each, all linked with two or three different linkers (see also FIG. 1, examples 1-4 and FIG. 2 for additional preferred domain topologies of the disclosure). In i and k, the exemplified preferred proteinaceous molecules of the disclosure further comprise an effector moiety linked to the single chain polypeptide comprising different binding domains (additional preferred proteinaceous molecules of the disclosure comprising at least one effector moiety are provided in examples 5 and 6, in FIG. 1). In j and k, the exemplified preferred proteinaceous molecules of the disclosure further comprise an Fc monomer linked to the different binding domains (see also FIG. 2). In 1, an example is provided of a preferred mono-specific proteinaceous molecule of the disclosure.

[0051] FIG. 4. Elisa results of 4th selection

[0052] The extinction results at OD450 showing the binding of Fab fragments to HCV/A2 antigens.

[0053] FIGS. 5A and 5B. Selections on combined PBL, Spleen and combinatorial Fab library.

[0054] The results of the four selection rounds using the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50) and a combined PBL, spleen and combinatorial Fab library are separately displayed for each selection round. Finally, after the fourth round, two specific clones were obtained.

DETAILED DESCRIPTION

[0055] An Fc fragment is a dimer composed of two linked Fc monomers. The two Fc monomers are covalently bound in the Fc fragment, preferably via one or more disulphide bonds between cystein residues, or are non-covalently bound. An Fc monomer is commonly composed of two or three constant domains C, commonly referred to as CH2-CH3 or CH2-CH3-CH4, respectively. More specifically, any functional fragment of an Fc fragment is part of the disclosure. An example of such a functional fragment of an Fc fragment is the CH2 domain or the CH3 domain.

[0056] A further aspect relates to a method for providing the binding molecules, according to the disclosure. As described hereinabove, it typically involves providing a nucleic acid construct encoding the desired binding molecule. The nucleic acid construct can be introduced, preferably via a plasmid or expression vector, into a prokaryotic host cell and/or in a plant cell and/or in a eukaryotic host cell capable of expressing the construct. In one embodiment, a method of the disclosure to provide a binding molecule comprises the steps of providing a host cell with one or more nucleic acid(s) encoding the binding molecule capable of recognizing and binding to multiple binding sites, and allowing the expression of the nucleic acids by the host cell.

[0057] Binding molecules of the disclosure are, for example, expressed in plant cells, eukaryotic cells or in prokaryotic cells. Non-limited examples of suitable expression systems are tobacco plants, Pichia pastoris, Saccharomyces cerevisiae. Also cell-free recombinant protein production platforms are suitable. Preferred host cells are bacteria, like, for example, bacterial strain BL21 or strain SE1, or mammalian host cells, more preferably human host cells. Suitable mammalian host cells include human embryonic kidney (HEK-293) cells or Chinese hamster ovary (CHO) cells, which can be commercially obtained. Insect cells, such as S2 or S9 cells, may also be used using baculovirus or insect cell expression vectors, although they are less suitable when the polypeptides include elements that involve glycosylation. The produced binding molecules can be extracted or isolated from the host cell or, if they are secreted, from the culture medium of the host cell. Thus, in one embodiment, a method of the disclosure comprises providing a host cell with one or more nucleic acid(s) encoding the binding molecule, allowing the expression of the nucleic acid(s) by the host cell. In another preferred embodiment, a method of the disclosure comprises providing a host cell with one or more nucleic acid(s) encoding two or more different binding molecules allowing the expression of the nucleic acids by the host cell. For example, in one embodiment nucleic acids encoding for two or more different binding molecules all comprising an Fc monomer are provided, enabling isolation of multiple single-chain binding molecules, and/or enabling isolation of homo-dimers and/or hetero-dimers formed through Fc dimerization. Methods for the recombinant expression of (mammalian) proteins in a (mammalian) host cell are well known in the art.

[0058] As will be clear, a binding molecule of the disclosure finds its use in many therapeutic applications and non-therapeutic applications, e.g., diagnostics. Proteinaceous molecules of the disclosure suitable for diagnostic purposes are of particular use for monitoring the expression levels of molecules exposing binding sites on pathogens or on cells infected by pathogens that are targeted by proteinaceous molecules of the disclosure applied for their therapeutic use. In this way, it is monitored whether the therapy remains efficacious or whether other proteinaceous molecules of the disclosure targeting one or more different binding sites on the pathogen or on the infected aberrant cells should be applied instead, in case the expression levels of the first targeted binding sites are below a certain threshold. Binding molecules of the disclosure may also be used for the detection of (circulating) tumor cells related to infection. Or for the target-pathogen, or target-cell specific delivery of cytotoxic compounds, or for the delivery of immune-stimulatory molecules.

[0059] Accordingly, also provided is the use of a binding molecule as medicament. In another aspect, provided is the use of a binding molecule for the manufacture of a medicament for the treatment of infections, aberrancies such as cancer related to infections. Viral infections that can be treated with the invented molecules and compositions include, but are not limited to, Hepatitis viruses (in particular HCV), RSV, HIV, influenza, herpes viruses and human papilloma viruses.

[0060] In one embodiment, proteinaceous molecules of the disclosure comprise binding domains mimicking pattern recognition receptors (PRRs) present on the cells of the body. These PRRs are part of the body's defense mechanism against invading pathogens. The PRRs recognize and bind to broadly shared molecular patterns specifically associated with (classes of) pathogens and not with molecules of the host. Examples of PRRs are the extra-cellular and intra-cellular Toll-like receptors (TLR) 1-13. Proteinaceous molecules of the disclosure comprising binding domains mimicking binding capacities of one or more different PRRs are particularly suitable for binding to pathogens exposing the at least one or more different binding sites for this/these PRR(s). An example is a proteinaceous molecule of the disclosure comprising at least one copy of a binding domain mimicking TLR-2, for binding to gram-positive bacteria exposing a lipoprotein binding site for TLR-2. Of course, in alternative binding molecules of the disclosure, binding domains mimicking binding capacities of PPRs are also combined with different binding domains binding to other pathogen-associated binding sites.

[0061] Antibody fragments of human origin can be isolated from large antibody repertoires displayed by phages. One aspect of the disclosure, known by the art, is the use of human antibody phage display libraries for the selection of two or more human antibody fragments specific for two or more selected different binding sites, e.g., epitopes. These antibody fragments usually display low affinity. It is an important aspect of the disclosure that binding domains specific for pathogens or pathogen-related antigen on aberrant cells are selected for their relatively high affinity. A method is provided that allows the generation of high avidity antibody domain chains able to bind and exert the modulating biological activity in a specific and efficient manner. An aspect of the disclosure is the development of a binding molecule comprising multiple binding domains. That is to say, preferably a human Vh domain, capable of binding to a certain binding site combined with a second, third, fourth, and so on copy of an identical binding domain (multi-valency), and at least one copy of one or more different human Vh domains with each different human Vh domain capable of binding to a separate binding site (multi-specificity). In this way, avidity regarding the first binding site and, if multiple binding domains are applied specific for a second, third, fourth, and so on binding site, avidity regarding this second, third, fourth, and so on binding site is enhanced.

[0062] Thus, a proteinaceous molecule is provided comprising at least two copies of a binding domain specific for a binding site functionally connected with at least one copy of a different binding domain specific for a different binding site or with a different affinity for the same binding site. Preferably, these different binding domains are functionally connected to each other via peptide bonds between amino-acid residues flanking the binding domains, providing a linear single chain proteinaceous molecule (FIG. 1). It is also part of the disclosure that the binding domains are linked together via bonds and/or binding interactions other than covalent peptide bonds between amino acid residues in a linear sequence. Alternative methods for linking proteinaceous molecules to each other are numerous and well known to those skilled in the art of protein linkage chemistry. Protein linkage chemistry not based on peptide bonds in a single chain amino acid sequence can be based on covalent interactions and/or on non-covalent interactions.

[0063] A multi-specific proteinaceous molecule in a monovalent or multivalent binding molecule form of the disclosure capable of modulating a biological process such as an infection is, for example, composed of at least copies of two different human Vh domains or functional fragments thereof, which are multimerized at the DNA level in order to obtain a single-chain polypeptide construct upon expression.

[0064] Isolated human Vh domains usually do not meet the standards for stability and efficient expression that are required by the field. They tend to be unstable, poorly soluble and poorly expressed. A process called camelization may be used to convert human Vh into more stable antibody fragments.

[0065] The human antibody germ-line region Vh-3 displays high homology with antibody Vh fragments of llamas. Llamas have two types of antibodies, those composed of heavy and light chains, and antibodies that only contain heavy chains. These heavy-chain only antibodies bind antigens similar to classical antibodies composed of heavy and light chains. The smallest functional llama antibody binding domain, the Vhh domain, also called (single) domain antibodies ((s)dAb), have been shown to be expressed well and may bind antigen with high affinity. In addition, it has been shown that some of the characteristics, such as ease of expression and stability, of llama sdAb can be transferred to, e.g., human Vh by replacing a few amino acids in the human Vh for those of llama Vhh. Antibody molecules with multi-specificity can then be generated by ligation of one or more copies of several different camelized human Vh domains each with affinity for different binding sites, into one single molecule. Moreover, high avidity antibody molecules can then be generated by ligation of several of the camelized human Vh domains binding to the same binding site, into one single molecule.

[0066] For each of the at least two binding sites, the molecules of the disclosure comprise one to twelve and more preferably one to six and even more preferably one to three camelized human Vh domains interspersed by short linkers, for example, short Gly-Ser linkers, and connected through peptide bonds to the camelized human Vh domains interspersed by short linkers, specific for the other target binding sites of the binding molecules. In another embodiment, for at least one of the at least two binding sites, the molecules of the disclosure comprise preferably four to six camelized human Vh domains interspersed by short linkers, herewith providing the molecules with the capacity to cross-link four to six target molecules exposing this targeted binding site. In an even more preferred embodiment, this cross-linking of molecules induces apoptosis in infected cells expressing surface molecules (e.g., MHCpathogen-derived antigen peptide complex) exposing the targeted binding site for the four to six binding domains.

[0067] Compared to binding molecules specific for a single binding site, the proteinaceous molecules of the disclosure have amongst others the following advantages regarding efficacy and specificity. The proteinaceous binding molecules of the disclosure have an increased specificity for infected aberrant cells by targeting multiple binding sites specific for the aberrant cell simultaneously and/or by targeting combinations of binding sites unique to the aberrant cell simultaneously. In this way, aberrant cells are targeted more efficiently, avoiding (excessive) targeting of healthy cells, and, thus, lowering the risk for toxic and undesired side-effects significantly. This high specificity for infected aberrant cells is achieved with proteinaceous molecules of the disclosure bearing relatively low affinity for binding sites present on both aberrant cells and healthy cells, while bearing relatively high avidity for aberrant cells exposing a combination of different binding sites unique to the aberrant cells. Below, examples are provided for these combinations of binding sites that provide suitable therapeutic targets for the molecules of the disclosure. Moreover, with the multi-specific proteinaceous molecules of the disclosure, difficult to target and/or difficult to reach aberrant cells have a higher chance of being hit by at least one of the binding domains, thereby providing at least in part the therapeutic activity and increasing the success rate when compared to single molecule/single target therapies.

[0068] Examples of various preferred domain topologies in the proteinaceous molecules of the disclosure, as exemplified below, are provided in FIGS. 1 through 3. For example, a proteinaceous molecule of the disclosure that is suitable for specifically targeting aberrant B-cells in Epstein-Barr virus (EBV) infection has the following characteristics: four to six binding domains endowed with high affinity for the infected B-cell specific EBV antigen LMP-1 and/or LMP-2A and/or LMP-2B, linked to one or two binding domains endowed with low affinity for one or more of the adhesion molecules LFA1, CD54, and/or CD58 and/or B-cell activation markers CD23, CD39, CD40, CD44, and/or HLA class II, specific for the infected B-cells. The affinity for LFA1, CD54, CD58, CD23, CD39, CD40, CD44, and/or HLA class II is then selected to be so low that no binding occurs to healthy low-expressing non-infected B-cells lacking an EBV-specific antigen LMP. These exemplified proteinaceous molecules of the disclosure are highly specific for the infected aberrant cells, compared to the healthy (neighboring/circulating) cells. Low affinity and avidity for the proteins also present on the non-infected B-cells prevents binding of the binding molecules to healthy cells. High avidity for the infected B-cells exposing the LMP receptors directs the binding molecules to the aberrant cells. Subsequently, avidity, and, thus, specificity of the binding molecules is even increased due to sequential binding of the low-affinity/low-avidity binding domains specific for the proteins specific for the B-cells. Therefore, in a preferred embodiment, the desired high specificity for infected aberrant cells and concomitant high efficacy regarding infected cell eradication, leaving healthy cells in essence unaltered, of the proteinaceous molecules of the disclosure, are tunable (mix & match approach) by selecting for, for example:

[0069] i) optimal target binding sites,

[0070] ii) optimal number of different binding sites,

[0071] iii) optimal number of binding domains for each selected binding site,

[0072] iv) optimal domain topologies,

[0073] v) optimal affinity of each binding domain,

[0074] vi) optimal avidity for each binding site and for the proteinaceous molecule as a whole,

[0075] vii) optimally facilitating cellular uptake of the proteinaceous molecules of the disclosure (for example, when the binding molecule comprises apoptin),

[0076] viii) optimally facilitating clustering of molecular complexes of targeted surface molecules with bound proteinaceous molecules of the disclosure at the outer membrane surface of infected cells (for example, when the targeted binding sites on surface molecules are complexes of MHC 2 with pathogen derived peptides).

[0077] Abbreviations Used:

[0078] Ab, antibody; CH, constant domain of the heavy chain of an antibody; CHO, Chinese hamster ovary; DAMPs, damage associated molecular patterns; HEK, human embryonic kidney; HPV, human papilloma virus; IEP, iso-electric point; Ig, immunoglobulin; MAGE, melanoma-associated antigen; MHC, major histocompatibility complex; PAMPs, pathogen associated molecular patterns; RA, rheumatoid arthritis; sc-Fv, single-chain variable fragment; V.sub.HH or sdAb, single domain antibodies; VH, Vh or V.sub.H, variable amino-acid sequence of an antibody heavy domain.

EXAMPLES

[0079] Examples of at least two different binding sites each targeted in a monovalent or multivalent manner by proteinaceous molecules of the disclosure comprising at least two different binding domains, such as depicted in FIGS. 1 through 3, are provided in the specification and in the examples, below.

Example 1

[0080] Non-exhaustive examples of proteinaceous molecules of the disclosure comprising binding domains binding to at least two different binding sites, which are each targeted in a monovalent or multivalent manner by the different binding domains, with binding domain topologies as outlined, for example, in FIGS. 1 through 3, are:

[0081] Proteinaceous molecules of the disclosure comprising binding domains binding to:

[0082] a. one or more epitopes in human immunodeficiency virus-1 (HIV-1) envelope protein gp41, and to one or more epitopes in HIV-1 envelope protein gp120, for the treatment of HIV-1 infection or acquired immune-deficiency syndrome, or for opportunistic infection prophylaxis, for example, by neutralizing HIV-1;

[0083] b. one or more epitopes in human immunodeficiency virus-1 (HIV-1) envelope protein gp120, for example, an epitope in the CD4 binding site of gp120 and an epitope in the V3 region of gp120, for the treatment of HIV-1 infection or acquired immune-deficiency syndrome, or for opportunistic infection prophylaxis, for example, by neutralizing HIV-1;

[0084] c. one or more epitopes in human immunodeficiency virus-1 (HIV-1) envelope protein gp41, for example, an epitope encompassing any of the gp41 amino-acid sequences 656-671 (656-NEKELLELDKWASLWN-671, SEQ ID NO:1), 659-673 (659-ELLELDKWASLWNWF-673, SEQ ID NO:2), 660-667 (660-LLELDKWA-667, SEQ ID NO:3), 660-670 (660-LLELDKWASLW-670, SEQ ID NO:4), 661-670 (LELDKWASLW, SEQ ID NO:5), 662-ELDKWA-667 (SEQ ID NO:6) or 662-ELDKWAS-668, (SEQ ID NO:7), for the treatment of HIV-1 infection or acquired immune-deficiency syndrome, or for opportunistic infection prophylaxis, for example, by neutralizing HIV-1;

[0085] d. at least one epitope encompassing any of the gp41 amino-acid sequences 656-671 (656-NEKELLELDKWASLWN-671, SEQ ID NO.1), 659-673 (659-ELLELDKWASLWNWF-673, SEQ ID NO:2), 660-667 (660-LLELDKWA-667, SEQ ID NO:3), 660-670 (660-LLELDKWASLW-670, SEQ ID NO:4), 661-670 (661-LELDKWASLW-670, SEQ ID NO:5), 662-ELDKWA-667 (SEQ ID NO:6) or 662-ELDKWAS-668 (SEQ ID NO:7) of HIV-1 and/or to at least one epitope in the conserved V3 region of gp120 of HIV-1 and/or to at least one epitope in the conserved CD4 binding site of gp120 of HIV-1, for the treatment of HIV-1 infection or acquired immune-deficiency syndrome, or for opportunistic infection prophylaxis, for example, by neutralizing HIV-1;

[0086] e. one or more epitopes in two or more antigens, or to two or more binding sites in a single antigen, exposed by, for example, lipid-A, lipo-polysaccharides, toxins, Rabies, Hepatitis virus, Herpes virus, Rubella virus, Varicella-zoster virus, Staphylococcus, Streptococcus, Hemophilus, Actinomycetes, Pseudomonas, Neisseria, for the treatment of diseases or health problems related to infections by these pathogens and/or related to infections accompanied by the exposure to these molecules;

[0087] f. one or more epitopes in two or more antigens, or to two or more binding sites in a single antigen for which the antigen is (part of) an agent of use in biological warfare, including toxins, plague, smallpox, anthrax, hemorrhagic fever virus, ricin, for the prevention of devastating health effects upon exposure to these agents;

[0088] g. one or more conserved epitopes in the F subdomain of influenza A virus hemagglutinin glycoprotein, for the neutralization of influenza A virus comprising any of the (sixteen) known hemagglutinin subtypes of group 1 and group 2 influenza A viruses, for use as a universal prophylactic or therapeutic flu vaccine;

[0089] h. one or more conserved epitopes in the F subdomain of influenza A virus hemagglutinin glycoprotein and/or to one or more conserved epitopes in the virus' M protein and/or to one or more conserved epitopes in the virus' neuramidase protein, for the treatment of influenza A virus infection, for use as a universal flu vaccine;

[0090] i. one or more epitopes in the CD4-binding site in the gp120 subunit of human immunodeficiency virus type 1 (HIV-1)'s trimeric gp120-gp41 envelope spike and to one or more epitopes in the membrane-proximal external region (MPER) of gp41, for neutralizing HIV-1 strains;

[0091] j. two or more epitopes in a capsular polysaccharide of Streptococcus pneumonia, or to one or more epitopes in two or more different capsular polysaccharides of Streptococcus pneumonia, for the protection against infection (prophylaxis) or for the treatment of infection;

[0092] k. one or more epitopes in the Epstein-Barr virus proteins Epstein-Barr nuclear antigen 1 and/or in latent membrane protein 1 and/or in latent membrane protein 2, for the treatment of Hodgkin's lymphoma associated with Epstein-Barr virus infection;

[0093] l. two or more epitopes in soluble IL-1-binding proteins produced by cowpox or vaccinia, to prevent binding to secreted IL-1 in the infected body, and, thus, to prevent the inhibitory activity on the inflammatory response of the body;

[0094] m. two or more epitopes in TNF-receptor mimic of the Shope fibroma virus, for inhibiting the binding of the TNF-receptor mimic to TNF in the infected body, thereby inhibiting the anti-inflammatory activity of the TNF-receptor mimic;

[0095] n. two or more epitopes in Epstein-Barr virus BCRF1 protein, for inhibiting the stimulatory effect of this human IL-10 analog BCRF1 on production of T-helper 2 cells. Stimulating T-helper 2 cells simultaneously down-regulates T-helper 1 activation, thereby inhibiting T-helper 1 inflammatory response beneficial for infection suppression;

[0096] o. at least two binding sites in the complex of peptide E2(614-622) of Hepatitis C virus with HLA-A2, for the treatment of Hepatitis C virus infection;

[0097] p. at least two conserved (conformational) epitopes on surface E2 glycoprotein present in all major genotypes (1a, 1b, 2a, 2b, 3a, 4, 5, 6) of Hepatitis C virus, for treatment of Hepatitis C virus infections;

[0098] q. one or more epitopes in EBV receptors LMP-1, LMP-2A, LMP2B on infected B-cells and one or multiple copies of binding domains neutralizing EBV derived interleukin-10 homologue BCRF-1 and/or EBV derived BCL-2 homologue BHRF-1 and/or EBV derived C-FMS receptor homologue BARF-1, for the eradication of (primary) EBV-infected cells;

[0099] r. one or more epitopes in EBV surface molecules gp220 and/or gp340 and/or gp350, for the eradication of EBV from the body;

[0100] s. the HLA B8 restricted epitope from EBV nuclear antigen 3, FLRGRAYGL (SEQ ID NO:8), complexed with MHC I, and one or more domains binding to a second surface molecule specific for EBV infected cells, for the clearance of EBV infected cells;

[0101] t. one or more IgE binding sites on a food allergen, for the prevention of an allergic reaction by neutralizing the IgE binding sites.

[0102] Of particular interest are of course combinations of surface molecules exposed by pathogens or by infected aberrant cells exposing pathogen-specific antigens. Targeting binding sites on one of such exposed molecules unique to the infected aberrant cell by proteinaceous molecules of the disclosure would already provide high specificity for aberrant cells over healthy cells not expressing the pathogen-specific antigen. Specificity and efficacy is then even further improved when binding domains are combined in proteinaceous molecules of the disclosure that target binding sites in two or more pathogen-specific antigens uniquely exposed by the infected aberrant cell. Such molecules of the disclosure provide even a higher specificity than molecules of the disclosure targeting two different antigens which are co-expressed on aberrant cells, with one of the two antigens also expressed on healthy cells. Combining binding domains with relatively high affinity for pathogen-specific antigens exposed by aberrant cells with binding domains with relatively low affinity for other surface markers of the particular infected cell type further improves the specificity of the proteinaceous molecules of the disclosure for the aberrant cells. Especially when the affinity for the surface markers specific for the type of cells is below a certain threshold prohibitive for binding of the proteinaceous molecules of the disclosure to healthy cells via binding interactions with these surface markers alone.

[0103] Target binding sites suitable for specific targeting of infected aberrant cells by proteinaceous molecules of the disclosure are combinations of pathogen-derived antigen peptides complexed with MHC molecules. Examples of T-cell epitopes of the E6 and E7 protein of human papilloma virus, complexed with indicated HLA molecules, are provided below. Any combination of pathogen-derived T-cell epitope and bound HLA molecule provides a specific target on infected cells for molecules of the disclosure. An example of an infected cell is a keratinocyte in the cervix infected by human papilloma virus (HPV), presenting T-cell epitopes derived from, for example, E6 or E7 protein, in the context of MHC.

[0104] For example, provided is binding molecules that comprise low-affinity binding domains binding to immune-reactive thrombomodulin expressed on suprabasal spinous layer keratinocytes, low-affinity binding domains binding the squamous cell-marker SPRR1 and high-affinity binding domains binding to one or several, for example, one to three MHC I-HPV 16 E6 T-cell epitope complexes, expressed on epithelial tumor cells, for the targeting of squamous tumors induced upon HPV infection. Examples of suitable target HPV 16 E6 T-cell epitopes are peptides FQDPQERPR (SEQ ID NO:9), TTLEQQYNK (SEQ ID NO:10), ISEYRHYCYS (SEQ ID NO:11) and GTTLEQQYNK (SEQ ID NO:12) binding to HLA A1, KISEYRHYC (SEQ ID NO:13) and YCYSIYGTTL (SEQ ID NO:14) binding to HLA A2, LLRREVYDF (SEQ ID NO:15) and IVYRDGNPY (SEQ ID NO:16) binding to HLA A3, TTLEQQYNK (SEQ ID NO:10) binding to HLA All, CYSLYGTTL (SEQ ID NO:17), KLPQLCTEL (SEQ ID NO:18), HYCYSLYGT (SEQ ID NO:19), LYGTTLEQQY (SEQ ID NO:20), EVYDFAFRDL (SEQ ID NO:21) AND VYDFAFRDLC (SEQ ID NO:22) binding to HLA A24, 29-TIHDIILECV-38 (SEQ ID NO:23) binding to HLA A*0201. Equally suitable are HPV 16 E7 T-cell epitopes such as 86-TLGIVCPI-93 (SEQ ID NO:24), 82-LLMGTLGIV-90 (SEQ ID NO:25), 85-GTLGIVCPI-93 (SEQ ID NO:26) and 86-TLGIVCPIC-94 (SEQ ID NO:27) binding to HLA A*0201, HPV 18 E6 T-cell epitopes and HPV 18 E7 T-cell epitopes, binding to HLA A1, A2, A3, All or A24. Yet additional examples of T-cell epitopes related to HPV infected cells are HPV E7 derived peptides 1-MHIGDTPTLHEYD-12 (SEQ ID NO:28), 48-DRAHYNIVTFCCKCD-62 (SEQ ID NO:29) and 62-DSTLRLCVQSTHVD-75 (SEQ ID NO:30) binding to HLA DR, 7-TLHEYMLDL-15 (SEQ ID NO:31), 11-YMLDLQPETT-20 (SEQ ID NO:32), 11-YMLDLQPET-19 (SEQ ID NO:33) and 12-MLDLQPETT-20(SEQ ID NO:34) binding to HLA A*201, 16-QPETTDLYCY-25 (SEQ ID NO:35), 44-QAEPDRAHY-52 (SEQ ID NO:36) and 46-EPDRAHYNIV-55 (SEQ ID NO:37) binding to HLA B18, 35-EDEIDGPAGQAEPDRA-50 (SEQ ID NO:38) binding to HLA DQ2, 43-GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-77 (SEQ ID NO:39) binding to HLA DR3, 50-AHYNIVTFCCKCD-62 (SEQ ID NO:40) binding to HLA DR15, 58-CCKCDSTLRLC-68 (SEQ ID NO:41) binding to HLA DR17 and 61-CDSTLRLCVQSTHVDIRTLE-80 (SEQ ID NO:42) binding to HLA-DRB1*0901. Examples of alternative keratinocyte markers to which low-affinity binding domains in binding molecules of the disclosure can bind are human gene encoding keratinocyte proline-rich protein, glycoprotein-80 and 174H.64.

[0105] For the treatment of health problems related to exposure to agents used for acts of bioterrorism and biological warfare, multi-specific binding molecules are designed as part of the disclosure. For example, binding molecules of the disclosure comprise different binding domains with specificity for two or more agents used for biological warfare. Examples are the combination of one or more binding domains specific for anthrax, combined with one or more binding domains specific for botulinum neurotoxin. In this way, a few multi-specific binding molecules are designed, which are useful for the prophylaxis or treatment of all the commonly acknowledged biological warfare threats, and which can be stock piled. This provides the benefits of being prepared for attacks by the common agents, by producing, purifying and stock-piling only a few different multi-specific proteinaceous molecules of the disclosure.

[0106] It is one of the advantages of the disclosure that immune escape mechanisms of pathogens are effectively counteracted upon use of the binding molecules of the disclosure. For example, the binding molecules are multi-specific, in a monovalent or multivalent manner, for pathogen associated molecular patterns (PAMPs). In this way, the probability for occurrence of immune escape is strongly reduced. Proteinaceous molecules of the disclosure will only then not be able to bind to the targeted pathogen anymore in the unlikely situation when all binding sites on the PAMP(s) are mutated simultaneously in a way that binding affinity is lost completely. Thus, the proteinaceous molecules of the disclosure can still exert at least partially a desired therapeutic effect as long as at least one binding site on a PAMP remains unaltered while the other, or one or more of the other, or even all other binding sites are mutated on the pathogen surface.

[0107] One example of proteinaceous molecules of the disclosure are molecules comprising multiple different Vh domains, in monovalent or multivalent form, specific for multiple isotypes or serotypes of the same virus. By doing so, the area of therapeutic use of the binding molecules is expanded, covering a broader range of viral subtypes. An example that is provided are proteinaceous molecules of the disclosure comprising binding domains specific for conserved epitopes in the F subdomain of influenza A virus hemagglutinin glycoprotein. Such proteinaceous molecules of the disclosure are of particular use in the treatment of infection with influenza virus of any of the known A subtypes.

Example 2: Selection of Antibody Fragments

[0108] Multi-specific proteinaceous molecules of the disclosure are built from any antigen binding domain, such as, but not limited to, antibodies, alpha-helices and T-cell receptors. Antibody Vh fragments specific for pathogens or for pathogen associated surface antigens are derived from hybridoma cells producing mouse, rat, rabbit, llama or human antibodies. Antibody fragments can also be obtained after immunization of animals with pathogen (cells) or (partly) purified pathogen antigen. Alternatively, antibody fragments of human, mouse, rat or llama origin can be obtained from antibody phage, yeast, lymphocyte or ribosome display libraries. Such antibody libraries (scFv, Fab, Vh or Vhh) may be constructed from non-immunized species as well as immunized species.

[0109] 2.1: Selection of human antibody fragments specific for pathogen antigens or cell-surface expressed pathogen-associated antigens.

[0110] To obtain human antibody fragments specific for a surface molecule on a pathogen or for a pathogen associated antigen expressed at the surface of an invaded cell, a Human antibody Fab, VHCH or Vh phage display library will be used for selections essentially as described by Chames et al. Human Fab phages (10.sup.13 colony forming units) are first pre-incubated for 1 h at room temperature in PBS containing 2% non-fat dry milk (PB SM). In parallel, 200 l Streptavidin-coated beads (Dynal) are equilibrated for 1 h in PB SM. For subsequent rounds, 100 l beads are used. Equilibrated beads are added, and the mixture is incubated for 15 minutes under rotation. Beads are drawn to the side of the tube using magnetic force. To the depleted phage fraction, subsequently decreasing amounts of biotinylated target antigen are added and incubated for 1 h at room temperature, with continuous rotation. Equilibrated streptavidin-coated beads are added, and the mixture incubated for 15 minutes under rotation. Phages are selected by magnetic force. Non-bound phages will be removed by 5 washing steps with PBSM, 5 steps with PBS containing 0.1% TWEEN, and 5 steps with PBS. Phages are eluted from the beads by 10 minutes incubation with 500 l freshly prepared tri-ethylamine (100 mM). The pH of the solution is then neutralized by the addition of 500 l 1 M Tris (pH 7.5). The eluted phages are incubated with logarithmic growing E. coli TG1 cells (OD.sub.600 nm of 0.5) for 30 minutes at 37 C. Bacteria are grown overnight on 2TYAG plates. Next day, colonies are harvested, and a 10 l inoculum is used in 50 ml 2TYAG. Cells are grown until an OD.sub.600 nm of 0.5, and 5 ml of this suspension is infected with M13k07 helper phage (510.sup.11 colony forming units). After 30 minutes incubation at 37 C., the cells are centrifuged, resuspended in 25 ml 2TYAK, and grown overnight at 30 C. Phages are collected from the culture supernatant, as described previously, and used for the next round panning. After two, three or four selection rounds enrichment of specific binders is obtained, and individual clones are analyzed for binding to specific influenza virus by ELISA.

Example 3: Production of Multi-Specific Proteins Comprising Camelized Single Domain Vh Domains

[0111] 3.1: Design of Genes for Production of Multi-Specific Vh Proteins.

[0112] Human antibody germline gene VH3 demonstrates high homology to llama single domains VHH. Exchange of amino-acids 44, 45 and 47 in the human VH3 genes by amino-acids present in llama VHH at these positions has shown to enhance stability and expression of the human VH3 genes [Riechmann, Muyldermans, 199]. For expression and stability many of the selected human Vh might benefit from the exchange of amino-acids 44, 45 and 47 by llama VHH amino-acids, a process called camelization. A gene comprising at least two distinct human Vh domains binding to at least two distinct pathogen epitopes or pathogen-associated cell surface epitopes of invaded cells will be compiled such that upon expression it would comprise six Vh domains. To this end, a gene will be designed comprising the pelB secretion signal, which will be operatively linked to six codon-optimized, camelized Vh domains with linkers ((Gly.sub.4Ser).sub.n (SEQ ID NO:43), (GSTSGS).sub.n (SEQ ID NO:44), GSTSGSGKPGSGEGSTKG (SEQ ID NO:45), EFAKTTAPSVYPLAPVLESSGSG (SEQ ID NO:46) or any other linker that provides flexibility for protein folding, or, EPKSCDKTHT (IgG1) (SEQ ID NO:47), ELKTPLGDTTHT (IgG3) (SEQ ID NO:48), or ESKYGPP (IgG4) (SEQ ID NO:49)) between each Vh domain. This gene will, for example, be synthesized by Geneart (Regensburg, Germany) and cloned into the pStaby 1.2 vector (Delphi genetics, Belgium) for expression in E. coli.

Example 4: Production and Purification of Hexameric Vh Protein

[0113] For expression of multi-specific Vh proteins the pStaby-multispecific-protein vectors will be introduced via electroporation into SE1 bacteria. Positive clones will be grown in the presence of 2% glucose at 25-30 C. until OD.sub.600=0.8. Bacterial TYAG medium will then be replaced with TY medium containing 0,1-1 mM IPTG to induce expression. After overnight culture at 25-30 C. bacteria and medium will be harvested. The periplasm fraction will be collected after incubation of bacteria with PBS/EDTA/NaCl for 30 minutes on ice. Protein expression will then be analyzed by SDS-PAGE.

[0114] Multi-specific Vh proteins will be isolated from media and bacteria using Ni-affinity purification. To this end medium will be incubated with Ni-coupled Sepharose-beads and incubated overnight while stirring gently. To obtain intracellular proteins bacteria will be lysed and cellular debris removed by centrifugation. After overnight dialysis with PBS multi-specific Vh proteins will be purified with Ni-Sepharose. Purity of the multi-specific Vh proteins will be analyzed by SDS-PAGE and protein concentration determined by BCA protein assay (Pierce).

Example 5: Construction of Multi-Specific Genes to Improve Circulation

[0115] The pharmacokinetic properties of therapeutic proteins, e.g., their distribution, metabolism and excretion are dependent on factors such as shape, charge and size. Most small plasma molecules (MW<50-60 kDa) possess very short half-life, whereas larger plasma proteins such as human serum albumin (HSA) and immunoglobulins (Ig) have very long half-lives (19 days for HSA, 1-4 weeks for Ig). Indeed, addition of IgG-Fc or Human serum albumin has shown to extend circulation time when linked to therapeutic proteins. In addition the coupling of IgG-Fc to the multi-specific proteins will allow recruitment of immune cells to the site of infected cells allowing immune specific responses against the colonized tissue.

[0116] 5.1: Construction of Multi-Specific Proteins with IgG1-Fc and Human Serum Albumin.

[0117] The multi-specific construct will be linked to the IgG1-Fc region or to human serum albumin, codon optimized for expression in eukaryotic cells and cloned into the pcDNA-3.1+ vector (Geneart, Regensburg, Germany).

Example 6: Isolation of a Binding Domain for a HCV Epitope (KLVALGINAV (SEQ ID NO:50)) in the Context of HLA A02.01 from a FAB Phage Display Library

[0118] 6.1: Selection of Human Antibody Fragments Specific for Pathogen Antigens Presented by HLA-A02.01.

[0119] For selection of human antibody fragments specific for the HCV epitope KLVALGINAV (SEQ ID NO:50) presented by HLA-A02.01 on the surface of infected cells, essentially the protocol as outlined in example 2, was conducted (see above). Thus, to obtain human antibody fragments specific for a HCV epitope (KLVALGINAV (SEQ ID NO:50)) presented by HLA-A02.01 at the surface of an infected cell, a human antibody Fab phage display library was used for selections. For the first selection round, human Fab phages (10.sup.13 colony forming units) were first pre-incubated for 1 h at room temperature in PBS containing 2% non-fat dry milk (PBSM). In parallel, 200 l Streptavidin-coated beads (Dynal) were equilibrated for 1 h in PBSM. For a subsequent second, third and fourth selection round, 100 l beads were used. Equilibrated beads were added, and the mixture was incubated for 15 minutes under rotation. Beads were drawn to the side of the tube using magnetic force. To the depleted phage fraction, subsequently decreasing amounts of biotinylated target antigen were added and incubated for 1 h at room temperature, with continuous rotation. Equilibrated Streptavidin-coated beads were added, and the mixture incubated for 15 minutes under rotation. Phages were selected by magnetic force. Non-bound phages were removed by 5 washing steps with PBSM, 5 steps with PBS containing 0.1% TWEEN, and 5 steps with PBS. Phages were eluted from the beads by 10 minutes incubation with 500 l freshly prepared tri-ethylamine (100 mM). The pH of the solution was then neutralized by the addition of 500 l 1 M Tris (pH 7.5). The eluted phages were incubated with logarithmic growing E. coli TG1 cells (OD.sub.600 nm of 0.5) for 30 minutes at 37 C. Bacteria were grown overnight on 2TYAG plates. Next day, colonies were harvested, and a 10 l inoculum was used in 50 ml 2TYAG. Cells are grown until an OD.sub.600 nm of 0.5, and 5 ml of this suspension was infected with M13k07 helper phage (510.sup.11 colony forming units). After 30 minutes incubation at 37 C., the cells were centrifuged, resuspended in 25 ml 2TYAK, and grown overnight at 30 C. Phages were collected from the culture supernatant as described previously, and used for the next round panning. After four selection rounds individual clones were analyzed for specific binding to the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50) by ELISA (FIG. 4). The ELISA results revealed that after the fourth selection round two antibody Fab molecules were selected, enriched for specific binding to the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50).

Example 7: Production of a Mono-Specific Protein Comprising Six Camelized Single Domain Vh Domains

[0120] 7.1: Design of Genes for Production of a Hexameric Mono-Specific Vh Protein Specific for the Complex of HLA-A02.01 and the HCV Peptide Epitope KLVALGINAV (SEQ ID NO:50).

[0121] A hexameric monospecific protein comprising six Vh domains specific for the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50) and obtained with the selection procedure outlined in example 6.1, above, will be constructed essentially as outlined in example 3.1 (see above).

[0122] 7.2: Production and Purification of Hexameric Monospecific Vh Protein.

[0123] Production and purification of the hexameric mono-specific Vh protein specific for the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50) as will be obtained, according to Example 7.1, will essentially be performed according to the protocol provided in Example 4.

Example 8: Apoptosis Inducing Activity of the Hexameric Mono-Specific Vh Protein Specific for the Complex of HLA-A02.01 and the HCV Peptide Epitope KLVALGINAV (SEQ ID NO:50) Towards Cells Presenting this Complex

[0124] The apoptosis inducing capacity of purified hexameric monospecific binding molecules specific for the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50) towards mammalian cells presenting this complex are demonstrated with HLA-A02.01 expressing HCV infected cells. To this end, HCV-infected mammalian cells are either exposed to the hexameric monospecific binding molecules specific for the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50), or to a negative control (e.g., a non-binding hexameric Vh molecule and/or assay buffer only). In addition, as a negative control, non-infected cells are exposed to the hexameric monospecific binding molecules. Apoptosis inducing activity of the hexameric monospecific binding molecules towards HCV-infected cells is quantified by analyzing (the) amount(s) of apoptotic marker molecule(s) exposed or secreted by these infected cells (e.g., caspase) exposed to the hexameric monospecific binding molecules specific for the complex of HLA-A02.01 and the HCV peptide epitope KLVALGINAV (SEQ ID NO:50), compared to (the) amount(s) of apoptotic marker molecules exposed or secreted by the controls.

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