ABERRANT CELL-RESTRICTED IMMUNOGLOBULINS PROVIDED WITH A TOXIC MOIETY

Abstract

Described are immunoglobulins provided with a toxic moiety, comprising at least an immunoglobulin variable region that specifically binds to an MHC-peptide complex preferentially associated with aberrant cells. These immunoglobulins provided with a toxic moiety may be used in selectively modulating biological processes. These immunoglobulins provided with a toxic moiety are of particular use in pharmaceutical compositions for the treatment of diseases related to cellular aberrations, such as cancers and autoimmune diseases.

Claims

1. An immunoglobulin provided with a toxic moiety, comprising at least an immunoglobulin variable region that specifically binds to an MHC-peptide complex preferentially associated with aberrant cells.

2. The immunoglobulin according to claim 1 wherein said immunoglobulin variable region is a Vh or Vhh.

3. The immunoglobulin according to claim 2 wherein said immunoglobulin variable region further comprises a Vl.

4. The immunoglobulin according to claim 3, which is a human IgG.

5. The immunoglobulin of claim 1, wherein the MHC-peptide complex is specific for aberrant cells.

6. The immunoglobulin of claim 1, wherein the toxic moiety is chemically linked to the immunoglobulin.

7. The immunoglobulin of claim 1, wherein the toxic moiety is a fusion protein, fused to the immunoglobulin at the DNA level.

8. A pharmaceutical composition comprising: the immunoglobulin of claim 1, and suitable diluents and/or excipients.

9. A method of treating a host suffering from a disease associated with aberrant cells, the method comprising: utilizing an immunoglobulin provided with a toxic moiety of claim 1, for the treatment of the host suffering from a disease associated with aberrant cells.

10. The method according to claim 9, wherein the toxic moiety is internalized into the aberrant cell.

11. A method of treating a subject determined to be suffering from cancer, the method comprising: utilizing the immunoglobulin of claim 1 to treat cancer.

12. The method according to claim 11, wherein at least the toxic moiety is internalized into an aberrant cell of the subject.

13. An immunoglobulin provided with a toxic moiety according to FIG. 5B.

14. The immunoglobulin of claim 5, wherein the MHC-peptide complex is specific for aberrant cells through a peptide derived from MAGE.

15. The immunoglobulin of claim 14, wherein the MAGE is MAGE-A.

16. The immunoglobulin of claim 7, wherein the toxic moiety is a fusion protein fused to the immunoglobulin at the DNA level through a linking sequence.

17. An immunoglobulin chemically linked with a toxic moiety comprising: at least a Vh or Vhh immunoglobulin variable region that specifically binds to an MHC-peptide complex, which is derived from MAGE, preferentially associated with aberrant cells, wherein the immunoglobulin is a human IgG.

18. The immunoglobulin of claim 17 wherein the immunoglobulin variable region is a Vl.

19. The immunoglobulin of claim 17, wherein the MAGE is MAGE-A.

20. The immunoglobulin of claim 1, wherein the toxic moiety is a fusion protein fused to the immunoglobulin at the DNA level through a linking sequence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1: Specific binding of HLA-A0201/multi-MAGE-A specific phage clones isolated from a large human non-immune antibody Fab phage library. Individual antibody Fab expressing phages that were selected against biotinylated HLA-A0201/multi-MAGE-A were analyzed by ELISA for their capacity to bind the relevant peptide/MHC complex only. Streptavidin coated 96 well plates were incubated with soluble HLA-A0201/multi-MAGE-A (A2/multiMage) or HLA-A0201/JCV (A2/JC) peptide/MHC complexes (10 μg/ml), washed to remove non-bound complexes and incubated with individual phage clones. Non-binding phages were first removed by three washes with PBS/Tween, followed by incubation with anti-M13 antibody (1 μg/ml, Amersham) for one hour by room temperature. Finally, the wells were incubated with an HRP-labeled secondary antibody and bound phages detected.

[0024] FIG. 2: Phages AH5, CB1 and CG1 specifically bind cells presenting the multi-MAGE-A peptide. Phages AH5, CB1, CG1, BD5 and BC7 that had shown specific binding in ELISA using the relevant HLA-A201/multi-MAGE-A complex and an irrelevant HLA-A201 complex loaded with a JCV peptide were analyzed for their capacity to bind cells presenting the multi-MAGE-A peptide in HLA-A0201 molecules. To this end, human B-LCL (BSM) were loaded with multi-MAGE-A peptide (10 μg in 100 μl PBS) for 30 minutes at 37° C., followed by incubation with the Fab phages AH5, CB1, CG1, BD5 and BC7 and analyzed by flow-cytometry using anti-phage antibodies and a fluorescently labeled secondary antibody.

[0025] FIG. 3: Phages expressing HLA-A2/multi-MAGE-A specific Fab bind tumor cells of distinct histologic origin. Phages AH5, CB1 and CG1 specific for HLA-A0201/multi-MAGE-A and a positive control phage specific for HA-0101/MAGE-A1 were used for staining of distinct tumor cell lines. To this end the prostate cancer cell line LNCaP, the multiple myeloma cell line MDN, the melanoma cell lines MZ2-MEL43 and G43, and the breast cancer cell line MDA-MD157 were incubated with the different phages (30 minutes at 4° C.), bound phages were then detected by flow cytometry using anti-phage antibodies and fluorescently labeled secondary antibodies.

[0026] FIG. 4: Phage AH5 specifically binds HLA-A0201/multi-MAGE-A complexes only. To determine specificity of the phage AH5 an ELISA was performed using relevant and irrelevant peptide/MHC complexes. HLA-A0201 with multi-MAGE-A, gp100, JCV and MAGE-C2 peptides, as well as HLA-A1 with MAGE-A1 peptide were coated on streptavidin 96 well plates and incubated with phage AH5.

[0027] FIG. 5: Cartoon displaying examples of preferred immunoglobulins provided with a toxic moiety, according to the disclosure.

[0028] A. Cartoon displaying the topology of the twelve immunoglobulin domains assembled in an immunoglobulin G. B. Examples are provided of preferred immunoglobulins provided with a toxic moiety, according to the disclosure. Shown are immunoglobulins provided with a single toxic moiety such as, e.g., a cytostatic agent, linked to the immunoglobulin with a chemical linker (exemplified by I. and II.; immunoglobulin-toxic moiety conjugates), or immunoglobulins provided with a single toxic moiety, linked to the immunoglobulin with a peptide linker (exemplified by III.; fused immunoglobulin-toxic moiety molecule). In IV., an immunoglobulin provided with a toxic moiety hereof is shown, comprising one immunoglobulin heavy chain comprising a fused proteinaceous toxic moiety, comprising immunoglobulin variable regions specific for a certain binding site, and comprising a second immunoglobulin heavy chain comprising immunoglobulin variable regions specific for a different binding site. Of course, also part hereof are bi-specific immunoglobulins provided with a toxic moiety hereof comprising two heavy chains comprising different immunoglobulin variable regions specific for different binding sites and further comprising the same or different proteinaceous toxic moieties fused two the heavy chains. Of course, as part hereof, more than one and typically two to six toxic moiety molecules can be fused or conjugated to an immunoglobulin molecule.

[0029] FIG. 6: Human Fab phage F9 specifically binds HLA-A2/FLWGPRALV positive CMT64 mouse lung tumor cells.

[0030] Human Fab clone F9 was analyzed for its capacity to bind mouse lung tumor cells (CMT64) stably expressing the HLA-A2/FLWGPRALV [SEQ ID NO:23] complex. Purified Clone F9 Fab fragments (3 μg total) were incubated with 0.5×10.sup.6 CMT64 cells that do not express human HLA, that express HLA-A2/YLEYRQVPG [SEQ ID NO:3] or that express HLA-A2/FLWGPRALV [SEQ ID NO:23]. After one hour incubation on ice CMT64 cells were incubated with a fluorescently labeled secondary antibody and analyzed by flow cytometry.

[0031] FIG. 7: Llama VHH specifically binds CMT64 mouse lung tumor cells expressing human HLA-A2/multi-MAGE-A.

[0032] Llama VHH specific for A2/FLW or A2/YLE were analyzed by flow cytometry for their binding capacity to CMT64 cells expressing these human HLA-A0201/multi-MAGE-A complexes. Purified VHH fragments (3 μg total) were incubated with 0.5×10.sup.6 CMT64 cells that do not express human HLA, that express HLA-A2/YLEYRQVPG [SEQ ID NO:3] or that express HLA-A2/FLWGPRALV [SEQ ID NO:23]. After one hour incubation on ice CMT64 cells were incubated with a fluorescently labeled secondary antibody and analyzed by flow cytometry.

DETAILED DESCRIPTION

[0033] One aspect of the disclosure relates to a method for providing the described antibodies. As described herein above, it typically involves providing a nucleic acid construct encoding the desired immunoglobulin part of antibodies hereof, or encoding the desired immunoglobulin fused to a proteinaceous toxic moiety. 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 hereof to provide an immunoglobulin or to provide an immunoglobulin fused to a proteinaceous toxic moiety, comprises the steps of providing a host cell with the nucleic acid(s) encoding the immunoglobulin or the immunoglobulin fused to a proteinaceous toxic moiety, and allowing the expression of the nucleic acid(s) by the host cell.

[0034] Included herein is that nucleic acids encoding selected (human) immunoglobulin Vh(h) domains, according to any of the described embodiments, are combined with nucleic acids encoding human immunoglobulin heavy chain constant domains, providing nucleic acid molecules hereof enencoding a heavy chain of a human antibody. The human antibody heavy chain protein product of such a nucleic acid molecule hereof then may be hetero-dimerized with a universal human antibody light chain. It is also part of the disclosure that nucleic acids encoding (jointly) selected human immunoglobulin Vl domains and Vh domains, according to any of the described embodiments, are combined with nucleic acids encoding a human immunoglobulin light chain constant domain and are combined with nucleic acids encoding human immunoglobulin heavy chain constant domains, respectively, providing nucleic acid molecules enencoding a light chain and for a heavy chain of a human antibody. In yet another embodiment, the nucleic acids encoding the complementarity determining regions 1, 2 and 3 (CDR1, CDR2, CDR3), forming together the immunoglobulin variable region of a selected immunoglobulin Vh domain and/or a selected immunoglobulin Vl domain, according to any of the described embodiments, are combined with nucleic acids encoding human immunoglobulin Vh domain frame work regions and/or human immunoglobulin Vl domain frame work regions, respectively, providing nucleic acid molecules hereof enencoding a heavy chain variable domain (Vh) of a human antibody and/or enencoding a light chain variable domain (Vl) of a human antibody (a method known in the art as “grafting”). These nucleic acid molecules enencoding variable domains Vh and/or Vl are, as part hereof, then combined with nucleic acids encoding human immunoglobulin constant domains, providing a nucleic acid molecule enencoding a human antibody heavy chain and/or providing a nucleic acid molecule enencoding a human antibody light chain.

[0035] Immunoglobulins or immunoglobulins fused to a proteinaceous toxic moiety are, e.g., 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, e.g., 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, PerC6 cells or preferably 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 immunoglobulins or the fused immunoglobulins-toxic moiety molecules hereof include elements that involve glycosylation. The produced immunoglobulins or fused immunoglobulin-toxic moiety molecules hereof 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 hereof comprises providing a host cell with one or more nucleic acid(s) encoding the immunoglobulin or the fused immunoglobulin-toxic moiety molecule, allowing the expression of the nucleic acids by the host cell. In another preferred embodiment, a method hereof comprises providing a host cell with one or more nucleic acid(s) encoding two or more different immunoglobulins or two or more different fused immunoglobulin-toxic moiety molecules, allowing the expression of the nucleic acids by the host cell. For example, in one embodiment, nucleic acids enencoding a so-called universal immunoglobulin light chain and nucleic acids enencoding two or more different immunoglobulin heavy chains are provided, enabling isolation of mono-specific immunoglobulins or mono-specific fused immunoglobulin-toxic moiety molecules comprising homo-dimers of heavy chains and/or enabling isolation of bi-specific immunoglobulins or bi-specific fused immunoglobulin-toxic moiety molecules comprising hetero-dimers of heavy chains, with all different heavy chains complexed with a universal light chain. Methods for the recombinant expression of (mammalian) proteins in a (mammalian) host cell are well known in the art.

[0036] As said, it is preferred that the immunoglobulins hereof are linked with the toxic moieties via bonds and/or binding interactions other than peptide bonds. Methods for linking proteinaceous molecules such as immunoglobulins to other proteinaceous molecules or non-proteinaceous molecules are numerous and well known to those skilled in the art of protein linkage chemistry. Protein linkage chemistry not based on peptide bonds can be based on covalent interactions and/or on non-covalent interactions. A typical example of linkage chemistries applicable for linking toxic moieties to immunoglobulins hereof are the various applications of the Universal Linkage System disclosed in patent applications WO92/01699, WO96/35696, WO98/45304, WO03040722.

[0037] As will be clear, an antibody finds its use in many therapeutic applications and non-therapeutic applications, e.g., diagnostics, or scientific applications. Antibodies, or more preferably the immunoglobulin part of the antibodies hereof, suitable for diagnostic purposes are of particular use for monitoring the expression levels of molecules exposing binding sites on aberrant cells that are targeted by antibodies hereof. In this way, it is monitored whether the therapy remains efficacious or whether other antibodies hereof targeting one or two different binding sites on the aberrant cells should be applied instead. This is beneficial when the expression levels of the first or the first two targeted binding site(s) are below a certain threshold, whereas another or new binding sites (still) can serve as newly targeted binding sites for antibodies hereof comprising the appropriate specific immunoglobulin variable regions for these alternative binding site(s). Antibodies hereof may also be used for the detection of (circulating) tumor cells, and for the target-cell specific delivery of immune-stimulatory molecules. For these later two uses, the sole immunoglobulins hereof without the fused or conjugated toxic moiety may also be used.

[0038] Provided herein is a method for inducing ex vivo or in vivo a modulating effect on a biological process in a target cell, comprising contacting the cell with an antibody hereof in an amount that is effective to induce the modulating effect. Preferably, the antibody hereof is used for a modulating effect on a biological process of aberrant cells in a subject, more preferably a human subject. For therapeutic applications in humans, it is, of course, preferred that an antibody hereof does not contain amino acid sequences of non-human origin. More preferred are antibodies hereof, which only contain human amino acid sequences. Therefore, a therapeutically effective amount of an antibody hereof capable of recognizing and binding to one or two disease-specific binding sites and subsequently inducing a modulating effect on a biological process in the cell, can be administered to a patient to stimulate eradication of aberrant cells expressing the binding site(s) without affecting the viability of (normal) cells not expressing the disease-specific binding site(s). The specific killing of aberrant cells while minimizing or even avoiding the deterioration or even death of healthy cells will generally improve the therapeutic outcome of a patient after administration of the antibodies hereof.

[0039] Accordingly, also provided is the use of an antibody hereof as medicament. In another aspect, provided is the use of an antibody hereof for the manufacture of a medicament for the treatment of cancer, autoimmune disease, infection or any other disease of which the symptoms are reduced upon targeting aberrant cells expressing disease-specific binding sites with antibodies hereof. For example, an antibody hereof is advantageously used for the manufacture of a medicament for the treatment of various cancers (e.g., solid tumors, hematologic malignancies).

[0040] An example of a preferred antibody is an antibody comprising at least an immunoglobulin variable region specifically binding to the complex between MHC-1 HLA-0201 and a multi-MAGE-A epitope, conjugated with a toxic moiety, using, e.g., Universal Linkage System linker chemistry for conjugation. A second example of a preferred antibody is an antibody comprising at least an immunoglobulin variable region specifically binding to the complex between MHC-1 HLA-CW7 and a multi-MAGE-A epitope, conjugated with a toxic moiety, using, e.g., Universal Linkage System linker chemistry for conjugation. With the bi-specific antibodies hereof, difficult to target and/or difficult to reach aberrant cells have a higher chance of being “hit” by at least one of the two different immunoglobulin variable regions in the bi-specific antibodies hereof, thereby providing at least in part the therapeutic activity. An example of a preferred bi-specific antibody hereof is an immunoglobulin comprising an immunoglobulin variable region specific for the complex between MHC-1 HLA-0201 and a multi-MAGE-A epitope and comprising a second immunoglobulin variable region specific for the complex between MHC-1 HLA-CW7 and a second multi-MAGE-A epitope, conjugated with a toxic moiety.

[0041] Antibody fragments of human origin can be isolated from large antibody repertoires displayed by phages. Included herein is the use of human antibody phage display libraries for the selection of human antibody fragments specific for a selected binding site, e.g., an epitope. Examples of such libraries are phage libraries comprising human Vh repertoires, human Vh-Vl repertoires, and human Vh-Ch1 or human antibody Fab fragment repertoires.

[0042] Although the disclosure contemplates many different combinations of MHC and antigenic peptides, the most preferred is the combination of MHC-1 and an antigenic peptide from a tumor related antigen presented by the MHC-1, exclusively expressed by aberrant cells and not by healthy cells. Because of HLA restrictions, there are many combinations of MHC-1-peptide complexes as well as of MHC-2-peptide complexes that can be designed based on the rules for presentation of peptides in MHC. These rules include size limits on peptides that can be presented in the context of MHC, restriction sites that need to be present for processing of the antigen in the cell, anchor sites that need to be present on the peptide to be presented, etc. The exact rules differ for the different HLA classes and for the different MHC classes. We have found that MAGE derived peptides are very suitable for presentation in an MHC context. An MHC-1 presentable antigenic peptide with the sequence Y-L-E-Y-R-Q-V-P-G in MAGE-A [SEQ ID NO:3] was identified, that is present in almost every MAGE-A variant (multi MAGE peptide) and that will be presented by one of the most prevalent MHC-1 alleles in the Caucasian population (namely HLA A0201). A second MAGE peptide that is presented by another MHC-1 allele (namely HLA-CW7) and that is present in many MAGE variants, like, e.g., MAGE-A2, -A3, -A6 and -A12, is E-G-D-C-A-P-E-E-K [SEQ ID NO:4]. These two combinations of MHC-1 and MAGE peptides together would cover 80% of the Caucasian population. The same approach can be followed for other MHC molecules, other HLA restrictions and other antigenic peptides derived from tumor-associated antigens. Relevant is that the chosen antigenic peptide to elicit the response to must be presented in the context of an MHC molecule and recognized in that context only. Furthermore, the antigenic peptide must be derived from a sufficiently tumor specific antigen and the HLA restriction must occur in a relevant part of the population. One of the important advantages of the disclosure is that tumors that down regulate their targeted MHC-peptide complex can be treated with a second immunoglobulin comprising at least one variable region binding to a different MHC-peptide complex based on the same antigen. If this one is down regulated, a third one will be available. For heterozygotes six different targets on MHC-1 may be available. Since cells need to be “inspected” by the immune system from time to time, escape through down regulation of all MHC molecules does not seem a viable escape route. In the case that MAGE is the antigen from which the peptide is derived escape through down regulation of the antigen is also not possible, because MAGE seems important for survival of the tumor [8]. Thus, the disclosure, in an important aspect reduces or even prevents escape of the tumor from the therapy. Thus, provided is in a preferred embodiment an antibody hereof whereby the immunoglobulin variable region is capable of binding to an MHC-I-peptide complex. In a further preferred embodiment, provided is an immunoglobulin whereby the immunoglobulin variable region is capable of binding to MHC-I-peptide complexes comprising an antigenic peptide derived from a tumor related antigen, in particular MHC-I-peptide complexes comprising an antigenic peptide present in a variety of MAGE antigens, whereby the immunoglobulin is provided with a toxic moiety.

[0043] Because in one embodiment, the disclosure uses MHC molecules as a target, and individuals differ in the availability of MHC targets, also provided is a so-called companion diagnostic to determine the HLA composition of an individual. Although the disclosure preferably uses a more or less universal (MAGE) peptide, also provided is a diagnostic for determining the expression of the particular antigen by the tumor. In this manner the therapy can be geared to the patient (personalized medicine, patient stratification), particularly, also in the set-up to prevent escape, as described hereinbefore. It is known that the HLA restriction patterns of the Asian population and the black population are different from the Caucasian population. For different populations different MHC-peptide complexes can be targeted.

[0044] Although the disclosure presents more specific disclosure on tumors, it must be understood that other aberrant cells can also be targeted by the antibodies of the disclosure. These other aberrant cells are typically cells that also proliferate without sufficient control. This occurs in autoimmune diseases. It is typical that these cells start to show expression of tumor antigens. In particular, MAGE polypeptides have been identified in rheumatoid arthritis [7].

[0045] In literature it is shown that a single nine amino-acid (A.A.) peptide in MAGE-A2, -A3, -A4, -A6, -A10, and -A12 is presented by HLA-A0201 on tumor cells, and can be recognized by cytotoxic T-lymphocytes [1]. This nine amino acid residues peptide with sequence Y-L-E-Y-R-Q-V-P-G [SEQ ID NO:3] is almost identical to the HLA-A0201 presented MAGE-A1 peptide Y-L-E-Y-R-Q-V-P-D [SEQ ID NO:5], except for the anchor residue at position 9. Replacement of the anchor residue with Valine results in a 9 amino acid residues peptide with enhanced binding capacity to HLA-A0201 molecules [1]. Human and mouse T-lymphocytes recognizing the Y-L-E-Y-R-Q-V-P-V [SEQ ID NO:6] peptide presented by HLA-0201 also recognize the original MAGE-A Y-L-E-Y-R-Q-V-P-G [SEQ ID NO:3] and Y-L-E-Y-R-Q-V-P-D [SEQ ID NO:5] peptides presented on tumors of distinct origin. As diverse tumors may each express at least one MAGE-A gene, targeting of this so-called multi-MAGE-A epitope includes the vast majority of tumors. As an example, MAGE-A expression in human prostate tumor cell lines and in human xenographs was analyzed and shown to be highly diverse, but in each individual sample tested at least one MAGE-A gene was expressed (Table 2), confirming that targeting this multi-MAGE-A epitope serves as a universal HLA-A0201 restricted target for therapy.

[0046] Of course, several other multi-MAGE or multi-target epitopes may be designed. In principle, the disclosure contemplates combinations of tumor specific antigen derived MHC presented epitopes in different HLA restrictions of both MHC-I and MHC-II, targeted by immunoglobulins linked to a toxic moiety, to induce apoptosis in aberrant cells. Examples of MHC-MAGE peptide combinations that can be targeted by antibodies hereof are peptide IMPKAGLLI (MAGE-A3) [SEQ ID NO:8] and HLA-DP4 or peptide 243-KKLLTQHFVQENYLEY-258 (MAGE-A3) [SEQ ID NO:9] and HLA-DQ6. Other non-limiting examples of tumor specific complexes of HLA and antigen peptide are: HLA A1-MAGE-A1 peptide EADPTGHSY [SEQ ID NO:10], HLA A3-MAGE-A1 SLFRAVITK [SEQ ID NO:11], HLA A24-MAGE-A1 NYKHCFPEI [SEQ ID NO:12], HLA A28-MAGE-A1 EVYDGREHSA [SEQ ID NO:13], HLA B37-MAGE-A1/A2/A3/A6 REPVTKAEML [SEQ ID NO:14], expressed at aberrant cells related to melanoma, breast carcinoma, SCLC, sarcoma, NSCLC, colon carcinoma (Renkvist, N. et al., Cancer Immunol. Immunother. (2001) V50:3-15 (ref. 13)). Further examples are HLA B53-MAGE-A1 DPARYEFLW [SEQ ID NO:15], HLA Cw2-MAGE-A1 SAFPTTINF [SEQ ID NO:16], HLA Cw3-MAGE-A1 SAYGEPRKL [SEQ ID NO:17], HLA Cw16-MAGE-A1 SAYGEPRKL [SEQ ID NO:18], HLA A2-MAGE A2 KMVELVHFL [SEQ ID NO:19], HLA A2-MAGE-A2 YLQLVFGIEV [SEQ ID NO:20], HLA A24-MAGE-A2 EYLQLVFGI [SEQ ID NO:21], HLA-A1-MAGE-A3 EADPIGHLY [SEQ ID NO:22], HLA A2-MAGE-A3 FLWGPRALV [SEQ ID NO:23], HLA B44-MAGE-A3 MEVDPIGHLY [SEQ ID NO:24], HLA B52-MAGE-A3 WQYFFPVIF [SEQ ID NO:25], HLA A2-MAGE-A4 GVYDGREHTV [SEQ ID NO:26], HLA A34-MAGE-A6 MVKISGGPR [SEQ ID NO:27], HLA A2-MAGE-A10 GLYDGMEHL [SEQ ID NO:28], HLA Cw7-MAGE-A12 VRIGHLYIL [SEQ ID NO:29], HLA Cw16-BAGE AARAVFLAL [SEQ ID NO:30], expressed by, e.g., melanoma, bladder carcinoma, NSCLC, sarcoma, HLA A2-DAM-6/-10 FLWGPRAYA [SEQ ID NO:31], expressed by, e.g., skin tumors, lung carcinoma, ovarian carcinoma, mammary carcinoma, HLA Cw6-GAGE-1/-2/-8 YRPRPRRY [SEQ ID NO:32], HLA A29-GAGE-3/-4/-5/-6/-7B YYWPRPRRY [SEQ-ID 33], both expressed by, e.g., melanoma, leukemia cells, bladder carcinoma, HLA B13-NA88-A MTQGQHFLQKV [SEQ ID NO:34], expressed by melanoma, HLA A2-NY-ESO-1 SLLMWITQCFL [SEQ ID NO:35], HLA A2-NY-ESO-1a SLLMWITQC [SEQ ID NO:36], HLA A2-NY-ESO-1a QLSLLMWIT [SEQ ID NO:37], HLA A31-NY-ESO-1a ASGPGGGAPR [SEQ ID NO:38], the latter four expressed by, e.g., melanoma, sarcoma, B-lymphomas, prostate carcinoma, ovarian carcinoma, bladder carcinoma.

[0047] The disclosure is further exemplified by the non-limiting Examples.

ABBREVIATIONS USED

[0048] A.A., amino acid; Ab, antibody; β2-M, CDR, complementarity determining region; CHO, Chinese hamster ovary; CT, cancer testis antigens; CTL, cytotoxic T-lymphocyte; E4orf4, adenovirus early region 4 open reading frame; EBV, Epstein-Barr virus; ELISA, enzyme linked immunosorbent assay; HAMLET, human α-lactalbumin made lethal to tumor cells; HEK, human embryonic kidney; HLA, human leukocyte antigen; Ig, immunoglobulin; i.v., intravenously; kDa, kilo Dalton; MAGE, melanoma-associated antigen; Mda-7, melanoma differentiation-associated gene-7; MHC, major histocompatibility complex; MHC-p, MHC-peptide; NS1, parvovirus-H1 derived non-structural protein 1; PBSM, PBS containing 2% non-fat dry milk; TCR, T-cell receptor; VH, Vh or V.sub.H, amino-acid sequence of an immunoglobulin variable heavy domain; Vl, amino-acid sequence of an immunoglobulin variable light domain; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

EXAMPLES

Example 1

[0049] Non-exhaustive examples of immunoglobulins comprising at least an immunoglobulin variable region that specifically binds to an MHC-peptide complex preferentially associated with aberrant cells or to an aberrant cell surface marker preferentially associated with aberrant cells, with domain topologies as outlined, e.g., in FIG. 5B, are:

[0050] Antibodies hereof comprising immunoglobulin variable regions that specifically bind to:

[0051] a. a complex comprising a T-cell epitope selected from 146-KLQCVDLHV-154 [SEQ ID NO:74], 141-FLTPKKLQCV-150 [SEQ ID NO:75], 154-VISNDVCAQV-163 [SEQ ID NO:76], 154-YISNDVCAQV-163 [SEQ ID NO:77] of PSA, presented by HLA-A2 and/or 162-QVHPQKVTK-170 [SEQ ID NO:78] of PSA, presented by HLA-A3, and/or 152-CYASGWGSI-160 [SEQ ID NO:79], 248-HYRKWIKDTI-257 [SEQ ID NO:80] of PSA, presented by HLA-A24, and/or 4-LLHETDSAV-12 [SEQ ID NO:81], 711-ALFDIESKV-719 [SEQ ID NO:82], 27-VLAGGFFLL-35 [SEQ ID NO:83] of PSMA, presented by HLA-A2, and/or 178-NYARTEDFF-186 [SEQ ID NO:84], 227-LYSDPADYF-235 [SEQ ID NO:85], 624-TYSVSFDSL-632 [SEQ ID NO:86] of PSMA, presented by HLA-A24, and/or 299-ALDVYNGLL-307 [SEQ ID NO:87] of PAP, presented by HLA-A2 and/or 213-LYCESVHNF-221 [SEQ ID NO:88] of PAP, presented by HLA-A24 and/or 199-GQDLFGIWSKVYDPL-213 [SEQ ID NO:89], 228-TEDTMTKLRELSELS-242 [SEQ ID NO:90] of PAP, presented by MHC-2 and/or 14-ALQPGTALL-22 [SEQ ID NO:91], 105-AILALLPAL-113 [SEQ ID NO:92], 7-ALLMAGLAL-15 [SEQ ID NO:93], 21-LLCYSCKAQV-30 [SEQ ID NO:94] of PSCA, presented by HLA-A2 and/or 155-LLANGRMPTVLQCVN-169 [SEQ ID NO:95] of Kallikrein 4, presented by DRB1*0404 and/or 160-RMPTVLQCVNVSVVS-174 [SEQ ID NO:96] of Kallikrein 4, presented by DRB1*0701 and/or 125-SVSESDTIRSISIAS-139 [SEQ ID NO:97] of Kallikrein 4, presented by DPB1*0401, for the treatment of prostate cancer;

[0052] b. the HLA B8 restricted epitope from EBV nuclear antigen 3, FLRGRAYGL [SEQ ID NO:98], complexed with MHC I, for the clearance of EBV infected cells;

[0053] c. the MAGE-A peptide YLEYRQVPG [SEQ ID NO:3] presented by MHC 1 HLA-A0201, for treatment of cancers accompanied by tumor cells expressing these MHC-peptide complexes (see Table 1);

[0054] d. the MAGE-A peptide EGDCAPEEK [SEQ ID NO:4] presented by MHC-1 HLA-CW7, for treatment of cancers accompanied by tumor cells expressing these MHC-peptide complexes (see Table 1);

[0055] e. complexes of HLA-A2 and HLA-A2 restricted CD8.sup.+ T-cell epitopes, e.g., nonamer peptides FLFLLFFWL [SEQ ID NO:99] (from prostatic acid phosphatase (PAP, also prostatic specific acid phosphatase (PSAP))), TLMSAMTNL [SEQ ID NO:100] (from PAP), ALDVYNGLL [SEQ ID NO:101] (from PAP), human HLA-A2.1-restricted CTL epitope ILLWQPIPV [SEQ ID NO:102] (from PAP-3), six-transmembrane epithelial antigen of prostate (STEAP), or complexes of HLA-A2.1 and HLA-A2.1-restricted CTL epitope LLLGTIHAL [SEQ ID NO:103] (from STEAP-3), epitopes from mucin (MUC-1 and MUC-2), MUC-1-32mer (CHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA [SEQ ID NO:104]), epitopes from Globo H, Lewis.sup.y, Tn(c), TF(c) clusters, GM2, prostate-specific membrane antigen (PSMA), Kallikrein 4, prostein, or complexes of HLA-A2.1 and HLA-A2.1-restricted epitopes from BA46, PTH-rP, HER-2/neu, hTERT, and MAGE-A8, for the treatment of prostate cancer;

[0056] f. an aberrant cell specific epitope in aberrant cell-specific altered MUC-1 complexed with MHC, or to an aberrant cell specific epitope in aberrant cell-specific altered MUC-1 for, the targeting of aberrant cells in, e.g., breast cancer or for the treatment of colorectal cancer;

[0057] g. an aberrant cell specific epitope of the aberrant-cell specific epidermal growth factor receptor mutant form vIII complexed with MEW, or to an aberrant cell specific epitope of the epidermal growth factor receptor mutant form vIII, for the treatment of the brain neoplasm glioblastoma multiforme;

[0058] h. the complex of MEW with T-cell epitope peptide 369-376 from human Her-2/neu, for the treatment of malignancies related to Her-2 and/or Her-1 over-expression; and

[0059] i. an epitope of the aberrant-cell specific surface marker CD44 splice variants known as CD44-v6, CD44-v9, CD44-v10, complexed with MHC, or to an aberrant cell specific epitope of an aberrant-cell specific CD44 splice variant, for the treatment of multiple myeloma.

[0060] Target binding sites suitable for specific and selective targeting of infected aberrant cells by antibodies hereof are pathogen-derived antigen peptides complexed with MEW 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 an HLA molecule complexed with a pathogen-derived T-cell epitope provides a specific target on infected aberrant cells for antibodies hereof. An example of an infected aberrant 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. Examples of suitable target HPV 16 E6 T-cell epitopes are peptides FQDPQERPR [SEQ ID NO:39], TTLEQQYNK [SEQ ID NO:40], ISEYRHYCYS [SEQ ID NO:41] and GTTLEQQYNK [SEQ ID NO:42] binding to HLA A1, KISEYRHYC [SEQ ID NO:43] and YCYSIYGTTL [SEQ ID NO:44] binding to HLA A2, LLRREVYDF [SEQ ID NO:45] and IVYRDGNPY [SEQ ID NO:46] binding to HLA A3, TTLEQQYNK [SEQ ID NO:47] binding to HLA A11, CYSLYGTTL [SEQ ID NO:48], KLPQLCTEL [SEQ ID NO:49], HYCYSLYGT [SEQ ID NO:50], LYGTTLEQQY [SEQ ID NO:51], EVYDFAFRDL [SEQ ID NO:52] and VYDFAFRDLC [SEQ ID NO:53] binding to HLA A24, 29-TIHDIILECV-38 [SEQ ID NO:54] binding to HLA A*0201. Equally suitable are HPV 16 E7 T-cell epitopes such as 86-TLGIVCPI-93 [SEQ ID NO:55], 82-LLMGTLGIV-90 [SEQ ID NO:56], 85-GTLGIVCPI-93 [SEQ ID NO:57] and 86-TLGIVCPIC-94 [SEQ ID NO:58] binding to HLA A*0201, HPV 18 E6 T-cell epitopes and HPV 18 E7 T-cell epitopes, binding to HLA A1, A2, A3, A11 or A24. Yet additional examples of T-cell epitopes related to HPV infected cells are HPV E7 derived peptides 1-MHGDTPTLHEYD-12 [SEQ ID NO:59], 48-DRAHYNIVTFCCKCD-62 [SEQ ID NO:60] and 62-DSTLRLCVQSTHVD-75 [SEQ ID NO:61] binding to HLA DR, 7-TLHEYMLDL-15 [SEQ ID NO:62], 11-YMLDLQPETT-20 [SEQ ID NO:63], 11-YMLDLQPET-19 [SEQ ID NO:64] and 12-MLDLQPETT-20 [SEQ ID NO:65] binding to HLA A*201, 16-QPETTDLYCY-25 [SEQ ID NO:66], 44-QAEPDRAHY-52 [SEQ ID NO:67] and 46-EPDRAHYNIV-55 [SEQ ID NO:68] binding to HLA B18, 35-EDEIDGPAGQAEPDRA-50 [SEQ ID NO:69] binding to HLA DQ2, 43-GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-77 [SEQ ID NO:70] binding to HLA DR3, 50-AHYNIVTFCCKCD-62 [SEQ ID NO:71] binding to HLA DR15, 58-CCKCDSTLRLC-68 [SEQ ID NO:72] binding to HLA DR17 and 61-CDSTLRLCVQSTHVDIRTLE-80 [SEQ ID NO:73] binding to HLA-DRB1*0901.

[0061] A good source for selecting binding sites suitable for specific and selective targeting of aberrant cells by antibodies hereof, is the Peptide Database listing T-cell defined tumor antigens and the HLA's binding the T-cell epitopes [9-12; on the World Wide Web at cancerimmunity.org/peptidedatabase/Tcellepitopes.htm]. The database provides combinations of antigen peptides complexed with MHC molecules comprising the indicated class of HLA, unique to tumor cells or over-expressed by tumor cells.

Example 2: Selection of Human Antibody Fragments Specific for HLA-A0201/Multi-MAGE-A

[0062] To obtain human antibody fragments comprising immunoglobulin variable regions specific for the HLA-A0201 presented multi-MAGE-A epitope Y-L-E-Y-R-Q-V-P-V [SEQ ID NO:6] and FLWGPRALV [SEQ ID NO:23] a Human Fab phage display library was constructed according to the procedure previously described by de Haard et al. (2) and used for selections 1) essentially as described by Chames et al. using biotinylated MHC/p complexes (3), or 2) on cells expressing the relevant antigen.

2.1: Selection of Human Antibody Fragments Specific for HLA-A0201/YLEYRQVPV [SEQ ID NO:6] Using Biotinylated MHC-Peptide Complexes

[0063] Human Fab phages (10.sup.13 colony forming units) were first pre-incubated for one hour at room temperature in PBS containing 2% non-fat dry milk (PBSM). In parallel, 20 μl Streptavidin-coated beads (DYNAL™) were equilibrated for one hour in PBSM. For subsequent rounds, 100 μl beads were used. To deplete for pan-MHC binders, each selection round, 200 nM of biotinylated MHC class I-peptide (MHC-p) complexes containing an irrelevant peptide (Sanquin, the Netherlands) were added to the phages and incubated for 30 minutes under rotation. 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 MHC-p complexes (200 nM for the first round, and 20 nM for the second and third round) were added and incubated for one hour at room temperature, with continuous rotation. Simultaneously, a pan-MHC class I binding soluble Fab (D3) was added to the phage-MHC-p complex mixture (50, 10, and 5 μg for rounds 1-3, respectively). Equilibrated streptavidin-coated beads were added, and the mixture was 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 μl freshly prepared tri-ethylamine (100 mM). The pH of the solution was neutralized by the addition of 500 μl 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 2×0 TYAG plates. Next day, colonies were harvested, and a 10 μl inoculum was used in 50 ml 2×TYAG. Cells were grown until an OD.sub.600 nm of 0.5, and 5 ml of this suspension was infected with M13k07 helper phage (5×10.sup.11 colony forming units). After 30 minutes incubation at 37° C., the cells were centrifuged, resuspended in 25 ml 2×TYAK, and grown overnight at 30° C. Phages were collected from the culture supernatant, as described previously, and were used for the next round panning. After three selection rounds a 261-fold enrichment was obtained, and 46 out of 282 analyzed clones were shown to be specific for the HLA-A2-multi-MAGE-A complex (FIG. 1). ELISA using the HLA-A0201/multi-MAGE-A complexes as well as HLA-A0201 complexes with a peptide derived from JC virus was used to determine the specificity of the selected Fab.

2.2: Selection of Human Fab Specific for HLA-A0201/FLWGPRALV [SEQ ID NO:23] Using Cells

[0064] Selections of Fab phages specifically binding to HLA-A0201/FLWGPRALV [SEQ ID NO:23] were performed using mouse CMT64 lung tumor cells. To obtain CMT64 cells stably expressing HLA-A0201/FLWGPRALV [SEQ ID NO:23] (A2/FLW) complexes, the CMT64 cells were retroviral infected with a vector encoding a single chain peptide-β2M-HLA-A0201 heavy chain construct [SEQ ID NO:105]. Human Fab phages (10.sup.13 colony forming units) were first pre-incubated for one hour at room temperature in PBS containing 2% FCS (PBSF). In parallel, 1.0×10.sup.6 CMT64-A2/FLW cells were equilibrated for one hour in PBSF. The phages were first incubated for one hour with 10×10.sup.6 CMT 64 cells expressing HLA-A0210/YLEYRQVPG [SEQ ID NO:3] to deplete non-specifically binding phages. The non-bound fraction was then incubated (1 hr at 4° C.) with HLA-A0201/FLWGPRALV [SEQ ID NO:23] expressing CMT64 cells. After extensive washing, bound phages were eluted by adding 500 μl freshly prepared tri-ethylamine (100 mM). The pH of the solution was neutralized by the addition of 500 μl 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 2×TYAG plates. Next day, colonies were harvested. After four rounds of selection individual clones were selected and tested for specificity of binding.

2.3: Human Fab Specific for HLA-A0201/Multi-MAGE-A Epitopes Bind Antigen Positive Cells

[0065]

TABLE-US-00001 Multi-MAGE-A; [SEQ ID NO: 6] Y-L-E-Y-R-Q-V-P-V

[0066] Fab phages were analyzed for their capacity to bind HLA-A0201 positive EBV-transformed B-LCL loaded with the multi-MAGE-A peptide Y-L-E-Y-R-Q-V-P-V [SEQ ID NO:6]. The B-LCL line BSM (0.5×10.sup.6) was loaded with multi-MAGE-A peptide (10 μg in 100 μl PBS) for 30 minutes at 37° C., followed by incubation with the Fab phages AH5, CB1, CG1, BD5 and BC7 and analyzed by flow-cytometry. As shown in FIG. 2, Fab AH5, CB1 and CG1, specifically bound to the peptide loaded cells only, whereas Fab BD5 and BC7 displayed non-specific binding to BSM that was not loaded with the multi-MAGE-A peptide. No binding was observed by AH5, CB1 and CG1 to non-peptide loaded cells.

[0067] Phages presenting AH5, CB1 and CG1, as well as the HLA-A0101/MAGE-A1 specific Fab phage G8 (4) were then used to stain tumor cell lines of distinct histologic origin. To this end prostate cancer cells (LNCaP), multiple myeloma cells (MDN), melanoma cells (MZ2-MEL43 and G43), and breast cancer cells (MDA-MB157) were stained and analyzed by flow cytometry (FIG. 3). The Fab AH5 specifically bound multiple myeloma cells MDN, and not the HLA-A0201 negative melanoma and breast cancer cells. Both CB1 and CG1 displayed non-specific binding on the melanoma cell line G43. The positive control Fab G8 demonstrated binding to all cell lines tested.

TABLE-US-00002 Multi-MAGE-A: [SEQ ID NO: 23] F-L-W-G-P-R-A-L-V

[0068] To determine the cell-binding capacity of the HLA-A0201/FLWGPRALV selected Fab clone F9 soluble Fab fragments were made by induction of TG-1 bacteria. TG-1 containing pCes-F9 were grown until OD=0.8 and Fab production was induced by addition of 1 mM IPTG. After 13 hours induction the bacterial periplasmic fraction was isolated and dialyzed overnight. Next day soluble Fab F9 fragments were purified by IMAC.

[0069] Purified Fab F9 was added to 0.5×10.sup.6 CMT 64 cells expressing either HLA-A0210/YLEYRQVPG [SEQ ID NO:3], HLA-A0201/FLWGPRALV [SEQ ID NO:23], or CMT 64 cells that do not express human HLA. As shown in FIG. 6 the Fab clone F9 specifically binds HLA-A0201/FLWGPRALV [SEQ ID NO:23] expressing CMT64 cells and not CMT 64 cells that do not express human HLA or that do express the irrelevant HLA-A0201/YLEYRQVPG [SEQ ID NO:3] molecules.

2.4: Fab AH5 Binds HLA-A0201/Multi-MAGE-A Complexes Only

[0070] ELISA using multiple peptide/MHC complexes then confirmed the specificity of Fab-AH5. To this end HLA-A0201 complexes presenting peptides multi-MAGE-A, gp100, JCV and MAGE-C2, as well as a HLA-A1/MAGE-A1 complex were immobilized on 96 well plates and incubated with phages displaying Fab AH5 and control Fab G8. As shown in FIG. 4, AH5 only binds HLA-A0201/multi-MAGE-A and not the irrelevant complexes HLA-A0201/gp100, HLA-A0201/MAGE-C2, HLA-A0201/JCV and HLA-A0101/MAGE-A1. The positive control Fab G8 only binds to its relevant target HLA-A0101/MAGE-A1.

[0071] The nucleic acids enencoding the HLA-A0201-multi-MAGE-A complex binding Fab AH5 will be combined with nucleic acids enencoding human antibody Ch2-Ch3 domains, providing nucleic acid molecules enencoding a human antibody light chain encompassing the selected Cl-Vl encoding nucleic acids and enencoding a human antibody heavy chain encompassing the selected Ch-Vh encoding nucleic acids. These nucleic acid molecules encoding the desired immunoglobulin will be introduced, via a plasmid or via an expression vector, into a eukaryotic host cell such as a CHO cell. After expression of the immunoglobulin, it will be isolated from the cell culture and purified. Then, a selected toxic moiety will be linked to the immunoglobulin, e.g., using Universal Linkage System linker chemistry.

Example 3: Cell Binding and Internalization of an Immunoglobulin Provided With a Toxic Moiety

[0072] Binding capacity of an antibody hereof is analyzed by flow-cytometry. For example, an antibody comprising immunoglobulin variable regions specific for complexes of HLA-A0201 and the multi-MAGE-A peptide is analyzed. HLA-A0201/multi-MAGE-A positive tumor cells (Daju, MDN and mel 624) and HLA-A0201/multi-MAGE-A negative cells (BSM, G43 and 293) are incubated on ice with purified antibody and detected by addition of fluorescently labeled antibodies. Cells bound by the antibody are quantified and visualized by flow-cytometry. Internalization of antibody is analyzed by confocal microscopy. To this end cells are incubated with the antibody, kept on ice for 30 minutes to allow binding but no internalization. Next, fluorescently labeled antibodies specific for the antibody are added. To induce internalization cells are transferred to 37° C. and fixed with 1% PFA after 5, 10 and 15 minutes.

Example 4: Apoptosis Induction by Antibodies Hereof in Diverse Tumor Cells

4.1: Killing of Diverse Tumor Cells by Immunoglobulin Provided With a Toxic Moiety

[0073] Antibodies hereof are analyzed for their capacity to induce apoptosis by incubation with diverse tumor cells, known to express the antigens comprising the binding sites for the immunoglobulin variable regions. For example, an antibody comprising immunoglobulin variable region VH specific for complexes of HLA-A0201 and the multi-MAGE-A peptide, AH5-BTX, is coupled to a synthetic HPMA polymer containing the BTX peptide and Doxorubicin (as we described in WO2009131435) and analyzed. To this end antibodies hereof coupled to doxorubicin are analyzed for their capacity to induce apoptosis by incubation with diverse tumor cells known to express both HLA-A0201 and MAGE-A genes. The cell-lines Daju, Mel 624 (melanoma), PC346C (prostate cancer), and MDN (multiple myeloma) as well as MAGE-A negative cells (911 and HEK293T) are incubated with different concentrations of the antibodies (in DMEM medium, supplemented with pen/strep, Glutamine and non-essential amino acids). Several hours later, cells are visually inspected for classical signs of apoptosis such as detachment of the cells from tissue culture plates and membrane blebbing. In addition, cells are stained for active caspase-3 to demonstrate apoptosis. It is accepted that the antibodies induce apoptosis in the Daju Mel 624, PC346C and MDN cells. Cells that are not treated with the antibodies are not affected, as well as cells that do not express HLA-A0201 (HEK293T) and MAGE-A genes (911 and HEK293T).

[0074] Another antibody, comprising Vh and Vl domains (scFv) with specificity for complexes of HLA-A01, presenting a MAGE-A1 peptide was also analyzed. The scFv-BTX construct was coupled to the HPMA polymer containing doxorubicin and incubated with MAGE-A1 positive and MAGE-A1 negative cells. Apoptosis is shown by staining for active caspase-3.

4.2: Detection of Active Caspase-3

[0075] A classical intra-cellular hallmark for apoptosis is the presence of active caspase-3. To determine whether or not the antibodies induce active caspase-3, Daju, Mel624 and MDN cells are incubated with various concentrations of antibodies hereof. After four and 13 hours FAM-DEVD-FMK, a fluorescently caspase-3/7 inhibitor, is added and positively stained cells are visualized by fluorescent microscopy and flow-cytometry. Caspase-3 activity is shown in antigen positive cells and not in antigen negative cells, with the (fragment of the) antigen providing the specific target-binding site for the antibodies hereof.

4.3 Treatment of Tumor Bearing Mice With Immunoglobulins Provided With a Toxic Moiety

[0076] Nude mice (NOD-scid, 8 per group) with a palpable subcutaneous transplantable human tumor (Daju or MDN) are injected with different doses of immunoglobulins provided with a toxic moiety. As a control mice are treated with standard chemotherapy or receive an injection with PBS. Mice receiving an optimal dose of the immunoglobulins provided with a toxic moiety survive significantly longer that those mice receiving chemotherapy or PBS, when the aberrant cells expose the target binding sites for the antibodies hereof.

Example 5: Selection of Llama VHH With Specificity for HLA-A0201/FLWGPRALV and HLA-A0201/YLEYRQVPG

[0077] Selection of Llama VHH fragments with specificity for HLA-A0201/FLWGPRALV [SEQ ID NO:23] (A2/FLW) and HLA-A0201/YLEYRQVPG [SEQ ID NO:3] (A2/YLE) were performed on CMT64 cells stably expressing these HLA/peptide complexes. Llama VHH phages (10.sup.11 colony forming units) were first pre-incubated for one hour at room temperature in PBS containing 2% FCS (PBSF). In parallel, 1.0×10.sup.6 CMT64-A2/FLW and 1.0×10.sup.6CMT64 A2/YLE cells were equilibrated for one hour in PBSF. To deplete for non-specific binding phages 10×10.sup.6 CMT 64 cells expressing either A2/FLW or A2/YLE were incubated for one hour with the llama VHH. The non-bound fractions were then incubated (1 hr at 4° C.) with A2/FLW or A2/YLE expressing CMT64 cells. After extensive washing, bound phages were eluted by adding 500 μl freshly prepared tri-ethylamine (100 mM). The pH of the solution was neutralized by the addition of 500 μl 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 2×TYAG plates. Next day, colonies were harvested. After four rounds of selection individual clones were selected and tested for specificity of binding.

5.2: Llama VHH Specific for HLA-A0201/Multi-MAGE-A Epitopes Bind Antigen Positive Cells

[0078] To determine the cell-binding capacity of the A2/FLW and A2/YLE selected VHH soluble VHH fragments were made by induction of TG-1 bacteria. TG-1 containing pHen-VHH were grown until OD=0.8 and Fab production was induced by addition of 1 mM IPTG. After 13 hours induction, the bacterial periplasmic fraction was isolated and dialyzed overnight. Next day soluble VHH fragments were purified by IMAC.

[0079] CMT 64 cells (0.5×10.sup.6) expressing either HLA-A0210/YLEYRQVPG [SEQ ID NO:3], HLA-A0201/FLWGPRALV [SEQ ID NO:23], or CMT 64 cells that do not express human HLA were incubated with purified VHH fragments for one hour at 4° C. As shown in FIG. 7 the A2/FLW specific VHH bind HLA-A0201/FLWGPRALV [SEQ ID NO:23] expressing CMT64 cells and not CMT 64 cells that do not express human HLA or that do express the irrelevant HLA-A0201/YLEYRQVPG [SEQ ID NO:23] molecules. The A2/YLE specific VHH only bind HLA-A2/YLEYRQVPG [SEQ ID NO:23] expressing CMT64 cells and not A2/FLW positive CMT64 cells and CMT64 cells that do not express human HLA.

TABLE-US-00003 TABLE 1 Examples of the frequency of MAGE-A expression by human cancers. Frequency of expression (%) Cancer MAGE-A1 MAGE-A2 MAGE-A3 MAGE-A4 MAGE-A6 MAGE-A10 MAGE-A11 Melanoma 16 E 36 E 64 E 74 Head and neck 25 42 33 8 N N N Bladder 21 30 35 33 15 N 9 Breast 6 19 10 13 5 N N Colorectal N 5 5 N 5 N N Lung 21 30 46 11 8 N N Gastric 30 22 57 N N N N Ovarian 55 32 20 E 20 N N Osteosarcoma 62 75 62 12 62 N N hepatocarcinoma 68 30 68 N 30 30 30 Renal cell 22 16 76 30 N N N carcinoma E, expressed but the frequency is not known; N, expression by tumors has never been observed

TABLE-US-00004 TABLE 2 MAGE-A expression in human prostate cancer cell lines and prostate cancer xenografts. MAGE- Cell line/ Xenograft A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 LNCaP + ++ ++ ++ + PC346C + ++ ++ + ++ + + ++ OVCAR + + + + JON ++ ++ ++ + + PNT 2 C2 + + + + + SD48 + + + + PC-3 + + + PC 374 + PC 346p + ++ ++ ++ + ++ + PC 82 + + PC 133 ++ + + PC 135 + PC 295 + PC 324 + + + PC 310 + ++ + ++ + PC 339 ++ ++ + ++ + + + Expression of the MAGE-A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11 and A12 genes in diverse prostate tumor cell lines and prostate xenografts was analyzed by RT-PCR. Shown are expression levels in individual samples tested. Blank = no expression, + = low expression, ++ = high expression. All cell lines/xenografts express at least one MAGE-A gene.

TABLE-US-00005 SEQUENCE IDENTIFIERS SEQ ID NO: 1. Amino acid sequence Vh AH5 QLQLQESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKEREGVAV ISYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAGGS YYVPDYWGQG TLVTVSSGST SGS SEQ ID NO 3. Amino acid sequence MHC-1 HLA-A0201 presentable peptide in MAGE-A YLEYRQVPG SEQ ID NO 4. Amino acid sequence MHC-1 HLA-CW7 presentable peptide in MAGE-A EGDCAPEEK SEQ ID NO 5. Amino acid sequence MHC-1 HLA-A0201 presentable peptide in MAGE-A1 YLEYRQVPD SEQ ID NO 6. Amino acid sequence MHC-1 HLA-A0201 presentable peptide in MAGE-A1, with enhanced binding capacity for HLA-A0201 YLEYRQVPV SEQ ID NO 7. Amino acid sequence Vh binding domain 11H EVQLVQSGGG LVKPGGSLRL SCAASGFTFS DYYMSWIRQA PGKGLEWLSY ISSDGSTIYY ADSVKGRFTV SRDNAKNSLS LQMNSLRADD TAVYYCAVSP RGYYYYGLDL WGQGTTVTVS S SEQ ID NO 8, amino acid sequence of MAGE-A3 peptide epitope binding to HLA IMPKAGLLI SEQ ID NO 9, amino acid sequence of MAGE-A3 peptide epitope binding to HLA KKLLTQHFVQENYLEY SEQ ID NO 10, amino acid sequence of MAGE peptide epitope binding to HLA EADPTGHSY SEQ ID NO 11, amino acid sequence of MAGE peptide epitope binding to HLA SLFRAVITK SEQ ID NO 12, amino acid sequence of MAGE peptide epitope binding to HLA NYKHCFPEI SEQ ID NO 13, amino acid sequence of MAGE peptide epitope binding to HLA EVYDGREHSA SEQ ID NO 14, amino acid sequence of MAGE peptide epitope binding to HLA REPVTKAEML SEQ ID NO 15, amino acid sequence of MAGE peptide epitope binding to HLA DPARYEFLW SEQ ID NO 16 amino acid sequence of MAGE peptide epitope binding to HLA SAFPTTINF SEQ ID NO 17, amino acid sequence of MAGE peptide epitope binding to HLA SAYGEPRKL SEQ ID NO 18, amino acid sequence of MAGE peptide epitope binding to HLA SAYGEPRKL SEQ ID NO 19, amino acid sequence of MAGE peptide epitope binding to HLA KMVELVHFL SEQ ID NO 20, amino acid sequence of MAGE peptide epitope binding to HLA YLQLVFGIEV SEQ ID NO 21, amino acid sequence of MAGE peptide epitope binding to HLA EYLQLVFGI SEQ ID NO 22, amino acid sequence of MAGE peptide epitope binding to HLA EADPIGHLY SEQ ID NO 23, amino acid sequence of MAGE peptide epitope binding to HLA FLWGPRALV SEQ ID NO 24, amino acid sequence of MAGE peptide epitope binding to HLA MEVDPIGHLY SEQ ID NO 25, amino acid sequence of MAGE peptide epitope binding to HLA WQYFFPVIF SEQ ID NO 26, amino acid sequence of MAGE peptide epitope binding to HLA GVYDGREHTV SEQ ID NO 27, amino acid sequence of MAGE peptide epitope binding to HLA MVKISGGPR SEQ ID NO 28, amino acid sequence of MAGE peptide epitope binding to HLA GLYDGMEHL SEQ ID NO 29, amino acid sequence of MAGE peptide epitope binding to HLA VRIGHLYIL SEQ ID NO 30, amino acid sequence of BAGE peptide epitope binding to HLA AARAVFLAL SEQ ID NO 31, amino acid sequence of DAM-6 and DAM-10 peptide epitope binding to HLA FLWGPRAYA SEQ ID NO 32, amino acid sequence of GAGE-1/-2/-8 peptide epitope binding to HLA YRPRPRRY SEQ ID NO 33, amino acid sequence of GAGE-3/-4/-5/-6/-7B peptide epitope binding to HLA YYWPRPRRY SEQ ID NO 34, amino acid sequence of NA88-A peptide epitope binding to HLA MTQGQHFLQKV SEQ ID NO 35, amino acid sequence of NY-ESO-1 peptide epitope binding to HLA SLLMWITQCFL SEQ ID NO 36, amino acid sequence of NY-ESO-1a peptide epitope binding to HLA SLLMWITQC SEQ ID NO 37, amino acid sequence of NY-ESO-1a peptide epitope binding to HLA QLSLLMWIT SEQ ID NO 38, amino acid sequence of NY-ESO-1a peptide epitope binding to HLA ASGPGGGAPR SEQ ID NO 39, HPV 16 E6 T-cell epitope binding to HLA A1 FQDPQERPR SEQ ID NO 40, HPV 16 E6 T-cell epitope binding to HLA A1 TTLEQQYNK SEQ ID NO 41, HPV 16 E6 T-cell epitope binding to HLA A1 ISEYRHYCYS SEQ ID NO 42, HPV 16 E6 T-cell epitope binding to HLA A1 GTTLEQQYNK SEQ ID NO 43, HPV 16 E6 T-cell epitope binding to HLA A2 KISEYRHYC SEQ ID NO 44, HPV 16 E6 T-cell epitope binding to HLA A2 YCYSIYGTTL SEQ ID NO 45, HPV 16 E6 T-cell epitope binding to HLA A3 LLRREVYDF SEQ ID NO 46, HPV 16 E6 T-cell epitope binding to HLA A3 IVYRDGNPY SEQ ID NO 47, HPV 16 E6 T-cell epitope binding to HLA A11 TTLEQQYNK SEQ ID NO 48, HPV 16 E6 T-cell epitope binding to HLA A24 CYSLYGTTL SEQ ID NO 49, HPV 16 E6 T-cell epitope binding to HLA A24 KLPQLCTEL SEQ ID NO 50, HPV 16 E6 T-cell epitope binding to HLA A24 HYCYSLYGT SEQ ID NO 51, HPV 16 E6 T-cell epitope binding to HLA A24 LYGTTLEQQY SEQ ID NO 52, HPV 16 E6 T-cell epitope binding to HLA A24 EVYDFAFRDL SEQ ID NO 53, HPV 16 E6 T-cell epitope binding to HLA A24 VYDFAFRDLC SEQ ID NO 54, HPV 16 E6 T-cell epitope binding to HLA A*0201 29-TIHDIILECV-38 SEQ ID NO 55, HPV 16 E7 T-cell epitope binding to HLA A*0201 86-TLGIVCPI-93 SEQ ID NO 56, HPV 16 E7 T-cell epitope binding to HLA A*0201 82-LLMGTLGIV-90 SEQ ID NO 57, HPV 16 E7 T-cell epitope binding to HLA A*0201 85-GTLGIVCPI-93 SEQ ID NO 58, HPV 16 E7 T-cell epitope binding to HLA A*0201 86-TLGIVCPIC-94 SEQ ID NO 59, HPV E7 T-cell epitope binding to HLA DR 1-MHGDTPTLHEYD-12 SEQ ID NO 60, HPV E7 T-cell epitope binding to HLA DR 48-DRAHYNIVTFCCKCD-62 SEQ ID NO 61, HPV E7 T-cell epitope binding to HLA DR 62-DSTLRLCVQSTHVD-75 SEQ ID NO 62, HPV E7 T-cell epitope binding to HLA A*201 7-TLHEYWILDL-15 SEQ ID NO 63, HPV E7 T-cell epitope binding to HLA A*201 11-YMLDLQPETT-20 SEQ ID NO 64, HPV E7 T-cell epitope binding to HLA A*201 11-YMLDLQPET-19 SEQ ID NO 65, HPV E7 T-cell epitope binding to HLA A*201 12-MLDLQPETT-20 SEQ ID NO 66, HPV E7 T-cell epitope binding to HLA B18 16-QPETTDLYCY-25 SEQ ID NO 67, HPV E7 T-cell epitope binding to HLA B18 44-QAEPDRAHY-52 SEQ ID NO 68, HPV E7 T-cell epitope binding to HLA B18 46-EPDRAHYNIV-55 SEQ ID NO 69, HPV E7 T-cell epitope binding to HLA DQ2 35-EDEIDGPAGQAEPDRA-50 SEQ ID NO 70, HPV E7 T-cell epitope binding to HLA DR3 43-GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-77 SEQ ID NO 71, HPV E7 T-cell epitope binding to HLA DR15 50-AHYNIVTFCCKCD-62 SEQ ID NO 72, HPV E7 T-cell epitope binding to HLA DR17 58-CCKCDSTLRLC-68 SEQ ID NO 73, HPV E7 T-cell epitope binding to HLA-DRB1*0901 61-CDSTLRLCVQSTHVDIRTLE-80 SEQ ID NO 74, PSA T-cell epitope binding to HLA-A2 146-KLQCVDLHV-154 SEQ ID NO 75, PSA T-cell epitope binding to HLA-A2 141-FLTPKKLQCV-150 SEQ ID NO 76, PSA T-cell epitope binding to HLA-A2 154-VISNDVCAQV-163 SEQ ID NO 77, PSA T-cell epitope binding to HLA-A2 154-YISNDVCAQV-163 SEQ ID NO 78, PSA T-cell epitope binding to HLA-A3 162-QVHPQKVTK-170 SEQ ID NO 79, PSA T-cell epitope binding to HLA-A24 152-CYASGWGSI-160 SEQ ID NO 80, PSA T-cell epitope binding to HLA-A24 248-HYRKWIKDTI-257 SEQ ID NO 81, PSMA T-cell epitope binding to HLA-A2 4-LLHETDSAV-12 SEQ ID NO 82, PSMA T-cell epitope binding to HLA-A2 711-ALFDIESKV-719 SEQ ID NO 83, PSMA T-cell epitope binding to HLA-A2 27-VLAGGFFLL-35 SEQ ID NO 84, PSMA T-cell epitope binding to HLA-A24 178-NYARTEDFF-186 SEQ ID NO 85, PSMA T-cell epitope binding to HLA-A24 227-LYSDPADYF-235 SEQ ID NO 86, PSMA T-cell epitope binding to HLA-A24 624-TYSVSFDSL-632 SEQ ID NO 87, PAP T-cell epitope binding to HLA-A2 299-ALDVYNGLL-307 SEQ ID NO 88, PAP T-cell epitope binding to HLA-A24 213-LYCESVHNF-221 SEQ ID NO 89, PAP T-cell epitope binding to MHC-2 199-GQDLFGIWSKVYDPL-213 SEQ ID NO 90, PAP T-cell epitope binding to MHC-2 228-TEDTMTKLRELSELS-242 SEQ ID NO 91, PSCA T-cell epitope binding to HLA-A2 14-ALQPGTALL-22 SEQ ID NO 92, PSCA T-cell epitope binding to HLA-A2 105-AILALLPAL-113 SEQ ID NO 93, PSCA T-cell epitope binding to HLA-A2 7-ALLMAGLAL-15 SEQ ID NO  94, PSCA T-cell epitope binding to HLA-A2 21-LLCYSCKAQV-30 SEQ ID NO 95, Kallikrein 4 T-cell epitope binding to DRB1*0404 155-LLANGRMPTVLQCVN-169 SEQ ID NO 96, Kallikrein 4 T-cell epitope binding to DRB1*0701 160-RMPTVLQCVNVSVVS-174 SEQ ID NO 97, Kallikrein 4 T-cell epitope binding to DPB1*0401 125-SVSESDTIRSISIAS-139 SEQ ID NO 98, EBV nuclear antigen 3 T-cell epitope binding to MHC I HLA B8 FLRGRAYGL SEQ ID NO 99, HLA-A2 restricted CD8.sup.+ T-cell epitope of PAP binding to HLA-A2 FLFLLFFWL SEQ ID NO 100, HLA-A2 restricted CD8.sup.+ T-cell epitope of PAP binding to HLA-A2 TLMSAMTNL SEQ ID NO 101, HLA-A2 restricted CD8.sup.+ T-cell epitope of PAP binding to HLA-A2 ALDVYNGLL SEQ ID NO 102, human HLA-A2.1-restricted CTL epitope of PAP-3 binding to HLA A2.1 ILLWQPIPV SEQ ID NO 103, HLA-A2.1-restricted CTL epitope of STEAP-3 binding to HLA-A2.1 LLLGTIHAL SEQ ID NO 104, HLA-A2.1-restricted CTL epitope of MUC-1 and MUC-2 binding to HLA-A2.1 CHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA SEQ ID NO 105, single chain HLA-A0201/FLWGPRALV construct. MAVMAPRTLVLLLSGALALTQTWAFLWGPRALVGGGGSGGGGSGGGGSGGGSGIQRT PKIQVYSRHP AENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTE KDEYACRVNH VTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYV DDTQFVRFDSDA ASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSESHTVQRMY GCDVGSDWRFLRG YHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWL RRYLENGKETLQRT DSPKAHVTHHPRSKGEVTLRCWALGFYPADITLTWQLNGEELTQDMELVETRPAGDGT FQKWASVVVPLG KEQNYTCRVYHEGLPEPLTLRWEPPPSTDSYMVIVAVLGVLGAMAIIGAVVAFVMKRR RNTGGGDYALAPGSQSSEMSLRDCKA

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