Tumor-specific antibody

Abstract

The present invention relates to an antibody or fragment thereof specifically binding to meningeosis carcinoma-specific antigen, such as DDX53, KIF3C, and AKR1A1, an anti-body or fragment thereof for use in the prophylaxis and / or treatment of cancer, preferably melanoma, further preferably melanoma with leptomeningeal spread, highly preferably melanoma with meningeosis carcinomatosa, a pharmaceutical composition comprising said antibody or fragment thereof, a nucleic acid encoding said antibody or fragment thereof, a vector comprising said nucleic acid, a prokaryotic or eukaryotic host cell comprising said vector, and to a method for the prophylaxis and / or treatment of cancer, preferably melanoma, further preferably melanoma with leptomeningeal spread, highly preferably melanoma with meningeosis carcinomatosa, in a living being, comprising the admin- istration of a prophylactically and / or therapeutically effective amount of said antibody or fragment thereof or said pharmaceutical composition.

Description

Tumor-specific antibody

[0001] The present invention relates to an antibody or fragment thereof specifi-cally binding to meningeosis carcinoma-specific antigen, such as DDX53, KIF3C, andAKR1A1, an antibody or fragment thereof for use in the prophylaxis and / or treatment of cancer, preferably melanoma, further preferably melanoma with leptomeningeal spread, highly preferably melanoma with meningeosis carcinomatosa, a pharmaceutical composi- tion comprising said antibody or fragment thereof, a nucleic acid encoding said antibody or fragment thereof, a vector comprising said nucleic acid, a prokaryotic or eukaryotic hostcell comprising said vector, and to a method for the prophylaxis and / or treatment of can-cer, preferably melanoma, further preferably melanoma with leptomeningeal spread, highly preferably melanoma with meningeosis carcinomatosa, in a living being, comprising the administration of a prophylactically and / or therapeutically effective amount of said anti- body or fragment thereof or said pharmaceutical composition. BACKGROUND

[0002] Melanoma, also redundantly known as malignant melanoma, is a type ofcancer that develops from the pigment-producing cells known as melanocytes.

[0003] Melanomas typically occur in the skin, but may rarely occur in the mouth,intestines, or eye. The primary cause of melanoma is ultraviolet light (UV) exposure in those with low levels of the skin pigment melanin. According to GLOBOCAN 2020, malig- nant melanoma was diagnosed in 324,635 people and caused 57,043 deaths in 2020. Melanoma is the most lethal skin cancer worldwide due to its high metastasis potential.Patients with metastatic melanoma have reduced survival compared with patients in ear- lier disease stages.

[0004] Chemotherapy drugs such as dacarbazine have been the backbone ofmetastatic melanoma treatment since FDA approval in 1975; however, its efficacy in terms of survival has never been proven in a randomized controlled trial.

[0005] Small-molecule targeted therapies work by blocking the genes involvedin pathways for tumor proliferation and survival. The main treatments are BRAF, C-Kit and NRAS inhibitors. These inhibitors work to inhibit the downstream pathways involved in cell proliferation and tumor development due to specific gene mutations. However, melanoma tumors can develop resistance during therapy which can make therapy no longer effec- tive.

[0006] An overview of the current options for adjuvant therapy for melanomacan be found in Lao et al. (2022), Current state of adjuvant therapy for melanoma: less is more, or more is better? Am. Soc. Clin. Oncol. Educ Book 42, 1-7.

[0007] Meningeosis carcinomatosa (MC) is a diffuse dissemination of tumorcells in the cerebrospinal fluid (CSF) and / or meninges and occurs in around 5 % of pa-tients with malignant melanoma. The patients with such distant metastasis have a poor prognosis and are characterized by rapid disease progression and death from neurologi- cal causes.

[0008] Established treatments for singular brain metastases are neurosurgicalresection and stereotactic radiotherapy, which can prolong survival. In patients with asymptomatic BRAF V600E-mutant brain metastases, the BRAF inhibitors dabrafenib, vemurafenib, and immunotherapy with ipilimumab are used. In the case of multiple symp- tomatic brain metastases, palliative whole-brain radiotherapy is used for treatment, alt- hough it has failed to show an overall survival benefit.

[0009] Overall, it can therefore be said that the treatment options for patientswith MC in melanoma are extremely unsatisfactory.SUMMARY

[0010] Against this background, it is the object of the present invention to pro-vide an alternative or new agent or compound with which the disadvantages of the prior art can be avoided or at least reduced. In particular, such an agent or compound is to be provided with which cancer, in particular melanoma, especially melanoma with lep-tomeningeal spread, such as meningeosis carcinomatosa (MC), can be prevented ortreated in a targeted manner.

[0011] The problem underlying the invention is solved by the provision of an an-tibody or fragment thereof specifically binding to meningeosis carcinoma-specific antigen (MC-specific antigen), characterized in comprising -as heavy chain variable domain a CDR3 region, said CDR3 region has an aminoacid sequence being at least 90 % identical to one selected from those in SEQ IDNO: 1, 2 or 3, and / or -as light chain variable domain a CDR3 region, said CDR3 region has an aminoacid sequence being at least 90 % identical to one selected from those in SEQ IDNO: 4, 5 or 6.

[0012] The inventors were able to isolate B cells from the cerebrospinal fluid ofmelanoma patients with meningeosis carcinomatosa (MC), obtain antibodies from themand identify tumor-specific antibody binding sites. These tumor-specific antibody binding sites can be used to construct recombinant antibodies with a high therapeutic potential in an advantageous way.

[0013] In several melanoma patients with meningeosis, the inventors couldgenerate a number of recombinant antibodies from clonally expanded B cells and detecttumor specificity of the antibodies. The recombinant antibodies were examined and veri- fied with microarrays, immunofluorescence, flow cytometry and ELISA tests for tumor specificity.

[0014] The inventors' findings were not to be expected in particular because it isextremely difficult to isolate B cells or antibodies from body fluids that recognize certaintumor cells in a highly specific and selective manner, as – especially in the peripheralblood - latent infections of the individual from which the B cells originate cause a "back-ground noise" by which the immune responses against the tumor are masked. Since the Bcells are nearly absent in the cerebrospinal fluid under physiological conditions the inven- tors took advantage of the cerebrospinal fluid compartment being a separate immune compartment where B cells specifically maturated against the melanoma cells.

[0015] The term "antibody" is intended to include any polypeptide chain-con-taining molecular structure with a specific shape thatfits to and recognizes an epitope, where one or more non-covalent binding interactions may stabilize the complex between the molecular structure and the epitope. The term includes both polyclonal and monoclo- nal antibodies. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g., human, rodent, rab- bit, cow, sheep, pig, dog, camelid, other mammals, chicken, other avians, etc., are consid- ered to be "antibodies". A preferred source for producing antibodies useful as starting ma- terial according to the invention is rabbits.

[0016] According to the invention, "CDR" refers to the complementary determin-ing regions which, along with the framework regions (FR), are parts of the variable regions of the immunoglobulin and T cell receptor chains. CDRs and FRs are defined according to the International ImMunoGeneTics (IMGT) information system; see https: / / www.imgt.org / .

[0017] In an embodiment of the invention the antibody is a humanized antibody,i.e., an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., a "humanized immunoglobulinlight chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that in- cludes a variable framework region substantially from a human immunoglobulin or anti- body and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The term "humanized variable region" (e.g., "humanized lightchain variable region" or "humanized heavy chain variable region") refers to a variable re-gion that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-hu- man immunoglobulin or antibody.

[0018] "Antibody fragments" comprise a portion of a full-length antibody, prefer-ably the variable domain thereof, or at least the antigen binding site thereof, such as any of CDR3, CDR2 or CDR1. Examples of antibody fragments include diabodies, single- chain antibody molecules (scFv or scFab), and multispecific antibodies (e.g. bispecific) formed from antibody fragments.

[0019] The antibody or fragment thereof according to the invention can be pro-duced by recombinant means. Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody or fragment and usually purification to a pharma- ceutically acceptable purity. For the expression of the antibodies or fragments as afore- mentioned in a host cell, nucleic acids encoding the respective light and / or heavy chains or fragments are inserted into expression vectors by standard methods. Expression is per- formed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NSO cells,SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the anti-body or fragment is recovered from the cells (supernatant or cells after lysis). General methods for recombinant production of antibodies are well-known in the state of the artand described, for example, in the review articles of Makrides, S.C. (1999), Protein Expr.Purif.17, 183-202; Geisse et al. (1996), Protein Expr. Purif.8, 271-282; Kaufman, R.J. (2000), Mol. Biotechnol.16, 151-161; Werner, R.G. (1998), J. Drug Res.48, 870-880.

[0020] As used herein, the term "binding" or "specifically binding" refers to thebinding of the antibody or fragment thereof to an epitope of the antigen, e.g., with purifiedwild-type antigen in an in vitro assay such as a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden). Preferably, a specifically binding antibody or fragment does not exhibit significant cross-reactivity. An antibody or fragment thereof that "does notexhibit significant cross-reactivity" is one that will not appreciably bind to an undesirableentity, e.g., an undesirable proteinaceous entity. For example, an antibody or fragment that specifically bind to meningeosis carcinoma(MC)-specific antigen will appreciably bind MC-specific antigen but will not significantly react with non-MC-specific proteins or pep- tides, e.g., non-MC-specific proteins or peptides located in tumors. An antibody specific for a preferred epitope will, for example, not significantly cross-react with remote epitopeson the same protein or peptide. Specific binding can be determined according to any art-recognized means for determining such binding. Preferably, specific binding is determined according to Scatchard analysis and / or competitive binding assays. The affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody / antigen complex), kD (dissociation constant), and KD (kD / ka). Binding or specifically binding means a binding affinity (KD) of 10-8mol / l or less, preferably 10-9M to 10-13mol / l.

[0021] In an embodiment of the invention the antibody or fragment thereof isisolated. The term "isolated" means that the material is removed from its original environ- ment (e.g., the natural environment, if it is naturally occurring). For example, a naturally occurring antibody or fragment thereof present in a living animal is not isolated, but the same antibody or fragment thereof, separated from some or all of the coexisting materials in the natural system, is isolated.

[0022] The inventors have recognized that, in one embodiment of the invention,the antibodies or fragments thereof need not necessarily be sequence identical with the indicated sequences, e.g., of the CDRs or CDR3 respectively. Specific and affine bindingof the antibody or fragment is also possible if the binding regions are at least 90 % identi-cal with the indicated specific sequences.

[0023] "Percent identity" or "percent identical" in turn, when referring to a se-quence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the de- scribed or claimed sequence (the "Reference Sequence"). The percent identity is then de- termined according to the following formula: percent identity = 100 [1 -(C / R)] wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein (i) each amino acid or base in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned amino acid or base in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference and (iv) the alignment has to start at position 1 of the aligned sequences; and R is the number of amino acids or bases in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Se- quence also being counted as an amino acid or base.

[0024] According to the invention, throughout the description and with respectto all embodiments, "at least 90 %" identity includes a sequence identity of 90, 91, 92, 93,94, 95, 96, 97, 98, 99, and 100 %.

[0025] The antibody or fragment thereof disclosed in accordance with the pre-sent invention may also be in "purified" form. The term "purified" does not require absolute purity; rather, it is intended as a relative definition, and can include preparations that arehighly purified or preparations that are only partially purified, as those terms are under- stood by those of skill in the relevant art. For example, individual clones isolated from cel- lular material have been conventionally purified to electrophoretic homogeneity. Purifica- tion of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly con- templated. Furthermore, a claimed antibody or fragment thereof which has a purity of pref-erably 99.999 %, or at least 99.99 % or 99.9 %; and even desirably 99 % by weight orgreater is expressly encompassed.

[0026] The antibody or fragment thereof according to the invention may be in"enriched form". As used herein, the term "enriched" means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for exam-ple), advantageously 0.01 %, by weight, preferably at least about 0.1 % by weight. En-riched preparations of about 0.5 %, 1 %, 5 %, 10 %, and 20 % by weight are also contem-plated. The sequences, constructs, vectors, cells, and other materials comprising the pre- sent invention can advantageously be in enriched or isolated form.

[0027] A "meningeosis carcinoma-specific antigen" or "MC-specific antigen" re-fers to an antigen that is specifically and selectively found in meningeosis carcinoma, pref- erably of human melanoma origin. Examples of such MC-specific antigens are the pro- teins DDX53, AKR1A1 and KIF3C.

[0028] These findings by the inventors were surprising, as the prior art does notdescribe the proteins DDX53, AKR1A1 and KIF3C as such specific tumor markers, in par-ticular those associated with meningeosis carcinoma.

[0029] The testis / cancer antigen DDX53 also known as CAGE (cancer-associ-ated gene) is an antigen that is expressed in various cancers but not in normal tissue ex-cept testis (Cho et al., Identification and characterization of a novel cancer / testis antigen gene CAGE. Biochem Biophys Res Commun.2002, 292(3):715-26. The expression of DDX53 is related with the cell cycle suggesting that it might play a role in cellular prolifera- tion (Cho et al., ibid.). Another group showed co-expression of DDX53 with CD133, amarker for cancer stem cells and DDX53 directly regulated the SOX-2 expression as afactor for maintaining self-renewal in drug-resistant Malme3MR cells (Kim et al., DDX53Regulates Cancer Stem Cell-Like Properties by Binding to SOX-2. Mol Cells.2017,40(5):322-330). DDX53 might therefore serve as an immunotherapeutic target for regulat-ing cancer stem-like properties (Kim et al., ibid.). Another study showed that DDX53 pro- motes stem cell-like properties, autophagy, and confers resistance to anti-cancer drugs in breast cancer cells (Kim et al., 2017). DDX53 has also been shown to possess oncogenicpotential and promotes cell cycle progression by inducing AP-1- and E2F-dependent ex-pression of cyclins D1 and E (Por et al., The cancer / testis antigen CAGE with oncogenicpotential stimulates cell proliferation by up-regulating cyclins D1 and E in an AP-1- andE2F-dependent manner. J Biol Chem. 2010, 285(19):14475-85).

[0030] Aldo-keto reductases (AKRs), belong to a highly conserved enzyme su-perfamily and are key enzymes for detoxification of reactive aldehydes. AKR1A1 has been shown to be widely distributed in cancer cells with relatively stable abundances and sug- gests involvement in tumorigenesis (Zhang et al., Quantitative analysis of the human AKR family members in cancer cell lines using the mTRAQ / MRM approach. J Proteome Res. 2013,12(5):2022-33). The AKR1A1 gene is also closely related to the prognosis of pa- tients with lung adenocarcinoma (Liu et al., Identification of a novel glycolysis-related gene signature that can predict the survival of patients with lung adenocarcinoma. Cell Cycle. 2019,18(5):568-579) and has also been shown to be significantly overexpressed in breastcancer (Hlaváč et al., The role of cytochromes p450 and aldo-keto reductases in progno-sis of breast carcinoma patients. Medicine (Baltimore).2014, 93(28)).

[0031] KIF3C, a member of kinesin superfamily, was highly overexpressed inmalignant melanoma (He et al., Identification of differentially expressed methylated genesin melanoma versus nevi using bioinformatics methods. Peer J.2020, 8:e9273) and inbreast cancer tissues also associated with tumor recurrence and lymph node metastasis (Wang et al., Suppression of motor protein KIF3C expression inhibits tumor growth and metastasis in breast cancer by inhibiting TGF-β signaling. Cancer Lett.2015, 368(1):105- 114). Silencing of KIF3C by shRNA inhibited epithelial-mesenchymal transition and me- tastasis by inhibiting TGF-β signaling and suppressed breast cancer cell proliferation (Wang et al., ibid.). Furthermore, KIF3C pathway promoted proliferation, migration, andinvasion of glioma cells by activating the PI3K / AKT pathway (Gao et al., KIF3C promotes proliferation, migration, and invasion of glioma cells by activating the PI3K / AKT pathway and inducing EMT. Biomed Res Int.2020, 6349312.).

[0032] The disclosed amino acid sequences have been identified by the inven-tors in such tumor- or MC-specific antibodies in the respective particular important CDR3regions, in each case of the heavy and light chain, which are particularly suitable accord- ing to the invention to be applied prophylactically or therapeutically for the treatment of cancer, in particular melanoma.

[0033] In another embodiment of the invention said antibody or fragmentthereof is further characterized in that- the heavy chain variable domain comprises a CDR2 region, said CDR2region has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 7, 8 or 9, and / or -the light chain variable domain comprises a CDR2 region, said CDR2 re-gion has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 10, 11, or 12.

[0034] In still another embodiment of the invention said antibody or fragmentthereof is further characterized in that -the heavy chain variable domain comprises a CDR1 region, said CDR1region has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 13, 14, or 15, and / or -the light chain variable domain comprises a CDR1 region, said CDR1 re-gion has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 16, 17, or 18.

[0035] This measure provides the two further CDRs, i.e., CDR2 and CDR1, re-sponsible for direct interaction with and binding to the antigen or epitope of the MC- specific antigen. By including these additional CDRs the affinity, specificity and selectivity and thus also prophylactic and therapeutic suitability of the antibody according to the in- vention and fragment thereof are thus further increased.

[0036] Another embodiment of the invention provides the antibody or fragmentthereof which comprises -the heavy chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 1 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 7 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 13 and / or -the light chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 4 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 10 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 16.

[0037] With this measure, the antigen-binding sites responsible for antigenbinding and therapeutic efficacy of the particular affine and MC-specific antibody desig- nated by the inventors as 'clone P1-B4' are made available. Said clone and, thus, the anti- body and fragment thereof according to this embodiment, specifically and selectively bindto protein DDX53. The antibody or fragment thereof according to this embodiment can bereadily produced in a known manner.

[0038] Another embodiment of the invention provides the antibody or fragmentthereof which comprises -the heavy chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 2 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 8 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 14 and / or -the light chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 5 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 11 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 17.

[0039] With this measure, the antigen-binding sites responsible for antigenbinding and therapeutic efficacy of the particular affine and MC-specific antibody desig- nated by the inventors as 'clone P1-F6' are made available. Said clone and, thus, the anti- body and fragment thereof according to this embodiment, specifically and selectively bindto protein KIF3C. The antibody or fragment thereof according to this embodiment can bereadily produced in a known manner.

[0040] Yet, another embodiment of the invention provides the antibody or frag-ment thereof which comprises -the heavy chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 3 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 9 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 15 and / or -the light chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 6 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 12 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 18.

[0041] With this measure, the antigen-binding sites responsible for antigenbinding and therapeutic efficacy of the particular affine and MC-specific antibody desig- nated by the inventors as 'clone P3-F1' are made available. Said clone and, thus, the anti- body and fragment thereof according to this embodiment, specifically and selectively bindto protein AKR1A1. The antibody or fragment thereof according to this embodiment canbe readily produced in a known manner.

[0042] In another embodiment of the invention said antibody or fragmentthereof comprises -a heavy chain having an amino acid sequence being at least 90 % identical tosaid in SEQ ID NO:19, and / or -a light chain having an amino acid sequence being at least 90 % identical to saidin SEQ ID 20.

[0043] In this embodiment, the entire amino acid sequences of the hypervaria-ble regions of the heavy (IgH) and light chains (IgK) of the antibody of the invention (clone P1-B4; target DDX53) are provided, i.e., not only the sequences of CDR1, 2, and 3, but also the sequences of the intervening 'framework regions' (FRs) FR1, FR2, FR3, and FR4. The production is thus considerably simplified, and the resulting antibody and frag- ment are characterized by particular suitability according to the invention for the prophy- laxis and treatment of cancer and in particular melanoma.

[0044] In another embodiment of the invention the antibody or fragment thereofcomprises -a heavy chain having an amino acid sequence being at least 90 % identical tosaid in SEQ ID NO:21, and / or -a light chain having an amino acid sequence being at least 90 % identical to saidin SEQ ID 22.

[0045] In this embodiment, the entire amino acid sequences of the hypervaria-ble regions of the heavy (IgH) and light chains (IgK) of the antibody of the invention (clone P1-F6; target KIF3C) are provided, i.e., not only the sequences of CDR1, 2, and 3, but also the sequences of the intervening 'framework regions' (FRs) FR1, FR2, FR3, and FR4. The production is thus considerably simplified, and the resulting antibody and frag- ment are characterized by particular suitability according to the invention for the prophy- laxis and treatment of cancer and in particular melanoma.

[0046] In yet another embodiment of the invention the antibody or fragmentthereof comprises -a heavy chain having an amino acid sequence being at least 90 % identical tosaid in SEQ ID NO:23, and / or -a light chain having an amino acid sequence being at least 90 % identical to saidin SEQ ID 24.

[0047] In this embodiment, the entire amino acid sequences of the hypervaria-ble regions of the heavy (IgH) and light chains (IgK) of the antibody of the invention (clone P3-F1; target AKR1A1) are provided, i.e., not only the sequences of CDR1, 2, and 3, but also the sequences of the intervening 'framework regions' (FRs) FR1, FR2, FR3, and FR4. The production is thus considerably simplified, and the resulting antibody and frag- ment are characterized by particular suitability according to the invention for the prophy- laxis and treatment of cancer and in particular melanoma.

[0048] In an embodiment of the invention said antibody or fragment thereofspecifically bind to the MC-specific antigen, which is selected from the group consisting of DDX53, KIF3C, and AKR1A1.

[0049] Since no antibody is yet available in the prior art, which specifically andselectively binds to MC-specific antigen, and which is suitable for a prophylactic and / or therapeutic use in medicine, especially in the treatment of cancer and in particularmelanoma, a further object of the invention relates precisely to such an antibody or frag- ment thereof configured for a specific binding to MC-specific antigen, preferably DDX53, KIF3C, and AKR1A1. Further preferably, this antibody or fragment thereof are the anti- body or fragment thereof according to the invention, which is described in detail above.

[0050] The features, characteristics, advantages and embodiments mentionedfurther above apply mutatis mutandis also for this subject-matter.

[0051] As used herein, "prophylaxis" describes the totality of all measures takenfor this purpose to prevent impairment of health by risk factors, diseases or accidents. The prevention of secondary diseases or maldevelopments by timely treatment of a primary disease is also a form of prophylaxis. In relation to the invention is thought, in particular, to the prevention of the development of cancer, especially melanoma.

[0052] Prophylaxis of cancer also includes "prevention of metastasis" or "pre-vention of secondary tumors", i.e., measures aiming to prevent the transmission of can- cerous cells from the primary tumor to one or more sites elsewhere in a patient wherethen secondary tumors develop. This means that the metastasis of the primary, tumor orcancer is prevented, delayed, or reduced and thus the development of secondary tumors is prevented, delayed, or reduced. Preferably the metastasis, i.e., secondary tumors, ofthe lung are prevented or reduced, which means that metastatic transmission of cancer-ous cells from the primary tumor to the lung is prevented or reduced.

[0053] According to the invention "treatment", as used in this context, refers totherapeutic measures aimed either at eliminating the cause of the disease (causal ther- apy) or at eliminating the symptoms (symptomatic therapy). The invention, in particular,refers to the treatment of cancer, especially melanoma. Also included is the use of the an-tibody or fragment thereof according to the invention for use in adjuvant therapy. Adjuvant therapy generally refers to supplementary or supportive therapeutic measures, but in par- ticular after surgical removal of the tumor, e.g., melanoma.

[0054] Another subject-matter of the invention relates to an antibody or frag-ment thereof, which specifically bind to MC-specific antigen, characterized in that it specif-ically binds to an epitope comprising an amino acid sequence being at least 90 % identicalto one selected from those in SEQ ID NOS: 49-55.

[0055] Herein, a further antibody or fragment thereof according to the inventionis provided which can specifically bind to the epitopes of the new tumor markers recog- nized for the first time by the inventors. The amino acid sequences SEQ ID Nos: 49, 50and 51 form epitopes of the target protein DDX5. The amino acid sequences SEQ ID Nos:52 and 53 form epitopes of the target protein KIFC3. The amino acid sequences SEQ IDNos: 54 and 55 form epitopes of the target protein AKR1A1. Thereby, "specific binding"means that the antibody or fragment selectively bind to the epitopes mentioned understringent conditions, but not to other epitopes or targets.

[0056] A yet further subject-matter of the invention relates to a polypeptidecomprising an amino acid sequence being at least 90 % identical to one selected fromthose in SEQ ID NOS: 49-55.

[0057] Such a polypeptide can be used as an active ingredient of a peptide vac-cine, aiming at inducing an immune response capable of treating and / or preventing a tu-mor, such as a melanoma, preferably melanoma with meningeosis carcinomatosa (MC).In another approach such a polypeptide can be used to generate CAR T cells specifically binding to the epitopes identified by the inventors.

[0058] Still another subject-matter of the invention relates to a pharmaceuticalcomposition characterized in comprising an antibody of fragment thereof or a polypeptide according to the invention.

[0059] The features, characteristics, advantages and embodiments mentionedin relation with the antibody or fragment thereof apply mutatis mutandis also for the phar-maceutical composition.

[0060] The pharmaceutical composition may comprise a pharmaceutical carrier.As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intrave- nous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).

[0061] A pharmaceutical composition of the present invention can be adminis-tered by a variety of methods known in the art. As will be appreciated by the skilled arti- san, the route and / or mode of administration will vary depending upon the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include ster- ile aqueous solutions or dispersions and sterile powders for the extemporaneous prepara- tion of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

[0062] These pharmaceutical compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlo- robutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0063] Actual dosage levels of the active ingredient, i.e., antibody or fragmentthereof or polypeptide, in the pharmaceutical composition of the present invention may bevaried so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of admin- istration, without being toxic to the patient. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treat- ment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0064] In an embodiment the antibody and / or fragment thereof or polypeptideare the only active ingredients of the pharmaceutical composition. In another embodiment, the pharmaceutical composition may contain additional active agents, such as anti-canceror anti-melanoma compounds, e.g., traditional chemotherapeutics, interferons, imatinib,anti-PD-1 antibodies, BRAF and / or MEK inhibitors, etc. The pharmaceutical compositioncomprising the polypeptide according to the invention can be an anti-tumor vaccine and,in an embodiment, may contain an adjuvant as activity enhancer.

[0065] A still further subject-matter according to the invention relates to a kitcomprising: a) a container comprising the antibody or fragment thereof according to the in-vention in solution or in lyophilized formulation; b) optionally, a second container containing a diluent or reconstituting solutionfor the lyophilized formulation; c) optionally, instructions for (i) use of the solution or (ii) reconstitution and / oruse of the lyophilized formulation.

[0066] The kit may further comprise one or more of (iii) a buffer, (iv) a diluent,(v) a filter, (vi) a needle, or (v) a syringe. The container is preferably a bottle, a vial, a sy- ringe or test tube; and it may be a multi-use container. The pharmaceutical composition is preferably lyophilized.

[0067] The kit of the present invention preferably comprises a lyophilized formu-lation of the present invention in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (e.g., dual chamber vi- als), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. Preferably the kit and / or con- tainer contain / s instructions on or associated with the container that indicates directions for reconstitution and / or use. For example, the label may indicate that the lyophilized for- mulation is to be reconstituted to peptide concentrations as described above. The labelmay further indicate that the formulation is useful or intended for subcutaneous admin-istration.

[0068] The features, characteristics, advantages and embodiments mentionedin relation with the antibody or fragment thereof apply mutatis mutandis also to the kit.

[0069] Still another subject-matter of the invention relates to a nucleic acid en-coding an antibody or fragment thereof or a polypeptide according to the invention.

[0070] This measure allows the recombinant production of the antibody or frag-ment thereof or polypeptide, according to the invention, in an advantageous manner.Methods for recombinant production are widely known in the state of the art as has been described further above.

[0071] The features, characteristics, advantages and embodiments mentionedin relation with the antibody or fragment thereof apply mutatis mutandis also for the nu-cleic acid.

[0072] The terms "nucleic acid" or "nucleic acid molecule", as used herein inter-changeably, are intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. It includes oligonucleotide molecules.

[0073] The nucleic acid molecule or oligonucleotide according to the invention,in an embodiment, comprise a nucleotide sequence being at least 90 % identical to oneselected from those in SEQ ID NO: 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and / or 48.

[0074] Yet, another subject-matter of the present invention is a vector, prefera-bly an expression vector, comprising the nucleic acid or oligonucleotide according to the invention, and, optionally, regulatory elements necessary for expression in a prokaryotic and / or eukaryotic cell.

[0075] A "vector" is a nucleic acid molecule, in particular self-replicating, whichtransfers an inserted nucleic acid molecule into and / or between host cells. The term in- cludes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromo- somal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and / or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as de- scribed.

[0076] An "expression vector" is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into a polypeptide. An "expres- sion system" usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product. "Regulatory elements" of expression vectors are well known to the skilled artisan and include, e.g., promotors, enhancers, poly- adenylation signals and transcription termination sequence, etc.

[0077] The features, characteristics, advantages and embodiments mentionedin relation with the antibody or fragment thereof apply mutatis mutandis also for the vector.

[0078] Still another subject-matter of the invention is a prokaryotic or eukaryotichost cell comprising the vector according to the invention.

[0079] The term "host cell" as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodies according to the current invention. "Host cell” herein typically refers to a cell or non-human organism genet- ically engineered to produce the antibodies or fragments thereof according to the inven- tion. These hosts and host cells include mammalian cells, bacterial, yeast, insect cells, plant cells and transgenic plants or animals such as rodents, plants and bovines. Typi- cally, antibodies or antibody fragments are expressed in mammalian, bacterial and yeast cells. In one embodiment HEK293 cells and CHO cells are used as host cells.

[0080] The features, characteristics, advantages and embodiments mentionedin relation with the antibody or fragment thereof apply mutatis mutandis also for the pro-karyotic or eukaryotic host cell.

[0081] Another subject-matter of the invention relates to a method for theprophylaxis and / or treatment of cancer, preferably melanoma, in a living being, comprising the administration of a prophylactically and / or therapeutically effective amount of the anti- body or fragment thereof or polypeptide and / or the pharmaceutical composition according to the invention.

[0082] The features, characteristics, advantages and embodiments mentionedin relation with the antibody or fragment thereof apply mutatis mutandis also for themethod according to the invention.

[0083] A "living being" refer to any subject or organism, including mammals, inparticular humans.

[0084] The invention is now further explained by means of embodiments andexamples resulting in additional features, characteristics and advantages of the invention. The embodiments and examples are of pure illustrative nature and do not limit the scope or range of the invention. The features mentioned in the specific embodiments and exam- ples are features of the invention and may be seen as general features which are notapplicable in the specific embodiment or example but also in an isolated manner in the context of any embodiment or example of the invention.

[0085] The invention is now further described and explained in more detail byreferring to the following non-limiting examples and figures. BRIEF DESCRIPTION OF THE FIGURESFigure 1: Immunohistochemistry staining with fixed Malme-3M melanoma cellsand the recombinant antibodies P1-B4, P1-B8, P3-F1 and P1-F6. Addi- tionally, the proliferation marker Ki-67 and DAPI was stained.Figure 2: Binding of recombinant antibodies to selected targets tested by customELISA. Additionally, serum and liquor supernatant of the respective pa- tients was tested. Optical density (OD) was measured at 450 nm. ODs above 0.1 were considered as positive for binding.Figure 3: Predicted binding clusters of each antibody with its respective potentialtarget using AbAdapt. The first five top hits of the antibody binding are shown. The Top hits are colored in the following order beginning with cluster 0 (highest rate) red, blue, yellow, magenta and cyan. The respec- tive target is displayed in grey.Figure 4: Predicted binding clusters of each antibody with its respective potentialtarget. Predictions were performed using AlphaFold3. Panel A depicts the highest ranked binding model of each antibody (heavy chain in blue; light chain in magenta) with its potential target (green). Panel B shows the prediction accuracy of AlphaFold3 by the pLDDT values. Blue color- ing indicates a precise prediction whereas red indicates poor prediction. Panel C shows the potential epitopes (red) found via close contacts in the structure of each target. The Epitope found by the microarray isdisplayed in yellow. Overlapping residues of both epitopes are marked in orange.Figure 5: Co-staining of rec. antibodies and MHCI. No Co-localization was ob-served.Figure 6: SDS-PAGE of the IP with B4 antibody from Malme-3M lysate. L: ladder;E: eluate; C: negative control. EXAMPLES 1. Introduction

[0086] The concept of immuno-surveillance in humans was first described inthe late 1950s by Thomas and Burnet who suggested that lymphocytes act as sentinels that continuously eliminate neo-transformed cells to prevent manifestation of neoplasms. Decades later, this theory has been widely accepted and the use of checkpoint inhibitors has revolutionized treatment strategies in cancer.

[0087] Regarding the role of lymphocytes during carcinogenesis, T cells havebeen in the focus of research whereas systemic studies on B cells are scarce. Most stud- ies on B cell infiltration and tumor specific antibody production in humans concentrated on tumors outside the CNS with partially controversial results. Whereas several studies found correlations between tumor B cell infiltrates and favorable clinical outcomes, other resultssuggested tumor promoting roles of B cells. Concerning CNS tumors, studies on B cell in-filtration and the cerebrospinal fluid (CSF) in cancer patients with gliomas, brain or menin- geal metastasis are generally scarce and the role of B cells is poorly understood. Detailed studies on the humoral immune reaction showed that tumor specific antibodies can be identified in the peripheral blood and tumor tissues of cancer patients. A major drawback has been that the analysis of blood-derived B cells is highly susceptible to "background noise" from latent infections and the immune response to everyday pathogens, which can mask acute immune responses against the tumor. As a result, clonally expanded B cellsin peripheral blood do not usually target malignant tissue, and the search for peripheral tu- mor-specific B cells resembles the proverbial search for a needle in a haystack.

[0088] In a previous study it was shown that elevated B-cell levels in the CSFare detectable in a subset of patients with CNS neoplasia, including brain metastases and meningeal carcinomatosis. B cells are largely absent from the CSF of healthy individuals, and an elevated CSF B cell fraction is usually associated with defined immune responses. Furthermore, CSF B cell derived recombinant antibodies in several diseases such as sub- acute sclerosing panencephalitis, multiple sclerosis and neuromyelitis optica spectrum disorder have been shown to be disease relevant. Taking advantage of the CNS as a sep-arate compartment, the present invention aims to determine whether the observed CSF Bcell population in patients with meningeal carcinomatosis represents a specific response against tumor cells.

[0089] In order to further analyze the CSF B cell reaction during meningeal car-cinomatosis, three melanoma patients with leptomeningeal spread and an elevated B cell fraction were investigated. By applying single-cell sequencing, paired heavy and light chain immunoglobulin sequences were recovered, 13 recombinant antibodies were gener- ated and tested for tumor specificity. This proof-of-concept study demonstrates antibodies binding to defined targets, identifies potential epitopes of these targets, and presents com- pelling evidence for a tumor-targeted B cell response in melanoma patients with lep- tomeningeal spread. 2. Methods

[0090] Three melanoma patients with leptomeningeal spread and a presumableCSF B cell reaction were selected by analyzing CSF standard parameters including in- trathecal immunoglobulin (Ig) synthesis. Single cell sorting of CSF B cells was performed followed by conventional sequencing of Ig heavy and light chain transcripts.13 recombi- nant antibodies were generated from CSF B cells and were tested for antigen specificity using antigen microarrays, ELISA and Immunocytochemistry as well as bioinformatic tools for binding predictions.3. Results

[0091] Single cell immunoglobulin repertoires were assessed in all three se-lected patients. On average, 46 heavy chain (range 24 – 73) and 41 light chain (range 13– 75) sequences were recovered. In total, 60 paired heavy and light chain sequenceswere found in patient 1, 32 in patient 2 and 10 in patient 3. The average assignment of se-quences to clonal groups was 61 % for heavy chain (range 34 % – 77 %) and 52 % forlight chain sequences (range 28 % - 73 %). The absolute number of clones was 7 in pa-tient 1 and 3 for each patient 2 and patient 3 (Table 1). Clonal groups showed several mu- tations to germline which is indicative for ongoing somatic hypermutations. Previous treatment Patient Age Primary tumorwith Checkpoint Inhib- Antibodies itors P1-B4 P1-B8 P1-C4 P1 69 Melanoma NoP1-C7 P1-F6 P1-H6 P1-H7 P2-C2 P2 29 Melanoma YesP2-C3 P2-G4 P3-E1 P3 33 Melanoma NoP3-E6 P3-F1Table 1: Metadata of analysed Meningeosis patients with their corresponding recombinantantibodies. Antigen recognition and epitope mapping by microarrays

[0092] In order to identify the potential target antigens of the produced recombi-nant antibodies, pooled antibodies (batch 1: 7 antibodies from patient 1; batch 2: 6 anti-bodies from patients 2 and 3) were screened by the HuProt™ Human Proteome Microar-ray v4.0 (PEPperPRINT). A prominent cut-off after the first two proteins (AKR1A1, GART) was detectable in antibody batch 2 from patients 2 and 3 (Table 3) so that those were considered to be the most likely candidate target antigens for these antibodies. Since thecut-off for antibody batch 1 of patient 1 was not as clear (Table 2), the first 50 protein hits were further analyzed according to similarity, biological function and tumor association.Within these first hits three peptidyl prolyl isomerases (PPIL1, PPIE, PPIA) and two phos-phatases (PPP1R12B, MDP1) were identified. Both types of enzymes are heavily involved in oncogenic processes. In addition, 6 antigens (LASP1, MTUS2, CCR1, KIFC3, LMNA, DDX53) were found to be listed as either cancer enhanced or cancer enriched in the Hu- man Protein Atlas. Protein UniProt ID Full NameCorrected In- tensity Ratio PPP1R12B O60237-2protein phosphatase 1 regulatory subunit 12B 1309.2 PPIL1 Q9Y3C6 peptidylprolyl isomerase like 1 1309.2SHFM1 P60896 SEM1 26S proteasome complex subunit 1309.1RAD23A P54725-3RAD23 homolog A, nucleotide excision re- pair protein 1309.1 LASP1 Q14847 LIM and SH3 protein 1 1309.0CCR1 P32246 C-C motif chemokine receptor 1 1308.6RAD23A P54725RAD23 homolog A, nucleotide excision re- pair protein 1304.6 PPIE Q9UNP9 peptidylprolyl isomerase E 1297.1NACA Q13765nascent polypeptide associated complex subunit alpha 1295.7 SETD7 Q8WTS6SET domain containing 7, histone lysinemethyltransferase APBB1 O00213amyloid beta precursor protein bindingfamily B member 1 ACBD3 Q9H3P7 acyl-CoA binding domain containing 3 1269.5WASF2 Q9Y6W5 WASP family member 2 1206.0PPIA P62937 peptidylprolyl isomerase A 1205.9UBL7 Q96S82 ubiquitin like 7 1203.9GPBP1_frag GC-rich promoter binding protein 1 1175.1NAPB Q9H115 NSF attachment protein beta 1172.1DBNL Q9UJU6 drebrin like 1081.8MTUS2 Q5JR59-3 microtubule associated scaffold protein 2 1068.7KCTD5 Q9NXV2potassium channel tetramerization domain containing 5 1051.5 LRRFIP1 Q32MZ4 LRR binding FLII interacting protein 1 1048.3SCL-70 autoantigen 1007.5CCDC102B_frag coiled-coil domain containing 102B 984.8family member C3 942.5MDP1 Q86V88 magnesium dependent phosphatase 1 919.9DBNL Q9UJU6-2 drebrin like 918.3PEX19 P40855 peroxisomal biogenesis factor 19 917.0SF3A1 Q15459 splicing factor 3a subunit 1 914.2CALCOCO2 Q13137 calcium binding and coiled-coil domain 2 899.4LMNA lamin A / C 894.4SLC7A6OS Q96CW6solute carrier family 7 member 6 oppositestrand ACRC Q96QF7 germ cell nuclear acidic peptidase 893.9Hep B Protein X autoantigen 865.0DDX53 Q86TM3 DEAD-box helicase 53 864.5ACY3 Q96HD9 aminoacylase 3 863.6CRK P46108 CRK proto-oncogene, adaptor protein 821.9TEX13A Q9BXU3 testis expressed 13A 819.2PARVA Q9NVD7 parvin alpha 816.2PA2G4 Q9UQ80 proliferation-associated 2G4 776.7CEP85 Q6P2H3-3 centrosomal protein 85 730.4BRD2 P25440 bromodomain containing 2 706.4NACA2 Q9H009nascent polypeptide associated complex subunit alpha 2 701.7 PSMD7 P51665 proteasome 26S subunit, non-ATPase 7 683.1GIPC1 O14908-2GIPC PDZ domain containing family mem- ber 1 666.3 CABP4 P57796-2 calcium binding protein 4 651.7KIFC3 Q9BVG8- 5kinesin family member C3 646.1SWI / SNF related, matrix associated, actin SMARCC1 Q92922dependent regulator of chromatin subfam- 592.2 ily c member 1 PACSIN2 Q9UNF0protein kinase C and casein kinase sub-strate in neurons 2 ASCC2 Q9H1I8activating signal cointegrator 1 complexsubunit 2 OTUD6B Q8N6M0 OTU domain containing 6B 577.6Table 2: Top 50 hits of HuProt™ Human Proteome Microarray v4.0 of antibody pool frompatient 1. Selected proteins for further analyses are highlighted.Corrected ProteinFull Name Intensity Ra- tio AKR1A1 P14550 aldo-keto reductase family 1 member A1 1309.3GART P22102-2phosphoribosylglycinamide formyltrans- ferase, phosphoribosylglycinamide 1308.9synthetase, phosphoribosylaminoimida- zole synthetaseRHOA P61586 ras homolog family member A 786.9SHFM1 P60896 SEM1 26S proteasome complex subunit 718.2RAC3 P60763 Rac family small GTPase 3 715.4RAB11A P62491 RAB11A, member RAS oncogene family 709.4SNX9 Q9Y5X1 sorting nexin 9 638.4OFD1_frag OFD1 centriole and centriolar satellite pro- tein 606.5NAPB Q9H115 NSF attachment protein beta 582.7SLC7A6OS Q96CW6solute carrier family 7 member 6 opposite strand 548.9 OFD1_frag OFD1 centriole and centriolar satellite pro- tein 519.2RAP1GDS1 P52306-2Rap1 GTPase-GDP dissociation stimula- tor 1 506.6 ING3 Q9NXR8- 2inhibitor of growth family member 3 471.9PMEPA1 Q969W9-2prostate transmembrane protein, andro- gen induced 1 442.0XRCC4 Q13426 X-ray repair cross complementing 4 440.7CCDC102B_frag coiled-coil domain containing 102B 438.4ACSL4 O60488-2acyl-CoA synthetase long chain family member 4 403.1ARFGAP1 Q8N6T3-2ADP ribosylation factor GTPase activating protein 1 377.3MED22 Q15528-2 mediator complex subunit 22 362.8USP5 P45974 ubiquitin specific peptidase 5 360.9PPM1B O75688-2protein phosphatase, Mg2+ / Mn2+ depen- dent 1B 348.5RAC1 P63000 Rac family small GTPase 1 323.9PPM1J Q5JR12protein phosphatase, Mg2+ / Mn2+ depen- dent 1J 312.9NAP1L1 P55209 nucleosome assembly protein 1 like 1 312.6XRCC4 Q13426-3 X-ray repair cross complementing 4 292.5ZFYVE16 Q7Z3T8-3 zinc finger FYVE-type containing 16 289.2TLE3 Q04726TLE family member 3, transcriptional core- pressor 283.5PPM1D O15297-2protein phosphatase, Mg2+ / Mn2+ depen- dent 1D 272.7ARFGAP1 Q8N6T3ADP ribosylation factor GTPase activating protein 1 272.6APBB1 O00213amyloid beta precursor protein binding family B member 1 269.8ARHGEF16 Q5VV41-2Rho guanine nucleotide exchange factor 16 268.6PDCL Q13371 phosducin like 267.9TSEN15 Q8WW01 tRNA splicing endonuclease subunit 15 241.3SCYL3 Q8IZE3-2 SCY1 like pseudokinase 3 236.9RBBP7 Q16576RB binding protein 7, chromatin remodel- ing factor 235.5 CSNK1G1 Q9HCP0- 2casein kinase 1 gamma 1 221.6PVRL3_frag nectin cell adhesion molecule 3 210.7C14orf37 Q86TY3 armadillo like helical domain containing 4 210.2RAB2B Q8WUD1 RAB2B, member RAS oncogene family 208.8EEF2K O00418 eukaryotic elongation factor 2 kinase 208.7USP5 P45974-2 ubiquitin specific peptidase 5 200.6LDLRAD4 O15165-7low density lipoprotein receptor class A domain containing 4 196.9 AKT3 Q9Y243-2 AKT serine / threonine kinase 3 195.5RAC1 P63000 Rac family small GTPase 1 192.5LOC105372277 Ensembl ID: ENSP00000394047 188.9KRT8 keratin 8 187.8CALU O43852 calumenin 187.7MDP1 Q86V88 magnesium dependent phosphatase 1 185.4GPBP1_frag GC-rich promoter binding protein 1 185.4PRDM4 Q9UKN5 PR / SET domain 4 179.0Table 3: Top 50 hits of HuProt™ Human Proteome Microarray v4.0 of antibody pool frompatient 2 and patient 3. Selected proteins for further analyses are highlighted. Selected recombinant antibodies present binding to the melanoma cell line Malme-3M

[0093] Flow cytometry was performed with all 13 recombinant antibodiesagainst the Malme-3M cell line to evaluate general binding properties to melanoma cells. Since the potential targets detected were intracellular proteins, cells were permeabilized. The recombinant antibodies P1-F6 presented positive staining (38 % positive cells), whileall other antibodies were considered as negative (0 – 1 % positive cells). Interestingly,when testing non-permeabilized Malme-3M cells not only P1-F6 but also P1-B4, P1-B8 and P3-F1 showed a huge proportion of cells bound to the respective antibody (78.0 – 99.6 % positive cells). Additionally, the three antibodies P3-E6, P1-H6 and P1-H7 pre-sented a small but not neglectable proportion of bound cells (21.8 – 33.9 % positive cells).These results indicate an extracellular binding of the antibodies. One can hypothesise thatthese target proteins are presented on MHC I and induce a T-cell directed B-cell activation and antibody production against them.

[0094] Antibodies that bound to Malme-3M cells in the flow cytometric experi-ments were further analyzed in fixed Malme-3M cells to further evaluate the binding pat- terns. Intracellular binding mostly found on the edges of the cells was observed for all four antibodies tested, even though P1-B8 and P3-F1 presented only weak staining (Figure 1). P1-B4 had the most intense staining. Co-staining with KI-67, a marker for cell proliferation, was also performed which mostly showed intra-nuclear staining patterns. In co-stainingwith our recombinant antibodies and KI-67, the inventors observed a partial overlap at theborder of cell nuclei. Confirming DDX53, KIFC3 and AKR1A1 as antibody targets

[0095] To validate the possible antigen targets found by the microarrays, cus-tom ELISAs were performed to test the binding of the recombinant antibodies to ACY3, AKR1A1, DDX53, GART, KIFC3, LMNA, MDP1, MTUS2 and PPIL1 (Figure 2). Addition- ally, also serum and liquor supernatant of the respective patients was tested. Two anti- bodies (P1-B4 and P1-H7) from patient 1 were found to bind the known tumor-associated DDX53 protein. Also, serum and liquor from this patient were tested positive. Besides this,P3-F1 also binds only one target, AKR1A1. In this case only the serum but not the liquorof this patient was positive. The antibodies P1-F6 and P1-B8 from patient 1 were found to bind to KIFC3 and ACY3 as well as ACY3 and MTUS2, respectively. Probably those twoare rather unspecific antibodies. All other antibodies did not present any binding for any ofthe proteins tested. Determining potential epitope candidates

[0096] To further refine the determination of the binding of each antibody,epitope mapping was performed for Aldo-keto reductase family 1 member A1 (AKR1A1), phosphoribosylglycinamide synthetase (GART) and for one protein of each type of en- zyme, namely peptidyl-prolyl isomerase-like 1 (PPIL1) and magnesium dependent phos- phatase 1 (MDP1). In addition, the following cancer associated antigens were tested: mi- crotubule associated tumor suppressor candidate 2 (MTUS2), DEAD-box helicase 53 (DDX53), LIM and SH3 domain protein 1 (LASP1), Kinesin family member C3 (KIFC3)and Lamin-A / C (LMNA). It was found that AKR1A1, DDX53, KIFC3 and MDP1 are the most prominent targets (Table 4, Table 5). potential Antibodybinding stretch (SEQ ID NO:) potential epitopetarget EAKVRIFGNREMKAK (56) RIFGNREMKAK KVRIFGNREMKAKAK (57) (SEQ ID NO: 49) RIFGNREMKAKAKAA (58) LKSGEKRLIPKPTCR (59) SGEKRLIPKPTCRB4 DDX53SGEKRLIPKPTCRFK (60)(SEQ ID NO: 50)SRGLDLNDVTHVYNY (61) HVYNY (SEQ ID GLDLNDVTHVYNYDF (62) NO: 51) HVYNYDFPRNIDVYV (63) ASDWEYTITVSAAEI (64) EYTITVSAAEI (SEQ DWEYTITVSAAEIYN (65) EYTITVSAAEIYNEV (66)ID NO: 52) F6 KIFC3AELGSWSSQEHLEWE (67) SWSSQEHLEWE LGSWSSQEHLEWEPA (68) (SEQ ID NO: 53) SWSSQEHLEWEPACQ (69) RKTLADLQLEYLDLY (70) TLADLQLEYLDLYLM (71) LEYLDLY (SEQ ID ADLQLEYLDLYLMHW (72) NO: 54) LQLEYLDLYLMHWPY (73) LEYLDLYLMHWPYAF (74) F1 AKR1A1WRYIVPMLTVDGKRV (75) YIVPMLTVDGKRVPR (76) TVDGKRV (SEQ ID VPMLTVDGKRVPRDA (77) NO: 55) MLTVDGKRVPRDAGH (78) TVDGKRVPRDAGHPL (79) Table 4: Binding patterns found in the microarray analysis. Marked sequences are identi- cal with the potential epitopes found by the bioinformatics analysis. Bioinformatics approach for epitope recognition

[0097] Since it is hypothesized that the target proteins are presented on MHC I,an IEDB analysis was performed, which revealed a set of possible epitopes for each tar- get that could be presented on MHC I. For each target potential epitopes were isolated when comparing the microarray analysis with the predictions and experimental data from IEDB. One potential epitope for AKR1A1 is YLMHWPYAF (aa 110-118; SEQ ID NO: 80) which was detected in the microarray analysis of P3-F1 (Table 4) and also the IEDB pre- dictions suggested an IC50 below 50 nM indicating a high binding potential to MHC I. Ad- ditionally, this exact epitope was already experimentally confirmed for patients suffering from brain cancer, adenocarcinoma or leukemia. Two epitope candidates were found for DDX53. The epitopes GESEAKVRIF (aa 87-96; SEQ ID NO: 81) and HVYNYDFPR (aa 538-546; SEQ ID NO: 82) were both found in the microarray for P1-B4 (Table 4) but only the later motif was found to have a predicted IC50 below 50 nM, whereas the first motif is predicted to have an intermediate binding affinity to MHCI. For both epitopes no experi- mental data is available in the IEDB. Lastly, SAAEIYNEV (aa 573-581; SEQ ID NO: 83) was found as a potential epitope candidate for KIFC3. P1-F6 binds to this epitope in the microarray analysis (Table 4) and also the IEDB prediction presents very low IC50 values around 5 nM for this epitope. Additionally, this epitope was already experimentally con- firmed for patients with different kinds of cancer including brain cancer and melanoma.

[0098] In order to further determine the epitope of the recombinant antibodies,the binding of P1-B4, P1-F6, and P3-F1 to their respective targets (DDX53, KIFC3, AKR1A1) was analyzed using AbAdapt. In Figure 3, the top five clusters are presented for each antibody-antigen pair, whereas the antibodies are colored, and the antigens are shown in grey. In general, but especially for DDX53, it can be recognized that most of the predicted antibody binding sides are in the same region. Besides that, in KIFC3 it looks like there are two regions capable of antibody binding. Interestingly, the highest rated cluster (red) for AKR1A1 and F1 binds on a very different side as all other antibodies. To further evaluate the possible binding epitope for each antibody close interactions for each of the clusters from the antibody-antigen pair were determined. For DDX53 and P1-B4 most of the binding clusters present close interactions for the amino acids of theGESEAKVRIF (aa 87-96; SEQ ID NO: 81) motif. Taken together with the previous results,this suggests that this motif may be the epitope of this target. In the analysis of AKR1A1 and P3-F1, none of the amino acid residues of YLMHWPYAF (aa 110-118; SEQ ID NO:80) were found to be close interaction partners. However, amino acids in proximity to this motif were close interactors. For KIFC3 and P1-F6, very different interacting amino acids were found in the different clusters. There was also no overlap with previously identified epitopes. Antibody Microarray FACS ICC ELISAP1-B4 DDX53, + + DDX53AKR1A1 P1-B8 AKR1A1 + + ACY3, MTUS2P1-C4 - - n.d. -P1-C7 MDP1 - - -P1-F6 KIFC3, MDP1, + + ACY3, KIFC3AKR1A1 P1-H6 KIFC3, - - -AKR1A1, MDP1 P1-H7 - - - DDX53P2-C2 - - n.d. -P2-C3 - - n.d. -P2-G4 n.d. - n.d. -P3-E1 n.d. - n.d. -P3-E6 n.d. - - -P3-F1 AKR1A1 + + AKR1A1Table 5: Overview of antigen targets bound by each of the produced antibodies in the dif- ferent experimental approaches. Highlighted are the antibodies with consistent results in all experiments. Update on the determination of potential epitope candidates

[0099] To ascertain the precise binding of each antibody, epitope mapping(PEPperPRINT) was conducted by analyzing the binding characteristics to peptide stretches of 15 amino acids from the potential target proteins. Binding to defined epitopes was further analyzed by looking at the length of amino acid stretches, the intensity of bind- ing to amino acid stretches, and the occurrence of basic amino acids to discriminate be- tween the most likely binding sites and unspecific binding. Consistent antibody binding toepitopes was found for the proteins AKR1A1, DDX53, KIFC3 (Table 4).

[0100] Results were then compared to the outcome of protein structure / bind-ing prediction tools (AlphaFold 3 and ElliPro). As indicated in Figure 4 (B), the structuralpredictions generated by AlphaFold 3 exhibited overall high accuracy, although theprecision of the predictions was notably lower for unstructured loop regions. However, an- tibody binding is not taking place in those regions (Figure 4A). Consistent overlaps be- tween epitope detection methods were observed (Table 4, Figure 4C). The epitopeSGEKRLIPKPTCR (aa 210 – 222) was identified by both the microarray and ElliPro BCRprediction for DDX53, with AlphaFold 3 structure predictions supporting its role as the P1-B4 binding site. Similarly, analysis of AKR1A1 identified TVDGKRV (aa 304 – 310) as thepotential binding epitope for P3-F1. This epitope was identified in both the ElliPro and Al-phaFold 3 predictions. For KIFC3, SWSSQEHLEWE (aa 781 – 791) was found as thebinding epitope of P1-F6 by the ElliPro analysis, though no correspondence was found in the AlphaFold 3 structure. No correlation of rec. AB binding and MHCI expression

[00101] It was hypothesized that the targets of the recombinant antibodies arepresented on MHCI to induce the immune response. To verify these immunocytochemis- try staining of Malme-3M cells with rec. Abs and anti-MHC I antibody was performed. No co-staining was observed (Figure 5). It is now theorized that the immune response mightbe mediated via the cancer-immunity cycle (Mellman et al., The cancer-immunity cycle:Indication, genotype, and immunotype. Immunity.2023 Oct 10;56(10):2188-2205). Immunoprecipitation with B4 antibody

[00102] IP was performed to isolate the target of B4 for further analysis. SDS-PAGE presents the successful pull-down of the B4 antibody target (Figure 6). 4. Conclusion

[00103] The aim of the study underlying the invention was to determine whetheran intrathecal B cell response in patients with malignant melanoma and meningeal spread can be directed against tumor cells in the CSF and what potential targets can be identifiedby CSF B cell-derived recombinant antibodies. These antibodies and their correspondingtumor antigens may be extremely useful as they appear to be sufficiently immunogenic toelicit an immune response and may target specific epitopes, thereby reducing off-target effects through B cell receptor selection. In this study, an intrathecal B cell response in 3 patients with malignant melanoma was identified and 13 recombinant antibodies were generated for further analysis (Table 5).

[00104] Although antigen detection of antibodies is often difficult due to cross-reactivity and the conformational nature of many antigens, two target antigens were identi- fied in patient 1 and one target antigen in patient 3. It was demonstrated by flow cytometry that the antibodies P1-B4, P1-B8, P1-F6, and P3-F1 bind extracellularly to the Malme-3M cell line, which represents molecular characteristics of the tumor cells of melanoma pa-tients. Further immunostaining in fixed cells showed a cytosolic staining pattern and ex-pression at the edge of the cells. Microarrays of pooled antibodies revealed several poten- tial antigens, which were further selected according to their biological function and tumor association. For the selected antigens AKR1A1, GART, PPIL1, MDP1, MTUS2, DDX53, LASP1, KIFC3 and LMNA, additional epitope mapping by permutation of linear peptides of 13 AA was performed. The antibody P1-B4 could be associated with binding to a potential specific epitope for DDX53, the antibody P1-F6 with an epitope for the antigen KIF3C and the antibody P3-F1 with an epitope for the antigen AKR1A1. Potential epitope sequences were also isolated with bioinformatic simulations of binding patterns between the antibodyand the antigen as well as MHC I. For DDX53 the potential epitopes wereRIFGNREMKAK (SEQ ID NO: 49), SGEKRLIPKPTCR (SEQ ID NO: 50), and HVYNY(SEQ ID NO: 51), for AKR1A1 LEYLDLY (SEQ ID NO: 54) and TVDGKRV (SEQ ID NO:55) was found and lastly EYTITVSAAEI (SEQ ID NO: 52) and SWSSQEHLEWE (SEQ IDNO: 53) might be the epitope for KIFC3. Finally, custom ELISA tests were established thatconfirmed binding of the antibody P1-B4 against DDX53, and P1-F6 against KIF3C in pa- tient 1; serum and CSF of this patient was also tested positive against DDX53 whereasserum was positive against KIF3C only. In patient 3, the inventors could confirm AKR1A1as a target of antibody P3-F1 with serum also showing positive signal in the ELISA test.

[00105] Altogether all three proteins, DDX53, AKR1A1, and KIFC3, are highly in-teresting and promising targets for an anti-cancer treatment. In addition, tumor antigens recovered by CSF may be extremely useful since they seem to be sufficientlyimmunogenic to trigger an immune response and may target specific epitopes reducing off-target effects by B cell receptor selection.Sequences Part of the application and disclosure is a sequence listing in WIPO St.26-Forrmat. In the following, all sequences are again listed which are subject invention. In the event of dis- crepancies between the sequences in the attached sequence listing and the sequences listed in the following, the sequences listed in the following shall take precedence. ANTIBODIES AMINO ACIDS CDR3Clone P1-B4 heavy chain CDR3 amino acidARSRGIVGSTLDDN (SEQ ID NO: 1)Clone P1-F6 heavy chain CDR3 amino acidAHRPIASSRDGFDY (SEQ ID NO: 2)Clone P3-F1 heavy chain CDR3 amino acidSHHRLTDGRLR (SEQ ID NO: 3)Clone P1-B4 light chain CDR3 amino acidQQYHSTPRT (SEQ ID NO: 4)Clone P1-F6 light chain CDR3 amino acidQQYNGFPFT (SEQ ID NO: 5)Clone P3-F1 light chain CDR3 amino acidQQYDTSPWT (SEQ ID NO: 6)CDR2Clone P1-B4 heavy chain CDR2 amino acidISVYKSDT (SEQ ID NO: 7)Clone P1-F6 heavy chain CDR2 amino acidIYWNDDK (SEQ ID NO: 8)Clone P3-F1 heavy chain CDR2 amino acidVYWDDDK (SEQ ID NO: 9)Clone P1-B4 light chain CDR2 amino acidWAS (SEQ ID NO: 10)Clone P1-F6 light chain CDR2 amino acidKAS (SEQ ID NO: 11)Clone P3-F1 light chain CDR2 amino acidRAS (SEQ ID NO: 12)CDR1Clone P1-B4 heavy chain CDR1 amino acidGYTFTSYG (SEQ ID NO: 13)Clone P1-F6 heavy chain CDR1 amino acidGFSLSTNGVS (SEQ ID NO: 14)Clone P3-F1 heavy chain CDR1 amino acidGFSLNTRAVG (SEQ ID NO: 15)Clone P1-B4 light chain CDR1 amino acidQSVLYSSNNRNY (SEQ ID NO: 16)Clone P1-F6 light chain CDR1 amino acidQSVSGW (SEQ ID NO: 17)Clone P3-F1 light chain CDR1 amino acidQSITSSY (SEQ ID NO: 18)Full-length clone P1-B4 Heavy chain QVQLVESGPEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISVYKSDT NDAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSRGIVGSTLDDNWGQGTL VTVSS (SEQ ID NO: 19) Light chain HPQMTQSPDSLAVSLGERATINCKSSQSVLYSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYHSTPRTFGQGTKVEIK (SEQID NO: 20)Full-length clone P1-F6 Heavy chain QVPLKESGPALVKPTRTLTLTCTFSGFSLSTNGVSVGWIRQPPGKALDWLALIYWNDDK RYSPSLKNRLTITKDTSKNLVVLRMTNMDPVDTATYYCAHRPIASSRDGFDYWGQGTLV TVSS (SEQ ID NO: 21) Light chain DIQMTQFPSTLSASVGETVTISCRASQSVSGWLAWYQQKPGKAPKLLMYKASALENGVP SRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNGFPFTFGPGTKVDIN (SEQ ID NO: 22) Full-length clone P3-F1 Heavy chain QVQLVESGPTLVKPTQTLTVTCTFSGFSLNTRAVGVGWIRQPPGGALEWLALVYWDDD KRYSPSLKNRLTITKDTSRNQVVLTLTNVAPVDTGTYYCSHHRLTDGRLRWGQGTLVTVSS (SEQ ID NO: 23)Light chain QVQLVQSPGTLSLSPGERATLSCRASQSITSSYLAWYQQKFGQAPRLLIYRASNRATGIPDRFSGSGSGTDFTLTISSLESEDFAVYYCQQYDTSPWTFGQGTKVEIK (SEQ ID NO: 24)NUCLEIC ACIDSCDR3Clone P1-B4 heavy chain CDR3 nucleic acidGCC CGC TCT AGG GGC ATC GTG GGC AGT ACA CTG GAC GAC AAC (SEQ ID NO:25)Clone P1-F6 heavy chain CDR3 nucleic acidGCC CAC AGG CCC ATC GCC TCC TCC CGA GAC GGC TTC GAC TAC (SEQ ID NO:26)Clone P3-F1 heavy chain CDR3 nucleic acidAGC CAC CAC AGG CTG ACC GAC GGA AGA CTG AGG (SEQ ID NO: 27)Clone P1-B4 light chain CDR3 nucleic acidCAG CAG TAC CAC TCC ACC CCC AGA ACC (SEQ ID NO: 28)Clone P1-F6 light chain CDR3 nucleic acidCAG CAG TAC AAT GGC TTT CCC TTC ACA (SEQ ID NO: 29)Clone P3-F1 light chain CDR3 nucleic acidCAG CAG TAC GAC ACC TCT CCC TGG ACC (SEQ ID NO: 30)CDR2Clone P1-B4 heavy chain CDR2 nucleic acidATC TCC GTG TAT AAG AGC GAT ACA (SEQ ID NO: 31)Clone P1-F6 heavy chain CDR2 nucleic acidATC TAC TGG AAC GAC GAC AAA (SEQ ID NO: 32)Clone P3-F1 heavy chain CDR2 nucleic acidGTG TAC TGG GAC GAC GAC AAG (SEQ ID NO: 33)Clone P1-B4 light chain CDR2 nucleic acidTGG GCA TCC (SEQ ID NO: 34)Clone P1-F6 light chain CDR2 nucleic acidAAA GCC TCC (SEQ ID NO: 35)Clone P3-F1 light chain CDR2 nucleic acidAGG GCC TCC (SEQ ID NO: 36)CDR1Clone P1-B4 heavy chain CDR1 nucleic acidGGC TAC ACC TTC ACC TCC TAC GGC (SEQ ID NO: 37)Clone P1-F6 heavy chain CDR1 nucleic acidGGC TTC TCC CTG TCC ACC AAC GGC GTG TCC (SEQ ID NO: 38)Clone P3-F1 heavy chain CDR1 nucleic acidGGC TTC TCA CTG AAC ACA AGG GCC GTG GGC (SEQ ID NO: 39)Clone P1-B4 light chain CDR1 nucleic acidCAG TCC GTC CTC TAC TCC TCC AAC AAC CGC AAC TAC (SEQ ID NO: 40)Clone P1-F6 light chain CDR1 nucleic acidCAG TCC GTG TCC GGC TGG (SEQ ID NO: 41)Clone P3-F1 light chain CDR1 nucleic acidCAG TCA ATC ACA TCC TCC TAC (SEQ ID NO: 42)Full-length clone P1-B4 Heavy chain CAGGTGCAGCTGGTGGAAAGTGGCCCCGAGGTGAAAAAGCCCGGCGCCTCCGTGA AGGTGTCCTGCAAAGCAAGCGGCTACACCTTCACCTCCTACGGCATCTCCTGGGTGA GGCAGGCCCCCGGACAGGGACTGGAGTGGATGGGATGGATCTCCGTGTATAAGAG CGATACAAATGACGCCCAGAAGTTCCAGGGCCGCGTGACAATGACCACCGACACCT CCACCAGCACCGCTTATATGGAGCTGAGATCCCTGAGGAGCGACGACACCGCCGTG TACTACTGTGCCCGCTCTAGGGGCATCGTGGGCAGTACACTGGACGACAACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCC (SEQ ID NO: 43)Light chain CACCCTCAGATGACCCAGTCCCCCGACTCCCTGGCCGTCTCCCTCGGAGAAAGAGC CACCATCAACTGCAAATCCTCCCAGTCCGTCCTCTACTCCTCCAACAACCGCAACTA CCTCGCCTGGTACCAGCAGAAACCCGGCCAGCCCCCCAAACTGCTCATCTACTGGG CATCCACCAGAGAATCCGGCGTGCCCGACCGCTTCTCCGGCTCTGGAAGCGGCACC GACTTCACCCTGACTATCTCCTCCCTGCAGGCTGAGGACGTCGCCGTTTACTACTGC CAGCAGTACCACTCCACCCCCAGAACCTTCGGCCAGGGCACCAAGGTGGAAATCAAA (SEQ ID NO: 44)Full-length clone P1-F6 Heavy chain CAGGTGCCACTGAAGGAAAGCGGCCCCGCCCTGGTGAAACCCACCAGAACACTGAC CCTGACCTGCACCTTCTCCGGCTTCTCCCTGTCCACCAACGGCGTGTCCGTGGGCT GGATCAGGCAGCCCCCCGGAAAAGCCCTGGACTGGCTGGCTCTGATCTACTGGAAC GACGACAAAAGATACTCCCCCTCCCTGAAGAACCGGCTGACCATCACAAAAGACACC AGCAAGAACCTGGTGGTGCTGAGGATGACCAACATGGACCCTGTGGACACCGCCAC CTACTACTGCGCCCACAGGCCCATCGCCTCCTCCCGAGACGGCTTCGACTACTGGGGACAGGGAACCCTGGTGACCGTGTCCAGC (SEQ ID NO: 45)Light chain GACATTCAGATGACCCAGTTTCCCTCCACCCTGTCCGCCTCCGTGGGCGAAACCGT GACCATCTCCTGCAGGGCCTCCCAGTCCGTGTCCGGCTGGCTGGCTTGGTACCAGC AGAAACCCGGCAAAGCCCCCAAACTGCTGATGTACAAAGCCTCCGCCCTGGAGAAC GGCGTGCCCTCACGCTTTAGCGGTTCCGGCTCCGGGACCGAGTTCACCCTGACTAT CTCCTCACTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAATGGCTTTCCCTTCACATTCGGTCCCGGCACAAAGGTGGATATTAAC (SEQ ID NO: 46)Full-length clone P3-F1 Heavy chain CAGGTGCAGCTGGTGGAAAGTGGCCCCACCCTGGTGAAGCCCACACAGACACTGAC AGTGACATGCACATTCTCCGGCTTCTCACTGAACACAAGGGCCGTGGGCGTGGGAT GGATCAGGCAGCCCCCCGGAGGCGCACTGGAATGGCTGGCTCTGGTGTACTGGGACGACGACAAGAGGTACTCCCCTAGTCTGAAGAACAGGCTGACAATCACAAAGGATAC CAGCAGGAACCAGGTGGTGCTGACACTGACCAACGTGGCTCCCGTGGACACCGGC ACCTACTACTGCAGCCACCACAGGCTGACCGACGGAAGACTGAGGTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCT (SEQ ID NO: 47)Light chain CAGGTCCAGCTCGTCCAGAGCCCCGGCACACTCTCCCTCTCCCCCGGAGAAAGAGC CACACTCTCATGCCGGGCCTCCCAGTCAATCACATCCTCCTACCTCGCCTGGTACCA GCAGAAATTCGGCCAGGCCCCCAGGCTCCTGATCTACAGGGCCTCCAACAGGGCCA CCGGAATCCCCGACAGGTTTTCCGGCTCCGGCTCCGGAACCGACTTCACCCTGACT ATCTCCTCCCTCGAGTCTGAGGACTTCGCAGTTTACTACTGCCAGCAGTACGACACCTCTCCCTGGACCTTCGGCCAGGGTACCAAGGTGGAGATCAAG (SEQ ID NO: 48)EPITOPES AMINO ACIDS RIFGNREMKAK (SEQ ID NO: 49) SGEKRLIPKPTCR (SEQ ID NO: 50) HVYNY (SEQ ID NO: 51) EYTITVSAAEI (SEQ ID NO: 52) SWSSQEHLEWE (SEQ ID NO: 53) LEYLDLY (SEQ ID NO: 54) TVDGKRV (SEQ ID NO: 55) YLMHWPYAF (SEQ ID NO: 80) GESEAKVRIF (SEQ ID NO: 81) HVYNYDFPR (SEQ ID NO: 82) SAAEIYNEV (SEQ ID NO: 83) BINDING STRETCHES AMINO ACIDS EAKVRIFGNREMKAK (SEQ ID NO: 56) KVRIFGNREMKAKAK (SEQ ID NO: 57)RIFGNREMKAKAKAA (SEQ ID NO: 58) LKSGEKRLIPKPTCR (SEQ ID NO: 59) SGEKRLIPKPTCRFK (SEQ ID NO: 60) SRGLDLNDVTHVYNY (SEQ ID NO: 61) GLDLNDVTHVYNYDF (SEQ ID NO: 62) HVYNYDFPRNIDVYV (SEQ ID NO: 63) ASDWEYTITVSAAEI (SEQ ID NO: 64) DWEYTITVSAAEIYN (SEQ ID NO: 65) EYTITVSAAEIYNEV (SEQ ID NO: 66) AELGSWSSQEHLEWE (SEQ ID NO: 67) LGSWSSQEHLEWEPA (SEQ ID NO: 68) SWSSQEHLEWEPACQ (SEQ ID NO: 69) RKTLADLQLEYLDLY (SEQ ID NO: 70) TLADLQLEYLDLYLM (SEQ ID NO: 71) ADLQLEYLDLYLMHW (SEQ ID NO: 72) LQLEYLDLYLMHWPY (SEQ ID NO: 73) LEYLDLYLMHWPYAF (SEQ ID NO: 74) WRYIVPMLTVDGKRV (SEQ ID NO: 75) YIVPMLTVDGKRVPR (SEQ ID NO: 76) VPMLTVDGKRVPRDA (SEQ ID NO: 77) MLTVDGKRVPRDAGH (SEQ ID NO: 78)TVDGKRVPRDAGHPL (SEQ ID NO: 79)

Claims

Claims1. An antibody or fragment thereof specifically binding to meningeosis carcinoma-specific antigen (MC-specific antigen), characterized in comprising- as heavy chain variable domain a CDR3 region, said CDR3 region has anamino acid sequence being at least 90 % identical to one selected fromthose in SEQ ID NO: 1, 2 or 3, and / or -as light chain variable domain a CDR3 region, said CDR3 region has anamino acid sequence being at least 90% identical to one selected from those in SEQ ID NO: 4, 5 or 6.

2. The antibody or fragment of claim 1, characterized in that- the heavy chain variable domain comprises a CDR2 region, said CDR2region has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 7, 8 or 9, and / or -the light chain variable domain comprises a CDR2 region, said CDR2 re-gion has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 10, 11, or 12.

3. The antibody or fragment of claim 1 or 2, characterized in that- the heavy chain variable domain comprises a CDR1 region, said CDR1region has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 13, 14, or 15, and / or- the light chain variable domain comprises a CDR1 region, said CDR1 re-gion has an amino acid sequence being at least 90 % identical to one se-lected from those in SEQ ID NO: 16, 17, or 18.

4. The antibody or fragment of any of claims 1-3, characterized in comprising- the heavy chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 1 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 7 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 13 and / or -the light chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 4 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 10 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 16.

5. The antibody or fragment of any of claims 1-3, characterized in comprising- the heavy chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 2 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 8 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 14 and / or -the light chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 5 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 11 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 17.

6. The antibody or fragment of any of claims 1-3, characterized in comprising- the heavy chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 3 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 9 and / or- CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 15 and / or -the light chain variable domains- CDR3 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 6 and / or -CDR2 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 12 and / or -CDR1 region having an amino acid sequence being at least 90 % identicalto said in SEQ ID NO: 18.

7. The antibody of fragment thereof or any of the preceding claims comprising- a heavy chain having an amino acid sequence being at least 90 % identical tosaid in SEQ ID NO:19, and / or -a light chain having an amino acid sequence being at least 90 % identical to saidin SEQ ID 20.

8. The antibody of fragment thereof or any of the preceding claims comprising- a heavy chain having an amino acid sequence being at least 90 % identical tosaid in SEQ ID NO:21, and / or -a light chain having an amino acid sequence being at least 90 % identical to saidin SEQ ID 22.

9. The antibody of fragment thereof or any of the preceding claims comprising- a heavy chain having an amino acid sequence being at least 90 % identical tosaid in SEQ ID NO:23, and / or -a light chain having an amino acid sequence being at least 90 % identical to saidin SEQ ID 24.

10. The antibody or fragment thereof of any of the preceding claims, characterized inthat the MC-specific antigen is selected from the group consisting of DDX53, KIF3C, and AKR1A1.

11. An antibody or fragment thereof specifically binding to MC-specific antigen, char-acterized in that it specifically binds to an epitope comprising an amino acid se- quence being at least 90% identical to one selected from those in SEQ ID NOS: 49-55.

12. An antibody or fragment thereof configured for a specific binding to meningeosiscarcinoma-specific antigen for use in the prophylaxis and / or treatment of cancer, preferably melanoma, further preferably melanoma with leptomeningeal spread, highly preferably melanoma with meningeosis carcinomatosa.

13. A pharmaceutical composition characterized in comprising an antibody or fragmentthereof according to any of claims 1-12.

14. A nucleic acid, preferably a vector, encoding an antibody or fragment thereof ac-cording to any of claims 1-13.

15. A prokaryotic or eukaryotic host cell comprising the nucleic acid of claim 14.

Images