Targeting abcb5 for cancer therapy

[0001] Human malignant melanoma is a highly chemically resistant cancer. There are currently not many effective treatment options. Malignant skin melanoma is highly prevalent in the United States, affecting 1 in 63 men and women during their lifetime. Of these, 11% are diagnosed after the cancer has spread to regional lymph nodes or directly outside the original site, and 3% after the tumor has metastasized (late stage), with a 5-year relative survival rate of 63.8% and 16, respectively 0%.

SUMMARY OF THE INVENTION [0002] The invention is based at least in part on the discovery that chemically resistant ABCB5 + tumor stem cells contribute to the development of cancers such as melanoma and these cells may be targeted for cancer treatment. Targeting ABCB5 can be used as a separate therapeutic approach to disseminated disease or as an adjunct therapy to sensitize cancer cells to chemotherapeutic agents, especially in those patients with current refractory metastatic disease. The advantage of therapeutic approaches targeted at ABCB5 is that they target oncogenic stem cells, while conventional therapies only target the majority of the cancer cell population.

[0003] In some aspects, there is a described method for delivering a therapeutic agent to the intracellular compartment of a cell by contacting the cell with an isolated molecule that selectively binds to ABCB5 conjugated to the therapeutic agent in an amount effective to deliver the therapeutic agent to the intracellular compartment of the cell.

[0004] In some embodiments, an isolated molecule that selectively binds to ABCB5 is an isolated peptide. In other embodiments, it is a small molecule. An isolated peptide may be, for example, an antibody or fragment binding that antigen or scFv fragment.

[0005] The therapeutic agent may in some embodiments be a toxin, siRNA or a chemotherapeutic agent or therapeutic antibody.

[0006] The method includes, in other embodiments, the step of contacting the cell with an isolated molecule that selectively binds to a surface marker such as CD49e, CD133, CD166, BMPR1a, TIR-1, VE-cadherin (CD144) or nestin.

[0007] Disclosed herein is a composition provided from an isolated peptide that selectively binds to ABCB5 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or functionally equivalent thereof

-2 variants containing conservative substitutions where the isolated peptide is not mAb 3C2-ID12.

[0008] In other disclosures, an isolated peptide composition is provided that selectively binds to ABCB5 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or functionally equivalent variants thereof containing conservative substitutions. The isolated antibody or antibody fragment is present in an effective amount to enhance chemosensitivity in a human subject.

[0009] According to yet another disclosure, an isolated peptide composition is provided that selectively binds to ABCB5 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or functionally equivalent variants thereof containing conservative substitutions. The isolated peptide is preferably co-formulated with a therapeutic agent.

[0010] The isolated peptide in some embodiments is conjugated to a therapeutic agent. In other embodiments, the teraepeutic agent is selected from the group consisting of camptothecin 9-NH2, mitoxantrone, camptothecin 7-Cl, pyrazofurin, menogaril, camptothecin ester 20, camptothecin, amsacrine, etoposide, camperotinazole, antrapyrazine derivative camptothecin, daunorubicin, doxoroxorubicin, doxorubicin, oxantrazole, 11HOMe camptothecin, zorubicin, uracil mustard, piperazinedione, hepsulfame, melphalan, bisantrene, triethylenemelamine, spiromustine, Yoshi-864, chlorambucil, piperazine mustard, hydroxycarbamide, porphyromycin, mechlorethamine, fluorodopane, mitomycin, cytarabine (araC), dianhydrogalacticol, gemcitabine, thiotepa, N, teroxynycin

[0011] A kit is provided herein according to other disclosures. The kit includes a container containing an isolated peptide that selectively binds to ABCB5 and contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or functionally equivalent variants thereof conservative substitutions and instructions for administering the isolated peptide to a human subject.

[0012] Provided herein is a method of treating a subject according to other disclosures. The method comprises administering a systematically isolated peptide that selectively binds to ABCB5 and contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or functionally equivalent variants thereof containing conservative substitutions to a subject having cancer in an effective amount for cancer treatment.

[0013] A method of treating a subject is provided by administering any composition described herein to a subject having the cancer in an effective amount for treating the cancer.

[0014] Provided herein is a method of treating a subject according to other disclosures. The method comprises administering an isolated peptide that selectively binds to ABCB5 and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or functionally equivalent variants thereof containing conservative substitutions and a chemotherapeutic agent, to an individual having the cancer in an effective amount for cancer treatment.

[0015] According to other disclosures herein, there is provided a method of treating a subject by systemically administering to the subject having a tumor an effective amount for treating the cancer of an isolated antibody or antibody fragment that selectively binds to ABCB5 and a chemotherapeutic agent.

[0016] An isolated immunoglobulin heavy chain variable domain peptide is disclosed herein, wherein: (i) CDR1-H1 comprises the amino acid sequence of SEQ ID NO 3; (ii) CDR2-H2 comprises the amino acid sequence of SEQ ID NO 4; and (iii) the CDR3-H3 sequence, wherein the isolated peptide is not mAb 3C2-1D12. In some embodiments, the CDR3-H3 has the amino acid sequence of SEQ ID NO 3. The isolated peptide may bind to human ABCB5 and may be an antibody. Optionally, the isolated peptide further comprises a light chain variable domain, where CDR1-L1 has the amino acid sequence of SEQ ID NO 6 and CDR2-L2 which has the amino acid sequence of SEQ ID NO 7 and / or CDR3-L3 which has the amino acid sequence of SEQ ID NO 8.

[0017] An isolated peptide having an immunoglobulin light chain variable domain is also described, wherein: (i) CDR1-L1 has the amino acid sequence of SEQ ID NO 6; (ii) CDR2-L2 has the amino acid sequence of SEQ ID NO 7; and (iii) the CDR3-H3 sequence, wherein the isolated peptide is not mAb 3C2-1D12. In some embodiments, CDR3-L3 has the amino acid sequence of SEQ ID NO 8.

[0018] According to other disclosures, an isolated peptide having at least two antibody variable domains is provided herein: (a) an antibody heavy chain variable domain comprising the isolated peptide as described herein and (b) an antibody light chain variable domain comprising the isolated peptide as described herein. In some embodiments, the isolated peptide is a single chain Fv. In other embodiments, the isolated peptide is an isolated Fab peptide. In yet other embodiments, the isolated peptide is fully human isolated peptide.

[0019] The isolated peptide may further comprise FR1, FR2, FR3 and / or FR4 framework regions for the isolated peptide variable domain corresponding to the variant CDR1-H1, CDR2-H2, CDR3-H3, wherein the framework regions are obtained from a single matrix

-4polipeptydowej. Each framework may have an amino acid sequence corresponding to the amino acid sequences of the SEQ ID NO: 1 peptide framework.

[0020] In some embodiments, the isolated peptide further comprises a dimerization domain linked to the C-terminal region of the heavy chain variable domain of the polypeptide. The dimerization domain may be a leucine zipper domain or a sequence having at least one cysteine ​​residue. The dimerization domain has a hinge region in some embodiments. In other embodiments, the dimerization domain is a single cysteine.

[0021] In some embodiments, the isolated peptide is monoclonal antibody antibodies. In other embodiments, it is a bispecific antibody. In yet other embodiments, the isolated peptide is a synthetic antibody.

[0022] According to yet another disclosure herein, an anti-ABCB5 antibody or antigen binding fragment thereof is provided. The antibody has a human constant region, wherein the anti-ABCB5 antibody or antigen-binding fragment competitively inhibits mAb 3C2-1D12 to ABCB5 binding. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab') 2, Fv, scFv, dsFv, Fd, VH dAb and VL dAb. In other embodiments, the antibody or antigen-binding fragment is an IgA, IgGb 1, IgG2, IgG3, IgG4 or IgM immunoglobulin. In yet other embodiments, the antibody or antigen binding fragment comprises a human constant region and the human variable framework region or antigen binding fragment is a single chain antibody. The single chain antibody is optionally a camelid antibody.

[0023] A variable domain of a humanized antibody having a functional antigen binding region according to other disclosures herein is provided. The variable domain of a humanized antibody has non-human CDR1-H1, CDR2-H2, CDR3-H3, CDR1-L1, CDR2-L2 and CDR3-L3 having at least 90% homology to CDR1-H1, CDR2-H2, CDR3-H3, CDR1L1, CDR2-L2 and CDR3-L3 from mAb 3C2-1D12 incorporated into the human antibody variable domain.

[0024] In other disclosures, chimeric antibodies are provided herein. Chimeric antibodies have a variable domain that specifically binds to ABCB5 and a constant domain, wherein the variable domain and the constant domain are from different species.

[0025] In some embodiments, the isolated peptide has an ABCD5 binding CDR3-H3 amino acid sequence or a functionally equivalent variant thereof. In other embodiments, the isolated peptide has the ABCB5 binding CDRH2 amino acid sequence or a functionally equivalent variant thereof. In other embodiments, the isolated peptide has the ABCB5 binding CDR-H1 amino acid sequence or a functionally equivalent variant thereof. In other embodiments, the isolated peptide has the ABCB5 binding CDR-L3 amino acid sequence or a functionally equivalent variant thereof. In other embodiments, the isolated peptide has a sequence

An amino acid CDR-L2 binding ABCB5 or a functionally equivalent variant thereof. In other embodiments, the isolated peptide has the ABCB5 binding CDR-L1 amino acid sequence or a functionally equivalent variant thereof.

[0026] In other embodiments, the isolated peptide is an antibody or antibody fragment. The isolated antibody or antibody fragment may optionally be an intact soluble monoclonal antibody. In some embodiments, the isolated antibody or antibody fragment is an isolated monoclonal antibody fragment selected from the group consisting of Fab, Fab ', F (ab') 2, Fv, scFv, dsFv, Fd, VH dAb and VL dAb. In yet other embodiments, the isolated antibody or antibody fragment enhances chemosensitization. In a preferred embodiment, the isolated peptide selectively binds to ABCB5. In yet other embodiments, the isolated antibody or antibody fragment is a humanized antibody. The isolated peptide may optionally be scFv.

[0027] The isolated peptide in other embodiments is conjugated to a detectable label.

[0028] The composition may also contain a pharmaceutically acceptable carrier and is optionally a sterile formulation.

[0029] The invention is not limited in its application to the details of the structure and arrangement of the components further defined in the following description or illustrated in the figures. The invention is suitable for other embodiments and for being used or practicing in many ways. In addition, the phraseology and terminology used herein is for the purpose of description and should not be construed as limiting. Use of the term "including"; "Containing" or "having"; "Including"; "Exploiting" and their variations here is intended to cover the elements listed below and their equivalents, as well as additional elements.

BRIEF DESCRIPTION OF THE FIGURES [0030] The accompanying figures are not to be drawn to scale. In the figures, each identical or nearly identical component that is illustrated in the various figures is represented as a numeral. For clarity, not every component can be marked on each figure. On the figures:

Fig. 1 is a series of images and graphs showing analysis of melanoma progression in tissue microarray for ABCB5, as well as characterization of the ABCB5 melanoma population⁺. Fig. 1 (a) is a graph illustrating the analysis performed by the Chromavision Automated Cellular Image System, showing significant differences in ABCB5 staining intensities for thin and thick melanocytic nevi relative to thin and thick primary melanomas, relative to melanoma lymph node and organ metastases parenchymal (thin or thick birthmark vs. thin or thick primary melanomas or vs. metastases of melanoma to lymph nodes or parenchymal organs, all P values ​​<0.001; thin

-6 primary melanomas vs. thick primary melanomas P = 0.004; thin and thick primary melanomas vs. lymph node metastases, P = 0.001; lymph node metastases vs. metastases to parenchymal organs, P = 0.025). Fig. 1 (bc) show many characterizations of the ABCB5 melanoma population⁺. Fig. 1 (b) shows a single-color flow cytometry analysis of clinical melanoma samples for the expression of ABCB5, CD20, nestin, TIE-1, VE-cadherin, CD31 or BMPR1a. Illustrated is% positive cells for n = 6 patients with melanoma (level bars indicate average expression). Figure 1 (c) shows the expression of CD20, nestin, TIE-1, VE-cadherin, CD31 or BMPR1a by clinical ABCB5 melanoma cells⁺ or ABCB5^- as determined by two-color flow cytometry. % positive cells (mean ± SEM) are illustrated for n = 3-6 patients with melanoma.

Fig. 2 is a series of graphs and images showing in vivo carcinogenicity of ABCB5 melanoma cell subsets⁺ in human tumor xenograft methods to mouse tumors. Fig. 2 (a) (left panel) is a graph showing the in vivo tumor formation capacity (%) of unsegregated (US) melanoma cells, ABCB5^- or ABCB5⁺ G3361 after subcutaneous xenograft (10⁷, 10⁶, or 10⁵ cells / inoculum) into NOD / SCID mice. (Middle panel) is a graph showing% inoculum without tumor formation expressed as a function of the number of inoculated cells for unsegregated melanoma cells (US), ABCB5^- or ABCB5⁺ G3361 for NOD / SCID mice, to determine 50% Cancer Forming Capacity (TF50). (Right panel) shows tumor volumes (mean ± SEM) of primary melanoma xenografts 8 weeks after subcutaneous xenograft melanoma xenograft cells (10⁷/ inoculum) unsorted (US), ABCB5^- or ABCB5⁺ G3361. Fig. 2 (b) (left panel) is a graph showing the in vivo tumor formation capacity (%) of unsegregated (US) melanoma cells, ABCB5or ABCB5⁺ A375 after subcutaneous xenografts (2x10⁶, 2x10⁵ or 2x10⁴cells / inoculum) into NOD / SCID mice. (middle panel) shows% inoculum without tumor formation expressed relative to the number of inoculated cells for unsegregated melanoma cells (US), ABCB5^- or ABCB5⁺ A375 in NOD / SCID mice, to determine 50% Cancer Forming Ability (TF50). (Right panel) shows tumor volumes (mean ± SEM) of primary melanoma xenografts 5 weeks after subcutaneous xenograft melanoma xenograft cells (2x10⁶/ inoculum) unsorted (US), ABCB5^- or ABCB5⁺A375. Fig. 2 (c) (Left panel) shows immunohistochemistry for ABCB5 expression in a representative primary, unsegregated xenograph obtained from melanoma cells in NOD / SCID mice, illustrating three separate zones delimited by dashed lines: ABCB5^-/ melanin-negative (upper left part of the panel), ABCB5^-/ melanin-positive (upper right of the panel) and ABCB5⁺/ melanin negative (bottom half of panel). (Right panel) is a series of images of double immunofluorescence staining of frozen melanoma xenograft sections for co-expression of ABCB5 (FITC) and VE-cadherin

-7 (Texas red). The nuclei are imaged with 4 ', 6-diamidino-2-phenylindole staining (DAPI, blue). Fig. 2 (d) is a graph showing secondary tumor formation capacity (%) in NOD / SCID mice with ABCB5 cells^- or ABCB5⁺(10⁷/ inoculum) isolated from primary ABCB5 melanoma tumors⁺cellular origin. Fig. 2 (e) contains two graphs showing in vivo tumor formation capacity (%) (left panel) and tumor volume (mean ± SEM, right panel) of unsegregated melanoma (US) cells, ABCB5^- or ABCB5⁺ freshly obtained from the patient (10⁶/ inoculum), 8 weeks after subcutaneous hetero-transplantation into NOD / SCID mice.

Fig. 3 shows in vivo tumorigenesis tracking, self-reproduction and differentiation of ABCB5 human melanoma cells⁺ in NOD / SCID mouse recipients. Fig. 3 (a) (left panels) shows two-color flow cytometry (F11 (EYFP) vs. F12 (DsRed2) - dot plots) tumor cell inoculum consisting of 10% ABCB5⁺ G3361 / DsRed2 and 90% ABCB5^- G3361 / EYFP cells before xenograft (shown on large panel). Controls (shown on small panels) are non-transfected human G3361 melanoma cells (top), G3361 / DsRed2 cells (center) and G3361 / EYFP cells (bottom). (Right panels) show two-color flow cytometry (F11 (EYFP) vs. F12 (DsRed2) dot plots) of a disseminated xenograft tumor formed 6 weeks after inoculation with 10% ABCB5 cells⁺ G3361 / DsRed2 and 90% ABCB5^- G3361 / EYFP (shown on the large panel). Controls (shown on small panels) are non-transfected human G3361 melanoma cells (top), G3361 / DsRed2 cells (center) and G3361 / EYFP cells (bottom). Fig. 3 (b) is a graph of the average percent (mean ± SEM) of DsRed2 cells⁺ (% DsRed2⁺ / (% DsRed2⁺ +% EYFP⁺) x 100) from ABCB5 cells⁺ or EYFP⁺ (% EYFP⁺ / (% DsRed2⁺ +% EYFP⁺) x 100) of ABCB5 origin^- as a function of weeks after inoculation of melanoma cells for tumors obtained in vivo at t = 4 or 6 weeks (n = 3 copies, respectively) and xenografted cell inocula (n = 6), respectively. FIG. 3 (c) is a series of images of two-channel fluorescence microscopy of G3361 / DsRed2 and G3361 / EYFP cells (upper and middle rows) and cross-section of frozen tissue (bottom row) obtained from tumors formed in vivo 6 weeks after subcutaneous xenograft to NOD / SCID mice with 10% G3361 / DsRed2 ABCB5 cell inocula⁺ and 90% G3361 / EYFP ABCB5^-. The left panels represent a bright field, the middle left panels represent DsRed2 (of ABCB5 origin⁺), the middle right panels depict EYFP (ABCB5 origin^-) and the panels to the right are the combined images (size bars: 25 μm). Fig. 3 (d) (left panels) show flow cytometry analysis of DsRed2 and EYFP expression in ABCB5 cells⁺ (top) and ABCB5 cells^- (bottom) obtained from tumors formed in NOD / SCID mice 6 weeks after inoculation with 10% ABCB5 cells⁺ G3361 / DsRed2 and 90% ABCB5G3361 / EYFP. (Right panel) is a graph showing the average percentage (mean ± SD) of either DSRed2 or EYFP fluorescent cells (calculated as

-8% DsRed2⁺ / (% DsRed2⁺ +% EYFP⁺) x 100 or% EYFP⁺ / (% DsRed2⁺ +% EYFP⁺) x 100) in subsets of ABCB5 cells⁺ and ABCB5^- obtained with n = 3 tumor copies.

Fig. 4 is a series of graphs and images showing an analysis of the effect of ABCB5 mAb on melanoma xenograft growth. Fig. 4 (a) is a graph measuring tumor volumes (mean ± SEM) of melanoma xenografts as a function of days after subcutaneous inoculation of melanoma cells into Balb / c nude mice (10⁷ cells / inoculum) for untreated (n = 18), mAb-treated control isotype (n = 10) or anti-ABCB5 mAb-treated animals (n = 11). [Days of intraperitoneal mAb administration are indicated by arrows. ] Fig. 4 (b) is a graph measuring tumor formation (%) 58 days after subcutaneous inoculation of melanoma cells into Balb / c nude mice (10)⁷cells / inoculum) in untreated (n = 18), mAb-treated control isotype (n = 10) or anti-ABCB5 mAb-treated animals (n = 11). Figure 4 (c) shows the immunohistochemistry ABCB5 (left panel) and conventional histology (H&E) (right panel) of human melanoma xenografts in nude mice. (Panels show adjacent sections.) ABCB5 regions⁺ are sorted with non-melanized areas (to the left of the central dotted line), while ABCB5^- correlate with regions representing a particular brown-black melanization (to the right of the central dotted line). Fig. 4 (d) shows flow cytometry analysis (FITC, Fl1) for surface-bound antibody in melanoma xenografts, 1 day after intraperitoneal administration of anti-ABCB5 mAb (solid line) and mAb isotype control (shaded). Representative melanoma xenograft isolated from anti-ABCB5 mAb treated mouse showed 20.5% positivity compared to that obtained from an animal treated with isotype control. FIG. 4 (e) summarizes the assessment of cell mediated antibody-dependent cytotoxicity (ADCC) by two-color flow cytometry in targeted anti-ABCB5 mAb or mAb melanoma cell cultures treated with isotype control or untreated DiO-labeled, stained with propidium iodide (PI) after 24h co-culture with unlabeled immune effector cells obtained from the spleens of Balb / c nude mice (target to effector ratio 1:40). (Left panels) is a series of representative results of two-color ADCC flow cytometry with lysed DIO target cells⁺PI⁺ located in the upper right target / effector quadrants of anti-ABCB5 mAb-treated (top), mAb-treated control isotype (middle) or Abused (bottom) cultures. (Right panel) presents the ADCC analysis (% mean ± SEM) in n = 6 copies of the experiment in treatment groups as shown above ([ADCC (%) = (percentage of DIO sample positive)⁺PI⁺) - (average positive percentage of Ab-untreated DIO sample⁺PI⁺).

Fig. 5 summarizes the characteristics of non-segregated human ABCB5 melanoma cells⁺ or ABCB5^- before xenograft. Fig. 5 (a) shows representative surface expression of ABCB5 in flow cytometry or control staining (FITC, Fl1) as a function of forward scatter (FSC) determined

-9 in unsorted human A375 melanoma cell cultures. Fig. 5 (b) shows an analysis of representative single-color flow cytometry on cell survival for non-split human melanoma cells (left panels), ABCB5⁺ (middle panels) and ABCB5^- (right panels) as determined by cellular incorporation and enzymatic activation of the calcein-AM fluorescent dye. Top panels show calcein-AM samples, bottom panels control without calcein-AM. Viable cells were found in R1 gates of FSC vs Fl2 plots. Fig. 5 (c) is a graph depicting the expression of ABCB5 human melanoma cells in unsected and purified ABCB5⁺ or ABCB5⁺- deleted (ABCB5^-) G3361.

Fig. 6 is a graph summarizing the analysis of the correlation of relative ABCB5 gene expression with times of doubling melanoma cell culture. Pearson correlation relative ABCB5 gene expression determined by real-time RT-PCR (mean ± SD, n = 3 independent experiments) and culture doubling times of 10 melanoma cell lines (1, LOX IMVI; 2, SK-MEL-5; 3, M14; 4, A375; 5, G3361; 6, UACC-62; 7, SK-MEL-28; 8, UACC-257; 9, SK-MEL-2; 10, MALME-3M); r is the Pearson correlation coefficient.

Fig. 7 is a gel showing cDNA strips that were obtained from heavy chain (HC) variable regions (VR) and RNA light chain (LC) by reverse transcription. Both PCR HC and LC, VR products were cloned into the Invitrogen pCR2.1 sequence vector and transformed into TOP10 cells.

Fig. 8 is the amino acid sequence of HC VR of the 3C2-1D12 antibody.

Fig. 9 is the nucleotide sequence of HC VR of the 3C2-1D12 antibody.

Fig. 10 is the amino acid sequence of LC VR of the 3C2-1D12 antibody.

Fig. 11 is the nucleotide sequence of LC VR of the 3C2-1D12 antibody.

Figure 12 is the full length nucleotide sequence of the 3C2-1D12 antibody heavy chain.

Figure 13 is the full length nucleotide sequence of the 3C2-1D12 antibody light chain.

DETAILED DESCRIPTION [0031] Tumor initiating cells capable of self-reproducing and differentiating that are responsible for tumor growth have been identified in human hematological malignancies and solid tumors. If such minority populations are associated with tumor progression in human patients, specific targeting of tumor initiating cells may provide novel strategies for the eradication of cancers currently resistant to systemic therapy. A subpopulation enriched with human tumor initiators determined by the expression of the chemo-resistant ABCB5 mediator has been

-10zidentyfikowane. As shown in the examples below, the specific targeting of this carcinogenic minority population frogs tumor growth.

[0032] The inventors have recently cloned and characterized ABCB5, a novel multi-drug resistance transporter that has been shown to be preferentially expressed by cells from melanocytic lines. Inhibition of ABCB5 makes normally resistant melanoma cells susceptible to doxorubicin. We have demonstrated that ABCB5 expression 1) means carcinogenic melanoma cells with phenotype and stem cell function; and 2) specific targeting of the ABCB5 + melanoma stem cell compartment represents a novel, highly promising approach for targeting stem cells in melanoma therapy. The data is described in more detail in the examples chapter.

[0033] In addition, in serial human to mouse xenograft experiments, ABCB5 + melanoma cells were more carcinogenic than populations with the majority of ABCB5-. Furthermore, following the genetic fate of the cells in vivo showed that ABCB5 + and ABCB5- offspring were able to generate ABCB5 + offspring, while ABCB5- cells only gave offspring to ABCB5- offspring. This identification of the specific relationship between the mechanism of chemo-resistance and cancer stem cells in human malignancy has important implications for stem cell targeting approaches in cancer therapy.

[0034] It has also been discovered that ABCB5 + melanoma cell ablation by immunotherapeutic targeting approaches can present a new strategy to achieve more stable clinical responses than those obtained by therapeutic strategies directed predominantly at the dominant tumor cell population. Therefore, we examined whether the selective ablation of chemo-resistant, carcinogenic human ABCB5 + melanoma stem cells by systemic administration of anti-ABCB5 monoclonal antibody (clone mAb 3C2-1 D 12) allows inhibition of tumor formation / tumor eradication in an appropriate preclinical human melanoma animal model Plasmatic involving human xenograft tumor to nude mice.

[0035] As set out in more detail below, we examined the bioavailability and efficacy / specificity of melanoma binding of in vivo anti-ABCB5 mAbs administered in a human to mouse xenograft model. To investigate whether administration of anti-ABCB5 mAb resulted in in vivo detectable serum levels, mouse sera were incubated with freshly harvested human melanoma cell cultures followed by counterstaining of goat anti-mouse Ig FITC-conjugated secondary Ig followed by single-color cytometry analysis flow. Significant binding of FITC-conjugated goat anti-mouse Ig secondary to FITC was observed with these melanoma cultures pre-incubated with sera at all tested dilutions obtained from anti-ABCB5 mAb- treated mice. Binding was not observed with sera obtained from animals treated with isotype control or untreated. Detection of 5.4% ABCB5 positive at

Serum dilutions at 1: 100 (Figure 1A) were consistent with previously reported ABCB5 + cell frequency among in vitro cultured G3361 melanoma cells (Frank, NY et al. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res 65 , 4320-33 (2005); Frank, NY et al. Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette transporter. J Biol Chem 278, 47156-65 (2003)). These findings demonstrate that systemic administration of anti-ABCB5 mAbs results in effective serum mAb levels in vivo. The data presented here further demonstrate that systemic administration of anti-ABCb5 mAbs efficiently and preferentially binds ABCB5 + xenografts to human melanoma cells in vivo, providing evidence of its suitability for targeted therapeutic in vivo approaches. Using human melanoma cell xenografts to nude mice, it was demonstrated that specific targeting of the ABCB5 + melanoma stem cell compartment with antibodies was an effective approach based on stem cell targeting in melanoma therapy.

[0036] The invention is based in part on the discovery, isolation and characterization of ABCB5 binding molecules, such as human monoclonal antibodies, which bind to ABCB5 and are useful in the treatment of cancer. ABCB5 is a multi-drug resistance transporter that is present in cancer stem cells.

[0037] In this connection, the compositions described herein may be useful in the treatment of an individual having or having a risk of developing cancer. The subject should be a human or vertebrate animal, including but not limited to a dog, cat, horse, goat, and primate, e.g. monkey. Accordingly, the compositions described herein can also be used to treat diseases or ailments in non-human subjects. For example, cancer is one of the dominant causes of death in pets (i.e. cats and dogs). Preferably the individual is human.

[0038] As used herein, the term treat, treat or treat when used to refer to a disorder such as cancer refers to prophylactic treatment that increases the subject's resistance to the development of the disease or, in other words, reduces the likelihood that the subject will develop disease, as well as treatment after the subject has contracted the disease to combat the disease, preventing the disease from getting worse, or slowing the progression of the disease compared to the lack of therapy.

[0039] A subject at risk of developing a cancer is one who has a high probability of developing cancer. Such individuals include, for example, individuals having a genetic disorder whose presence has been shown to correlate with a higher likelihood of developing cancer, and individuals exposed to agents that cause cancer such as tobacco, asbestos or other chemical toxins, or an individual who has previously been treated for cancer and is in visible remission. An individual at risk of developing cancer includes an individual having precancerous lesions. A precancerous lesion is an area of ​​tissue that has changed properties and carries the risk of becoming a cancer of the skin. Pre-cancerous lesions can be caused by, for example, UV radiation,

Genetics, exposure to carcinogens such as arsenic, tar or X-rays.

[0040] A subject having a cancer is an individual who has detectable cancer cells. The cancer may be malignant or benign. Tumors or tumors include, but are not limited to bile duct cancer, brain cancer; breast cancer; cervical cancer; choriocarcinoma; colorectal cancer; endometrial cancer; esophageal cancer; stomach cancer; endothelial cancers; lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell carcinoma); melanoma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer as well as other cancers and sarcomas. Preferably the cancer comprises tumor stem cells expressing ABCB5.

[0041] Optionally, prior to treatment, the presence of ABCB5 positive stem cells can be detected using the binding molecules described herein. Provided detection or diagnostic methods generally include contacting one or more molecules with a sample in or with a subject. Preferably, the sample is first taken from the subject, although in vivo detection methods are also provided. The sample may include any body tissue or fluid that is expected to be a hotbed of cancer stem cells. For example, stem cells are commonly found at or near tumor mass.

[0042] Disclosed herein is the binding of molecules such as peptides, antibodies, antibody fragments, and small molecules. The molecules bind to ABCB5 and increase tumor killing. Binding molecules are referred to herein as isolated molecules that selectively bind to ABCB5. It is understood that such antibodies are able to bind ABCB5 regardless of its source. Consequently, antibodies that are defined as binding to, for example, ABCB5 melanoma cells and capable of detecting and / or enhancing anti-tumor effects in, for example, melanoma cells as well as in other cancers such as breast cancer.

[0043] Although not wanting to be limited by any particular theory, it is believed that the treatment of tumors and tumors may fail because carcinogenic stem cells are not effectively targeted by conventional treatments. ABCB5 binding molecules specifically target and are involved in the destruction of these cells. Therefore, when these molecules are used alone or in combination with conventional therapies, the most aggressive tumor cells can be killed.

[0044] There are many possible mechanisms by which treatment of anti-ABCB5 mAbs can inhibit in vivo tumorigenic growth and tumor survival from human melanoma xenografts in this nude mouse model, including antibody mediated cell mediated cytotoxicity (ADCC), complement mediated cytotoxicity ( CDC) or antibody-mediated macrophage mediated cytotoxicity (ABMC) and / or inhibition or function of ABCB5, which may contribute to

-13 stem cell carcinogenicity. Any of these mechanisms are predicted to target only subsets of tumor cells expressing ABCB5 compared to controls. We also expect therapeutic anti-ABCB5 mediated mAb targeting ABCB5 + melanoma stem cells by chemosensitization - or immunotoxin mediated cell ablation strategies. Because targeted ABCB5 delivery of toxins (chemical or biological toxins, radionuclides) or ABCB5 mAb conjugated siRNA towards additional tumor stem cell specific gene targets may require cellular toxin internalization, we also examined cell internalization of anti-ABCB5 mAb after surface binding to human ABCB5 + melanoma cells . The results indicate that anti-ABCB5 mAb conjugated toxins can be specifically delivered to intracellular compartments in chemically resistant human ABCB5 + melanoma cells, highlighting the therapeutic benefit of this novel approach to the clinical treatment of melanoma and other cancers.

[0045] A molecule that selectively binds to ABCB5 as used herein refers to a molecule, e.g., a small molecule, peptide, antibody, fragment that interacts with ABCB5 and optionally interferes with ABCB5 activity. In some embodiments, the molecules are peptides.

[0046] The peptides minimally contain regions that bind to ABCB5. ABCB5 binding regions in some embodiments are derived from ABCB5 antibody binding regions, or alternatively, they are functionally equivalent variants of such regions. Consequently, two particularly important classes of antibody-binding ABCB5 binding regions are the variable regions and CDRs of the antibodies described herein. CDRs and nucleic acids of variable regions can be cloned from antibody-producing cells or synthetically prepared based on the sequences described herein.

[0047] The term "antibody" is used herein in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments, as long as they exhibit desired biological activity and similar molecules to antibodies such as scFv. The native antibody usually refers to heterotetrameric glycoproteins consisting of two identical light (L) chains and two identical heavy (H) chains. Each heavy and light chain has regularly separated intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the light chain constant domain is set with the first heavy chain constant domain and the light chain variable domain is set with the heavy chain variable domain. Specific amino acid residues are believed to form a link between light and heavy chain variable domains.

[0048] Certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each specific antibody for its particular antigen. However, the variability is not evenly distributed between antibody variable domains. It is concentrated in three or four segments called "complementarity determining regions" (CDRs) or "hypervariable regions" in both light and heavy chain variable domains. Stronger parts of variable domains are called framework (FR). The variable domains of native heavy and light chains contain four or five FR regions each, largely adopting the β-card configuration, connected by CDRs, which form loops connecting and in some cases forming part of the β-card structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the second chain, contribute to the formation of the antigen binding site of the antibodies (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647 -669 (1991)). Constant domains are not necessarily directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity.

[0049] The hypervariable region or CDR as used herein defines a subregion within the variable region of extreme antibody sequence variation that form the antigen binding site and are the main determinants of antigen specificity. According to one definition, they can be residues (Kabat naming) 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region and residues (naming Kabat 31-35 (H1), 5065 ( H2), 95-102 (H3) in the heavy chain variable region. Kabat et al., Sequences of Proteins of Immunological Interest, ed. 5 Public Health Service, National Institute of Health, Bethesda, Md. [1991]).

[0050] An "intact" antibody is one that comprises an antigen binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)) . However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically paired to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ') 2 fragments can be isolated directly from recombinant host cell culture.

[0051] "Antibody fragments" comprise a portion of an intact antibody, preferably an antigen binding or variable region of the intact antibody. Examples of fragments

Antibodies include Fab, Fab ', F (ab') 2 and Fv fragments; bifunctional antibodies; single chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site and a residual "Fc" fragment whose name reflects its ability to easily crystallize. Pepsin treatment provides an F (ab ') 2 fragment that has two antigen-binding sites and is still able to cross-link antigen.

[0052] "Fv" is the minimal antibody fragment that contains the complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain in tight non-covalent association. It is in this configuration that three CDRs from each variable domain interact to determine the antigen binding site on the surface of the VH-VL dimer. Together, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0053] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab 'in which the cysteine ​​residue (s) from the constant domains contains a free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0054] The term "Fc region" is used to define an immunoglobulin C-terminal region of a heavy chain that can be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or an Fc variable region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined to extend from the amino acid residue at approximately the Cys226 position or from about the Pro230 position to the carboxyl terminus of the Fc region. An immunoglobulin Fc region typically contains two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. By "Fc region chain" one or two polypeptide chains of the Fc region are considered herein.

[0055] "Hinge region" and its variations, as used herein, include the meaning known in the art, which is illustrated in, for example, Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th edition, 1999) [0056] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and many of them can be further divided

Into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of heavy chains that correspond to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0057] The "light chains" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one or two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains.

[0058] Preferably, ABCB5 binding peptides minimally contain at least one CDR of those described herein or those that can be obtained from the sequences described herein. As used herein, ABCB5 binding CDR is the CDR described herein. The ABCB5 binding region may be CDC1 binding ABCB5, ABCB5 binding CDR2 or ABCB5 binding CDR3, all of which are obtained from the antibodies and antibody variable chains disclosed herein.

[0059] As used herein, "ABCB5 binding CDR1" is CDR1, which binds, preferably specifically, to ABCB5 and is obtained from variable regions from the heavy or light chain of the antibodies described herein. It may contain an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO .: 6. "ABCB5 binding CDR2" is a CDR2 that binds, preferably specifically, to ABCB5 and is obtained from variable regions from the heavy or light chain of the antibodies described herein. It may contain an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO .: 7. "ABCB5 binding CDR3" is a CDR3 which binds, preferably specifically, to ABCB5 and is obtained from heavy variable regions, or from the light chain of antibodies described herein. It may contain an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO .: 8.

[0060] In addition to the sequences listed herein, there are functionally equivalent equivalent variants of these sequences, including conservative substitution variants in the amino acid sequence or nucleotide sequence as described in more detail below.

[0061] Peptides are useful inter alia in diagnostic methods for detecting, in a sample or in a subject, ABCB5 antigen or ABCB5 expressing cells. Minimally, peptides useful in these methods must only recognize and bind to ABCB5 regardless of whether they also increase tumor killing. Antibodies can be used, for example, in FACS diagnostic analysis, Western blotting and immunohistochemistry. Such antibodies can also be used for in vivo diagnostic applications where mAb conjugated can be used to assess tumor load, tumor location or tumor residual mass after chemotherapy or surgery for ABCB5 expressing tumors. In important embodiments, the antibodies and fragments thereof selectively bind to ABCB5. In some embodiments, they contain one or more CDRs derived from the antibody clones described herein. In preferred embodiments, the peptides comprise ABCB5 binding CDR3 and even more preferably, the peptides comprise heavy chain ABCB5 binding CDR3.

It is understood that not all of the CDRs are required for effective binding to ABCB5.

However, in some embodiments, the peptides contain all of the CDRs of a given antibody clone disclosed herein.

[0062] In addition, it should be understood that CDR exchange between the variable regions described herein is enabled. Preferably, the heavy chain CDR is exchanged with another heavy chain variable region CDR and similarly, the light chain CDR is exchanged with another light chain variable region CDR.

[0063] The peptides may also contain an ABCB5 binding variable region. The variable region binding ABCB5 is a variable region (preferably an antibody variable region as described herein). SEQ ID NO: 1 corresponds to the amino acid sequences of the heavy chain variable region. SEQ ID NO: 9 corresponds to the nucleotide sequence of the heavy chain variable region. SEQ ID NO: 2 corresponds to the amino acid sequences of the light chain variable region. SEQ ID NO: 10 corresponds to the nucleotide sequence of the light chain variable region.

[0064] It is understood that the nucleic acids or peptides may be derived from the sequence provided herein. These sequences can be cloned (e.g., by PCR) and introduced into the vector and / or cells to produce peptides corresponding to full-length variable regions or fragments of full-length variable regions, and antibodies containing variable regions. Therefore, it is possible to produce antibodies or fragments thereof that contain a combination of light and heavy chain variable regions.

[0065] The compositions and methods are intended to capture the antibody and antibody fragments of different isotypes. The antibody may be IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM, IgA1, IgA2 or sIgA isotypes. Compositions and methods to capture isotypes found in non-human species as well as, but not limited to, IgY in birds and sharks. Vectors encoding constant regions of different isotypes are known and described previously. (See, for example, Coloma et al. Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. J Immunol Methods. 1992 Jul 31; 152 (1): 89-104; Guttieri et al. Cassette vectors for conversion of Fab fragments into full-length human IgG1 monoclonal antibodies by expression in stably transformed insect cells. Hybrid Hybridomics. 2003 Jun; 22 (3): 135-45; McLean et al. Human and murine immunoglobulin expression vector cassettes. Mol Immunol. 2000 Oct; 37 (14): 837-45; Walls et al. Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions. Nucleic Acids Res. 1993 Jun 25; 21 (12): 2921-9; Norderhaug et al. Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells. J Immunol Methods. 1997 May 12; 204 (1): 77-87.) [0066] These peptides are isolated peptides. As used herein, the term "isolated peptides" means that the peptides are substantially pure and are substantially free of

- other substances with which they may be found in nature or in vivo in a practical and appropriate way for their intended use. In particular, the peptides are sufficiently pure and sufficiently free from other biological components of their host cells to be useful in, for example, pharmaceutical preparation or sequencing. Since the isolated peptide can be mixed with a pharmaceutically acceptable carrier in a pharmaceutical formulation, the peptide may contain only a small percentage by weight of the formulation. The peptide is nevertheless substantially pure in the sense that it has been substantially separated from the substances with which it can be associated in living systems.

[0067] The peptides bind to ABCB5, preferably in a selective manner. As used herein, the terms "selective binding" and "specific binding" are used interchangeably to refer to the ability of a peptide to bind with greater affinity to ABCB5 and to fragments thereof than to non-ABCB5 compounds. That is, peptides that bind selectively to ABCB5 will not bind to non-ABCB5-derived compounds to the same extent and with the same affinity as they bind to ABCB5 and its fragments, except for the cross-reactivity of antigens or molecules produced to mimic ABCB5. such as carbohydrate peptide mimetics or anti-idiotypic antibody variable regions that bind to ABCB5 binding peptides in the same way as ABCB5. In some embodiments, the peptides bind only to ABCB5 and fragments thereof. As used herein, a binding peptide that binds selectively or specifically to an ABCB5 tumor cell can also bind ABCB5 from other sources and will bind with less affinity (if at all) to non-ABCB5 derived compounds. Lower affinity may include at least 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or 95% less.

[0068] "Isolated antibodies" as used herein refer to antibodies that are substantially physically isolated from residual cellular material (e.g., separated from cells that produce antibodies) or from other material that impairs their use in diagnostic or therapeutic methods. described here. Preferably, the isolated antibodies are present in a homogeneous population of antibodies (e.g., monoclonal antibody populations). Isolated antibody compositions may, however, be combined with other components such as, but not limited to, pharmaceutically acceptable carriers, adjuvants and the like.

[0069] "Isolated antibody producing cells" including isolated hybridomas and isolated recombinant cells (such as those described herein), as used herein, refers to antibody producing cells that are substantially physically separated from other cells, other body material (e.g. ascites, tissues and fluid) and other material that impedes their use in the production of, for example, an isolated and preferably homogeneous population of antibodies.

[0070] Therefore, in one embodiment, the peptide is an isolated, intact monoclonal antibody specific for ABCB5. As used here,

The term "monoclonal antibody" refers to a homogeneous population of immunoglobulins that specifically bind to an identical epitope (ie, an antigenic determinant). The peptide in one embodiment is, for example, a monoclonal antibody having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 2. Monoclonal antibodies having any combination of light chain variable regions and heavy chains are included herein.

[0071] Antibodies other than, for example, 3C2-1D12 sequences are described as long as such antibodies have the binding characteristics of the monoclonal antibodies described herein. Optionally, these additional antibodies also increase tumor killing of ABCB5-expressing tumor cells. One skilled in the art can readily recognize antibodies having functional characteristics of this monoclonal antibody using the screening and binding assays described in detail hereinafter.

[0072] Unless otherwise indicated, the term "3C2-1D12 monoclonal antibody" or "mAb3C2-1D12" refers to an antibody that has antigen-binding residues from or obtained from murine antibodies and 3C2-1D12.

[0073] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, separate antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma cultures not contaminated by other immunoglobulins. "Monoclonal" modifiers indicate the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be interpreted as requiring the production of the antibody in any particular manner.

[0074] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the separate antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Accordingly, "monoclonal" modifiers indicate the nature of antibodies as not being a mixture of separate antibodies.

[0075] For example, monoclonal antibodies can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975) or can be made by recombinant DNA methods (US Pat. No. 4,816,567).

[0076] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used.

-20 for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then combined with myeloma cells using a suitable binding agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0077] The hybridoma cells prepared in this way are seeded and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-linked parent myeloma cells. For example, if the myeloma parent cells do not contain the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the hybridoma culture medium will typically contain hypoxanthine, aminopterin and thymidine (HAT medium), which prevent the growth of non-HGPRT containing cells.

[0078] Preferred myeloma cells are those that fuse efficiently, support high level stable antibody production by selected antibody producing cells, and are sensitive to a medium such as HAT medium. Among them, preferred myeloma cell lines are mouse myeloma lines, such as those derived from the MOPC-21 and MPC-11 mouse tumors available at the Salk Institute Cell Distribution Center, San Diego, Calif. USA and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and murine-human heterozymoma cell lines have also been described in the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0079] Culture medium in which hybridoma cells are growing is assayed for the production of anti-antigen monoclonal antibodies. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as a radioimmunoassay (RIA) or enzyme immunoassay (ELISA).

[0080] The binding affinity of the monoclonal antibody can, for example, be determined by Scatchard analysis according to Munson et al., Anal. Biochem., 107: 220 (1980).

[0081] Once the hybridoma cells that produce the antibodies with the desired specificity, affinity and / or activity have been identified, the clones can be subcloned by limiting dilution procedures and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo as ascites tumors in an animal.

[0082] Monoclonal antibodies secreted by subclones are appropriately separated from culture medium, ascites fluid or serum by conventional antibody purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0083] DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (e.g., with oligonucleotide probes that are capable of specifically binding to genes encoding heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be inserted into expression vectors, which are then transfected into host cells such as E cells. coli, monkey COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that otherwise do not produce antibody protein to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on the recombinant expression of bacterial DNA encoding antibodies include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151188 (1992).

[0084] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describing the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity human antibodies (nM range) by means of chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combination infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)). Therefore, these techniques are realistic alternatives to traditional hybridoma monoclonal antibody techniques for isolating monoclonal antibodies.

[0085] Typically, such non-immunoglobulin polypeptides are substituted into antibody constant domains or are substituted into variable domains of one antigen-binding antibody site to produce a chimeric bivalent antibody containing one antigen-binding site having antigen specificity and another antigen-connecting site having other antigen specificity. .

[0086] In some embodiments, the peptide is an antibody fragment. As is well known in the art, only a small portion of the antibody molecule, paratope, is involved in binding the antibody to its epitope (see, in general, Clark, WR (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th ed., Blackwell Scientific Publications, Oxford; and Pier GB, Lyczak JB, Wetzler LM, (eds). Immunology, Infection and Immunity (2004) ed. 1 American Society for Microbiology Press, Washington DC). Antibody pFc 'and Fc regions, for example, are complement cascade effectors and may mediate Fc receptor binding on phagocytic cells, but are not involved in antigen binding. The antibody from which the pFc 'region has been enzymatically cleaved or which was produced without the pFc' region, designated the F (ab ') 2 fragment, retains both antigen binding sites from the intact antibody. The isolated F (ab ') 2 fragment is referred to as a bivalent monoclonal fragment because of its two binding sites

-22antygen. Similarly, an antibody from which the Fc region has been enzymatically cleaved or that has been produced without the Fc region of the Fab fragment being fragmented retains one of the antigen binding sites from the intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain designated Fd (heavy chain variable region). Fd fragments are a significant determinant of antibody specificity (a single Fd fragment can be associated with up to ten different light chains without altering antigen specificity) and Fd fragments retain their ability to bind epitope in isolation.

[0087] The terms Fab, Fc, pFc ', F (ab') 2 and Fv are used with standard immunological meanings [Klein, Immunology (John Wiley, New York, NY, 1982); Clark, WR (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, ed. 7, (Blackwell Scientific Publications, Oxford); and Pier GB, Lyczak JB, Wetzler LM, (eds). Immunology, Infection and Immunity (2004) ed. 1 American Society for Microbiology Press, Washington DC].

[0088] In other embodiments, the Fc portions of the antibodies can be replaced to produce IgM as well as human IgG antibodies comprising some or all of the CDRs from the monoclonal antibodies described herein. It is particularly important to include the ABCB5 binding CDR3 region and, to a lesser extent, other CDRs and parts of the framework regions of the monoclonal antibodies described herein. Such human antibodies will have particular clinical utility such that they will recognize and bind, preferably selectively, to ABCB5, but will not elicit an immune response from humans against the antibody itself.

[0089] It is intended to include functionally equivalent variants of ABCB5 binding peptides. A "functionally equivalent variant" is a compound having the same functionality (ie, ABCB5 binding capacity) as peptides. A functionally equivalent variant may be a peptide in nature, but is not so limited. For example, it may be a hydrocarbon, peptidomimetic, etc. In important embodiments, the functionally equivalent variant is a peptide having the variable region or CDR amino acid sequence with conservative substitutions there that is still capable of binding to ABCB5. An example of a functionally equivalent CDR3 variant binding ABCB5 from a heavy chain variable region (i.e. SEQ ID NO: 1) is a peptide having conservative substitutions in SEQ ID NO: 1 that bind, preferably specifically, to ABCB5 and optionally that increases tumor killing of ABCB5 expressing cells.

[0090] The term "amino acid sequence variant" refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Amino acid sequence variants contain substitutions, deletions and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence.

[0091] "Homology" is defined as the percentage of residues in an amino acid sequence variant that are identical when the sequence is set and the breaks introduced, provided that

-23 necessary to achieve the maximum percentage of homology. Methods and computer programs for setting are well known in the art.

[0092] Modification of the amino acid sequence of the antibodies described herein is predicted. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants from antibodies a are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions to and / or substitution of residues within the amino acid sequence of antibodies a. Any combination of deletion, insertion and substitution is performed to obtain the final construct, as long as the final construct contains the desired characteristics. Amino acid changes can be introduced into the substantive amino acid sequence at the time this sequence is produced.

[0093] A useful method for identifying certain residues or regions of the antibody that are preferred mutagenesis sites is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. Here, the residue or group of target residues are recognized (e.g. charge-containing residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. These amino acid positions demonstrating functional sensitivity to substitutions are then refined by introducing further or other variants at or into substitution sites. Therefore, although the sites for introducing amino acid sequence variations are predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze mutation efficiency at a given site, ala scanning or random mutagenesis at a target codon or region is performed, and expressed immunoglobulins are screened for the desired activity.

[0094] Insertions of the amino acid sequence contain amino- and / or carboxy-terminal fusions covering the length from one residue to polypeptides containing a hundred or more residues, as well as insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody linked to a cytotoxic polypeptide. Other inserted variants of the antibody molecule include fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or polypeptide which increases the serum half-life of the antibody.

[0095] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by another residue. Sites of greatest substitution mutagenesis interest include hypervariable regions, but FR changes are also predicted.

[0096] As used herein, "conservative substitution" refers to an amino acid substitution that does not change the relative characteristics of the charge or size of the peptide in which the amino acid substitution is carried out. Conservative substitutions

-24 amino acids include substitutions made among amino acids with the following groups: (1) M, I, L, V; (2) F, Y, W; (3) K, R, H; (4) A, G; (5) S, T; (6) Q, N; and (7) E, D.

[0097] Significant modifications in the biological properties of the antibody are achieved by choosing a substitution that differs significantly in its effect on (a) the polypeptide core structure in the area of ​​substitution, for example, as a conformation of a sheet or helical, (b) the charge or hydrophobicity of the molecule target site or (c) side chain size. Amino acids can be grouped according to similarities in the properties of their side chains (in AL Lehninger, in Biochemistry, second ed., Pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) acid: Asp (D), Glu (E) (4 ) basic: Lys (K), Arg (R), His (H) [0098] Alternatively, naturally occurring residues can be divided into groups based on the common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0099] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues may also be introduced into conservative substitution sites or, more preferably, into other (non-preserved) sites.

[0100] One type of substitution variant includes substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant (s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, many hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus produced are expressed from the filamentous phage molecules as fusions into the M13 gene III product packaged within each molecule. Phage display variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To recognize potential hypervariable region sites for modification, alanine scanning mutagenesis may be performed to recognize hypervariable region residues contributing significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to recognize contact points between

-25 antibody and antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques developed herein. When such variants are generated, the variant panel is screened as described herein and antibodies with better properties in one or more appropriate tests can be selected for further development.

[0101] Nucleic acid molecules encoding antibody amino acid sequence variants are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared variant or a non-variant version of the antibody .

[0102] It may be desirable to introduce one or more amino acid modifications in the Fc region of immunoglobulin polypeptides, thereby producing a variant of the Fc region. A variant Fc region may comprise a sequence of a human Fc region (e.g., human Fc region IgG1, IgG2, IgG3 or IgG4) containing an amino acid modification (e.g. substitution) at one or more amino acid positions, including hinge cysteine.

[0103] In accordance with this description and the prior art, it is envisioned that in some embodiments, the antibody used in the methods described herein may contain one or more lesions compared to the wild type counterpart of the antibody, e.g., in the Fc region, in addition to the hinge mutation described here. Nevertheless, these antibodies would retain essentially the same characteristics required for therapeutic utility as compared to their wild-type counterpart. Eg. it is believed that some changes in the Fc region can be made that would result in altered (i.e., improved or reduced) C1q binding and / or Complement-Dependent Cytotoxicity (CDC), e.g. as described in WO99 / ​​51642. See also Duncan & Winter Nature 322: 738-40 (1988); US Pat. Well. 5,648,260; US Pat. Well. 5,624,821; and WO94 / 29351 regarding other examples of Fc region variants.

[0104] Any cysteine ​​residue not involved in maintaining the appropriate conformation of the anti-ABCB5 antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bond (s) may be added to the antibody to improve its stability (especially where the antibody is an antibody fragment such as an Fv fragment).

[0105] Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. The change is considered to be the deletion of one or more carbohydrate moieties present in the antibody and / or the addition of one or more glycosylation sites that are not present in the antibody, the glycosylation of the antibodies is typically N-linked, or O-linked. The N-connection refers to the attachment of the moiety

Carbohydrate to the side chain of asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Accordingly, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of Nacetylgalactosamine, galactose or xylose sugars to hydroxyamic acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0106] Addition of glycosylation sites to the antibody is conveniently achieved by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The change can also be accomplished by addition or substitution with one or more serine or threonine residues to the primary antibody sequence (for O-linked glycosylation sites).

[0107] Functional equivalence refers to equivalent activity (e.g., binding to ABCB5 or enhancing the killing of cells expressing ABCB5), but also includes variation in the level of such activity. For example, a functional equivalent is a variant that binds to ABCB5 with less, equal or greater affinity than the monoclonal antibody clones described herein, as long as the variant is still useful (i.e., binds to ABCB5 and optionally increases tumor killing).

[0108] Such substitutions can be carried out by a number of methods known to a person skilled in the art. For example, amino acid substitutions can be performed by PCR-directed mutation, site-directed mutagenesis according to the Kunkel method (Kunkel, Proc. Nat. Acad. Sci. USA 82: 488-492, 1985), or by chemical synthesis of a coding gene specific CDRs or a peptide comprising the CDR amino acid sequences described herein. These and other methods for altering a CDR-containing peptide will be known to those skilled in the art and can be found in sources that list such methods, e.g., Sambrook or Ausubel, mentioned above. However, in some embodiments, due to the size of the CDRs, it may be more convenient to synthesize peptide variants using a peptide synthesizer such as those commercially available. The activity of functionally equivalent ABCB5 binding CDR variants can be tested by binding assays and in some cases biological assays, discussed in more detail below. As used herein, the term "functional variant"; "Functionally equivalent variant" and "functionally active variant" are used interchangeably.

[0109] As used herein, the term "functionally active antibody fragment" means a fragment of the antibody molecule comprising an ABCB5 binding region that retains the ability to bind ABCB5, respectively, preferably in a specific manner. Such fragments can be used both in vitro and in vivo. In particular, well-known functionally active antibody fragments include, but are not limited to, F (ab ') 2, Fab, Fv and Fd fragments of antibodies. These fragments do not contain the Fc fragment

Intact antibodies, are removed more rapidly from the circulation and may show less non-specific tissue binding than intact antibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). As another example, single chain antibodies can be made according to the methods described in US Patent No. 4,946,778 for Ladner et al. Such single chain antibodies contain the variable region of the light and heavy chains linked by a flexible linker moiety. Methods for obtaining single-domain ("Fd") antibodies that contain an isolated heavy chain single variable domain have also been described (see, for example, Ward et al., Nature 341: 644-646 (1989), disclosing a screening method for variable region recognition heavy chain antibody (VH single domain antibody) with sufficient affinity for its target epitope to bind to it in isolated form). Methods for producing recombinant Fv fragments based on known sequences of heavy chain variable regions and light chains are known in the art and have been described, e.g., Moore et al., US Patent No. 4,462,334. Other sources describing the use and production of antibody fragments include, e.g. Fab fragments (Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)), Fv fragments (Hochman et al., Biochemistry 12: 1130 (1973); Sharon et al., Biochemistry 15: 1591 (1976); Ehrlich et al., US Patent No. 4,355,023) and parts of antibody molecules (AudiloreHargreaves, US Patent No. 4,470,925). Accordingly, one of skill in the art can prepare antibody fragments from various parts of intact antibodies without destroying the specificity of the antibodies to ABCB5.

[0110] In important disclosures herein, a functionally active antibody fragment also retains the ability to enhance the killing of ABCB5 expressing cells. In the latter case, the antibody fragment contains the Fc region as well as the epitope binding domain. The Fc region allows the antibody fragment to bind to Fc receptor positive cells, which then phagocyte epitope bound by the antibody Fab region.

[0111] Anti-ABCB5 peptides may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2, or other antigen-binding antibody subsections) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient are replaced by residues from CDRs of non-human species (donor antibody) such as a mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the human immunoglobulin Fv framework residues are replaced by the corresponding non-human residues. Humanized antibodies may also contain residues that are not found in the recipient antibody, nor in imported CDRs or framework sequences. Generally, a humanized antibody will contain substantially all of at least one, typically two, variable domains in which all or substantially all of the CDR regions correspond to those of non-human immunoglobulin and all or substantially all of the FR regions are those of the immunoglobulin consensus sequence

-28ludzkiej. The humanized antibody will optimally also contain at least a portion of the immunoglobulin (Fc) constant region, typically human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biot, 2: 593-596 (1992)].

[0112] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues that are typically derived from an "import" variable domain. Humanization can basically be performed using the Winter method and co-workers [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting the CDR or rodent CDR sequence with appropriate human antibody sequences. Consequently, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567), where substantially less than the variable domain of the intact antibody has been substituted by the appropriate sequence from non-human species. In practice, humanized antibodies are typically human antibodies in which CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0113] The choice of human variable domains, both light and heavy, for use in the production of humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the rodent antibody variable domain sequence is screened against the entire library of known human variable domain sequences. The human sequence that is closest to this rodent is accepted as a human framework (FR) for a humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a specific framework region obtained from the consensus sequence of all human antibodies with a specific subgroup of light or heavy chains. The same framework can be used for many different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).

[0114] It is further important that the antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of parental sequence analysis of various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are widely available and are known to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected potential immunoglobulin sequences. Inspection of these presentations allows the analysis of the likely role of residues in the functioning of a potential immunoglobulin sequence, i.e. the analysis of residues that affect the ability of the potential immunoglobulin to bind its antigen. In this way, FR residues can be selected and linked to the recipient sequence and

Imported such that desired antibody characteristics, such as increased affinity for the target antigen (s) are achieved. In general, the hypervariable region residues are directly and most involved in influencing antigen binding.

[0115] An exemplary humanized antibody of interest herein comprises heavy variable domain residues determining DYYMY complementarity (SEQ ID NO: 3); TINDGGTHTY (SEQ ID NO: 4); and / or DDYYYGSHFDAMDY (SEQ ID NO: 5), optionally including amino acid modifications of these CDR residues, e.g., where the modifications substantially retain or improve the affinity of the antibody. For example, the variant antibody of interest may have from about one to about seven or about five amino acid substitutions in the above variable heavy CDR sequences. Such antibody variants can also be prepared by affinity maturation.

[0116] The humanized antibody may contain variable light domains determining the complementarity of the RASKSVSTSGYSYMH residue (SEQ ID NO: 6); LVSNLES (SEQ ID NO: 7); and / or QHIRELTR (SEQ ID NO: 8), e.g. in addition to those CDR residues of the variable heavy domains in the previous paragraph. Such humanized antibodies optionally contain amino acid modifications of the above CDR residues, e.g., where the modifications substantially retain or increase the affinity of the antibody. For example, the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the above variable light CDR sequences.

[0117] The application also predicts the affinity of mature antibodies that bind ABCB5. The parent antibody may be a human antibody or a humanized antibody, e.g. one containing light and / or heavy variable sequences of SEQ ID No. 2 and 1, respectively. The affinity matured antibody preferably binds to ABCB5 with an affinity greater than that of mouse mAb3C2-1D12.

[0118] Various forms of humanized or mature affinity antibodies are envisaged. For example, a humanized antibody or an affinity matured antibody may be an antibody fragment, such as a Fab, which is optionally conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody or mature affinity antibody may be an intact antibody, such as an intact IgG1 antibody.

[0119] European Patent Application 0239400 provides exemplary instruction on the production and use of humanized monoclonal antibodies in which at least a portion of the CDR of a murine (or other non-human mammalian) antibody is contained in a humanized antibody. Briefly, the following methods are useful for preparing a humanized CDR monoclonal antibody, including at least a portion of mouse CDRs. A first replicable expression vector is prepared containing the appropriate promoter operably linked to the co-coding DNA sequence

At least the Ig heavy or light chain variable domain and variable domains containing the human antibody framework regions and the murine antibody CDR regions. Optionally, a second replicable expression vector was prepared containing the appropriate promoter operably linked to the DNA sequence encoding at least the variable domain of the complementary human Ig light or heavy chain, respectively. The cell line is then transformed with vectors. Preferably, the cell line is an immortalized mammalian cell line of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line, or is a normal lymphoid cell that has been immortalized by transformation with a virus. The transformed cell line is then cultured under conditions known to those skilled in the art to produce a humanized antibody.

[0120] As further established in European Patent Application 0239400, many techniques are well known in the art for producing specific antibody domains for insertion into a replicable vector. (Preferred vectors and recombination techniques are discussed in more detail below.) For example, the DNA sequence encoding the domain may be prepared by oligonucleotide synthesis. Alternatively, a synthetic gene that lacks CDR regions in which four framework regions are joined together with their respective junction restriction sites, such that double-stranded synthetic or restriction subcloned sticky CDR cassettes can be ligated at the junction of framework regions. Another method involves preparing a DNA sequence encoding a CDR-containing domain by site-directed oligonucleotide mutagenesis. Each of these methods is well known in the art. In this regard, those skilled in the art can prepare humanized antibodies containing the mouse CDR region without destroying the specificity of the antibody for its epitope.

[0121] As an alternative to humanization, human antibodies can be made. A "human antibody" is one that contains an amino acid sequence that corresponds to that of a man-made antibody and / or has been produced using techniques for making human antibodies. The definition of a human antibody specifically excludes a humanized antibody containing non-human antigen binding residues. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable, after immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been reported that homozygous deletion of the antibody heavy chain (JH) region linking gene in chimeric and germline mouse mutants results in complete inhibition of endogenous antibody production. Transferring an array of human germline immunoglobulin genes to such mutated germline mice will result in the production of human antibodies after any antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and US Pat. Nose. 5,591,669,, 5,589,369 and 5,545,807.

[0122] Alternatively, phage display technology (McCafferty et al., Nature 348: 552553 (1990)) can be used to produce human antibodies and

-31 antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from non-immunized donors. According to this technique, the antibody V domain genes are cloned in stroma into the large or small coat protein gene of a filamentous bacteriophage such as M13 or fd and exposed as functional antibody fragments on the surface of phage particles. Because the filamentous molecule contains a copy of single-stranded DNA from the phage genome, choices based on the functional properties of the antibody also result in the selection of the gene encoding the antibody exhibiting these properties. Therefore, the phage mimics some of the properties of the B cell. Phage display can be performed in a number of formats; for their review see e.g. Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Many sources of the V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated differentiated anti-oxazole antibody arrays from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be prepared, and antibodies to differentiated antigen arrays (including autoantigens) can be isolated essentially following the techniques described in Marks et al., J. Mol. Biol. 222: 581-597 (1991) or Griffith et al., EMBO J. 12: 725-734 (1993). See also US Pat. Well. 5,565,332 and 5,573,905. Human antibodies can also be generated using in vitro activated B cells (see US Pat. No. 5,567,610 and 5,229,275).

[0123] Human monoclonal antibodies can also be made using any of the methods known in the art, such as disclosed in US Patent No. 5, 567, 610, issued to Borrebaeck et al., US Patent No. 565, 354, issued to Ostberg, US Patent No. 5,571,893, published to Baker et al, Kozber, J. Immunol. 133: 3001 (1984), Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc, new York, 1987), and Boerner el al., J. Immunol., 147: 86-95 (1991).

[0124] The use of single chain variable region (svFv) fragments is also disclosed. Single chain variable region fragments are produced by joining light and / or heavy chain variable regions using a short linking peptide. Any peptide having sufficient flexibility and length can be used as a linker in scFv. Usually the linker is selected to have the least immunogenicity. An example of a connecting peptide are numerous GGGGS residues that form a bridge between the carboxy terminus of one variable region and the amino terminus of another variable region. Other linker sequences can also be used.

[0125] All or any part of the heavy or light chain may be used in any combination. Typically, all variable regions are included in scFv. For example, the light chain variable region can be linked to the heavy chain variable region. Alternatively, a portion of the light chain variable region can be connected to the heavy chain variable region or part thereof. ScFvs are also envisaged in which the heavy chain variable region is of antibodies of interest and the light chain variable region is of another immunoglobulin.

[0126] sFVs may be assembled in any order, for example, VH-linker-VL or VLlinker-VH. There may be a difference in the level of expression of these two configurations in specific expression systems, in which case one of these forms may be beneficial. Tandem scFvs, such as (X) -linker- (X) -linker- (X), in which X are polypeptides of the antibodies of interest, or combinations of these polypeptides with other polypeptides can also be produced. In another embodiment, single chain antibody polypeptides do not have any polypeptide linker or simply have a short, inflexible linker. Possible configurations are VL - VH and VH - VL. The connection is too short to allow interaction between VL and VH within the chain, and the chains form homodimers with the VL / VH antigen binding site at each end. Such molecules are referred to in the art as "diabodies".

[0127] Single chain variable regions can be produced recombinantly or synthetically. An automated synthesizer can be used to synthesize scFv. For recombinant production of scFv, a suitable plasmid containing a polynucleotide that encodes scFv can be introduced into a suitable host cell, eukaryotic, such as yeast, plant, insect or mammalian or prokaryotic, such as E. coli and expressed protein can be isolated using standard protein purification techniques.

[0128] The expression conditions should be such that the scFv polypeptide can adopt an optimal tertiary structure. Depending on the plasmid and host cell used, production may need to be modulated. For example, the use of a weaker promoter or expression at lower temperatures may be necessary to optimize the production of suitably folded scFv in prokaryotic systems; or it may be advantageous for scFv to be expressed in eukaryotic cells.

[0129] The term "diabodies" refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen binding sites. Diabodies are described more completely in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad Sci. USA, 90: 6444-6448 (1993).

[0130] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to the respective sequences in the antibodies obtained from specific species or belonging to a particular class or subclass of antibodies, while the remaining chain (s) is identical to or homologous to the respective antibody sequences obtained from other species or belonging to another class or subclass of antibodies, as well as with fragments of such antibodies as long as they exhibit the desired biological activity.

[0131] Bispecific antibodies are monoclonal, preferably human or humanized antibodies, which have binding specificities for at least two different antigens. In the case shown, one binding specificity is for ABCB5, the other is for any other antigen and preferably for cell surface protein or the receptor or receptor subunit. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305: 537-539 (1983)]. Due to the random selection of immunoglobulin heavy and light chains, these hybridomas (quadromes) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by means of affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

[0132] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

[0133] In addition, small peptides, including those containing the ABCB5 binding CDR3 region can be easily synthesized or produced by recombinant methods for peptide production. Such methods are well known to those skilled in the art. Peptides can be synthesized, for example, using automated peptide synthesizers that are commercially available. Peptides can be produced by recombination techniques by incorporating DNA expressing the peptide into an expression vector and transforming the cells with an expression vector to produce the peptide.

[0134] Peptides, including antibodies, can be tested for their ability to bind ABCB5 using standard binding assays known in the art. As an example of a suitable test, ABCB5 can be immobilized on the surface (such as in a well or multiwell plate) and then contacted with the labeled peptide. The amount of peptide that binds to ABCB5 (and thereby becomes immobilized on the surface itself) can be measured to determine if a particular peptide binds to ABCB5. Alternatively, the amount of unbound surface peptide can also be measured. In a variation of this assay, the peptide may be tested for its ability to bind directly to an ABCB5 expressing cell.

[0135] Peptide binding can also be tested using a competition assay. If the test peptide (including antibody) competes with the monoclonal antibodies or antibody fragments described herein, as shown by the decrease in binding of the monoclonal antibody or fragment, then it is likely that the peptide and monoclonal antibody bind to the same or at least an overlapping epitope. In this test system, the o antibody or antibody fragment is labeled and ABCB5 is immobilized on a solid surface. In this way, competing peptides, including competing antibodies, can be recognized. There are described peptides as well

In particular antibodies (and fragments thereof) that compete with the 3C2 1D12 antibody for binding to ABCB5 (i.e., antibodies that recognize and bind to the same epitopes as 3C2 1D12).

[0136] There are also disclosed small molecules that bind to ABCB5 and increase tumor killing. Such binding molecules can be recognized by conventional screening methods, such as phage display procedures (e.g. methods described in Hart et al., J. Biol. Chem. 269: 12468 (1994)). Hart et al. describe a filamentous phage expression library for recognizing novel peptide ligands. In general, phage display libraries, e.g. M13 or fd phage are prepared using conventional procedures such as those described in the source above. In libraries, inserts containing 4 to 80 amino acid residues are generally expressed. The inserts optionally represent completely degenerate or erroneous peptide sets. Ligands having appropriate binding properties are obtained by selecting those phages that express the ligand on their surface that binds to the target molecule. These phages are then subjected to multiple re-selection cycles to recognize the phage with the most useful binding characteristics that express the peptide ligand. Typically, phages that show the best binding characteristics (e.g. highest affinity) are further characterized by nucleic acid analysis for recognition of specific amino acid sequences from the expressed peptide on the surface of the phage at the optimal length of the expressed peptide to achieve optimal binding. The phage display library of a peptide or antibody is also described in Brissette R et al Curr Opin Drug Discov Devel. 2006 May; 9 (3): 363-9.

[0137] Alternatively, binding molecules can be recognized from combinatorial libraries. Many types of combinatorial libraries have been described. For example, US Patent Nos. 5,712,171 (which describes methods for preparing a matrix of synthetic molecular constructs by creating multiple molecular constructs having a scaffolding core in the form of a chemical molecule and modifying at least one place on the molecule in a logically arranged matrix); 5, 962, 412 (which describes methods for preparing polymers having specific physiochemical properties); and 5, 962, 736 (which specifically describes matrices of compounds).

[0138] Other binding molecules can be identified by those skilled in the art by following the directions described herein. Library technology can be used to recognize small molecules, including small peptides, that bind to ABCB5 and interfere with its function. One advantage of using libraries for antagonist recognition is the easy manipulation of millions of different putative candidates of small size in small reaction volumes (i.e. in synthesis and screening reactions). Another advantage of libraries is the ability to synthesize antagonists that may not otherwise be achievable using naturally occurring sources, in particular for non-peptide molecules.

[0139] Small molecule libraries can be screened for their modulating effect on the efflux transport of ABCB5-mediated rhodamine-123, from which binding to ABCB5 can be implicated. Potential substrates or inhibitors of ABCB5 function can also be recognized by correlating the ABCB5 gene or protein expression in the NCI-60 panel of tumor cell lines from the National Cancer Institute with established potentials> 100,000 compounds for these cell lines, as described in Frank et al. Cancer Research 2005 for 119 selected anticancer agents.

[0140] Many, if not all of these compounds can be synthesized using recombinant or chemical libraries. A wide range of potential compounds can be generated from libraries of synthetic or natural compounds. Libraries of natural compounds in the form of bacterial, fungal, plant or animal extracts are available or can be easily created. Naturally and artificially produced libraries and compounds can be easily modified using conventional chemical, physical and biochemical methods. In addition, compounds known to bind to and therefore act as calcium channel antagonists may undergo targeted or accidental chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogues that can act similarly or perhaps with greater specificity.

[0141] Small molecule combinatorial libraries can also be created. The combinatorial library of small organic compounds is a collection of closely related analogues that differ from each other by one or more diversity points and are synthesized by organic techniques using multi-step methods. Combinatorial libraries contain a wide number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to generate a compound matrix. A "compound matrix" as used herein is a set of compounds identifiable by their spatial addresses in Cartesian coordinates and set so that each compound has a common molecular core and one or more structural variable diversity elements. Compounds in such a matrix of compounds are produced in parallel in separate reaction vessels, with each compound recognized and localized by a spatial address. Examples of parallel synthesis of mixtures and parallel synthesis methods are provided in published PCT patent application WO95 / 18972, published July 13, 1995 and in US Patent No. 5,712,171 issued January 27, 1998 and the corresponding published PCT Patent Application WO96 / 22529.

[0142] Standard binding assays are well known in the art, and a number of them are suitable, including ELISA, competitive binding assay (as described above), sandwich assays, radioreceptor assays using radioactively labeled peptides or radiolabeled antibodies, immunotests, etc. The nature of the test need not necessarily be described, as long as it is sensitive to detect binding of small amounts of peptides.

[0143] A number of other reagents may also be included in the binder mixture. These include reagents such as salts, buffers, neutral proteins (e.g. albumin), detergents, etc., which can be used to facilitate optimal binding. Such a reagent may also reduce non-specific or background interactions of reaction components. Other reagents that increase the efficiency of the test can also be used. The mixture of said test materials is incubated under conditions in which the monoclonal antibody usually binds specifically to ABCB5. Such conditions will preferably mimic physiological conditions. The order in which components are added, incubation temperature, incubation time and other test parameters can also be easily determined. Such an experiment almost does not contain optimization of test parameters or the fundamental composition of the test. Incubation temperatures are typically between 4 ° C and 40 ° C. Incubation times are preferably minimized to allow rapid high-screening and typically last from 0.1 to 10 hours. After incubation, the presence or absence of specific binding between the peptide and ABCB5 is detected by any conventional method available to the user.

[0144] Typically, many test mixtures are tested in parallel with different peptides or different concentrations of peptides to obtain a different response to different concentrations. One of these concentrations serves as a negative control, i.e. at zero ABCB5 concentration or at ABCB5 concentration below the detection limits of the test.

[0145] The separation step is often used to separate bound or unbound peptide or antibody. The separation step can be achieved in a number of ways. Suitably, at least one of the components (e.g., peptide or antibody) is immobilized on a solid support by binding to ABCB5. Unbound components can be easily separated from the bound fraction. A solid support can be made of a number of materials and a range of shapes, e.g. polyacrylamide columns or gels, agarose or sepharose, microtitre plates, microgranules, resin particles, etc. The separation step preferably includes multiple rinses or washes. For example, when the solid support is a microtiter plate, the wells can be washed many times with a washing solution, which usually contains those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, nonspecific protein, etc. Where the substrate is a magnetic granule, the granules can be washed one or more times with a washing solution and isolated using a magnet.

[0146] The molecules described herein may be used alone or in conjugates with other molecules such as detection or cytotoxic agents in the methods of detection and treatment described herein as described in more detail herein.

[0147] Typically, one of the components typically includes or is paired or coupled to a detectable label. A detectable tag is a grouping whose presence can be ascertained directly or indirectly. Generally, tag detection includes the emission of energy by the tag. The label can be detected directly by the ability to emit and / or absorb photons or other atomic molecules o

- specific wavelength (e.g. radioactivity, luminescence, optical or electron density, etc.). The label can be recognized indirectly by its ability to bind, recruit and, in some cases, cleave another moiety that itself can emit or absorb light at a specific wavelength (e.g., epitope tag such as FLAG epitope, enzyme tag such as peroxidase horseradish, etc.). An example of indirect detection is the use of the first enzyme label that cleaves the substrate into visible products. The label may be of a chemical, peptide or nucleic acid nature, although this is not so limited. Other detectable labels include radioactive isotopes such as P³² or H³, luminescence markers such as fluorochromes, optical or electron density markers, etc., or epitope tags such as FLAG epitope or HA epitope, biotin, avidin, and enzyme tags such as horseradish peroxidase, β-galactosidase, etc. The marker can be bound to a peptide in during or after its synthesis. There are many different markers and labeling methods known to those of skill in the art. Examples of the types of labels that can be used include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Skilled artisans will know about other suitable labels for the peptides described herein or will be able to determine this using routine testing. In addition, conjugation or conjugation of these labels to peptides can be performed using standard techniques known to those of skill in the art.

[0148] Another labeling technique that can result in greater sensitivity consists of coupling the molecules described herein to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which works with avidin or dinitrophenol, pyridoxal or fluoroscein, which can react with specific anti-hapten antibodies.

[0149] The conjugation of peptides, including antibodies or fragments thereof, with a detectable label allows, inter alia, the use of such agents in diagnostic tests. Another category of detectable labels includes diagnostic and imaging labels (generally referred to as in vivo detectable labels) such as, for example, magnetic resonance imaging (MRI). Gd (DOTA); for nuclear medicine²⁰¹Tl, gamma-emitting 99mTc radionuclide; for positron emission tomography (PET), positron emitting isotopes; (18) F-fluorodeoxyglucose ((18) FDG), (18) F-fluoride, copper-64, gadodiamide and Pb (II) radioisotopes such as 203Pb; 111 In.

[0150] The conjugations or modifications described herein utilize routine chemistry, which chemistry is not part of the invention, and which chemistry is well known to those skilled in the art of chemistry. The use of protecting groups and known linkers such as hetero-bifunctional monoorase linkers are well documented in the literature and will not be repeated here.

[0151] As used herein, "conjugated" means two units stably attached to each other by any physicochemical method. It is important that the nature of the combination is such that it does not significantly affect the effectiveness of any of the entities. While providing these parameters, any covalent bond or may be used

Non-covalent known to those skilled in the art. In some embodiments, covalent bonding is preferred. Non-covalent coupling includes hydrophobic interactions, ionic interactions, high affinity interactions such as biotin-avidin and biotin-streptavidin complexation and other affinity interactions. Such combination means and methods are well known to those skilled in the art.

[0152] A variety of tag detection methods can be used, depending on the nature of the tag and other assay components. For example, the label can be detected after binding to a solid support or after separation from a solid support. Labels can be directly detected via optical or electron density, radioactive emissions, non-radiative energy transfer, etc., or indirectly detected by antibody conjugates, streptavidin-biotin conjugates, etc. Methods for detecting labels are well known in the art.

[0153] Conjugates also include an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or small molecule toxin), or a radioactive isotope (i.e., radioconjugate) . Other anti-cancer agents that can be conjugated to antibodies include BCNU, streptozocin, vincristine and 5-fluorouracil, a family of agents known collectively as the LL-E33288 complex described in US Patent 5,053,394, 5,770,710, as well as esperamycin (US Patent No. 5,877,296). Enzymatically active toxins and fragments thereof that can be used in conjugates include diphtheria A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha sarcin, Aleurites fordii, diantyin, Phytolaca americana (PAPI, PAPII and PAP-S), Momordica charantia, curcine, crotin, Sapaonaria officinalis, gelonin, mitogelin, restrictorocin, phenomycin, enomycin and tricotecenes.

[0154] For the selective destruction of tumors, the antibody may contain a highly radioactive atom. A number of radioactive isotopes are available for the production of radioconjugate antibodies. Examples include At²¹¹, And¹³¹, And¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, 212 32 212

Bi, P, Pb and radioactive isotopes Lu. When the conjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example Tc^99m likes¹²³, or a spin tag for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging (MRI), such as iodine-123, iodine 131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17 , gadolinium, manganese or iron.

[0155] Radioactive or other labels can be incorporated into the conjugate by known methods. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis, using appropriate amino acid precursors, including, for example, fluorine-19 instead of hydrogen. Tags such as Tc^99m or 123 186 188 111

I, Re, Re and In can be attached via a cysteine ​​residue in the peptide. Itr90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to attach iodine-123.

-39 "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes in detail other methods.

[0156] Antibody and cytotoxic conjugates can be prepared using a number of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridylthio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolate (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (pazidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (pdiazoniobenzoyl) ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate) and bisactive fluorine compounds (such as 1, 5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for coupling a radionucleotide to an antibody. See. WO94 / 11026. The linker may be a "cleavage linker" facilitating the release of the cytotoxic drug in the cell. For example, an acid labile linker, peptidase sensitive linker, photolabile linker, dimethyl linker or disulfide containing linker may be used (Chari et al., Cancer Research 52: 127-131 (1992); US Patent No. 5,208,020).

[0157] Sequences responsible for the specificity of monoclonal antibodies have been determined. Accordingly, peptides can be obtained using recombinant DNA technology. There are entities in the United States that will perform this function commercially, such as Thomas Jefferson University and the Scripps Protein and Nucleic Acids Core Sequencing Facility (La Jolla, California). For example, a cDNA variable region can be obtained by polymerase chain reaction using degenerate or non-degenerate primers (derived from an amino acid sequence). The cDNA can be cloned to obtain sufficient double-stranded DNA for sequencing using conventional reactions or sequencing equipment.

[0158] With knowledge of the nucleic acid sequence of the variable domains of the heavy chain and light chain of the anti-ABCB5 monoclonal antibody, one of ordinary skill in the art will be able to produce nucleic acids that encode this antibody or that encode various antibody fragments, humanized antibodies, or polypeptides described above. It is contemplated that such nucleic acids will be operably linked to other nucleic acids forming a recombinant vector for cloning or expression of peptides. The disclosure includes any recombinant vector containing coding sequences, or part thereof, whether for prokaryotic or eukaryotic transformation, transfection or gene therapy. Such vectors may be obtained using conventional molecular biology techniques known to those skilled in the art and will contain coding DNA sequences for the CDR region (and preferably the CDR3 region) and additional variable sequences contributing to the specificity of the antibodies or parts thereof, as well as other non-specific peptide and corresponding promoter from (Whittle et al., Protein Eng. 1: 499, 1987 and Burton et al., Science

-40266: 1024-1027, 1994) or without (Marasco et al., Proc. Natl. Acad. Sci. (USA) 90: 7889, 1993 and Duan et al., Proc. Natl. Acad. Sci. (USA) 91: 5075-5079,1994) a signal sequence for export or isolation purposes. Prokaryotic cells can be transformed or transfected with such vectors (Huse et al., Science 246: 1275, 1989, Ward et al., Nature 341: 644646, 1989; Marks et al., J. Mol. Biol. 222: 581, 1991 and Barbas et al., Proc. Natl. Acad Sci. (USA) 88: 7978,991) or eukaryotic (Whittle et al., 1987 and Burton et al., 1994) or used for gene therapy purposes (Marasco et al., 1993 and Duan et al., 1994) using conventional techniques known to those of skill in the art.

[0159] As used herein, "vector" can be any of a number of nucleic acids into which the desired sequence can be inserted by restriction cleavage and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector means one that has the ability to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites in which the vector can be predictably cleaved and to which the desired DNA sequence can be ligated, that the new recombinant vector retains the ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur repeatedly as the number of plasmid copies increases within the bacterial host or just once per host, prior to the reproduction of the host by mitosis. In the case of phage, replication can take place actively during the lytic phase or passively during the lysogenic phase. An expression vector is one into which the desired DNA sequence can be introduced by restriction cleavage and ligation such that it is operably linked to regulatory sequences and can be expressed as an RNA transcript. The vectors may additionally contain one or more marker sequences suitable for use to identify cells that have been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detected by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes that clearly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors mean those capable of autonomous replication and expression of structural gene products present in DNA segments with which they are operably linked.

[0160] Expression vectors of the disclosure include regulatory sequences operably linked to a nucleotide sequence encoding one of the peptides. As used herein, the term "regulatory sequences" means nucleotide sequences that are necessary for or conducive to transcription of a nucleotide sequence that encodes the desired polypeptide and / or which are necessary for or favor the translation of the resulting transcript into the desired polypeptide. Regulatory sequences include, but are not limited to, 5 'sequences such as operators, promoters and ribosome binding sequences, and 3' sequences such as polyadenylation signals. The vectors may optionally contain 5 'leader sequences

Or 5 'or 3' signal sequences coding for fusion products to aid in protein purification, and various markers that help in the identification or selection of transformants. Choosing and designing the right vector is within the skill and freedom of the expert. Subsequent purification of the peptides can be carried out by any of a number of methods known in the art.

[0161] A preferred peptide screening vector, but not necessarily preferred for mass production of peptides, is a recombinant DNA molecule containing a nucleotide sequence that encodes and is capable of expressing a fusion polypeptide containing, from the amino to carboxyl terminus, a (1) prokaryotic domain secretory signal, (2) polypeptide, and, optionally, (3) fusion protein domain. The vector includes DNA regulatory sequences for expressing the fusion polypeptide, preferably prokaryotic regulatory sequences. Such vectors can be constructed by one of skill in the art and have been described by Smith et al. (Science 228: 1315-1317, 1985), Clackson et al. (Nature 352: 624-628, 1991); Kang et al. (in "Methods: A Companion to Methods in Enzymology: volume 2", RA Lerner and DR Burton, ed. Academic Press, NY, pp. 111-118, 1991); Barbas et al. (Proc. Natl. Acad Sci. (USA) 88: 7978-7982, 1991), Roberts et al. (Proc. Natl. Acatl. Sci. (USA) 89: 2429-2433, 1992) [0162] The fusion polypeptide may be useful for purifying peptides. The fusion domain may, for example, include a poly-His tail that allows purification on Ni + columns or maltose binding protein from a commercially available pMAL vector (New England BioLabs, Beverly, MA). At present, preferably, although not in any way necessary, the fusion domain is a filamentous phage membrane anchor. This domain is particularly useful for screening phage display libraries of monoclonal antibodies, but may be less suitable for mass production of antibodies. The filamentous phage membrane anchor is preferably a cpIII or cpVIII domain of a coat protein capable of binding to the filamentous phage particle matrix, thereby incorporating the fusion polypeptide into the surface of the phage to allow binding of the solid phase to specific antigens or epitopes, and thereby enabling enrichment and selection of specific antibodies or fragments encoded by the phagemid vector.

[0163] The secretory signal is the protein leader peptide domain, which is directed to the membrane protein of the host cell, such as the periplasmic membrane of Gram-negative bacteria. A preferred secretion signal for E. coli is a pelB secretion signal. The predicted amino acid residue sequences of the secretory domain from two pelB genes forming variants from Erwinia carotova are described in Lei, et al. (Nature 381: 543-546,1988). The leader pelB protein sequence has previously been used as a secretion signal for fusion proteins (Better, et al., Science 240: 1041-1043, 1988; Sastry, et al., Proc. Natl. Acad. Sci (USA) 86: 5728-5732, 1989; and Mullinax, et al., Proc. Natl. Acad Sci. (USA) 87: 8095-8099, 1990). The amino acid residue sequences for other useful polypeptide secretion domains from E. coli can be found in Oliver, in Neidhard, FC (ed.), Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington, DC, 1: 56-69 (1987).

[0164] To achieve high levels of gene expression in E. coli, it is not only necessary to use strong promoters to generate large amounts of mRNA, but also ribosome binding sites, to ensure efficient mRNA translation. In E. coli, the ribosome binding site includes an initiation codon (AUG) and a 3-9 nucleotide sequence located 3-11 nucleotides upstream of the initiation codon (Shine, et al., Nature 254: 34, 1975). The AGGAGGU sequence, which is referred to as the Shine-Dalgarno (SD) sequence, is complementary to the 3 'end of E. coli 16S rRNA. Several factors can affect ribosome binding to mRNA and sequence at the 3 'end of the mRNA: (i) the degree of complementarity between the SD sequence and the 3' end of 16S rRNA; (ii) the spacing and potential DNA sequence between the SD and AUG sequences (Roberts, et al., Proc. Natl. Acad Sci. (USA) 76: 760., 1979a: Roberts, et al., Proc. Natl. Acad Sci. (USA) 76: 5596, 1979b; Guarente, et al., Science 209: 1428, 1980; and Guarente, et al., Cell 20: 543, 1980). Optimization is achieved by measuring the level of gene expression in plasmids in which this interval is systematically changed. Comparison of different mRNAs shows that there are statistically favorable sequences from position -20 to +13 (where A of AUG is position 0) (Gold, et al., Annu. Rev. Microbiol. 35: 365, 1981). Leader sequences have been shown to dramatically affect translation (Roberts, et al., 1979a, b supra); and (iii) the nucleotide sequence behind AUG that affects ribosome binding (Taniguchi, et al., J. Mol. Biol., 118: 533, 1978).

[0165] 3 'regulatory sequences define at least one termination (stop) codon in the reading frame with and operably linked to a heterologous fusion polypeptide.

[0166] In a prokaryotic expression host, the vector used includes a prokaryotic replication or replicon origin, i.e. a DNA sequence having the ability to direct autonomous replication and maintain the recombinant DNA molecule outside chromosomally in a prokaryotic host cell, such as a bacterial host cell that has been transformed with it. Such replication initiation sites are well known in the art. Preferred replication initiation sites are those that are effective in the host. A prokaryotic host cell, for example, is E. coli. For use of the vector in E. coli, the preferred origin of replication is ColE1 found in pBR322 and in many other commonly used plasmids. Also preferred is the p15A origin of replication found in pACYC and its derivatives. ColEl and p15A replicons are widely used in molecular biology, are available in many plasmids and are described in Sambrook. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989).

[0167] Additionally, those embodiments that include the prokaryotic replicon preferably also include a gene whose expression confers a selective benefit, such as drug resistance, to the bacterial host transformed with it. Typical bacterial drug resistance genes are those that confer resistance to ampicillin, tetracycline, neomycin / kanamycin or chloramphenicol. The vectors also typically contain convenient restriction sites for the introduction of the translated DNA sequences. Exemplary vectors are plasmids pUC18 and pUC19 and derived vectors such as pcDNAII available from Invitrogen (San Diego, CA).

[0168] When the peptide is an antibody comprising both heavy chain and light chain sequences, the sequences may be encoded on separate vectors or, more conveniently, may be expressed from a single vector. The heavy and light chain may, after translation or isolation, form the heterodimeric structure of natural antibody molecules. Such heterodimeric antibody may or may not be stabilized by disulfide bonds between heavy and light chains.

[0169] The expression vector of heterodimeric antibodies, such as intact F (ab ') 2, Fab or Fv antibody fragments, is a recombinant DNA molecule adapted to receive and express first and second translated DNA sequences. That is, the DNA expression vector for expressing the heterodimeric antibody provides a system of independent cloning (inserting) two translated DNA sequences into two separate cassettes present in the vector to form two separate cistrons for expressing the first and second of the heterodimeric antibody polypeptides. A DNA expression vector for expressing two cistrons is referred to as a dicistronic expression vector.

[0170] Preferably, the vector comprises a first cassette which comprises upstream and downstream DNA regulatory sequences operably linked via a nucleotide sequence adapted for directed ligation with the DNA insert. The upstream translated sequence preferably encodes a secretion signal as described above. The cassette includes DNA regulatory sequences for expressing the first antibody polypeptide that is produced when the insert of the translated DNA sequence is directed in the cassette via a nucleotide sequence adapted for directed ligation.

[0171] The dicistronic expression vector also includes a second cassette for expressing the second antibody polypeptide. The second cassette comprises a second translated DNA sequence that preferably encodes a secretion signal, as described above, operably linked at the 3 'end via a nucleotide sequence adapted for directed ligation to the underlying vector DNA sequence, which typically defines at least one stop codon in cassette reading frame. The second translated DNA sequence is operably linked at the 5 'end to the DNA regulatory sequences forming the 5' elements. The second cassette has the ability, after introducing the translated DNA sequence (insert DNA), to express a second fusion polypeptide containing a secretion signal from the polypeptide encoded by the insert DNA.

[0172] Peptides can also be produced by eukaryotic cells such as CHO cells, human hybridomas, immortalized B-lymphoblastoid cells and the like. In this case, a vector is constructed in which eukaryotic regulatory sequences are operably linked to nucleotide sequences encoding the peptide. The selection and design of the appropriate eukaryotic vector is within the skill and freedom of action of a person skilled in the art. Subsequent purification of the peptides can be carried out by any of a number of methods known in the art.

[0173] In another embodiment, host cells, both prokaryotic and eukaryotic, are transformed or transfected with vectors, and therefore also vectors.

[0174] Suitable host cells for expressing glycosylated anti-ABCB5 antibody are from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. A number of viral strains for transfection are publicly available, e.g. the L-1 variant NPV Autographa californica and the Bm-5 NPV strain Bombyx mori, and such viruses can be used as the virus of the invention, especially for transfection of Spodoptera frugiperda cells.

[0175] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0176] Vertebrate cells as host cells are also of particular interest. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); Sertoli mouse cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Norwegian rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse breast cancer (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383: 4468 (1982)); MRC 5 cells; FS4 cells; and human malignant liver tumor line (Hep G2).

[0177] Host cells are transformed with the expression or cloning vectors described above for the production of anti-ABCB5 antibodies and cultured in conventional nutrient media modified as appropriate for promoter induction, selection of transformants or amplification of genes encoding the desired sequences.

[0178] Host cells used to produce anti-ABCB5 antibody can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM, (Sigma)) are suitable for growing host cells. In addition, any media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Pat. No. 4,767,704; 4,657,866; 4,927,762;

-454,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or US Patent Re. 30,985 can be used as host cell culture media. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ™), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or an equivalent source of energy. Any other necessary additions may also be included at appropriate concentrations that will be known to those skilled in the art. Culture conditions such as temperature, pH and the like are those previously used for the host cell selected for expression, and will be apparent to those skilled in the art.

[0179] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the first step is to remove molecular contaminants, host cells or lysed fragments, for example, by centrifugation or ultrafiltration. The antibody composition obtained from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, where affinity chromatography is the preferred purification technique. The matrix to which the affinity ligand is attached is usually agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly (styrenedivinyl) benzene, allow faster flow rates and shorter processing times than can be achieved with agarose. After any preliminary purification step, the mixture may be subjected to hydrophobic interaction chromatography at low pH using an elution buffer with a pH of about 2.5 to 4.5, preferably carried out at a low salt concentration (e.g. about 0-0.25 M salt).

[0180] As used herein with respect to nucleic acids, the term "isolated" means:

(i) amplified in vitro by, for example, polymerase chain reaction (PCR);

(ii) recombinantly produced by cloning; (iii) purified, by cleavage and gel separation; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is one that is easily manipulated by recombinant DNA techniques well known in the art. Hence, the nucleotide sequence contained in the vector in which 5 'and 3' restriction sites are known, or for which the polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated, but the nucleic acid sequence found in its native state in the natural host is not is. Isolated nucleic acid can be extensively purified, but this is not necessary. For example, a nucleic acid that is isolated within a cloning vector or expression vector is not pure in the sense that it may contain only a small percentage of the material in the cell in which it is located. Such nucleic acid is, however, isolated as the term is used in the specification because of the ease of manipulation of standard techniques known to the skilled person.

[0181] As used herein, the coding sequence and regulatory sequences are considered "operably linked" when they are covalently linked in such a way that the expression or transcription of the coding sequence is influenced or controlled by regulatory sequences. It is desirable that the coding sequences be translated into a functional protein, two DNA sequences are considered operably linked if induction of the promoter in the 5 'regulatory sequences results in transcription of the coding sequence, and if the nature of the binding between the two DNA sequences (1) does not result in the introduction of a shift mutation the reading frame, (2) does not interfere with the promoter region's ability to direct the transcription of coding sequences, or (3) does not interfere with the ability of the corresponding RNA transcript to translate into protein. Hence, the promoter region will be operably linked to the coding sequence if the promoter region is able to transcribe this DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide.

[0182] The exact nature of the regulatory sequences necessary for gene expression may vary between cell species or types, but should generally include, as required, 5 'non-transcriptional and 5' non-translated sequences associated with transcription initiation and translation, respectively like the TATA box, the hat sequence, the CAAT sequence and the like. Specifically, such 5 'non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably linked gene. Regulatory sequences may also include upstream enhancer sequences or activator sequences as desired.

[0183] The compositions and methods described herein can be strengthened by use in conjunction with other procedures relating to cancer and pre-cancerous lesions. In some cases, the treatment procedure involves the administration of another therapeutic agent, such as an anti-cancer agent, including but not limited to chemotherapeutic agents and radiation therapy. The chemotherapeutic agents can be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, taxol, paclitaxel, sugar-free chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxicine, valcillin, taxoline , MMI270, BAY 12-9566, RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, dacarbazine, LY294002, PX866, MTA / LY231514, LY264618 / Lometexol, Glamolec, CI-994, TNP-470, Hycamtin / Topotecan, PKC412, Valspodar / PSC833, Novantrone / Mitroxantrone,

Metaret / Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel / VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516 / Marmistat, BB2516 / Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil / OK-432, AD 32 / valrubicin, Metastron / strontium derivative, Temodal / Temozolomide, Evacet / liposomal doxorubicin, taxis from cis / paclitaxel, paclitaxel , Xeloda / capecitabine, Furtulon / doxifluridine, Cyclopax / oral paclitaxel, oral taxoid, SPU077 / cisplatin, HMR 1275 / flavopyridol, CP-358 (774) / EGFR, CP-609 (754) / inhibitor

-47oncogen RAS, BMS-182751 / oral platinum, UFT (Tegafur / Uracil),

Ergamisol / Levamisole, Eniluracil / 776C85 / 5FU enhancer, Campto / Levamisole, Camptosar / Irinotecan, Tumodex / Ralitrexed, Leustatin / Cladribine, Paxex / Paclitaxel, Doxil / liposomal doxorubicubin, Focalinic / Lipocomorubicin / Focalarabine / Fecalxabine , ZD1839, LU 79553 / bis-naphthalimide, LU 103793 / dolastatin, Caetyx / liposomal doxorubicin, Gemzar / gemcitabine, ZD 0473 / Anormed, YM 116, iodine grains, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809 / Dexifosamide Ifes / Mesnex / Ifosamide, Vumon / teniposide, Paraplatin / carboplatin, Plantinol / cisplatin, Vepeside / etoposide, ZD 9331, Taxotere / docetaxel, prodrug guanine arabinoside, taxane analogue, nitrosoureas, cyclophosphamide, such as cyclophosphamide, and such as aminophosphamide , busulfan, carboplatin, chlorombucil, cytarabine HCl, dactinomycin, daunorubicin HCl, sodium phosphate estramustine, etoposide (VP16-213), floxuridine, fluorouracil (5-FU), flutamide, hydroxycarbamide (hydroxycarbamide), ifosfamide, interferon alfa-2a, alpha-2b, leuprolide acetate (LHRH releasing factor analog), lomustine (CCNU), mechloretamine HCl (nitrogen mustard), mercaptopurine, mesna, mitotane), mitoxantrone HCl, octamycretin , procarbazine HCl, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacytidine, erythropoietin, hexamethylmelamine (HMM), interleukin 2, mitoguazone; bis-guanylhydrazone methyl glyoxal; MGBG), pentostatin (2'-deoxycoformycin), semustine (methyl-CCNU), teniposide (VM26) and vindesine sulfate.

[0184] The methods described herein can be carried out with respect to therapies for the treatment of cancer, such as surgery and radiation therapy. The methods described herein can also be used in conjunction with a therapeutic that is an isolated short RNA that directs sequence-specific degradation of tumor-specific mRNA via a process known as RNA interference (RNAi). In some embodiments, the tumor-specific mRNA is ABCB5. The method is known to occur in a wide group of organisms, including embryos of mammals and other vertebrates. It has been shown that dsRNA is processed to 21-23 nucleotide (nt) RNA segments and in subsequent order that they are involved in RNA interference in the absence of longer dsRNAs. Hence, these 21-23 nt fragments are sequence specific mediators of RNA degradation and are referred to herein as siRNA or RNAi. The methods described herein include using these fragments (or those obtained by recombination or chemical synthesis of oligonucleotides of the same or similar nature) to enable the targeting of tumor-specific mRNAs for degradation in mammalian cells useful in the therapeutic applications discussed herein.

[0185] RNA design methods that regulate RNAi and methods for RNA transfection into cells and animals are well known in the art, and RNAi molecules are readily available commercially (Verma NK et al, J. Clin. Pharm. Ther., 28 (5 ): 395-404 (2004), Mello CC et al. Nature, 431 (7006) 338-42 (2004), Dykxhoom DM et al., Nat. Rev. Mol. Cell Biol. 4 (6): 45767 (2003) Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA),

-48ChemGenes (Ashland, MA, USA) and Cruachem (Glasgow, UK)). RNAs are preferably chemically synthesized using appropriately protected ribonucleosides phosphoramidate and a conventional DNA / RNA synthesizer. Most conveniently siRNA is obtained by commercial oligo RNA synthesis from the suppliers specified in the description. In general, RNAs are not too difficult to synthesize and are easily delivered in quality appropriate for RNAi. Typical 0.2 μmol RNA synthesis provides approximately 1 milligram of RNA, sufficient to perform 1000 transfection experiments using 24-well tissue culture plate format.

[0186] Tumor-specific cDNA specific siRNA is preferably designed by selecting a sequence that is not within 50-100 bp of the start codon and termination codon, intron regions are avoided, 4 or more base sequences such as AAAA are avoided , CCCC, regions with GC contents <30% or> 60% are avoided, repetitions and sequences with low complexity are avoided, and sites with single nucleotide polymorphisms are avoided. The target sequence may have a GC content of about 50%. The siRNA target sequence may be further evaluated by homology search in the BLAST program to avoid interaction other than targeted to other genes or sequences. Negative controls are designed by coding targeted siRNA sequences. The control RNA preferably has the same length and nucleotide composition as siRNA, but has at least 4-5 bases that do not match siRNA. RNA molecules may contain a 3 'hydroxyl group. RNA molecules can be single-stranded or double-stranded; such molecules may have blunt ends or have protruding ends (e.g., 5 ', 3') from about 1 to about 6 nucleotides in length (e.g., pyrimidine nucleotides, purine nucleotides). To further increase RNA stability, the 3 'protruding ends can be protected from degradation. RNA can be stabilized by the inclusion of purine nucleotides, such as adenosine or guanosine nucleotides.

Alternatively, substitution of pyrimidine nucleotides with modified analogues, e.g., substitution of 2-nucleotide uridine with 3'-protruding ends on 2'-deoxythymidine is tolerated and does not affect RNAi efficacy. The absence of 2 'hydroxyl clearly increases the protruding end resistance to nucleases in tissue culture medium.

[0187] RNA molecules used in the methods can be obtained using a variety of techniques known to a person skilled in the art. For example, RNA can be chemically synthesized or recombinantly produced using methods known in the art. Such methods are described in US Published Patent Applications Nos. US2002-0086356A1 and US2003-0206884A1.

[0188] The methods described herein are used to identify or obtain RNA molecules that are useful as sequence specific mediators of tumor specific mRNA degradation and, consequently, to inhibit proteins that contribute to the functioning of cancer cells. Expression of ABCB5, for example, can be inhibited in humans to prevent protein translation and hence prevent its functioning in vivo.

[0189] Any RNA can be used in the methods, as long as it has sufficient homology to the tumor specific gene to mediate RNAi. RNA may correspond to the entire tumor specific gene or part thereof. There is no upper limit to the length of RNA that can be used. For example, RNA may range from about 21 base pairs (bp) of the gene to the full length of the gene or more. In one embodiment, the RNA used in the methods is about 1000 bp in length. In another embodiment, the RNA is about 500 bp in length. In yet another embodiment, RNA is about 22 bp in length. In some embodiments, the preferred RNA length is 21 to 23 nucleotides. The ABCB5 sequence is known, for example, see US Patent No. 6846883 (which refers to ABCB5 as 7β P-glycoprotein).

[0190] ABCB5 binding molecules are administered to an individual in an effective amount to treat cancer. "Effective amount for treating cancer" means the amount necessary or sufficient to obtain the desired biological effect. For example, an effective amount of a compound can be the amount necessary to (i) kill a cancer cell; (ii) inhibiting further tumor growth, i.e. stopping or slowing its development; and / or (iii) sensitizing the tumor cell to an anti-cancer agent or therapeutic.

According to some disclosures, as used herein, an effective amount means that amount of a compound, alone or in combination with a cancer drug, which when combined or co-administered or administered alone results in a response to cancer treatment, in preventing or treating cancer. The biological effect can be improvement of the condition and / or complete removal of symptoms caused by cancer. In another embodiment, the biological effect is to combat the tumor completely, as demonstrated, for example, by the absence of a tumor or a biopsy or blood smear that is free of cancer cells.

[0191] The effective amount of a compound in the treatment of cancer or in reducing the risk of cancer may vary, depending on the use of the particular compound, the mode of delivery of the compound, and whether it is used alone or in combination. The effective amount for any particular application may also vary depending on factors such as the cancer being treated, the particular compound being administered, the size of the individual, or the severity of the disease or condition. The skilled person can empirically determine the effective amount of a particular molecule without having to experiment unnecessarily. In conjunction with the content provided in the description by choosing among a variety of active compounds and after considering factors such as potency, relative bioavailability, patient weight, severity of adverse effects, and favorable route of administration, an effective prophylactic or therapeutic regimen can be planned that does not causes significant toxicity and is, however, fully effective for the treatment of a particular individual.

[0192] The subject doses of compounds described herein typically range from about 0.1 μg to 10,000 mg, more typically from about 1 μg / day up to 8,000 mg, and most typically from about 10 μg to 100 μg. Given in relation to body weight

-50 subjects, typical dosages range from about 0.1 μg to 20 mg / kg / day, more typically from about 1 to 10 mg / kg / day and most typically from about 1 to 5 mg / kg / day. The absolute amount will depend on a number of factors, including concomitant treatment, number of doses, and individual patient parameters, including age, physical condition, size, and body weight. These factors are well known to those skilled in the art and can be solved by no more than routine experimentation. It is generally advantageous to use the maximum dose, i.e. the highest safe dose according to sound medical knowledge.

[0193] Multiple doses of molecules are also contemplated. In some cases, when the molecules are administered with a cancer drug, a subtherapeutic dose of the molecules or cancer drug, or a subtherapeutic dose of both is used to treat the subject having the cancer or at risk of having it. When two classes of drugs are used together, the cancer drug can be administered in a sub-therapeutic dose to achieve the desired therapeutic result. The "subtherapeutic dose" as described herein refers to a dosage that is lower than the dosage that will provide a given therapeutic result in the subject when administered in the absence of another agent. Hence, a sub-therapeutic dose of a cancer drug is one that will not provide the desired therapeutic effect to the subject in the absence of administration of the molecules. Therapeutic doses of cancer drugs are well known in the medical field for cancer treatment. These dosages are extensively described in references such as Remington's Pharmaceutical Sciences, 18th Edition, 1990; as well as in many other medical references cited by doctors as guidelines for the treatment of cancer. Therapeutic doses of antibodies have also been described in the art.

[0194] A number of routes of administration are available. The particular route chosen will of course depend on the choice of the particular anti-ABCB5 antibody, the particular condition being treated and the dosage required to achieve therapeutic efficacy. The methods, in general, can be practiced using any mode of administration that is medically acceptable, which means any mode that gives effective levels of protection without causing clinically unacceptable side effects. Preferred modes of administration are parenteral. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intra-arterial injection or infusion techniques. Other routes include, but are not limited to, oral, nasal, cutaneous, sublingual and local.

[0195] The formulations are administered in pharmaceutically acceptable solutions that can routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

[0196] The compounds may be administered by any of the usual modes of drug administration. Depending on the type of cancer being treated, the compounds can be administered by inhalation, ingestion or systemic administration. Systemic routes include oral and parenteral. Inhaled medications are preferred in some embodiments because of direct use

-Lung delivery, especially in patients with lung cancer. Several types of metered dose inhalers are regularly used for inhalation use. These types of devices include metered dose inhalers (MDI), breath activated MDI, dry powder inhalers (DPI), spacer / extension chambers in combination with MDI and nebulizers. Preferred routes of administration include, but are not limited to, oral, parenteral, intramuscular, intranasal, intratracheal, intrathecal, intravenous, inhaled, intraocular, vaginal and rectal. For use in therapy, an effective amount of compounds can be administered to a subject by any route that provides nucleic acid to the affected organ or tissue. "Administering" the pharmaceutical composition can be achieved by any method known to a person skilled in the art.

[0197] According to the methods described herein, the peptide may be administered in a pharmaceutical composition. In general, the pharmaceutical composition comprises a peptide and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers for peptides, monoclonal antibodies and antibody fragments are well known to the skilled person. As used herein, a pharmaceutically acceptable carrier means a non-toxic material that does not affect the effectiveness of the biological activity of active ingredients, e.g. ability of the peptide to bind to ABCB5.

[0198] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizing agents and other materials that are well known in the art. Exemplary pharmaceutically acceptable carriers for peptides are described in particular in US Patent No. 5,211,657. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicaments, salts should be pharmaceutically acceptable, but pharmaceutically unacceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the disclosure. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those obtained from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, lemon, formic, malonic, succinic and the like. Also, pharmaceutically acceptable salts can be obtained as alkali or alkaline earth metal salts, such as sodium, potassium or calcium salts.

[0199] The peptides may be formulated as solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalants and injectables, and for ordinary routes of oral parenteral administration or surgical. Pharmaceutical compositions that are formulated for topical administration, such as by implants, are also described.

[0200] Compositions suitable for oral administration may be in the form of discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or emulsion.

[0201] When the compounds described herein (including peptide and non-peptide variants) are therapeutically used, in some embodiments, a pulmonary aerosol may be the desired route of administration. The techniques used to prepare aerosol delivery systems containing compounds are well known to those of skill in the art. In general, such systems should utilize components that will not significantly interfere with the biological properties of the peptides (see, for example, Sciarra and Cutie, "Aerosols" in Remington's Pharmaceutical Sciences, 18th Edition, 1990, pp. 1694-1712). One skilled in the art can easily determine the various parameters and conditions for aerosol production without the need for unnecessary experimentation.

[0202] Peptides can be administered directly to tissue. Preferably, the tissue is one in which tumor stem cells have been found. Alternatively, tissue means one in which the tumor is likely to occur. Direct administration to tissue can be achieved by direct injection. The peptides can be administered once, or alternatively they can be administered multiple times. When administered multiple times, the peptides can be administered by various routes. For example, the first (or first few) administration may be directly to the diseased tissue, whereas the subsequent administration may be systemic.

[0203] For oral administration, the compounds can be easily formulated by combining active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained with a solid excipient, optionally by trituration of the resulting mixture, and processing the mixture into granules, after addition of suitable excipients, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate may be added. Optionally, oral formulations may also be formulated in physiological saline or buffers to neutralize the acidic conditions inside or may be administered without any carriers.

[0204] Dragee cores, with appropriate coatings, provided. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, coating solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings to identify or characterize different combinations of active compound doses.

[0205] Pharmaceutical preparations that can be used orally include hard gelatin capsules as well as soft gelatin and plasticizer capsules such as glycerol or sorbitol. The hard capsules may contain the active ingredients in admixture with a filler such as lactose, binders such as starches and / or moisturizers such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or liquid polyethylene glycols. In addition, stabilizers can be added. Formulated microspheres for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should have a dosage appropriate to such administration.

[0206] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0207] For inhalation administration, the compounds may conveniently be provided in the form of an aerosol spray, administered from pressurized packs or a nebulizer, using a suitable propellant gas, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges e.g. gelatin, for use in an inhaler or nebulizer can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Techniques for obtaining aerosol delivery systems are well known to those of skill in the art. In general, such systems should utilize ingredients that will not significantly interfere with the biological properties of the active agent (see, for example, Sciarra and Cutie, "Aerosols" in Remington's Pharmaceutical Sciences, 18th Edition, 1990, pp. 1694-1712). One skilled in the art can easily determine the various parameters and conditions for aerosol production without the need for unnecessary experimentation.

[0208] The compounds, when it is desired to provide them systemically, can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0209] Preparations for parenteral administration include aqueous or non-aqueous sterile solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral excipients include sodium chloride solutions, Ringer's dextrose solution, dextrose and sodium chloride, lactated Ringer's or vegetable oils. Intravenous excipients

-54 include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose solution), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that the subject's response is insufficient for the initial doses used, higher doses (or effectively higher doses using other, more local routes of administration) may be used to the extent that the patient's tolerance allows. Many doses are considered daily to achieve appropriate systemic levels of compounds.

[0210] In yet other embodiments, the preferred excipient is a biocompatible microparticle or implant that is suitable for implantation in a mammalian recipient. Exemplary erosionable implants that are useful in this way are described in PCT International Application No. PCT / US / 03307 (Publication No. WO 95/24929, titled "Polymeric Gene Delivery System", claiming priority from US Patent Application No. 213,668 , filed on March 15, 1994). PCT / US / 0307 describes a biocompatible, preferably biodegradable polymer matrix for containing biological macromolecules. A polymer matrix can be used to achieve sustained release of a given agent in an individual. The agent described herein may be encapsulated or dispersed within a biocompatible, preferably biodegradable polymer matrix disclosed in PCT / US / 03307. The polymer matrix is ​​preferably in the form of a microparticle, such as a microsphere (wherein the agent is dispersed within a solid polymer matrix) or a microcapsule (wherein the agent is stored in the core of the polymer coating). Other forms of the polymer matrix to contain the agent include films, coatings, gels, implants and stents. The size and composition of the polymer matrix device was chosen so as to obtain favorable release kinetics in the tissue into which the device - matrix will be implanted. The size of the polymer matrix device is further selected according to the delivery method to be used, typically injection into tissue or administration of an aerosol suspension into the nasal and / or lung region. The composition of the polymer matrix can be selected to obtain both favorable degradation rates as well as the formation of a material that is bioadhesive to further increase the efficiency of the transfer when the device is applied to a vascular, pulmonary or other surface. The composition of the matrix can also be chosen so that it does not degrade, but rather releases content by diffusion over an extended period of time.

[0211] Both non-biodegradable and biodegradable polymer matrices can be used to deliver agents to a subject. Biodegradable matrices are preferred. Such polymers can be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally on the order of several hours to a year or more. Typically, the release is most desirable for a period ranging between several hours and three to twelve months. The polymer is optionally in the form of a hydrogel that can

Absorb up to about 90% of its mass in water and additionally is cross-linked with polyvalent ions or other polymers.

[0212] In general, agents can be delivered using a bioerodible implant, by diffusion, or more preferably, by degradation of the polymer matrix. Exemplary synthetic polymers that can be used to form a biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidone, polyglycols, polyglycols, polyglycols, polyglycols, polyols polymers of acrylic and methacrylic esters, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate phthalate, carboxyl ethyl cellulose, cellulose triacetate, sodium cellulose sulfate, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate) ) , poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate), polyethylene, polypropylene, polyethylene glycol, polyethylene oxide, polyethylene terephthalate, polyvinyl alcohols, polyvinyl acetate, polyvinyl chloride, polystyrene and polyvinylpyrrolidone.

[0213] Examples of non-biodegradable polymers include ethylene vinyl acetate, poly (meth) acrylic acid, polyamides, copolymers and mixtures thereof.

[0214] Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly (ortho) esters, polyurethanes, poly (butyric acid), poly (valeric acid), and poly (lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides, including dextran and cellulose, collagen, its chemical derivatives (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylation, oxidations and other modifications routinely carried out by one of ordinary skill in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, materials are degraded by enzymatic hydrolysis or exposure to water in vivo, by surface or mass erosion.

[0215] Bioadhesive polymers of particular interest include the bioerodible hydrogels described by HS Sawhney, CP Pathak and JA Hubell in Macromolecules, 1993,26, 581-587, the content of which is incorporated herein, polyhialuronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly (methyl methacrylates), poly (ethyl methacrylates), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate).

[0216] Other delivery systems may include timed release, sustained release, or sustained release delivery systems. Such systems can bypass multiple peptide administration, increasing patient and physician convenience. Many types of release delivery systems are available and known to those skilled in the art. These include polymer-based systems such as poly (lactide-glycolide), copolyvate, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides. Microcapsules of the above polymer-containing drugs are described in, for example, US Patent No. 5,075,109. Delivery systems also include non-polymer systems, i.e., lipids, including sterols, such as cholesterol, cholesterol esters and fatty acids, or neutral fats, such as monodi- and triglycerides; hydrogel release systems; silicone elastomer systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially coupled implants; and similar. Specific examples include, but are not limited to: (a) erosive systems in which the platelet reducing agent is contained within a matrix within the form such as those described in US Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusion systems in which the active ingredient controlled-speed filtrate from polymer such as described in US Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, delivery systems with pump-based equipment can be used, some of which are implantable.

[0217] The use of a long-term sustained release implant may be particularly suitable for the prophylactic treatment of individuals at risk of recurrent cancer. Prolonged release, as used herein, means that the implant is constructed and adapted to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

[0218] Therapeutic antibody formulations can be prepared for storage by mixing the antibodies of the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents,

-57 like EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

[0219] The following examples are provided to illustrate specific practical cases of the present invention and are not intended to limit the scope of the invention. As will be apparent to those skilled in the art, the invention will find application in a number of compositions and methods.

EXAMPLES

Materials and methods:

[0220] Melanoma cells and culture methods. The human malignant melanoma G3361 cell line, derived from a single tumor cell cloned in soft agar, was obtained from Dr. Emil Frei III (Dana-Farber Cancer Institute, Boston, MA), A375 cell line is commercially available from the American Type Culture Collection (ATCC; American Pure Culture Collection) (Manassas, VA). All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 6 mmol / L HEPES, 2 mmol / L L-glutamine, and 100 IU / ml penicillin / streptomycin at 37 ° C and 5% CO2 in a humidified incubator as previously described. G3361 / DsRed2 and G3361 / EYFP cell lines were generated by stable transfection of G3361 melanoma cells with red fluorescent protein from Discosoma sp. (DsRed2) or the enhanced yellow-green variant (EYFP) of the green fluorescence protein from Aequorea victoria (GFP) in combination with the retention signal in the nucleus of the large monkey T virus antigen 40 (Kalderon, D., Roberts, BL, Richardson, WD &amp; Smith , AE A short amino acid sequence able to specify nuclear location. Cell 39, 499-509 (1984)), using mammalian expression vectors pDsRed2Nuc or pEYFP-Nuc also containing a neomycin resistance cassette (BD Biosciences, Palo Alto, CA.) and the lipofectamine 2000 reagent (Invitrogen) as previously described. Cultures of clones G3361 / DsRed and G3361 / EYFP were generated from stably transfected cultures by limiting dilution. Clinical melanoma cells (n = 6 patients) obtained fresh from surgical samples according to human study protocols approved by bioethics committees from the University of Wurzburg Medical School or Wistar Institute, Philadelphia, PA.

[0221] Antibodies. IgG1K specific anti-ABCB5 mAb 3C2-1D12 was used as described in expression studies. FITC 3C2-1D12 mAb conjugated was used to determine the purity of ABCB5 sorted melanoma subsets⁺ and ABCB5^-. Unconjugated or FITC conjugated mouse MOPC-31C mAbs isotype controls, FITC conjugated goat anti-mouse IgG secondary Ab, conjugated with phycoerythrin (PE) anti-human CD20, anti-human CD31 and mAbs - isotype control purchased from PharMingen, San Diego , CA. Allophycocyanin (APC) conjugated and PE conjugated secondary mAbs were purchased from eBioscience, San Diego, CA. Unlinked anti-human TIE-1, anti-human BMPR1a, PE conjugated anti-human VE-cadherin mAbs and anti-human mAbs for nestin were from R&amp;D Systems,

-58 Minneapolis, MN. The following antibodies were used for immunohistochemistry and ABCB5, TIE-1 and VE-cadherin immunofluorescence staining: mouse anti-ABCB5 mAb (Frank, NY et al. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res 65, 4320-33 (2005); Frank, NY et al. Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette transporter. J Biol Chem 278, 47156-65 (2003)), HRP-conjugated horse anti-mouse IgG secondary Ab (Vector Laboratories, Burlingame, CA), FITC-conjugated rabbit anti-mouse IgG secondary Ab (ZYMED Laboratories, San Francisco, CA), uncoupled rabbit anti-human Ab VE-cadherin (courtesy of Cell Signaling Technology, Danvers, MA), mouse IgG Abs control (DAKO, Carpinteria, CA), uncoupled rabbit anti-human mAb TIE-1 (Santa Cruz Biotechnologies, Santa Cruz, CA) FITC-conjugated anti-mouse IgG secondary Ab, Texas-conjugated anti-rabbit IgG secondary Ab, conjugated to Cy3-donkey anti-rabbit IgG secondary Ab, and rabbit control IgG Ab (all from Jackson ImmunoResearch, West Grove, PA) .

[0222] Histopathology and immunohistochemistry. 5 microns thick melanoma cryoparticles were fixed at -20 ° C in acetone for 5 minutes. Air dried sections were incubated with 10 μg / ml ABCB5 mAb at 4 ° C overnight; 10 μg / ml mouse IgG was used as a negative control. Sections were washed with PBS x 3 for 5 minutes and incubated with 1: 200 peroxidase conjugated horse anti-mouse IgG Ab for ABCB5 staining. For fluorescent double labeling ABCB5 / VE-cadherin or ABCB5 / TIE-1, 5 μm melanoma sections were fixed at -20 ° C in acetone for 5 minutes. Air-dried sections were incubated with 10 μg / ml ABCB5 mAb and 2.5 μg / ml VE-cadherin or TIE-1 Abs at 4 ° C overnight; 10 μg / ml mouse IgG and 2.5 μg / ml rabbit IgG were used as negative controls. Sections were washed with PBS containing 0.05% Tween 20 for 5 minutes x 3 and incubated with a 1: 150 dilution of Texas Red conjugated or Cy3 conjugated donkey anti-rabbit IgG Ab and FITC conjugated rabbit anti-mouse IgG Ab for 30 minutes in room temperature. After later washings, sections were fastened with VECTASHIELD (Vector Laboratories) mounting medium and covered with a coverslip. Immunofluorescence reactivity was observed using an Olympus BX51 / 52 system microscope coupled to a Cytovision system (Applied Imaging, San Jose, CA).

[0223] Construction and analysis of tissue microarrays. Melanocytic Tumor Progression TMA Microarray is a product of joint effort of three Skin SPORES units (Harvard, MD Anderson, University of Pennsylvania). This matrix contains 480 x 0.6 mm tumor tissue cores, representing four main diagnostic types of tumors: benign nevi, primary cutaneous melanoma, lymph node metastasis and parenchymal organ metastasis. Cases were collected using the services of facilities operating in the field of pathology within the three institutions participating in the study. To ensure quality control, two replicate cores were selected from each separate region. Birthmarks and basal melanomas had one or three regions of tissue block sampling (2 or 6 cores), whereas metastatic tumors had one region of sampling from each block. Consequently, 480 cores represent 2 adjacent cores of 240

-59 separate histological regions. This matrix includes 130 cores with 35 moles, 200 cores with 60 primary melanomas, and 150 cores with 75 metastatic lesions. From an operational point of view, thin birthmarks and thin melanomas related only to superficial / papillary dermis, while the increase in thick birthmarks and thick melanomas affected both papillary and deep (reticular) dermis. This matrix was constructed in the laboratory of Dr. Mark Rubin (Brigham and Women's Hospital Department of Pathology and Dana Farber Cancer Institute, Boston). Histological slides of the slide with tissue matrix were baked at 58 ° C for 20 minutes and then treated as follows: xylene x 2 (1 hour, 10 minutes), 100% ethanol x 2 for 2 minutes, 95% ethanol for 2 minutes, and dH2O x 3 for 2 minutes. Antigen recovery was carried out in 10 mMol citrate buffer, pH 6.0 with cooking in a pressure cooker for 10 minutes and then cooling to room temperature. After washing with PBS x for 5 minutes, the tissue was blocked with 10% horse serum and 1% BSA in PBS at room temperature for 1 hour, followed by incubation with 5 μg / ml ABCB5 mAb at 4 ° C overnight. The tissue was then washed with PBS-0.05% Tween 20 x 3 for 5 minutes, and then treated with 3% H2O2 / PBS for 15 minutes. After washing in PBS, sections were incubated with 1: 200 biotinylated horse anti-mouse IgG Ab at room temperature for 30 minutes, washed in PBS-Tween x 3 for 5 minutes, and incubated with the avidinibin-horseradish peroxidase complex (Vector Laboratories) for 30 minutes at room temperature. Immunoreactivity was detected using a NovaRed substrate (Vector Laboratories). The Chromavision Automated Cellular Imaging System (ACIS) was used to quantify the intensity of ABCB5 and mIgGIR immunostaining on HTMA 84 tissue matrix. The intensity values ​​from the control slide (background plus internal melanin conversion) were subtracted from the experimental slide and the difference in intensity for each the core was taken for true staining. This graph (see fig. 1) shows with a 95% confidence interval the difference in intensity for each diagnosis of a pathological condition. The p-values ​​between the respective groups were calculated using the independent Student's t-test for samples. The number above each error bar shows the number of cases within each group.

[0224] Analysis of ABCB5 expression by flow cytometry. Analysis of the co-expression of ABC5 with CD20, CD31, VE-cadherin or BMPR1a surface markers or nestin or TIE-1 intracellular markers in clinical patient-derived melanoma cell suspensions was carried out using bicolor flow cytometry as described previously. Clinical melanoma cells were incubated with anti-ABCB5 mAb or mAb isotype control or without Ab, with subsequent APC-conjugated anti-mouse IgG staining. Cells were then fixed in PBS containing 2% paraformaldehyde (30 min at 4 ° C), and then incubated with PE-conjugated anti-CD20, anti-CD31, anti-VE-cadherin, anti-nestin or PE-conjugated mAbs isotype controls. or unconjugated anti-BMPR1a, anti-TIE-1 or unconjugated mAbs isotype controls with subsequent anti-staining of PE- or FITC conjugated anti-60immunoglobulin secondary antibodies. Washing steps with staining buffer or permeabilizing buffer with 1% saponin were carried out between each step. Bicolor flow cytometry was then performed, with fluorescence emission acquisition using FI1 (FITC) or F12 (PE) and F14 (APC) spectra on Becton Dickinson FACScan (Becton Dickinson, San Jose, CA) as described. Statistical differences in the expression levels of the markers listed above, relative to ABCB5 cells⁺ and ABCB5^- determined using a non-parametric Mann-Whitney test. A two-sided p-value of p &lt;0.05 was considered significant. A375 melanoma cells were analyzed for surface expression of ABCB5 by incubation with anti-ABCB5 mAb or mAb isotype control (10 μg / ml) with subsequent anti-staining with FITC-conjugated goat anti-mouse secondary antibody immunoglobulin and monochromatic flow cytometry (FI1) as described.

[0225] Cell isolation. Single cell suspensions were generated from human melanoma xenografts after surgical treatment of tumors from killed Balb / c NOD / SCID or Balb / c nude mice 8 weeks after tumor cell inoculation. Each tumor was cut into small pieces (approx. 1 mm³) and tumor fragments were then incubated in 10 ml sterile PBS containing 0.1 g / l calcium chloride and 5 mg / ml Serva NB6 collagenase (SERVA Electrophoresis GmbH, Heidelberg, Germany) for 3 hours at 37 ° C on a shaking platform at 200 rpm ./min to generate single cell suspensions. Subsequently, tumor cells were washed with PBS to remove excess collagenase. ABCB5 cells⁺ were isolated by positive selection and ABCB5 cell populations^- generated by removing ABCB5 cells⁺ using anti-ABCB5 mAb labeling and cell sorting with magnetic beads as described. Briefly, human G3361 or A375 melanoma cells or single cell suspensions derived from human melanoma xenografts or clinical melanoma samples were labeled with anti-ABCB5 mAb (20 μg / ml) for 30 min at 4 ° C, washed to remove excess antibody, and then incubated with magnetic microspheres coated with secondary anti-mouse IgG mAb (Miltenyi Biotec, Auburn, CA) and with subsequent cell separation with double passage through the columns to the MiniMACS separation (Miltenyi Biotec) according to the manufacturer's instructions. Purity of ABCB5 cell isolates⁺ and human ABCB5^- G3361 melanoma cells were determined by analysis of ABCB5 expression by flow cytometry (Fl1) on a FACSCalibur device (Becton Dickinson, Sunnyvale, CA) after incubation with FITC-conjugated anti-ABCB5 mAb, followed by incubation of anti-mouse IgG mAb and magnetic coated microspheres cell sorting. Statistical differences in ABCB5 expression between unsorted, ABCB5⁺, and ABCB5^- human G3361 melanoma cells were determined using one-way ANOVA followed by Bonferroni correction. A two-sided p-value of p &lt;0.05 was considered statistically significant.

[0226] Animals. Balb / c nude mice and Balb / c NOD / SCID mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were bred according to the institutional guidelines of Children's Hospital Boston and Harvard Medical School, and experiments were carried out according to approved experimental protocols.

[0227] Human melanoma xenotransplantation. Unsorted, ABCB5⁺, or ABCB5human G3361 (10⁷, 10⁶, or 10⁵/ vaccine, respectively), or human A375 (2 x 10⁶, 2 x 10⁵, or 2 x 10⁴/ vaccine, respectively), or clinical patient-derived melanoma cells (10⁶/ vaccine, respectively), or ABCB5 cells⁺ or ABCB5^- isolated from derived from ABCB5⁺ primary G3361 hetero tumor transplants (10⁷/ vaccine, respectively) were injected sc [subcutaneously] into one or both sides of the Balb / c NOD / SCID recipient mice. Tumor formation / growth was determined weekly as a course of time, at least up to an endpoint of 8 weeks, unless excessive tumor size required faster protocol-determined euthanasia, by determining tumor volume (TV) according to a fixed formula [TV (mm³) = π / 6 x 0.5 x length x (width)²]. Regarding tumor formation, mice were considered negative for tumor if no tumor tissue was identified after autopsy. Statistically significant differences in the formation of primary and secondary tumors were assessed using Fisher's proportional test. Differences in tumor volumes were determined using one-way ANOVA with subsequent Bonferroni correction or Kruskal-Wallis test with subsequent Dun correction, with two-sided p &lt;0.05 considered significant.

[0228] Tracking genetic pedigree in vivo. ABCB5 populations⁺/ DsRed2 and ABCB5^-/ EYFP human G3361 tumor cells, generated using cell sorting using magnetic beads as above, were reconstituted with relative numbers of 1x10⁶ and 9 x 10⁶ cells, respectively, with the subsequent determination of the resulting cell proportions in the grafts by two-color flow cytometry (Fl1 (EYFP) vs. Fl2 (DsRed2) plots) before xenotransplantation. Co-cultures G3361 / DsRed2 and G3361 / EYFP were implanted sc (10⁷cells / graft) to the right flank of Balb / c NOD / SCID recipient mice. At 4 or 6 weeks after xenotransplantation, tumors were harvested and single cell suspensions or sections of frozen tissue were obtained as above for the purpose of determining the relative in vivo number of DsRed2 melanoma cells⁺ and EYFP⁺ by two-color flow cytometry or fluorescence microscopy of tumor-derived single cell suspensions (after adhering to tissue culture attachment plates), and for analysis of 5 μm thick frozen tissue sections by fluorescence microscopy. In additional experiments, the relative number of DsRed2 melanoma cells⁺ and EYFP⁺ determined in ABCB5 cell subsets⁺ or ABCB5^- derived from xenografts, using bicolor flow cytometry as above, and DsRed2 tumor cell percentages⁺and EYFP⁺ were statistically compared using an unpaired student T-test, with a two-sided p-value of p &lt;0.05 considered statistically significant.

[0229] Anti-ABCB5 mAb targeting. Un-segregated human G3361 melanoma cells were sc x-transplanted into Balb / c nude recipient mice (10⁷/graft). The animals were injected with anti-ABCB5 mAb ip (clone 3C2-1D12), mAb isotype control (500 μg / injection) twice weekly or no Ab injected from 24 hours. before melanoma xenotransplantation. Tumor growth was determined twice a week as a time course, determining tumor volume (TV) as described above. Differences in tumor volumes were determined using nonparametric parameters

-62 one-way ANOVA (Kruskal-Wallis test) followed by Dun correction for comparisons between three experimental groups, with two-sided p values ​​&lt;0.05 considered significant. For the purpose of determining the in vivo binding efficacy of anti-ABCB5 mAb administered to established human to mouse nude melanoma xenografts, single cell suspensions and frozen sections were generated from melanoma xenografts 24 hours after ip administration antiABCB5 mAb, mouse IgG1 κ mAb isotype control, or in no treatment conditions. The resulting single cell suspensions were then incubated with FITC conjugated goat anti-mouse Ig secondary Ab for 30 min at 4 ° C and analyzed by one-color flow cytometry as above, and frozen sections were incubated with HRP-conjugated horse anti-mouse Ab secondary Ig analyzed as above.

[0230] ADCC and CDC assessment. ADCC or CDC was determined by bicolor flow cytometry as previously described. Briefly, suspensions of human G3361 melanoma cells in serum-free Dulbecco's Eagle modified media (DMEM) (BioWhittaker, Walkersville, MD) were assayed with 3,3'-dioctadecyloxacarbocyanine (DiO) (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. DiO-labeled melanoma cells were then placed at a density of 300,000 cells per well in 6-well flat bottom culture plates in 3 ml and grown in standard medium in a greenhouse with humidification overnight. Then, DiO-labeled target melanoma cells were preincubated in the presence or absence of anti-ABCB5 or mAbs isotype control (20 μg / ml, respectively) for 30 min at 37 ° C, 5% CO₂, and then co-cultured for an additional 24 hours at 37 ° C, 5% CO2 with or without freshly isolated effector splenocytes of Balb / c nude mice (12 x 10⁶ cells / well, 1:40 ratio of target to effector cells) for ADCC assessment, or in the presence or absence of 5% Balb / c nude mouse serum for CDC determination. Then, cells and their supernatants were harvested and analyzed by two-color flow cytometry on a FACSCalibur (Becton Dickinson) device immediately after the addition of 10 μg / ml propidium iodide (PI) (Sigma, Milwaukee, WN), with lyzed target cells recognized by the DiO phenotype⁺PI⁺. ADCC levels for the three treatment groups were calculated as follows: [ADCC (%) = (DIO⁺PI⁺ percentage of positive sample) - (average Ab-untreated DIO⁺PI⁺ positive sample percentage)]. Differences in ADCC levels were determined using nonparametric one-way ANOVA (KruskalWallis test) followed by Dun correction, with two-sided p &lt;0.05 considered significant.

[0231] Cell viability measurements. Cell viability was measured in tumor cell inoculations prior to xenotransplantation using calcein-AM staining. Briefly, 1x10⁶ non-segregated ABCB5 melanoma cells⁺, or ABCB5^- incubated with calceinAM (Molecular Probes, Eugene, OR) for 30 min at 37 ° C and 5% CO2 to allow substrate capture and enzymatic activation into a fluorescent derivative. Cells were then washed and fluorescence measurements obtained by flow cytometry on Fl2 emission spectra on Becton Dickinson FACScan. Showing cells

The generation of fluorescent calcein-AM compared to unexposed samples was considered viable. Cell viability was also determined in all samples using the trypan blue dye exclusion method.

[0232] RNA extraction and real-time quantitative reverse transcription-PCR. Extraction of RNA from G3361 and A375 human melanoma cells and standard cDNA synthesis reactions were performed using the SuperScript First-Strand Synthesis for reverse transcription-PCR (Invitrogen) as described previously. Total RNA obtained from 8 additional NCI-60 panel melanoma cell lines panel (LOX IMVI, SK-MEL-5, M14, UACC-62, SK-MEL-28, UACC-257, SK-MEL-2, MALME-3M) maintained at the National Cancer Institute under conditions and with a number of passages as described previously was provided by the NCI / NIH Developmental Therapeutics Program. Real-time quantitative reverse transcription-PCR for relative expression of ABCB5 genes was carried out as described previously. Expression of ABCB5 was assessed by the ratio of the expression level in the sample relative to the average expression in all samples, with n = 3 independent experiments. Growth data (culture doubling time) for 8 human melanoma cell lines from panel NCI-60 were those obtained by the National Cancer Institute that can be found online (<a href="http://dtp.nci.nih.gov/docs/misc/">http://dtp.nci.nih.gov/docs/misc/</a> common_files / cell_list.html). Growth kinetics for melanoma cell lines G3361 and A375 were determined in our laboratory by cell counting according to the formula: population doubling time (in hours) = T2 - T1 / (log2 (number of T2 cells / number of T1 cells)), where T2 and T1 are two separate time points (h) in the logarithmic growth phase of the culture. The linear correlation of ABCB5 relative mRNA expression and culture doubling times (h) were analyzed and the Pearson correlation coefficient was counted, and criteria p <0.05 and r> 0.3 or r <-0.3 were used to identify significant correlations as described previously.

Example 1 [0233] The association of ABCB5 with clinical progression of malignant melanoma was first investigated because of its close association with CD 166, a marker for a more advanced disease. This was estimated by means of immunohistochemical staining of ABCB5 and quantitative image analysis for microarray established tissue melanoma progression (TMA) containing 480 patient-derived melanoma tissue cores (0.6 mm), representing the four main diagnostic types of tumors: benign pigmented nevi, primary cutaneous melanoma , lymph node metastases and melanoma metastases to parenchymal organs (Fig. 1a). It was found that primary or metastatic melanomas expressed significantly more ABCB5 than benign pigmented moles (p &lt;0.001), thick primary melanomas expressed more ABCB5 than thin primary melanomas (p = 0.004), and lymph node metastatic melanomas expressed more ABCB5 than primary lesions ( p = 0.001), identifying ABCB5 as a new molecular marker of cancer progression in human malignant melanoma. Clear heterogeneity in ABCB5 expression was noted in metastases, with stronger staining in lymph nodes than in metastases in parenchymal organs (p = 0.025).

Example 2 [0234] When evaluated by flow cytometry in single cell suspensions freshly obtained from a smaller series of surgically prepared clinical melanomas (n = 6 patients, Table 1), ABCB5 was also found to be consistently expressed in 6 out of 6 samples, with a range of frequencies for ABCB5 tumor cells⁺ 1.6 to 20.4% (9.2 ± 3.2%, mean ± SEM) (Figure 1b, Table 1). Further phenotypic characterization with respect to antigens associated with the more primary molecular phenotype revealed significant CD20 expression in 3 of 6 samples (frequency in all samples: 0.3 ± 0.2%, mean ± SEM), nestin in 6 out of 6 (31, 9 ± 7.8%), TIE-1 in 6 out of 6 (24.9 ± 6.9%), VEkadherin in 4 out of 6 (0.2 ± 0.1%), BMPR1a in 6 out of 6 (1, 8 ± 1.0%), and the CD31 framework marker in 5 of 6 samples (0.8 ± 0.4%) (Fig. 1b). Preferential expression of ABCB5⁺ compared to the ABCB5 subpopulation^-, as previously identified for CD133 stem cell determinants, has been shown herein in these samples expressing relevant nestin markers (49.4 ± 6.6% vs. 26.6 ± 4.9%, respectively, mean ± SEM; p = 0.026) , TIE-1 (59.4 ± 7.8% vs. 23.8 ± 7.5%, p = 0.015), VE-cadherin (6.4 ± 1.2% vs. 0.1 ± 0.1 %, p = 0.029) and BMPR1a (37.0 ± 4.4 vs. 2.0 ± 0.2%, p = 0.002), but not for CD20 (0.2 ± 0.2% vs. 1.1 ± 0.7%, NS), or CD31 (2.4 ± 1.2% vs. 0.5 ± 0.3%, NS) (Fig. 1c). In situ immunohistochemistry revealed single ABCB5 cells⁺ or assemblies responsible for a smaller subpopulation within clinical tumors with positive-stained cells correlating primarily with non-stained, undifferentiated regions or TIE-1 expression, and non-reactive zones corresponding to stained, more differentiated areas.

[0235] Table 1 summarizes the tumor characteristics of six patients with a melanoma site (metastasis or primary recurrent). Tumors were quantified using% ABCB5 + present. A summary of results (number of mice with tumors) is also shown for nine groups of NOD / SCID mice transplanted (n = 2-10) with unsegregated, ABCB5 vaccines⁺ or ABCB5^- human melanoma cells.

Table 1 Patient and tumor characteristics

<td></td><td>No. patient</td><td>Place melanoma</td><td>ABCB5⁺ in the tumor</td><td colspan="3">Number of mice with tumors after transplantation</td>

<td></td><td></td><td></td><td> (%)</td><td>unsorted</td><td>ABC B5-</td><td>AB CB5 +</td>

<td> 1</td><td>P</td><td>Metastasis</td><td> 8,5</td><td> 0/2</td><td> 0/2</td><td> 2/2</td>

<td> 2</td><td>P</td><td>Metastasis</td><td> 1,6</td><td> 1/2</td><td> 0/2</td><td> 2/2</td>

<td> 3</td><td>P</td><td>Metastasis</td><td> 3,2</td><td> 5/5</td><td> 1/5</td><td> 5/5</td>

<td> 4</td><td>P</td><td>Metastasis</td><td> 20,4</td><td>ON</td><td>ON</td><td>ON</td>

<td> 5</td><td>P</td><td>Metastasis</td><td> 17,4</td><td>ON</td><td>ON</td><td>ON</td>

<td></td><td>No. patient</td><td>Place melanoma</td><td>ABCB5⁺ in the tumor</td><td colspan="3">Number of mice with tumors after transplantation</td>

<td></td><td></td><td></td><td> (%)</td><td>unsorted</td><td>ABC B5-</td><td>AB CB5 +</td>

<td> 6</td><td>P</td><td>Original</td><td> 4,2</td><td>ON</td><td>ON</td><td>ON</td>

Example 3 [0236] To determine if the subset of melanoma cells defined by ABCB5 was enriched in MMIC, we compared ABCB5 abilities⁺- cleaned (ABCB5⁺) vs. ABCB5⁺- depleted (ABCB5^-) melanoma cells to initiate tumor formation in vivo using established clonal cutaneous human melanomas from culture (G3361: 2-10% ABCB5 positivity; A375: 1-10% positivity, Fig. 5a) or freshly derived patient cells melanoma (Fig. 1b, Table 1) in humans for experiments with xenotransplantation of murine NOD / SCID tumors. Groups of NOD / SCID mice were transplanted (n = 2-10) with unsorted segregations, ABCB5⁺ or ABCB5^- human melanoma cells in the logarithmic range of times doses of cells unable to successfully initiate tumor growth (G3361: 10⁵ cells, A375: 2 x 10⁴ cells) relative to doses that consistently initiated tumor formation when ABCB5 cells were used⁺ (G3361: 10⁷cells, A375: 2 x 10⁶ cells, fresh isolates from patients: 10⁶ cells). Cell viability determined by calcein-AM staining exceeded 90% in all tumor cell inoculations and did not change significantly between isolates (Fig. 5b).

[0237] From 22 grouped mice injected with ABCB5 melanoma cells^- G3361 only 1 mouse transplanted with the highest dose of cells developed a tumor (Fig. 2a, left panel). Contrast, 13 out of 20 with injected ABCB5 cells⁺ formed tumors (p <0.0001), including all mice injected with the highest cell doses (Fig. 2a, left panel, additional p values ​​for individual dose-specific comparisons provided in Fig.), indicating> 2 logarithmic enrichment fold in MMIC in this subset of cells as determined by inoculated dose comparisons required for 50% tumor formation (TF50) (Fig. 2a, middle panel).

[0238] Similarly, of 21 grouped mice injected with ABCB5 melanoma cellsA375, only 8 mice developed a tumor, while 16 of 22 mice injected with ABCB5 cells⁺ formed tumors (P <0.05), indicating> 1 logarithmic fold enrichment for MMIC among ABCB5 cells⁺ A375 (Fig. 2b, left and middle panels). Purification of ABCB5 cells⁺ resulted in a 19.8-fold increase in frequency for ABCB5 cells⁺ from 5.0 ± 0.4% for unsorted cultures to 98.8 ± 0.8% (mean ± SD, n = 3, p <0.001) when determined in representative samples using G3361 melanoma cells, and depletion in ABCB5⁺ resulted in a 4.75 fold reduction in frequency for ABCB5 cells⁺ from 5.0 ± 0.4% to 1.1 ± 0.3% (mean ± SD, n = 3, p <0.001) (Fig. 5c). This is a residual contamination (22% of naturally occurring ABCB5 frequency⁺) from

-66 ABCB5 cells⁺ may be responsible for the observed tumor formation with ABCB5 vaccines^- at the highest doses, and suggests a potential underestimation of MMIC enrichment among the ABCB5 population⁺. Notably, in those cases where tumor formation occurred as a result of injection of ABCB5 cells^- at the highest cell doses, tumors have consistently been shown to be smaller than those resulting from ABCB5 xenografts⁺ (G3361: tumor volume (TV) = 15 ± 15 vs. 286 ± 90 mm³mean ± SEM, p &lt;0.01, respectively; A375: TV = 239 ± 70 vs. 832 ± 121 mm³mean ± SEM, p &lt;0.05, respectively) (Figures 2a and 2b).

[0239] Melanoma xenograft culture was heterogeneous and contained ABCB5 cells⁺ mostly correlating with non-stained regions and expression of VE-cadherin, and ABCB5 zones^- corresponding to the colored areas (Fig. 2c). ABCB5 cells⁺again purified from derived from ABCB5⁺ primary tumors formed secondary tumors more effectively than their ABCB5 'counterparts in 11 of 11 vs. 7 out of 12 recipients, respectively (p = 0.037) (Fig. 2d) and re-established the heterogeneity of the primary tumors. According to results obtained using the clonal melanoma model system, only 1 out of 9 recipient mice injected with 10⁶ freshly derived ABCB5 melanoma cells^- a tumor developed, while all 9 recipients of 10⁶ABCB5 melanoma cells⁺ formed tumors (p &lt;0.001), with mean TV less in recipients of ABCB5 vaccinations^- Vs. ABCB5⁺ (TV = 2 ± 2 vs. 35 ± 11 mm³mean ± SEM, p &lt;0.01, respectively) (Figure 2e, Table 1). Tumors generated from ABCB5 melanoma cells⁺ re-establish naturally occurring tumor heterogeneity with respect to ABCB5 expression, as determined by immunohistochemistry and flow cytometry of the dissociated tumor sample, with an ABCB5 positivity in the range of 2 to 8% (results not shown). These statements establish that the frequency of MMIC is clearly enriched in the minority melanoma population defined by ABCB5.

Example 4 [0240] To directly investigate the relative proportion of tumor growth of co-transplanted ABCB5 subpopulations⁺ and ABCB5^-, and for further study of ABCB5 self-reproduction and differentiation capabilities⁺, ABCB5 melanoma cells were isolated⁺or ABCB5^- from stably transfected variants of the G3361 cell line expressing red fluorescence protein (DsRed2) or enhanced yellow-green fluorescence protein (EYFP), respectively, a model system designed in our laboratory that allows tracking of the genetic pedigree in vivo. We have found that ABCB5 co-culture xenotransplantation⁺ G3361 / DsRed2 and ABCB5Fluorochrome transfected G3361 / EYFP restored, at 14.0 ± 3.0% and 86.0 ± 3.0%, the relative abundance (mean ± SD, n = 6), respectively, in NOD / SCID mice resulting in time-dependent , serially increasing relative frequencies for DsRed2 tumor cells⁺ on the origin of ABCB5⁺ (linear regression slope 6.4 ± 1.0, p &lt;0.0001) in experimental tumors compared to vaccines, up to a frequency of 51.3 ± 1.4% for the experimental endpoint of 6 weeks (mean ± SD, n = 3, p = 0.024) (Figs. 3a, 3b, and 3c upper and lower panels). These statements establish greater tumorigenicity

-67ABCB5⁺ Vs. co-xenografted mass populations of ABCB5 melanoma^- in a competitive tumor development model. Importantly, these results additionally indicate that tumor-initiating cells may additionally direct more diverse and spontaneously non-tumor mass tumor populations to also, although less effectively, contribute to tumor mass growth. The experimental tumors also contained DsRed2 / EYFP double positive melanoma cells (Fig. 3c middle panels), indicating that ABCB5 derived⁺ tumor cells, such as physiological ABCB5 progenitors⁺ from skin (Frank, NY et al. Regulation of progenitor cell fusion by ABCB5 Pglycoprotein, a novel human ATP-binding cassette transporter. J Biol Chem 278, 47156-65 (2003)), are involved in the fusion of cells with ABCB5 subgroups^-.

Example 5 [0241] When ABCB5 melanoma cells were purified⁺ from experimental tumors resulting from 10% ABCB5 co-xenotransplantation⁺ G3361 / DsRed2 and 90% ABCB5^-G3361 / EYFP fluorochrome transfectants, it was found that 92.9 ± 6.4% (mean ± SD, n = 3) of fluorescent cells have the DsRed2 phenotype⁺ (on the origin of ABCB5⁺) (Fig. 3d, upper left panel), which demonstrated the ability to self-renew this subset of cells. EYFP cells⁺ not found in significant amounts (7.1 ± 6.4%, mean ± SD, n = 3) among ABCB5 isolates⁺, and the low frequency observed was fully responsible for the scale of residual ABCB5 cell contamination measured⁺ among ABCB5 co-transplanted populations^- EYFP⁺ (1.1% of 90% EYFP cells⁺ = 0.99% vs. 10% ABCB5 cells⁺DsRed2⁺ in vaccinations), which indicates that ABCB5 tumor cells⁺ arose only from ABCB5 vaccines⁺ and that ABCB5 cells^- they only lead to ABCB5 offspring^-. In addition, fluorescent ABCB5 tumor cell isolates^- showed 52.5 ± 0.8% (mean ± SD, n = 3) of DsRed2 positivity (ABCB5 origin⁺) and 47.5 ± 0.8% of EYFP positivity (ABCB5 origin^-) (Fig 3d, lower left panel), indicating that ABCB5 melanoma cells⁺ have the ability to differentiate and lead to the formation of an ABCB5 tumor population^-. These statements show the existence of a tumor hierarchy in which ABCB5 melanoma cells⁺, enriched in MMIC, are self-renewing and lead to the emergence of more diverse ABCB5 tumor progeny^-.

Example 6 [0242] To mechanistically consider whether ABCB5-defined, MMIC-enriched minority population is required for tumor formation when non-segregated tumor mass populations are xenografted, it was examined whether selective killing of this subset of cells could inhibit tumor growth and formation . So far, the prospective molecular marker of tumor initiating cells has not been targeted for inhibition of tumor growth in vivo. We administered monoclonal antibody (mAb) directed to ABCB5 in a human model to nude xenograft melanoma because nude mice, in contrast to NOD / SCID, are able to kill tumor cells by mediated antibody-dependent cellular cytotoxicity (ADCC). Melanoma cells were sc x transplanted into nude Balb / c recipient mice, animals were injected with anti-ABCB5 mAb ip or control mAb twice in

-68 week starting 24 hours. before melanoma xenotransplantation, and tumor formation and growth were evaluated in series using TV measurements as the time course. Anti-ABCB5 mAb administration resulted in significantly inhibited tumor growth compared to that determined for control mAb-treated or untreated mice over the 58 day observation period (mean TV at endpoint 58 days for anti-ABCB5 mAb treated (n = 11 mice, no death) during the observation period) vs. control treated mAbs (n = 10 mice, excluding 1 death during the follow-up period) or vs. untreated (n = 18 mice, excluding 1 death during the follow-up period): 23 ± 16 vs. 325 ± 78 mm³, p &lt;0.01, or vs. 295 ± 94 mm³, p &lt;0.001, mean ± SEM, respectively) (Fig. 4a). Control, treated mAbs showed no significant difference in tumor growth compared to no treatment (Figure 4a). Anti-ABCB5 mAb treatment also significantly inhibited tumor formation, as assessed 58 days after melanoma cell xenotransplantation, with tumors detected only in 3 of 11 mice treated with anti-ABCB5 mAb, vs. 10 from 10 control mAb treated mice and 18 from 18 untreated control animals (p &lt;0.01 and p &lt;0.001, respectively) (Fig. 4b).

Example 7 [0243] Human melanoma xenografts growing in untreated nude mice, like those in NOD / SCID recipients, present tumor heterogeneity and contain a minority population of ABCB5 cells⁺ correlating primarily with undifferentiated, non-colored regions and ABCB5 zones^- corresponding to different colored regions (Fig. 4c). In vivo binding efficiency analysis revealed that systemically administered anti-ABCB5 mAb, but not control mAb, bound to a subset of tumor cells in established melanoma xenografts (Fig. 4d) according to the scale for the ABCB5 tumor cell subset.⁺ (Fig. 4c) as quantified in xenograft cell suspensions by flow cytometry (Fig. 4d) as well as by immunohistochemistry by detecting a positive staining cell group.

Example 8 [0244] To determine the mechanism of anti-ABCB5 mAb mediated inhibition of tumor formation and growth, ADCC immuno-effector responses and complement-dependent cytotoxicity (CDC) were assessed using bicolor flow cytometry as previously described. Anti-ABCB5 mAb treated, control mAb treated or untreated melanoma target cultures were labeled with a DiO green fluorescent membrane dye and counterstained with red fluorescent propidium iodide (PI for which only lyzed cells are permeable), following a culture with unlabeled immune effector cells or from spleen serum from Balb / c nude mice. Anti-ABCB5 mAb, but not mAb isotype control significantly induced ADCC-mediated melanoma target cell death (2.1 ± 0.4% vs. 0.2 ± 0.2%, respectively, p &lt;0.05) in a comparable melanoma subpopulation size to the ABCB5 expressing subset (Frank, NY et al. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res 65, 4320-33

-69 (2005)), as determined on the basis of the percentage of double-positive DiO / PI cells (Fig. 4e). Addition of serum to Ab-treated cultures in the absence of effector cells, or addition of anti-ABCB5 mAb alone under these experimental conditions did not induce a significant level of cell death compared to controls (results not illustrated), suggesting that CDC or the direct toxic effects of mAbs are not important causes of tumor inhibition in this experimental system.

[0245] The effect of targeting ABCB5 on established human to nude mice xenograft melanomas (n = 13 from three separate patients and in = 10 from established melanoma cultures) was investigated to examine the hypothesis that negative selection for MMIC mediated by ADCC mediated ABCB5 + cell ablation inhibits tumor growth. Such a result would be observable in a dynamic situation in vivo if the ABCB5 + melanoma subset was critical for intensive tumor formation.

[0246] Characteristics of ABCB5 + or ABCB5- human melanoma cells used in xenotransplantation experiments were undertaken. In vivo administration of anti-ABCB5 mAb, started 14 days after tumor cell inoculation after xenograft establishment (day 0), abolished the significant tumor growth observed in the mAb treated or untreated isotype control groups during the 21-day treatment period (p &lt;0.001 ip &lt; 0.001 respectively) and significantly inhibited the mean tumor volume compared to that determined for control mAb-treated or untreated mice (TV for anti-ABCB5 mAb treated (n = 23 mice) vs. control mAb treated (n = 22 mice) or untreated (n = 22 mice): 32.7 ± 9.4 vs. 226.6 ± 53.8 mm3, p &lt;0.001, or 165.4 ± 36.9 mm³, respectively, mean ± sem, p &lt;0.01). The inhibitory activity of ABCB5 mAb was also statistically significant when subsets of freshly derived patient melanoma tumor xenografts were analyzed independently, with abolition of significant tumor growth observed in isotype control groups treated with mAbs treated or untreated (p &lt;0.05 and p &lt;0.001, respectively) and significantly reduced mean TV compared to that determined for control mAb-treated or untreated mice (anti-ABCB5 mAb treated (n = 13 mice) vs. control treated with mAb (n = 12 mice) or vs. untreated (n = 12 mice): 29.6 ± 9.2 vs. 289.2 ± 91.8 mm³, p &lt;0.05, or vs. 222.9 ± 57.5 mm³, respectively, mean ± sem, p &lt;0.001). Control mAb treatment showed no significant effect on tumor growth or tumor volume compared to no treatment in any of the analyzed groups. The animals were sacrificed after the treatment period, in accordance with the requirements of the relevant experimental protocols for animals, due to tumor burden and disease status in control groups with a patient-derived tumor (measured maximum TV: 971.5 mm³).

[0247] Immunohistochemical analysis of anti-ABCB5 mAb treated patient-derived melanoma xenografts revealed only small foci of ABCB5 expression (total &lt;1% of cells) corresponding to the in vivo anti-ABCB5 mAb bound in the adjacent section. An additional adjacent section stained for CD11b revealed infiltration of macrophages corresponding to anti-ABCB5 mAb localization regions, often on the border of zones

-70 cellular degeneration and necrosis. In contrast, control treated mAb xenografts revealed that 10-15% of ABCB5-reactive cells, secondary anti-Ig mAb did not reach localization in the respective regions in the adjacent section, but murine immunoglobulin regions were detected and CDR11b + macrophages failed to infiltrate tumor tissue. Similar effects were observed in melanoma xenograft cell lineages with increased tumor necrosis in anti-ABCB5 mAb treated animals vs. mAb-treated isotype control (30-40% vs. &lt;5% of necrotic cells, respectively). These statements further support the concept that the ABCB5-enriched MMIC minority population is required for tumor formation.

[0248] Characteristics of G3361 melanoma xenografts were performed in nude Balb / c mice. ABCB5 + regions segregated with non-stained areas, while ABCB5 regions correlated with regions presenting granular brown-black color. Immunohistochemistry of melanoma xenograft treated with anti-ABCB5 mAb and stained with anti-ABCB5 mAb, secondary anti-Ig Ab or CD11b mAb revealed results consistent with those described above. As in the primary patient-derived xenografts, immunohistochemical analysis of adjacent tumor sections revealed that systemically administered anti-ABCB5 mAbs bound to ABC5 + tumor regions, which also correlated with CD11b + cell infiltration. Rare ABCB5 expression areas have also been detected to which the in vivo antibody failed to localize and to which CD11b positive cells failed to infiltrate.

Example 9 [0249] Sequencing of the 3C1 1D12 antibody: Extracted total RNA from pellets using the Fusion Antibodies Ltd RNA extraction protocol in-house RNA extraction protocol. cDNA was generated from RNA by reverse transcription with the oligo (dT) primer. PCR reactions using variable domain primers to amplify the variable region (VR) heavy chain (HC) and VR regions of the light chain (LC) of the monoclonal DNA of the antibody gave the bands shown in Figure 7. Both HC and LC VR PCR products were cloned into Invitrogen pCR2.1 sequencing vector and transformed into TOP10 cells. Positive heavy and light chain clones were selected for sequencing analysis. The following sequences were obtained.

1. Full length HC DNA sequence including signal sequence (underlined)

-71ATGGACTTTGGGCTGAGCTTGGTTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAG

TGTGAAGTGCAACTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAA GCTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGTC AGACTCCGGAAAAGAGGCTGGAGTGGGTCGCCACCATTAATGATGGCGGTACTCACACC TACTATCCAGACAGTCTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAT CCTGTACCTGCAAATGAGCAGTCTGATGTCTGAGGACACAGCCATGTATTATTGTGCAA GAGATGATTATTACTACGGTAGTCACTTCGATGCTATGGACTACTGGGGTCAAGGAACC TCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTC CAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGT CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG GTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTATĆCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGA SEQ ID NO: 17

2. Full length LC DNA sequence including signal sequence (underlined)

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCC

ACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAG GGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGC ACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAAC CTAGAATCTGAGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGG ncrTTnrnrcTTrccaccnnnrrjrrnBcrTrcassTfiisnccsrTCTccrTrrsrrn n XN- XXX "XX x Αίφφ WHWł 1Χ Χ ~ \ μΙ X \ J \ Ji X S" XXX \ JVJV A "XA

TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACC TACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTA CGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTGA SEQ ID NO: 18

3. VR HC DNA sequence, including CRDs (underlined)

GAAGTGCAACTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTG

AAGCTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCG

TCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCCACCATTAATGATGGCGGTACTCACA CCTACTATCCAGACAGTCTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAAC ATCCTGTACCTGCAAATGAGCAGTCTGATGTCTGAGGACACAGCCATGTATTATTGTGC AAGAGATGATTATTACTACGGTAGTCACTTCGATGCTATGGACTACTGGGGTCAAGGAA CCTCAGTCACCGTCTCCTCA SEQ ID NO: 9

4. DNA sequence of VR LC, including CRDy (underlined)

-72GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGG GCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCA CTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACC lAGAATCTGAGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTC AACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGA GCTTACACGTTCGGAGGGGGGCACCAAGCTGGAAATCAAACGG SEQ ID NO: 10

5. The amino acid sequence of VR HC, including framework regions (F1, F2, F3, and F4) and CRDs (CDR-H1, CDR-H2 and CDR-H3) as indicated. Framework regions and CDR regions were determined according to the Kabat nomenclature (EA Kabat et al. Sequences of Proteins of Immunological Interest, fifth edition, 1991, NIH).

HC-F1. CDR-H1

EVQLVESGGDLVKPGGSLKLSCAASGFTFS DYYMY

HC-F2, CDR_rH2. HC-F3.

WVRQTPEKRLEWVA TINDGGTHTY YPDSLKGRFTISRDNAKNILYLQMSSL

CDR-H3 HC-F4

MSEDTAMYYCAR DDYYYGSHFDAMDY WGQGTSVTVSS SEQ ID NO: 1

6. The amino acid sequence of VR LC, including framework regions (F1, F2, F3, and F4) and CRDs (CDR-L1, CDR-L2 and CDR-L3) as indicated. Framework regions and CDR regions were determined according to the Kabat nomenclature (EA Kabat et al. Sequences of Proteins of Immunological Interest, fifth edition, 1991, NIH).

LC-F1 CDR-L1 LC-F2

DIVLTQSPASLAVSLGQRATISY RASKSVSTSGYSYMH WNQQKPGQPPRLLIY

CDR-L2 LC-F3 CDR-L3

LVSNLES EVPARFSGSGSGDTFTLNIHPVEEEDAATYYC QHIRELTR

LC-F4

SEQIDNO SEGGTKLEIKR: 2

7. CDR-H1, CDR-H2 and CDR-H3 sequences:

CDR-H1: DYYMY SEQ ID NO: 3 CDR-H2: TINDGGTHTY SEQ ID NO: 4 CDR-H3: DDYYYGSHFDAMDY SEQ ID NO: 5

8. CDR-L1, CDR-L2 and CDR-L3 sequences:

CDR-L1: RASKSVSTSGYSYMH SEQ ID NO: 6 CDR-L2: LVSNLES SEQ ID NO: 7 CDR-L3: QHIRELTR SEQ ID NO: 8

Example 10: Future studies [0250] The ability to influence melanoma growth and progression by using a) two complementary sources of human melanoma will be investigated (established cell lines

-73 human melanoma and freshly isolated melanoma cells from primary and metastatic human tumors); b) two model systems for studying these cells (subcutaneous tumorigenic screening in immunodeficient mice, and the more significant tumor genesis found in authentic human skin xenografts); and c) two alternative strategies to combat melanoma stem cells (chemosensitization mediated by ABCB5 functional blockade, and killing of stem cells using immunotoxin or inhibitory siRNAs specifically delivered to ABCB5 + stem cell targets).

[0251] We will investigate whether ABCB5-directed reversal of chemo-resistance of melanoma stem cells can also inhibit tumor initiation / progression in chimeric Rag2 - / - mouse / human skin xenografts in vivo.

[0252] Tumor-targeted immunotoxins have been successfully constructed by coupling mAbs targeted to tumor-specific antigens to otherwise non-specific cytotoxic agents such as toxins, radionuclides and growth factors. In the proposed research, we will initially focus on the use of one such molecule, gelonin, 29 kDa ribosome inactivating plant toxin, because gelonin, when used in immunoconjugates targeting melanoma-specific antigens, has already shown that it exerts tumor-specific cytotoxicity in human xenograft models A375 melanoma, also used in this proposal, which indicates that gelonin immunoconjugates are excellent candidates for clinical development. In other future studies, we are also considering the study of radionuclide immunoconjugates involving, for example, yttrium, which is known to exert anti-melanoma. When using ABCB5-targeted gelonin immunotoxins as a strategy for selectively controlling ABCB5 + melanoma xenograft sub-populations in vivo, ABCB5-targeted gelonin immunotoxins will contain chemical gelonin / anti-ABCB5 3C2-1D12 mAb or gelonin / isotype control conjugates as synthesized as previously described and synthesized. In addition, due to the potential limitations of intact mAb immunoconjugates with respect to tumor penetration, we will also use recombinant anti-ABCB5 3C2-ID12 sFv / gelonin fusion proteins that will be constructed by fusing the anti-ABCB5 3C21D12 sFv gene, generated as above into gelonin DNA , using a PCR method with extension of overlapping splice sites. The recombinant fusion immunotoxin will be expressed in E. Cola and purified as previously described. Recombinant sFv / gelonin control fusion proteins will be generated in an identical manner from mAb isotype control producing mouse hybridoma cell lines.

[0253] ABCB5-mediated antibody specific for the delivery of siRNA to specific oncogenes to the target cell will also be developed and used as a strategy for selectively inhibiting ABCB5 + parent melanoma tumor populations in vivo. While the delivery of small interfering RNAs (siRNAs) to cells has until recently been a key obstacle to their therapeutic use in vivo, the efficacy of a new approach involving fusion proteins has recently been demonstrated.

Antibody / protamine as carriers for siRNA delivery, in systemic cell type specific siRNA delivery to melanoma tumors in experimental animal models in vivo, and have been shown to be effective in inhibiting melanoma growth in vivo when siRNAs directed to MYC, MDM2 and VEGF have an antibody directed to a receptor model expressed on B16 melanoma cells. This approach uses the advantage of protamine - the nucleic acid binding property that normally binds to DNA in semen to bind siRNAs with different specificities and delivers them to cells bearing a specific cell surface marker when protamine is conjugated with Fab or sFv antibody fragments specifically directed to such a marker. In order to use this strategy to target the ABCB5-expressing melanoma stem cells, a recombinant anti-ABCB5 3C2-1D12 sFv / protamine fusion protein (ABCB5 sFv-P) will be constructed by fusing the anti-ABCB5 3C2-1D12 gene fusion with protamine DNA using the PCR method of extending overlapping splice sites. The recombinant ABCB5 sFv-P fusion protein will be expressed and purified as previously described. ABCB5 sFv-P will initially be used to deliver MYC-targeted siRNAs, as the MYC-targeted expression reduction inhibits tumor growth in vivo not only in B16 mouse melanoma, but also in mice bearing established human melanoma xenografts, leading to extensive cell apoptosis. tumor by induction of p53 and inhibition of Bcl-2 proteins. MYC has already been found to be coherently expressed in subcopulations of human ABCB5 + melanoma. In addition, expression of human MYC genes can be effectively inhibited by RNAi approaches, and MYC-targeted siRNA oligonucleotides validated in these studies are commercially available from Dharmacon, Inc. (Chicago, IL.). The proposed studies will first analyze the ABCB5 sFv-P binding capacity for MYC siRNA, ABCB5 sFv-P mediated delivery of MYC siRNA to target cells and the resulting inhibition of the MYC gene, and ABCB5 sFv-P / MYC mediated siRNA blockade of tumor cell proliferation. in vitro in human G3361 and A375 melanoma cultures exactly as described previously.

[0254] The in vivo study protocol for targeting ABCB5 + melanoma stem cells will use tumor xenograft models, human to mouse, using both NOD-SCID mice as well as chimeric Rag2 - // human skin mice as recipients of human melanoma xenografts derived from established cell lines or freshly isolated patients - just as described above. In the first set of experiments aimed at assessing the effect of immunotoxins (ABCB5 mAb / gelonin or sFv / gelonin) or ABCB5 sFv-P / MYC siRNA on tumor initiation, immunotoxins (ABCB5 mAb / gelonin or sFv / gelonin or controls) will be administered in 0.25 ml sterile PBS by injection into the tail vein, and ABCB5 sFv-P complexed with MYC siRNA or controls will be administered on days 0.1 and 3 after tumor implantation by tail vein injection (80 μg siRNA in the injected volume of 100 μl in molar ratio ABCB5 sFv-P / total siRNA 1: 6 ) to murine recipients of human melanoma cell xenografts randomized on day 0 after xenotransplantation to the treatment and control groups below (n = 10

-75 animal samples for each melanoma cell line and for each tumor cell sample, freshly isolated from each of n = 10 primary melanomas, and = 10 metastases of melanoma, sc x-transplanted NOD-SCID mice or intradermally to the human skin chimera / Rag2 - / - mice: 1) ABCB5 mAb / gelonin 500 μg / mouse ivqod starting on day 0; 2) mAb / gelonin 500 μg / ivqod mouse isotype control starting on day 0; 3) ABCB5 sFv / gelonin 500 μg / mouse ivqod starting on day 0; 4) sFv / gelonin control 500 μg / ivqod mouse starting on day 0; 5) ABCB5 sFv-P / MYC siRNA iv on days 0.1 and 3; 6) ABCB5 sFv-P / control siRNA iv on days 0.1 and 3; 7) ABCB5 sFv-P iv on days 0.1 and 3. The treatment protocol is summarized in Table 2:

Table 2

<td>Group</td><td>number mice</td><td>Treatment</td>

<td> 1</td><td> 10</td><td>ABCB5 mAb / gelonin 500 μg / ivqod mouse starting on day 0</td>

<td> 2</td><td> 10</td><td>isotype control mAb / gelonin 500 μg / ivqod mouse starting on day 0</td>

<td> 3</td><td> 10</td><td>ABCB5 sFv / gelonin 500 μg / mouse ivqod starting on day 0</td>

<td> 4</td><td> 10</td><td>sFv / gelonin control 500 μg / mouse ivqod starting on day 0</td>

<td> 5</td><td> 10</td><td>ABCB5 sFv-P / MYC siRNA iv on days 0.1 and 3</td>

<td> 6</td><td> 10</td><td>ABCB5 sFv-P / siRNA iv control on days 0.1 and 3</td>

<td> 7</td><td> 10</td><td>ABCB5 sFv-P iv on days 0.1 and 3</td>

[0255] In a second set of experiments aimed at assessing the effect of an immunotoxin (ABCB5 mAb / gelonin or sFv / gelonin) or ABCB5 sFv-P / MYC siRNA on tumor progression of established tumors, mouse recipients of human melanoma cell xenografts will be randomized on day 7 after xenotransplantation (after tumor formation) to the treatment and control groups summarized in Table 7 (n = 10 animal copies for each melanoma cell line and for each tumor cell sample freshly isolated from each n = 10 primary melanomas and n = 10 metastases of melanoma, xenografted sc to NOD-SCID mice or intradermally to the human skin chimera / Rag2 - / - mice:

Table 3

<td>Group</td><td>number mice</td><td>Treatment</td>

<td> 8</td><td> 10</td><td>ABCB5 mAb / gelonin 500 μg / mouse ivqod starting on day 7</td>

<td>Group</td><td>number mice</td><td>Treatment</td>

<td> 9</td><td> 10</td><td>isotype control mAb / gelonin 500 μg / ivqod mouse starting on day 7</td>

<td> 10</td><td> 10</td><td>ABCB5 sFv / gelonin 500 μg / mouse ivqod starting on day 7</td>

<td> 11</td><td> 10</td><td>sFv / gelonin control 500 μg / mouse ivqod starting on day 7</td>

<td> 12</td><td> 10</td><td>ABCB5 sFv-P / MYC siRNA iv on days 7.8 and 10</td>

<td> 13</td><td> 10</td><td>ABCB5 sFv-P / iv siRNA control on days 7.8 and 10</td>

<td> 14</td><td> 10</td><td>ABCB5 sFv-P iv on days 7.8 and 10</td>

[0256] Clinical tumor formation / growth will be assessed daily as a time course by determining tumor volume (TV) according to the established formula [TV (mm3) = n / 6 x 0.5 x length x (width) 2] for the length of the experiment period (45 days). Statistically significant differences in tumor formation as a function of the treatment regimen used will be assessed using Fisher's proportional test. Differences in tumor volumes between experimental groups will be determined using nonparametric ANOVA. Bilateral p-values ​​<0.05 will be considered statistically significant. Immunofluorescence and immunohistochemical analysis of each transplanted tumor xenograft prepared from animals from all treatment groups, killed initially on day 45 of the experiment (subsequent killings [e.g. on days 10, 20, 30 and 45] will be conducted based on the findings of day 45, and in addition to primary tumor testing, killed animals will be necropsied, all metastases evaluated, and all tissues pathologically assessed for evidence of toxicity associated with the pattern used treatment). Expression of ABCB5 and co-expression of ABCB5 with CD133 will be assessed by sequential double staining of HRP / APimmunoenzymatic frozen melanoma xenograft sections as previously described. Tumor sections will be analyzed by light field microscopy, and the mean percentage of cells staining positive for each marker will be classified, semi-quantitatively (no positivity: -; <10% positivity: +; 10-50% positivity: ++;> 50 % positivity: +++) based on the number of cells in three microscopic fields (400x magnification) for each staining state as previously described. Using fluorescence microscopy and separate filters for each fluorochrome, RFP positive cells (ABCB5 + origin) and GFP positive cells (ABCB5- origin) will be counted (100 cells / sample) and the ratio of RFP / GFP cells within each tumor will be calculated . The average ratios from subsequent specimens of animals undergoing each treatment regimen will be statistically compared using non-parametric ANOVA. To assess the effectiveness of the ABCB5 + targeting strategy, apoptotic melanoma cells growing in murine subcutaneous tissue,

Human skin transplants at metastases will be identified according to established criteria used for light microscopy and confirmed in the TUNEL assay. Protein expression related to apoptosis pathways, including Bax, Bcl-2 and Bcl-XL will also be screened for immunohistochemistry. Finally, these results will correlate with the results of screening for markers associated with cell proliferation (MIB-1, PCNA, and cyclin D1 / D3). Positive cells will be counted manually for cross-sectional profiles, and by using computer-assisted imaging programs available in the co-PI's laboratory (GFM), which should significantly increase the efficiency of quantification.

Piotr Godlewski

Patent Attorney