ENDOSIALIN-BINDING ANTIBODY

Abstract

The present disclosure relates to the generation of an antibody that specifically recognizes and binds Endosialin, a cell surface antigen characteristic of tumor pericytes and cells of tumor stroma. The antibody has the ability to become internalized in Endosialin expressing cells and to block the activation of MAPK in PDGF stimulated human pericytes. The antibody is able to block angiogenesis induced by LGALS3BP, a known Endosialin interactor and to inhibit tumor growth alone and in combination with 1959, a humanized antibody against LGALS3BP in human osteosarcoma xenograft. Furthermore, upon conjugation of the hu manized version of the anti-Endosialin antibody with a duocarmycin derivative, the resulting ADC displays potent and antigen de pendent in vitro tumor cell cytotoxicity and effective antitumor efficacy in vivo. The disclosure is also related to nucleotides encoding the antibodies of the disclosure and cell expressing the antibodies.

Claims

1. An antibody or functional fragment thereof, which is directed against an epitope between amino acids 477-488 of human Endosialin according to SEQ ID No: 1.

2. The antibody of claim 1 which comprises (i) a heavy chain comprising: a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence as shown in SEQ ID No: 2 or a sequence differing in 1 or 2 amino acids therefrom, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence as shown in SEQ ID No: 3 or a sequence differing in 1 or 2 amino acids therefrom, and/or a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence as shown in SEQ ID No: 4 or a sequence differing in 1 or 2 amino acids therefrom, and/or (ii) a light chain comprising: a light chain complementarity determining region 1 (CDRL1) having the amino acid sequence as shown in SEQ ID No: 5 or a sequence differing in 1 or 2 amino acids therefrom, a light chain complementarity determining region 2 (CDRL2) having the amino acid sequence as shown in SEQ ID No: 6 or a sequence differing in 1 or 2 amino acids therefrom, and/or a light chain complementarity determining region 3 (CDRL3) having the amino acid sequence as shown in SEQ ID No: 7 or a sequence differing in 1 or 2 amino acids therefrom, or an antibody recognizing the same epitope on human FGRF4.

3. The antibody of claim 1 or 2 which comprises (i) a heavy chain comprising a CDRH1 as shown in SEQ ID NO: 2, a CDRH2 as shown in SEQ ID NO: 3 and a CDRH3 as shown in SEQ ID NO: 4, and (ii) a light chain comprising a CDRL1 as shown in SEQ ID NO: 5, a CDRL2 as shown in SEQ ID NO: 6 and a CDRL3 as shown in SEQ ID NO: 7.

4. The antibody of any one of the previous claims comprising: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID No: 8, or an amino acid sequence having a sequence identity of at least 90% thereto, and/or a light chain variable region comprising an amino acid sequence as shown in SEQ ID No: 9, or an amino acid sequence having a sequence identity of at least 90% thereto.

5. The antibody of any one of the previous claims, which is a Fab fragment, a Fab′ fragment, a F(ab′) fragment, a Fv-fragment, a diabody, ScFv, SMIP, single chain antibody, affibody, avimer, nanobody and/or a domain antibody.

6. The antibody of any one of the previous claims, which is of the IgG1-, IgG2-, IgG3-or IgG4-type or an IgM, IgA1, IgA2, IgAsec, IgD or IgE-type antibody.

7. The antibody of any one of the previous claims, which is a monoclonal antibody, in particular a murine antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, in particular a bispecific antibody, or a fragment thereof.

8. The antibody of any of the previous claims comprising a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 or an amino acid sequence having a identity of at least 90% thereto, and/or a light light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 or an amino acid sequence having a sequence identity of at least 90% thereto.

9. The antibody of any one of the previous claims, wherein a labeling group and/or to an effector group, preferably a therapeutic group is coupled to the antibody.

10. The antibody of claim 9, wherein the antibody is linked to a paramagnetic, radioactive or fluorogenic ion that is detectable upon imaging.

11. The antibody according to claim 9, wherein the antibody is linked to an anticellular agent, in particular an anti-mitotic or DNA damaging agent capable of killing or suppressing growth or cell division of endothelial cells.

12. The antibody of claim 11, wherein the anticellular agent comprises a chemotherapeutic agent, radioisotope or cytotoxin.

13. The antibody of claim 12, wherein the anticellular agent comprises an antimetabolite, an anthracycline, a vinca alkaloid, an antibiotic, an alkylating agent or a plant-, fungus- or bacteria-derived toxin.

14. The antibody of claim 12, wherein the anticellular agent comprises a DNA damaging agent, in particular a Minor Grove Binder duocarmycin derivative.

15. The antibody of claim 12, wherein the cytotoxin comprises an A chain toxin, a ribosome inactivating protein, a-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin or Pseudomonas exotoxin.

16. The antibody of claim 12, wherein the cytotoxin comprises deglycosylated ricin A chain.

17. The antibody of any one of claims 1-16, wherein the antibody recognizes human Endosialin that is expressed on the cell surfaces of tumor vascular cells to a greater degree than on the surfaces of normal endothelial cells.

18. The antibody of any one of claims 1-17, wherein the antibody is a bispecific antibody that recognizes the human tumor-associated antigen LGALS3BP (aka Mac-2 BP or 90K).

19. An isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid sequence encoding an antibody, antibody fragment or a derivative thereof of any of claims 1-18, preferably a nucleic acid sequence as shown in any one of SEQ ID NO: 14 to SEQ ID NO. 25 (b) a nucleic acid sequence complementary to any of the sequences in (a), and (c) a nucleic acid sequence capable of hybridizing to (a) or (b) under stringent conditions.

20. A vector comprising the nucleic acid sequence of claim 19, preferably an expression vector, wherein the nucleic acid sequence is operably linked to a control sequence.

21. A host comprising the nucleic acid of claim 19 or the vector of claim 20, which is preferably a human, bacteria, animal, fungal, amphibian or plant cell, or a non-human transgenic animal.

22. A process of manufacturing a monoclonal antibody according to anyone of claims 1-18 comprising the step of obtaining said antibody from the host of claim 21.

23. A pharmaceutical composition comprising an antibody of anyone of claims 1-18, a nucleic acid molecule of claim 19, a vector of claim 20, a host of claim 21, or an antibody generated by the process of claim 22, optionally in combination with a pharmaceutically acceptable carrier, diluent, and/or excipient.

24. The pharmaceutical composition according to claim 23, comprising a further active agent, such as a further antibody or antibody fragment, in particular an anti-neoplastic agent, preferably selected from the group consisting of antibodies, small molecules, antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule-targeting agents, kinase inhibitors, protein synthesis inhibitors, immuno-therapeutics, hormones or analogs thereof.

25. A compound selected from the antibody according to any one of claims 1-18, the nucleic acid molecule of claim 19, the vector of claim 20, the host of claim 21 and the pharmaceutical composition according to claim 24, for use in the prevention or treatment of a neoplastic disease or cancer.

26. The compound for the use of claim 25, wherein the disease is neuroblastoma, sarcoma (synovial sarcoma, fibrosarcoma, MFH, liposarcom, osteosarcoma), high-grade glioma, brain tumor, carcinoma (bladder, breast, colon, renal, gastric cancer, endometrial cancer, lung cancer, ovarian cancer) and/or a tumor expressing Endosialin in tumor vasculature and stroma and/or in tumor cells.

27. The compound for the use of claim 25 or 26, which is to be administered intravenously, intramuscularly, and/or subcutaneously.

28. The compound for the use of any one of claims 25-27, for administration in combination with a further therapeutic composition and/or irradiation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] FIG. 1 shows: (A) protein sequence of the target protein. (B) sequence of the different peptides use for immunization of the mice.

[0121] FIG. 2 shows that mMP-E-8.3, cMP-E-8.3 and the selected humanized antibody hMP-E-8.3 recognize human recombinant Endosialin by ELISA (2A) and flow cytometer (2B); and for mMP-E-8-3, also by laser scanning confocal microscopy (2C).

[0122] FIG. 3 shows that mMP-E-8.3 internalizes in Sjsa-1 Endosialin positive cells by flow cytometer (3A) and by laser scanning confocal microscopy (3B).

[0123] 15

[0124] FIG. 4 shows that mMP-E-8.3 inhibits the phosphorylation of MAPK Erk1/2 in PDGF stimulated human pericytes.

[0125] FIG. 5 shows that Endosialin status (positive versus negative) identifies patients with shorter DFS (A) and OS rate (B). The prognostic role of Endosialin status on survival (DFS and OS) was also examined in the context of LGALS3BP status (high versus low) (C,D).

[0126] FIG. 6 shows that cMP-E-8.3 inhibits LGALS3BP-induced tube formation by pericytes on matrigel.

[0127] FIG. 7 shows that cMP-E-8.3 restrains growth of the human osteosarcoma Sjsa-1 xenograft in nude mice. Also, the figure shows that the inhibitory effect of cMP-E-8.3 is potentiated by 1959, a humanized antibody against the tumor secreted protein, LGALS3BP.

[0128] FIG. 8 shows hMP-E-8.3 humanized sequences.

[0129] FIG. 9 shows hMP-E-8.3/ADC characterization

[0130] FIG. 10 shows hMP-E-8.3/ADC internalization in Sjsa-1 Endosialin positive cells by flow cytometer (2A) and by laser scanning confocal microscopy (2B).

[0131] FIG. 11 shows the correlation of hMP-E-8.3/ADC in vitro antitumor activity and Endosialin surface expression

[0132] FIG. 12 shows hMP-E-8.3/ADC in vitro antitumor activity is lost/reduced in SjSa cells knocked down for Endosialin surface expression by

[0133] CRISPR/Cas9 technology.

[0134] FIG. 13 shows hMP-E-8.3/ADC in vivo antitumor activity in SjSA-1 cells.

[0135] FIG. 14 shows that hMP-E-8.3/ADC in vivo antitumor activity is superior to the naked antibody in SjSA-1 cells.

EXAMPLES

Example 1: Production of the Monoclonal Antibody mMP-E-8.3

[0136] Four-weeks old Balb/c mice were immunized by intraperitoneal injection as emulsions in Complete Freund's Adjuvant (CFA) or Incomplete Freund's Adjuvant (IFA). Seven days later, mice were given an additional intraperitoneal injection of the immunogen. After additional seven days, mice were boosted intravenously with the immunogen, and spleens were removed for cell fusion 3 days later. Somatic cell hybrids were prepared by fusion of immune splenocytes with the murine non-secreting myeloma cell line NS-1. Hybridoma supernatants were selected with Elisa assay towards the respective peptide. All positive hybridoma cell colonies were cloned twice by limiting dilution and further characterized.

[0137] In FIG. 1A shows the sequence of the target protein; in FIG. 1B, a list of the peptides used for immunization (sequence of peptide of mMP-E-8.3 highlighted). All positive hybridoma supernatants were checked in ELISA for antigen affinity, and mMP-E-8.3 was selected as the antibody that recognised the antigen with higher affinity.

Example 2: mMP-E-8.3, cMP-E-8.3 and hMP-E-8.3 are Able to Recognize Endosialin by ELISA; mMP-E-8.3 by Flow Cytometer and Confocal Microscopy

[0138] Materials and Methods: (FIG. 2). (A) Ninety-six well plates (NUNC Maxisorp modules) were pre-coated with human recombinant Endosialin (1 pg/ml) overnight at 4° C. After blocking with 1% BSA in PBS+0.1% Tween-20 for 1 hour at 4° C., mMP-E-8.3, cMP-E-8.3, hMP-E-8.3 and a commercial antibody against Endosialin at the indicated concentration were added and incubated for 2 hours at room temperature. After several washes with PBS+0.1% Tween-20, a goat anti-mouse or anti-human IgG-HRP solution was added to each well and incubated for 1 hour at 37° C. After washes, stabilized chromogen was added to each well for at least 10 minutes in the dark, then the reaction was stopped with the addition of 1 N H.sub.2SO4 and the absorbance was read at 450 nm with an ELISA reader. (B) Sjsa-1 human osteosarcoma cell line were stained with 1 μg/ml of mMP-E-8.3 antibody, or cMP-E-8.3 and hMP-E-8.3 at 100 ng/ml (blue line) and 1 ug/ml (green line) or with 1 μg/ml of a commercial antibody against Endosialin on ice for 30 minutes after incubation with a Goat anti-mouse/anti-human Alexa-488 conjugated antibodies for 1 hour on ice, cells were analyzed by flow cytometer (FACS). (C) Sjsa-1 human osteosarcoma cells were grown on glass coverslips for 24 hours. Cells were then fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.25% Triton X-100 for 5 minutes, and blocked with 0.1% BSA for 1 hour at room temperature. Coverslips were incubated for 2 hours at room temperature with mMP-E-8.3 or a commercial antibody, followed by goat anti-mouse secondary antibody Alexa Fluor 488 conjugated. DRAQ5 was used to visualize nuclei. Images were acquired with a Zeiss LSM 510 meta-confocal microscope using 488and 633-nm lasers. The yellow arrows indicate that mMP-E-8.3 recognize Endosialin present on the cell plasma membrane.

[0139] Results: mMP-E-8.3, cMP-E-8.3 and the selected humanized variant hMP-E-8.3 recognize Endosialin by ELISA and flow-cytometer; murine, antibody was able to recognize human Endosialin expressed by Sjsa-1 cells by laser scanning confocal microscopy (FIG. 2C)

Example 3:mMP-E-8.3 Internalization in Sisa-1 Human Osteosarcoma Cell Line

[0140] Materials and Methods: (FIG. 3). Sjsa-1 cells were plated in 12 well-plates and grown in 10% FBS RPMI-1640 for 24 hours. Cells were then incubated with 10 μg/ml of mMP-E-8.3, for 30 minutes on ice and returned at 37° C. for 6 hours. (A) After 6 hours, cells were stained with a goat anti-mouse Alexa 488-conjugated secondary antibody and analysed by FACS. (B) After 6 hours, cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton-X100 in PBS and then stained with a fluorescein-labeled goat anti-mouse/anti-human antibody (green staining). Cell nuclei were counterstained in blue. The yellow and the white arrows indicate antibody localization at the cell membrane and the cytoplasm, respectively.

[0141] Results: (A) Sjsa-1 cells show goat anti mouse membrane positivity after 30 minutes of mMP-E-8.3 incubation on ice indicating that the antibody is completely localized on the plasma membrane. After 6 hours at 37° C., the goat anti-mouse signals is reduced by 60% indicating that mMP-E-8.3 has been internalized by cells. (B) Sjsa-1 cells show goat anti-mouse membrane positivity (yellow arrows) after 30 minutes of mMP-E-8.3 incubation on ice indicating that the antibody is completely localized on the plasma membrane. After 6 hours at 37° C., the goat anti mouse signals present inside the cells, in particular in the peri-nuclear region (white arrows).

Example 4: mMP-E-8.3 Blocked PDGF Signaling in Human Pericytes

[0142] Materials and methods: (FIG. 4). T/G HA-VSMC, a human vascular smooth muscular cell line were seeded for 24 hours in 12 well-plates, then were serum starved for 2 hours in pericyte's culture medium lacking serum and growth factors. Cells were then incubated with 10 μg/ml of mMP-E-8.3 antibody or a negative control antibody for 2 hours and then stimulated for 15 minutes with PDGF-BB (100 ng/mL). Cells were lysed directly with RIPA buffer and 30 pg of total lysates were subjected to western blot analysis to detect Endosialin, the phosphorylated form of Akt and MAPK. Actin was using as a loading control.

[0143] Results: Cells pre-treated with mMP-E-8.3 exhibit an inhibition of MAPK phosphorylation induced by PDGF treatment (FIG. 4)

Example 5: Production of Chimerized and Humanized Versions of the mMP-E-8.3 Antibody

[0144] Methods for humanizing non-human antibodies are well known in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers.sup.29-32, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework region (FR) residues are substituted by residues from analogous sites in rodent antibodies.

[0145] To produce the chimerized version of mMP-E-8.3 antibody (called cMP-E-8.3), hybridoma cells producing the mMP-E-8.3 were expanded, total RNA extracted and RT-PCR performed to clone and sequence the variable regions of the antibody using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

[0146] For antibody chimerization, the murine constant regions were replaced with the human constant regions. It is a G1m17 IgG1 allotype with a human km3 kappa LC.

[0147] For antibody humanization, Complementarity Determining Regions (CDRs) from the murine were grafted in to a human antibody framework. Four humanized version of the heavy chain (HC) and light chain (LC) were designed and combined, obtaining the following antibody variants:

[0148] 8.3-LIBR-H1L1 (No. E02999)

[0149] 8.3-LIBR-H1L2 (No. E03000)

[0150] 8.3-LIBR-H1L3 (No. E03001)

[0151] 8.3-LIBR-H1L4 (No. E03002)

[0152] 8.3-LIBR-H2L1 (No. E03003)

[0153] 8.3-LIBR-H2L2 (No. E03004)

[0154] 8.3-LIBR-H2L3 (No. E03005)

[0155] 8.3-LIBR-H2L4 (No. E03006)

[0156] 8.3-LIBR-H3L1 (No. E03007)

[0157] 8.3-LIBR-H3L2 (No. E03008)

[0158] 8.3-LIBR-H3L3 (No. E03009)

[0159] 8.3-LIBR-H3L4 (No. E03010)

[0160] 8.3-LIBR-H4L1 (No. E03011)

[0161] 8.3-LIBR-H4L2 (No. E03012)

[0162] 8.3-LIBR-H4L3 (No. E03013)

[0163] 8.3-LIBR-H4L4 (No. [03014)

[0164] 8.3-LIBR-H1L2 (No. E03000) was chosen as the best candidate based on affinity, antibody titer and stability.

Example 6. Prognostic Value of Endosialin in Human Colorectal Cancer

[0165] Materials and methods: Endosialin expression was analyzed in human primary colorectal cancer, diagnosed without lymph-node or distant metastases, from 175 patients by immunohistochemistry on Tissue Micro Arrays (TMAs). Results were correlated with patients outcome. One hundred forty-two (81.1%) patients had colon cancer and 33 (18.9%) had rectal cancer. One hundred twelve patients were males (64.0%) and 63 patients were females (36.0%). The median age of the patients at the time of diagnosis was 70 years (range 36-90). The median follow-up time was 54.0 months (range 3-238). Five-micron TMA sections were prepared for immunohistochemical staining. Staining was made by using anti-endosialin (TEM1) rabbit polyclonal antibody (Novus Biological) and anti-LGALS3BP mouse monoclonal antibody 1A422. Antigen retrieval was performed by microwave treatments at 750 W (10 min) in citrate buffer (pH 6.0). The anti-rabbit or anti-mouse EnVision kit (Dako) was used for signal amplification. To exclude unspecific staining, non-immune serum was included. The relationship between Endosialin expression and clinicopathologic characteristics of the patients was assessed by χ2 test. Survival analysis was done by the Kaplan-Meier method and the groups were compared with the log-rank test. Statistical procedures were done using SPSS version 15.0 (SPSS Inc.). P<0.05 was considered as statistically significant.

[0166] Results: Thirty-seven out of 175 (21.1%) cases expressed Endosialin in the cytoplasm of tumor cells which also coexisted with a specific positive staining of stromal cells in 11 out of 37 (29.7%) positive cases. The proportion of Endosialin positive tumor cells was in the range of 4 to 100%, with a mean±SE of 45.4±5.3. All these cases were considered Endosialin positive. Statistical analysis revealed no relationship between Endosialin protein expression and any of the clinicopathological parameters evaluated. A disease relapse was observed in 37.8% (14/37) of patients with Endosialin positive, and in 21.0% (29/138) of those with Endosialin negative tumors. Death occurred in 29.7% and 13.9% of patients with positive and negative Endosialin tumors, respectively. At Kaplan-Meier analyses, expression of Endosialin was significantly associated with a lower OS (P=0.037) (FIG. 5A) and DFS (P=0.038) (FIG. 5B).

[0167] As LGALS3BP is an Endosialin binding partner.sup.6 and the inventors developed a humanized monoclonal antibody against LGALS3BP (Use of anti-90k monoclonal antibodies for the prevention and treatment of tumors and metastases thereof WO 2010097825 Al), the prognostic role of Endosialin expression on survival (DFS and OS) was also examined in the context of LGALS3BP status. LGALS3BP was found to be a negative prognostic factor in the majority of human cancers, except in colon carcinoma where LGALS3BP lower expression in CRC tissues was found as a marker of poor prognosis.

[0168] Endosialin positivity identified patients with lower OS and DFS rate (FIGS. 5C and D) in LGALS3BP low expression cases (P=0.015 and P=0.040, respectively). Conversely, LGALS3BP high expression identified patients at significantly lower probability of relapse and death in Endosialin negative cases.

Example 7: Effect of cMP-E-8.3 on Tube Formation on Matrigel

[0169] Materials and methods: (FIG. 6). T/G HA-VSMC human vascular smooth muscular cells were seeded at a density of 5×10.sup.4 cells/well in F12K serum free medium. Cells were maintained in F12K serum free medium containing 10 μg/ml recombinant LGALS3BP in the absence or presence of cMP-E-8.3 at the concentrations of 20 or 40 μg/ml. PDGF (100 ng/ml) was used as a positive control. (A) Representative phase-contrast photographs of capillary-like tube formation by T/G HA-VSMC on Cultrex (Matrigel)-coated chamber slides. (B) Histograms show quantitative determination of tube formation by counting number of branch points in 4 different fields. Data are represented as mean±SEM of three independent experiments. *p<0.05.

[0170] Results: The chimeric antibody cMP-E-8.3 is able to inhibit pericyte's tube formation on matrigel induced by LGALS3BP in a dose dependent manner.

Example 8: Effect of cMP-E-8.3 on Osteosarcoma Cancer Xenografts

[0171] Materials and Methods: (FIG. 7) Human osteosarcoma cancer xenografts were established by injecting subcutaneously 5×10.sup.6 Sjsa-1 cells in 5-week old CD1 female nude mice. Three days after cell injection, mice randomly divided into four groups of 10 animals. One group received intraperitoneal injection twice per week of 15 mg/kg of 1959 (a humanized antibody against LGALS3BP) in PBS buffer, or cMP-E-8.3 antibody at 15 mg/kg or a combination of both antibodies, each at 15 mg/kg. One group received PBS only (control group). Tumor volume was monitored two times a week by a caliper.

[0172] Results: cMP-E-8.3 treated mice show up to 40% reduction of tumor volume compared to the control mice, while the group receiving 1959 and cMP-E-8.3 show up to 70% reduction of tumor volume. *p≤0.05; **p≤0.01.

Example 9: Production and Characterization of hMP-E-8.3/ADC

[0173] ADC preparation: The hMP-E-8.3/ADC was generated by partially reducing the hMP-E-8.3 antibody and conjugating the drug, a potent Minor Grove Alkylating Agent derivative of duocarmycin bearing an enzymatically cleavable linker (valine-citrulline) to the available reduced inter-chain cysteine residues. The produced hMP-E-8.3/ADC was characterized by SDS-PAGE under reducing and non reducing conditions. Three pg of naked mAb or ADC both for reducing (R) and non reducing (NR) were loaded (FIG. 9A). Size Exclusion Chromatography (SEC) was performed to determine the aggregation state. Signal was detected at two different wavelengths 220 (Blue) and 320 nm (Red) to monitor antibody and drug, respectively (FIG. 9B). Hydrophobic interaction chromatography (HIC) was performed to evaluate the presence of differently loaded isoforms in native conditions; PLRP LC/MS in reducing conditions was performed to determine the Drug Antibody Ratio (DAR). Results:No antibody degradation or aggregation was detected in the tested preparation (FIGS. 9A and B). The calculated DAR was 3.6 (FIG. 9C).

Example 10: hMP-E-8.3/ADC is Internalized by SjSA-1 Cells

[0174] Materials and Methods: (FIG. 10) Sjsa-1 cells were plated in 12 well-plates and grown in 10% FBS RPMI-1640 for 24 hours. Cells were then incubated with 10 μg/ml of hMP-E-8.3/ADC, for 30 minutes on ice and returned at 37° C. for 6 hours. (A) After 2 hours, cells were stained with a goat anti-human Alexa 488-conjugated secondary antibody and analysed by FACS. (B) After 2 hours, cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton-X100 in PBS and then stained with a fluorescein-labeled goat anti-human antibody (green staining). Cell cytoplasm was counterstained in red using Alexa Fluor phalloidin. The white arrows indicate antibody localization in the cytoplasm in cells returned at 37° C.

[0175] Results: (A) Sjsa-1 cells show goat anti human membrane positivity after 30 minutes of mMP-E-8.3 incubation on ice indicating that the antibody is completely localized on the plasma membrane. After 2 hours at 37° C., the goat anti-human signals is reduced by 80% indicating that hMP-E-8.3 has been internalized by cells. (B) Sjsa-1 cells show goat anti-human membrane positivity after 30 minutes of hMP-E-8.3 incubation on ice indicating that the antibody is completely localized on the plasma membrane. After 2 hours at 37° C., the goat anti human signals present inside the cells, in particular in the peri-nuclear region (white arrows).

Example 11: hMP-E-8.3/ADC in vitro antitumor activity correlates with Endosialin Surface Expression Level

[0176] Materials and Methods: Human Osteosarcoma Cancer (SjSa-1), Ewing's sarcoma (A673), neuroblastoma (SKNAS) and melanoma (A375) cells were plated in 24 wells (1×10.sup.3 per well) and growth in media supplemented with 10% serum in the presence or not of increasing amount of hMP-E-8.3/ADC (0.03 to 1.6 μg/ml). After 144 hrs from the beginning of treatment cells were harvested and processed for MTT staining. Results are shown as % of control (PBS treated cells). Results: hMP-E-8.3/ADC shows a strong and dose-dependent ability to inhibit cell growth. Moreover, this in vitro antitumor activity of hMP-E-8.3/ADC correlates with the amount of Endosialin receptor expression on cell surface (FIG. 11).

Example 12: hMP-E-8.3/DC54 Activity is Nearly Lost in Endosialin Knocked Down SjSa-1 Cells

[0177] Materials and Methods: TEM-1 expression was ablated in SJSA-1 cells by means of CRISPR-Cas9 system of genome editing, in accordance with the protocol developed by Zhang and co-workers.sup.33. After transient transfection Endosialin not-expressing cells were sorted by FACS and single cell clones isolated and propagated. Using FACS and WB clones were analyzed for Endosilain expression. Clone #3 resulted with a complete knock down for Endosialin expression. Gene destruction of both alleles was confirmed by genomic DNA sequencing.

[0178] Results: loss of Endosialin expression on surface of SjSa-1 cells dramatically reduced hMP-E-8.3/ADC killing activity, indicating that ADC efficacy is target-dependent (FIG. 12) 30

Example 13: hMP-E-8.3/ADC Shows a Potent and Durable Antitumor Activity in Human Osteosarcoma Cancer (SjSa-1) Xenograft

[0179] Materials and Methods: Human osteosarcoma cancer xenografts were established by injecting subcutaneously 2.5×10.sup.6 Sjsa-1 cells in 5-week old CD1 female nude mice. Once tumor become palpable (Tumor Volume range 100 mm.sup.3), mice were randomly divided into two groups of 6 animals. One group received intravenous injection once/weekly for two weeks of 10 mg/kg of hMP-E-8.3/ADC in PBS buffer, whereas the control group received PBS only. Tumor volume was monitored every week by a caliper. For Kaplan Meier survival curve the cut-off value for this study was volume of 1500 mm.sup.3.

[0180] Results: hMP-E-8.3/ADC treated mice show a significant and durable reduction of tumor growth. Moreover, two complete remission were observed in treated mice up to 100 days form starting of treatment. Kaplan Mayer survival curve demonstrate a significant increase of survival in hMP-E-8.3/ADC treated mice (Log-rank (Mantel-Cox) Test p=0.02) (FIG. 12). Of note, hMP-E-8.3/ADC at the dosage used in this study resulted well tolerated by the animals, as no toxicity was observed in terms of weight loss.

Example 14: hMP-E-8.3/ADC Shows Superior Antitumor Activity Over the Naked Antibody in Human Osteosarcoma Cancer (SjSa-1) Xenograft

[0181] Materials and Methods: Human osteosarcoma cancer xenografts were established by injecting subcutaneously 2.5×10.sup.6 Sjsa-1 cells in 5-week old CD1 female nude mice. Once tumor become palpable (Tumor Volume range 100 mm.sup.3), mice were randomly divided into three groups of 6 animals. One group received intravenous injection twice/weekly for two weeks of 10 mg/kg of hMP-E-8.3/ADC or naked hMP-E-8.3 antibody in PBS buffer, whereas the control group received PBS only. Tumor volume was monitored every week by a caliper. For Kaplan Meier survival curve the cut-off value for this study was volume of 1500 mm.sup.3.

[0182] Results: The naked antibody slightly reduced tumor growth, although the reduction in tumor size was not statistically significant. On the other hand, a significant and durable tumor growth inhibition was observed in mice treated with the ADC, demonstrating that the cytotoxic compound confers a far superior antitumor activity to the hMP-8.3 mAb (FIG. 13). Kaplan Mayer survival curve demonstrate a significant increase of survival in hMP-E-8.3/ADC treated mice (Log-rank (Mantel-Cox) Test p=0.002). Of note, hMP-E-8.3/ADC at the dosage used in this study resulted well tolerated by the animals, as no toxicity was observed in terms of weight loss.

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