BINDING PROTEIN DRUG CONJUGATES COMPRISING ANTHRACYCLINE DERIVATIVES

Abstract

The present invention relates to an anthracycline (PNU) derivative conjugate comprising a derivative of the anthracycline PNU-159682 having the formula (i) or formula (ii) which further comprises a linker structure X-L1-L2-L3-Y.

Claims

1. An anthracycline (PNU) derivative conjugate or a binding protein-drug conjugate (BPDC) the same, said conjugate comprising a derivative of the anthracycline PNU-159682 having the following formula (i) or formula (ii) ##STR00006## said conjugate comprising at its wavy line a linker structure X-L.sub.1-L.sub.2-L.sub.3-Y, wherein L.sub.1-L.sub.3 represent linkers, and two of L.sub.1-L.sub.3 are mandatory, and wherein X and Y further represent each one or more optional linkers.

2. A binding protein-drug conjugate (BPDC), having the following formula: ##STR00007## wherein a) L.sub.1-L.sub.3 represent linkers, and two of L.sub.1-L.sub.3 are mandatory, b) X any Y each represent one or more optional linkers, c) BP is a binding protein, and d) n is an integer ≧1 and ≦10.

3. The anthracycline (PNU) derivative conjugate according to claim 1, wherein the linker structure comprises, as L.sub.2, an oligo-glycine peptide (Gly).sub.n coupled to said anthracycline derivative, directly or by means of another linker L.sub.1, in such a way that the oligo-glycine (Gly).sub.n peptide has a free amino terminus, and wherein n is an integer ≧1 and ≦21.

4. The anthracycline (PNU) derivative conjugate or the binding protein-drug conjugate (BPDC) according to claim 1, wherein the oligo-glycine peptide (Gly).sub.n is conjugated to the anthracycline derivative of formula (i) by means of an alkylenediamino linker (EDA), designated as L.sub.1, which alkylenediamino linker is conjugated to the anthracycline derivative by means of a first amide bond, while it is conjugated to the carboxy terminus of the oligo-glycine peptide by means of a second amide bond, said conjugate of alkylenediamino linker and oligo-glycine peptide having the following formula (v), ##STR00008## wherein the wavy line indicates the linkage to the anthracycline derivative of formula (i), wherein m is an integer ≧1 and ≦11 and n is an integer ≧1 and ≦21.

5. The anthracycline (PNU) derivative conjugate or the binding protein-drug conjugate (BPDC) according to claim 3, wherein the oligo-glycine peptide (Gly).sub.n is, directly or by means of another linker L.sub.1, coupled to Ring A of the anthracycline derivative of formula (ii).

6. The anthracycline (PNU) derivative conjugate or the binding protein-drug conjugate (BPDC) according to claim 3, wherein the oligo-glycine peptide (Gly.sub.n) is conjugated to the anthracycline derivative of formula (ii) by means of an alkyleneamino linker (EA), designated as L.sub.1, which alkyleneamino linker is conjugated to the carboxy terminus of the oligo-glycine peptide by means of an amide bond, said conjugate of alkyleneamino linker and oligo-glycine peptide having the following formula (vi) ##STR00009## wherein the wavy line indicates the linkage to the anthracycline derivative of formula (ii), wherein m is an integer ≧1 and ≦11 and n is an integer ≧1 and ≦21.

7. The binding protein-drug conjugate (BPDC) according to claim 3, wherein the linker structure L.sub.3 comprises a peptide motif that results from specific cleavage of a sortase enzyme recognition motif.

8. The binding protein-drug conjugate (BPDC) according to claim 7, wherein said sortase enzyme recognition motif comprises a pentapeptide.

9. The binding protein-drug conjugate (BPDC) according to claim 7, wherein said sortase enzyme recognition motif comprises at least one of the following amino acid sequences LPXTG, LPXSG, and/or LAXTG.

10. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the anthracycline (PNU) derivative is conjugated, by means of the one or more linkers, to the carboxy terminus of the binding protein, or to the carboxy terminus of at least one domain or subunit thereof.

11. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the binding protein is conjugated to the free amino terminus of the oligo-glycine peptide (Gly.sub.n) by means of an amide bond.

12. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the binding protein is at least one selected from the group consisting of an antibody, modified antibody format, antibody derivative or fragment, antibody-based binding protein, oligopeptide binder and an antibody mimetic.

13. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the binding protein binds at least one entity selected from the group consisting of a receptor, an antigen, a growth factor, a cytokine, and/or a hormone.

14. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the binding protein has at least two subunits.

15. The binding protein-drug conjugate (BPDC) according to claim 14, wherein at least one subunit comprises a derivative of the anthracycline PNU-159682.

16. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the binding protein binds HER-2.

17. The binding protein-drug conjugate (BPDC) according to claim 16, wherein the binding protein is an antibody that binds HER-2.

18. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the antibody is characterized as follows: a) comprises the CDR regions 1-6 of trastuzumab; b) comprises the heavy chain variable domain and the light chain variable domain of trastuzumab; c) has an amino acid sequence identity of 90% or higher with the regions or domains of a) or b); d) is trastuzumab, or a target binding fragment or derivative thereof, and/or e) competes with trastuzumab for binding to HER-2.

19. The binding protein-drug conjugate (BPDC) according to claim 2, wherein the binding protein binds CD30.

20. The binding protein-drug conjugate (BPDC) according to claim 19, wherein the binding protein is an antibody that binds CD30.

21. The binding protein-drug conjugate (BPDC) according to claim 19, wherein the antibody is characterized as follows: a) comprises the CDR regions 1-6 of brentuximab; b) comprises the heavy chain variable domain and the light chain variable domain of brentuximab; c) has an amino acid sequence identity of 90% or higher with the regions or domains of a) or b); d) is brentuximab or a target binding fragment or derivative thereof, and/or e) competes with brentuximab for binding to CD30.

22. A method of producing a binding protein-drug conjugate (BPDC) according to claim 2, wherein a binding protein carrying a sortase enzyme recognition motif is conjugated, by means of a sortase enzyme, to at least one anthracycline derivative conjugate according to claim 2, which carries, as L.sub.2, an oligo-glycine peptide (Gly).sub.n.

23. Use of a binding protein drug conjugate (BPDC) according to claim 2, for the treatment of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a given pathologic condition.

24. Use according to claim 23, wherein the pathologic condition is a neoplastic disease.

25. Use according to claim 24, wherein the neoplastic disease is a) a cancer that has an HER-2 expression score of 1+, 2+ or 3+, as determined by IHC or ISH, which cancer is preferably a breast cancer, or b) a cancer that is CD30 positive as determined by IHC, ELISA or flow cytometry, preferably a lymphoma, more preferably a Hodgkin lymphoma (HL) or a systemic anaplastic large cell lymphoma (sALCL).

26. A pharmaceutical composition comprising a binding protein drug conjugate (BPDC) according to claim 2 and at least one other pharmaceutically acceptable ingredient.

Description

EXPERIMENTS AND FIGURES

[0179] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.

[0180] In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0181] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5′->3′.

Example 1: Generation of Site-Specifically C-Terminally PNU-EDA-Gly.SUB.n.-Payload Conjugated Monoclonal Antibodies Brentuximab and Trastuzumab by Sortase Mediated Antibody Conjugation Technology (SMAC-Technology)

[0182] The heavy and light chain variable region sequences of monoclonal antibody brentuximab (clone cAc10) specific for the human CD30 target were obtained from patent US2008213289A1, those of the human HER-2 specific trastuzumab antibody contained in the commercial antibody Herceptin (trastuzumab), or the ADC Kadcyla® derived thereof, were derived from the online IMGT database (V.sub.H: http://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=7637&Part=Chain&Chain=7637H & V.sub.L: http://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=7637&Part=Chain&Chain=7637L. Chimeric mAb cAc10 and humanized mAb trastuzumab were produced with their heavy and light chains C-terminally tagged with a Sortase A recognition sequence and an additional Strep II affinity purification tag (HC tag sequence: LPETGGWSHPQFEK; LC tag sequence: GGGGSLPETGGWSHPQFEK) using methods known to those skilled in the art. (see FIGS. 11A & 11B).

[0183] The anthracycline derivative PNU-EDA-Gly.sub.5 (FIG. 3A) was provided by Levana Biopharma, San Diego, Calif., which synthesized a pentaglycin peptide to the carbonyl group of PNU159682 via an ethylenediamino (EDA) linker according to the synthesis scheme of FIG. 3B. For this, commercially available PNU159682 was first oxidized to obtain a carboxylic acid thereof (1 on FIG. 3B) with NaIO.sub.4 in 60% methanol at RT for 3 hours. Thereafter, N-hydroxysuccidimide (NHS, 46 mg, 400 μmol) and ethyl(dimethylaminopropyl) carbodiimide (EDC, 100 mg, 523 μmol) in dichloromethane (DCM) were added to a solution of 1 (51 mg, 81 μmol) in 6 mL of DCM. After 30 min, the mixture was washed with water (2×6 mL), dried over Na.sub.2SO.sub.4 and evaporated. The residue was then dissolved in 2 mL of dimethylformamide (DMF) prior to addition of the amine (2 on FIG. 3B, 55 mg, 81 μmol, as trifluoroacetate salt), followed by addition of N,N-diisopropylethylamine (DIEA, 504). The mixture was stirred for 1 h prior to addition of piperidine (404), followed by 20 min of additional stirring. The mixture was purified by HPLC to give PNU-EDA-Gly.sub.5 (3 on FIG. 3B, 34 mg, 44%) as a red solid; MS m/z 955.2 (M+H).

[0184] PNU-EDA-Gly.sub.5 was conjugated to mAbs by incubating LPETG-tagged mAbs [10 μM] with PNU-EDA-Gly.sub.5, [200 μM] in the presence of 0.62 μM Sortase A in 50 mM Hepes, 150 mM NaCl, 5 mM CaCl.sub.2, pH 7.5 for 3.5 h at 25° C. The reaction was stopped by passing it through a Protein A HiTrap column (GE Healthcare) equilibrated with 25 mM sodium phosphate pH 7.5, followed by washing with 5 column volumes (CVs) of buffer. Bound conjugate was eluted with 5 CVs of elution buffer (0.1M succinic acid, pH 2.8) with 1 CV fractions collected into tubes containing 25% v/v 1M Tris Base to neutralise the acid. Protein containing fractions were pooled and formulated in 10 mM Sodium Succinate pH 5.0, 100 mg/mL Trehalose, 0.1% % w/v Polysorbate or phosphate20 by G25 column chromatography using NAP 25 (GE Healthcare) columns according to the manufacturer's instructions.

[0185] The aggregate content of each conjugate was assessed by chromatography on a TOSOH TSKgel G3000SWXL 7.8 mm×30 cm, 5 μm column run at 0.5 mL/min in 10% IPA, 0.2M Potassium Phosphate, 0.25M Potassium Chloride, pH 6.95. The drug loading was assessed both by Hydrophobic Interaction Chromatography (HIC) and Reverse-Phase Chromatography. HIC was performed on a TOSOH Butyl-NPR 4.6 mm×3.5 cm, 2.5 μm column run at 0.8 mL/min with a 12 minute linear gradient between A—1.5M (NH.sub.4).sub.2SO.sub.4, 25 mM NaPi, pH=6.95±0.05 and B—75% 25 mM NaPi, pH=6.95±0.05, 25% IPA. Reverse phase chromatography was performed on a Polymer Labs PLRP 2.1 mm×5 cm, 5 μm column run at 1 mL/min/80° C. with a 25 minute linear gradient between 0.05% TFA/H.sub.2O and 0.04% TFA/CH.sub.3CN. Samples were first reduced by incubation with DTT at pH 8.0 at 37° C. for 15 minutes. Both PNU-EDA-Gly.sub.5-based ADCs were predominantly monomeric and had drug-to-antibody-ratios close to the theoretical maximum of, respectively, 4. Table 2 summarizes the results of the ADC manufacturing.

TABLE-US-00002 TABLE 2 Summary of PNU-EDA-Gly.sub.5-based ADCs manufactured. HC, heavy chain; LC, light chain; % mono, % monomer content; DAR, drug-to-antibody-ratio. mAb target HC tag LC tag % mono DAR Brentuximab CD30 Yes Yes 99.6 4.0 Trastuzumab HER-2 Yes Yes 98.2 3.9

Example 2. In Vitro Cytotoxicity Assay with Sortase A-Conjugated Brentuximab-PNU-EDA-Gly.SUB.5 .and Trastuzumab-PNU-EDA-Gly.SUB.5 .ADCs

[0186] Cytotoxicity of Brentuximab-PNU-EDA-Gly.sub.5 was investigated using Karpas-299, a non-Hodgkin's lymphoma cell line expressing high levels of CD30, and L428, a Hodgkin's lymphoma cell line expressing low to moderate levels of CD30 (FIG. 4). As controls, efficacy of cAc10-PNU-EDA-Gly.sub.5 was compared to that of the commercially available CD30-specific cAc10-vcPAB-MMAE conjugate Adcetris® (as positive control) and the commercially available HER-2-specific Trastuzumab-DM1 conjugate Kadcyla® (as negative control). For this, cells were plated on 96-well plates in 100 μl RPMI/10% FCS at a density of 10.sup.4 cells per well and grown at 37° C. in a humidified incubator at 5% CO.sub.2 atmosphere. After one day incubation, 25 μl medium was carefully removed from each well and replaced by 25 μl of 3.5-fold serial dilutions of each ADC in growth medium, resulting in final ADC concentrations ranging from 20 μg/ml to 0.25 ng/ml. Each dilution was done in duplicate. After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 minutes, 100 μl CellTiter-Glo® Luminescent Solution (Promega, Cat. No G7570) was added to each well and, after shaking the plates at 450 rpm for 5 min followed by a 10 min incubation without shaking, luminescence was measured on a Tecan Infinity F200 with an integration time of 1 second per well.

[0187] As expected, the anti-CD30 ADC Adcetris® used as a positive control potently killed CD30.sup.HI Karpas-299 cells with an EC50 of 8.2 ng/ml (FIG. 4A), while being inefficient at killing CD30.sup.LO L428 cells (FIG. 4B). In contrast, the anti-HER-2 ADC Kadcyla® used as a negative control displayed no specific cell killing and was ineffective on either cell line (FIG. 4). Significantly, Sortase-conjugated ADC cAc10-PNU-EDA-Gly.sub.5 potently killed the CD30.sup.HI Karpas-299 cells with an EC50 value of 6.9 ng/ml (FIG. 4A). cAc10-PNU-EDA-Gly.sub.5 killed the CD30.sup.LO L428 cells only at higher concentrations, similar to the control ADCs employed, indicating that the efficacy of this ADC is indeed specific and mediated by CD30 binding (FIG. 4B). Thus, Sortase-mediated conjugation of PNU-EDA-Gly.sub.5 yielded an ADC with a very high potency, even exceeding that of the reference ADC Adcetris®.

[0188] The potency for tumor cell killing of a SMAC-generated Trastuzumab-PNU-EDA-Gly.sub.5 ADC was investigated using SKBR3 cells, a human breast cancer cell line overexpressing HER-2, and T47D cells, a breast cancer cell line naturally expressing low levels of HER-2, and this was compared to the commercially available HER-2-specific ADC Trastuzumab-DM1 conjugate Kadcyla® (FIG. 5). For this, cells were plated on 96 well plates in 100 μl DMEM/10% FCS at a density of 10.sup.4 cells per well and assays were performed exactly as described above.

[0189] As expected, the positive control ADC Kadcyla® potently killed HER-2-overexpressing human SKBR3 breast cancer cells, with an EC50 of 23.7 ng/ml (FIG. 5A), while being ineffective at killing HER-2.sup.LO T47D cells (FIG. 5B). Significantly, Trastuzumab-PNU-EDA-Gly.sub.5 generated by SMAC-technology displayed superior cytotoxicity and not only killed HER-2-overexpressing SKBR3 cells, but also HER2.sup.LO T47D cells, with EC50 values of, respectively, 4.8 and 11.0 ng/ml (FIG. 5). Thus, Sortase-mediated conjugation of PNU-EDA-Gly.sub.5 to Trastuzumab yields an ADC with a very high potency, exceeding that of the commercially available and FDA-approved reference ADC Kadcyla®, and is even effective on HER2.sup.LO human breast cancer cells.

Example 3: In Vitro Serum Stability of Sortase A-Conjugated cAc10-PNU-EDA-Gly.SUB.5 .ADC as Compared to Maleimide Linker Containing Trastuzumab Emtansine (Kadcyla®)

[0190] The in vitro serum stability of brentuximab-PNU-EDA-Gly.sub.5 (cAc10-PNU-EDA-Gly.sub.5) and Kadcyla ADCs was evaluated in an ELISA-based serum stability assay. Briefly, cAc10-PNU-EDA-Gly.sub.5 was diluted in mouse (Sigma, M5905), rat (Sigma, R9759) and human serum (Sigma, H6914), and incubated at 37° C. Samples were snap-frozen in liquid nitrogen on days 0, 3, 7, 14 and stored at −80° C. until ELISA analysis. For rodent sera, dilution series of cAc10-PNU-EDA-Gly.sub.5 serum samples were captured on ELISA plates coated with 2 μg/ml of a mouse anti-PNU mAb (produced in-house by immunizing mice with a human IgG-PNU conjugate and screening with a BSA-PNU conjugate) to bind ADC, or with anti-human Fc F(ab′)2 (Jackson Immunoresearch) to bind total IgG, and detected with a 1:2500 dilution of an HRP-conjugated anti-human IgG F(ab′)2 (Jackson Immunoresearch). For primate sera, 2 μg/ml of recombinant human CD30 (Sino Biologicals, 10777-H08H) was coated on ELISA plates and a 1:2500 dilution of HRP-conjugated anti-human IgG F(ab′)2 (Jackson Immunoresearch) or 1 μg/ml of a mouse anti-PNU IgG (produced in-house) followed by HRP-conjugated anti-mouse Fc F(ab′)2 (Jackson Immunoresearch) was used for detection of total IgG and ADC, respectively. In the case of Kadcyla, the same protocol was used as above to determine stability in mouse, rat and human serum but with an in-house produced anti-maytansine mAb to bind ADC. Serum concentrations of ADC and total IgGs were calculated from half maximal values of the sample titrations by comparison with a sample of the same ADC of known concentration.

[0191] FIG. 7 A shows the excellent stability of cAc10-PNU-EDA-Gly.sub.5 ADC, particularly as compared to that of maleimide linker containing Kadcyla (FIG. 7 B), with virtually no decrease in ADC levels throughout the entire experiment in any serum of the four species tested. By fitting the time points between day 0 and 14 to a one-phase exponential decay function constrained to reach a final concentration of 0, the half-life values of cAc10-PNU-EDA-Gly.sub.5 and Kadcyla were determined in each serum. The half-life of Kadcyla was of 3.7 days, 4.4 days and 2.9 days in mouse, rat and human serum, respectively, whereas the half-life of cAc10-PNU-EDA-Gly.sub.5 was greater than 14 days in mouse, rat and human serum.

Example 4: In Vivo Stability of Sortase A-Conjugated Ac10-Gly5-PNU in Mice

[0192] Ac10-Gly5-PNU ADC was thawed at room temperature and diluted to 0.2 mg/ml in sterile PBS for a dosing concentration of 1 mg/kg. The samples were injected i.V. at a volume of 5 mL/kg in nine female Swiss Webster mice. Blood was collected from animals after 1 h, 24 h, 72 h, 7 days, 14 days, and 21 days. Individual animals according to ethical standards were only used for two blood draw time points at least a week apart. Thus, three mice had blood drawn after 1 h and 7 days, three different mice had blood drawn after 24 h and 14 days, and three additional different mice had blood drawn after 72 h and 21 days for a total of nine mice per group. For each group of animals, approximately 2004, of blood was collected by lancet-puncture of the submandibular vein during the first collection, and approximately 6004, of blood by lancet-puncture of submandibular vein during the final collection (terminal bleed). All blood was collected into tubes containing K2-EDTA. Plasma was isolated from blood by centrifugation at 1500 g for 10 minutes, and transferred to sterile cryovials for storage at −80° C. until analysis by ELISA as described in Example 4.

[0193] The data in FIG. 8 shows the high stability of the ADC generated by SMAC-technology. For the entire duration of the experiment, concentrations of ADC are only marginally lower than those measured for total IgG, which implies that the linker between drug and antibody is stable in vivo. By fitting the time points between day 3 and 21 to a one-phase exponential decay function constrained to reach a final concentration of 0, in vivo half life in the slow phase was determined with 8.3 and 7.8 days for total IgG and ADC, respectively.

Example 5: Description and Characterization of EMT-6 Clones Expressing HER-2

[0194] Cytotoxicity of anti-HER-2 ADCs was investigated using the murine mammary tumor cell line EMT-6 engineered to overexpress human HER-2. EMT-6 cells were cultured as monolayers in DMEM (Dulbecco's Modified Eagle Medium—high glucose) supplemented with 10% (v/v) of FCS (Fetal Calf Serum), 1% (v/v) of 10,000 IU/mL penicillin-streptomycin and 1% (v/v) of 200 mM L-glutamine.

[0195] EMT-6 cells were electroporated with an expression vector encoding the human HER-2 gene and a puromycin resistance marker and cell pools stably expressing human HER-2 were selected using methods known to those skilled in the art.

[0196] HER-2 expression was confirmed by flow cytometry. Briefly, following trypzinization, 10.sup.6 cells were centrifuged in FACS tubes; obtained pellets were resuspended in PBS (phosphate-buffered saline) supplemented with 2% of FCS. Cells were then incubated with the anti-HER-2 antibody trastuzumab (30 min, 4° C.), followed by centrifugation and washing (3 mL of PBS with 2% FCS). Cells were then resuspended as previously and incubated with anti-human IgG antibody (F.sub.c gamma-specific) PE (Ebioscience) in the dark (30 min, 4° C.), prior to washing (4 mL PBS with 2% FCS). Flow cytometry was then performed on a FACS Calibur (BD).

[0197] HER-2-transfected EMT-6 cells were single cell-sorted by flow cytometry using a FACS ARIA II to isolate single cell clones. These were expanded and HER-2 expression was verified by flow cytometry.

[0198] FIG. 9 shows the FACS analysis data of the clone selected for in vivo studies (Example 6).

Example 6: In Vivo Efficacy of Sortase A-Conjugated Trastuzumab-PNU-EDA-Gly.SUB.5 .ADC in an Orthotopic Breast Cancer Model

[0199] The in vivo efficacy of Trastuzumab-PNU-EDA-Gly.sub.5 was evaluated in an immunocompetent orthotopic mouse model of HER-2-positive breast cancer. For this, 10.sup.6 EMT6 mouse breast cancer cells expressing human HER-2 (Example 6), previously determined to be suitable for in vivo growth, were implanted into the right mammary fat pads of female Balb/c mice. In addition, control animals were implanted with HER-2-negative EMT6 cells. In the following, primary tumor volumes were measured by calipering. After 13 days, when a mean tumor volume of 100-150 mm.sup.3 was reached, tumor-bearing animals were randomized into groups of 6 animals each according to tumor sizes. Animals were treated on the same day (day 13, i.e. day of randomization) and 7 days later (day 20) by intravenous injection of the reference ADC Kadcyla® (15 mg/kg), Trastuzumab-PNU-EDA-Gly.sub.5 (1 mg/kg) or vehicle control. Tumor sizes were monitored by calipering and animals whose tumor volume reached 1000-1500 mm.sup.3 were terminated (FIG. 10).

[0200] Tumors in vehicle control mice grew rapidly and reached an average size of approximately 1000 mm.sup.3 within 30 days after transplantation of cells (FIG. 10A). Treatment with Kadcyla® had little effect on tumor growth in most animals. Only one out of six animals displayed a significant delay in tumor growth (FIG. 10C). In striking contrast, in all animals treated with Trastuzumab-PNU-EDA-Gly.sub.5, the tumors continuously regressed during treatment and were essentially undetectable by day 30 after transplantation of the cells (FIG. 10D). No tumor was detectable most animals until day 60, and tumor recurrence was observed in only one animal around day 40. Significantly, the anti-tumor activity of Trastuzumab-PNU-EDA-Gly.sub.5 was highly specific and treatment of mice bearing HER-2-negative tumors did not lead to tumor regression (FIG. 10B). Taken together, the data demonstrate that sortase-mediated site-specifically conjugated of Trastuzumab-EDA-Gly.sub.5-PNU ADCs yielded an ADC with in vivo tumor cell killing activity far superior to the benchmark ADC Kadcyla®.

FIGURE LEGENDS

[0201] FIG. 1: Schematic drawing of site-specific sortase mediated antibody conjugation (SMAC-technology). The monoclonal antibodies need to be produced with C-terminal LPXTG sortase tags. The toxic payload needs to be produced to contain an oligoglycine peptide stretch (Gly.sub.n-stretch) with a certain number of glycine residues in a row (n≧1 and ≦21, preferably n≧3 and ≦10, preferably n=3 or n=5, most preferably n=5). Sortase A enzyme from Staphylococcus aureus specifically recognizes the LPXTG pentapeptide motif and catalyzes the transpeptidation of the oligo-glycine peptide stretch to the threonine-glycine peptide bond of LPXTG, thereby generating a new stabile peptide bond between the threonine and the N-terminal glycine of the oligo-glycine stretch.

[0202] FIG. 2. Structure of PNU-159682 as described in the prior art (e.g. WO2009099741, or Quintieri et al (2005)), including the official anthracycline numbering system for reactive carbons of the tetracyclic aglycone structure.

[0203] FIG. 3. (A) Structure of PNU derivative-EDA-Gly.sub.5, called “PNU-EDA-Gly.sub.5” herein, as utilized for the SMAC-technology conjugation to C-terminally LPETG sortase tagged monoclonal antibodies using sortase enzyme as disclosed in the Examples herein. (B) Synthesis scheme of anthracycline derivative PNU-EDA-Gly.sub.5.

[0204] FIG. 4. Dose response of the cytotoxic effects of the indicated ADCs on human Non-Hodgkin lymphoma cell line Karpas-299, expressing high levels of CD30 target on the cell surface (A), and on human Hodgkin lymphoma cell line L428 cells expressing very low levels of CD30 target in the cell surface (B). Adcetris refers to commercially available anti-CD30 ADC brentuximab-vedotin. Kadcyla refers to commercially available anti-HER-2/neu ADC T-DM1 (trastuzumab-emtansine). Both cell lines are negative for HER-2/neu, and therefore Kadcyla acts as a negative control ADC, that should not effect cell killing in a target-specific way. Cells were incubated with serial dilutions of ADCs for 4 days, after which CellTiter-Glo® Luminescent Solution (Promega) was added and viable cells were quantified by measuring the luminescence on a Tecan Infinity F200.

[0205] FIG. 5. Dose response of the cytotoxic effects of the indicated ADCs on human breast cancer cell line SKBR3, expressing high levels of HER-2/neu (A) and human breast cancer cell line T47D expressing low levels of HER-2/neu (B). Cells were incubated with serial dilutions of ADCs for 4 days, after which CellTiter-Glo® Luminescent Solution (Promega) was added and viable cells were quantified by measuring the luminescence on a Tecan Infinity F200.

[0206] FIG. 6. Additional PNU-159682 related anthracycline derivatives useful for site-specific-conjugation to LPXTG-tagged binding proteins or antibodies by SMAC-technology to produce BPDCs or ADCs. Only the preferred versions with Gly5-stretch are depicted. 6A depicts a derivative, in which the Gly5 amino acid stretch is directly coupled via its carboxy terminus to the A-Ring of the tetracyclic aglycone structure of the PNU derivative. 6B depicts a derivative in which a preferred ethylene-amino linker and Gly5 amino acid stretch is directly coupled to the A-Ring of the tetracyclic aglycone structure of the PNU derivative

[0207] FIG. 7 (A) Measurement of in vitro concentration of brentuximab-PNU-EDA-Gly.sub.5 ADC (labeled as “cAc10-PNU ADC”) and total IgG in mouse (A), rat (B), human (C) serum over 14 days. (B) Measurement of in vitro concentration of trastuzumab-emtansine (Kadcyla®) ADC and total IgG in mouse (A), rat (B) and human (C) serum over 14 days.

[0208] FIG. 8: In vivo plasma concentrations of ADC and total IgG measured at 6 time-points over a 21-day period following administration of Ac10-Gly5-PNU ADC in mice.

[0209] FIG. 9: Data of FACS analysis of EMT-6 HER-2 clone selected for in vivo studies following incubation with anti-HER-2 antibody trastuzumab and then incubation with flurophore-containing anti-human IgG antibody (Fc gamma-specific) PE.

[0210] FIG. 10: In vivo evaluation of HER-2-specific ADCs in an immunocompetent orthotopic mouse model of HER2-positive breast cancer. EMT6 mouse breast cancer cells expressing human HER-2 (A, C, D) or irrelevant antigen ROR-1 were grown in the mammary fat pads of Balb/c mice. On days 13 and 20, animals were treated i.v. with vehicle control (A), 1 mg/kg Trastuzumab-PNU159682 (B, D), or 15 mg/kg Kadcyla (C). Tumor growth was monitored until animals had to be sacrificed due to ethical reasons.

[0211] FIGS. 11 A & B: Amino acid compositions of the C-terminally SMAC-Technology™ conjugated IgH and IgL chains of the trastuzumab (A) and brentuximab (B) PNU-toxin derivative containing ADCs used for the studies, comprising the PNU derivative depicted in FIG. 3B linked through the amino group of the Gly5-stretch to the 4th amino acid of the sortase tag (highlighted in boldface print) via a peptide bond following sortase enzyme conjugation.

REFERENCES

[0212] Beerli et al. (2015) PloS One 10, e0131177 [0213] Dorr et al. (2014) PNAS 111, 13343-8 [0214] Quintieri L et al (2005), Clin Cancer Res. 2005 Feb. 15; 11(4):1608-17. [0215] Roguska et al. (1994) PNAS 91, pp 969-971 [0216] Perez et al. (2014) Drug Discovery Today 19, pp. 869-881 [0217] Alley et al. (2008) Bioconjug. Chem. 19, pp. 759-765 [0218] Panowski et al. (2014) mAbs 6, pp. 34-45 [0219] Wolff A C et al (2013), J Clin Oncol. 2013 Nov. 1; 31(31):3997-4013 [0220] Young K H (2014) Clinical Advances in Hematology & Oncology, Volume 12, Issue 4, Supplement 10 [0221] Spirig et al. (2011) Mol. Microbiol. 82, 1044-1059 [0222] Ducry & Stump (2010) Bioconjug. Chem. 21, 5-13, [0223] McCombs et al. (2015) The AAPS Journal 17, 339-351 [0224] Mullard (2013) Nature Rev Drug Disc 12, 329-332 [0225] Doktor et al. (2014) Mol Cancer Ther 13, 2618-2629 [0226] Hartley & Hochhauser (2012) Curr. Opin. Pharmacol. 12, 398-402 [0227] Minotti (2004) Pharmacol. Rev 56, 185-229 [0228] Cancer and Chemotherapy—Antineoplastic Agents Vol. III, Stanley T. Crooke and Archie W. Prestayko (eds.), Academic Press 1981).