Conjugates of proteins and multivalent cell-penetrating peptides and their uses

Abstract

The present invention relates to conjugates comprising a protein and multivalent cell-penetrating peptide(s), each multivalent cell-penetrating peptide comprising at least two cell-penetrating peptides, wherein the multivalent cell-penetrating peptide(s) is/are covalently attached to the protein. The present invention furthermore relates to a method of generating the conjugates and to their medical uses, in particular their use in the diagnosis, prevention and/or treatment of diseases. The present invention relates to methods of diagnosis, prevention and/or treatment of diseases, comprising administering the conjugates of the invention to a patient.

Claims

1. A conjugate comprising: a protein, and one or more multivalent cell-penetrating peptide(s) (multivalent CPP(s)) each multivalent CPP comprising at least two cell-penetrating peptides (CPPs), wherein the multivalent CPP(s) is/are covalently attached to the protein, wherein the one or more multivalent CPP is a dendrimer of cell-penetrating peptides (cell-penetrating peptide dendrimer, dCPP) comprising a dendrimer core and at least two cell-penetrating peptides (CPPs), which are coupled to the dendrimer core, said dendrimer core being a peptidyl dendrimer core comprising lysines as branching points and cysteine(s) as the anchoring group(s), and comprises: ##STR00002## wherein K is Lys, A is Ala and C is Cys, wherein the CPPs each comprises the amino acid sequence of penetratin (SEQ ID NO: 1), TAT (47-60) (human immunodeficiency virus-derived trans-activator of transcription, SEQ ID NO: 2), PreS2-TLM (hepatitis B virus-preS2-domain-derived translocation motif, SEQ ID NO: 3), R9 (SEQ ID NO: 4), MTS (membrane translocation signal, SEQ ID NO: 5), SynB1 (synthetic porcine protegrin 1-derived CPP, SEQ ID NO: 6), pVEC (vascular endothelial cadherin-derived CPP, SEQ ID NO: 7), or NLS (nuclear localization signal, SEQ ID NO: 8), or a combination thereof, and/or the wherein the CPPs each comprises an amino acid sequence of SEQ ID NOs: 9 to 77 or a combination thereof, and/or wherein the CPPs each comprises an amino acid sequence of SEQ ID NOs: 78 to 760 or a combination thereof.

2. The conjugate of claim 1, wherein the one or more multivalent CPPs comprise 2 to 50 CPPs.

3. The conjugate of claim 1, wherein the one or more multivalent CPPs comprise a dendrimer of cell-penetrating peptides or multiple copies of CPPs.

4. The conjugate of claim 1, wherein the dendrimer core comprises anchoring group(s), branching point(s), and, optionally, a spacer between the anchoring group(s) and the branching point(s).

5. The conjugate of claim 1, wherein the one or more multivalent CPPs each comprises an amino acid sequence having 5 to 30 amino acids.

6. The conjugate of claim 1, wherein the one or more multivalent CPPs comprise one or more of natural amino acids, amino acid derivatives, D-amino acids, modified amino acids, -amino acid derivatives, ,-disubstituted amino acid derivatives, N-substituted -amino acid derivatives, aliphatic or cyclic amines, amino- and carboxy-substituted cycloalkyl derivatives, amino- and carboxy-substituted aromatic derivatives, -amino acid derivatives, aliphatic -amino acid derivatives, diamines and polyamines.

7. The conjugate of claim 1, wherein the CPPs each is a peptide capable of being internalized into a cell and/or wherein the CPPs each comprises in its amino acid sequence at least 25% positively charged amino acid residues, and/or wherein the CPPs each is internalized into a cell with an efficacy being at least 50% of the internalization efficacy of the TAT peptide having the amino acid sequence of SEQ ID NO: 2.

8. The conjugate of claim 1, wherein the CPPs each comprise the amino acid sequence of penetratin (SEQ ID NO: 1), and/or R9 (SEQ ID NO: 4).

9. The conjugate of claim 1, wherein the one or more cell-penetrating peptide(s) each comprise an amino acid sequence selected from SEQ ID NOs. 1 to 760 or amino acid sequences having at least 90% sequence identity to an amino acid sequence of SEQ ID NOs. 1 to 760.

10. The conjugate of claim 1, wherein the one or more multivalent CPPs each comprises 2 more different CPPs.

11. The conjugate of claim 1, wherein the protein is a biological or clinically active or therapeutic protein.

12. The conjugate of claim 1, furthermore comprising a linker connecting the protein and the one or more multivalent CPP(s).

13. The conjugate of claim 12, wherein the linker is a bifunctional (cross)linker covalently coupling the protein with the one or more multivalent CPPs.

14. The conjugate of claim 1, comprising an antibody, one or more cell-penetrating peptide dendrimer(s) (dCPP), and one or more linkers each covalently coupling the antibody with a dCPP.

15. The conjugate of claim 1, further comprising a label, a drug or prodrug, and/or a further biologically active component.

16. A method for generating a conjugate according to claim 1, comprising the steps of (a) providing multivalent cell-penetrating peptide(s) (multivalent CPP(s)) comprising anchoring group(s), (b) generating a chemically activated protein by using a linker, or providing a protein comprising coupling site(s), (c) coupling the multivalent CPP(s) of step (a) to the protein of step (b), (d) obtaining the conjugate, (e) purifying the conjugate.

17. The method of claim 16, wherein in step (a) cell-penetrating peptide dendrimer(s) (dCPPs) comprising one or more anchoring group(s) are provided.

18. The method of claim 16, wherein the linker is a bifunctional (cross) linker and/or comprising using in step (b) excess of the linker to generate a chemically activated protein having one or more maleimide molecule(s) on the surface.

19. The method of claim 17, comprising using in step (c) excess of the cell-penetrating peptide dendrimer(s).

20. The method of claim 16, wherein the coupling site(s) of the protein are the side chains of cysteine(s), glutamine(s) and/or lysine(s) and/or unnatural amino acids.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Overview of cell-penetrating dendrimer syntheses.

(2) A) Schematic presentation of the dendrimer showing branching (lysine) and focal (cysteine) points.

(3) B) Chemical structure of the dendrimer core. Lysine branching points are shown in dark orange and the crosslinking-referring sulfhydryl group of cysteine in red.

(4) C) Schematic dCPP structure in one letter code exemplified by penetratin.

(5) FIG. 2: Conjugation of the mAb with dCPPs.

(6) A) Schematic overview of antibody activation by SMCC and subsequent coupling to the dCPP. In the first step, the mAb is activated as maleimide, which is attacked by the free sulfhydryl-group of the dCPP in the second step. Excess dCPP is removed by gelfiltration.

(7) B) Influence of SMCC excesses in the conjugation reaction analyzed by SDS-PAGE. The image is shown in reverse grayscale to improve contrast.

(8) FIG. 3: Binding of matuzumab and of the mAb-dCPP conjugates

(9) to A) A431 cells and B) to the control cell line DU-145. The internalized portion of the activity is illustrated in the lower part of the bars in black, the membrane bound activity by the upper part shown in gray. For clarity reasons the error bars are omitted.

(10) FIG. 4:

(11) A) Organ and tissue distribution of the .sup.131I-labeled antibody, mAb-dPenetratin and mAb-dR9 96 hours after injection into A431 xenografts.

(12) B) Time-dependent development of the tumor-to-blood ratio of the naked antibody and the immunoconjugates.

(13) FIG. 5:

(14) A) Planar scintigraphic images of A431 tumor-bearing nude mice 48 hours after injection of .sup.125I-labeled mAb, mAb-dPenetratin and mAb-dR9. B) As FIG. 5A, except that the radioiodination was carried out with iodine-124 and that static PET images were recorded.

(15) FIG. 6: Possible mechanisms for target cell binding by the mAb-dCPPs.

(16) A) For little internalized conjugates, mainly with amphipathic dCPPs, a dock and lock mechanism is hypothesized. The antibody binds to its antigen, but the internalization is disturbed, as a transitory structure necessary for uptake, is not formed by the CPP dendrimer, but the dCPP still locks onto the cell surface.

(17) B) Internalization mechanism: The antibody binds to the target structure on the cell surface and then the dCPP promotes cell uptake either by direct translocation or endocytosis.

(18) FIG. 7: Schematic overview of the structures of the synthesized dCPPs.

(19) FIG. 8: Western blot analysis of EGFR-expression by A431 and DU-145 cells.

(20) Matuzuinab was used as primary antibody. Horseradish peroxidase conjugated goat anti-human IgG served as secondary antibody.

(21) FIG. 9:

(22) Planar scintigraphic images of A431 xenografts injected with .sup.125I-labeled matuzumab, mAb-dPenetratin and mAb-dR9. The images were recorded at 1, 4, 24, and 48 hours p.i.

(23) FIG. 10: Cytotoxicity assays with Penetratin antibody conjugates

(24) A) Cytotoxicity assay with EGFR-positive cells

(25) B) Cytotoxicity assay with EGFR-negative cells

(26) The cells were treated for 72 hours with different concentrations of a toxin-antibody-Penetratin (4-mer) conjugate (Herceptin-ADC-Penetratin) or a toxin-antibody-conjugate without Penetratin (Herceptin-ADC), respectively. Then, the cell viability was tested via BrdU incorporation. The tables show the molar EC50 concentrations.

(27) FIG. 11: Cellular uptake of Penetratin antibody conjugates

(28) Incubation of A431 cells (EGFR positive cells) with

(29) A) Native EGFR antibody

(30) B) EGFR antibody Pen10-conjugate (4-mer) (Pen-10=10-mer-Penetratin partial structure, a control peptide causing reduced internalization)

(31) C) EGFR antibody Penetratin-conjugate (4-mer)

(32) Experimental conditions: Incubation for 1 hour with a concentration of 1 g/ml of all 3 antibodies, fixation of the cells with PFA after incubation, staining with fluorescence-labelled secondary anti-huIgG antibody (AlexaFluor-488), image acquisition with 100 objective lens, fluorescence microscope Keyence BioRevo BZ-9000

(33) Bar: 20 m

EXAMPLES

1. Materials and Methods

(34) 1.1 General.

(35) All chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany) at the highest available purity unless otherwise stated. Fmoc-protect amino acid building blocks were purchased from Bachem (Bubendorf, Switzerland). Anti-EGFR antibody Matuzumab (EMD72000) was provided by Merck KGaA (Darmstadt, Germany) (see also EP 0 531 472 B1, U.S. Pat. No. 5,558,864, WO 2009/043490 A1). Radioactive iodine 1-125 and 1-131 isotopes were purchased from Perkin-Elmer (Rodgau, Germany) and Eckert & Ziegler (Berlin, Germany) for I-124.

(36) 1.2 Synthesis of Dendritic Cell-Penetrating Peptides (dCPPs).

(37) Branched structures of the CPPs were obtained by manual generation of a solid phase peptide synthesis (SPPS) resin, presenting four amino groups- and -amines of lysinesas branching points, one alanine residue as spacer and a cysteine as focal group for crosslinking with the antibody using 9-fluorenylmethoxycarbonyl (Fmoc)-protected L--amino acids. The desired sequenceK.sub.2KACwas yielded on resin by subsequent incubation of 1.5 g Amphisphere 40 RAM (0.4 mmol/g), swollen in dichloromethane (DCM) and washed four times using dimethyl formamide (DMF), with 4 eq of Fmoc-Cys(Trt)-OH, 4 eq Fmoc-Ala-OH, 3 eq Fmoc-Lys(Fmoc)-OH and 6 eq Fmoc-Lys(Fmoc)-OH respectively. For the coupling steps (75 min) equal amounts of O-(Benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluoro-phosphate (HBTU) and 500 L N,N-Diisopropylethylamine (DIPEA) were added. After each coupling the resin was washed five times with DMF and Fmoc was cleaved by consecutive incubation with 10 mL (1 min) and 7 mL (10 min) of 25% (v/v) piperidine solution in DMF followed by DMF washing steps (5).

(38) This dried resin (150 mg, for each dCPP), containing the four-branch core molecule, was then used in a fully automated peptide synthesizer (Applied Biosystems 433 A, Carlsbad, Calif., USA) with 10 eq (Fmoc)-protected amino acid building blocks. HBTU/DIPEA in NMP was used as a coupling reagent. Fmoc deprotection efficiency was monitored at 301 nm. Cleavage from solid support was performed with TFA/H.sub.2O/triisopropylsilane (95:2.5:2.5) for 2 h at ambient temperature. The peptide was precipitated with cold diethyl ether and pelletized (4000 rpm, 4 C., 5 min), washed with diethyl ether, dried in vacuo, and subject to preparative HPLC purification (Waters, Eschbom, Germany; XBridge BEH 130 PREP; C18 column, 5 m pore size, 15019 mm). Fractions containing the product were identified by using HPLC-ESI-MS with Orbitrap technology (Exactive, Thermo Fisher Scientific, Waltham, Mass., USA) equipped with an Agilent 1200 HPLC system and a Hypersil Gold C18 column (Thermo Fisher Scientific, Bonn, Germany; 1.9 m, 2002.1 mm), pooled and freeze-dried.

(39) 1.3 Conjugation of dCPPs with the Monoclonal Antibody.

(40) Conjugation was carried out using the commercially available, hetero-bifunctional crosslinker SMCC (Thermo Fisher Scientific, Bonn, Germany). Crosslinking was carried out according to the manufacturer's protocol. In brief, 7, 15 and 30 equivalents of the crosslinker (15 mg/mL in DMF) were added to the monoclonal antibody (mAb) solution (1.5 mg/mL in PBS) and incubated at room temperature for 30 min. Excess crosslinker was removed by using pre-equilibrated (PBS, pH 7.4) NAP-10 columns (GE Healthcare, Freiburg, Germany). The maleimide-activated mAb solutions were concentrated to 1 mg/mL (concentration determination using Bradford test (Bradford, 1976)) using 100 k Amicon Ultra-0.5 mL centrifugal filters (Merck Millipore, Darmstadt, Germany). Then the maleimide-activated mAb was incubated with 15 eq of the eight different dCPPs (20 mg/mL) at room temperature for 45 min. Reactions with 15 eq SMCC in the first coupling yielded the best results, i.e. predominantly a single attachment of a dCPP to the mAb. Excess dCPP and in part unmodified mAb were removed by size exclusion chromatography using a FPLC manifold equipped with a Superdex 200, 10/300 GL (GE Healthcare) column and PBS as the mobile phase. Fractions containing the antibody-dCPP conjugates were identified by UV-monitoring (=280 nm), pooled and concentrated to approximately 0.1 mg/mL using 30 k Amicon Ultra-15 centrifugal filters (Merck Millipore).

(41) 1.4 Western Blot.

(42) A431 and DU-145 cells were grown to 80% confluency. Cells were washed twice with 10 mL ice-cold PBS pH 7.4, scraped off and centrifuged (3 min, 1000 rpm). The pellet was washed with 5 mL PBS (3 min, 1000 rpm) and lysed by addition of 2 mL 1% Triton X-100 followed by centrifugation (10 min, 2700 rpm). The supernatant was loaded onto a polyacrylamide gel and SDS-PAGE was performed. Proteins were transferred from the gel to a nitrocellulose membrane using a Mini Trans-Blotter (100 V for 90 min) Non-specific binding sites were blocked by 5% non-fat milk powder in TBST buffer (1 h, RT). Matuzumab (anti-EGFR-antibody; 1:1000 dilution) was used as primary antibody and incubated with the membrane overnight at 4 C. After washing in TBST, the nitrocellulose membrane was incubated with horseradish peroxidase conjugated goat polyclonal anti-human IgG antibody (Thermo Fisher Scientific, Bonn, Germany, 1:1000 dilution) in blocking buffer at room temperature for 60 min. Antibody binding was determined using an enhanced chemiluminescence detection system (Western Lightening Plus ECL, Perkin-Elmer) according to the manufacturer's protocol and exposures were recorded on hyperfilms (10 s to 3 min).

(43) 1.5 Radiolabeling.

(44) A modified version of the established chloramine-T method (Hunter & Greenwood, 1962) employing [.sup.125I]NaI or [.sup.131I]NaI was used to introduce the radioactive iodine at random tyrosine side-chains of the mAb-dCPP conjugate. In brief, 30 L of the conjugates (0.1 mg/mL) was mixed with 20 L of phosphate buffer (0.25 M, pH 7.5). A solution containing 1-30 MBq [.sup.125I]NaI or [.sup.131I]NaI in 10 M NaOH was added and the labeling reaction was started by addition of an aqueous chloramine-T solution (10 mM, 5 L). After 30 s, the labeling reaction was quenched by adding a saturated aqueous solution of methionine (10 L). The labeling reaction mixture was passed over a PBS equilibrated buffer exchange column (NAP-10, GE Healthcare) and 300 L fractions were collected. 5 L of each fraction was transferred to a new vial and analyzed for -radiation using a -counter (LB 2111, Berthold Technologies, Bad Wildbad, Germany). Fractions 4-6 usually contained the radioactively labeled immunoreagent and were pooled. For in vivo experiments, the volume was reduced to 100 L in vacuo, and for cell binding experiments the solution was used as was.

(45) 1.6 Cell Binding and Uptake Assays.

(46) For binding experiments approximately 510.sup.5 cells of the EGFR-positive cell line A431 or the EGFR-negative, control cell line DU-145 cells were seeded into six-well plates and cultivated in 3 mL/well of RPMI-1640 (with 10% fetal calf serumFCS) at 37 C. in a 5% CO.sub.2 incubator. After 24 h the medium was replaced with 1 mL fresh medium (without FCS) containing 0.8-1.210.sup.6 cpm of .sup.125I-labeled mAb-dCPP conjugate and incubated for 60, 150 or 240 min at 37 C. After incubation the medium was removed and cells were washed three times with 1 mL ice cold PBS in order to remove unbound radiolabeled mAb-dCPP conjugates. To determine membrane bound activity, each well was incubated with 1 mL glycine buffer (50 mm glycine-HCl, pH 2.2) for 10 min at room temperature. The cells were washed again with PBS and subsequently lysed using 0.5 mL 0.3 mm sodium hydroxide solution. Radioactivity of the membrane bound fraction (glycine wash) and the internalized fraction (sodium hydroxide lysis) was measured using a -counter (LB 951 G, Berthold Technologies). The radioactivity was calculated as percentage applied dose per 10.sup.6 cells.

(47) 1.7 In Vivo Experiments.

(48) All animal experiments were carried out in conformity with German and European animal protection laws.

(49) 1.8 Biodistribution Studies.

(50) Radioactivity amounts of approximately 1 MBq of .sup.131I-labeled mAb-dCPPs in PBS were administered intravenously into female six-week old, immunodeficient, A431 tumor-bearing BALB/c nude mice (Charles River, Sulzfeld, Germany). After 1, 4, 24 and 48 h the mice (n=3; 18-22 g) were sacrificed, and selected tissue/blood samples (heart, lung, spleen, liver, kidney, muscle, intestine, brain and tumor) were removed, drained of blood, weighed, and the radioactivity determined using a -counter (LB 951G, Berthold Technologies). The percentage of injected dose per gram of tissue (% ID/g) was calculated.

(51) 1.9 Small Animal Imaging.

(52) Planar scintigraphy studies were performed using female BALB/c nude mice (Charles River, Sulzfeld, Germany), carrying subcutaneously transplanted A431 tumors. A cell suspension of 510.sup.6 cells in 100 L OPTI-MEM (Life Technologies, Darmstadt, Germany) was injected subcutaneously into the hind leg of the animals and the tumors were grown to a size of 1.0 cm.sup.3 in 10-12 days. Selected .sup.125I-labeled mAb-dCPP conjugates (7-10 MBq) were injected into the tail vein of the animals and planar scintigraphic images were recorded, while mice were anesthetized by 3-4% sevoflurane (Abbott, Wiesbaden, Germany), at defined time points using a -imager (Biospace Lab, Paris, France).

(53) Small-animal PET imaging was carried out using female BALB/c nude mice (Charles River) with xenografted A431 tumors. A suspension of 510.sup.6 A431 cells in 100 L OPTI-MEM (Life Technologies) was injected subcutaneously into the hind leg of the mice. Tumors were grown to a size of 0.5-0.7 cm.sup.3 in 11 days. PET scans, of anesthetized mice (3-4% sevoflurane, Abbott) in prone position, were performed using a Inveon microPET system (Siemens, Knoxville, Tenn., USA). .sup.124I-labeled mAb-CPP conjugates (10-12 MBq in 100 L PBS) were injected intravenously, and static images were acquired after 4, 24 and 48 h. Image data reconstructions and analyses were carried out using Inveon Research Workplace software (Siemens, Knoxville, Tenn., USA).

2. Results

(54) 2.1 Syntheses of Cell-Penetrating Peptides Dendrimers.

(55) In the first step the core of the dendrimer was synthesized using Fmoc-based solid phase peptide synthesis (SPPS). A cysteine residue as orthogonal, chemically addressable sulfhydryl group for crosslinking was coupled via its carboxyl-group onto the solid support (FIG. 1A), followed by an alanine residue, which served as a linker. In the next step the branching points were coupled to the resin. Due to their - and -amino groups, lysine qualifies to fulfill this task (Tam, 1988). By simultaneous deprotection of both amino groups two coupling positions are generated for the next amino acid during SPPS. In the final step of the core peptide synthesis, an additional lysine residue is coupled, and a tetravalent resin was gained, i.e. dendrimers with four branches will be obtained (FIG. 1B). This core peptide containing resin was used as a scaffold in SPPS for the generation of eight different tetravalent, dendritic cell-penetrating peptides (Table 2). After cleavage from solid support and side chain deprotection, the dendrimers were obtained as C-terminally amides for enhanced stability and their identities were proven by high-resolution mass spectrometry (Table 2). The structures of the generated dCPPs are exemplified by the dendritic tetravalent penetratin (FIG. 1C). An overview on the schematic structures of the synthesized dCPPs can be found in FIG. 7.

(56) TABLE-US-00003 TABLE2 SynthesizeddCPPs,sequences,molecularweight andreferences. Calc.Mw Observed Sequenceofthe ofdCPP (M+ H).sup.+ dCPP Monomer [Da] [Da] Reference dPenetratin RQIKIWFQNRRMKWKK 9486 9487 Dupontet (SEQIDNO:1) al.,2011 dTAT YGRKKRRQRRRPPQ 8029 8030 Vivesetal., (47-60) (SEQIDNO:2) 1997 dPreS2- PLSSIFSRIGDP 5656 5657 Oessetal., TLM (SEQIDNO:3) 2000 dR9 RRRRRRRRR 6197 6198 Mitchellet (SEQIDNO:4) al.,2000 dMTS AAVALLPAVLLALLAP 6565 6566 Kersemans (SEQIDNO:5) etal.,2008 dSynB1 RGGRLSYSRRRFSTSTGR 8901 8902 Rousselleet (SEQIDNO:6) al.,2000 dpVEC LLIILRRRIRKQAHAHSK 9338 9340 Elmquistet (SEQIDNO:7) al.,2001 dNLS PKKKRKV 4035 4036 Kalderonet (SEQIDNO:8) al.,1984 Abbreviations: Prefix d: dendritic; CPP: cell-penetrating peptide; TAT: human immunodeficiency virus-derived trans-activator of transcription; preS2-TLM: hepatitis B virus-preS2-domain-derived translocation motif; MTS: membrane translocation signal; SynB1: synthetic porcine protegrin 1-derived CPP; pVEC: vascular endothelial cadherin-derived CPP; NLS: nuclear localization signal.
2.2 Conjugation of the dCPPs with a Monoclonal Antibody.

(57) The epidermal growth factor receptor (EGFR) specific, humanized, monoclonal antibody Matuzumab (EMD72000, Merck, see also EP 0 531 472 B1, U.S. Pat. No. 5,558,864, WO 2009/043490 A1) was used in conjugation experiments. Crosslinking of the individual dCPPs was carried out employing the heterobifunctional crosslinker SMCC (FIG. 2A). To determine, the optimal crosslinker-to-antibody ratio the mAb was incubated with 7, 15 or 30 equivalents of SMCC. Amine groups of the mAb attacked the activated ester of SMCC, resulting in maleimide-activated antibody. Excess crosslinker was then removed by size exclusion chromatography using buffer exchange columns. Subsequently, the conjugation experiment was carried out using the maleimide-activated mAb and a 15-fold excess of the corresponding dCPP to allow efficient coupling between the mAb-maleimide and the sulfhydryl group of the dCPP. Exemplary, the reaction is shown for dendritic, tetravalent penetratin (dPenetratin, FIG. 2B). 15 equivalents of SMCC in the first reaction turned out to be ideal for the desired 1:1 or less coupling of the mAb to the dCPP.

(58) 2.3 Western Blot.

(59) To test the binding of the immunoconjugates to EGFR-expressing cells, the expression of the target antigen was validated by western blot. Therefore, the whole cell lysates of the EGFR-positive human epidermoid carcinoma cell line A431 and the EGFR-negative human prostate carcinoma cell line DU-145 were prepared and used in western blot analysis. The binding of the EGFR-targeting antibody matuzumab was visualized by a HRP-conjugated goat anti-human IgG antibody. The western blot (FIG. 8) revealed that matuzumab exclusively binds to A431, where a clear band at approximately 130 kDa is visible, which is in good agreement with the molecular weight of EGFR (132 kDa). No binding of the mAb to the control cell line DU-145 was observed.

(60) 2.4 Cell Binding and Internalization Experiments.

(61) The antibody and the conjugates were labeled with iodine-125 at a random tyrosine side chain using chlorarnine-T and [.sup.125I]NaI, as outlined in the methods section. The labeled antibody and the eight immunoconjugates respectively were added to the cell culture medium and incubated with the EGFR-positive cell line A431 for 60, 150 or 240 min. Control experiments were carried out accordingly with the EGFR-negative cell line DU-145. To be able to distinguish between membrane bound activity and internalized activity, the cells were washed with glycine buffer (pH 2.2) first to remove membrane-bound activity. In the second step the cells were lysed and internalized activity was measured. The measurement revealed that 220.2% of the applied dose of the radiolabeled, unmodified matuzumab bound to the EGFR-positive cells A431 (FIG. 3A), whereas no binding was observed for the control cell line DU-145. In addition only a small portion of the activity was internalized into the EGFR-positive cells (3%, FIG. 3A, black part of column). For the antibody, if modified with cell-penetrating peptide dendrimers, these values were in general higher and binding of the applied dose to the cells ranged from 470.5% (mAb-dSynB1) to 921.2% (mAb-dR9). Internalization rates were also higher, with a maximum of 380.9% (mAb-dR9) and a minimum of 90.7% (mAb-dSynB1). Binding of the immunoconjugates to the control cell line (FIG. 3B) was low (<10%) to moderate (161.1% for mAb-dTAT, 140.6% for mAb-dR9 and 130.6% for mAb-dNLS). The detailed results are visualized in FIG. 3.

(62) Furthermore, FIG. 10 shows that a toxin-conjugated antibody furthermore conjugated with Penetratin (4-mer) exhibits increased cell toxicity compared to the toxin-conjugated antibody without Penetratin (FIG. 10A).

(63) In addition, FIG. 11 shows fluorescence imaging results showing the cellular uptake of Penetratin antibody conjugates into A431 cells. As can be seen, EGFR antibody Penetratin-conjugate (4-mer) is internalized in A431 cells (FIG. 11C), whereas native EGFR antibody is not internalized (FIG. 11A) and the conjugate with Pen10 (4-mer), the control peptide, known to cause lower internalization rates, shows only reduced internalization (FIG. 11B).

(64) 2.5 Biodistribution Studies.

(65) The two most promising immunoconjugates, based on the cell binding and internalization experiments, were chosen for biodistribution studies. These two were mAb-dPenetratin and mAb-dR9. Although mAb-R9 was more unspecific140.6% binding to the control cell linethan other conjugates, it had the highest binding value of the eight different conjugates (921.2% binding). The immunoconjugate of matuzumab and dendritic penetratin showed good binding to A431 cells (680.9% binding) and low affinity for the control cell line (maximum 50.2%). As control, the unmodified antibody was used. All three compounds were labeled with iodine-131 and injected into A431 tumor-bearing mice. At different time points the mice were sacrificed, dissected and organs were examined for radioactivity uptake. This is exemplified by FIG. 4A, where the biodistribution after 96 hours is shown. At this time point the differences between the dCPP-modified antibody and the wildtype became most obvious. Tumor binding was high for all three compounds (8.90.2% for mAb-dR9, 8.70.3% for mAb and 6.20.2% for mAb-dPenetratin), whereas binding to other organs is less than 1.7%, except for the unmodified mAb. There, high values of 2.00.1%, 2.60.4% and 5.10.2% were observed for the kidneys, the heart and the lung, respectively. Most significantly, the blood contained 7.10.8% of the injected dose for the unmodified mAb; for mAb-dPenetratin and mAb-dR9 the blood contained 3.10.2% and 3.30.6% respectively, resulting in favorable tumor-to-blood ratios for the conjugates. As depicted in FIG. 4B, within the first four hours after administration of the antibody and the immunoconjugates, similar values for the tumor-to-blood ratios were observed. However, after 24 h there was a significant difference between the conjugates and the unmodified antibody for this ratio, which was even higher after 48 h. Over the full course of the 96 h examination period, the tumor-to-blood ratio reached a value of 2.7 for mAb-dR9 and 2.0 for mAb-dPenetratin, whereas the unmodified antibody did not exceed 1.3.

(66) 2.6 Small Animal Imaging Experiments.

(67) In the first set of experiments, planar scintigraphic images were recorded of athymic nude mice, with A431 tumors xenografted into the upper hind limb. The antibody and the immunoconjugates mAb-dR9 and mAb-dPenetratin were labeled with iodine-125. Then the radioactive compounds were administered intravenously into individual rodents, in order to record planar images after 1, 4, 24 and 48 h (FIG. 9). After 48 h (FIG. 5A) the differences between the unmodified and the conjugated antibody became obvious. In all mice high accumulation of the different radiopharmaceuticals was observed at the upper hind limb tumor site. A significant amount of the applied dose was still in circulation for the unmodified antibody, whereas almost no background was observed for the conjugates mAB-dPenetratin and mAb-dR9.

(68) In the second experimental setup, the antibody and its conjugates were radiolabeled with the positron emitter iodine-124 as outlined in the material and methods section. Again, these radiopharmaceuticals were administered intravenously into individual A431 nude mice xenografts and static PET images were recorded. As observed for the planar .sup.125I-images, the PET images showed that the conjugates had a faster clearance from the blood than the unmodified antibody. In addition, accumulation of radioactivity in the urinary tract was observed for mAb and mAb-dPenetratin. For all three examined radiopharmaceuticals a significant amount of the applied dose was found in the thyroid with the unconjugated antibody showing the highest value.

(69) The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

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