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. A method of producing a binding protein-drug conjugate (BPDC) comprising an anthracycline (PNU) derivative, the BPDC having the following formula: ##STR00006## wherein: a) each of L.sub.1-L3 represents a linker, wherein L.sub.1 is optional and both L.sub.2 and L.sub.3 are mandatory, b) L.sub.1, when present, represents an alkylene-amino linker or an alkylene-diamino linker, c) L.sub.2 represents an oligo-glycine peptide (Gly.sub.p), wherein p is an integer 1 and 21, d) L.sub.3 represents the amino acid residues 1-4 of a processed sortase tag pentapeptide motif, e) X and Y each represent one or more optional linkers, f) BP represents a binding protein selected from the group consisting of an antibody, a modified antibody format, an antigen-binding antibody derivative or fragment, and an antibody-based binding protein, and g) n is an integer 1 and 10, the method comprising conjugating, by means of a sortase enzyme, a binding protein carrying a sortase enzyme recognition motif, to at least one anthracycline (PNU) derivative conjugate comprising a derivative of the anthracycline PNU-159682 having the following formula (i) or formula (ii) ##STR00007## to form the BPDC, said anthracycline (PNU) derivative conjugate comprising at its wavy line a linker structure X-L.sub.1-L.sub.2.

2. The method of claim 1, wherein Y, when present, is (N-terminal to C-terminal) Gly-Gly-Gly-Gly-Ser (G.sub.4S) (SEQ ID NO: 8).

3. The method according to claim 1, wherein the oligo-glycine peptide is conjugated to the anthracycline (PNU) derivative by means of an ethylenediamino (EDA) linker, designated as L.sub.1, which ethylenediamino linker is conjugated to the anthracycline (PNU) derivative by means of a first amide bond, and it is conjugated to the carboxy terminus of the oligo-glycine peptide by means of a second amide bond.

4. The method according to claim 1, wherein said sortase enzyme recognition motif comprises a pentapeptide.

5. The method according to claim 4, wherein said sortase enzyme recognition motif comprises at least one amino acid sequence selected from the group consisting of (N-terminal to C-terminal) NPQTN (SEQ ID NO: 9), LPXTG (SEQ ID NO: 5), LPXSG (SEQ ID NO: 6), and LAXTG (SEQ ID NO: 7), wherein X in each of SEQ ID NOs: 5-7 represents any amino acid.

6. The method according to claim 1, wherein the anthracycline (PNU) derivative is conjugated, by means of the two or more linkers, to the carboxy terminus of the binding protein, or to the carboxy terminus of at least one domain or subunit thereof.

7. The method according to claim 1, 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 a hormone.

8. The method according to claim 1, wherein the binding protein is an antibody.

9. The method according to claim 1, wherein the binding protein is an antibody that binds HER-2.

10. The method according to claim 1, wherein the binding protein is an antibody that binds CD30.

11. The method according to claim 1, wherein the BPDC has the following formula: ##STR00008## wherein a) X is absent, b) L.sub.1 represents ethylenediamine (EDA), c) L.sub.2 represents an oligo-glycine peptide (Gly.sub.p), wherein p is an integer 1 and 21, d) L.sub.3 consists of amino acid residues 1-4 of a processed sortase enzyme recognition motif (N-terminal to C-terminal) Leu-Pro-Xaa-Thr-Gly (LPXTG) (SEQ ID NO: 5, wherein Xaa is any amino acid), e) Y, when present, is (N-terminal to C-terminal) Gly-Gly-Gly-Gly-Ser (G.sub.4S) (SEQ ID NO: 8), f) BP is an IgG antibody, and g) n is an integer 1 and 10.

12. The method according to claim 11, wherein the N-terminal Gly of Y is linked to the last C-terminal Cys amino acid of at least one light chain of the antibody.

13. The method according to claim 11, wherein the N-terminal Gly of Y is linked to the last C-terminal Ser amino acid of at least one light chain of the antibody.

Description

FIGURE LEGENDS

(1) 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 (n1 and 21, preferably m3 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.

(2) 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.

(3) FIGS. 3A-3B: (FIG. 3A) 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. (FIG. 3B) Synthesis scheme of anthracycline derivative PNU-EDA-Gly.sub.5.

(4) FIGS. 4A-4B: 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 (FIG. 4A), and on human Hodgkin lymphoma cell line L428 cells expressing very low levels of CD30 target in the cell surface (FIG. 4B). 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.

(5) FIGS. 5A-5B: Dose response of the cytotoxic effects of the indicated ADCs on human breast cancer cell line SKBR3, expressing high levels of HER-2/neu (FIG. 5A) and human breast cancer cell line T47D expressing low levels of HER-2/neu (FIG. 5B). 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.

(6) FIGS. 6A-6B: 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. FIG. 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. FIG. 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.

(7) FIGS. 7A-7B: (FIG. 7A) 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. (FIG. 7B) 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.

(8) 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.

(9) 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 fluorophore-containing anti-human IgG antibody (Fc gamma-specific) PE.

(10) FIGS. 10A-D: 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 (FIGS. 10A, 10C, 10D) 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 (FIG. 10A), 1 mg/kg Trastuzumab-PNU159682 (FIG. 10B, 10D), or 15 mg/kg Kadcyla (FIG. 10C). Tumor growth was monitored until animals had to be sacrificed due to ethical reasons.

(11) FIGS. 11A-B: Amino acid compositions of the C-terminally SMAC-technology conjugated IgH and IgL chains of the trastuzumab (FIG. 11A) and brentuximab (FIG. 11B) 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 4.sup.th amino acid of the sortase tag (highlighted in boldface print) via a peptide bond following sortase enzyme conjugation.

REFERENCES

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