Novel bacterial translocation domains and recombinant polypeptides containing same for use in cell delivery - Patents.com

Abstract

In general, the present disclosure provides novel bacterial translocation domains for use in cellular delivery. One example is a translocation domain having the amino acid sequence of SEQ ID NO:3, from the Austwickia chelonae protein of SEQ ID NO:2. Translocase domains from other bacterial strains and species are also described (e.g., SEQ ID NOs:4-17 and 36-48). Recombinant polypeptides comprising those translocation domains are described. The recombinant polypeptides are intended for use in delivery of cargo molecules, including therapeutic polypeptides that can be used in the treatment of disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from Greek Patent Application No. 20210100770, filed November 4, 2021, entitled "Novel bacterial translocation domains and recombinant polypeptides comprising same for use in cellular delivery."

[0002] Field The present disclosure relates generally to a delivery platform. In particular, the present disclosure relates to a bacterial toxin-based platform for delivering cargo molecules to cells.

[0003] background Therapeutic molecules are often difficult to deliver to cells. They often do not easily penetrate biological membranes. Immunotoxins are a class of biologics consisting of bacterial toxins, such as diphtheria toxin (DT), that have been repurposed into targeted cancer therapies, both by retargeting their receptor-binding domains (RBDs) to target cancer receptors and by delivering enzymatic cargos that target intracellular cancer proteins. However, global vaccination programs against diphtheria have led to population-level immunity to DT and DT-based therapeutics.

[0004] There remains a need for delivery platforms that offer the possibility of delivering therapeutics, including protein-based therapeutics, to cells.

[0005] Abstract It is the subject of the present disclosure to obviate or mitigate at least one of the disadvantages of the previous approaches.

[0006] In a first aspect, the present disclosure provides a compound of general formula (I): A B C (i) (In the formula, A is a cargo molecule, B is, a) Austwickia chelonae protein of SEQ ID NO:2, Streptosporangium nondiastaticum protein with GenBank accession number PSJ28985.1; Streptomyces sp. TLI053 protein, GenBank accession number SDT83331.1; Streptomyces sp. SLBN-118 protein, GenBank accession number WP_160159328.1; Streptomyces sp. AA8 protein, GenBank accession number WP_168096531.1; Streptomyces roseoverticillatus protein, GenBank accession number WP_078659863.1; Streptomyces piniterrae protein, GenBank accession number JZ58907.1; Streptomyces MBT76 protein, GenBank accession number WP_079110321.1; Streptomyces klenkii protein, GenBank accession number WP_120757473.1; Streptomyces albireticuli protein, GenBank accession number WP_095582082.1; Streptacidiphilus pinicola protein, GenBank accession number WP_133259917.1; Seinonella peptonophila protein, GenBank accession number WP_073156187.1; Longimycelium tulufanense protein with GenBank accession number WP_189053160.1; Austwickia sp. TVS96-490-7B protein, GenBank accession number WP_219106995.1; Austwickia chelonae LK16-18 protein, GenBank accession number WP_162873017.1; Klebsiella aerogenes protein, GenBank accession number EIZ2913133.1; Streptomyces sp. MUM178J protein, GenBank accession number MCH0551590.1; Crossiella cryophila protein, GenBank accession number MBB4677777.1; Allokutzneria sp. NRRL B-24872 protein, with GenBank accession number WP_143261759.1; Allokutzneria albata protein, GenBank accession number WP_156051914.1; Streptomyces sp. AV19 protein, GenBank accession number WP_199893204.1; Streptomyces sp. NRBC_110611 protein with GenBank accession number WP_147264604.1; Streptomyces syringium protein, GenBank accession number WP_209513619.1; Pseudonocardiaceae bacterium YIM PH21723 protein with GenBank accession number RJQ69589.1; Actinokineospora bangkokensis protein with GenBank accession number WP_143218892.1; Streptomyces eurocidicus protein, GenBank accession number MBF6055834.1; Streptomyces pathocidini protein, GenBank accession number WP_169790908.1, or Streptomyces caatingaensis protein with GenBank accession number WP_157868472.1 the transition domain from which it originated, or b) a transition domain that is at least 80% identical to the transition domain defined in a); a transition polypeptide comprising C is a targeting moiety The present invention provides a recombinant polypeptide of the present invention.

[0007] In one aspect, a nucleic acid encoding a recombinant polypeptide as defined herein is provided.

[0008] In one aspect, there is provided a vector comprising a nucleic acid as defined herein.

[0009] In a further aspect, the present disclosure provides a composition comprising a recombinant polypeptide as defined herein, together with an acceptable excipient, diluent or carrier.

[0010] In one aspect of the invention, there is provided a pharmaceutical composition comprising a recombinant polypeptide as defined herein, together with a pharma- ceutically acceptable excipient, diluent or carrier.

[0011] In one aspect, there is provided a method of delivering a cargo molecule to a cell comprising contacting the cell with a recombinant polypeptide as defined herein.

[0012] In one aspect there is provided a use of a recombinant polypeptide as defined herein for delivering a cargo molecule to a cell.

[0013] In one aspect there is provided the use of a recombinant polypeptide as defined herein for the preparation of a medicament for delivering a cargo molecule to a cell.

[0014] In one aspect, there is provided a recombinant polypeptide as defined herein for use in delivering a cargo molecule to a cell.

[0015] In one aspect, there is provided a method of treating cancer in a subject comprising administering to the subject a recombinant polypeptide as defined herein.

[0016] In one aspect, there is provided a use of a recombinant polypeptide as defined herein for treating cancer in a subject.

[0017] In one aspect, there is provided a use of a recombinant polypeptide as defined herein for the preparation of a medicament for treating cancer in a subject.

[0018] In one aspect there is provided a recombinant polypeptide as defined herein for use in the treatment of cancer in a subject.

[0019] Other aspects and features of the present disclosure will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments in conjunction with the accompanying drawings.

[0020] Embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0021] [Figure 1-1] FIG. 1A is a schematic representation of the cell entry mechanism of diphtheria toxin. [Figure 1-2]FIG. 1B is a diagram showing the domain organization of DT and CT1, showing the catalytic domain (C) and the bridging furin recognition site (F) followed by the translocase (T) and receptor binding domains (R). [Figure 1-3] FIG. 1C shows the crystal structures of diphtheria toxin and CT1. [Figure 2-1] Figure 2A is a graph showing the results of experiments assessing the function and release of the CT1 catalytic domain. DT and DT chimeras containing the C domain of CT1 show similar toxicity towards HEK293T cells (b). [Figure 2-2] FIG. 2B is a graph showing that DT and DT chimeras containing the C domain of CT1 have no effect on cell viability in DPH4− / − cells. [Figure 2-3] FIG. 2C shows that incubation of DT and CT1 with cell lysates resulted in cleavage at the furin recognition site. [Figure 3-1] FIG. 3A is a graph showing that DT and a DT chimera containing the furin site from CT1 are equipotent on Vero cells, indicating efficient cargo release by both toxins. [Figure 3-2] FIG. 3B is a graph showing the reduction of protein synthesis in Vero cells by CT1-T mediated delivery of DT-C. [Figure 3-3] Figure 3C is a graph showing CT1-T mediated delivery of the unnatural cargo RRSP, which is toxic to RAS mutant cells such as CFPAC-1. [Diagram 3-4] FIG. 3D shows that delivery was confirmed by cleavage of intracellular RAS by RRSP. [Figure 3-5] Figure 3E is a graph showing CT1-T-mediated delivery of DT-C as measured by cell viability in HPAF II cells. Unexpectedly, CT1-T is a more efficient translocase than DT-T when complexed with a targeting domain other than DT-R. [Diagram 3-6]FIG. 3F is a graph showing CT1-T mediated delivery of RRSP by measuring cell viability in HPAF II cells. [Figure 4-1] Figure 4A is a graph showing the results of an experiment evaluating human serum binding and neutralization of CT1.To quantify the level of pre-existing anti-DT or anti-CT1 antibodies in human serum, DT or CT1 was immobilized on Nunc MaxiSorp™ plates and incubated with human serum at different dilutions.The wells were then incubated with anti-human IgG antibody conjugated to HRP, and HRP was developed using TMB reagent.The absorbance was read at 630 nm. [Figure 4-2] Figure 4B is a graph showing the results of further experiments evaluating human serum binding and neutralization of CT1. To determine the effect of neutralizing antibodies on the ability of DT or CT1 to intoxicate cells, various DT / CT1 toxin chimeras (as indicated) were incubated with human serum (or PBS) and then added to Vero cells and protein synthesis levels were measured. EC50 values ​​were measured and fold-difference from the PBS control was plotted. Human serum had no effect on the ability of CT1 to intoxicate cells, demonstrating that CT1 is not neutralized by human serum. [Diagram 5] FIG. 5 is a schematic representation of key residues conserved between DT and CT1. [Figure 6-1] 6A and 6B are graphs showing the results of the functional characterization of the translocase. [Figure 6-2] 6A and 6B are graphs showing the results of the functional characterization of the translocase. [Figure 7-1] FIG. 7A is a graph showing that antibodies in human serum recognize DT but show no binding to CT1. [Figure 7-2] FIG. 7B is a graph showing that antibodies in human serum recognize the translocase derived from DT but not from CT1. [Figure 8]FIG. 8 is a graph showing that anti-DT antibodies do not neutralize CT1-based immunotoxins. [Figure 9] FIG. 9 shows a phylogenetic tree illustrating the relationships of the various translocases.

[0022] Detailed Description Generally, the present disclosure provides novel bacterial translocation domains for use in cellular delivery. Recombinant polypeptides comprising those translocation domains are described. The recombinant polypeptides are intended for use in the delivery of cargo molecules, including therapeutic polypeptides.

[0023] Recombinant Polypeptides In one embodiment, a compound of general formula (I): A B C (i) (In the formula, A is a cargo molecule, B is, a) Austwickia chelonae protein of SEQ ID NO:2, Streptosporangium nondiastaticum protein, GenBank accession number PSJ28985.1; Streptomyces sp. TLI053 protein, GenBank accession number SDT83331.1; Streptomyces sp. SLBN-118 protein, GenBank accession number WP_160159328.1; Streptomyces sp. AA8 protein, GenBank accession number WP_168096531.1; Streptomyces roseoverticillatus protein, GenBank accession number WP_078659863.1; Streptomyces piniterrae protein, GenBank accession number JZ58907.1; Streptomyces MBT76 protein, GenBank accession number WP_0791103f21.1; Streptomyces klenkii protein, GenBank accession number WP_120757473.1; Streptomyces albireticuli protein, GenBank accession number WP_095582082.1; Streptacidiphilus pinicola protein, GenBank accession number WP_133259917.1; Seinonella peptonophila protein, GenBank accession number WP_073156187.1; Longimycelium tulufanense protein with GenBank accession number WP_189053160.1; Austwickia sp. TVS96-490-7B protein, GenBank accession number WP_219106995.1; Austwickia chelonae LK16-18 protein, GenBank accession number WP_162873017.1; Klebsiella aerogenes protein, GenBank accession number EIZ2913133.1; Streptomyces sp. MUM178J protein, GenBank accession number MCH0551590.1; Crossiella cryophila protein, GenBank accession number MBB4677777.1; Allokutzneria sp. NRRL B-24872 protein, with GenBank accession number WP_143261759.1; Allokutzneria albata protein, GenBank accession number WP_156051914.1; Streptomyces sp. AV19 protein, GenBank accession number WP_199893204.1; Streptomyces sp. NRBC_110611 protein with GenBank accession number WP_147264604.1; Streptomyces syringium protein, GenBank accession number WP_209513619.1; Pseudonocardiaceae bacterium YIM PH21723 protein with GenBank accession number RJQ69589.1; Actinokineospora bangkokensis protein with GenBank accession number WP_143218892.1; Streptomyces eurocidicus protein, GenBank accession number MBF6055834.1; Streptomyces pathocidini protein, GenBank accession number WP_169790908.1, or Streptomyces caatingaensis protein with GenBank accession number WP_157868472.1 the transition domain from which it originated, or b) a transition domain that is at least 80% identical to the transition domain defined in a); a transition polypeptide comprising C is a targeting moiety Recombinant polypeptides of the invention are provided.

[0024] In one embodiment, in a), the transition domain is - the Austwickia chelonae protein of SEQ ID NO: 2, - the Streptosporangium nondiastaticum protein with GenBank accession number PSJ28985.1, - Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1, - the Streptomyces sp. SLBN-118 protein, with GenBank accession number WP_160159328.1; - the Streptomyces sp. AA8 protein with GenBank accession number WP_168096531.1; - Streptomyces roseoverticillatus protein with GenBank accession number WP_078659863.1, - the Streptomyces piniterrae protein with GenBank accession number JZ58907.1, - the Streptomyces MBT76 protein, with the GenBank accession number WP_079110321.1; - the Streptomyces klenkii protein with the GenBank accession number WP_120757473.1, - the Streptacidiphilus pinicola protein with the GenBank accession number WP_133259917.1, - the Longimycelium tulufanense protein with the GenBank accession number WP_189053160.1, - the Austwickia sp. TVS96-490-7B protein with GenBank accession number WP_219106995.1, or - Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1 It comes from.

[0025] In one embodiment, in a), the transition domain is - the Austwickia chelonae protein of SEQ ID NO: 2, - Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1, - the Streptomyces klenkii protein with the GenBank accession number WP_120757473.1, - the Austwickia sp. TVS96-490-7B protein with GenBank accession number WP_219106995.1, or - Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1 It comes from.

[0026] In one embodiment, in a), the transition domain is - the Austwickia chelonae protein of SEQ ID NO: 2, - Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1 It comes from.

[0027] In one embodiment, in a), i. the translocation domain from Austwickia chelonae protein, referred to herein as CT1, has the amino acid sequence of SEQ ID NO:3; ii. The translocation domain from Streptosporangium nondiastaticum protein of GenBank accession number PSJ28985.1 has the amino acid sequence of SEQ ID NO:4; iii. The translocation domain from Streptomyces sp. TLI053 protein, GenBank accession number SDT83331.1, has the amino acid sequence of SEQ ID NO:5; iv. The translocation domain from Streptomyces sp. SLBN-118 protein, having GenBank accession number WP_160159328.1, has the amino acid sequence of SEQ ID NO:6; v. The translocation domain from Streptomyces sp. AA8 protein of GenBank accession number WP_168096531.1 has the amino acid sequence of SEQ ID NO:7; vi. The translocation domain from Streptomyces roseoverticillatus protein of GenBank accession number WP_078659863.1 has the amino acid sequence of SEQ ID NO:8; vii. The translocation domain from Streptomyces piniterrae protein having GenBank accession number JZ58907.1 has the amino acid sequence of SEQ ID NO:9; viii. The translocation domain from Streptomyces MBT76 protein of GenBank accession number WP_079110321.1 has the amino acid sequence of SEQ ID NO: 10; ix. The translocation domain from Streptomyces klenkii protein of GenBank accession number WP_120757473.1 has the amino acid sequence of SEQ ID NO: 11; x. The translocation domain from Streptomyces albireticuli protein of GenBank accession number WP_095582082.1 has the amino acid sequence of SEQ ID NO: 12; xi. The translocation domain from Streptacidiphilus pinicola protein of GenBank accession number WP_133259917.1 has the amino acid sequence of SEQ ID NO: 13; xii. The translocation domain from Seinonella peptonophila protein of GenBank accession number WP_073156187.1 has the amino acid sequence of SEQ ID NO: 14; xiii. The translocation domain from the Longimycelium tulufanense protein of GenBank accession number WP_189053160.1 has the amino acid sequence of SEQ ID NO: 15; xiv. The translocation domain from Austwickia sp. TVS96-490-7B protein of GenBank accession number WP_219106995.1 has the amino acid sequence of SEQ ID NO: 16; xv. The translocation domain from Austwickia chelonae LK16-18 protein of GenBank accession number WP_162873017.1 has the amino acid sequence of SEQ ID NO: 17; and xvi. The translocation domain has an amino acid sequence of any one of SEQ ID NOs: 36 to 48.

[0028] In one embodiment, in a), i. the translocation domain from Austwickia chelonae protein, referred to herein as CT1, has the amino acid sequence of SEQ ID NO:3; ii. The translocation domain from Streptosporangium nondiastaticum protein of GenBank accession number PSJ28985.1 has the amino acid sequence of SEQ ID NO:4; iii. The translocation domain from Streptomyces sp. TLI053 protein, GenBank accession number SDT83331.1, has the amino acid sequence of SEQ ID NO:5; iv. The translocation domain from Streptomyces sp. SLBN-118 protein, having GenBank accession number WP_160159328.1, has the amino acid sequence of SEQ ID NO:6; v. The translocation domain from Streptomyces sp. AA8 protein of GenBank accession number WP_168096531.1 has the amino acid sequence of SEQ ID NO:7; vi. The translocation domain from Streptomyces roseoverticillatus protein of GenBank accession number WP_078659863.1 has the amino acid sequence of SEQ ID NO:8; vii. The translocation domain from Streptomyces piniterrae protein having GenBank accession number JZ58907.1 has the amino acid sequence of SEQ ID NO:9; viii. The translocation domain from Streptomyces MBT76 protein of GenBank accession number WP_079110321.1 has the amino acid sequence of SEQ ID NO: 10; ix. The translocation domain from Streptomyces klenkii protein of GenBank accession number WP_120757473.1 has the amino acid sequence of SEQ ID NO: 11; xi. The translocation domain from Streptacidiphilus pinicola protein of GenBank accession number WP_133259917.1 has the amino acid sequence of SEQ ID NO: 13; xiii. The translocation domain from the Longimycelium tulufanense protein of GenBank accession number WP_189053160.1 has the amino acid sequence of SEQ ID NO: 15; xiv. The translocation domain from Austwickia sp. TVS96-490-7B protein of GenBank accession number WP_219106995.1 has the amino acid sequence of SEQ ID NO: 16; and xv. The translocation domain from Austwickia chelonae LK16-18 protein, having GenBank accession number WP_162873017.1, has the amino acid sequence of SEQ ID NO:17.

[0029] A "translocation polypeptide" as referred to herein is intended to refer to a polypeptide that comprises a translocation domain.

[0030] A "translocation domain," as referred to herein, is a polypeptide sequence that functions to facilitate the transport of a protein across a cell membrane, thereby facilitating cell entry. This activity can be assessed, for example, using the assays described herein.

[0031] In one embodiment, the transition polypeptide is a) a translocation domain from the Austwickia chelonae protein CT1, having the amino acid sequence of SEQ ID NO:3 (herein referred to as "CT1-T"), or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0032] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 4 from the Streptosporangium nondiastaticum protein having GenBank accession number PSJ28985.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0033] In one embodiment, the transition polypeptide is a) a translocation domain from the Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1, having the amino acid sequence of SEQ ID NO: 5; or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0034] In one embodiment, the transition polypeptide is a) a translocation domain from the Streptomyces sp. SLBN-118 protein having the GenBank accession number WP_160159328.1 and having the amino acid sequence of SEQ ID NO: 6, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0035] In one embodiment, the transition polypeptide is a) a translocation domain from the Streptomyces sp. AA8 protein of GenBank accession number WP_168096531.1, having the amino acid sequence of SEQ ID NO: 7, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0036] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 8 from the Streptomyces roseoverticillatus protein of GenBank accession number WP_078659863.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0037] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 9 from the Streptomyces piniterrae protein with GenBank accession number JZ58907.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0038] In one embodiment, the transition polypeptide is a) a translocation domain from the Streptomyces MBT76 protein of GenBank accession number WP_079110321.1, having the amino acid sequence of SEQ ID NO: 10, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0039] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 11 from the Streptomyces klenkii protein with GenBank accession number WP_120757473.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0040] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 12 from the Streptomyces albireticuli protein of GenBank accession number WP_095582082.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0041] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 13 from the Streptacidiphilus pinicola protein of GenBank accession number WP_133259917.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0042] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 14 from the Seinonella peptonophila protein of GenBank accession number WP_073156187.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0043] The transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 15, derived from the Longimycelium tulufanense protein having GenBank accession number WP_189053160.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0044] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 16 from the Austwickia sp. TVS96-490-7B protein of GenBank accession number WP_219106995.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0045] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 17 from the Austwickia chelonae LK16-18 protein of GenBank accession number WP_162873017.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0046] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 36 from the Klebsiella aerogenes protein with GenBank accession number EIZ2913133.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0047] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 37 from the Streptomyces sp. MUM178J protein with GenBank accession number MCH0551590.1; or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0048] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 38 from the Crossiella cryophila protein with GenBank accession number MBB4677777.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0049] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 39 from the Allokutzneria sp. NRRL B-24872 protein of GenBank accession number WP_143261759.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0050] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 40 from the Allokutzneria albata protein of GenBank accession number WP_156051914.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0051] In one embodiment, the transition polypeptide is a) a translocation domain from the Streptomyces sp. AV19 protein with GenBank accession number WP_199893204.1, having the amino acid sequence of SEQ ID NO: 41; or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0052] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 42 from the Streptomyces sp. NRBC_110611 protein with GenBank accession number WP_147264604.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0053] In one embodiment, the transition polypeptide is a) a translocation domain from the Streptomyces syringium protein with GenBank accession number WP_209513619.1, having the amino acid sequence of SEQ ID NO: 43; or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0054] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 44 from the Pseudonocardiaceae bacterium YIM PH21723 protein with GenBank accession number RJQ69589.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0055] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 45 from the Actinokineospora bangkokensis protein of GenBank accession number WP_143218892.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0056] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 46 from the Streptomyces eurocidicus protein with GenBank accession number MBF6055834.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0057] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 47 from the Streptomyces pathocidini protein with GenBank accession number WP_169790908.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0058] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 48 from the Streptomyces caatingaensis protein with GenBank accession number WP_157868472.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a); Includes.

[0059] The Austwickia chelonae LK16-18 protein, GenBank Accession No. WP_162873017.1, may be referred to herein as "CT2." The Austwickia sp. TVS96-490-7B protein, GenBank Accession No. WP_219106995.1, may be referred to herein as "CT3."

[0060] In one embodiment, the transition domain is as defined in b) of any one of the above embodiments and is at least at least 85% identical over its entire length to the transition domain defined in a). In one embodiment, the transition domain is as defined in b) of any one of the above embodiments and is at least at least 90% identical over its entire length to the transition domain defined in a). In one embodiment, the transition domain is as defined in b) of any one of the above embodiments and is at least at least 95% identical over its entire length to the transition domain defined in a). In one embodiment, the transition domain is as defined in b) of any one of the above embodiments and is at least at least 98% identical over its entire length to the transition domain defined in a). In one embodiment, the transition domain is as defined in b) of any one of the above embodiments and is at least at least 99% identical over its entire length to the transition domain defined in a).

[0061] In one embodiment, the transition polypeptide is as defined under a) in any one of the above embodiments.

[0062] In one embodiment, the transition polypeptide is a) a translocation domain from the Austwickia chelonae CT1 protein having the amino acid sequence of SEQ ID NO:3; Includes.

[0063] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO:4 from the Streptosporangium nondiastaticum protein of GenBank accession number PSJ28985.1; Includes.

[0064] In one embodiment, the transition polypeptide is a) A translocation domain having the amino acid sequence of SEQ ID NO:5 from the Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1 Includes.

[0065] In one embodiment, the transition polypeptide is a) A translocation domain having the amino acid sequence of SEQ ID NO: 6 from the Streptomyces sp. SLBN-118 protein of GenBank accession number WP_160159328.1 Includes.

[0066] In one embodiment, the transition polypeptide is a) A translocation domain having the amino acid sequence of SEQ ID NO: 7 from the Streptomyces sp. AA8 protein of GenBank accession number WP_168096531.1 Includes.

[0067] In one embodiment, the transition polypeptide is a) Translocation domain from Streptomyces roseoverticillatus protein with GenBank accession number WP_078659863.1 Includes.

[0068] In one embodiment, the transition polypeptide is a) Translocation domain from the Streptomyces piniterrae protein with GenBank accession number JZ58907.1 Includes.

[0069] In one embodiment, the transition polypeptide is a) A translocation domain having the amino acid sequence of SEQ ID NO: 10 from the Streptomyces MBT76 protein of GenBank accession number WP_079110321.1 Includes.

[0070] In one embodiment, the transition polypeptide is a) Translocation domain from the Streptomyces klenkii protein with GenBank accession number WP_120757473.1 Includes.

[0071] In one embodiment, the transition polypeptide is a) A translocation domain having the amino acid sequence of SEQ ID NO: 12 from the Streptomyces albireticuli protein of GenBank accession number WP_095582082.1. Includes.

[0072] In one embodiment, the transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 13 from the Streptacidiphilus pinicola protein of GenBank accession number WP_133259917.1; Includes.

[0073] In one embodiment, the transition polypeptide is a) A translocation domain having the amino acid sequence of SEQ ID NO: 14, derived from the Seinonella peptonophila protein of GenBank accession number WP_073156187.1. Includes.

[0074] The transition polypeptide is a) a translocation domain having the amino acid sequence of SEQ ID NO: 15, derived from the Longimycelium tulufanense protein of GenBank accession number WP_189053160.1; Includes.

[0075] In one embodiment, the transition polypeptide is a) A transition domain having the amino acid sequence of SEQ ID NO: 16 from the Austwickia sp. TVS96-490-7B protein of GenBank accession number WP_219106995.1 Includes.

[0076] In one embodiment, the transition polypeptide is a) A translocation domain having the amino acid sequence of SEQ ID NO: 17 from the Austwickia chelonae LK16-18 protein of GenBank accession number WP_162873017.1. Includes.

[0077] In some embodiments, the translocation domains having the amino acid sequence of SEQ ID NO: 12 (from the Streptomyces albireticuli protein with GenBank accession number WP_095582082.1) and the amino acid sequence of SEQ ID NO: 14 (from the Seinonella peptonophila protein with GenBank accession number WP_073156187.1) are excluded from the above embodiments.

[0078] In some embodiments, a translocation polypeptide can comprise a functional truncation of a full-length protein that includes any one of the corresponding translocation domains described above, provided that the function of the translocation domain is maintained.

[0079] In one embodiment, A and B are separated by a linker. In one embodiment, A and B are separated by an amino acid linker. In one embodiment, the amino acid linker comprises (G4S)2. In one embodiment, the linker is cleavable. In one embodiment, the linker comprises a protease recognition site. In one embodiment, the protease recognition site is a furin protease recognition site. In one embodiment, the protease recognition site is bracketed by cysteine ​​residues that allow for the formation of a disulfide bond forming an intramolecular loop. In one embodiment, the amino acid linker comprises a furin protease recognition site bracketed by cysteine ​​residues. In one exemplary embodiment, the amino acid linker comprises SEQ ID NO: 32, which comprises a furin protease recognition site and cysteine ​​residues flanking it. In one embodiment, said (G4S)2 is located N-terminally with respect to said SEQ ID NO: 32. In one embodiment, the linker is self-cleaving. In one embodiment, the linker is self-clearing, hi one embodiment, the linker comprises an autoprocessing domain.

[0080] In one embodiment, B and C are separated by a linker. In one embodiment, B and C are separated by an amino acid linker. In one embodiment, the amino acid linker comprises (G4S)2. In one embodiment, the amino acid linker comprises (G4S)2. In one embodiment, the amino acid linker comprises SEQ ID NO: 33. In one embodiment, said (G4S)2 is located N-terminally with respect to said SEQ ID NO: 33.

[0081] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 821 of SEQ ID NO:22, preferably 90% identical to amino acids 1 to 821 of SEQ ID NO:22, more preferably 95% identical to amino acids 1 to 821 of SEQ ID NO:22, and even more preferably 100% identical to amino acids 1 to 821 of SEQ ID NO:22.

[0082] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:22.

[0083] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO:23, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO:23, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO:23, and even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO:23.

[0084] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:23.

[0085] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO:24, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO:24, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO:24, and even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO:24.

[0086] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:24.

[0087] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 821 of SEQ ID NO:25, preferably 90% identical to amino acids 1 to 821 of SEQ ID NO:25, more preferably 95% identical to amino acids 1 to 821 of SEQ ID NO:25, and even more preferably 100% identical to amino acids 1 to 821 of SEQ ID NO:25.

[0088] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:25.

[0089] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 806 of SEQ ID NO:26, preferably 90% identical to amino acids 1 to 806 of SEQ ID NO:26, more preferably 95% identical to amino acids 1 to 806 of SEQ ID NO:26, and even more preferably 100% identical to amino acids 1 to 806 of SEQ ID NO:26.

[0090] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:26.

[0091] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 811 of SEQ ID NO:27, preferably 90% identical to amino acids 1 to 811 of SEQ ID NO:27, more preferably 95% identical to amino acids 1 to 811 of SEQ ID NO:27, and even more preferably 100% identical to amino acids 1 to 811 of SEQ ID NO:27.

[0092] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:27.

[0093] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO:28, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO:28, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO:28, and even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO:28.

[0094] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:28.

[0095] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 796 of SEQ ID NO:29, preferably 90% identical to amino acids 1 to 796 of SEQ ID NO:29, more preferably 95% identical to amino acids 1 to 796 of SEQ ID NO:29, and even more preferably 100% identical to amino acids 1 to 796 of SEQ ID NO:29.

[0096] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:29.

[0097] In one embodiment, the recombinant polypeptide comprises an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO: 30, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO: 30, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO: 30, and even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO: 30.

[0098] In one embodiment, the recombinant polypeptide comprises the sequence of SEQ ID NO:30.

[0099] In one embodiment, the targeting moiety comprises a targeting polypeptide or an aptamer.

[0100] In one embodiment, the targeting polypeptide comprises an antibody, a binding fragment of an antibody, an affibody, an affitin, a DARPin, or a receptor ligand.

[0101] In one embodiment, the targeting polypeptide comprises an affibody against Her3. In one embodiment, the affibody against Her3 comprises the amino acid sequence of SEQ ID NO:19.

[0102] In one embodiment, the targeting polypeptide comprises a receptor ligand for avβ6 integrin. In one embodiment, the receptor ligand for avβ6 integrin comprises the amino acid sequence of SEQ ID NO:20.

[0103] In one embodiment, the targeting moiety comprises at least two targeting polypeptides, at least two aptamers, or a combination of targeting polypeptides and aptamers. In one embodiment, the at least two targeting polypeptides are selected from the group of antibodies, binding fragments of antibodies, affibodies, peptides, affitins, DARPins, receptor ligands, and combinations thereof. In one embodiment, the at least two targeting polypeptides comprise an affibody for Her3. In one embodiment, the affibody for Her3 comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, the at least two targeting polypeptides comprise a receptor ligand for avβ6 integrin. In one embodiment, the receptor ligand for avβ6 integrin comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, the at least two targeting polypeptides comprise both an affibody for Her3 and a receptor ligand for avβ6 integrin, preferably the former comprises the amino acid sequence of SEQ ID NO: 19 and the latter comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, at least two targeting polypeptides, at least two aptamers, or a combination thereof are separated by an amino acid linker. In one embodiment, the amino acid linker comprises (G4S)2.

[0104] In one embodiment, the targeting moiety binds to a cell surface protein.

[0105] In one embodiment, the cell surface protein is lineage-specific or tissue-specific.

[0106] In one embodiment, the cell surface protein is ubiquitously expressed.

[0107] In one embodiment, the cell surface protein is expressed in a diseased cell.

[0108] In one embodiment, the cell surface protein is specific to a diseased cell and is not expressed in a corresponding healthy cell.

[0109] In one embodiment, the cell surface protein has elevated expression in diseased cells relative to corresponding healthy cells.

[0110] In one embodiment, the diseased cells are cancer cells.

[0111] In one embodiment, the cargo molecule comprises a cargo polypeptide.

[0112] The cargo polypeptide may comprise any polypeptide for which cellular delivery is desired. The cargo polypeptide may comprise an enzyme, or an active fragment thereof having substantially the same activity. By "substantially the same activity" it is meant that the core function of the enzyme is substantially unchanged in the fragment.

[0113] The cargo polypeptide may have a molecular weight of less than 10 kDa, more than 10 kDa, more than 20 kDa, more than 30 kDa, more than 50 kDa, more than 100 kDa, or more than 150 kDa.

[0114] The cargo polypeptide comprises a genome-modifying protein. The genome-modifying protein comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR (clustered regularly interspaced short palindromic repeats) protein. The CRISPR protein can be Cas9. The cargo polypeptide can comprise a complex of a genome-modifying protein and a nucleic acid, such as a guide nucleic acid. For example, Cas9 can be complexed with a nucleic acid, such as a guide RNA, such as a crRNA, a trRNA, and / or a sgRNA.

[0115] In one embodiment, the cargo molecule comprises a therapeutic polypeptide.

[0116] "Therapeutic polypeptide" refers to any protein whose cellular delivery can be used for therapeutic purposes. For example, it is well known that many human diseases or disorders are caused by or characterized by protein deficiencies. Therapeutic proteins include proteins whose delivery can improve or correct such deficiencies. Therapeutic proteins can act to replace proteins that are defective in a disease or disorder. Therapeutic proteins can be proteins that are defective in a disease or disorder. However, therapeutic proteins do not necessarily have to be identical to the proteins that are defective in a disease or disorder. For example, therapeutic proteins can be active fragments or modified versions of defective proteins. Therapeutic proteins can also partially or completely compensate for the protein deficiency that is the basis of a disease or disorder. Therapeutic proteins can also improve or correct downstream or secondary effects of cellular deficiencies of a particular protein.

[0117] In one embodiment, the therapeutic polypeptide comprises a cytotoxic polypeptide, preferably a polypeptide toxin or a functional fragment thereof. In one embodiment, the cytotoxic polypeptide comprises a catalytic domain derived from diphtheria toxin. In one embodiment, the cytotoxic polypeptide comprises a catalytic domain derived from Chelona Toxin, such as CT1 (SEQ ID NO: 2), CT2 (SEQ ID NO: 21), or CT3 (SEQ ID NO: 35) as described herein.

[0118] In one embodiment, the catalytic domain is derived from CT1. In one embodiment, the catalytic domain from Chelone toxin set forth in SEQ ID NO:2 (CT1) has the amino acid sequence according to amino acids 1 to 186 of SEQ ID NO:2.

[0119] In one embodiment, the catalytic domain is derived from CT2. In one embodiment, the catalytic domain from Chelone toxin set forth in SEQ ID NO:21 (CT2) has the amino acid sequence according to amino acids 1 to 186 of SEQ ID NO:21.

[0120] In one embodiment, the catalytic domain is derived from CT3. In one embodiment, the catalytic domain from Chelone toxin set forth in SEQ ID NO:35 (CT3) has the amino acid sequence according to amino acids 1 to 191 of SEQ ID NO:35.

[0121] In one embodiment, a therapeutic polypeptide comprises a protein that is deficient is a disease state, or a functional fragment thereof.

[0122] In one embodiment, the therapeutic polypeptide comprises a Ras / Rap1 specific endopeptidase (RRSP) from Vibrio vulnificus, e.g., as set forth in SEQ ID NO: 18. In some embodiments, the therapeutic polypeptide may be at least 80% identical to RRSP. In some embodiments, the therapeutic polypeptide may be at least 90% identical to RRSP. In some embodiments, the therapeutic polypeptide may be at least 95% identical to RRSP. In some embodiments, the therapeutic polypeptide may be at least 98% identical to RRSP. Such sequence variants may retain substantially the same activity as full-length RRSP.

[0123] In one embodiment, the cargo molecule comprises an N-terminal cysteine ​​residue for use in "click" chemistry bioconjugation.

[0124] In one embodiment, the cargo molecule comprises a nucleic acid molecule.

[0125] The percent sequence identity described herein can be calculated over the entire length of the alignment.

[0126] The amino acid sequences referred to herein may, in some embodiments, include sequence differences compared to a reference sequence (such as those listed in Table 1, below). They may be variants, mutations, insertions or deletions. In some applications, it may be important to ensure that the primary function of the protein is not substantially altered or abrogated, which can be easily tested, for example, using the assays described herein. The amino acid sequences described herein may include sequences with 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the reference sequence. The amino acid sequences may include conservative amino acid substitutions. Conservative amino acid substitutions known in the art are as follows, with the conservatively substituted candidate amino acid shown in parentheses: Ala (Gly, Ser); Arg (Gly, Gln); Asn (Gln; His); Asp (Glu); Cys (Ser); Gln (Asn, Lys); Glu (Asp); Gly (Ala, Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gln); Met (Leu, Ile); Phe (Met, Leu, Tyr); Ser (Thr; Gly); Thr (Ser; Val); Trp (Tyr); Tyr (Trp; Phe); Val (Ile; Leu). Some so-called "functional" variants, mutations, insertions, or deletions include sequences whose function is substantially the same as, for example, the function of the reference sequence from which they are derived. They can be easily tested using assays similar to those described herein.

[0127] Nucleic acids and vectors In one aspect, a nucleic acid is provided that encodes a recombinant polypeptide as defined herein. In one embodiment, the nucleic acid is DNA or RNA. The RNA can be mRNA.

[0128] Those skilled in the art will readily recognize that, for example, due to the degeneracy of the genetic code, there are many ways to code the recombinant polypeptides described herein, all of which are included in certain embodiments. Deletion, insertion, and substitution may also be possible, provided that protein function remains substantially intact. For example, nucleic acids may be included that have 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with wild-type or reference sequences. The nucleic acids may also be codon-optimized depending on the organism or expression system in which they are intended to be expressed.

[0129] In one aspect, there is provided a vector comprising a nucleic acid as defined herein.

[0130] In one embodiment, there is provided a host cell comprising a nucleic acid as defined herein or a vector as defined herein. The host cell may be transformed or transfected.

[0131] composition In one aspect, there is provided a composition comprising a recombinant polypeptide as defined herein, together with an acceptable excipient, diluent or carrier.

[0132] In one aspect, there is provided a pharmaceutical composition comprising a recombinant polypeptide as defined herein, together with a pharma- ceutically acceptable excipient, diluent or carrier.

[0133] Pharmaceutically acceptable carriers include solvents, diluents, liquid vehicles, dispersions, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders or lubricants. Carriers may be selected to extend the residence time of the sustained release appropriate for the selected route of administration. Exemplary carriers include sugars such as glucose and sucrose, starches such as corn starch and potato starch, fibers such as cellulose and its derivatives, sodium carboxymethylcellulose, ethylcellulose, acetylcellulose, powdered tragacanth, malt, gelatin, talc, cocoa butter, suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol, esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffers, non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, coating agents, sweeteners, flavors, fragrances, preservatives and antioxidants.

[0134] The composition may be administered to a subject by any acceptable route, for example, topically (powders, ointments or drops), orally, rectally, mucosally, sublingually, parenterally, intracisternally, intravaginally, intraperitoneally, bucally, ocularly or intranasally.

[0135] Liquid dosage forms for oral administration may include emulsions, microemulsions, solutions, suspensions, syrups and elixirs.Liquid dosage forms may contain inert diluents, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol fatty acid esters, and mixtures thereof.Besides inert diluents, oral compositions may further include adjuvants, such as wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, and aromatics.

[0136] Dosage forms for topical or transdermal administration of a pharmaceutical composition according to the invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active agent is mixed under sterile conditions with a pharma- ceutically acceptable carrier and any needed preservatives or buffers which may be required.

[0137] Injectable preparations, such as aqueous or oily sterile injectable suspensions, can be formulated using suitable dispersing or wetting agents and suspending agents according to known art. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution (USP) and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as solvents or suspension media. For this purpose, any non-irritating fixed oils can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectable solutions. Injectable preparations can be sterilized prior to the addition of spores, for example by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0138] It is often desirable to slow the absorption of drugs from subcutaneous or intramuscular injections. Delayed absorption of parenterally administered active agents can be accomplished by dissolving or suspending the agent in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0139] Compositions for rectal or vaginal administration are preferably suppositories, which may be prepared by mixing the active agent of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity to release the active agent.

[0140] Solid dosage forms for oral, mucosal, or sublingual administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one pharma- ceutically acceptable inert excipient or carrier, such as, for example, sodium citrate or dicalcium phosphate; fillers or extenders, such as, for example, starches, sucrose, glucose, mannitol, and silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; wetting agents, such as glycerol; disintegrating agents, such as, for example, agar-agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarders, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as, for example, kaolin and bentonite clay; and lubricants, such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.

[0141] Similar types of solid compositions can also be utilized as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose and high molecular weight polyethylene glycols. Tablets, capsules, pills, and granules solid dosage forms can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active agent can be mixed with at least one inert diluent, such as sucrose or starch. Such dosage forms can also contain, as is customary, additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms can also contain buffering agents. These dosage forms can also be of a composition that optionally contains opacifying agents and releases only the active agent, preferentially in a certain part of the intestinal tract, and optionally with a delayed release. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0142] The therapeutically effective amount can be determined individually or based on established requirements.The dosage for an individual subject is selected taking into consideration the subject to be treated.The dosage and administration can be adjusted to provide sufficient levels of active agent or to maintain desired effects.Factors that can be considered include the degree of pathology, past contact with infectious agents, and possible future contact; the age, weight, and sex of the subject; diet, time and frequency of administration, drug combinations, reaction sensitivity, and tolerance / response to therapy.Slow-release compositions can be administered less frequently than fast-acting compositions.

[0143] Methods and Uses In one aspect, there is provided a method of delivering a cargo molecule to a cell comprising contacting the cell with a recombinant polypeptide as defined herein.

[0144] In one aspect there is provided the use of a recombinant polypeptide as defined herein for use in delivering a cargo molecule to a cell.

[0145] In one aspect there is provided the use of a recombinant polypeptide as defined herein for the preparation of a medicament for delivering a cargo molecule to a cell.

[0146] In one aspect, there is provided a recombinant polypeptide as defined herein for use in delivering a cargo molecule to a cell.

[0147] In one aspect, there is provided a method of treating cancer in a subject comprising administering to the subject a recombinant polypeptide as defined herein.

[0148] In one aspect, there is provided a use of a recombinant polypeptide as defined herein for treating cancer in a subject.

[0149] In one aspect, there is provided a use of a recombinant polypeptide as defined herein for the preparation of a medicament for treating cancer in a subject.

[0150] In one aspect there is provided a recombinant polypeptide as defined herein for use in the treatment of cancer in a subject.

[0151] In one aspect, a method of alleviating an enzyme or protein deficiency in a cell is provided, comprising contacting the cell with a recombinant polypeptide described herein.

[0152] In one aspect, there is provided a use of a recombinant polypeptide described herein to alleviate an enzyme or protein deficiency in a cell.

[0153] In one aspect, there is provided a use of a recombinant polypeptide as described herein for the preparation of a medicament for ameliorating an enzyme or protein deficiency in a cell.

[0154] In one aspect, there is provided a recombinant polypeptide as described herein for use in alleviating an enzyme or protein deficiency in a cell.

[0155] As used herein, "alleviate" means that the cargo molecule corrects or at least partially improves a protein or enzyme deficiency, an aspect of the function of the defective protein or enzyme, or one or more of its downstream or secondary cellular effects or consequences.

[0156] In embodiments of the above methods and uses, the cargo molecule may be released.

[0157] Working Example Example 1 Introduction Engineered chimeric toxins have led to the emergence of novel therapeutics for challenging diseases such as cancer. Immunotoxins are a class of biologics consisting of bacterial toxins, e.g., diphtheria toxin (DT), that have been repurposed into targeted cancer therapy by both retargeting their receptor-binding domains (RBDs) to targeted cancer receptors and delivering enzymatic cargos that target intracellular cancer proteins. However, a global vaccination program against diphtheria has led to population-level immunity to DT, and DT-based therapeutics. To circumvent the problem of pre-existing neutralizing antibodies against DT, we investigated whether a distant homolog of DT, which shares only minor sequence identity, could retain the function of DT but evade DT-specific neutralizing antibodies. Here, we structurally and functionally characterized a putative gene sequence from Austwickia chelonae that is only 38% identical to DT. We named it chelonae toxin 1 (CT1). Its x-ray crystal structure was resolved to 2.50 Å, and its structure was found to be highly similar to DT. Using various biochemical assays, a domain-by-domain analysis was performed to explore the ability of DT-like proteins to function both as toxins and as novel platforms for therapeutic protein delivery. It was demonstrated that each domain of this novel protein can perform its respective function as a toxin, while the translocase of CT1 (CT1-T) can be engineered to target non-natural receptors and deliver non-natural cargos into cells. Importantly, CT1 is not recognized by pre-existing anti-DT antibodies found in human sera, and is unexpectedly superior to DT in delivering cargo into cells. Chelone toxins provide novel insights into toxin biology and represent an improved platform for therapeutic protein delivery.

[0158] material and method Crystallization of a DT-like protein from A. chelonae Due to the conservation of key residues identified as important for DT functionality, the closest DT-like protein from the species Austwickia chelonae (herein referred to as CT1 for "Chelone toxin 1") (SEQ ID NO: 2) was chosen as a candidate for an alternative immunotoxin scaffold (Figure 5).

[0159] SEQ ID NO:2 is derived from the combination of two ORFs (see GenBank accession numbers WP_143115263.1 and WP_040322835.1) representing two fragments of the toxin. When compared to the genome sequence of Austwickia chelonae (see GenBank accession number NZ_BAGZ01000024.1), a one base pair (bp) frameshift in the genome sequence appeared to have caused the separation of the full-length toxin into two ORFs. The reading frame was restored by deleting 1 bp (NZ_BAGZ01000024.1 C41398), resulting in the full-length toxin, subsequently referred to as "Chelone toxin 1 (CT1)" (SEQ ID NO:2). It is unclear whether the 1 bp insertion was a sequence error in Austwickia chelonae or reflected a genuine mutation. In any event, the 1 bp insertion was removed to produce the protein and translocation domain used in the experiments described herein.

[0160] To determine the structure of CT1, an E. coli codon-optimized gBlock gene fragment was ordered from Integrated DNA Technologies and cloned into the Champion™ pET SUMO E. coli Expression System by Gibson assembly.

[0161] A 50 mL seed of NiCo21(DE3) E. coli cells (New England Biolabs) was inoculated into 1 L of LB medium and induced with 0.1 mM IPTG for 18 hours at 18° C. Cells were centrifuged at 5000 rpm and resuspended in lysis buffer (1% protease inhibitor cocktail, 1 mg / mL lysozyme, 0.01% Pierce™ Universal Nuclease Inhibitor, 20 mM imidazole, 500 mM NaCI, 20 mM Tris-HCI pH 7.5). Cells were lysed by three passes through an Emulsiflex C3 (Avestin) at 15000 psi. Total cell lysate was centrifuged at 18000×g and the supernatant was filtered through a 0.45 μm filter and passed over a HisTrap FF crude column (Cytiva). The protein was eluted with 50-75 mM imidazole, buffer exchanged into 150 mM NaCI, 20 mM Tris-HCI pH 7.5, and incubated overnight at 4°C with SUMO protease to cleave the 6xHis-SUMO affinity tag. The protein was loaded onto a HisTrap FF crude column and the flow-through (protein) was collected and concentrated to 8 mg / mL by centrifugation.

[0162] Crystals were grown using the hanging drop vapor diffusion method. The conditions under which CT1 crystals were obtained contained 2 μL of mother liquor (0.2 M calcium chloride, 0.1 M Tris-HCI pH 8.5, 25% (w / v) PEG4000) and 1 μL of 8 mg / mL protein. Droplets were dehydrated with 130 μL of 2 M (NH4)2PO4 for 45 min and then frozen in liquid nitrogen. Data were collected at the 23-ID-D beamline at the Advanced Photon Source.

[0163] Initial phases were determined using a multicomponent search model with the individual DT domains (C domain residues 13-167, R domain residues 391-531, T domain residues 205-378) with promiscuous loops removed, using Phaser in the Phenix software package. The structure was refined using iterative cycles of phenix.refine and autobuild.

[0164] Protein synthesis assay Vero-nLucP cells (nanoluciferase reporter line of Vero cells) were plated at 5000 cells / well in 96-well white clear bottom plates (Corning). The next day, protein toxins were added and incubated for 24 hours, after which the luminescence signal of the cells was read on a SpectraMax M5e plate reader (Molecular Devices) using the NanoGlo® Luciferase Assay Kit (Promega). Data was normalized to untreated cells (100% nanoluciferase signal).

[0165] Liposomal dye release assay Unilamellar liposomes (DOPC, 0.8% DGS-NTA, Avanti Polar Lipids) were prepared as previously described. Briefly, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids) was combined with 0.8% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid) succinyl] (nickel salt) (DGS-NTA[Ni]) (Avanti Polar Lipids) and dried in a vacuum desiccator with N2 for 1 h. Lipids were resuspended in 20 mM Tris pH 8, 35 mM 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), 50 mM p-xylene-bis-pyridinium bromide (DPX) (Thermo Fischer), subjected to 10X freeze-thaw cycles in dry ice and a 42°C water bath, and extruded 10X using a 200 μm filter. Liposomes were then purified by gel filtration using a Hi Prep 16 / 60 Sephacryl S-300 HR column (GE Healthcare) and 150 mM NaCl, 20 mM Tris pH 8 buffer. Protein was added at a ratio of 1:10,000 with liposomes, for a final liposome concentration of approximately 400 μM in 150 mM citrate phosphate buffer at pH intervals of 0.5, spanning pH 4.0-7.5. Assays were performed in 96-well opaque plates (Corning) and fluorescence was monitored at 20 min intervals with readings taken every 30 s (excitation at 403 nm, emission at 510 nm). Data were normalized to % of total HPTS fluorescence by addition of 0.3% Triton X-100.

[0166] Serum antibody binding ELISA Nunc MaxiSorp™ plates (Thermo Fisher Scientific) were blocked with 1% BSA before immobilizing 2000ng of protein and subsequently incubating with various concentrations of human serum (frozen pooled human serum, Cedarlane) for 1 hour. Wells were washed with PBST (0.01% tween) and then incubated with anti-human IgG antibody conjugated to HRP (Abcam, ab102420) and HRP was developed using TMB reagent (Thermo Fisher Scientific). Absorbance was read at 630nm and protein wells were corrected against control wells (no protein, + human serum).

[0167] Serum toxicity assay Protein toxins were incubated with either human serum (frozen pooled human serum, Cedarlane) or mouse serum (frozen sterile mouse serum, Cedarlane) in a 1:1 ratio for 30 min at room temperature. Samples were added to Vero-nLucP cells plated the day before at 5000 cells / well in 96-well white clear bottom plates (Corning). Cells were incubated for 24 h before cells were lysed and luminescence signal was assessed. Values ​​were normalized to cells treated with serum only, which represents 100% nanoluciferase signal and 100% protein synthesis.

[0168] result Structural characterization of a DT-like protein from A. chelonae With the aim of finding an alternative DT-like immunotoxin platform that is less susceptible to existing anti-DT antibodies but is functionally active, we selected the evolutionarily closest DT-like protein outside the genus Corynebacterium. Based on the sequence, the DT-like protein from A. chelonae (CT1) is 38% identical to DT. The catalytically active residue (E148) as well as residues important for substrate (NAD) binding and coordination (H21, Y54, Y65) as well as the furin recognition site are all conserved, and the disulfide bond formed between C186 and C201 (in DT) is also present in the CT1 sequence (Table 1). Furthermore, key histidine and charged residues involved in the pH-dependent unfolding and pore formation of the translocation domain were found to be conserved in the predicted translocation domain of CT1-derived sequences (Table 1). To test whether the putative domain of CT1 is functional, the protein was cloned, expressed, purified, and structurally and functionally tested (Fig. 1B).

[0169] The hanging drop vapor diffusion method was used to obtain the x-ray crystal structure of CT1. The protein was successfully crystallized and diffracted to 2.50 Å. It was not possible to use the full-length DT structure (pdb 1MDT) as a search model for molecular replacement. However, a partial search model with 1MDT was used to resolve the structure (Figure 1C). CT1 has an RMSD of 2.4 Å relative to DT and retains the same three-domain Y-shaped structure as DT.

[0170] The C domain of CT1 is functional and has the same intracellular targets as DT CT1-C (catalytic domain of CT1), DT C The structural alignment to the DT catalytic domain is C It shows good structural conservation among key residues required for functionality. To test whether CT1-C is functional, DT C A chimera was generated in which CT1-C was replaced with CT1-C (CT1-C-DT T -DT R(called DPH4 gene knockout (DPH4 - / - The chimeric proteins were tested using HEK293T cells with and without CT1-C-DT. These cells are completely resistant to DT because they are defective in the diphthamide synthesis pathway and produce eukaryotic elongation factor 2 (eEF-2) without diphthamide modification (Figures 2A and 2B). Wild-type HEK293T cells were resistant to both wild-type DT and CT1-C-DT. T -DT R In contrast to diphthamide knockout cells, CT1 was sensitive to both DT and CT2, indicating that the catalytic domain of CT1 has the same intracellular target as DT.

[0171] The C domain of CT1 is efficiently released A key part of the intoxication mechanism of DT is the release of the C domain from the rest of the molecule upon entry into the cytosol. The furin protease recognition site is conserved in CT1 (RAKR in CT1). To confirm that the furin site is recognized and the C domain is released from the rest of the molecule, DT and CT1 were incubated with mammalian cell lysates overnight at 37°C. Both DT and CT1 were cleaved between the C and T domains (Figure 2C). DTC-CT1-F-DTT-DTR was as toxic as DT when tested on Vero cells (Figure 3A), suggesting that it is efficiently cleaved by furin.

[0172] CT1 contains a functional translocase Endosome acidification induces DT T This process results in the refolding and insertion of the C domain into the endosomal membrane and subsequent translocation of the C domain to the cytosol. TThe pore-forming potential of CT1-T appears to be initiated and driven by nine charged residues in the pore, six of which are conserved in CT1-T. To test whether CT1-T forms a DT-like pore, the isolated T domain was purified and its ability to release dye from liposomes was tested in vitro (data not shown). CT1-T showed a pH-dependent increase in dye release, with the onset of dye release (interpreted as pore formation) at pH 5.5 (DT T (Similar to ).

[0173] We further investigated whether the translocase delivers cargo into cells. To this end, DT T To generate chimeric DT, R ) and its effect on protein synthesis was measured. CT1-T inhibited the DT C The DT was effectively delivered into the cells (Figure 3B). C Exchange of CT1-T for a non-native cargo (Ras / Rap1-specific protease; RRSP) targeting the Ras oncoprotein was shown to effectively deliver this structurally distinct cargo (as shown by cell viability and Western blots assessing intracellular Ras levels in CFPAC-1 cells; Figure 3C and Figure 3D). Collectively, these experiments demonstrated that CT1-T is a functional translocase capable of delivering a variety of cargoes to the cytosolic compartment of cells.

[0174] The translocation domain of CT1 can accommodate diverse receptor-binding domains CT1-T has been demonstrated to be able to tolerate engineering at the N-terminus (DT C and RRSP), we next assessed whether CT1-T could tolerate such manipulations on the C-terminus. C -CT1-T-DT R and RRSP-CT1-T-DT R The receptor binding domain (DT RWe generated chimeras in which CT1-T was replaced with a Her3 (human epithelial receptor 3)-targeted affibody (ZHer3:08699, hereafter referred to as ZHer3) and tested the construct on HPAF II cells (Figures 3E and 3F). Not only could CT1-T be retargeted to Her3-expressing cells, but surprisingly, the CT1-T-containing constructs also demonstrated a DT1-dependent effect on cargo delivery. T It was found that the 10-fold increase in the β-amino acid concentration appeared to be more efficient than the 10-fold increase in the β-amino acid concentration.

[0175] CT1 is not recognized or neutralized by human sera To quantify the levels of pre-existing anti-DT or anti-CT1 antibodies in human serum, an ELISA assay was used in which either DT or CT1 was immobilized on a high-binding plate and incubated with different amounts of pooled human serum, after which the level of antibody binding was determined using an anti-IgG antibody conjugated to HRP (Figure 4A). DT showed a dose-dependent increase in signal in the presence of human serum, whereas CT1 did not, indicating that CT1 is not recognized by antibodies in human serum.

[0176] To confirm that DT is neutralized by antibodies in human serum, DT was incubated with pooled human serum for 30 minutes before adding it to Vero cells. A 6 log shift in DT toxicity was observed, indicating neutralization by human serum (Figure 4B). In contrast, CT1 was not neutralized by human serum, indicating that CT1 is not neutralized by anti-DT antibodies. This gives CT1-based delivery vectors an advantage over DT-based vectors by avoiding the problem of existing neutralizing anti-DT antibodies.

[0177] In addition to the translocation domain of CT1 (SEQ ID NO:3), it has been established that the translocation domains of SEQ ID NOs:12 and 14 are also functional.

[0178] Based on the results obtained, the related polypeptide sequences set forth in SEQ ID NOs: 4-11, 13, 15-17 and 36-48 (and related sequences) from other bacterial strains and species described herein are also expected to be functional translocation domains that are active within the polypeptide constructs described herein. Figure 9 presents a phylogenetic tree showing the relationships between translocase domains.

[0179] Example 2 To evaluate the function of the novel translocases on DT, each translocase sequence "T" was cloned between the intracellular RAS cleavage enzyme RRSP (Ras / Rap1 specific peptidase, SEQ ID NO: 18; i.e. cargo) and a dual receptor binding domain known as ZHer3-A20 (consisting of an affibody against Her3, SEQ ID NO: 19, and a peptide against avβ6 integrin (A20), SEQ ID NO: 20, known as A20FMDV2), resulting in the construct RRSP-T-ZHer3-A20 (where "T" denotes translocase). A range of protein concentrations of each identified construct was incubated with human pancreatic adenocarcinoma (HPAF-II) cells for 72 hours (Figure 6A) and / or epithelial-like cells (H358 cells) for 72 hours (Figure 6B). Because RRSP induces apoptosis after delivery into cells, translocation was quantified by measuring the ability of each construct to kill human pancreatic adenocarcinoma cells after 72 hours of incubation.

[0180] Figures 6A and 6B show the results of functional characterization of the translocases. The functionality of each translocase was quantified using the RRSP-T-ZHer3-A20 template. The concentration of a given construct that results in a decrease in cell viability with 50% of maximum toxicity (EC50) is represented in a bar graph. Lower values ​​represent more efficient translocases. The translocase from S. pinicola was not expressed and therefore could not be evaluated. The translocases from S. piniterrae and L. tulafanense were determined to be non-functional in this assay since no toxicity was observed up to the maximum dose tested (100 nM). The remaining five translocases were functional in this assay. The translocases of the proteins from A. chelonae (SEQ ID NO: 2) and A. chelonae LK16-18 (SEQ ID NO: 17) were the most active on the cells and were superior to the translocase of DT. The observed activity levels indicate that RRSP was efficiently released.

[0181] Translocase domains from Streptomyces albireticuli and Seinonella peptonophila have also been shown to be functional (see Sugiman-Marangos et al. 2022, incorporated by reference in its entirety).

[0182] Diphtheria toxoid vaccines are part of a global vaccination program that serves to protect against diphtheria disease. Anti-DT antibodies in human serum prevent the action of DT by binding to it and neutralizing its function. Unfortunately, these same antibodies also bind to and neutralize DT-based therapeutics. ELISA was performed to assess the extent to which anti-DT antibodies in human serum recognize full-length toxins DT and CT1 (SEQ ID NO: 2), as well as translocases DT-T and CT1-T (SEQ ID NO: 3). As shown in the ELISA data in Figure 7A, antibodies in human serum recognize DT but show no binding to CT1. Similarly, high titers are observed against the translocase derived from DT, but not against CT1 (Figure 7B).

[0183] Figure 7A and Figure 7B together show that pre-existing anti-DT antibodies in human serum do not bind or neutralize CT1-based immunotoxins. Each protein was immobilized on a plate and incubated with different amounts of pediatric human serum samples, and then the level of antibody binding was determined using an anti-IgG antibody conjugated to HRP. Both DT and DT-T showed a dose-dependent signal increase with human serum, but CT1 and CT1-T did not. This indicates that full-length CT1, and indeed its translocase, do not contain epitopes recognized by antibodies in human serum.

[0184] The DT- and CT-based immunotoxins from A. chelonae LK16-18 were then incubated with human serum to assess the extent to which pre-existing anti-DT antibodies in human serum neutralize DT-based therapeutics, as well as corresponding therapeutics based on the novel transition domain. The C and T domains of the respective toxins were recombinantly attached to ZHer3-A20 (DT 1~389 -ZHer3-A20 or CT2 1~391 As shown in Figure 8, the expression levels of the DT-ZHer3-A20 and DT-ZHer3-A20 mutants were significantly increased in the absence of human serum (PBS), DT, and DT.1~389 -ZHer-3-A20, and CT2 1~391 Both -ZHer3-A20 and -ZHer3-A20 were toxic to human cells. In the presence of human serum (human), DT and DT 1~389 For -ZHer3-A20, a rightward shift in the toxicity curve was observed, indicating that human serum inhibited / neutralized their function. 1~391 -ZHer3-A20 had no effect on the toxicity curve, indicating that human serum did not recognize or inhibit / neutralize the CT-based therapeutic.

[0185] Figure 8 shows that anti-DT antibodies do not neutralize CT-based immunotoxins. Immunotoxins were incubated with either PBS control or human serum for 30 minutes before being added to cells. At 72 hours, cell viability was assessed and no significant difference was observed between DT and human serum incubation. 1~389 -ZHer3-A20 showed approximately 3-log reduction in toxicity and CT2 1~391 -ZHer3-A20 was found not to shift.

[0186] Example 3 Example of annotated construct sequence RRSP-T (C. diphtheriae)-ZHer3-A20 (SEQ ID NO: 22)

[0187] [ka]

[0188] In the above Residues 1-510 = RRSP Residues 511-724 = T domain from C. diphtheriae Residues 725-734 = G4S2 linker Residues 735-792 = ZHer3:08699 affibody Residues 793-802 = G4S2 linker Residues 803-822 = A20FMDV2 peptide Residues 823-840 = thrombin cleavage site and strep tag II

[0189] RRSP-T (A. chelonae)-ZHer3-A20 (SEQ ID NO: 23)

[0190] [ka]

[0191] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-711 = T domain from A. chelonae Residues 712-723 = linker sequence from C. diphtheriae Residues 724-733 = G4S2 linker Residues 734-791 = ZHer3:08699 affibody Residues 792-801 = G4S2 linker Residues 802-821 = A20FMDV2 peptide Residues 822-839 = thrombin cleavage site and strep tag II

[0192] RRSP-T (A. chelonae LK16-18)-ZHer3-A20 (SEQ ID NO: 24)

[0193] [ka]

[0194] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-712 = T domain from A. chelonae LK16-18 Residues 713-724 = linker sequence from C. diphtheriae Residues 725-734 = G4S2 linker Residues 735-792 = ZHer3:08699 affibody Residues 793-802 = G4S2 linker Residues 803-822 = A20FMDV2 peptide Residues 823-840 = thrombin cleavage site and strep tag II

[0195] RRSP-T (A.TVS96-490-7B)-ZHer3-A20 (SEQ ID NO: 25)

[0196] [ka]

[0197] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-711 = A. T domain from TVS96-490-7B Residues 712-723 = linker sequence from C. diphtheriae Residues 724-733 = G4S2 linker Residues 734-791 = ZHer3:08699 affibody Residues 792-801 = G4S2 linker Residues 802-821 = A20FMDV2 peptide Residues 822-839 = thrombin cleavage site and strep tag II

[0198] RRSP-T (S. klenkii-ZHer3-A20 (SEQ ID NO: 26)

[0199] [ka]

[0200] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-700 = T domain from S. klenkii Residues 701-712 = linker sequence from C. diphtheriae Residues 713-722 = G4S2 linker Residues 723-780 = ZHer3:08699 affibody Residues 781-790 = G4S2 linker Residues 791-810 = A20 FMDV2 peptide Residues 811-828 = thrombin cleavage site and strep tag II

[0201] RRSP-T (S. species TLI053)-ZHer3-A20 (SEQ ID NO: 27)

[0202] [ka]

[0203] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-701 = T domain from S. species TLI053 Residues 702-713 = linker sequence from C. diphtheriae Residues 714-723 = G4S2 linker Residues 724-781 = ZHer3:08699 affibody Residues 782-791 = G4S2 linker Residues 792-811 = A20FMDV2 peptide Residues 812-829 = thrombin cleavage site and strep tag II

[0204] RRSP-T (L. tulufanense)-ZHer3-A20 (SEQ ID NO: 28)

[0205] [ka]

[0206] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-714 = T domain from L. tulufanense Residues 715-726 = linker sequence from C. diphtheriae Residues 727-736 = G4S2 linker Residues 737-794 = ZHer3:08699 affibody Residues 795-804 = G4S2 linker Residues 805-824 = A20FMDV2 peptide Residues 825-842 = thrombin cleavage site and strep tag II

[0207] RRSP-T (S. piniterrae)-ZHer3-A20 (SEQ ID NO: 29)

[0208] [ka]

[0209] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-695 = T domain from S. piniterrae Residues 696-707 = linker sequence from C. diphtheriae Residues 708-717 = G4S2 linker Residues 718-775 = ZHer3:08699 affibody Residues 776-786 = G4S2 linker Residues 786-805 = A20FMDV2 peptide Residues 806-823 = thrombin cleavage site and strep tag II

[0210] RRSP-T (S. pinicola)-ZHer3-A20 (SEQ ID NO: 30)

[0211] [ka]

[0212] In the above Residues 1-510 = RRSP Residues 511-520 = G4S2 linker Residues 521-536 = C. diphtheriae sequence containing the furin protease recognition site Residues 537-712 = T domain from S. pinicola Residues 713-724 = linker sequence from C. diphtheriae Residues 725-734 = G4S2 linker Residues 735-792 = ZHer3:08699 affibody Residues 793-802 = G4S2 linker Residues 803-822 = A20FMDV2 peptide Residues 823-840 = thrombin cleavage site and strep tag II

[0213] DT-Her3-A20 (SEQ ID NO: 31)

[0214] [ka]

[0215] In the above Residues 1-389 = sequence from C. diphtheriae Residues 390-399 = G4S2 linker Residues 400-457 = Her3:08699 affibody Residues 458-467 = G4S2 linker Residues 468-487 = A20FMDV2 peptide Residues 487-505 = thrombin cleavage site and strep tag II

[0216] CT2-ZHer3-A20 (SEQ ID NO: 32)

[0217] [ka]

[0218] In the above Residues 1-391 = sequence from A. chelonae LK16-18 Residues 392-401 = G4S2 linker Residues 402-459 = Her3:08699 affibody Residues 460-469 = G4S2 linker Residues 470-489 = A20FMDV2 peptide Residues 491-507 = thrombin cleavage site and strep tag II

[0219]

Table 1-1

[0220]

Table 1-2

[0221]

Table 1-3

[0222]

Table 1-4

[0223]

Table 1-5

[0224]

Table 1-6

[0225]

Table 1-7

[0226]

Table 1-8

[0227]

Table 1-9

[0228]

Table 1-10

[0229] [Table 1-11]

[0230] References

[0231] [Table 2]

[0232] In the above description, for purposes of explanation, numerous details are set forth to provide a thorough understanding of the embodiments. However, it will be apparent to those skilled in the art that such specific details are not necessary. In other instances, well-known electrical structures and circuits are shown in block diagram form in order to avoid obscuring the understanding. For example, specific details are not provided regarding whether the embodiments described herein are implemented as software routines, hardware circuits, firmware, or a combination thereof.

[0233] The above embodiments are intended to be merely examples. Changes, modifications, and variations may be achieved by those skilled in the art to the specific embodiments. The claims should not be limited to the specific embodiments described herein, but should be interpreted in a manner consistent with the specification as a whole.

Claims

1. General formula (I): A-B-C(i) (In the formula, A is a cargo molecule, B is the following: a) i. Austwickia chelonae protein of SEQ ID NO: 2; ii. Streptosporangium nondiastaticum protein with GenBank accession number PSJ28985.1; iii. Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1; iv. Streptomyces sp. SLBN-118 protein with GenBank accession number WP_160159328.1; v. Streptomyces sp. AA8 protein with GenBank accession number WP_168096531.1; vi. Streptomyces roseoverticillatus protein with GenBank accession number WP_078659863.1; vii. Streptomyces piniterrae protein with GenBank accession number JZ58907.1; viii. Streptomyces MBT76 protein, GenBank accession number WP_079110321.1; ix. Streptomyces klenkii protein with GenBank accession number WP_120757473.1; x. Streptomyces albireticuli protein with GenBank accession number WP_095582082.1; xi. Streptacidiphilus pinicola protein with GenBank accession number WP_133259917.1; xii. Seinonella peptonophila protein with GenBank accession number WP_073156187.1; xiii. Longimycelium tulufanense protein with GenBank accession number WP_189053160.1; xiv. Austwickia sp. TVS96-490-7B protein with GenBank accession number WP_219106995.1; xv. Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1; xvi. Klebsiella aerogenes protein with GenBank accession number EIZ2913133.1; xvii. Streptomyces sp. MUM178J protein with GenBank accession number MCH0551590.1; xviii. Crossiella cryophila protein with GenBank accession number MBB4677777.1; xix. Allokutzneria sp. NRRL B-24872 protein with GenBank accession number WP_143261759.1; xx. Allokutzneria albata protein with GenBank accession number WP_156051914.1; xxi. Streptomyces sp. AV19 protein with GenBank accession number WP_199893204.1; xxii. Streptomyces sp. NRBC_110611 protein, GenBank accession number WP_147264604.1; xxiii. Streptomyces syringium protein with GenBank accession number WP_209513619.1; xxiv. Pseudonocardiaceae bacterium YIM PH21723 protein with GenBank accession number RJQ69589.1; xxv. Actinokineospora bangkokensis protein with GenBank accession number WP_143218892.1; xxvi. Streptomyces eurocidicus protein with GenBank accession number MBF6055834.1; xxvii. The Streptomyces pathocidini protein of GenBank accession number WP_169790908.1, or xxviii. Streptomyces caatingaensis protein with GenBank accession number WP_157868472.1 the transition domain of origin, or b) a transition domain that is at least 80% identical to the transition domain defined in a) above. a transition polypeptide comprising C is a targeting moiety Recombinant polypeptide of.

2. General formula (I): A-B-C(i) (In the formula, A is a cargo molecule, B is the following: a) i. Austwickia chelonae protein of SEQ ID NO: 2; ii. Streptosporangium nondiastaticum protein with GenBank accession number PSJ28985.1; iii. Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1; iv. Streptomyces sp. SLBN-118 protein with GenBank accession number WP_160159328.1; v. Streptomyces sp. AA8 protein with GenBank accession number WP_168096531.1; vi. Streptomyces roseoverticillatus protein with GenBank accession number WP_078659863.1; vii. Streptomyces piniterrae protein with GenBank accession number JZ58907.1; viii. Streptomyces MBT76 protein, GenBank accession number WP_079110321.1; ix. Streptomyces klenkii protein with GenBank accession number WP_120757473.1; x. Streptacidiphilus pinicola protein with GenBank accession number WP_133259917.1; xi. Longimycelium tulufanense protein with GenBank accession number WP_189053160.1; xii. Austwickia sp. TVS96-490-7B protein, GenBank accession number WP_219106995.1; or xiii. Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1 Transition domain of origin; b) a transition domain that is at least 80% identical to the transition domain defined in a) above; c) a translocation domain comprising the amino acid sequence of any one of SEQ ID NOs: 36-48; or d) a transition domain that is at least 80% identical to the transition domain defined in c) above. a transition polypeptide comprising C is a targeting moiety Recombinant polypeptide of.

3. General formula (I): A-B-C(i) (In the formula, A is a cargo molecule, B is the following: a) i. Austwickia chelonae protein of SEQ ID NO: 2; ii. Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1; iii. Streptomyces klenkii protein with GenBank accession number WP_120757473.1; iv. Austwickia sp. TVS96-490-7B protein with GenBank accession number WP_219106995.1, or v. Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1 the transition domain of origin; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above. a transition polypeptide comprising C is a targeting moiety Recombinant polypeptide of.

4. General formula (I): A-B-C(i) (In the formula, A is a cargo molecule, B is the following: a) i. Austwickia chelonae protein of SEQ ID NO: 2; ii. Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1 the transition domain of origin; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above. a transition polypeptide comprising C is a targeting moiety Recombinant polypeptide of.

5. In a), The translocation domain from the Austwickia chelonae protein has the amino acid sequence of SEQ ID NO:

3. The translocation domain from the Streptosporangium nondiastaticum protein of GenBank accession number PSJ28985.1 has the amino acid sequence of SEQ ID NO:

4. The translocation domain from Streptomyces sp. TLI053 protein, GenBank accession number SDT83331.1, has the amino acid sequence of SEQ ID NO:

5. The translocation domain from Streptomyces sp. SLBN-118 protein, GenBank accession number WP_160159328.1, has the amino acid sequence of SEQ ID NO:

6. The translocation domain from Streptomyces sp. AA8 protein, GenBank accession number WP_168096531.1, has the amino acid sequence of SEQ ID NO:

7. The translocation domain from Streptomyces roseoverticillatus protein of GenBank accession number WP_078659863.1 has the amino acid sequence of SEQ ID NO:

8. The translocation domain from the Streptomyces piniterrae protein of GenBank accession number JZ58907.1 has the amino acid sequence of SEQ ID NO:

9. The translocation domain from Streptomyces MBT76 protein, GenBank accession number WP_079110321.1, has the amino acid sequence of SEQ ID NO:

10. The translocation domain from Streptomyces klenkii protein with GenBank accession number WP_120757473.1 has the amino acid sequence of SEQ ID NO:

11. The translocation domain from Streptomyces albireticuli protein of GenBank accession number WP_095582082.1 has the amino acid sequence of SEQ ID NO:

12. The translocation domain from the Streptacidiphilus pinicola protein of GenBank accession number WP_133259917.1 has the amino acid sequence of SEQ ID NO:

13. The translocation domain from the Seinonella peptonophila protein of GenBank accession number WP_073156187.1 has the amino acid sequence of SEQ ID NO:

14. The translocation domain from the Longimycelium tulufanense protein having GenBank accession number WP_189053160.1 has the amino acid sequence of SEQ ID NO:

15. The translocation domain from Austwickia sp. TVS96-490-7B protein, GenBank accession number WP_219106995.1, has the amino acid sequence of SEQ ID NO: 16; and The translocation domain from Austwickia chelonae LK16-18 protein, GenBank accession number WP_162873017.1, has the amino acid sequence of SEQ ID NO:

17. A recombinant polypeptide according to any one of claims 1 to 4.

6. the transit polypeptide a) a translocation domain from the Austwickia chelonae protein having the amino acid sequence of SEQ ID NO: 3, or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

5. The recombinant polypeptide of claim 1, comprising:

7. the transit polypeptide a) a translocation domain from the Streptosporangium nondiastaticum protein having GenBank accession number PSJ28985.1 and having the amino acid sequence of SEQ ID NO: 4; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

8. the transit polypeptide a) a translocation domain from the Streptomyces sp. TLI053 protein with GenBank accession number SDT83331.1, having the amino acid sequence of SEQ ID NO: 5; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

4. The recombinant polypeptide of claim 1, comprising:

9. the transit polypeptide a) a translocation domain from the Streptomyces sp. SLBN-118 protein having GenBank accession number WP_160159328.1 and having the amino acid sequence of SEQ ID NO: 6; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

10. the transit polypeptide a) a translocation domain from the Streptomyces sp. AA8 protein having GenBank accession number WP_168096531.1 and having the amino acid sequence of SEQ ID NO: 7, or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

11. the transit polypeptide a) a translocation domain from the Streptomyces roseoverticillatus protein with GenBank accession number WP_078659863.1, having the amino acid sequence of SEQ ID NO: 8; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

12. the transit polypeptide a) a translocation domain from the Streptomyces piniterrae protein with GenBank accession number JZ58907.1, having the amino acid sequence of SEQ ID NO: 9; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

13. the transit polypeptide a) a translocation domain from the Streptomyces MBT76 protein of GenBank accession number WP_079110321.1, having the amino acid sequence of SEQ ID NO: 10; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

14. the transit polypeptide a) a translocation domain from the Streptomyces klenkii protein with GenBank accession number WP_120757473.1, having the amino acid sequence of SEQ ID NO: 11; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

4. The recombinant polypeptide of claim 1, comprising:

15. the transit polypeptide a) a translocation domain from the Streptomyces albireticuli protein with GenBank accession number WP_095582082.1, having the amino acid sequence of SEQ ID NO: 12; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

2. The recombinant polypeptide of claim 1, comprising:

16. the transit polypeptide a) a translocation domain from the Streptacidiphilus pinicola protein with GenBank accession number WP_133259917.1, having the amino acid sequence of SEQ ID NO: 13; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

17. the transit polypeptide a) a translocation domain from the Seinonella peptonophila protein with GenBank accession number WP_073156187.1, having the amino acid sequence of SEQ ID NO: 14; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

2. The recombinant polypeptide of claim 1, comprising:

18. the transit polypeptide a) a translocation domain from the Longimycelium tulufanense protein having GenBank accession number WP_189053160.1 and having the amino acid sequence of SEQ ID NO: 15; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

3. The recombinant polypeptide of claim 1 or 2, comprising:

19. the transit polypeptide a) a translocation domain having the amino acid sequence of SEQ ID NO: 16 from the Austwickia sp. TVS96-490-7B protein with GenBank accession number WP_219106995.1, or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

4. The recombinant polypeptide of claim 1, comprising:

20. the transit polypeptide a) a translocation domain from the Austwickia chelonae LK16-18 protein with GenBank accession number WP_162873017.1, having the amino acid sequence of SEQ ID NO: 17; or b) a transition domain that is at least 80% identical to the transition domain defined in a) above.

5. The recombinant polypeptide of claim 1, comprising:

21. 5. A recombinant polypeptide according to any one of claims 1 to 4, wherein the translocation domain is as defined in b) and is at least 85% identical over its entire length to the translocation domain defined in a).

22. The recombinant polypeptide of claim 6, wherein the transition domain is as defined in b) and is at least 90% identical over its entire length to the transition domain defined in a).

23. 5. A recombinant polypeptide according to any one of claims 1 to 4, wherein the translocation domain is as defined in b) and is at least 90% identical over its entire length to the translocation domain defined in a).

24. 5. A recombinant polypeptide according to any one of claims 1 to 4, wherein the translocation domain is as defined in b) and is at least 95% identical over its entire length to the translocation domain defined in a).

25. 5. A recombinant polypeptide according to any one of claims 1 to 4, wherein the translocation domain is as defined in b) and is at least 98% identical over its entire length to the translocation domain defined in a).

26. 5. A recombinant polypeptide according to any one of claims 1 to 4, wherein the translocation domain is as defined in b) and is at least 99% identical over its entire length to the translocation domain defined in a).

27. 5. The recombinant polypeptide of any one of claims 1 to 4, wherein the transit polypeptide is as defined in a).

28. 5. The recombinant polypeptide of claim 1, wherein A and B are separated by an amino acid linker.

29. The amino acid linker is ４ S) ２ 29. The recombinant polypeptide of claim 28, comprising:

30. 29. The recombinant polypeptide of claim 28, wherein the amino acid linker is cleavable, preferably the amino acid linker comprises a protease recognition site, preferably the protease recognition site is flanked by cysteine residues, more preferably the protease recognition site is a furin protease recognition site.

31. 31. The recombinant polypeptide of claim 30, wherein the amino acid linker comprises SEQ ID NO: 32, which contains a furin protease recognition site.

32. The above (G ４ S) ２ is located N-terminally with respect to said SEQ ID NO:

32.

33. 5. The recombinant polypeptide of claim 1, wherein B and C are separated by an amino acid linker.

34. The amino acid linker is ４ S) ２ 29. The recombinant polypeptide of claim 28, comprising:

35. 34. The recombinant polypeptide of claim 33, wherein the amino acid linker comprises SEQ ID NO:

33.

36. A recombinant polypeptide described in claim 34, wherein the amino acid linker comprises sequence number 33.

37. The above (G ４ S) ２ is located N-terminally with respect to said SEQ ID NO:

33.

38. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO:23, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO:23, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO:23, even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO:

23.

39. 5. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

23.

40. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO:24, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO:24, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO:24, even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO:

24.

41. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

24.

42. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 821 of SEQ ID NO:25, preferably 90% identical to amino acids 1 to 821 of SEQ ID NO:25, more preferably 95% identical to amino acids 1 to 821 of SEQ ID NO:25, and even more preferably 100% identical to amino acids 1 to 821 of SEQ ID NO:

25.

43. 5. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

25.

44. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 806 of SEQ ID NO:26, preferably 90% identical to amino acids 1 to 806 of SEQ ID NO:26, more preferably 95% identical to amino acids 1 to 806 of SEQ ID NO:26, even more preferably 100% identical to amino acids 1 to 806 of SEQ ID NO:

26.

45. 5. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

26.

46. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 811 of SEQ ID NO:27, preferably 90% identical to amino acids 1 to 811 of SEQ ID NO:27, more preferably 95% identical to amino acids 1 to 811 of SEQ ID NO:27, and even more preferably 100% identical to amino acids 1 to 811 of SEQ ID NO:

27.

47. 5. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

27.

48. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO:28, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO:28, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO:28, even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO:

28.

49. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

28.

50. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 796 of SEQ ID NO:29, preferably 90% identical to amino acids 1 to 796 of SEQ ID NO:29, more preferably 95% identical to amino acids 1 to 796 of SEQ ID NO:29, even more preferably 100% identical to amino acids 1 to 796 of SEQ ID NO:

29.

51. 5. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

29.

52. 5. The recombinant polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 80% identical to amino acids 1 to 822 of SEQ ID NO: 30, preferably 90% identical to amino acids 1 to 822 of SEQ ID NO: 30, more preferably 95% identical to amino acids 1 to 822 of SEQ ID NO: 30, even more preferably 100% identical to amino acids 1 to 822 of SEQ ID NO:

30.

53. 5. A recombinant polypeptide according to any one of claims 1 to 4, comprising the sequence of SEQ ID NO:

30.

54. 5. The recombinant polypeptide of claim 1, wherein the targeting moiety comprises a targeting polypeptide or an aptamer.

55. 55. The recombinant polypeptide of claim 54, wherein the targeting polypeptide comprises an antibody, a binding fragment of an antibody, an affibody, a peptide, an affitin, a DARPin, or a receptor ligand.

56. 56. The recombinant polypeptide of claim 55, wherein the targeting polypeptide comprises an affibody against Her3.

57. 57. The recombinant polypeptide of claim 56, wherein the affibody against Her3 comprises the amino acid sequence of SEQ ID NO:

19.

58. 54. The recombinant polypeptide of claim 53, wherein the targeting polypeptide comprises a receptor ligand for avβ6 integrin.

59. 59. The recombinant polypeptide of claim 58, wherein the receptor ligand for the avβ6 integrin comprises the amino acid sequence of SEQ ID NO:

20.

60. 55. The recombinant polypeptide of claim 54, wherein the targeting moiety comprises at least two targeting polypeptides, at least two aptamers, or a combination of targeting polypeptides and aptamers.

61. 61. The recombinant polypeptide of claim 60, wherein the at least two targeting polypeptides are selected from the group of antibodies, binding fragments of antibodies, affibodies, peptides, affitins, DARPins, receptor ligands, and combinations thereof.

62. 61. The recombinant polypeptide of claim 60, wherein the at least two targeting polypeptides comprise an affibody against Her3.

63. 63. The recombinant polypeptide of claim 62, wherein the affibody against Her3 comprises the amino acid sequence of SEQ ID NO:

19.

64. 61. The recombinant polypeptide of claim 60, wherein the at least two targeting polypeptides comprise a receptor ligand for avβ6 integrin.

65. 65. The recombinant polypeptide of claim 64, wherein the receptor ligand for the avβ6 integrin comprises the amino acid sequence of SEQ ID NO:

20.

66. 61. The recombinant polypeptide of claim 60, wherein the at least two targeting polypeptides, the at least two aptamers, or the combination are separated by an amino acid linker.

67. The amino acid linker is ４ S) ２ 67. The recombinant polypeptide of claim 66, comprising:

68. 5. The recombinant polypeptide of claim 1, wherein the targeting moiety binds to a cell surface protein.

69. 69. The recombinant polypeptide of claim 68, wherein the cell surface protein is lineage- or tissue-specific.

70. 69. The recombinant polypeptide of claim 68, wherein the cell surface protein is ubiquitously expressed.

71. 69. The recombinant polypeptide of claim 68, wherein the cell surface protein is expressed in a diseased cell.

72. 69. The recombinant polypeptide of claim 68, wherein the cell surface protein is specific to diseased cells and is not expressed in corresponding healthy cells.

73. 69. The recombinant polypeptide of claim 68, wherein the cell surface protein has elevated expression in diseased cells compared to corresponding healthy cells.

74. 72. The recombinant polypeptide of claim 71, wherein the diseased cells are cancer cells.

75. The recombinant polypeptide described in claim 72, wherein the diseased cells are cancer cells.

76. The recombinant polypeptide described in claim 73, wherein the diseased cells are cancer cells.

77. The recombinant polypeptide of claim 1 , wherein the cargo molecule comprises a therapeutic polypeptide.

78. 78. The recombinant polypeptide of claim 77, wherein the therapeutic polypeptide comprises a cytotoxic polypeptide, preferably a polypeptide toxin or a functional fragment thereof.

79. 79. The recombinant polypeptide of claim 78, wherein the cytotoxic polypeptide comprises a catalytic domain derived from diphtheria toxin.

80. 79. The recombinant polypeptide of claim 78, wherein the cytotoxic polypeptide comprises a catalytic domain derived from Chelona toxin.

81. the catalytic domain is Chelone toxin (CT1) as set forth in SEQ ID NO: 2; Chelone toxin (CT2) as set forth in SEQ ID NO: 21, or Chelone toxin (CT3) set forth in SEQ ID NO: 35 81. The recombinant polypeptide of claim 80, derived from

82. The catalytic domain from Chelone toxin (CT1) set forth in SEQ ID NO:2 has an amino acid sequence according to amino acids 1 to 186 of SEQ ID NO:2; The catalytic domain from Chelone toxin (CT2) set forth in SEQ ID NO: 21 has an amino acid sequence according to amino acids 1 to 186 of SEQ ID NO: 21; or 82. The recombinant polypeptide of claim 81, wherein the catalytic domain derived from Chelone toxin (CT3) set forth in SEQ ID NO: 35 has an amino acid sequence according to amino acids 1 to 191 of SEQ ID NO:

35.

83. 78. The recombinant polypeptide of claim 77, wherein the therapeutic polypeptide comprises a protein, or a functional fragment thereof, that is deficient in the pathological condition.

84. 5. The recombinant polypeptide of claim 1, wherein the cargo molecule comprises an N-terminal cysteine residue for use in "click" chemistry.

85. 5. The recombinant polypeptide of claim 1, wherein the cargo molecule comprises a nucleic acid molecule.

86. A nucleic acid encoding a recombinant polypeptide according to any one of claims 1 to 4.

87. A composition comprising a recombinant polypeptide according to any one of claims 1 to 4 together with an acceptable excipient, diluent or carrier.

88. A pharmaceutical composition comprising a recombinant polypeptide according to any one of claims 1 to 4 together with a pharmaceutically acceptable excipient, diluent or carrier.

89. 1. A method for delivering a cargo molecule to a cell, comprising: A method comprising contacting said cells with a recombinant polypeptide according to any one of claims 1 to 4.

90. 10. Use of a recombinant polypeptide according to any one of claims 1 to 4 for delivering said cargo molecule to a cell.

91. A recombinant polypeptide according to any one of claims 1 to 4 for use in delivering said cargo molecule to a cell.

92. 1. A pharmaceutical composition for use in a method of treating cancer in a subject, comprising: The pharmaceutical composition comprises a recombinant polypeptide according to any one of claims 1 to 4, The method comprises administering to the subject the recombinant polypeptide, a pharmaceutical composition.

93. A recombinant polypeptide according to any one of claims 1 to 4 for use in treating cancer in a subject.

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