Cell-targeting molecules comprising protease-cleavage resistant, Shiga toxin A subunit effector polypeptides and carboxy-terminal moieties

Patent metadata

Field	Value
Publication number	US-10815469-B2
Application number	US-201515317892-A
Country	US
Kind code	B2
Filing date	Jun 10, 2015
Priority date	Jun 11, 2014
Publication date	Oct 27, 2020
Grant date	Oct 27, 2020

Abstract

The present invention provides protease-cleavage resistant molecules comprising Shiga toxin effector polypeptides capable of exhibiting potent, Shiga toxin functions (e.g. subcellular routing and cytotoxicity). The present invention also provides protease-cleavage resistant, cell-targeting molecules for targeting specific cell types, e.g., infected or malignant cells. Certain molecules of the present invention are cytotoxic, and certain cell-targeting molecules of the present invention may be used for the targeted killing of specific cell types and the treatment of a variety of diseases, disorders, and conditions, including cancers, tumors, growth abnormalities, immune disorders, and microbial infections. Certain cell-targeting molecules of the invention exhibit improved, in vivo tolerability as compared to related cell-targeted molecules comprising protease-cleavage sensitive, wild-type, Shiga toxin effector polypeptides. The cell-targeting molecules of the invention can deliver additional materials, such as, e.g., antigens, cytotoxic agents, and detection-promoting agents, into the interiors of target cells.

First claim

The invention is claimed as follows:

1. A cytotoxic cell-targeting molecule comprising
i) a heterologous, binding region capable of specifically binding an extracellular target biomolecule,
ii) a cytotoxic, Shiga toxin effector polypeptide comprising
(a) a Shiga toxin A1 fragment region having a carboxy terminus, wherein said Shiga toxin A1 fragment region comprises an amino acid sequence that is at least 95% identical to a sequence selected from:
amino acids 75 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2;
amino acids 1 to 241 of SEQ ID NO: 1 or SEQ ID NO: 2; and
amino acids 1 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2; and

(b) a disrupted furin-cleavage motif at the carboxy terminus of the A1 fragment region comprising one or more mutations in a minimal furin-cleavage site relative to a wild-type Shiga toxin A Subunit, the one or more mutations comprising a substitution mutation of an arginine residue natively positioned at 248 or 251 of SEQ ID NO: 1 or SEQ ID NO: 2 with a non-positively charged amino acid residue; and

iii) a molecular moiety having a mass of at least 1.5 kDa and associated with the carboxy terminus of the Shiga toxin effector polypeptide;

wherein the cytotoxic cell-targeting molecule does not comprise a compensatory furin-cleavage site located carboxy-terminal to the Shiga toxin effector polypeptide; and
wherein the cytotoxic cell-targeting molecule is capable of exhibiting cytotoxicity equivalent to the cytotoxicity of a second cytotoxic cell-targeting molecule, wherein said second cytotoxic cell-targeting molecule consists of the cytotoxic cell-targeting molecule except for the Shiga toxin effector polypeptide consists of a wild-type Shiga toxin A1 polypeptide without a disrupted minimal furin-cleavage site.

2. The cytotoxic cell-targeting molecule of claim 1 , wherein the molecular moiety has a mass of at least 4.5 kDa, 6 kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, or 50 kDa.

3. The cytotoxic cell-targeting molecule of claim 2 , wherein the molecular moiety comprises the heterologous, binding region.

4. The cytotoxic cell-targeting molecule of claim 1 , wherein the molecular moiety comprises at least one amino acid residue which is fused, either directly or indirectly, to the Shiga toxin effector polypeptide.

5. The cytotoxic cell-targeting molecule of claim 1 , wherein the cytotoxic cell-targeting molecule is capable of exhibiting improved, in vivo tolerability compared to the second cytotoxic cell-targeting molecule.

6. The cytotoxic cell-targeting molecule of claim 5 , wherein the molecular moiety is cytotoxic.

7. The cytotoxic cell-targeting molecule of claim 3 , wherein the heterologous, binding region comprises a polypeptide comprising an immunoglobulin-type binding region.

8. The cytotoxic cell-targeting molecule of claim 7 , wherein the immunoglobulin-type binding region comprises a polypeptide selected from the group consisting of:
single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, Fd fragment, antigen-binding fragment, fibronectin-derived 10 th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality.

9. The cytotoxic cell-targeting molecule of claim 8 , wherein the extracellular target biomolecule is selected from the group consisting of:
CD20, CD22, CD40, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, endoglin, fibroblast activation protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133, gpA33, mucin, TAG-72, carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha V beta3, Alpha5betal, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, tenascin, CD64, mesothelin, BRCA1, tyrosinase, human tyrosinase-related protein 1, human tyrosinase-related protein 2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NYESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen, prostate stem cell antigen, human aspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100, tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, CD38, CD15, CD23, CD52, CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, galectin-9, mrp-14, Siglec-8, Siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC class II, CD284-TLR4, CD107-Mac3, CD195-CCR5, HLA-DR, CD16/32, CD282-TLR2, CD11c, and any immunogenic fragment of any of the foregoing.

10. The cytotoxic cell-targeting molecule of claim 9 , which comprises a polypeptide shown in any one of SEQ ID NOs: 50-55 and 57-61.

11. A cytotoxic cell-targeting molecule comprising
i) a heterologous, binding region capable of specifically binding an extracellular target biomolecule;
ii) a Shiga toxin effector polypeptide comprising
(a) a Shiga toxin A1 fragment region having a carboxy terminus, wherein said Shiga toxin A1 fragment region comprises an amino acid sequence that is at least 95% identical to a sequence selected from:
amino acids 75 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2;
amino acids 1 to 241 of SEQ ID NO: 1 or SEQ ID NO: 2; and
amino acids 1 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2; and

(b) a disrupted furin-cleavage motif at the carboxy terminus of the A1 fragment region comprising one or more mutations in a minimal furin-cleavage site relative to a wild-type Shiga toxin A Subunit, the one or more mutations comprising a substitution mutation of an arginine residue natively positioned at 248 or 251 of SEQ ID NO: 1 or SEQ ID NO: 2 with a non-positively charged amino acid residue; and

iii) a molecular moiety having a mass of at least 1.5 kDa and associated with the carboxy terminus of the Shiga toxin effector polypeptide; and

wherein the cytotoxic cell-targeting molecule is capable of exhibiting improved in vivo tolerability compared to a second cytotoxic cell-targeting molecule, wherein said second cytotoxic cell-targeting molecule consists of the cytotoxic cell-targeting molecule except for the Shiga toxin effector polypeptide consists of a wild-type Shiga toxin A1 polypeptide without a disrupted minimal furin-cleavage site.

12. The cytotoxic cell-targeting molecule of claim 11 , wherein the molecular moiety comprises the binding region.

13. The cytotoxic cell-targeting molecule of claim 11 , wherein the molecular moiety is cytotoxic.

14. The cytotoxic cell-targeting molecule of claim 13 ,
wherein the Shiga toxin effector polypeptide is not cytotoxic, and
wherein the Shiga toxin effector polypeptide exhibits at least one Shiga toxin effector function selected from: inducing cellular internalization, delivering an exogenous material into a cell, directing subcellular routing, and directing intracellular routing to a cytosol.

15. The cytotoxic cell-targeting molecule of claim 13 , wherein the Shiga toxin effector polypeptide comprises a mutation relative to a naturally occurring A Subunit of a member of the Shiga toxin family which reduces or elimina

Assignees

• Molecular Templates Inc

Inventors

• Poma Eric
• Willert Erin
• Higgins Jack
• Kim Jason

Classifications

• C07K16/1145

  Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site · CPC title

• G01N33/569

  for microorganisms, e.g. protozoa, bacteria, viruses · CPC title

• C12Y302/02022

  rRNA N-glycosylase (3.2.2.22) · CPC title

• C12N9/2497 Primary

  hydrolysing N- glycosyl compounds (3.2.2) · CPC title

• A61K47/66

  the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells · CPC title