De-immunized Shiga toxin a subunit effector polypeptides for applications in mammals

Patent metadata

Field	Value
Publication number	US-12065469-B2
Application number	US-202117345576-A
Country	US
Kind code	B2
Filing date	Jun 11, 2021
Priority date	Jan 27, 2014
Publication date	Aug 20, 2024
Grant date	Aug 20, 2024

Abstract

The present invention relates to Shiga toxin effector polypeptides with reduced antigenic and/or immunogenic potential. Immunogenicity can be a limitation for the repeated administration to mammals of proteins and polypeptides derived from Shiga toxins. The Shiga toxin effector polypeptides of the present invention have uses as components of therapeutics, diagnostics, and immunization materials. The cytotoxic proteins of the present invention have uses for selective killing of specific cell types and as therapeutics for the treatment of a variety of diseases, including cancers, immune disorders, and microbial infections. The proteins of the present invention also have uses for detecting specific cell types, collecting diagnostic information, and monitoring the treatment of a variety of diseases, such as, e.g., cancers, immune disorders, and microbial infections.

First claim

The invention claimed is:

1. A cell-targeting molecule comprising a protein as a component of the molecule, wherein the protein consists of:
(a) a binding region capable of specifically binding an extracellular target biomolecule physically coupled to the surface of a cell, wherein the extracellular target biomolecule is not CD38; and
(b) a Shiga toxin effector polypeptide comprising an amino acid sequence having at least 90% identity to amino acids 1 to 251 of SEQ ID NO: 1,
wherein the amino acid sequence comprises at least four endogenous B-cell epitope regions, and further comprises:
i) a plurality of disrupted endogenous B-cell epitope regions, wherein the disrupted endogenous B-cell epitope regions contain the following amino acid substitutions: S45 of SEQ ID NO: 1 to I, R55 of SEQ ID NO: 1 to L, 157 of SEQ ID NO: 1 to F, P59 of SEQ ID NO: 1 to F, E60 of SEQ ID NO: 1 to T, E61 of SEQ ID NO: 1 to L, G110 of SEQ ID NO: 1 to A, R188 of SEQ ID NO: 1 to A, R248 of SEQ ID NO: 1 to A and R251 of SEQ ID NO: 1 to A;
and ii) the amino acid substitution C242 of SEQ ID NO: 1 to S; and
wherein the amino acid sequence comprises an asparagine at the amino acid residue corresponding to position 75 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 77 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1, a glutamate at the amino acid residue corresponding to position 167 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 170 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 176 of SEQ ID NO: 1, and a tryptophan at the amino acid residue corresponding to position 203 of SEQ ID NO: 1.

2. The cell-targeting molecule according to claim 1 , wherein the amino acid sequence has at least 95% sequence identity to amino acids 1 to 251 of SEQ ID NO: 1.

3. The cell-targeting molecule of claim 1 , wherein the binding region is fused to the carboxy terminus of the Shiga toxin effector polypeptide to form a single, continuous polypeptide.

4. The cell-targeting molecule of claim 1 , wherein the binding region comprises an immunoglobulin-type binding region.

5. The cell-targeting molecule of claim 4 , wherein the immunoglobulin-type binding region comprises a polypeptide selected from: single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronectin-derived 10th 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, a heavy-chain antibody domain derived from a camelid V H H fragment, heavy-chain antibody domain derived from cartilaginous fish, immunoglobulin new antigen receptor (IgNAR), V NAR fragment, diabody, triabody, tetrabody, bivalent minibody, bispecific tandem scFv, bispecific tandem V H H, and bispecific minibody.

6. The cell-targeting molecule of claim 1 , wherein the binding region comprises a linker peptide (GxS)n wherein x is 1 to 6 and n is 1 to 30.

7. The cell-targeting molecule of claim 6 , wherein x is 4 and n is 1 (SEQ ID NO: 129.

8. A pharmaceutical composition comprising the cell-targeting molecule of claim 1 and a pharmaceutically acceptable excipient or carrier.

9. A cell-targeting molecule comprising a protein as a component of the molecule, wherein the protein consists of:
(a) a binding region capable of specifically binding an extracellular target biomolecule physically coupled to the surface of a cell, wherein the extracellular target biomolecule is not CD38;
(b) a Shiga toxin effector polypeptide comprising an amino acid sequence having at least 90% identity to amino acids 1 to 251 of SEQ ID NO: 1,
wherein the amino acid sequence comprises at least four endogenous B-cell epitope regions, and further comprises:
i) a plurality of disrupted endogenous B-cell epitope regions, wherein the disrupted endogenous B-cell epitope regions contain the following amino acid substitutions: S45 of SEQ ID NO: 1 to I, R55 of SEQ ID NO: 1 to L, 157 of SEQ ID NO: 1 to F, P59 of SEQ ID NO: 1 to F, E60 of SEQ ID NO: 1 to T, E61 of SEQ ID NO: 1 to L, G110 of SEQ ID NO: 1 to A, R188 of SEQ ID NO: 1 to A, R248 of SEQ ID NO: 1 to A and R251 of SEQ ID NO: 1 to A;
and ii) the amino acid substitution C242 of SEQ ID NO: 1 to S; and
wherein the amino acid sequence comprises an asparagine at the amino acid residue corresponding to position 75 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 77 of SEQ ID NO: 1, a tyrosine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1, a glutamate at the amino acid residue corresponding to position 167 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 170 of SEQ ID NO: 1, an arginine at the amino acid residue corresponding to position 176 of SEQ ID NO: 1, and a tryptophan at the amino acid residue corresponding to position 203 of SEQ ID NO: 1; and

(c) a linker between the binding region and the Shiga toxin effector polypeptide.

10. The cell-targeting molecule of claim 9 , wherein the amino acid sequence has at least 95% sequence identity to amino acids 1 to 251 of SEQ ID NO: 1.

11. The cell-targeting molecule of claim 9 , wherein the binding region comprises an immunoglobulin-type binding region.

12. The cell-targeting molecule of claim 11 , wherein the immunoglobulin-type binding region comprises a polypeptide selected from: single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, fibronectin-derived 10th 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, a heavy-chain antibody domain derived from a camelid V H H fragment, heavy-chain antibody domain derived from cartilaginous fish, immunoglobulin new antigen receptor (IgNAR), V NAR fragment, diabody, triabody, tetrabody, bivalent minibody, bispecific tandem scFv, bispecific tandem V H H, and bispecific minibody.

13. The cell-targeting molecule of claim 9 , wherein the linker is a peptide (GxS)n wherein x is 1 to 6 and n is 1 to 30.

14. A pharmaceutical composition comprising the cell-targeting molecule of claim 9 and a pharmaceutically acceptable excipient or carrier.

Assignees

• Molecular Templates Inc

Inventors

• Poma Eric
• Willert Erin
• Robinson Garrett Lee
• Rajagopalan Sangeetha
• Brieschke Brigitte

Classifications

• C07K16/1145

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

• C07K16/2866

  against receptors for cytokines, lymphokines, interferons · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C07K2319/55

  containing a fusion with a toxin, e.g. diphteria toxin · CPC title

• C07K2319/40

  containing a tag for immunodetection, or an epitope for immunisation · CPC title