Immunotoxins with albumin binding domain
Inventors
Pastan, Ira H. • Wei, Junxia • Onda, Masanori • Bera, Tapan • Ho, Mitchell
Assignees
US Department of Health and Human Services
Publication Number
US-12215164-B2
Publication Date
2025-02-04
Expiration Date
2038-09-18
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Abstract
Disclosed is a molecule comprising: (a) a first domain, which comprises a targeting moiety; (b) a second domain, which comprises an albumin binding domain (ABD), (c) a third domain, which comprises a furin cleavage sequence (“FCS”), which FCS is cleavable by furin; and (d) a fourth domain, which comprises an optionally substituted Domain III from Pseudomonas exotoxin A (“PE”). Related nucleic acids, recombinant expression vectors, host cells, populations of cells, pharmaceutical compositions, methods of producing the molecule, methods of treating or preventing cancer in a mammal, and methods of inhibiting the growth of a target cell are also disclosed.
Core Innovation
The invention provides a molecule comprising four domains: a first domain with a targeting moiety that is not an affibody; a second domain comprising an albumin binding domain (ABD); a third domain comprising a furin cleavage sequence (FCS) that is cleavable by furin; and a fourth domain containing an optionally substituted Domain III from Pseudomonas exotoxin A (PE). The molecule can optionally include substitutions in T-cell and/or B-cell epitopes or deletions of specified amino acid residues in the PE sequence. The invention further provides nucleic acids encoding these molecules, recombinant expression vectors, host cells, pharmaceutical compositions, and methods of treating or preventing cancer or inhibiting target cell growth using the molecules.
The problem addressed arises from limitations of native and prior modified Pseudomonas exotoxin A (PE) immunotoxins, which exhibit a short half-life in vivo, necessitating high doses that cause non-specific toxicity. While PE can be effective in destroying cancer cells, its clinical application is limited by rapid degradation and immunogenicity. The invention aims to improve immunotoxins by incorporating an albumin binding domain to increase serum half-life, maintain or improve cytotoxicity, and enhance anti-tumor activity with potentially lower doses.
Claims Coverage
The claims include two independent nucleic acid claims comprising nucleotide sequences encoding molecules with a modular structure involving targeting moiety, albumin binding domain, furin cleavage sequence, and Pseudomonas exotoxin A domain, as well as associated recombinant expression vectors and host cells. The main inventive features focus on the specific domain organization, inclusion of the albumin binding domain SEQ ID NO: 2, and modifications to reduce immunogenicity.
Nucleic acid encoding a molecule with ordered domains including an ABD from SEQ ID NO: 2
A nucleic acid encoding a molecule composed of (a) a first domain with a targeting moiety (not an affibody), (b) a second domain with an albumin binding domain comprising the amino acid sequence of SEQ ID NO: 2, (c) a third domain with a furin cleavage sequence cleavable by furin positioned between the ABD and the toxin domain, and (d) a fourth domain comprising Domain III from Pseudomonas exotoxin A or a substituted form thereof; with the second domain positioned between the first and third domains, optionally including substitutions in T-cell/B-cell epitopes or deletions in specified regions of SEQ ID NO: 1.
Presence of linkers between domains
The molecule optionally includes peptide linkers between the first and second domains, second and third domains, and/or third and fourth domains, where the linkers consist of 1 to 20 amino acid residues selected from glycine, serine, lysine, and alanine, preferably glycine and serine.
Specific amino acid substitutions in PE domain III
The fourth domain may include substitutions at amino acid residues R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552 of SEQ ID NO: 1, with preferred substitutions including R427A, F443A, R456A, D463A, R467A, L477H, R490A, R494A, R505A, R538A, and L552E to reduce immunogenicity and/or enhance function.
Targeting moiety selections
The targeting moiety is preferably a monoclonal antibody or an antigen binding portion thereof, selected from antibodies specific for cell surface markers such as CD19, CD21, CD22, CD25, CD30, CD33, CD79b, BCMA, glypican 2, glypican 3, transferrin receptor, EGFR, mutated EGFR, mesothelin, cadherin, Lewis Y, and specified known antibodies including HA22 and others.
A molecule sequence conforming to Formula (I)
A nucleic acid encoding a molecule with a formula TM-R1k-ABD-R2m-FCS-R3p-R4q-PE functional domain III, where TM is targeting moiety, ABD is sequence SEQ ID NO: 2, FCS is furin cleavage sequence cleavable by furin, R1, R2, and R3 are linkers of 1-20 amino acids, R4 is residues 365-394 of SEQ ID NO: 1, the PE functional domain III is residues 395-613 of SEQ ID NO: 1, with deletions of residues 253-273 and 285-364, and optionally having substitutions and deletions that reduce immunogenicity as described.
The independent claims cover nucleic acids encoding chimeric molecules with a non-affibody targeting domain, an albumin binding domain from SEQ ID NO: 2 positioned between targeting moiety and a furin cleavage sequence, and a modified Pseudomonas exotoxin A domain III, optionally including linkers and substitutions to reduce immunogenicity. The claims extend to recombinant expression vectors and host cells containing these nucleic acids, establishing the inventive molecular structuring that confers improved serum half-life and therapeutic properties.
Stated Advantages
High cytotoxicity of the immunotoxins despite inclusion of albumin binding domains.
Increased half-life of immunotoxin molecules relative to those lacking albumin binding domains, resulting in longer circulation times in blood.
Enhanced anti-tumor activity observed in animal models with albumin binding domain-containing immunotoxins.
High protein yields in production when ABD is inserted after the Fv and before the furin cleavage sequence.
Ability to kill target cancer cells and achieve therapeutic efficacy at potentially lower dosages due to prolonged serum half-life.
Documented Applications
The molecule is used for treating or preventing cancer in mammals, including various solid and hematologic malignancies characterized by expression of specific cell surface markers such as CD22, BCMA, and mesothelin.
Inhibiting growth of target cells in vitro or in vivo by specifically targeting cell surface markers on cancer cells, enabling delivery of cytotoxic Pseudomonas exotoxin A.
Production of recombinant immunotoxin proteins incorporating albumin binding domains to extend serum half-life and improve anti-tumor effects.
Use of the chimeric molecules in pharmaceutical compositions for parenteral administration to human or animal patients for cancer therapy.
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