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-11795235-B2
Publication Date
2023-10-24
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
Pseudomonas exotoxin A (PE) is a bacterial toxin that has cytotoxic activity potentially effective for destroying or inhibiting the growth of undesirable cells such as cancer cells. Although PE has been shown to produce positive clinical responses in some cancer patients, there is a recognized need for improved forms of PE with enhanced properties.
The invention discloses molecules comprising four domains: a targeting moiety (which is not an affibody), an albumin binding domain (ABD), a furin cleavage sequence (FCS) cleavable by furin, and a domain III from Pseudomonas exotoxin A, optionally substituted. The molecules may include substitutions within T-cell or B-cell epitopes and deletions of specific contiguous amino acid residues to reduce immunogenicity. The inventive molecules bind to serum albumin via the ABD, which is believed to prolong their half-life by reducing rapid degradation, thereby maintaining high cytotoxic activity, high anti-tumor activity, and high yields.
The inventive molecules are configured so that the ABD is positioned between the targeting moiety and the FCS, and the FCS between the ABD and the PE domain III. Various linkers connect the domains. The invention further provides related nucleic acids, recombinant vectors, host cells, cell populations, pharmaceutical compositions, and methods for producing the molecules. Methods of treating or preventing cancer in mammals, and inhibiting target cell growth by administering these molecules or compositions are also described.
Claims Coverage
The patent includes two independent claims describing molecules with specific domain architectures and sequences, focusing on the inclusion and positioning of an albumin binding domain (ABD) to enhance properties of immunotoxins.
Inclusion of albumin binding domain between targeting moiety and furin cleavage sequence
The molecule includes a first domain with a targeting moiety (not an affibody), a second domain comprising an albumin binding domain with amino acid sequence of SEQ ID NO: 2, a third domain containing a furin cleavage sequence cleavable by furin, and a fourth domain comprising domain III of Pseudomonas exotoxin A or its substituted form. The ABD is positioned between the targeting moiety and the furin cleavage sequence, which itself is between the ABD and the PE domain III.
Use of linkers connecting domains
The molecule optionally includes one or more peptide linkers positioned between the first and second domains, second and third domains, and/or third and fourth domains. These linkers consist of 1 to 20 amino acid residues selected independently from glycine, serine, lysine, and alanine, or specifically from glycine and serine.
Specific substitutions in domain III of Pseudomonas exotoxin A
The PE domain III in the molecule optionally contains amino acid substitutions at residues R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552 (referencing SEQ ID NO: 1), including specific substitutions R427A, F443A, R456A, D463A, R467A, L477H, R490A, R494A, R505A, R538A, and L552E to reduce immunogenicity or enhance function.
Molecule structure defined by Formula (I) with specified amino acid deletions and optional substitutions
An independent claim defines the molecule according to Formula (I): TM-R1k-ABD-R2m-FCS-R3p-R4q-PE functional domain III, where TM is the targeting moiety; ABD is the albumin binding domain of SEQ ID NO: 2; FCS is a furin cleavage sequence cleavable by furin; R1, R2, R3 are 1 to 20 amino acid residues linkers or sequences; R4 is one or more contiguous residues from SEQ ID NO: 1 (positions 365-394); PE domain III corresponds to residues 395-613 of SEQ ID NO:1. The molecule features deletions of amino acids 253-273 and 285-364 and optionally substitutions within T-cell and B-cell epitopes or deletions of residues 1-252.
Targeting moiety specificity
The targeting moiety is specified to be a monoclonal antibody or an antigen binding portion thereof that specifically binds to cell surface markers selected from CD19, CD21, CD22, CD25, CD30, CD33, CD79b, BCMA, GPC2, GPC3, transferrin receptor, EGFR, mutated EGFR, mesothelin, cadherin, and Lewis Y, including particular known antibodies.
The independent claims collectively disclose immunotoxin molecules characterized by the incorporation of an albumin binding domain between a targeting moiety and a furin cleavage sequence linked to domain III of Pseudomonas exotoxin A, with optional linkers and amino acid substitutions or deletions. These features are designed to improve pharmacokinetic properties, reduce immunogenicity, and maintain or improve cytotoxic activity for use in targeted cancer therapies.
Stated Advantages
High cytotoxicity against target cells.
High anti-tumor activity in vivo.
Improved purification yields for production.
Extended serum half-life compared to immunotoxins without albumin binding domains.
Potential to reduce rapid degradation and enhance therapeutic efficacy with lower dosages.
Documented Applications
Treatment or prevention of cancer in mammals by administering the inventive molecule, nucleic acid, vector, host cells, or pharmaceutical composition.
Inhibition of growth of target cells, including cancer cells, by contacting with the inventive molecule or related compositions.
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