Antibody derivatives
Inventors
Cianfriglia, Maurizio • Flego, Michela • Ascione, Alessandro • Fiori, Valentina • Domicini, Sabrina • Mallano, Alessandra • Moricoli, Diego • Zamboni, Silvia
Assignees
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Publication Number
US-8822648-B2
Publication Date
2014-09-02
Expiration Date
Abstract
An anti-CEA scFv having an uncleaved Pel B leader sequence is surprisingly stable and is highly specific for CEA and CEACAM1.
Core Innovation
The invention relates to an anti-CEA/CEACAM1 single chain variable fragment (scFv) fusion protein that uses an E. coli Pel B leader sequence comprising all, or substantially all, of the Pel B leader sequence. In the described forms, the leader sequence is retained in an uncleaved manner to form stable oligomers, enabling functional anti-CEA and anti-CEACAM1 binding. The scFv includes V_H and V_L portions defined by SEQ ID NO: 2, with specified amino-acid substitutions.
The fusion protein comprises an anti-CEA/CEACAM1 scFv and a leader sequence, with a linker sequence being optionally present between the leader sequence and the scFv. Engineered scFv sequence variants are described in which positions relative to SEQ ID NO: 2 are mutated, including Pro at position 200 mutated to Leu; additional mutations including Cys at position 242 mutated to Trp and Trp at position 208 mutated to Arg; and the combination of these mutations. Tagged constructs such as D3SD3-FLAG-His6 are described.
The document further describes production as inclusion bodies followed by refolding and purification to separate functional oligomers from aggregates. Multi-step chromatography is described, including methods that use salt-dependent separation to obtain functional oligomers versus aggregates. The described engineered fusion forms show improved stability and affinity compared to reference scFv forms, and are reported to have longer in vivo half-life, increased tumor localization, and PET imaging signal in xenograft mice while maintaining binding specificity for derivative effector fusions.
Claims Coverage
The independent claim defines the overall fusion protein architecture and the specific scFv identity, while the dependent claims refine scFv residue substitutions and further add optional structural features such as linker, tags, effector fusions, and narrowed ranges for certain components, plus process features for producing and separating functional oligomers.
Anti-CEA/CEACAM1 scFv fusion with Pel B leader retention
A fusion protein comprising a single chain variable fragment (scFv) specific for both carcinoembryonic antigen (CEA) and CEA related Cell Adhesion Molecule 1 (CEACAM1), and a leader sequence comprising all, or substantially all, of the E. coli Pel B leader sequence.
SEQ ID NO:2-defined scFv variant set with specified residue substitutions
The V_H and the V_L portion of the scFv are as shown in SEQ ID NO: 2 with one or more specified substitutions, including Pro at position 200 mutated to Leu; Trp at position 208 mutated to Arg; and/or Cys at position 242 mutated to Trp.
Leader-to-scFv linker optionality
A linker sequence being optionally present between said leader sequence and said scFv.
Effector selection for fusion variants
The fusion protein has an effector selected from HIV-1 Vpr, LLOΔPEST, Yeast Cytosine Deaminase (YCD), ubiquitin, or IL-2.
Specified linker length range
The fusion protein includes a linker that is between 5 and 15 amino acid residues long, inclusive.
Tag components including D3SD3, FLAG, and His6
The fusion protein includes a tag made from one or more of the components D3SD3, FLAG, and His6 (SEQ ID NO:16).
The claim set is centered on an anti-CEA/CEACAM1 scFv fusion protein with an E. coli Pel B leader sequence comprising all, or substantially all, of the Pel B leader sequence, where the scFv is defined by SEQ ID NO: 2 and specified residue substitutions. Dependent claims further constrain particular scFv variants and add optional linker, effector, and tag elements, including specified linker length bounds and selectable tag components.
Stated Advantages
Improved stability and affinity versus reference scFv forms.
Longer in vivo half-life.
Increased tumor localization.
Increased PET imaging signal in xenograft mice.
Maintained binding specificity for derivative effector fusions.
Documented Applications
Tumor localization and PET imaging in xenograft mice using a radioiodination approach (including 124I microPET) with CEA-related targeting.
Use of derivative effector fusions while maintaining binding specificity.
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