Frizzled5 protein-binding agents
Inventors
Pan, Guohua • Moffat, Jason • Sidhu, Sachdev • Angers, Stephane • Steinhart, Zachary • Wang, Xiaowei
Assignees
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Publication Number
US-10919968-B2
Publication Date
2021-02-16
Expiration Date
Abstract
The present disclosure is directed to novel FZD5-binding agents and methods and uses thereof for treating a disease or disorder associated with aberrant expression or activity of Frizzled protein.
Core Innovation
The invention relates to isolated antibody variable-region FZD5-binding agents and antibody variable-region variants that specifically bind human FZD5. The described antibody variable regions include complementarity determining regions with amino acid sequences provided for CDR-H1, CDR-H2, and CDR-H3, together with shared framework segments common across antibody variable region variants.
The invention provides an isolated FZD5-binding agent that binds human Frizzled-5 (FZD5) with an affinity (KD) less than or equal to 200 picomolar. The binding agent comprises an antibody variable region selected from antibody variable region IDs Fv-2898 to Fv-2936, and includes CDR sequence information and specified VH positions 39, 55, and 66.
The described FZD5-binding agents are linked to FZD5 epitope binding, including an FZD5 epitope segment such as Ala27–Pro167. The disclosure further includes downstream therapeutic and diagnostic use cases in cancers, particularly RNF43-mutant Wnt-pathway cancers, and examples of additional formats and downstream applications such as conjugates, immune-engagers, CARs, detection of FZD5-expressing cells, and screening assays inhibiting FZD5–Wnt7B binding.
Claims Coverage
The independent claim covers an isolated FZD5-binding agent with KD ≤ 200 picomolar, defined by an antibody variable region selected from Fv-2898 to Fv-2936 and containing CDR amino acid sequences provided in Tables 3A–C and Tables 4A–C. Dependent claims further narrow the antibody variable region by adding specified VH residue constraints at positions 39, 55, and 66 and by selecting a particular variant identified as Fv-2919.
Isolated FZD5-binding agent with KD ≤ 200 picomolar
An isolated FZD5-binding agent that binds FZD5 with affinity (KD) less than or equal to 200 picomolar, comprising an antibody variable region that specifically binds human FZD5.
CDRs from antibody variable regions selected from Fv-2898 to Fv-2936
The antibody variable region comprises the complementarity determining regions (CDRs) of an antibody variable region selected from antibody variable region IDs Fv-2898 to Fv-2936, wherein the amino acid sequences of the CDRs for each antibody variable region are shown in Tables 3A–C and Tables 4A–C.
VH residue constraints at positions 39, 55, and 66
The amino acid residues at VH positions 39, 55, and 66 of the VH domain are specified as listed in Tables 3A–C and Tables 4A–C.
Specific CDR sequences and VH residues (isoleucine/serine/serine at positions 39/55/66)
The amino acid sequences for the CDR regions are specified and require specific VH-domain residues at positions 39, 55, and 66 to be isoleucine, serine, and serine, respectively.
Selected antibody variable region variant Fv-2919
The FZD5-binding agent includes a selected antibody variable region identified as Fv-2919.
Overall, the claim set centers on an isolated human FZD5-binding antibody variable region (KD ≤ 200 picomolar) using CDRs from Fv-2898 to Fv-2936 with CDR sequence data provided in specific tables. Dependent claims progressively narrow sequence definition by imposing VH position 39/55/66 residue constraints and by specifying exact CDR sequence identities, culminating in selection of the Fv-2919 variant.
Stated Advantages
Binds human FZD5 with affinity (KD) less than or equal to 200 picomolar.
Provides binding epitope binding to an FZD5 epitope segment such as Ala27–Pro167.
Cross-reactivity/binding to selected other Frizzled proteins is described via binding specificity profiling.
Suppresses proliferation of pancreatic cancer cell lines harboring RNF43-inactivating mutations.
Shows no effect in RNF43 wild-type.
Dose-dependent tumor growth inhibition by IgG-2919 in vivo.
No toxicity.
Increases differentiation and mucin production.
Discriminates Frizzled family CRDs on cell surfaces.
Exhibits sub-nanomolar affinity.
Documented Applications
Therapeutic use in cancers, particularly RNF43-mutant Wnt-pathway cancers.
Diagnostic use.
Generation of anti-FZD5 Fab clones and binding validation (e.g., ELISA binding) is described.
Conjugates (effector agents; toxins/anti-neoplastic agents).
Immune-engagers including bispecific antibodies/BiTE targeting CD3.
CARs using a FZD5-binding scFv and CD3ζ signaling.
Detection of FZD5-expressing cells.
Screening assays inhibiting FZD5–Wnt7B binding.
Profiling anti-FZD5 Fab binders that discriminate Frizzled family CRDs on cell surfaces using flow cytometry and immunofluorescence.
Inhibiting pancreatic cancer proliferation in cell lines with RNF43-inactivating mutations.
In vivo xenograft use of IgG-2919 showing dose-dependent tumor growth inhibition with no toxicity, along with mucin/differentiation changes including increased differentiation.
Readouts associated with Wnt/β-catenin pathway inhibition, including AXIN2 and NKD1 mRNA.
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