Method of screening for compounds that inhibit proliferation of pancreatic cancer cells having a loss-of-function mutation in the RNF43 gene
Inventors
Pan, Guohua • Moffat, Jason • Sidhu, Sachdev • Angers, Stephane • Steinhart, Zachary • Wang, Xiaowei
Assignees
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Publication Number
US-12600785-B2
Publication Date
2026-04-14
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 of activity of Frizzled protein.
Core Innovation
The invention provides antibody variable regions and derived FZD5-binding agents that bind human FZD5, including agents that include CDRs and framework regions and exhibit binding and specificity profiles with Frizzled cross-reactivity and affinity profiles. The document also reports nucleotide and amino-acid sequence data for anti-FZD5 antibody variable regions, shared framework sequences for constructing full-length variable regions, and epitope mapping information for an FZD5 segment (Ala27–Pro167).
The invention addresses proliferation of pancreatic cancer cells having a loss-of-function mutation in the RNF43 gene through inhibition of binding between FZD5 and Wnt7B. The document describes a Wnt-pathway rationale in which RNF43-loss pancreatic cancer cell growth depends on FZD5 and Wnt7B, and where perturbation of FZD5 or WNT7B suppresses RNF43-mutant PDAC proliferation, downregulates Wnt target genes (AXIN2, NKD1), and induces a differentiation marker (MUC5AC).
The document further includes methods for identifying test agents that inhibit FZD5–Wnt7B binding and thereby inhibit proliferation of pancreatic cancer cells with an RNF43 loss-of-function mutation. In this screening approach, binding between FZD5 and Wnt7B in pancreatic cancer cells is measured, FZD5 and Wnt7B are exposed to a test agent, and inhibition of binding is determined; inhibition identifies the test agent as a compound that inhibits proliferation. Dependent refinements specify quantitative inhibition thresholds for the test agent to qualify.
Claims Coverage
The independent claim covers a screening method that identifies compounds by whether they inhibit FZD5–Wnt7B binding, thereby identifying compounds that inhibit proliferation of RNF43-loss pancreatic cancer cells. The dependent claims refine this by specifying quantitative inhibition thresholds.
Screening for compounds inhibiting FZD5–Wnt7B binding to identify proliferation inhibitors
A method of screening for compounds that inhibit proliferation of pancreatic cancer cells having a loss-of-function mutation in the RNF43 gene by inhibition of the binding between FZD5 and Wnt7B, comprising measuring binding between FZD5 and Wnt7B in the pancreatic cancer cells, exposing FZD5 and Wnt7B to a test agent, determining if the test agent inhibits binding between FZD5 and Wnt7B, wherein inhibition identifies the test agent as a compound that inhibits proliferation of the pancreatic cancer cells.
Quantitative binding inhibition threshold at least 5%
The method further comprises using a test agent that inhibits binding between FZD5 and Wnt7B by at least 5%.
Quantitative binding inhibition threshold at least 10%
The method further includes that a test agent inhibits binding between FZD5 and Wnt7B by at least 10%.
Quantitative binding inhibition threshold at least 25%
The method further includes that the test agent inhibits binding between FZD5 and Wnt7B by at least 25%.
Quantitative binding inhibition threshold at least 75%
The method further includes using a test agent that inhibits binding between FZD5 and Wnt7B by at least 75%.
Overall, the claims require a screening workflow that measures and tests whether a candidate agent inhibits FZD5–Wnt7B binding, where such inhibition is used to identify compounds that inhibit proliferation of RNF43-loss pancreatic cancer cells. The dependent claims further constrain the qualifying agents by specifying minimum quantitative inhibition thresholds.
Stated Advantages
Identifies test agents as compounds that inhibit proliferation of pancreatic cancer cells with a loss-of-function mutation in RNF43 by inhibition of binding between FZD5 and Wnt7B.
The document reports sub-nanomolar K_D values for several IgGs binding to FZD family members including FZD5 and FZD8.
Functional anti-proliferative activity is reported for selected IgGs and Fabs against pancreatic cancer cells carrying RNF43 loss-of-function, including dose-dependent effects against RNF43 loss-of-function mutant pancreatic cancer cells but not RNF43 wild-type lines.
In vivo xenograft efficacy is reported for IgG-2919, together with an absence of toxicity.
Documented Applications
Screening for compounds that inhibit proliferation of pancreatic cancer cells having a loss-of-function mutation in the RNF43 gene by inhibition of the binding between FZD5 and Wnt7B.
Use of anti-FZD5 binding agents to suppress RNF43-mutant PDAC proliferation in the context described in the document, including downregulation of Wnt target genes (AXIN2, NKD1) and induction of MUC5AC, with anti-tumor activity described in an in vivo xenograft context.
Binding validation using flow cytometry and immunofluorescence for a Frizzled profiler antibody panel that recognizes FZD family cysteine-rich domains.
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