Anti-ROR1 Antibodies Detecting C-Terminal Epitopes in FFPE Tissues
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Solution Overview
Problem
Current anti-ROR1 antibodies fail to effectively detect endogenously expressed ROR1 in formalin-fixed paraffin-embedded (FFPE) tissues and cannot distinguish between full-length and truncated ROR1 isoforms, limiting their utility in diagnosing ROR1-expressing tumors.
Innovation Solution
Development of binding proteins, such as immunoglobulin-like antibodies or antigen-binding fragments, that specifically bind to the C-terminal portion of ROR1, allowing for the detection of full-length ROR1 in FFPE tissues and distinguishing it from the truncated isoform.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If commercially available anti-ROR1 antibodies are used, then general ROR1 detection is possible, but they fail to effectively detect endogenously expressed ROR1 in FFPE tissues
Solution Approach 1:
The patent develops antibodies with altered binding parameters that specifically recognize ROR1 epitopes preserved in formalin-fixed tissues. The anti-ROR1 antibody (clone 6D4) was generated to detect conformational epitopes that remain intact after formalin fixation, changing the detection parameter from requiring native conformation to detecting fixation-resistant epitopes, thereby enabling reliable IHC detection in FFPE samples
Solution Approach 2:
The patent creates a surrogate detection system using antibodies that recognize conserved epitopes on ROR1 that are preserved through formalin fixation. The antibody clone 6D4 serves as a copy-detection tool that identifies ROR1 expression patterns in FFPE tissues without requiring the protein to be in its native, unfixed state, thus overcoming the limitations of commercial antibodies
2Measurement precision
If antibodies targeting the N-terminal portion of ROR1 are used, then ROR1 detection is possible, but they cannot distinguish between full-length and truncated ROR1 isoforms
Solution Approach 1:
The patent extracts the detection target from the N-terminal region (which is present in both isoforms) to the C-terminal region (which is unique to full-length ROR1). By designing antibodies that specifically bind to C-terminal epitopes such as those in the intracellular kinase domain or cytoplasmic tail, the method extracts the discriminatory information needed to distinguish full-length from truncated isoforms
Solution Approach 2:
The patent segments the ROR1 protein into functionally distinct regions for detection purposes: the N-terminal extracellular domain (present in both isoforms) and the C-terminal intracellular domain (unique to full-length). By targeting specific segments like the kinase domain or C-terminal tail with dedicated antibodies, the method achieves isoform-specific detection capability
3Measurement precision
If standard immunohistochemistry methods are used with commercial antibodies, then routine tissue analysis is possible, but accurate quantification of endogenous ROR1 is not achieved
Solution Approach 1:
The patent optimizes IHC detection parameters by using antibodies (clone 6D4) with enhanced affinity and specificity for ROR1 epitopes in FFPE tissues. The antibody shows optimal binding at controlled pH conditions and temperature, enabling accurate quantification of ROR1 expression levels while maintaining compatibility with routine IHC workflows and automated staining systems
Data Source
AI summary
The present disclosure relates to anti-ROR1 binding proteins, including those that bind to a ROR1 or portion thereof such as an intracellular C terminal portion of a ROR1 protein, and the use of such binding proteins in immunohistochemical and diagnostic methods. Related kits and methods of using the binding proteins are also provided, as are methods of treatment of subjects having diseases or conditions determined to be candidates for such treatments by the binding proteins or methods of this disclosure.