Universal Antibody Framework for Rabbit Humanization
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Solution Overview
Problem
Current methods for humanizing rabbit antibodies are inefficient due to structural differences between rabbit and human antibodies, leading to reduced antigen binding affinity and stability, and there is a lack of suitable human frameworks for accommodating rabbit CDRs.
Innovation Solution
A highly soluble and stable human antibody framework, FW 1.4, and its mutants rFW1.4 and rFW1.4(V2), are identified as universal acceptors for grafting rabbit CDRs, maintaining the spatial orientation and affinity of the original rabbit antibodies, allowing for high-throughput humanization with improved biophysical properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If classical grafting approaches are used to humanize rabbit antibodies, then immunogenicity is reduced, but antigen binding affinity and stability are compromised due to structural differences between rabbit and human antibodies
Solution Approach 1:
The patent applies parameter changes by systematically modifying framework region amino acid sequences in the human antibody acceptor to better accommodate rabbit CDR structures. Specific substitutions at positions like H31, H32, H33, H34, H35, H44, H45, H46, H47, H48, H49, H50, H51, H52, H53, H54, H55, H56, H57, H58, H59, H60, H61, H62, H63, H64, H65, H66, H67, H68, H69, H70, H71, H72, H73, H74, H75, H76, H77, H78, H79, H80, H81, H82, H83, H84, H85, H86, H87, H88, H89, H90, H91, H92, H93, H94, H95, H96, H97, H98, H99, H100, L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L25, L26, L27, L28, L29, L30, L31, L32, L33, L34, L35, L36, L37, L38, L39, L40, L41, L42, L43, L44, L45, L46, L47, L48, L49, L50, L51, L52, L53, L54, L55, L56, L57, L58, L59, L60, L61, L62, L63, L64, L65, L66, L67, L68, L69, L70, L71, L72, L73, L74, L75, L76, L77, L78, L79, L80, L81, L82, L83, L84, L85, L86, L87, L88, L89, L90, L91, L92, L93, L94, L95, L96, L97, L98, L99, L100 are made to optimize the framework for rabbit CDR accommodation while maintaining human-like sequence characteristics, thereby resolving the contradiction between reducing immunogenicity and preserving binding affinity
Solution Approach 2:
The patent creates a universal human antibody framework (FW 1.4 and its mutants rFW1.4, rFW1.4(V2)) that can accommodate CDRs from various rabbit antibodies with different binding specificities regardless of whether disulfide bridges are present. This universal framework serves multiple functions: it reduces immunogenicity like classical humanization approaches while simultaneously maintaining or improving antigen binding affinity and stability, unlike previous methods that had to choose one over the other
2Adaptability or versatility
If resurfacing strategies are used to remodel the solvent-accessible residues of the non-human framework, then human-like characteristics are achieved, but laborious development efforts are required for each immunobinder individually
Solution Approach 1:
The patent develops a universal human framework (FW 1.4 and mutants) that can be applied to humanize any rabbit antibody without requiring individualized remodeling efforts. This single universal framework replaces the need for custom resurfacing of each immunobinder, thereby maintaining human-like characteristics across all applications while dramatically improving productivity and reducing development time
Solution Approach 2:
The framework was preliminarily optimized through systematic evaluation of multiple human frameworks and targeted amino acid substitutions before being applied to rabbit antibody humanization. The framework rFW1.4 and its mutant rFW1.4(V2) were pre-engineered with specific amino acid changes that optimize their ability to accommodate rabbit CDRs, so that when applied to any rabbit antibody, the humanization process can proceed directly without requiring additional preliminary remodeling steps for each individual immunobinder
3Reliability
If rabbit antibodies are used directly for human therapy, then high affinity binding is achieved, but strong immune responses are elicited due to non-human sequences
Solution Approach 1:
The patent applies parameter changes by systematically modifying the amino acid sequence parameters of the framework regions in rabbit antibodies to create human-like sequences. The optimized human framework rFW1.4 and its mutant rFW1.4(V2) maintain structural parameters that support high-affinity antigen binding while changing the sequence composition parameters to resemble human antibodies, thereby reducing immune recognition while preserving binding functionality
Solution Approach 2:
The patent segments the rabbit antibody into functional components: the CDR regions that provide antigen binding specificity are retained from the original rabbit antibody, while the framework regions that provide structural support are replaced with human sequences. This segmentation allows the binding-affinity-determining CDRs to remain unchanged (preserving high affinity) while the framework is humanized (reducing immunogenicity)
Data Source
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Figure 3A~3C
AI summary
The present invention relates to a universal antibody acceptor framework and to methods for grafting non-human antibodies, e.g., rabbit antibodies, using a universal antibody acceptor framework. Antibodies generated by the methods of the invention are useful in a variety of diagnostic and therapeutic applications.