Humanized CD3 Complex Animal Models for Therapeutic Antibody Testing
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Solution Overview
Problem
There is a need for a convenient animal model for preclinical testing of CD3-based therapeutic antibodies, such as mono- and bispecific antibodies, to effectively activate T cells and target specific antigens.
Innovation Solution
Genetically modified non-human animals, such as mice, are engineered to express humanized CD3 proteins (CD3ε, CD3δ, and CD3γ) on their T cells by modifying their endogenous CD3 loci to encode human CD3 extracellular domains, optionally linked with animal-specific transmembrane and cytoplasmic domains, creating a chimeric CD3 protein for T cell surface expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If non-human animals are used for preclinical testing of CD3-based therapeutics, then testing can be performed, but the results may not accurately reflect human immune responses due to species differences in CD3 protein structure
Solution Approach 1:
The patent applies local quality by humanizing only the extracellular domain of the CD3 protein while retaining the transmembrane and cytoplasmic domains from the non-human animal. This allows the extracellular domain to interact with human-specific antibodies and therapeutics, improving testing accuracy, while the animal-specific domains maintain proper protein folding, membrane insertion, and intracellular signaling functions.
Solution Approach 2:
The invention creates a composite protein structure combining human and non-human animal sequences. The chimeric CD3 protein consists of a human extracellular domain (amino acids 1-130 for CD3ε, 1-105 for CD3δ, 1-116 for CD3γ) fused to non-human animal transmembrane and cytoplasmic domains, enabling both human therapeutic interaction and animal cellular function.
2Reliability
If the endogenous CD3 locus is genetically modified to encode human CD3 extracellular domains, then humanized CD3 proteins are expressed on T cells, but the complexity of genetic modification increases
Solution Approach 1:
The genetic modification is segmented into distinct functional modules: the endogenous promoter and regulatory elements are preserved, the extracellular domain coding sequence is replaced with human sequence, and the transmembrane/cytoplasmic domain coding sequences are retained from the animal genome. This modular approach simplifies the genetic engineering process while achieving the desired humanized protein expression.
3Adaptability or versatility
If chimeric CD3 proteins are expressed on T cell surfaces, then human CD3-based therapeutics can be tested, but T cell development and immune function must be maintained
Solution Approach 1:
The chimeric CD3 protein acts as an intermediary that bridges human therapeutics and animal T cell function. The human extracellular domain provides the interface for human antibody binding and therapeutic mechanism of action, while the animal transmembrane and cytoplasmic domains ensure proper integration into the animal T cell receptor complex and maintenance of normal T cell development, signaling, and immune responses.
Data Source
AI summary
Non-human animals, expressing humanized CD3 proteins are provided. Non-human animals, e.g., rodents, genetically modified to comprise in their genome humanized CD3 proteins are also provided. Additionally, provided are methods and compositions of making such non-human animals, as well as methods of using said non-human animals.