CAR Spacer Domain CD34 Extraction for Immune Specificity
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Solution Overview
Problem
Current chimeric antigen receptors (CARs) face challenges such as off-target immune responses and premature clearance by the host immune system, leading to reduced efficacy and specificity in targeting cancer cells, particularly due to the use of IgG1 or IgG4 hinge regions which can activate Fc receptor-positive cells independently of antigen presence.
Innovation Solution
The development of CARs with a spacer domain comprising part of the CD34 molecule, which allows for immune selection and avoids activation of unwanted immune responses, enabling efficient enrichment and persistence of transduced cells while maintaining cytotoxic activity, using sequences such as those shown in SEQ ID Nos. 5, 6, 7, or 8, and facilitating MACS-based selection and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If IgG1 or IgG4 hinge regions are used in CARs, then the structural stability and antigen binding are improved, but off-target immune responses are activated and premature clearance by the host immune system occurs
Solution Approach 1:
The patent extracts the problematic Fc region from the antibody structure and replaces it with a CD34-based spacer domain. This removal of the Fc portion eliminates the harmful Fc receptor interactions while preserving the antigen-binding functionality through the scFv domain, thereby resolving the contradiction between structural stability and off-target immune activation
Solution Approach 2:
The CD34 spacer domain serves as an intermediary element that connects the scFv antigen-binding domain to the transmembrane and intracellular signaling domains. This intermediary replaces the problematic IgG hinge region, providing necessary structural support and flexibility without triggering off-target immune responses, thus mediating between antigen recognition and cellular activation functions
2Stability of the object's composition
If IgG1 or IgG4 hinge regions are used in CARs, then the structural integrity is maintained, but persistence of transduced cells is reduced due to premature clearance
Solution Approach 1:
By extracting and removing the Fc region that targets CAR-expressing cells for premature clearance by host immune mechanisms, the patent extends the persistence of transduced cells while maintaining structural integrity through the alternative CD34 spacer domain configuration
Solution Approach 2:
The patent converts the harmful effect of Fc region-mediated immune clearance into a benefit by using the CD34 spacer domain, which not only eliminates premature clearance but also enables positive selection of transduced cells through CD34-specific antibodies, thereby improving both persistence and selection efficiency
3Reliability
If Fc region-containing CARs are used, then antigen binding capability is achieved, but activation of Fc receptor-positive cells occurs independently of antigen presence
Solution Approach 1:
The patent removes the Fc region from the CAR construct, extracting the harmful capability to activate Fc receptors while preserving the antigen binding function through the single-chain variable fragment (scFv) domain. This separation eliminates unwanted immune activation while maintaining target recognition
Solution Approach 2:
The CD34 spacer domain acts as an intermediary that connects antigen binding to cellular activation without involving Fc receptors. This intermediary structure ensures that cell activation occurs only when the antigen is bound, preventing the harmful effect of antigen-independent activation through Fc receptor engagement
Data Source
AI summary
In a first aspect, provided herein are chimeric antigen receptors (CAR) composed of at least an extracellular domain, a transmembrane domain and an intracellular domain, the extracellular domain comprises a spacer domain located C-terminally to a ligand-binding-domain also present in the extracellular domain, whereby the spacer domain comprises at least part of the CD34 molecule. Also provided are nucleic acid molecules encoding the same as well as vectors and cells containing the same. The cells include engineered T-cells and NK-cells and derivatives thereof. A pharmaceutical composition comprising the CAR e.g. in form of a vector, a polynucleotide encoding the CAR or the CAR itself and, in addition, cells, cell lines or host cells accordingly are provided. The CAR is useful in adoptive cell therapy. Finally, a method for enrichment or purification of CAR, in particular, of genetically engineered cells, cell lines or host cells expressing the CAR is provided.