Modified non-natural NKG2D ligands that selectively deliver attached heterologous molecules to non-natural NKG2D receptors on car-cells
Inventors
KIM, Kaman • Killeen, Nigel • HERZIG, Eytan • Greene, Warner
Assignees
J David Gladstone Institutes • Xyphos Biosciences Inc
Publication Number
US-11440948-B2
Publication Date
2022-09-13
Expiration Date
2040-01-28
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Abstract
This application relates generally to the production of modified, non-natural α1-α2 domains of NKG2D ligands with attached polypeptides having specific target-binding properties, for example, antibodies or variable fragments of antibodies, that are selectively delivered to Chimeric Antigen Receptors (CARs) comprised of modified, non-natural NKG2D receptors on engineered mammalian cells. The targeting of surface-expressed molecules includes those of virus-infected cells that can then be attacked and ablated by engineered cells of the immunity system expressing CARs cognate to the modified, non-natural α1-α2 domains of NKG2D ligands with attached polypeptides.
Core Innovation
This invention relates to modified, non-natural α1-α2 domains of NKG2D ligands that have attached heterologous polypeptides with specific target-binding properties, such as antibodies or their fragments. These modified ligands selectively bind to chimeric antigen receptors (CARs) comprising modified, non-natural NKG2D receptors expressed on engineered mammalian immune cells. The heterologous molecules attached to the ligands can bind specific molecules on target cell surfaces, thus directing immunity effector cells to target cells for elimination.
The problem addressed is that natural NKG2D ligands interact with natural NKG2D receptors broadly expressed in the immune system, leading to severe systemic toxicities and activation independent of targeting specificity in current CAR therapies. There is also a risk of tumor or virus-infected cells escaping immune attack due to antigen loss or immune evasion. The invention seeks to overcome these by engineering non-natural NKG2D ligands and receptors with selective, exclusive binding, allowing regulated activation and targeting control of CAR-modified immune cells.
The invention also solves problems related to manufacturing complexity and safety control of CAR therapies. The bispecific molecules composed of non-natural α1-α2 domains attached to targeting heterologous molecules can be administered separately, providing flexible and controlled activation of the CAR immune cells. This addresses limitations of current CAR-T approaches including lack of dose control, off-target effects, and antigen escape. The system enables multiplex targeting and better safety profiles for treating cancers and viral infections such as HIV.
Claims Coverage
The patent includes seven claims focused on the modified, non-natural ligands for modified, non-natural NKG2D receptors with attached heterologous molecules targeting HIV, particularly in CAR-cell contexts.
Modified ligand attached to heterologous molecule targeting HIV envelope proteins
A modified, non-natural ligand for a modified, non-natural NKG2D receptor with an attached heterologous molecule that selectively binds HIV proteins on infected cell surfaces, specifically to epitopes comprising SEQ ID NO: 169 or SEQ ID NO: 170, enabling selective binding to the modified NKG2D receptor and causing destruction of HIV-infected cells.
Specific amino acid sequences composing the modified ligand
The modified ligand comprises amino acid sequences selected from SEQ ID NOs: 68 through 72.
Binding specificity to modified NKG2D receptor sequences
The modified, non-natural ligand binds selectively to modified, non-natural NKG2D receptors comprising amino acid sequences SEQ ID NO: 54 or SEQ ID NO: 154.
Binding of modified ligand to CAR-cells
The modified, non-natural ligand is bound to CAR-cells expressing the modified NKG2D receptors.
Co-binding of different modified ligands on CAR-cells
CAR-cells expressing modified NKG2D receptors bind both a modified, non-natural ligand with an attached heterologous molecule that binds HIV proteins and a different modified ligand with an attached heterologous molecule or atom that does not bind HIV proteins.
Function modulation by heterologous molecules not targeting HIV
The heterologous molecule or atom attached to the modified ligand that does not bind HIV proteins modulates CAR-cell functions such as proliferation, differentiation, ablation, imaging, antagonism of immunosuppression, homing, or cytolysis of cells not infected by HIV.
The claims cover modified, non-natural ligands with heterologous molecules targeting HIV epitopes that selectively bind engineered, non-natural NKG2D receptors on CAR-cells to destroy infected cells, and also include modulation of CAR-cell functions by heterologous molecules not binding HIV.
Stated Advantages
The modified non-natural ligand–receptor pairs allow selective control of CAR immune cell activation, improving safety by avoiding off-target toxicities caused by binding natural ligands.
The system enables controlled activation of engineered immune effector cells via dosing of bispecific ligands, providing pharmacokinetic and pharmacodynamic regulation.
Multiplexing different bispecific molecules targeting distinct epitopes addresses tumor or virus antigen escape without needing multiple CAR cell products.
The invention simplifies manufacturing by using a single CAR design modulated by administered bispecific molecules with flexible targeting specificities.
Bispecific molecules with heterologous molecules that do not bind HIV can modulate CAR-cell function, allowing expansion of therapeutic applications and further control.
Documented Applications
Treatment and elimination of HIV-infected cells including reactivated latent reservoir cells using engineered CAR-T cells expressing non-natural NKG2D receptors selectively engaged by modified ligands with attached HIV-specific antibodies.
Selective targeting and killing of cells infected by HIV using CAR-T cells controlled by bispecific molecules linking modified NKG2D ligands and HIV envelope protein-binding heterologous molecules.
Potential treatment of viral infections and cancers by directing immune effector cells such as T-cells, NK-cells, macrophages, or other immunity cells via non-natural NKG2D ligands and receptors.
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