Human Granzyme B Based Targeted Cytolytic Fusion Proteins
Abstract
:1. Introduction to Targeted Therapy and Humanization of Immunotoxins
2. Granzyme B and Its Anti-Tumor Activity
Granzyme B in Targeted Therapy
3. Reducing the Off-Target Toxicity of Granzyme B for Future Targeted Therapies
4. Overcoming Natural Granzyme Inhibitors
5. Effective Cytosolic Delivery of Granzyme B to Target Cells: A Major Bottleneck
6. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Construct | Disease * | Target | Cell Line | P19 Expression in Cell Line | Cytotoxicity | Reference |
---|---|---|---|---|---|---|
GrB (wt)-H22(scFv) and GrBR201K-H22(scFv) | CMML | CD64+ | Cells from AMML and CMML patients. | Yes | Not specified | [35] |
CD64+ HL60 | No | 4–7 nM | ||||
GrB (wt)-ki4(scFv) and GrBR201K-Ki4(scFv) | cHL | CD30+ | L428 | Yes | [12] | |
L540cy | No | |||||
GrBR201K-scFv1711 | Epidermoid cancer cells | EGFR+ | A431 | Yes | 133.3 nM | [36] |
RD target cells | Yes | 21.1 nM | ||||
GrBR201K-αEpCAM(scFv) | TNBC | EpCAM+ | MDA-MB-231 | Yes | N/A | [37] |
MDA-MB-468 | yes | 221 nM | ||||
MDA-MB-453 | No | 307 nM |
Granzyme B Variant | Mutation | Implication of Mutation | Result | Reference |
---|---|---|---|---|
GzmBFacD | The kktmrkry sequence at the C-terminus was replaced with the acidic peptide DSVLA derived from human complement factor D | This sequence motif is not positively charged and should have little immunogenic potential because complement factor D occurs at relatively high levels in human plasma | Binding to HL60 cells was completely abolished | [21] |
GzmBKD | The region around K127 and K131 is known to function as a heparin binding site in thrombin. To stabilize this, both lysines were replaced with aspartate residues | Reduced HS binding | Reduced binding to HL60 cells compared to wild type GrB | [21] |
GzmBKD-FacD | Double mutant consisting with aspartate replacement at position K127 and K131 and the acidic C-terminal peptide DSVLA | Combined effect of mutation | The binding and internalization efficiency was completely abolished | [21] |
cs1 | Arginine in position 110, 114 and 116 (R110, R114, and R116) replaced with alanine. Constitutes an altered classical GAG-binding motif | Most proteins bind GAG. This is dependent on electrostatic interaction between the positively and negatively charged cells. Mutation in this region alters binding of GrB to negatively charged cells | Reduced cytotoxic activity. 20-fold less cytotoxic compared to wild type GrB. Abolished binding to Heparin region | [27] |
cs2 | Lysine in position 239, 240, 243 and 244 (K239, K240, K243, and K244) replaced with alanine. Constitutes an altered C-terminal helix | Amphipathic C-terminal helix that has paired basic residues that bind GAGs. Mutation in this region alters binding of GrB to negatively charged cells. | Reduced cytotoxic activity. 2.5-fold less cytotoxic compared to wild type GrB. Reduced binding to Heparin region | [27] |
cs1+2 | Combined mutation of cs1 and cs2 | Combined mutation of cs1 and cs3 | Reduced cytotoxic activity. 20-fold less cytotoxic compared to wild type GrB. Abolished binding to Heparin region | [27] |
Granzyme B Variant | Mutation | Implication of Mutation | References |
---|---|---|---|
R28A | Substitution of Arginine residue with Alanine (constitutes a neutral charge at position 28) | In the presence of PI-9 the GrBR28A mutant contains 54% activity | [54] |
R28E | Substitution of Arginine residue with Glutamate (constitutes an opposite charge at position 28) | In the presence of PI-9 the GrBR28E mutant contains 25% activity | [54] |
R28K | Substitution of Arginine residue with Lysine (constitutes an identical charge at position 28) | In the presence of PI-9, the GrBR28K and mutants retained 76% of their original activity | [54] |
R201A | Substitution of Arginine residue with Alanine (constitutes a neutral charge at position 201) | In the presence of PI-9, the GrBR201A mutants retained 46% of their original activity | [54] |
R201E | Substitution of Arginine residue with Glutamate (constitutes an opposite charge at position 201) | No activity in the presence of PI-9 | [54] |
R201K | Substitution of Arginine residue with Lysine (constitutes an identical charge at position 28) | In the presence of PI-9, the GrBR201K mutant retained 94% of its activity | [54] |
K27A | Substitution of Lysine residue with Alanine (constitutes a neutral charge at position 27) | Insensitive to P1-9 activity and K27A mutant showed a marked decrease in the ability to bind and cleave a substrate (substrate 3) containing P9 residues | [54] |
R28A & R201A | Double mutant; Arginine replaced with Alanine at position 28 and 201 | In the presence of PI-9 the double mutant contains 0.5% activity | [54] |
K27E & R28A (EA) | Double mutant; Lysine replaced with Glutamate at position 27 and Arginine replaced with Alanine at position 28 | In the presence of 50% human serum, the enzymatic activity of EA remained over 40% over 24 h | [41] |
K27L & R28A (LA) | Double mutant; Lysine replaced with Leucine at position 27 and Arginine replaced with Alanine at position 28 | LA double mutant appeared to behave intermediate to the wild-type protein (GrB/VEGF121) and the EA construct | [41] |
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Hlongwane, P.; Mungra, N.; Madheswaran, S.; Akinrinmade, O.A.; Chetty, S.; Barth, S. Human Granzyme B Based Targeted Cytolytic Fusion Proteins. Biomedicines 2018, 6, 72. https://doi.org/10.3390/biomedicines6020072
Hlongwane P, Mungra N, Madheswaran S, Akinrinmade OA, Chetty S, Barth S. Human Granzyme B Based Targeted Cytolytic Fusion Proteins. Biomedicines. 2018; 6(2):72. https://doi.org/10.3390/biomedicines6020072
Chicago/Turabian StyleHlongwane, Precious, Neelakshi Mungra, Suresh Madheswaran, Olusiji A. Akinrinmade, Shivan Chetty, and Stefan Barth. 2018. "Human Granzyme B Based Targeted Cytolytic Fusion Proteins" Biomedicines 6, no. 2: 72. https://doi.org/10.3390/biomedicines6020072