O’Sullivan JA, Youngblood BA, Schleimer RP, Bochner BS (2024) Siglecs as potential targets of therapy in human mast cell- and/or eosinophil-associated diseases. Semin Immunol 69:101799. doi: 10.1016/j.smim.2023.101799 PMID: 37413923
Objective: To review a subset of Siglecs and their various endogenous or synthetic sialoside ligands that regulate eosinophil and mast cell function and survival.
Summary: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are vertebrate glycan-binding cell-surface proteins. Many Siglecs mediate cellular inhibitory activity and are of interest as part of a strategy to therapeutically lessen unwanted cellular responses. Human eosinophils and mast cells express overlapping but distinct patterns of Siglecs, and certain Siglecs have become the focus of novel therapies for allergic and other eosinophil and mast cell-related diseases.
Usage: Saporin in conjunction with CD22 glycomimetic ligand BPCNeuAc leads to cells death induction in a ligand-dependent manner on B-lymphoma cells (Collins et al.). Incubation with anti-Siglec-8 monoclonal antibody conjugated to saporin led to the death of malignant mast cells and eosinophils (O’Sullivan et al.)
Lu S, Gan L, Lu T, Zhang K, Zhang J, Wu X, Han D, Xu C, Liu S, Yang F, Qin W, Wen W (2024) Endosialin in cancer: Expression patterns, mechanistic insights, and therapeutic approaches. Theranostics 14(1):379-391. doi: 10.7150/thno.89495 PMID: 38164138
Objective: To discuss the use of a saporin conjugate to 78Fc, an antibody fragment directed towards endosialin a.k.a. CD248, in the treatment of human sarcomas.
Summary: Endosialin/CD248 is over expressed in cancer and a human single-chain variable fragment -Fc fusion protein was created targeting this receptor, 78Fc. 78Fc was then conjugated to saporin creating an immunotoxin. 78Fc-SAP is discussed as a immunotoxic strategy for the treatment of endosialin-based treatment therapies in cancer.
Makarova AO, Kostenk VV, Ovsyanikova OV, Svirshchevskaya EV, Lutsenko GV, Lutsenko AM (2023) Heat shock proteins on tumor cell surface as target for anti-tumor therapy (a review). Russian Journal of Bioorganic Chemistry 50(3):644-656. doi: 10.1134/S1068162024030038
Objective: To present the characteristics of the main proteins of the heat shock proteins (HSP) family, the features of their expression in tumor cells, and the possibility of using monoclonal antibodies against these proteins as a guiding vector for anti-tumor immunotherapy.
Summary: Apart from targeted delivery of NPs and different therapeutic drugs into cancer cells, another advantage of mAbs against HSP70 is their ability to activate anti-tumor antibody-dependent cellular cytotoxicity.
Usage: Certain antitumor drugs can be delivered by mAbs not only as part of NPs, but also as a drug–antibody conjugate. For instance, anti-HSP65 mouse mAb ML30 conjugated to Saporin (PR-01) almost fully inhibited cell proliferation of cell lines U937 and Daudi that express HSP65 on their surfaces.
Goodman RL, Moore AM, Onslow K, Hileman SM, Hardy SL, Bowdridge EC, Walters BA, Agus S, Griesgraber MJ, Aerts EG, Lehman MN, Coolen LM (2023) Lesions of kndy and kiss1r neurons in the arcuate nucleus produce different effects on lh pulse patterns in female sheep. Endocrinology 164(11):bqad148. doi: 10.1210/endocr/bqad148 PMID: 37776515
Objective: To test the functional role of ovine KNDy neurons in pulse generation and identify the roles of nearby Kiss1 receptor (Kiss1R)-containing cells.
Summary: Injection of NK3-SAP (NKB-SAP) ablated over 90% of the KNDy cells, Kiss-SAP lesioned about two-thirds of the Kiss1R population. This led to a significant decrease in LH pulse amplitude and altering LH pulse patterns. NK3-SAP increased the interpulse interval without affecting the regularity of LH pulses, whereas Kiss-SAP disrupted their regular hourly occurrence but not the interpulse interval. The findings suggest that KNDy neurons are critical for GnRH pulse generation in ewes, while ARC Kiss1R cells support the amplitude and regularity of these pulses, possibly as part of a positive feedback loop involving GABA or glutamate.
Usage: Saporin conjugates were injected into the arcuate nucleus. Kiss-SAP (kisspeptin54-SAP) was diluted to 700 ng/μL in PBS immediately before use. In preliminary work to test the effectiveness of Kiss-SAP, a single unilateral injection (1 μL of 700 ng/μL) of this conjugate was made in the preoptic area of 3 ewes. The contralateral side was used as control and either received no injections or Blank-SAP (1 μL of 700 ng/μL) (IT-21).
Marquez J (2023) PTGFRN as a target for antibody-drug conjugate (ADC) development in mesothelioma and medulloblastoma. Univ Maryland Baltimore Thesis.
Objective: To investigate the role of Prostaglandin F2 Receptor Negative (PTGFRN) regulator in cancer progression and develop an antibody-drug conjugate (ADC) targeting PTGFRN for the treatment of mesothelioma and pediatric medulloblastoma.
Summary: This dissertation explores the expression and function of PTGFRN in mesothelioma and pediatric medulloblastoma, identifying its association with aggressive cancer phenotypes. The study further develops a potent ADC using a PTGFRN-specific monoclonal antibody conjugated to the cytotoxic compound Duocarmycin, demonstrating significant anti-cancer efficacy in both in vitro and in vivo models .
Usage: Both a custom direct conjugate and Fab-ZAP Mouse (IT-48) were used (up to 10 nM) on transfected HEK-293A cells.
Le Z, Pan Q, He A, Liu H, Shi Y, Liu L, Liu Z, Ping Y, Chen Y (2023) Direct cytosolic delivery of proteins and CRISPR-Cas9 genome editing by gemini amphiphiles via non-endocytic translocation pathways. ACS Cent Sci 9(7):1313-1326. doi: 10.1021/acscentsci.3c00207 PMID: 37521791
Objective: Create a library composed of 150 gemini amphiphiles (GAs), a molecule with polar and nonpolar regions, to identify means to facilitate delivery of saporin across the cell membrane.
Summary: The gemini amphiphiles studied differ in the spacing of functional groups, hydrophobicity and sterics. A Cas9 ribonucleoprotein conjugated to saporin showed highest efficacy for internalization. HeLa cells were used to study the efffects of this found gemini-amphiphile conjugated to saporin.
Usage: A1I2-1R2C18 [a gemini ampiphile] conjugated to saporin and treated against HeLa cells at 0.0625, 0.125, 0.25, 0.5, 1μg/mL for 4 hours and then assessed for cell viability.
Q: We recently spoke to you about performing a custom saporin conjugation using our antibody. Is 0.09% azide in PBS in the antibody stock acceptable?
A: There are a number of dialysis steps within the conjugation protocol that will ultimately remove the azide from your antibody solution. So as long as your antibody will be happy in PBS without azide during the procedure, sending the material in 0.09% azide is fine. The final conjugate will be returned to you in PBS, sterile-filtered, without azide.
Q: In general, how many saporin molecules are incorporated per antibody? Can we test this by HPLC?
A: We aim for 2-2.5 moles of saporin per mole of antibody. You should be able to see differences in HPLC between your antibody with one vs. two vs. three saporins attached, however we will provide you with a saporin molar ratio and a product that has had free saporin and free antibody removed from the final conjugate.
Masoumi KC, Huang X, Sime W, Mirkov A, Munksgaard Thorén M, Massoumi R, Lundgren-Åkerlund E (2021) Integrin α10-antibodies reduce glioblastoma tumor growth and cell migration. Cancers (Basel) 13(5):1184. doi: 10.3390/cancers13051184
Summary: The authors investigated the treatment effect of two antibodies that have been developed to target the protein integrin 10, which is present on the surface of Glioblastoma (GB) cells. Function-blocking integrin alpha10, beta1-antibodies inhibit GB tumor growth as well as the migration of GB cells. This further validates integrin alpha10, beta1 as a promising target in GB and suggests a novel therapeutic strategy for the treatment of GB and other high-grade gliomas.
Usage: Infusions of anti-integrin α10β1-SAP or Anti-ctrl-SAP were made icv (1 µg/2 L per infusion).
Marquez J, Dong J, Dong C, Tian C, Serrero G (2021) Identification of prostaglandin F2 receptor negative regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development. PLoS One 16(1):e0246197. doi: 10.1371/journal.pone.0246197
Summary: PTGFRN is a cell-surface protein that is upregulated in certain cancer types, including head and neck and, notably, pediatric medulloblastoma, an aggressive cancer with limited therapeutic options. With the selection of the mouse monoclonal antibody 33B7, the authors identified PTGFRN as a potential therapy target, and show that it is internalized by incubation with 33B7. Purified 33B7 antibody was sent to Advanced Targeting Systems where saporin was directly conjugated to the Fc region of 33B7 using their proprietary cleavable linker.
Usage: In a 96-well plate, 2000 cells/well were plated in triplicate in 100 μL of DMEM/F12 medium supplemented with 2.5% FBS, 0.4 ug/ml 33B7 antibody, and 0.9ug/ml of Fab-ZAP mouse. As an isotype control, cells were incubated with mouse Fab IgG-SAP as control (instead of 33B7) and Fab-ZAP.
Komatsu N, Komatsu M, Ohashi R, Horii A, Hoshi K, Takato T, Abe T, Hamakubo T (2020) Photosensitizer with illumination enhances in vivo antitumor effect of anti-robo1 immunotoxin on maxillary sinus squamous cell carcinoma. Anticancer Res 40(7):3792-3799. doi: 10.21873/anticanres.14368 PMID: 32620618
Objective: To focus on the axon guidance receptor roundabout guidance receptor 1 (ROBO1) as a target for monoclonal antibody therapy of Head and neck squamous cell carcinoma (HNSCC).
Summary: Pronounced anti-tumor effects were elicited by the administration of saporin-conjugated anti-Robo1 immunotoxin (IT-Robo1) and AlPcS2a with light illumination on tumor size and pathological characteristics. The results showed that photosensitizer treatment with illumination robustly enhanced the anti tumor effect of the IT-Robo1 immunotoxin.
Usage: IT-ROBO1 (16ug) was intraperitoneally administered to HSQ-89 xenografted mice.
