Garaudé S, Marone R, Lepore R, Devaux A, Beerlage A, Seyres D, Dell’ Aglio A, Juskevicius D, Zuin J, Burgold T, Wang S, Katta V, Manquen G, Li Y, Larrue C, Camus A, Durzynska I, Wellinger LC, Kirby I, Van Berkel PH, Kunz C, Tamburini J, Bertoni F, Widmer CC, Tsai SQ, Simonetta F, Urlinger S, Jeker LT (2024) Selective haematological cancer eradication with preserved haematopoiesis. Nature 630(8017):728-735. doi: 10.1038/s41586-024-07456-3 PMID: 38778101
Objective: To demonstrate that an antibody–drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specifc depletion of the entire haematopoietic system, including Haematopoietic stem cells ( HSC).
Summary: Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type.
Usage: For ADC killing assays involving saporin, a 100 nM stock was prepared by incubating the biotinylated antibody (BC8 or MIRG451 mAbs) and saporin–streptavidin (IT-27) at a 1:1 molar ratio for 30 min at room temperature
Simpson J, Starke CE, Ortiz AM, Ransier A, Darko S, Llewellyn-Lacey S, Fennessey CM, Keele BF, Douek DC, Price DA, Brenchley JM (2024) Immunotoxin-mediated depletion of Gag-specific CD8+ T cells undermines natural control of Simian immunodeficiency virus. JCI Insight e174168. doi: 10.1172/jci.insight.174168 PMID: 38885329
Objective: To investigate the role of gag epitope-specific CD8+ T cells in the immune control of Simian Immunodeficiency Virus (SIV) in a nonhuman primate model.
Summary: Antibody-mediated depletion studies suggest that CD8+ T cells suppress SIV replication, but bulk depletion of CD8+ T cells may increase SIV target cells. Authors selectively depleted CD8+ T cells specific to the CM9 epitope, but this didn’t suppress viral replication in SIV-infected rhesus macaques. The data indicate that CM9-specific CD8+ T cells alone are not sufficient for immune control of SIV.
Usage: Streptavidin-ZAP was added stepwise to purified CM9 monomers to a final molar ratio of 1:4 and administered intravenously at a doses of 350 pmol/kg, 500 pmol/kg, 1 nmol/kg, or 2 nmol/kg at various time intervals.
Hello, I am very interested in using your Fab-ZAP line. So Fab-ZAPs simply tells us what mAbs internalize best – is that right? Can you send me some example data? I’d love to see a cell line that has been evaluated and the data that has been generated.
Answer:
Yes, Fab-ZAPs will tell you what mAbs internalize best. We have a variety of different versions of these products (ZAP secondary conjugates) which you can find here.
These have been tested on many different cell lines, both in-house and by our customers.
I’ve included a graph that corresponds to Fab-ZAP human (IT-51). Here is a cytotox which tests Fab-ZAP human with Trastuzumab on SK-BR-3 cells (breast cancer cell line).
SK-BR-3 cells were plated at 1000 cells/90 μl/well and incubated overnight. Trastuzumab and Saporin dilutions were made in cell media and 10 μl was added to each well. The Trastuzumab was also diluted in cell media containing, at a final concentration, 4.5 nM/10 μl Fab-ZAP, and 10 μl was added to each well. The plates were incubated for 72 hours. The plates were developed using a solution of XTT/PMS and read at 450 nm. Cytotoxicity was analyzed by comparing well readings of the treated wells to those of the control wells, expressed as a percentage. The number of viable cells remaining on the day of development is measured via cell metabolism of a colorimetric molecule within the developing reagents. Analysis was performed using Prism software (GraphPad, San Diego).
Related Products
ZAP conjugates – These are non-targeted saporin conjugates that “piggyback” on to your primary targeting agent (biotinylated material or antibody) to eliminate specific cells and reveal cell function.
Where does the saporin payload release after internalization? For example, does it require trafficking into a late endosome/lysosomal compartment?
Answer:
Thank you for reaching out to us. Hopefully I can help answer some of your questions regarding what happens to saporin after being internalized.
I would first like to refer you to an article we published, titled “Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins”. In it we review the internalization of saporin and include a few references for support. To answer your question in general, yes the conjugate is typically endocytosed and makes its way to the late endosome.
As an overview of this debated topic, the Wensley, H.J. et al 2019 article (ref #4) studied the escape of saporin from the late endosome and examined the endocytic process to quantify the endosomal escape into the cytosol. The Holmes, S.E et al 2015 (ref #5) and Giansanti, F. et al 2018 (ref #6) articles describe additional research examining chemical and genetic strategies used in assisting in saporin’s escape from the endosome. After endocytocis, Vago, R. et al 2005 (ref #7) compared saporin and ricin A chain and other bacterial toxins looking at their different intracellular routes to enter the cytosol. These articles should provide a nice foundation and hopefully better answer any questions.
If you’re interested in visualizing lysosomal trafficking, you might consider our pHast product line. These are secondary pH-dependent fluorescent conjugates, meaning that they only fluoresce once inside the endosomes and lysosomes of cells (which are acidic compared to the cytosol).
Related Products
pHast Conjugates – one of our pHastest tools for quantitative testing.
Boucher A (2024) Unveiling cholera toxin binding and intoxication using enteroids and site-specific mutants. Univ Gothenburg Thesis.
Objective: To investigate the binding site requirements of cholera toxin in the human body.
Summary: The cause of cholera symptoms is cholera toxin secreted by bacteria once in the small intestine. Cholera toxin has multiple binding sites that lead to many different intake mechanisms. By identifying the binding sites responsible, the study seeks to lay the groundwork for better means of treatment.
Usage: Leukocytes were treated with biotinylated Cholera toxin B binding-deficient mutants mixed with Streptavidin-SAP (IT-27) and assessed for cell death.
Matsuda T, Morigaki R, Hayasawa H, Koyama H, Oda T, Miyake K, Takagi Y (2024) Striatal parvalbumin interneurons are activated in a mouse model of cerebellar dystonia. Dis Model Mech 17(5):dmm050338. doi: 10.1242/dmm.050338 PMID: 38616770
Objective: To examine the influence of cerebellar abnormalities on the basal ganglia circuitry to investigate dystonia pathophysiology.
Summary: Dystonia is a disorder characterized by twisting, repetitive movements, and abnormal postures induced by sustained muscle contractions. This study utilized a cerebellar dystonia mouse model to examine the cerebellum’s contribution. The authors found that modulating parvalbumin (PV) interneurons might provide a novel treatment strategy.
Usage: In order to selectively ablate dorsolateral striatal PV interneurons, Streptavidin-ZAP (Cat. #IT-27) was mixed equimolar with biotinylated anti-PV and diluted with PBS by 1:100 and 3 ul injected into the striatum of mice. BIgG-SAP Rabbit (Cat. #IT-75) was used as the control.
Komatsu N, Kosai A, Kuroda M, Hamakubo T, Abe T (2024) Cetuximab-toxin conjugate and npe6 with light enhanced cytotoxic effects in head and neck squamous cell carcinoma in vitro. Biomedicines 12(5):973. doi: 10.3390/biomedicines12050973 PMID: 38790935
Objective: Combine the use of antibody-directed saporin and a photosensitizer to exert directed and improved cytotoxicity towards carcinoma cells as compared to either by itself.
Summary: Photodynamic therapy uses photosensitizers and irradiation to exert cytotoxic effects on cancer. When combined with biotinylated anti-EGFR conjugated to Strep-ZAP (IT-27), an increased cytotoxicity was hypothesized. The method developed enhanced the cytotoxicity of anti-EGFR-Strep-ZAP by the photodynamic effect in Sa3 and HO-1-u-1 cells, which have moderate levels of EGFR expression.
Usage: Cells were seeded on a 96-well plate and delivered 1.34 pM to 4.2 nM of anti-EGFR or anti-EGFR-Strep-ZAP, and cell viability was measured with formazan.
Chen S (2024) Nanocapsule-based prodrugs for targeted treatment of AIDS-associated non-hodgkin lymphoma. Univ California Thesis.
Objective: To propose a novel nanocapsule based platform that encapsulates the native drugs using various monomers and crosslinkers through free radical polymerization.
Summary: This encapsulation technology modifies the surface properties of the encapsulated drugs, enhancing their penetration into deeper tumor tissues and across the blood-brain barrier (BBB). Moreover, it significantly improves the stability of the drugs in vivo, protecting them from rapid degradation or clearance by the immune system. By adjusting the composition of the monomers and crosslinkers, the surface charge, hydrophobicity, and size of the nanocapsules can be finely tuned to maximize their efficacy in reaching and penetrating the target tissues.
Usage: Conjugation of ch128.1Av (anti-TfR1 IgG3-avidin fusion protein) with biotinylated saporin 6 (b-SO6) to eliminate malignant cells, including NHL malignancies. However, safe systemic delivery of ch128.1Av/b-SO6 is limited by its non-specific toxicity to normal cells expressing TfR1.
Kitawi R, Ledger S, Kelleher AD, Ahlenstiel CL (2024) Advances in HIV gene therapy. Int J Mol Sci 25(5):2771. doi: 10.3390/ijms25052771 PMID: 38474018
Objective: This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach.
Summary: The long-term or permanent expression of anti-HIV genes and the modification of CD4+ and CD34+ cells to render them resistant to infection or to allow the disruption of the HIV life cycle are important strategies in the quest to achieve a HIV cure.
Usage: Authors referenced CD117 antibody conjugated to saporin via streptavidin (IT-27, IT-83), which enabled >99% depletion of endogenous HSCs in NSG mice and non-human primates.
Objective: Use biotinylated CD45.2 antibodies conjugated to Streptavidin to target hematopoietic stem cells (HSCs) for HSC transplantation.
Summary: Hematopoietic stem cell transplantation offers a promising alternative to standard cancer treatments. Using Click Chemistry, different toxic payloads were attached to streptavidin and observed.
Usage: 75 micrograms of CD45.2 delivered to HSCs deriving from B6 mice.
