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.
Zhao Q, Zong H, Zhu P, Su C, Tang W, Chen Z, Jin S (2024) Crosstalk between colorectal CSCs and immune cells in tumorigenesis, and strategies for targeting colorectal CSCs. Exp Hematol Oncol 13(1):6. doi: 10.1186/s40164-024-00474-x PMID: 38254219
Summary: Cancer immunotherapy has become a promising strategy in the treatment of colorectal cancer, and relapse after tumor immunotherapy. Cancer stem cells (CSCs) have the capabilities of self-renewal and differentiation and are also resistant to the traditional therapies of radiotherapy and chemotherapy. The authors review strategies for targeting colorectal CSCs, where one method described uses a biotinylated antibody against EpCAM (clone 3-171) conjugated to saporin via Streptavidin-ZAP (IT-27).
Allred CA, Gormley C, Venugopal I, Li S, McGuire MJ, Brown KC (2023) Tumor-specific intracellular delivery: peptide-guided transport of a catalytic toxin. Commun Biol 6(1):60. doi: 10.1038/s42003-022-04385-7 PMID: 36650239
Objective: The demonstration of a peptide optimized by chemical modifications for tumor specificity to deliver saporin, a catalytic toxin, specifically to cancer cells via both in vitro and in vivo.
Summary: Peptides rival antibodies in affinity and specificity and offer an alternative as cancer-targeting molecules. In comparison to antibodies, peptides have a faster development time and lower production cost. The authors isolated peptide MGS4, derived from a phage-displayed library using a non-small cell lung cancer (NSCLC) cell line as the target. MGS4 was modified to identify the minimal binding domain while also improving affinity and stability. Importantly, the authors provide data showing the peptide delivered saporin both in vitro and in vivo to cancer cells demonstrating anti-tumor efficacy in a mouse model.
Usage: In vitro delivery was performed by reacting biotinylated peptide with Streptavidin-ZAP (Cat. #IT-27) in a 1:1 molar ratio. Cells were treated for 6h at 37C. The drug was removed and replaced with media and after 72 hours, cell viability was measured with CellTiter-GLO. In vivo delivery was performed using biotinylated MGS4 reacted with Streptavidin-ZAP and administered via tail-veil injection (7.5 ug/100 ul) 2x/week for 2.5 weeks for a total of 5 treatments.
Ploeg E (2023) Novel approaches towards cancer-directed immune checkpoint inhibition. Univ Groningen Thesis. doi: 10.33612/diss.737906343
Objective: To evaluate a novel bispecific antibody, bsAb CD73xEGFR, that inhibits the immunosuppressive enzyme CD73 on cancer cells in an EGFR-directed manner.
Summary: The researchers constructed a bispecific antibody, bsAb CD73xEGFR, that binds to both CD73 and EGFR on cancer cells. In preclinical studies, they found that bsAb CD73xEGFR was more effective than the monospecific anti-CD73 antibody oleclumab at reducing tumor growth and enhancing anti-tumor immune responses, likely due to its ability to direct CD73 inhibition specifically to cancer cells overexpressing EGFR.
Usage: Cancer cells were incubated with bsAb CD73xEGFR (1 μg/ml) (or controls) in the presence of Fab-ZAP human (Cat. #IT-51). Apoptotic cancer cell death was evaluated after 24 h by flow cytometry using Annexin-V/PI staining.
Jin X, Yang GY (2023) Pathophysiological roles and applications of glycosphingolipids in the diagnosis and treatment of cancer diseases. Prog Lipid Res 101241. doi: 10.1016/j.plipres.2023.101241 PMID: 37524133
Objective: The authors review the tumor-related biological functions of GSLs and recent progress in using GSLs and related enzymes to diagnose and treat tumor diseases.
Summary: Glycosphingolipids (GSLs) are glycolipids present on the surface of living cell membranes. Specific GSLs and related enzymes are abnormally expressed in many cancer diseases and affect the malignant characteristics of tumors. The regulatory roles of GSLs in signaling pathways suggest that they are involved in tumor pathogenesis. GSLs have therefore been widely studied as diagnostic markers of cancer diseases and important targets of immunotherapy.
Usage: The stage-specific embryonic antigen-4 (SSEA4) mAb, MC-813-70, was mixed with Mab-ZAP at a molar ratio of approximately 3:1 with the complex used at nanomolar concentrations on MDA-MB-231 cells, a triple negative breast cancer cell known to express SSEA-4. The conjugate was able to reduce tumor viability in vitro.
Kfoury YS, Ji F, Jain E, Mazzola MC, Schiroli G, Papazian A, Mercier FE, Sykes DB, Kiem A, Randolph MA, Abdel-Wahab OI, Calvi LM, Sadreyev R, Scadden DT (2023) The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression. Blood Adv bloodadvances.2022008268. doi: 10.1182/bloodadvances.2022008268 PMID: 37450380
Objective: Evaluate mesenchymal cell molecular features searching for modifications that could impact Myelodysplastic syndrome (MDS) and offer potential therapeutics.
Summary: MDS is a heterogenous group of diseases affecting hematopoietic stem cells and are curable only by stem cell transplantation. Animal models of MDS indicate that changes in specific mesenchymal progenitor subsets in the BM can induce or select for abnormal hematopoietic cells. The authors identified that osteopontin (SPP1) is overexpressed in human bone marrow mesenchymal cells. SPP1 expression in comparable mesenchymal stromal cell populations plays protective roles in disease progression in an MDS mouse model.
Usage: Streptavidin-ZAP was combined with biotinylated CD117 (cKit) Ab in a 1:1 molar ratio. Mice were dosed with the conjugate at 3 mg/kg. The authors used the antibody-drug conjugate as a conditioning strategy that spares the non-hematopoietic microenvironment in the BM from genotoxic injury. This approach has been shown to deplete host hematopoietic stem cells with minimal toxicity effectively.
Konturek-Ciesla A, Dhapola P, Zhang Q, Säwén P, Wan H, Karlsson G, Bryder D (2023) Temporal multimodal single-cell profiling of native hematopoiesis illuminates altered differentiation trajectories with age. Cell Rep 42(4):112304. doi: 10.1016/j.celrep.2023.112304 PMID: 36961818
Objective: Using single-cell transcriptome and epitope profiling to study hematopoiesis and the effects from aging.
Summary: The contribution from Hematopoietic stem cells (HSCs) to mature blood cells decline with age. The authors used transcriptome and epitope profiling to reconstruct early hematopoiesis and assessed HSC-specific lineage tracing. Their analysis identified previously uncharacterized cell populations which included multipotent progenitor cells (MPP) Ly-1 and Ly-II. Flt3 is a marker indicative of early differentiation found on MPP cells and was targeted for elimination via an antibody to Flt3 combined with Streptavidin-ZAP. This cell depletion provided evidence for the lack of self-renewal of Ly-1 and Ly-II cells in a transplantation setting and suggests that they need to be continuously replenished by upstream HSPCs.
Usage: Biotinylated anti-CD135 (clone A2F10) was combined with Streptavidin-ZAP at a 1:1 molar ratio, diluted in PBS to 0.2 mg/ml and injected into mice at 3 mg/kg.
Summary: This manuscript describes the myriad of ways Streptavidin-ZAP is used and how this technology supports the scientific process of ‘Molecular Surgery’ and progress in research and drug development. Insights from publications and research performed using Streptavidin-ZAP and its impact on academia and industry for research and drug development are presented.
Question: I was wondering if you could elaborate on why the Streptavidin-ZAP product recommends to be used at an equimolar ratio with the targeting reagent, when it is capable of binding up to four biotins/molecule?
Answer: It’s a question we get asked sometimes and it’s a good question.
You are correct that streptavidin is capable of binding up to 4 biotin molecules. However, when we created streptavidin-ZAP with the purpose of being a modular way of creating targeted toxins, we learned that the best general rule to follow was using a equimolar reaction. In theory, it is a 1:1 ratio of targeting molecule to streptavidin-ZAP, where we are most likely seeing an average of 1:1, but there is also the possibility of mixed ratios.
The amount of publications using the equimolar approach gave the desired results whether they were using a small biotinylated peptide or whole IgG. You’ll notice that depending on the MW of your biotinylated targeting agent, the amount of streptavidin-ZAP needed for the experiment can vary drastically and through in-house characterization, the equimolar approach still worked best.
Another reason we recommend a 1:1 ratio is based on our experience with our other secondary conjugates. It may be intuitive to think that using a higher dose of targeting agent would induce more cell death, but we found the opposite effect, where the excess, un-reacted targeting agent competed with the conjugated material for surface binding sites, which in turn decreased the amount of saporin being delivered. We have a publication (PMCID: PMC8952126 ) that also describes this observation.
Once you’ve created a baseline using the equimolar protocol and are more accustomed to how streptavidin-ZAP works in your application, please contact us if you feel more optimization is needed. It will be easier to help trouble-shoot when we are all working off the same protocol.
