zap-conjugates

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.

Related Products: Mab-ZAP (Cat. #IT-04)

See Also:

• Ruggiero FM et al. Exploiting the internalization feature of an antibody against the glycosphingolipid SSEA-4 to deliver immunotoxins in breast cancer cells. Immunol Cell Biol 98(3):187-202, 2020.

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.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Ren X, Liu S, Virlogeux A, Kang SJ, Brusch J, Liu Y, Dymecki SM, Han S, Goulding M, Acton D (2023) Identification of an essential spinoparabrachial pathway for mechanical itch. Neuron 30:S0896-6273. doi: 10.1016/j.neuron.2023.03.013 PMID: 37023756

Objective: Study pathways of mechanical and chemical itch.

Summary: Mechanical and chemical itch are controlled by separate pathways within the body. Gpr83ligand PEN peptide conjugated to saporin (PEN-SAP) was used to selectively ablate CAlcrl projection neurons, alleviating mechanical itch sensitivity in wild-type mice. Substance-P saporin (SSP-SAP) was injected into mice and used as a negative control for the ablation of itch-sensitive neurons in the areas targeted. The ablated mice maintained mechanical itch sensitivity.

Usage: Intrathecal injections of SSP-SAP (IT-11; 100 ng/10 ml 0.9% sterile saline), biotinylated Gpr83 ligand (PEN) conjugated to Strepavidin-ZAP (IT-27), PEN-SAP, 3 ug/10 ml, 0.9% sterile saline, or control Blank-SAP (IT-21) in mice.

Related Products: SSP-SAP (Cat. #IT-11), Streptavidin-ZAP (Cat. #IT-27), Blank-SAP (Cat. #IT-21)

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.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Chan YY, Ho PY, Swartzrock L, Rayburn M, Nofal R, Thongthip S, Weinberg KI, Czechowicz A (2023) Non-genotoxic restoration of the hematolymphoid system in fanconi anemia. Transplant Cell Ther 29(3):164.e1-164.e9. doi: 10.1016/j.jtct.2022.08.015 PMID: 35995393

Objective: Evaluate the efficacy of different CD117 mAb agents and immunosuppression on hematopoietic stem cell (HSC) depletion, and their ability to safely establish therapeutic donor hematopoiesis post-HSCT in Fancomi Anemia (FA) disease models.

Summary: Patients with FA are particularly sensitive to genotoxic agents in the treatment of blood and immune diseases. By targeting the disease through use of a CD117 antibody conjugate to an antagonist agent, some of the sensitivities of Fancomi anemia can be circumvented compared to traditional therapeutics. The results demonstrated the efficacy of several different non-genotoxic monoclonal antibody-based conditioning strategies in Fancomi anemia.

Usage: Biotinylated CD117 mAb mixed with Streptavidin-ZAP (Anti-CD117-SAP) and was administered on Day 0. Anti-CD117-SAP was diluted with sterile PBS and administered at 1.5 mg/kg via i.v. injection.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Anti-CD117-SAP (Cat. #IT-83)

Ancheta LR, Shramm PA, Bouajram R, Higgins D, Lappi DA (2023) Streptavidin-saporin: Converting biotinylated materials into targeted toxins. Toxins 15(3):181. doi: 10.3390/toxins15030181 PMID: 36977072

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.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Read the full article.

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.

Related Products: Streptavidin-ZAP

References

1. Ancheta LR et al. Saporin as a commercial reagent: its uses and unexpected impacts in the biological sciences-tools from the plant kingdom. Toxins (Basel) 14(3):184, 2022.

Q: I read on your website that, “There are two types of RIPs: type I, which are much less cytotoxic due to the lack of the B chain and type II, which are distinguished from type I RIPs by the presence of the B chain and their ability to enter cells on their own.”

In the IT-27 Streptavidin-ZAP product, which type of saporin is there? Is it both type I and type II because the saporin is purified from the plant, or is it one specific type only in the product.

A: All saporin molecules are Type I ribosome-inactivating proteins. We only use saporin. An example of a Type II RIP is ricin, which can enter a cell on its own and has been used throughout history as a method of assassination.

Streptavidin-ZAP is streptavidin attached to saporin. On its own it has no way to get inside a cell. By mixing Streptavidin-ZAP with a biotinylated molecule that is recognized on the cell surface, the resulting conjugate is able to bind and internalize saporin into a cell. Once inside saporin inactivates the ribosomes which causes cell death.

Sonokawa T, Obi N, Usuda J, Sudo Y, Hamakubo T (2023) Development of a new minimally invasive phototherapy for lung cancer using antibody-toxin conjugate. Thorac Cancer 14(7):645-653. doi: 10.1111/1759-7714.14776 PMID: 36655546

Objective: Using a photo-sensitizer and red light to release Cetuximab-saporin to target cancer.

Summary: Photodynamic therapy is a non-invasive cancer treatment using a photo-sensitizer and laser irradiation to control the delivery of the chemotherapy agent. Streptavidin-ZAP mixed with biotinylated Cetuximab and a photo-sensitizer (mono-L-aspartyl chlorin e6) is used as an anti-cancer immunotoxin. In vitro analysis demonstrated that the novel photodynamic therapy method enhanced the cytotoxicity of Cetuximabe-SAP.

Usage: Streptavidin-ZAP (IT-27) mixed with biotinylated Cetuximab was used in vitro in A431, A549, and MCF7 cells.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Kim S, Shukla RK, Yu H, Baek A, Cressman SG, Golconda S, Lee GE, Choi H, Reneau JC, Wang Z, Huang CA, Liyanage NPM, Kim S (2022) CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells. Front Immunol 13:1011190. doi: 10.3389/fimmu.2022.1011190

Objective: To use a new murine testing model to demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment.

Summary: The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

Usage: Male C57BL/6J mice were injected into retro-orbital sinus with 15 μg S-CD3e-IT (Biotinylated Anti-CD3 mixed with Streptavidin-ZAP in sterile 200 μl PBS twice a day for four consecutive days.

Related Products: Streptavidin-ZAP (Cat. #IT-27)