zap-conjugates

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)

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.

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)

Gudgeon J, Marin-Rubio JL, Trost M (2022) The role of macrophage scavenger receptor 1 (MSR1) in inflammatory disorders and cancer. Front Immunol 13:1012002. doi: 10.3389/fimmu.2022.1012002 PMID: 36325338

Objective: Review the role of Macrophage scavenger receptor 1 (MSR1) in health and disease, focusing on the molecular mechanisms influencing expression, its effect on disease process, macrophage function and signaling pathways, and the therapeutic potential of targeting MSR1.

Summary: MSR1 is primarily found on the surface of various types of macrophages and affects processes such as astherosclerosis, innate and adaptive immunity, lung and liver disease and cancer. Recently, MSR1 has been implicated to trigger inflammatory and tumorigenic pathways. MSR1 is most often correlated with the anti-inflammatory M2 macrophage phenotype. Investigations into anti-inflammatory signalling downstream of MSR1 are vital to fully understand the influence of MSR1 expression in inflammatory disease. Manipulating M2 macrophages through MSR1 may represent a new targeted therapeutic approach for diseases such as cancer, arthritis, and other inflammatory diseases.

Usage: In reviewing therapeutic strategies, an antibody-based method was developed using the 2F8 anti-SR-A monoclonal antibody. The antibody conjugated to RAT-ZAP and unconjugated saporin was delivered to mice via intraperitoneal injection to deplete vascular leukocytes (VLCs) from the peritoneum to reduce the tumor burden. 4.8 μg of Rat-ZAP in the absence or presence of 4 μg of clone 2F8 antibody was incubated on ice for 30 min.

Related Products: Rat-ZAP (Cat. #IT-26)

Q: When using any of your Fab-Zap product line, the recommended final concentration is 4.5 nM. Is this based on experiments you have done? I question if at 4.5 nM my primary antibody will be saturated with Fab-ZAP secondary conjugate?

A: Yes, the 4.5 nM concentration is what we use to quality-control test our Fab-ZAP conjugates and why we recommend it in the literature.  We also recommend only titrating your primary antibody.  The 4.5 nM of Fab-ZAP should be enough to saturate your primary antibody.  If you have a test of ~10 nM of primary antibody and you experience less cell death than ~1 nM, this will indicate “antibody competition” (i.e., your primary antibody is not saturated).  The data sheet shows a cytotox with a nice example of this. (Fab-ZAP data sheet)

See: Fab-ZAP human (Cat. #IT-51)

Goodyer WR, Beyersdorf BM, Duan L, van den Berg NS, Mantri S, Galdos FX, Puluca N, Buikema JW, Lee S, Salmi D, Robinson ER, Rogalla S, Cogan DP, Khosla C, Rosenthal EL, Wu SM (2022) In vivo visualization and molecular targeting of the cardiac conduction system. J Clin Invest e156955. doi: 10.1172/jci156955

Objective: To engineer targeted antibody conjugates directed against the cardiac conduction system (CCS) to allow visualization of the CCS in vivo.

Summary: Accidental injury to the CCS, a specialized set of cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. They generated a fully human monoclonal Fab (hCNTN2) that targets the CCS with high specificity.

Usage: Streptavidin-ZAP was reacted with biotinylated hCNTN2 Fab to create hCNTN2-SAP. 100 ug of either hCNTN2-SAP and control-SAP were injected into wild-type mice with a single tail-vein injection and hearts were harvested after 2 days.

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

Van Hentenryck M, Li Z, Murphy PM, Czechowicz A (2022) Antibody-based preparative regimens for cell, tissue and organ transplantation. (eds. 162). OBM Transplantation 6(3):162. doi: 10.21926/obm.transplant.2203162

Objective: Provide a review of progress in the use of antibodies to support cell and tissue transplantation with a particular focus on induction of donor-specific tolerance for solid organ transplantation.

Summary: Antibody-based conditioning to prepare the recipient is a promising approach towards achieving transplant tolerance in both hematopoietic and solid organ transplant settings.

Usage: To enhance HSC depletion while avoiding bystander toxicity (neutropenia, lymphopenia, and thrombocytopenia) caused by CD45-radioimmunotherapy, Palchaudhuri et al. developed a saporin-based CD45 (CD45-SAP) immunotoxin using a biotinylated antibody and Streptavidin-ZAP.

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

See Also:

• Palchaudhuri R Featured Article: Targeted depletion of hematopoietic stem cells promises safer transplantation. Targeting Trends 17(3), 2016.
• Palchaudhuri R et al. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nat Biotechnol 34:738-745, 2016.

Takahashi JI, Nakamura S, Onuma I, Zhou Y, Yokoyama S, Sakurai H (2022) Synchronous intracellular delivery of EGFR-targeted antibody-drug conjugates by p38-mediated non-canonical endocytosis. Sci Rep 12(1):11561. doi: 10.1038/s41598-022-15838-8 PMID: 35798841

Objective: The binding of cetuximab to EGFR suppresses ligand-induced signaling events. The authors demonstrate that synchronous non-canonical EGFR endocytosis can increase the efficacy of EGFR-targeting ADCs.

Summary: Epidermal growth factor (EGFR) has been a popular target in the treatment of cancer via monoclonal antibodies, specifically cetuximab and panitumumab. They have been applied to antibody-drug conjugates (ADCs) and their clinical efficacy had been demonstrated, but this efficacy has also been limited by acquired resistance via secondary mutations or the activation of bypass pathways. To overcome these limiting factors, the authors investigated if non-canonical clathrin-mediated endocytosis (CME) of EGFR induced the internalization of membrane-bound EGFR-targeted mAbs. Their results show that tumor necrosis factor-alpha strongly induces endocytosis of the cetuximab-EGFR complex via the p38 phosphorylation of EGFR and that Hum-ZAP, a secondary antibody conjugated to saporin, will also undergo internalization with the complex and enhance anti-proliferative activity.

Related Products: Hum-ZAP (Cat. #IT-22)