We’re highlighting our ZAP antibody internalization kits and our line of secondary antibody saporin conjugates. We have given these products the moniker “ZAP” in place of saporin.
Our ZAP conjugates consist of a variety of secondary antibodies that allow a large number of targeting agents to be screened quickly and cost-efficiently for specificity, functional binding, internalization, and EC50 determination.
The conjugates are constructed using either species-specific secondary antibodies, or streptavidin (for use with biotinylated Targeting Agents), and they are chemically attached to Saporin, a potent plant ribosome-inactivating proteins.
This article by Marquez et al. showcases how our secondary conjugates can be used to screen antibodies and then from that choose the best candidate to create a custom conjugation.
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 were able to show that it is internalized by incubation with 33B7.
We’re highlighting our secondary saporin conjugate, Streptavidin-ZAP, which is streptavidinylated saporin. It combines with your biotinylated material to make a targeted toxin.
Unlike a secondary antibody binding to a primary antibody, the bond between streptavidin and biotin is rapid, essentially non-reversible, unaffected by most extremes of pH, organic solvents or denaturing reagents. It is essentially the strongest known noncovalent biological bond between protein and ligand. Streptavidin-ZAP is very modular and works with biotinylated antibodies, peptides, growth factor, aptamers, anything that will recognize a cell surface receptor and can be biotinylated.
This is a publication using a Streptavidin-ZAP reacted with an antibody as the targeting agent.
The authors’ objective was to investigate whether CD206-positive macrophages in the meninges play a role in regulating nociception and pain hypersensitivity. They injected rats intrathecally with conjugate made with biotinylated anti-CD206 reacted with Streptavidin-ZAP and looked at the effects on responses in naïve rats versus ones that received a skin incision, after depletion of CD206+ macrophages.
Their results indicated that depleting CD206+ meningeal macrophages did not regulate basal responses in naïve rats of either sex. However, ablation of these cells after skin injury induced mechanical hypersensitivity in male rats and not in females. Thus, they were able to conclude, that in a sex-dependent manner, CD206-positive meningeal macrophages prevent the spread of pain hypersensitivity after a minor injury.
Babatunde OO, Bibby MG, Atala A, Almeida-Porada G, Porada CD (2026) In utero HSC transplantation for sickle cell disease: A potential therapeutic approach that overcomes complications of current therapies. Prenat Diagn doi: 10.1002/pd.70142 PMID: 41936060
Objective: To examine current evidence and recent advances for treatment of sickle cell disease (SCD).
Summary: Biotinylated anti‐c‐kit/CD117 mAb coupled to a streptavidin‐conjugated saporin has been used to selectively deplete host HSC while preserving the host’s immune system. A single intravenous dose of the anti‐CD45‐saporin ADC enabled > 90% donor (congenic) hematopoietic engraftment and full correction of the SCD phenotype.
Serambeque B, Dias I, Mestre C, Marto CM, Botelho MF, Carvalho MJ, Laranjo M (2026) Photodynamic therapy-based strategies targeted at cancer stem cells: A scoping review. Cancers (Basel) 18(7):1162. doi: 10.3390/cancers18071162 PMID: 41976384
Objective: To examine strategies for targeting cancer stem cells using photodynamic therapy.
Summary: Photochemical internalization (PCI) is a widely adopted approach for targeting surface markers on cancer stem cells. Anti-CD133-SAP was evaluated in colorectal cancer, breast cancer, and melanoma. In CD133high colorectal cancer cells (WiDr), PCI with picomolar concentrations of AC133–saporin completely inhibited viability and colony formation, with no toxicity observed in the absence of light activation. Similar efficacy was observed in CD133+ breast (MDa-MB-231) and CD133high melanoma cells (FMEX-1) but not in CD133− breast cancer cells (MCF7), confirming target specificity. A similar strategy was employed to target CD44 in human cancer cell lines.
Xiang H, Mortenson GP, Lang SB, Jakkaraju S, Chirala A, Zhu Y, Jia M, Wen J, Chen Y, Baghela A, Chen YC, Sze MA, Hegde LG, Zhang-Hoover J, Willingham A, Handa M, Chi A, Baltus GA, Kamath RV, Levesque M, Vollmann EH (2026) uPAR-targeting cytotoxic antibody–drug conjugates selectively deplete proinflammatory myeloid cells for autoimmune indications. Cells 15(9):803. doi: 10.3390/cells15090803
Objective: To explore uPAR as a cell-surface marker to target and eliminate proinflammatory monocytes and macrophages using antibody–drug conjugates (ADCs).
Summary: Using recent scRNA-seq findings from RA patients, the authors identified the urokinase plasminogen activator receptor (uPAR), encoded by PLAUR, as a highly expressed cell surface marker on an inflammation-associated IL1B+ proinflammatory myeloid subset. an anti-uPAR mAb conjugated with monomethyl auristatin F (MMAF) was employed to demonstrate selective depletion of uPARhigh-expressing macrophages in a myeloid-rich rodent model.
Usage: Fab-ZAP rat (IT-55) was used to test the internalization capacity of anti-mouse uPAR mAbs. Saporin and Fab-IgG-ZAP were used as control.
Hamakubo S, Komatsu N, Kosai A, Kuroda M, Sawada M, Shimizu R, Ohashi R, Suenaga H, Hamakubo T, Abe T (2026) Enhanced antitumor efficacy of a combination of immunotoxin and photosensitizer under illumination in xenograft mice. Biomedicines 14:573. doi: 10.3390/biomedicines14030573
Objective: To investigate the in vivo therapeutic efficacy of intelligent Targeted Antibody Phototherapy (iTAP), utilizing light as a spatiotemporal trigger to promote the cytoplasmic release of toxins.
Summary: The authors investigated the in vivo therapeutic efficacy of iTAP using an EGFR-targeted IT composed of cetuximab conjugated to saporin (IT-Cmab), administered in combination with the clinically used photodynamic therapy (PDT) photosensitizer NPe6, in a xenograft mouse model. Findings indicate that iTAP represents a promising therapeutic strategy for HNSCC.
Usage: Biotinylated Cmab was mixed with streptavidin-saporin (IT-Cmab). Mice received intraperitoneal IT-Cmab (0.5 mg/kg), followed 72 h later by intravenous NPe6 (5 mg/kg).
Tendler S, De Gregorio R, Balderes P, Michel AL, Hoang TT, Bauer D, Tully KM, Korsen JA, Lorenz IC, Khan AG, Carter L, Vergnolle O, Lebedeva IV, Nyakatura EK, Bodei L, Morris MJ, Poirier JT, Rudin CM, Lewis JS (2026) Next-generation anti-DLL3 radiopharmaceuticals targeting high-grade neuroendocrine lung and prostate cancers. Proc Natl Acad Sci U S A 123(6):e2505785123. doi: 10.1073/pnas.2505785123 PMID: 41628333
Objective: To report on the development of anti-DLL3 radioimmunoconjugates for use as either a diagnostic imaging tracer or a therapeutic agent. Delta-like ligand 3 (DLL3) is a tumor-selective cell surface protein upregulated in high-grade neuroendocrine tumors, including small-cell lung cancer (SCLC) and neuroendocrine prostate cancer (NEPC).
Summary: The authors generated a panel of human monoclonal antibodies targeting human DLL3 by immunizing transgenic mice engineered with a human immunoglobulin repertoire. Six select candidates were reformatted as human IgG4 anti-DLL3 mAbs.
Usage: NCI-H82 cells were seeded in a 96-well plate and incubated with serial dilutions of tested antibodies, premixed with the Fab–ZAP (IT-48) for 72 h.
ZAP conjugates allow you to screen targeting agents in a quick and cost-efficient way, looking for specificity, functional binding, internalization, and EC50 determination.
Here we have a closer look at the typical in vitro data you can expect to see from a cytotoxicity assay. In this example, you are looking at the cytotoxicity curves using various secondary conjugates when compared to the direct conjugate, 192-IgG-SAP.
Mab-ZAP, our bivalent secondary antibody that recognizes whole IgG, reacted with primary antibody, produces a similar potency to the directly linked conjugate of saporin to the same antibody.
Fab-ZAP and FabFc-ZAP, which both use a monovalent secondary antibody, reacted with primary antibody produced a cytotoxic effect greater than 12-fold over the direct conjugate.
In this example, you will also see an interesting phenomenon with ZAP secondary conjugates. It may be intuitive to think that using a higher dose of primary antibody induces a higher amount of cell death, but at the highest concentration of 192-IgG (10 nM = Log (-8)) there is a lessened amount of killing, at a 25-fold lower concentration when compared to the antibody. The explanation is that at the higher concentrations of primary antibody, there are more unconjugated 192-IgG and fewer 192-IgG plus Fab-ZAP complexes. So, this free 192-IgG can then out-compete the conjugates for cell surface binding sites, which, in turn, decreases the amount of saporin being internalized, hence less cell death.
Our publication in the Journal of Toxins, provides a nice review of this phenomenon.
We are continuing to highlight our ZAP antibody internalization kits and our line of secondary antibody saporin conjugates.
ZAP conjugates allow you to screen targeting agents in a quick and cost-efficient way, looking for specificity, functional binding, internalization, and EC50 determination.
We also highlighted a publication that did a great job in showcasing how they can be used to screen antibodies for the best candidate, and then proceed with having us perform a custom conjugation of that antibody directly to saporin.
Here is information about the chemistry and why we offer conjugates that use whole, bivalent, IgG as well as monovalent Fab IgG.
Our first release of ZAP conjugates utilized whole-molecule IgG, bivalent secondary antibodies that recognized both heavy and light chains of primary antibodies. In theory though, this presented a possible limitation if the bivalent nature of an antibody suggested that cross-linking could occur on the cell surface and contribute to a phenomenon known as ‘cap formation’. The ‘cap’ could potentially induce some level of endocytosis that would lead to cytotoxicity and produce a false positive for internalization of a primary antibody.
We designed new secondary conjugates (given the moniker Fab-ZAP) which used monovalent antibodies that would continue to recognize whole IgG, but lacked components that would contribute to capping.
We now offer 3 main categories: (1) Conjugates made with bivalent antibodies that recognize whole IgG, (2) conjugates made with monovalent antibodies that recognize whole IgG, and (3) conjugates made with monovalent antibodies that recognize only the Fc region of IgG.
Objective: To develop and validate a modular streptavidin based antibody drug conjugate platform for optimizing antibody mediated hematopoietic stem cell transplant conditioning.
Summary: The authors demonstrate that a streptavidin drug conjugate system enables rapid comparison of antibody payload combinations for hematopoietic stem cell depletion and leukemia targeting. The study builds on prior work using CD45 targeted immunotoxins to achieve effective hematopoietic niche depletion in murine HSCT models.
Usage: This study references earlier conditioning experiments that utilized Streptavidin-ZAP (IT-27) in combination with biotinylated CD45 antibodies to selectively deplete hematopoietic stem cells in murine HSCT models, typically administered as a single intravenous dose of approximately 75 µg per mouse.
