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 are finishing off our topic of ZAP conjugates with 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.
Interestingly, in this example 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 as seen in the example, 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.
Diamantoudis SC, Miliotou AN, Galatou E, Telliou S, Sideris K, Grigoriadis N, Vizirianakis IS (2025) Assessing the hematological cancer stem cell landscape to improve immunotherapy clinical decisions. Biocell doi: 10.32604/biocell.2025.067216
Objective: To combine existing information and clinical evidence to assess and bring to the spotlight targets related to Hematological cancer stem cells (HCSCs) that can be considered for the improvement of therapeutic interventions.
Summary: Targeting HCSCs represents one of the most promising advances toward achieving lasting remission and potential cure in hematologic malignancies. Next-generation immunotherapies—enabled by advances in molecular profiling, synthetic biology, and systems immunology—can shift the paradigm in blood cancers by overcoming current limitations.
Usage: CD117-ADC (carrying streptavidin–saporin) has shown dose-dependent results in mice, with a range from 0.3–1.5 mg/kg, as depletion of stem cells was noted with the subsequent successful engraftment of allogenic transplants.
I am using your Fab-ZAP Human (IT-51) and I want to know how many antibody molecules can a secondary antibody conjugate theoretically bind to?
Answer
A whole IgG (bivalent) secondary antibody in theory is capable of binding two antigen molecules. Since our Fab-ZAP conjugates actually use a monovalent secondary antibody, in theory they would be able to each bind one antigen molecule. Our Fab-ZAP human conjugates use polyclonal monovalent secondary antibodies raised against both heavy and light chain of IgG and can cross-react across immunoglobulin classes and subclasses of the same species since they share the same light chain. In theory it would be possible that your primary antibody could have several secondary conjugates attached.
Question
When exploring the usage concentration for Fab-ZAP Human, what is the recommended concentration range (for example, is it a few times the initial concentration of the analyte antibody / the highest test concentration)?
Answer
Our standard protocol (in vitro only) for our Fab-ZAP secondary conjugates is to maintain a constant concentration of 4.5 nM of the Fab-ZAP while titrating the primary antibody, usually in a range starting at 10 nM with 1:5 to 1:10 serial dilutions. The goal would be to have the primary antibody as a ‘variable’ and the Fab-ZAP as the ‘constant’. Also, it’s important to saturate the primary antibody with Fab-ZAP secondary conjugate, as any ‘free unreacted primary antibody’ would compete with ‘antibody+Fab-ZAP conjugate’ for binding sites on the cell. I have attached a publication that discusses this topic.
Kolahdouzan M, Ghazisaeidi S, Tu Y, Muley M, Gambeta E, Salter M (2025) Meningeal macrophages mask incision pain sensitization in male rats. Mol Pain doi: 10.1177/17448069251383593
Objective: To investigate whether CD206+macrophages in the meninges play a role in regulating nociception and pain hypersensitivity.
Summary: The results indicate that while CD206+ meningeal macrophages do not regulate basal nociception in naïve rats, they mask mechanical hypersensitivity in male rats after skin incision injury. Thus, we conclude that in a sex-dependent manner, CD206+ meningeal macrophages prevent the spread of pain hypersensitivity after a minor injury.
Usage: Rats were injected intrathecally (30 μl) with saline, CD206-Saporin (20 μg mannose-receptor antibody and 7 μg of Streptavidin-ZAP in 30 μl), or Rabbit-IgG-Saporin (control).
Kofoed C, Erkalo G, Tay NES, Ye X, Lin Y, Muir TW (2025) Programmable protein ligation on cell surfaces. Nature 10.1038/s41586-025-09287-2. doi: 10.1038/s41586-025-09287-2 PMID: 40739351
Objective: To describe an autonomous decision-making device driven by proximity-gated protein trans-splicing that allows local generation of an active protein from two otherwise inactive polypeptide fragments
Summary: Authors showed that this protein-actuator platform can perform convergent protein ligation on designated cell surfaces, allowing highly selective generation of active proteins, which can either remain physically associated with the cell surface on which they were manufactured or be released into the surrounding milieu.
Usage: Flow cytometry: Mixed K562 cells (phenotypes indicated) were treated with a two-dose regimen of SMART-SpyCatcher/SpyTag003-biotin ([HER2 AND EGFR] logic,100 nM each) and Streptavidin–ZAP (20 nM) at a 24-h interval. Cell viability was assessed after 72 h by flow cytometry and normalized to untreated wild-type cells.
Ren X, Wang Y, Zhang Y (2025) Targeted depletion of dysfunctional hematopoietic stem cells mitigates myeloid-biased differentiation in aged mice. Cell Discov 11:56. doi: 10.1038/s41421-025-00810-3 PMID: 40490480
Objective: To develop and evaluate a targeted strategy for depleting dysfunctional, myeloid-biased CD150-high hematopoietic stem cells (HSCs) in aged mice to restore balanced hematopoiesis and mitigate aging-related blood disorders.
Summary: The study used an antibody-toxin conjugate to selectively eliminate CD150-high HSCs, improving lymphoid-to-myeloid ratios, reducing platelet hyperproduction, and restoring hematopoietic balance in aged mice. Treatment preserved functional CD150-low HSCs and showed minimal off-target or systemic toxicity.
Usage: Streptavidin-ZAP (IT-27) was combined with a biotinylated anti-CD150 antibody to generate Anti-CD150-SAP (IT-103). This conjugate was used at doses of 1–2 mg/kg in vivo and as low as 0.01 nM in vitro to specifically deplete CD150-high HSCs while sparing CD150-low populations.
Movahed AY, Bagheri R, Savatier P, Šarić T, Moradi S (2025) Elimination of tumorigenic pluripotent stem cells from their differentiated cell therapy products: An important step toward ensuring safe cell therapy. Stem Cell Reports 102543. doi: 10.1016/j.stemcr.2025.102543 PMID: 40541178
Objective: To review and evaluate current strategies for eliminating tumorigenic pluripotent stem cells (PSCs) from differentiated cell therapy products to improve the safety of PSC-based regenerative therapies.
Summary: Residual undifferentiated PSCs pose a tumorigenic risk in cell therapies. This review outlines genetic, antibody, toxin, and small molecule strategies for selectively removing PSCs, emphasizing the need for efficient, selective methods to ensure safety in regenerative medicine.
Usage: References a previous study that used Fab-ZAP to eliminate pluripotent stem cells by targeting specific surface markers, demonstrating its application as a targeted immunotoxin for PSC depletion.
Konturek-Ciesla A, Zhang Q, Kharazi S, Bryder D (2025) A non-genotoxic stem cell therapy boosts lymphopoiesis and averts age-related blood diseases in mice. Nat Commun 16(1):5129. doi: 10.1038/s41467-025-60464-3 PMID: 40456713
Objective: Application of Hematopoietic stem cell (HSC) transplantation leads to treatment toxicity. Therefore, authors employed a murine transplantation model based on low-intensity conditioning protocols using antibody-mediated HSC depletion to improve hematopoietic output and ameliorate age-compromised lymphopoiesis.
Summary: Authors demonstrate that young HSCs, once effectively engrafted in aged hosts, improve hematopoietic output and ameliorate age-compromised lymphopoiesis. This culminated in a strategy that robustly mitigates disease progression in a genetic model of myelodysplastic syndrome. These results suggest that non-genotoxic HSC transplantation could fundamentally change the clinical management of age-associated hematological disorders, offering a prophylactic tool to delay or even prevent their onset in elderly patients.
Usage: CD45-SAP (3 mg/kg) was administered to young (2 months) and aged (16 months) C57BL/6-CD45.2 mice
Park HB, Kim KH, Kim JH, Kim SI, Oh YM, Kang M, Lee S, Hwang S, Lee H, Lee T, Park S, Lee JE, Jeong GR, Lee DH, Youn H, Choi EY, Son WC, Chung SJ, Chung J, Choi K (2024) Improved safety of chimeric antigen receptor T cells indirectly targeting antigens via switchable adapters. Nat Commun 15(1):9917. doi: 10.1038/s41467-024-53996-7 PMID: 39557825
Objective: To show that switchable CAR-T cells with a tumor targeting adaptor can mitigate on-target off-tumor toxicity against a low selectivity tumor antigen that cannot be targeted by conventional CAR-T cells, such as CD40.
Summary: The system is composed of anti-cotinine murine CAR-T cells and cotinine-labeled anti-CD40 single chain variable fragments (scFv), with which the authors show selective tumor killing while sparing CD40-expressing normal cells including macrophages in a mouse model of lymphoma. The authors evaluated whether Cot CAR-T cells could be depleted by Cot-saporin in vivo in an allogeneic CAR-T cell transfer model. When Balb/C mice transplanted with B6 bone marrow cells were injected with B6 Cot CAR-T cells, the transferred Cot CAR-T cells expanded in the peripheral blood in response to Balb/C alloantigen. However, when Cot-saporin was administered during this expansion phase, the Cot CAR-T cells failed to expand and were subsequently eliminated in the blood. Thus, Cot-saporin-mediated CotCAR-T cell suicide was confirmed in vitro and in vivo.
Usage: in vitro Cot CAR-T cell depletion by cotinine-drug conjugates: Peptides were incubated with saporin-labeled streptavidin (IT-27) at a molar ratio of 4:1 to generate cotinine-saporin conjugate (Cot-saporin). For Cot-saporin-dependent cytotoxicity assays on Cot CAR-T cells, a 1:1 mixed population (50,000 cells each) of Cot CAR-T cells (target cells) and control T cells (bystander non-CAR-T cells) were incubated with various doses of Cot-saporin for 48 h in medium containing human IL-2. Seven days after CAR-T cell transfer, Cot-saporin was administered intraperitoneally three times at 3-day intervals.
Jiang H, Cui H, Chen M, Li F, Shen X, Guo CJ, Hoekel GE, Zhu Y, Han L, Wu K, Holtzman MJ, Liu Q (2024) Divergent sensory pathways of sneezing and coughing. Cell 187(21):5981-5997. doi: 10.1016/j.cell.2024.08.009 PMID: 39243765
Objective: To study the difference in sensory receptors and neurotransmission/modulation mechanisms between sneezing and coughing.
Summary: Sneezing and coughing are frequently associated with allergies and respiratory viral infections and it’s assumed both involve common sensory receptors and neurotransmission mechanisms. The author’s work show that the nasal mucosa is innervated by several discrete populations of sensory neurons, but only one population (MrgprC11+MrgprA3−) mediates sneezing. Although this same population innervates the trachea, it does not mediate coughing, and instead, a distinct sensory population (somatostatin SST) mediates coughing but not sneezing. NMB-SAP was used to ablate neruomedin B (NMB) receptor expressing and nucleus tractus solitarius (NTS) neurons. Deletion of these neurons did not affect the coughing responses to Ly344864 and IL-31 (agonists to SST neurons) suggesting that NMB-sensitive NTS neurons do not mediate coughing.
Usage: Neuronal ablation by SST-saporin and NMB-saporin. SST-saporin was made by mixing biotin-labeled somatostatin and Streptavidin-ZAP (IT-27) at a 1:1 molar ratio at room temperature for 20 minutes. SST-Saporin (10 μM, 50 nL), NMB-saporin (#IT-70; 50 ng in 50 nL) or Blank-SAP (#IT-21; 10 μM in 50 nL or 50 ng in 50 nL) was injected into the NTS region.
