Park JS, Kenum C, He L, Khan AG, Pohl MA, White TE, Kodangattil SR, Rudin CM, Balderes PJ, Lorenz IC, Massagué J, anesh K (2025) Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer. bioRxiv 2025.10.01.679843. doi: 10.1101/2025.10.01.679843 PMID: 41256399
Objective: To develop L1CAM-targeted antibody-drug conjugates (ADCs) using high-affinity monoclonal antibodies and assess their therapeutic potential against metastatic cancer.
Summary: L1CAM ADCs conjugated with the potent payload PNU-159682 achieved strong cytotoxicity, tumor regression, and prolonged survival in models of metastatic breast and lung cancer. Safety studies indicated a favorable therapeutic window for clinical translation.
Usage: FabFc-ZAP Human (IT-65) was used to evaluate antibody internalization. FabFc-ZAP was incubated with L1CAM candidate antibodies or isotype control and applied to L1CAM-HEK293T cells, with viability measured after five days.
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 49(10):1799-1858. 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 21. doi: 10.1177/17448069251383593 PMID: 40958149
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
Nakano T, Okita K, Okazaki S, Yoshimoto S, Masuko S, Yagi H, Kato K, Tomioka Y, Imai K, Hamada Y, Masuko K, Shimada-Takaura K, Nagai N, Saya H, Arai T, Ishiwata T, Masuko T (2025) CD44v, S1PR1, HER3, MET and cancer-associated amino acid transporters are promising targets for the pancreatic cancers characterized using mAb. FEBS Open Bio 15(5):867-884. doi: 10.1002/2211-5463.13963 PMID: 39757718
Objective: To analyze pancreatic ductal adenocarcinomas (PDAC) using novel rat mAbs against membrane proteins in conjunction with flow cytometry and immunohistochemistry
Summary: Internalization of membrane proteins by mAbs and growth inhibition by toxin-linked mAbs were demonstrated in many PDAC cell lines, and mAbs against S1PR1, ASCT2, HER3 and CD44v inhibited the growth of xenografted MIA PaCa-2PDAC cells. Furthermore, CD44v-high PDAC showed high mRNA expression of HER1–3, MET and CD44v, and was correlated with poor prognosis. Taken together, the results suggest that CD44v, S1PR1, HER3, MET and the above-mentioned cancer-associated amino acid transporters might be promising targets for the diagnosis and treatment of PDAC.
Usage: Growth inhibition by rat mAbs against PDAC cell lines with secondary antibodies conjugated to saporin (PR-01). Rat mAb solution (4 ug/mL), a PDAC cell suspension and Saporin-conjugated goat anti-rat IgG pAb (Rat-ZAP, IT-26 at 4 ug/mL) were added to each well of 96-well plates.
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.
