zap-conjugates

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)

Q: Does Mab-ZAP (Cat. #IT-04) bind to the FC portion of mouse IgG?

A: The antibody used to create our Mab-ZAP (IT-04), will react with whole molecule mouse IgG, which includes the Fc portion and the two antigen binding Fab portions.

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Q: Can your FabFc-ZAP human (Cat# IT-65) bind to the Fc portion of another species, such as mouse IgG? It looks like it binds to mouse IgG in our assay.

A: The antibody used to create our FabFc-ZAP Human (IT-65), can react with the Fc (gamma) portion of human IgG heavy chain and should not react with the Fab portion of human IgG. However, there could be minimal cross-reaction with mouse, horse, or bovine serum proteins, and it is possible to see cross-reaction with immunoglobulins from other species. 

Related Products: ZAP Conjugates

Kim S, Shukla RK, Kim E, Cressman SG, Yu H, Baek A, Choi H, Kim A, Sharma A, Wang Z, Huang CA, Reneau JC, Boyaka PN, Liyanage NPM, Kim S (2022) Comparison of CD3e antibody and CD3e-sZAP immunotoxin treatment in mice identifies szap as the main driver of vascular leakage. Biomedicines 10(6):1221. doi: 10.3390/biomedicines10061221

Objective: Investigate and identify the toxicity profiles of a CD3e-mAb and an immunotoxin of this CD3e antibody conjugated to saporin via a biotin-streptavidin bond, S-CD3e-IT.

Summary: The two agents had opposite effects on T cells, with the antibody alone able to modulate CD3e on the cell surface while the S-CD3e-IT caused depletion of the cell. The immunotoxin increased the infiltration of polymorphonuclear leukocytes (PMNs) into the tissue parenchyma of the spleen and lungs, a sign of vascular permeability while the antibody alone showed no signs of vascular leakage.

Usage: S-CD3e-IT was prepared by reacting biotinylated CD3e antibody with Streptavidin-ZAP in a 1:1 molar ratio. C57BL/6J mice received 25 μg of S-CD3e-IT in sterile 200 μL PBS, twice a day via retro-orbital injection for four days.

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

Q: Is the dosage of Fab-ZAP always enough for any level of antibody concentration?

A: The 4.5 nM dosage of Fab-ZAP is the recommended concentration.  We do not typically see unspecific killing at 4.5 nM on most cell lines.  If the concentration of Fab-ZAP is increased, it may undergo bulk-phase endocytosis and kill cells indiscriminately.  A lower concentration of Fab-ZAP may lead to antibody competition, resulting in a lack of killing of cells at the highest concentration of antibody.

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Q: Is there a recommended ratio between Fab-ZAP dosage and antibody concentration?

A: A recommended good starting point is 4.5 nM of Fab-ZAP, with a titration of the antibody starting at a concentration of 10 nM.

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Q: Each concentration is suggested to perform 6 replications, can it be adjusted more or less in practice?

A: Yes, the assay design is meant to be a thorough approach but can be adjusted by the user. We recommend 6 replications based on our 96-well plate template design. The concentration of Fab-ZAP is 4.5 nM in the suggested protocols.

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