zap-conjugates

Cua S, Tan HL, Fong WJ, Chin A, Lau A, Ding V, Song Z, Yang Y, Choo A (2018) Targeting of embryonic annexin A2 expressed on ovarian and breast cancer by the novel monoclonal antibody 2448. Oncotarget 9:13206-13221. doi: 10.18632/oncotarget.24152

Objective: To develop mAbs to potentially target oncofetal antigens and be repurposed for antibody or antibody drug conjugate (ADC) therapy.

Summary: The novel IgG1, 2448, was shown to target a unique glycosylated surface epitope on ANXA2. As a possible therapeutic candidate for ovarian and breast cancer, 2448 demonstrated anti-tumor activity via two independent mechanisms of action.

Usage: Cells were seeded in 96-well plates at 1000 or 2000 cells/well. Primary antibody, 2448 or ch2448 (10 μg/mL) was pre-mixed with appropriate secondary saporin conjugate, Mab-ZAP or Hum-ZAP.  The most significant decreases in cell viability (20% to 60%) were observed against the epithelial IGROV1 and MCF7 cell lines.  ATS created a Custom ADC by direct conjugation of saporin to ch2448 (ch2448-SAP).  As a control, an isotype chimeric IgG was also conjugated to saporin (IgG-SAP). Compared to using secondary saporin conjugates, ch2448-SAP induced and increase of  20–30% cytotoxicity.)

Related Products: Mab-ZAP (Cat. #IT-04), Hum-ZAP (Cat. #IT-22), Custom Conjugates

Su Y, Bidlingmaier S, Lee NK, Liu B (2018) Combine phage antibody display library selection on patient tissue specimens with laser capture microdissection to identify novel human antibodies targeting clinically relevant tumor antigens. (eds. Hust M, Lim T). In: Phage Display. Methods in Molecular Biology. 1701:331-347. Humana Press, New York, NY. doi: 10.1007/978-1-4939-7447-4_18

Objective: To develop a technology that allows selection of phage antibody display libraries on tumor cells in situ residing in their natural tissue microenvironment.

Summary: Intracellular delivery of Immunotoxin was determined as follows: Immunotoxin was prepared by mixing biotinylated scFv with Streptavidin-ZAP (Cat. #IT-27) at a molar ratio of 1:1 and incubated on ice for 30 min. 50 μl of serially diluted immunotoxin was added to each well and incubated for 96 h at 37°C in 5% CO2. Cell growth medium were carefully removed from each well.

Usage: 100 μl of diluted CCK-8 was added to each well in the 96-well plates and incubated for 1–4 h at 37°C in 5% CO2. The absorbance was measured at 450 nm using a microtiter plate reader and the EC50 value determined using GraphPad Prism.

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

Komatsu N, Mitsui K, Kusano-Arai O, Iwanari H, Hoshi K, Takato T, Abe T, Hamakubo T (2017) Enhancement of anti-Robo1 immunotoxin cytotoxicity to head and neck squamous cell carcinoma via photochemical internalization. Arch Can Res 5:157-163. doi: 10.21767/2254-6081.100157

Objective: To screen a monoclonal antibody to Robo1, an axon guidance receptor, for its suitability to target various cancers.

Summary: Conventional treatment exhibited an inadequate cytotoxic effect. With the addition of a photosensitizer and LED light illumination, the cytotoxic effect was remarkably improved.

Usage: Saporin-conjugated anti-Robo1 and Saporin-conjugated negative control antibody were prepared by incubating 2 mcl of 1.1 micromolar Streptavidin-ZAP (Biotin Z Internalization Kit) and 2 mcl of 1.1 micromolar biotinylated antibody for 30 min at room temperature.

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

Schiroli G, Ferrari S, Conway A, Jacob A, Capo V, Albano L, Plati T, Castiello M, Sanvito F, Gennery A, Bovolenta C, Palchaudhuri R, Scadden D, Holmes M, Villa A, Sitia G, Lombardo A, Genovese P, Naldini L (2017) Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1. Sci Transl Med 9(411):eaan0820. doi: 10.1126/scitranslmed.aan0820

Objective: To study potential approaches to gene therapy in mouse models of severe combined immunodeficiency.

Summary: The threshold of IL2RG gene editing can be reached for safe and efficient correction of SCID-X1 established in a preclinical model in human long-term repopulating HSPCs.

Usage: Biotinylated Anti-CD45 was mixed equimolar to Streptavidin-ZAP and administered as a single dose which caused substantial depletion (~70%) of the HSPC compartments and milder depletion of the more mature cell populations.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Anti-CD45.2-SAP (Cat. #IT-91)

Therapeutic Potential of Hematopoietic Stem Cell Gene Editing

Lowe D, Bivens C, Mobley A, Herrera C, McCormick A, Wichner T, Sabnani M, Wood L, Weidanz J (2017) TCR-like antibody drug conjugates mediate killing of tumor cells with low peptide/HLA targets. MAbs 9:603-614. doi: 10.1080/19420862.2017.1302630

Objective: To analyze the killing potential of TCR-like ADCs.

Summary: Data comprise proof-of-principle results that TCR-like ADCs mediate potent tumor cytotoxicity and support their continued development alongside agents that disrupt DNA replication.  Additionally, TCR-like antibody ligand binding appears to play an important role in ADC functionality and should be addressed during therapy development to avoid binding patterns that negate ADC killing efficacy.

Usage: TCR-like antibodies were indirectly bound to Saporin using Mab-ZAP.  Tumor cells (5×103) were plated in flat-bottom 96-well plates, then Mab-ZAP (100 ng) was added. Various dilutions of isotype control, BB7.2, TCR-like, and 4D5 antibodies were subsequently added to a final volume of 120 mcl and plates were incubated for 3– 5 d at 37 °C with 5% CO2.

Related Products: Mab-ZAP (Cat. #IT-04)

Huang L, Yuan T, Tan M, Xi Y, Hu Y, Tao Q, Zhao Z, Zheng J, Han Y, Xu F, Luo M, Sollars P, Pu M, Pickard G, So K, Ren C (2017) A retinoraphe projection regulates serotonergic activity and looming-evoked defensive behaviour. Nat Commun 8:14908. doi: 10.1038/ncomms14908 PMID: 28361990

Objective: To investigate how the dorsal raphe nucleus (DRN) and superior colliculus work in concert to extract and translate visual threats into defensive behavioural responses.

Summary: A dedicated population of RGCs signals rapidly approaching visual threats and their input to the DRN controls a serotonergic self-gating mechanism that regulates innate defensive responses.

Usage: Mice received bilateral intraocular injection (2 μg per eye) made between Streptavidin-Saporin and a biotinylated CTB antibody, or Anti-Melanopsin-SAP (2 μg per eye). For detection of melanopsin, retinas were incubated for 3 days at 4 °C with anti-melanopsin (1:600).

Related Products: Streptavidin-ZAP (Cat. #IT-27), Melanopsin-SAP (Cat. #IT-44), Melanopsin Rabbit Polyclonal (Cat. #AB-N38)

Ha K, Bidlingmaier S, Su Y, Lee N, Liu B (2017) Identification of novel macropinocytosing human antibodies by phage display and high-content analysis. Methods Enzymol 585:91-110. doi: 10.1016/bs.mie.2016.10.004

Objective: To describe a method for identifying antibodies that internalize via macropinocytosis by screening phage-displayed single-chain antibody selection outputs with an automated fluorescent microscopy-based high-content analysis platform.

Summary: Novel phage antibodies are identified by colocalization with macropinocytosis marker, converted into full-length human antibodies, and further characterized with regard to cell binding, pathway of internalization, and intracellular payload delivery.

Usage: Biotinylated IgG is mixed with Streptavidin-ZAP in 1:1 molar ratio to form an immunotoxin that is serially-diluted in a cytotoxicity assay.

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

Nohara S, Kato K, Fujiwara D, Sakuragi N, Yanagihara K, Iwanuma Y, Kajiyama Y (2016) Aminopeptidase N (APN/CD13) as a target molecule for scirrhous gastric cancer. Clin Res Hepatol Gastroenterol 40:494-503. doi: 10.1016/j.clinre.2015.11.003

Summary: Scirrhous gastric cancer has the worst prognosis of gastric carcinoma, and treatment with standard cancer therapies has had minimal success. In this work the authors target CD13 as a marker for scirrhous gastric cancer. A gastric cancer cell line was challenged with a CD13 antibody coupled to Mab-ZAP (Cat. #IT-04) in an in vitro cytotoxicity assay. The anti-CD13 complex was more cytotoxic than an anti-EpCAM-immmunotoxin. These data, combined with flow cytometry analysis and enzyme activity assays, demonstrate the expression of CD13 as a marker for scirrhous gastric cancer.

Related Products: Mab-ZAP (Cat. #IT-04)

Hay C, Sult E, Huang Q, Mulgrew K, Fuhrmann S, McGlinchey K, Hammond S, Rothstein R, Rios-Doria J, Poon E, Holoweckyj N, Durham N, Leow C, Diedrich G, Damschroder M, Herbst R, Hollingsworth R, Sachsenmeier K (2016) Targeting CD73 in the tumor microenvironment with MEDI9447. Oncoimmunology 5:e1208875. doi: 10.1080/2162402X.2016.1208875

Summary: MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here the authors show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences. MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. In vitro experiments validating the internalization of antibodies into cell lines MDA-MB-231 and 4T1 were measured using the Fab-ZAP human antibody internalization kit (Cat. #KIT-51-Z). Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosine-mediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).

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

Q: I’ve been looking at your secondary conjugates and want to see if my targeting agent is specific to certain cells. Which secondary conjugate should I use?

A: It depends on two factors: 1) the type of assay you want to use, and 2) the kind of targeting agent you want to use.

For in vitro assays, in particular, internalization assays, you can use any of the ZAP Antibody Internalization Kits (Z-Kits that include all the materials necessary to test your targeting agent).

For the Antibody Internalization Kits, you use your primary antibody and select the appropriate secondary antibody species, depending on the isotype of your primary antibody — e.g. for a human antibody, use Hum-ZAP (Cat. #KIT-22-Z), Fab-ZAP human (Cat. #KIT-51-Z), FabFc-Human (Cat. #KIT-65-Z), Hug-M-ZAP (Cat. #KIT-43-Z), or Fab-ZAP Hug-M (Cat. #KIT-78-Z).

Or you can biotinylate your antibody and use Biotin-Z Antibody Internalization Kit (Cat. #KIT-27-Z).

For in vivo applications, it depends on the kind of targeting agent you want to use. Regardless of whether you use an antibody, peptide or ligand, you will need to biotinylate the material first. ATS offers a biotinylation service that is efficient and economical.

If you are using a biotinylated peptide, you will use a kit that includes the appropriate control — Streptavidin-ZAP Peptide Kit (Cat. #KIT-27-B).

If you are using a biotinylated antibody, you will use the Streptavidin-ZAP Antibody Kit that includes the appropriate antibody species control: BIgG-SAP Human (Cat. #KIT-27-Ahu), BIgG-SAP Mouse (Cat. #KIT-27-Amu), BIgG-SAP Rabbit (Cat. #KIT-27-Arb), BIgG-SAP Rat (Cat. #KIT-27-Art). Select the kit that matches the species of your biotinylated primary antibody.

Related: ZAP Secondary Conjugates