ZAP Conjugate References


Marquez J, Dong J, Dong C, Tian C, Serrero G (2021) Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development. PLoS One 16(1):e0246197. doi: 10.1371/journal.pone.0246197

Summary: 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 show that it is internalized by incubation with 33B7. Purified 33B7 antibody was sent to Advanced Targeting Systems where saporin was directly conjugated to the Fc region of 33B7 using their proprietary cleavable linker. Dose: In a 96-well plate, 2000 cells/well were plated in triplicate in 100μL of DMEM/F12 medium supplemented with 2.5% FBS, 0.4 ug/ml 33B7 antibody, and 0.9ug/ml of Fab-ZAP mouse. As an isotype control, cells were incubated with mouse Fab IgG-SAP as control (instead of 33B7) and Fab-ZAP.


Gick GG, Arora K, Sequeira JM, Nakayama Y, Lai SC, Quadros EV (2020) Cellular uptake of vitamin B12: Role and fate of TCblR/CD320, the transcobalamin receptor. Exp Cell Res 396 (1):112256 396(1):112256. doi: 10.1016/j.yexcr.2020.112256

Summary: The increased and sustained expression of TCblR in proliferating cells has been used to target toxins preferentially to cancer cells and can be potentially used for targeted delivery of other anti-cancer drugs. In 2010 the authors published a paper which evaluated the potential of using immunotoxins to eliminate cancer cells expressing TCb1R the authors performed a series of in vitro experiments using their monoclonal antibody plus Mab-ZAP in varying concentrations. The results indicated that this is a viable therapeutic model that causes minimal peripheral damage.

London M, Gallo E (2020) The EphA2 and cancer connection: potential for immune-based interventions. Mol Biol Rep 47(10):8037-8048. doi: 10.1007/s11033-020-05767-y

Summary: The authors review the most current mAb-based therapies against EphA2 expressing cancers currently in pre-clinical and/or clinical stages. They reference Sakamoto et al. who performed in vitro testing of two different EPHA2 mAbs mixed with Mab-ZAP to discover their therapeutic potential against melanoma.


Cua S, Tan HL, Fong WJ, Chin A, Lau A, Ding V, Song Z, Yang Y, & Choo A. Targeting of Embryonic Annexin A2 Expressed on Ovarian and Breast Cancer by the Novel Monoclonal Antibody 2448. (2018). Oncotarget, 9 (17):13206-13221. 2018/03/24. 5862572

(IT-04: Mab-ZAP;  IT-22: Hum-ZAP; Custom Services)
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.
Dose: 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.  A Custom ADC was created 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.

Harvey BP, Cohen-Solal J, & Kaymakcalan Z. Adalimumab:TNF Complexes Are Cleared More Efficiently by Human Osteoclasts Than Those with Etanercept through Fcg-Receptor Binding and Internalisation. (2018).  Annals Rheumatic Diseases, 77 (Suppl 2):SAT0058  0893.

(IT-65: FabFc-ZAP human)
Objective:  To determine whether Fc-gamma receptor (FcgR)-mediated internalization of the biologic:TNF complexes is a contributing mechanism responsible for the difference in effectiveness between ADA and ETN in preventing TNF- enhanced OCgenesis.
Summary:  Human osteoclast  (OC) precursors can bind and internalise ADA:TNF complexes more efficiently than ETN:TNF complexes. In addition, this process is partially mediated through FcgRII.
Dose:  FcgR-mediated nternalization was assessed by monitoring a reduction in OC survival in response to preformed bio- logic: TNF complexes (25:1 ratio) bound with FabFC-ZAP human ± FcgR blocking antibodies.

Pore N, Borrok III MJ, Michelotti EF, Tice DA, Hollingsworth RE, Chang C-Y, & Chowdhury P. (2018). Binding molecules specific for ASCT2 and uses thereof.  USA Patent No. US20180273617A1: Assignee: Medimmune.

(IT-22: Hum-ZAP)
Objective:  A method of treating a cancer characterized by overexpression of ASCT2 in a subject, the method comprising administering to a subject in need of treatment an effective amount of the antibody or antigen binding fragment .
Summary:  Patent for versions of an antibody or antigen binding fragment, which specifically binds to an epitope of the neutral amino acid transporter 2 (ASCT2).
Dose:  To confirm the internalization of parental antibodies, and to predict whether they can deliver a cytotoxic payload, the parental antibodies were tested in the Hum-ZAP antibody internalization assay according to manufacturer’s instructions.

Sadhukhan R, Brown N, Ouellette D, Banach D, Filoti DI, Winarta D, Raghavendra R, Sousa S, Darcy A, Alessandri L, Ivanov A, Bose S, Eaton L, Preston G, Freeman J, & Correia I. Engineering Elastic Properties into an Anti-Tnfα Monoclonal Antibody. (2018). Cogent Biology, 4 (1):1469387. 

(IT-51: Fab-ZAP human)
Objective:  To engineer elastic properties into a TNFalpha antibody.
Summary:  The results presented in this report with an anti-TNFα ELP mAb are a foundation for building on a new generation of fusion ELP mAbs, or other formats, that are stable, active, responsive to cues in local environment, and, with the FcRn mutation, cleared rapidly from circulation. More detailed studies are warranted to identify the appropriate ELP sequences for IA delivery, calculate residence time in the IA space, and demonstrate pharmacodynamics effect of the ELP-fusion protein.
Dose:  Fab-ZAP human was mixed with anti-TNFα-ELP fusion monoclonal.

Sakamoto A, Kato K, Hasegawa T, & Ikeda S. An Agonistic Antibody to Epha2 Exhibits Antitumor Effects on Human Melanoma Cells. (2018). Anticancer Res, 38 (6):3273-3282. 2018/06/01.

(IT-04: Mab-ZAP)
Objective:  Investigate the therapeutic potential of antibody to EPHA2 against melanoma in vitro.
Summary:  Observations indicate a promising role for EPHA2 as a target in antibody treatments for melanoma, and demonstrate the potential therapeutic effects of an agonistic antibody to EPHA2.
Dose:  A375 cells were plated into a flat-bottom, 96-well plate (2,000 cells per well) and incubated for 4 days at 37˚C. Cell suspension included different concentrations of Mab-ZAP, along with either anti-EPHA2 mAb (SHM16, SHM17, or SHM20 at 2 μg/ml final concentration), or a control IgG1 mAb (2 μg/ml final concentration).

Tan HL, Yong C, Tan BZ, Fong WJ, Padmanabhan J, Chin A, Ding V, Lau A, Zheng L, Bi X, Yang Y, & Choo A. Conservation of Oncofetal Antigens on Human Embryonic Stem Cells Enables Discovery of Monoclonal Antibodies against Cancer. (2018). Scientific Reports, 8 (1):11608.

(IT-22: Hum-ZAP)
Objective:  To identify and characterize an antibody raised using human embryonic stem cells with potential as a cancer therapeutic.
Summary:  Antibody A19 not only binds to undifferentiated hESCs by flow cytometry, it also reacts with ovarian and breast cancer cell lines with low or no binding to normal cells.
DoseIn Vitro – Number of viable cells treated showed a decrease in cell number (Hum-ZAP mixed with A19; Streptavidin-ZAP mixed with biotinylated A19).  To determine if there were off-target effects, Hum-ZAP and chA19 were incubated with a non-binding cell line OVCAR10; no apparent cytotoxicity was observed.
In Vivo – 5 x 106 SKOV3 cells were implanted s.c. in NUDE mice and Biotinylated A19-Streptavidin-ZAP (ADC), administered ip.  The controls were free Saporin and naked A19.  By the end of 10 weeks, mice administered with the ADC saw a 60% reduction in tumor size compared to control groups.


Lowe DB, Bivens CK, Mobley AS, Herrera CE, McCormick AL, Wichner T, Sabnani MK, Wood LM, & Weidanz JA. TCR-Like Antibody Drug Conjugates Mediate Killing of Tumor Cells with Low Peptide/Hla Targets. (2017). mAbs, 9 (4):603-614.

(IT-04; Mab-ZAP)
:   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.
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.


Hay CM, Sult E, Huang Q, Mulgrew K, Fuhrmann SR, McGlinchey KA, Hammond SA, Rothstein R, Rios-Doria J, Poon E, Holoweckyj N, Durham NM, Leow CC, Diedrich G, Damschroder M, Herbst R, Hollingsworth RE, Sachsenmeier KF. (2016) Targeting Cd73 in the Tumor Microenvironment With Medi9447. Oncoimmunology 5(8):e1208875. PMID: 27622077 (Targeting Trends 17q1)

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(4):494-503. PMID: 26774363 (Targeting Trends 16q2)


Chua JX, Vankemmelbeke M, McIntosh RS, Clarke PA, Moss R, Parsons T, Spendlove I, Zaitoun AM, Madhusudan S, Durrant LG. (2015) Monoclonal Antibodies Targeting LecLex-Related Glycans with Potent Antitumor Activity. Clin Cancer Res 21(13):2963-2974. PMID: 25779947 (Targeting Trends 15q2)

Higgins SC, Fillmore HL, Ashkan K, Butt AM, Pilkington GJ. (2015) Dual targeting NG2 and GD3A using Mab-Zap immunotoxin results in reduced glioma cell viability in vitro. Anticancer Res 35(1):77-84. (Targeting Trends 15q2)

Pires ES, D’Souza RS, Needham MA, Herr AK, Jazaeri AA, Li H, Stoler MH, Anderson-Knapp KL, Thomas T, Mandal A, Gougeon A, Flickinger CJ, Bruns DE, Pollok BA, Herr JC. (2015) Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors. Oncotarget 6(30):30194-30211. PMID: 26327203 (Targeting Trends 15q4)


Leoni G, Rattray M, Fulton D, Rivera A, Butt AM. (2014) Immunoablation of cells expressing the NG2 chondroitin sulphate proteoglycan. J Anat 224(2):216-227. (Targeting Trends 14q1)


Daniotti JL. (2013) Antibodies to glycosphingolipids: An attractive tool for targeted delivery of cytotoxic agents to tumor cells. Targeting Trends 14(2).

Rust S, Guillard S, Sachsenmeier K, Hay C, Davidson M, Karlsson A, Karlsson R, Brand E, Lowne D, Elvin J, Flynn M, Kurosawa G, Hollingsworth R, Jermutus L, Minter R. (2013) Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type. Mol Cancer 12:11. (Targeting Trends 13q3)

Torres Demichelis V, Vilcaes AA, Iglesias-Bartolome R, Ruggiero FM, Daniotti JL. (2013) Targeted Delivery of Immunotoxin by Antibody to Ganglioside GD3: A Novel Drug Delivery Route for Tumor Cells. PLoS One 8(1):e55304. (Targeting Trends 13q2)


Tuscano JM, Kato J, Pearson D, Xiong C, Newell L, Ma Y, Gandara DR, O’Donnell RT. (2012) CD22 Antigen Is Broadly Expressed on Lung Cancer Cells and Is a Target for Antibody-Based Therapy. Cancer Res 72(21):5556-5565. (Targeting Trends 13q1)


Mitra N, Banda K, Altheide T, Schaffer L, Johnson-Pais TL, Beuten J, Leach RJ, Angata T, Varki N, Varki A. (2011) SIGLEC12: A human-specific segregating (PSEUDO) gene encodes a signaling molecule expressed in prostate carcinomas. J Biol Chem 286(26):23003-23011. (Targeting Trends 11q3)

Sawada R, Sun SM, Wu X, Hong F, Ragupathi G, Livingston PO, Scholz WW. (2011) Human Monoclonal Antibodies to Sialyl-Lewisa (CA19.9) with Potent CDC, ADCC, and Antitumor Activity. Clin Cancer Res 17(5):1024-1032. (Targeting Trends 11q2)


Hovinga KE, Shimizu F, Wang R, Panagiotakos G, Van Der Heijden M, Moayedpardazi H, Correia AS, Soulet D, Major T, Menon J, Tabar V (2010) Inhibition of Notch Signaling in Glioblastoma Targets Cancer Stem Cells via an Endothelial Cell Intermediate. Stem Cells 28:1019-1029. doi: 10.1002/stem.429

Summary:  The antibody CD105 (1:1,000) was incubated with Mab-ZAP, to allow binding and formation of a CD105 antibody-saporin complex, which was added to explants or to a human cerebral microvessel endothelial cell line as control.  CD105 antibody was also incubated with a Goat-IgG-SAP for control  that does not bind CD105.  The conjugates were injected into the explant under a dissecting microscope after gently incising the explant surface for better access.

Quadros EV, Nakayama Y, Sequeira JM (2010) Targeted delivery of saporin toxin by monoclonal antibody to the transcobalamin receptor, TCblR/CD320. Mol Cancer Ther 9(11):3033-3040. (Targeting Trends 11q1)


Rouleau C, Curiel M, Weber W, Smale R, Kurtzberg L, Mascarello J, Berger C, Wallar G, Bagley R, Honma N, Hasegawa K, Ishida I, Kataoka S, Thurberg BL, Mehraein K, Horten B, Miller G, Teicher BA (2008) Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma. Clin Cancer Res 14:7223-7236. (Targeting Trends 09q1)

Siva AC, Wild MA, Kirkland RE, Nolan MJ, Lin B, Maruyama T, Yantiri-Wernimont F, Frederickson S, Bowdish KS, Xin H (2008) Targeting CUB domain-containing protein 1 with a monoclonal antibody inhibits metastasis in a prostate cancer model. Cancer Res 68:3759-3766. (Targeting Trends 08q3)

Zhao XY, Liu HL, Liu B, Willuda J, Siemeister G, Mahmoudi M, Dinter H (2008) Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells. Transl Oncol 1:102-109. (Targeting Trends 08q4)


Bak SP, Walters JJ, Takeya M, Conejo-Garcia JR, Berwin BL (2007) Scavenger receptor-a-targeted leukocyte depletion inhibits peritoneal ovarian tumor progression. Cancer Res 67:4783-4789. (Targeting Trends 07q3)


Adam PJ, Terrett JA, Steers G, Stockwin L, Loader JA, Fletcher GC, Lu LS, Leach BI, Mason S, Stamps AC, Boyd RS, Pezzella F, Gatter KC, Harris AL (2006) CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding. Br J Cancer 95(3):298-306. (Targeting Trends 06q4)

Foehr ED, Lorente G, Kuo J, Ram R, Nikolich K, Urfer R (2006) Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model. Cancer Res 66(4):2271-2278. (Targeting Trends 06q2)

Fransson J, Borrebaeck CA (2006) The nuclear DNA repair protein Ku70/80 is a tumor-associated antigen displaying rapid receptor mediated endocytosis. Int J Cancer 119(10):2492-2496. (Targeting Trends 06q4)

Nguyen DH, Ball ED, Varki A (2006) Myeloid precursors and acute myeloid leukemia cells express multiple CD33-related Siglecs. Exp Hematol 34(6):728-735. (Targeting Trends 06q3)


Kohls MD, Lappi DA (2000) Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165. (Targeting Trends 01q1)  Published Online:

The assay for eliminating targeted cells, described in Kohls et al. (2000), uses the material listed below.

  • Mab-ZAP
  • your primary mouse monoclonal antibody
  • your cell line
  • medium
  • 96 well plate
  • PMS
  • MTS


Weltman JK, Melucci CL, Chen J, Davidson AE (1992) Internalization of monoclonal antibodies selected for immunotoxin activity against small-cell lung cancer. Hybridoma 1:547-559.


Barbieri L, Dinota A, Lappi DA, Soria M, Tazzari PL, Stirpe F (1989) Selective killing of CD4+ and CD8+ cells with immunotoxins containing saporin. Scand J Immunol 30:369-372.


Weltman JK, Pedroso P, Johnson S, Davignon D, Fast LD, Leone LA (1987)  Rapid screening with indirect immunotoxin for monoclonal antibodies against human small cell lung cancer.  Cancer Res 47(21):5552-5556.