ZAP Conjugate References

See complete product list of ZAP Secondary Conjugates

116 entries found for : zap-conjugates

CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells

Kim S, Shukla RK, Yu H, Baek A, Cressman SG, Golconda S, Lee GE, Choi H, Reneau JC, Wang Z, Huang CA, Liyanage NPM, Kim S (2022) CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells. Front Immunol 13:1011190. doi: 10.3389/fimmu.2022.1011190

Objective: To use a new murine testing model to demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment.

Summary: The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

Usage: Male C57BL/6J mice were injected into retro-orbital sinus with 15 μg S-CD3e-IT (Biotinylated Anti-CD3 mixed with Streptavidin-ZAP in sterile 200 μl PBS twice a day for four consecutive days.

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

In vivo visualization and molecular targeting of the cardiac conduction system

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)

Antibody-based preparative regimens for cell, tissue and organ transplantation

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)

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Comparison of CD3e antibody and CD3e-sZAP immunotoxin treatment in mice identifies szap as the main driver of vascular leakage

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)

Saporin as a commercial reagent: its uses and unexpected impacts in the biological sciences-tools from the plant kingdom

Ancheta LR, Shramm PA, Bouajram R, Higgins D, Lappi DA (2022) Saporin as a commercial reagent: its uses and unexpected impacts in the biological sciences-tools from the plant kingdom. Toxins (Basel) 14(3):184. doi: 10.3390/toxins14030184

Read complete article.

Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections

Hickerson BT, Daniels-Wells TR, Payes C, Clark LE, Candelaria PV, Bailey KW, Sefing EJ, Zink S, Ziegenbein J, Abraham J, Helguera G, Penichet ML, Gowen BB (2022) Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections. Nat Commun 13(1):558. doi: 10.1038/s41467-021-27949-3

Objective: Demonstrate that a fusion protein of the antibody (ch128.1/IgG1) directed against the apical domain of human transferrin receptor 1 (hTfR1) and the Machupo virus (MACV) can inhibit infection of attenuated and pathogenic New World mammarenaviruses (NWMs).

Summary: NWMs cause life-threatening hemorrhagic fever (HF) and these viruses enter into cells via hTfR1. Use of ch128.1/IgG1 with other promising direct-acting small molecule antivirals or antibodies targeting the viral envelope glycoprotein would provide a complementary therapeutic strategy that would increase efficacy and reduce the emergence of drug resistance.

Usage: References MonoBiotin-ZAP reacted with avidinylated anti-hTfR (ch128.1Av) in a 1:1 molar ratio on ice for 30 minutes.

Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP)

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Antibody-drug conjugates plus Janus kinase inhibitors enable MHC-mismatched allogeneic hematopoietic stem cell transplantation

Persaud SP, Ritchey JK, Kim S, Lim S, Ruminski PG, Cooper ML, Rettig MP, Choi J, DiPersio JF (2021) Antibody-drug conjugates plus Janus kinase inhibitors enable MHC-mismatched allogeneic hematopoietic stem cell transplantation. J Clin Invest 131(24):e145501. doi: 10.1172/JCI145501

Objective: To demonstrate that biotinylated anti-CD45-SAP or anti-cKit-SAP mixed with Streptavidin-Saporin along with Janus kinase 1/2, enables alloengraftment on murine allo-hematopoietic stem cell transplantation (HSCT) models.

Summary: HSCT has therapeutic potential. However, the transplantation requires first depletion and secondly, for allogeneic-HCST, host and immune responses need to be controlled to prevent graft rejection. The allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.

Usage: Antibodies were incubated with Streptavidin-ZAP (1:1 molar ratio) for 15 minutes at 20°C and . The doses of CD45.2 and cKit conjugates were injected retroorbitally (41.8 μg and 33.2 μg, respectively).

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

Enhancing the therapeutic potential of extracellular vesicles using peptide technology

Martin Perez C, Conceição M, Raz R, Wood MJA, Roberts TC (2022) Enhancing the therapeutic potential of extracellular vesicles using peptide technology. (eds. Langel Ü). In: Cell Penetrating Peptides. Methods in Molecular Biology 2383:119-141. Humana, New York, NY. doi: 10.1007/978-1-0716-1752-6_8

Objective: To modify EVs with peptides which confer specific advantageous properties, thus enhancing their therapeutic potential.

Summary: The authors provide an overview of the applications of peptide technology with respect to EV therapeutics. We focus on the utility of EV-modifying peptides for the purposes of promoting cargo loading, tissue-targeting and endosomal escape, leading to enhanced delivery of the EV cargo to desired cells/tissues and subcellular target locations. Both endogenous and exogenous methods for modifying EVs with peptides are considered.

Usage: Streptavidin-ZAP is combined with biotinylated peptides to make a targeted saporin conjugate.

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

Light-controlled elimination of PD-L1+ cells

Wong JJW, Selbo PK (2021) Light-controlled elimination of PD-L1+ cells. J Photochem Photobiol B 225:112355. doi: 10.1016/j.jphotobiol.2021.112355

Objective: To investigate novel strategies that simultaneously target both tumor cells and immunosuppressive cells in the tumor microenvironment. The focus was on the evaluation in vitro of Anti-PD-L1-SAP combined with photochemical internalization (PCI) as a therapeutic strategy to target and eliminate PD-L1 expressing tumor and immunosuppressive cells.

Summary: The authors show that the intracellular light-controlled drug delivery method induces specific and strongly enhanced cytotoxic effects of Anti-PD-L1-SAP in the PD-L1+ triple-negative breast cancer MDA-MB-231 cell line, while no enhanced efficacy was obtained in the PD-L1 negative control cell line MDA-MB-453. 

Usage: Anti-PD-L1-SAP and Streptavidin-ZAP (Control) were used in a cytotoxicity assay.

Related Products: Anti-PD-L1-SAP (Cat. #IT-45), Streptavidin-ZAP (Cat. #IT-27)

Probing transferrin receptor overexpression in gastric cancer mice models

Lodhi MS, Khan MT, Bukhari SMH, Sabir SH, Samra ZQ, Butt H, Akram MS (2021) Probing transferrin receptor overexpression in gastric cancer mice models. ACS Omega 6(44):29893-29904. doi: 10.1021/acsomega.1c04382

Objective: To investigate the role of the transferrin receptor, a glycoprotein receptor that is expressed many-folds on rapidly growing cells due to the greater demand of iron, in gastric cancer.

Summary: A mouse model of gastric cancer has the potential to be used in the future to study the therapeutic effects of cancer medicines, and overexpression of transferrin receptors could be identified through the designed probe to be used as diagnostics.

Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP)

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Identification and therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumorswith DS-6157a, a first-in-class antibody-drug conjugate

Iida K, Abdelhamid Ahmed AH, Nagatsuma AK, Shibutani T, Yasuda S, Kitamura M, Hattori C, Abe M, Hasegawa J, Iguchi T, Karibe T, Nakada T, Inaki K, Kamei R, Abe Y, Nomura T, Andersen JL, Santagata S, Hemming ML, George S, Doi T, Ochiai A, Demetri GD, Agatsuma T (2021) Identification and therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumorswith DS-6157a, a first-in-class antibody-drug conjugate. Cancer Discov 11(6):1508-1523. doi: 10.1158/2159-8290.Cd-20-1434

Objective: Introduce DS-6157a, an anti-GPR20 antibody-drug therapeutic for gastrointestinal stromal tumors (GIST).

Summary: The only approved treatments for GIST are currently tyrosine kinase inhibitors (TKI) which can be problematic. They lead to secondary resistance mutations in KIT or PDGFRA and disease progression. The authors identified G protein-coupled receptor 20 (GPR20) as a non-tyrosine kinase target and assessed its expression in cell lines, xenografts, and clinical samples. Preclinical pharmacokinetics and safety profile support its development as a novel GIST therapy.

Usage: Internalization activity of anti-GPR20 mAbs were evaluated by using Rat-ZAP. GPR20-expressing 293T cells were plated at 2500 cells/well in 96-well plates. Cells were treated with dilutions of various anti-GPR20 and 500 ng/ml of Rat-ZAP for 3 days. The percentage of living cells were measured using a CellTiter-Glo Luminescent Cell Viability Assay.

Related Products: Rat-ZAP (Cat. #IT-26)

Autosomal recessive osteopetrosis: mechanisms and treatments

Penna S, Villa A, Capo V (2021) Autosomal recessive osteopetrosis: mechanisms and treatments. Dis Model Mech 14(5):dmm048940. doi: 10.1242/dmm.048940

Summary: Autosomal recessive osteopetrosis (ARO) is a severe inherited bone disease characterized by defective osteoclast resorption or differentiation. Novel therapeutic approaches are needed for ARO patients. The authors review preclinical and proof-of-concept studies, such as gene therapy, systematic administration of deficient protein, in utero Hematopoietic stem cell transplantation (HSCT) and gene editing.

Usage: Efficacy in HSCT conditioning was demonstrated with CD45.2-SAP (biotinylated Anti-CD45 mixed with Streptavidin-ZAP). In mice, CD45.2–SAP preserved normal bone marrow architecture compared to total body irradiation, which instead reduced vascular integrity and bone marrow cellularity. Mice conditioned with CD45.2–SAP rapidly recovered their peripheral myeloid cells and had a survival advantage when exposed to infections (3 mg/kg iv; Palchaudhuri et al.). Additionally, conditioning with CD45.2–SAP resulted in significant chimerism after transplantation, even in a pathological mouse model (3 mg/kg iv; Castiello et al.).

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

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Activated factor X targeted stored in platelets as an effective gene therapy strategy for both hemophilia A and B

Wang D, Shao X, Wang Q, Pan X, Dai Y, Yao S, Yin T, Wang Z, Zhu J, Xi X, Chen Z, Chen S, Zhang G (2021) Activated factor X targeted stored in platelets as an effective gene therapy strategy for both hemophilia A and B. Clin Transl Med 11(3):e375. doi: 10.1002/ctm2.375

Summary: Treatment of hemophiliacs with inhibitors remains challenging, and new treatments are in urgent need. Coagulation factor X plays a critical role in downstream blood coagulation cascade, which could serve as a bypassing agent for hemophilia therapy. Target expression of the FXa precursor to platelets can generate a storage pool of FXa in platelet α-granules, the platelet-stored FXa is effective in treating HA and HB with inhibitors, suggesting that this could be a novel choice for hemophilia patients with inhibitors.

Usage: A single dose of CD45.2-SAP (biotinylated Anti-CD45 mixed with Streptavidin-ZAP) enabled efficient engraftment of donor cells (> 90%) and full correction of sickle-cell anemia. (3 mg/kg iv; Palchaudhuri et al.).

See: 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.

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

Bone marrow-derived myeloid progenitors in the leptomeninges of adult mice

Koeniger T, Bell L, Mifka A, Enders M, Hautmann V, Mekala SR, Kirchner P, Ekici AB, Schulz C, Wörsdörfer P, Mencl S, Kleinschnitz C, Ergün S, Kuerten S (2021) Bone marrow-derived myeloid progenitors in the leptomeninges of adult mice. Stem Cells 39(2):227-239. doi: 10.1002/stem.3311

Summary: This report confirms the presence of myeloid progenitors at the meningeal border of the brain and lays the foundation to unravel their possible functions in CNS surveillance and local immune cell production. Compared to bone marrow transfer after whole-body irradiation, chimerism developed more slowly in the CD45-SAP (biotinylated anti-CD45 mixed with Streptavidin-ZAP) model and only reached around 50% in the blood myeloid compartment 15 weeks after transplantation.

See: 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.

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

Identification of prostaglandin F2 receptor negative regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

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.

Usage: In a 96-well plate, 2000 cells/well were plated in triplicate in 100 μL 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.

Related Products: Fab-ZAP mouse (Cat. #IT-48), Fab IgG-SAP (Cat. #IT-67), Custom Conjugates

Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency.

Castiello MC, Bosticardo M, Sacchetti N, Calzoni E, Fontana E, Yamazaki Y, Draghici E, Corsino C, Bortolomai I, Sereni L, Yu HH, Uva P, Palchaudhuri R, Scadden DT, Villa A, Notarangelo LD (2021) Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency. J Allergy Clin Immunol 147(1):309-320.e6. doi: 10.1016/j.jaci.2020.04.033

Objective: To improve multi-lineage engraftment using non-genotoxic conditioning with Anti-CD45-Saporin.

Summary: Conditioning with Anti-CD45 antibody-drug conjugates may represent a novel and safe conditioning regimen for patients with RAG deficiency and other inborn errors of immunity.

Usage: Intravenous injection of Anti-CD45-SAP (3 mg/kg).

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

Pseudomonas Exotoxin A based toxins targeting epidermal growth factor receptor for the treatment of prostate cancer

Fischer A, Wolf I, Fuchs H, Masilamani AP, Wolf P (2020) Pseudomonas Exotoxin A based toxins targeting epidermal growth factor receptor for the treatment of prostate cancer. Toxins (Basel) 12(12):753. doi: 10.3390/toxins12120753

Summary: Refers to chimeric murine-human mAb cetuximab bound to Streptavidin-ZAP.

See: Yip WL et al. Targeted Delivery and Enhanced Cytotoxicity of Cetuximab-Saporin by Photochemical Internalization in EGFR-Positive Cancer Cells. Mol Pharm 4(2):241-251, 2007.

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

Cellular uptake of vitamin B12: Role and fate of TCblR/CD320, the transcobalamin receptor.

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. 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 TCblR 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.

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

The EphA2 and cancer connection: potential for immune-based interventions

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.

See: Sakamoto A et al. An Agonistic Antibody to EPHA2 Exhibits Antitumor Effects on Human Melanoma Cells. Anticancer Res 38:3273-3282, 2018.

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

ALPPL2 is a highly specific and targetable tumor cell surface antigen

Su Y, Zhang X, Bidlingmaier S, Behrens CR, Lee NK, Liu B (2020) ALPPL2 is a highly specific and targetable tumor cell surface antigen. Cancer Res 80(20):4552-4564. doi: 10.1101/2020.01.07.898122

Objective: To evaluate therapeutic potential of ALPPL2 targeting.

Summary: Exquisite tissue specificity and broad tumor type coverage suggest that ALPPL2 could be an excellent cell surface target for therapeutic development against mesothelioma.

Usage: Biotinylated M25 IgG1 and Streptavidin-ZAP were mixed at a molar ratio of 1:1.

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

Nongenotoxic antibody-drug conjugate conditioning enables safe and effective platelet gene therapy of hemophilia A mice.

Gao C, Schroeder JA, Xue F, Jing W, Cai Y, Scheck A, Subramaniam S, Rao S, Weiler H, Czechowicz A, Shi Q (2019) Nongenotoxic antibody-drug conjugate conditioning enables safe and effective platelet gene therapy of hemophilia A mice. Blood Adv 3(18):2700-2711. doi: 10.1182/bloodadvances.2019000516

Objective: To determine whether hematopoietic cell–targeted ADC preconditioning is effective for engraftments that are genetically manipulated by 2bF8 lentivirus (2bF8LV) and whether sustained therapeutic platelet FVIII expression is attainable in platelet-specific gene therapy utilizing ADC-based preconditioning.

Summary: The authors describe targeted nongenotoxic preconditioning for 2bF8 gene therapy utilizing  a hematopoietic cell–specific antibody-drug conjugate (ADC), which consists of saporin conjugated to CD45.2- and CD117-targeting antibodies.

Usage: ADCs were prepared by combining biotinylated antibody with Streptavidin-ZAP.  The combination of CD45.2-ADC (3 mg/kg) plus CD117-ADC (0.5 mg/kg), with or without additional CD4-ADC (0.5 mg/kg) or CD8-ADC (0.5 mg/kg), was administered IV to 5- to 6-week-old FVIIInull/CD45.2 recipients 2 days before transplantation.

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

Chemical strategies for antigen-selective targeting of autoreactive B Cells. Chapter 2: Sequential prodrug strategy to target and eliminate ACPA-selective autoreactive B cells.

Lelieveldt L (2019) Chemical strategies for antigen-selective targeting of autoreactive B Cells. Chapter 2: Sequential prodrug strategy to target and eliminate ACPA-selective autoreactive B cells. Radboud Universiteit Nijmegen Nijmegen, Netherlands 45-64. Thesis.

Objective: To develop a method to target and selectively eliminate autoreactive B cells that produce anti-citrullinated proteins antibodies (ACPA) using a sequential antigen prodrug targeting strategy, as a treatment for Rheumatoid Arthritis (RA).

Summary: The study used a synthesized cyclic citrullinated peptide (CCP) antigen suitable for BCR binding and demonstrated that binding by ACPA was impaired upon introduction of a carboxy-pnitrobenzyl (CNBz) caging group at the side chain of the citrulline residue. Enzymatic reduction of the CNBz moiety by nitroreductase fully restored citrulline-selective recognition by both ACPA and ACPA-expressing B cells and showed targeted cell death of CCP-recognizing B cells only. These results mark an important step towards antigen-selective B cell targeting in general and more specifically in RA.

Usage: Streptavidin-ZAP mixed with biotinylated CCP peptides was tested in cytotoxicity assays. The exposure of cells to CCP-SA-ZAP at 1 nM as well as the activated CCP(CNBz) induced death of up to 60% of ACPA-expressing B cells.

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

Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation.

Czechowicz A, Palchaudhuri R, Scheck A, Hu Y, Hoggatt J, Saez B, Pang WW, Mansour MK, Tate TA, Chan YY, Walck E, Wernig G, Shizuru JA, Winau F, Scadden DT, Rossi DJ (2019) Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation. Nat Commun 10:617. doi: 10.1038/s41467-018-08201-x

Objective: To investigate a safe and effective method for hematopoietic stem cell transplantation.

Summary: CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects.

Usage: The CD117-ADC was prepared by combining biotinylated anti-CD117 (clone 2B8) with Streptavidin–ZAP. A dose of 1.5 mg/kg of CD117-ADC (~12 µg Streptavidin-ZAP) optimally resulted in depletion of >99% of immunophenotypic and functional HSCs.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Anti-CD117-SAP (Cat. #IT-83)

Hematopoietic chimerism and donor-specific skin allograft tolerance after non-genotoxic CD117 antibody-drug-conjugate conditioning in MHC-mismatched allotransplantation.

Li Z, Czechowicz A, Scheck A, Rossi DJ, Murphy PM (2019) Hematopoietic chimerism and donor-specific skin allograft tolerance after non-genotoxic CD117 antibody-drug-conjugate conditioning in MHC-mismatched allotransplantation. Nat Commun 10:616. doi: 10.1038/s41467-018-08202-w

Objective: To develop a conditioning protocol for fully MHC-mismatched bone marrow allotransplantation in mice involving transient immunosuppression and selective depletion of recipient hematopoietic stem cells.

Summary: CD117-ADC conditioning promotes skin allograft tolerance.

Usage: Biotinylated monoclonal antibodies directed against mouse CD117 were coupled to Streptavidin-ZAP. Each mouse was injected with 1.5 mg/kg of ADC in a total volume of 300 mcl PBS.

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

Identification of lineage-specific markers for therapeutic targeting of mast cells.

Plum T (2019) Identification of lineage-specific markers for therapeutic targeting of mast cells. Ruperto-Carola University of Heidelberg, Germany Thesis. doi: 10.11588/heidok.00023555

Usage: Mice were injected i.v. with either 100 µg of 1:1 molar mixture of biotinylated CD63 antibody and Streptavidin-ZAP (60 µg mAb + 40 µg SAP) or with 40 µg SAP alone. All injections were performed in 200 µl PBS.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Saporin (Cat. #PR-01)

Human anti-NKp46 antibody for studies of NKp46-dependent NK cell function and its applications for type 1 diabetes and cancer research.

Berhani O, Glasner A, Kahlon S, Duev-Cohen A, Yamin R, Horwitz E, Enk J, Moshel O, Varvak A, Porgador A, Jonjic S, Mandelboim O. (2019) Human anti-NKp46 antibody for studies of NKp46-dependent NK cell function and its applications for type 1 diabetes and cancer research. Eur J Immunol 49(2):228-241. doi: 10.1002/eji.201847611

Objective: To investigate human NKp46 activity and its critical role in Natural Killer (NK) cell biology.

Summary: A unique anti-human NKp46 monoclocal antibody was developed and conjugated to Saporin. Targeted toxin inhibits growth of NKp46-positive cells; thus, exemplifying the potential as an immunotherapeutic drug to treat NKp46-dependent diseases, such as, type I diabetes and NK and T cell related malignancies.

Usage: Conjugation of the antibodies to Saporin, treatment of cells, and cell viability assay Biotin-Z Kit instructions.

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

Sequential prodrug strategy to target and eliminate ACPA-selective autoreactive B cells.

Lelieveldt LPWM, Kristyanto H, Pruijn GJM, Scherer HU, Toes REM, Bonger KM (2018) Sequential prodrug strategy to target and eliminate ACPA-selective autoreactive B cells. Mol Pharm 15(12):5565-5573. doi: 10.1021/acs.molpharmaceut.8b00741

Objective: To develop a method to target and selectively eliminate autoreactive B cells using a sequential antigen prodrug targeting strategy.

Summary: The selectivity of the antigen and the possibility to block binding toward circulation ACPA brings us a step closer to the specific elimination of autoreactive B cells for the treatment of patients with ACPA-positive RA.

Usage: Biotinylated CCP1, CArgP1, and CCP1(CNBz) were conjugated with Streptavidin-ZAP in a 4:1 ratio to make peptide-drug conjugates.

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

Antibody drug conjugates targeted to CD45 or CD117 enable allogeneic hematopoietic stem cell transplantation in animal models

Palchaudhuri R, Hyzy SL, Proctor JL, Adams HL, Pearse BR, Sarma G, Aslanian S, Gillard G, Lamothe TL, Burenkova O, Brooks ML, Gabros AD, McDonagh CF, Boitano AE, Cooke MP (2018) Antibody drug conjugates targeted to CD45 or CD117 enable allogeneic hematopoietic stem cell transplantation in animal models. Blood 132:3324. doi: 10.1182/blood-2018-99-119432

Objective: To further investigate and develop the utility of CD45-SAP and CD117-SAP, in combination with immunosuppression, in murine transplant models using i.v. administration in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients).

Summary: CD45-SAP or CD117-SAP in combination with immunosuppressants (30Fll and post-transplant Cytoxan) enabled >85% peripheral donor chimerism at 12 weeks post-transplantation. CD45-SAP and CD117-SAP were more effective at conditioning versus 2Gy TBI or pretransplant Cytoxan.

Usage: CD45-SAP (1.9 mg/kg, iv) and CD117-SAP (1mg/kg, iv) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients).

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

Screening targeting agents and their cell surface biomarkers for high specificity and rapid internalization via cell death and fluorescence

Ancheta L, Bouajram R, Lappi DA (2018) Screening targeting agents and their cell surface biomarkers for high specificity and rapid internalization via cell death and fluorescence. Neuroscience 2018 Abstracts 128.20 / M17. Society for Neuroscience, San Diego, CA.

Summary: Some of the most recent successes in the treatment of cancers or research into passive immunotherapies for neurodegenerative diseases, employ the use of antibodies. These treatments utilize antibodies that either: 1) interfere with cell surface proteins responsible for tumor cell proliferation, 2) act as immune checkpoint inhibitors, or 3) are re-engineered to allow transport of other molecules across the blood-brain barrier (BBB). There are a growing number of antibody and small molecule therapeutic candidates and this demands a quick and efficient technique to screen for biomarkers that internalize effectively upon binding. The method described provides for the efficient determination of internalization of cell surface biomarkers upon binding of antibodies or peptides. This one-step, robust method uses a targeting agent combined with both a fluorescent reporter and a cytotoxic payload. The construct that makes this method effective was formed by cross-linking a fluorescent reporter, in this case fluorescein (FITC) and streptavidin to the ribosome-inactivating protein, Saporin. The conjugate used in screening potential therapeutics is a mixture of a biotinylated targeting agent mixed in a 1:1 molar ratio with FITC-labeled Streptavidinylated-Saporin. The method provides a definitive assay readout: fluorescence within 1 hour and cell death in 72 hours. This method is designed for rapid screening, in a quick and reproducible manner, for specificity and internalization in various cell types to explore suitability of candidates as therapeutics.

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

Targeting CD46 for both adenocarcinoma and neuroendocrine prostate cancer

Su Y, Liu Y, Behrens CR, Bidlingmaier S, Lee NK, Aggarwal R, Sherbenou DW, Burlingame AL, Hann BC, Simko JP, Premasekharan G, Paris PL, Shuman MA, Seo Y, Small EJ, Liu B (2018) Targeting CD46 for both adenocarcinoma and neuroendocrine prostate cancer. JCI Insight 3(17):e121497. doi: 10.1172/jci.insight.121497

Objective: To investigate the suitability of a CD46 antibody as a metastatic castration-resistant prostate cancer (mCRPC) anti-tumor agent.

Summary: CD46 is an excellent candidate for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.

Usage: Biotinylated UA20 IgG and Streptavidin-ZAP were mixed at a molar ratio of 1:1. The conjugate was used in cytotoxicity assays and shown to specifically kill the mCRPC cells.

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

Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer

Tan HL, Yong C, Tan BZ, Fong WJ, Padmanabhan J, Chin A, Ding V, Lau A, Zheng L, Bi X, Yang Y, Choo A (2018) Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer. Sci Rep 8:11608. doi: 10.1038/s41598-018-30070-z

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.

Usage: in 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. invivo - 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.

Related Products: Hum-ZAP (Cat. #IT-22), Streptavidin-ZAP (Cat. #IT-27), Saporin (Cat. #PR-01)

Development and evaluation of T-Zap: a novel antibody-drug conjugate for the treatment of Her2 positive breast cancer

Hoffmann RM, Crescioli S, Thurston DE, Karagiannis SN (2018) Development and evaluation of T-Zap: a novel antibody-drug conjugate for the treatment of Her2 positive breast cancer. Cancer Res 78:LB-001. doi: 10.1158/1538-7445.AM2018-LB-001

Objective: Develop and Evaluate a novel ADC (T-Zap) for breast cancer.

Summary: Binding to target cells of T-Zap was confirmed. Comparison of T-Zap efficacy in breast cancer cell lines with and without resistance against trastuzumab showed a trend for higher efficacy of cell killing by T-Zap in trastuzumab resistant cells compared to T-DM1. Toxicity assays revealed no impact of T-Zap on cell viability in immune cells.

Usage: T-ZAP was made using Biotinylated monoclonal antibody trastuzumab mixed with Streptavidin-ZAP.

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

SAT0058 Adalimumab: TNF complexes are cleared more efficiently by human osteoclasts than those with etanercept through FCG-receptor binding and internalisation

Harvey BP, Cohen-Solal J, Kaymakcalan Z (2018) SAT0058 Adalimumab: TNF complexes are cleared more efficiently by human osteoclasts than those with etanercept through FCG-receptor binding and internalisation. Ann Rheum Dis 77:893. EULAR 2018, Amsterdam, The Netherlands doi: 10.1136/annrheumdis-2018-eular.3804

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.

Usage: 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.

Related Products: FabFc-ZAP human (Cat. #IT-65)

An agonistic antibody to EPHA2 exhibits antitumor effects on human melanoma cells

Sakamoto A, Kato K, Hasegawa T, Ikeda S (2018) An agonistic antibody to EPHA2 exhibits antitumor effects on human melanoma cells. Anticancer Res 38:3273-3282. doi: 10.21873/anticanres.12592

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.

Usage: 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).

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

Engineering elastic properties into an anti-TNFα monoclonal antibody

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 (2018) Engineering elastic properties into an anti-TNFα monoclonal antibody. Cogent Biol 4(1):1469387. doi: 10.1080/23312025.2018.1469387

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.

Usage: Fab-ZAP human was mixed with anti-TNFα-ELP fusion monoclonal.

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

Targeting prostate cancer: Prostate-specific membrane antigen based diagnosis and therapy.

Wüstemann T, Haberkorn U, Babich J, Mier W (2019) Targeting prostate cancer: Prostate-specific membrane antigen based diagnosis and therapy. Med Res Rev 39(1):40-69. doi: 10.1002/med.21508

Summary: Conjugation to the antibody was achieved by reacting the biotinylated humanized antibody to prostate-specific membrane antigen (PMSA) with Streptavidin-ZAP. Binding potency of the conjugate was comparable to that of the naked antibody and in vivo experiments proved potent for selective tumor growth inhibition in mice bearing LNCaP tumors.

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

See Also:

Enhanced targeting of triple-negative breast carcinoma and malignant melanoma by photochemical internalization of CSPG4-targeting immunotoxins

Eng MS, Kaur J, Prasmickaite L, Engesaeter BO, Weyergang A, Skarpen E, Berg K, Rosenblum MG, Maelandsmo GM, Hogset A, Ferrone S, Selbo PK (2018) Enhanced targeting of triple-negative breast carcinoma and malignant melanoma by photochemical internalization of CSPG4-targeting immunotoxins. Photochem Photobiol Sci 17:539-551. doi: 10.1039/C7PP00358G

Summary: The combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

Usage: To obtain the immunotoxin 225.28-saporin, Streptavidin-Saporin (Cat. #IT-27; Streptavidin-ZAP), with an average of 2.5 molecules of saporin per molecule of streptavidin, was combined with biotinylated 225.28, a CSPG4-specific mouse mAb, IgG2a.

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

Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Yuan X, Yang M, Chen X, Zhang X, Sukhadia S, Musolino N, Bao H, Chen T, Xu C, Wang Q, Santoro S, Ricklin D, Hu J, Lin R, Yang W, Li Z, Qin W, Zhao A, Scholler N, Coukos G (2018) Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy. Cancer Immunol Immunother 67:329-339. doi: 10.1007/s00262-017-2101-0

Objective: ScFv78 was conjugated with the ribosome-inactivating protein saporin (Streptavidin-ZAP) to evaluate whether scFv78 may be used as a vehicle for theTEM1-targeted delivery of toxins.

Summary: Site-specific, biotinylated scFv78 was conjugated with streptavidin-labeled saporin (Streptavidin-ZAP; Cat. #IT-27) by incubation at room temperature for 1h at a molar ratio of 4:1 (scFv78:ZAP).

Usage: Mouse endothelial cells (MS1) and MS1 cells transduced to express full-length human TEM1 (MS1-TEM1) were cultured in 96-well plates to 30% confluence and then incubated for 96h in the presence of 10-fold serially diluted Streptavidin-ZAP, scFv78, or scFv78-ZAP starting from 40nM down to 0.04nM. The data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

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

Synergistic cytotoxic effect on gastric cancer cells of an immunotoxin cocktail in which antibodies recognize different epitopes on CDH17

Kusano-Arai O, Iwanari H, Kudo S, Kikuchi C, Yui A, Akiba H, Matsusaka K, Kaneda A, Fukayama M, Tsumoto K, Hamakubo T (2018) Synergistic cytotoxic effect on gastric cancer cells of an immunotoxin cocktail in which antibodies recognize different epitopes on CDH17. Monoclon Antib Immunodiagn Immunother 37:1-11. doi: 10.1089/mab.2017.0043

Objective: To determine if an immunotoxin cocktail targeted to multiple epitopes has synergistic effects on low expression level cells, which would expand the applicable range of immunotoxin therapy for cancer.

Summary: The combination of immunotoxins with different mechanisms of action in an antibody cocktail will increase cytotoxic activities and decrease side effects.

Usage: The authors applied a monoclonal antibody (mAb) cocktail for one target protein with multiple epitopes. They generated anti-CDH17 mAbs recognizing different epitopes on CDH17 (Cadherin-17). CDH17 is expressed in gastric cancer, hepatocellular carcinoma, colorectal cancer, and pancreatic cancer and has limited distribution in normal tissues. For preparation of 3 immunotoxins, Streptavidin-ZAP was mixed with biotinylated mAbs in equimolar concentrations for 30 minutes at room temperature. The study provides data to demonstrate that the cocktail of different epitope-recognizing immunotoxins has synergistic cytotoxic effects on CDH17-expressing cells.

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

Targeting of embryonic annexin A2 expressed on ovarian and breast cancer by the novel monoclonal antibody 2448

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

Combine phage antibody display library selection on patient tissue specimens with laser capture microdissection to identify novel human antibodies targeting clinically relevant tumor antigens

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)

Enhancement of anti-Robo1 immunotoxin cytotoxicity to head and neck squamous cell carcinoma via photochemical internalization

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)

Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1.

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)

TCR-like antibody drug conjugates mediate killing of tumor cells with low peptide/HLA targets.

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 (5x103) 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)

A retinoraphe projection regulates serotonergic activity and looming-evoked defensive behaviour.

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

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)

Identification of novel macropinocytosing human antibodies by phage display and high-content analysis.

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)

Aminopeptidase N (APN/CD13) as a target molecule for scirrhous gastric cancer.

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)

Targeting CD73 in the tumor microenvironment with MEDI9447.

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)

Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin.

Palchaudhuri R, Saez B, Hoggatt J, Schajnovitz A, Sykes D, Tate T, Czechowicz A, Kfoury Y, Ruchika F, Rossi D, Verdine G, Mansour M, Scadden D (2016) Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nat Biotechnol 34:738-745. doi: 10.1038/nbt.3584

Summary: To demonstrate correction of a clinically relevant disease, we employed CD45-SAP in a mouse model of sickle cell anemia and demonstrated our method achieved >90% donor cell chimerism, all mice in three groups (18/18), resulting in complete disease correction (red blood cell counts, hemoglobin levels, hematocrit levels and reticulocyte frequencies were returned to normal). If these pre-clinical results can be successfully translated to the clinic, it would greatly reduce conditioning-related toxicities and expand the use of hematopoietic stem cell transplantation.

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

Read the featured article in Targeting Trends.

Method for confirming cytoplaintratumoral anti-HuD immunotoxinsmic delivery of RNA aptamers.

Dickey D, Thomas G, Dassie J, Giangrande P (2016) Method for confirming cytoplaintratumoral anti-HuD immunotoxinsmic delivery of RNA aptamers. (eds. Shum K, Rossi J). In: SiRNA Delivery Methods. Methods in Molecular Biology. 1364:209-217. Humana Press, New York, NY. doi: 10.1007/978-1-4939-3112-5_17

Objective: To describe a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA).

Summary: This publication details an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity.

Usage: The folded biotinylated aptamer was mixed at a 1:4 molar ratio of Streptavidin-ZAP, confirmed by agarose gel, a PSMA enzymatic activity (NAALADase) assay performed. FGF-SAP was used as a control.

Related Products: Streptavidin-ZAP (Cat. #IT-27), FGF-SAP (Cat. #IT-38)

Characterization of the first fully macropinocytosing human antibodies human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.

Yuan X, Yang M, Chen X, Zhang X, Sukhadia S, Musolino N, Bao H, Chen T, Xu C, Wang Q, Santoro S, Ricklin D, Hu J, Lin R, Yang W, Li Z, Qin W, Zhao A (2017) Characterization of the first fully macropinocytosing human antibodies human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy. Cancer Immunol Immunother 66:367-378.. doi: 10.1007/s00262-016-1937-z

Summary: Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. The authors previous isolated a single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library and evaluated potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. MS1 and MS1-hTEM1 cells were treated with site-specifically biotinylated scFv78 conjugated with the Streptavidin-ZAP (Cat. #IT-27) at a molar ratio of 4:1 (scFv78:ZAP) starting from 40 nM serially diluted down to 0.04 nM. The scFv78-saporin immunoconjugate exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cell in vitro. The data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has features advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.

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

Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors.

Pires E, D'Souza R, Needham M, Herr A, Jazaeri A, Li H, Stoler M, Anderson-Knapp K, Thomas T, Mandal A, Gougeon A, Flickinger C, Bruns D, Pollok B, Herr J (2015) Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors. Oncotarget 6:30194-30211. doi: 10.18632/oncotarget.4734

Summary: Ovastatin is a zinc matrix metallo-proteinase thought to play roles in sperm-egg interaction and the prevention of polyspermy in eutherians. This protein is not found in normal adult tissues, but is expressed by uterine carcinosarcomas. The authors investigated the possibility of targeting ovastatin as a tumor surface neoantigen for therapeutic purposes. SNU539 cells, a uterine malignant mixed Müllerian tumor-derived cell line, were challenged with 1 μM, 0.1 μM, and 0.01 μM rabbit polyclonal anti-ovastatin coupled to 5.42 nM Fab-ZAP rabbit (Cat. #IT-57). Rabbit IgG-SAP (Cat. #IT-35) was used as a control. The results indicate that for this form of uterine cancer, ovastatin is a viable therapeutic target.

Related Products: Fab-ZAP rabbit (Cat. #IT-57), Rabbit IgG-SAP (Cat. #IT-35)

Anti-EFNA4 calicheamicin conjugates effectively target triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regressions.

Damelin M, Bankovich A, Park A, Aguilar J, Anderson W, Santaguida M, Aujay M, Fong S, Khandke K, Pulito V, Ernstoff E, Escarpe P, Bernstein J, Pysz M, Zhong W, Upeslacis E, Lucas J, Lucas J, Nichols T, Loving K, Foord O, Hampl J, Stull R, Barletta F, Falahatpisheh H, Sapra P, Gerber H, Dylla S (2015) Anti-EFNA4 calicheamicin conjugates effectively target triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regressions. Clin Cancer Res 21:4165-4173. doi: 10.1158/1078-0432.CCR-15-0695

Summary: Triple-negative breast cancer (TNBC) is characterized by tumors lacking HER2, estrogen receptor, and progesterone receptor. TNBC has proved to be very difficult to treat, in large part because of the absence of consensus targets on the surface of the tumor cells. In this work the authors empirically established a set of surface markers associated with TNBC tumor initiating cells, as produced by patient-derived xenografts. Ephrin-A4 was selected as a therapeutic target, and a cell line transfected with the ephrin-A4 gene was challenged with two versions of biotinylated anti-ephrin-A4 coupled to Streptavidin-ZAP (Cat. #IT-27). Both the mouse monoclonal and the humanized antibodies reach an EC50 of 10 ng/ml, indicating that ephrin-A4 has promise as a therapeutic target for TNBC.

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

Monoclonal antibodies targeting LecLex-related glycans with potent antitumor activity.

Chua J, Vankemmelbeke M, McIntosh R, Clarke P, Moss R, Parsons T, Spendlove I, Zaitoun A, Madhusudan S, Durrant L (2015) Monoclonal antibodies targeting LecLex-related glycans with potent antitumor activity. Clin Cancer Res 21:2963-2974. doi: 10.1158/1078-0432.CCR-14-3030

Summary: In this work the authors characterized two monoclonal antibodies that target glycans containing Lewis carbohydrate antigens. One of the methods used was to combine varying concentrations of the antibodies with 50 ng mouse Fab-ZAP (Cat. #IT-48) and apply the conjugates to cells for 72 hours. The antibodies were demonstrated to have efficient internalization, supported by potent in vivo anti-tumor activity.

Related Products: Fab-ZAP mouse (Cat. #IT-48)

Dual targeting NG2 and GD3A using Mab-Zap immunotoxin results in reduced glioma cell viability in vitro.

Higgins S, Fillmore H, Ashkan K, Butt A, Pilkington G (2015) Dual targeting NG2 and GD3A using Mab-Zap immunotoxin results in reduced glioma cell viability in vitro. Anticancer Res 35:77-84.

Summary: Human glioma-derived cell lines were sequentially incubated with anti-NG2 and anti-GD3A coupled to Mab-ZAP (Cat. #IT-04) at 1 μg/ml and 5 μg/ml for 72 hours each. The combination therapy was significantly more effective than single therapy in eliminating the glioma cells.

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

Intratumoral anti-HuD immunotoxin therapy for small cell lung cancer and neuroblastoma.

Ehrlich D, Wang B, Lu W, Dowling P, Yuan R (2014) Intratumoral anti-HuD immunotoxin therapy for small cell lung cancer and neuroblastoma. J Hematol Oncol 7:91. doi: 10.1186/s13045-014-0091-3

Summary: HuD protein is a 40-kDa neuronal RNA-binding protein that is expressed in 100% of small cell lung cancer (SCLC) tumor cells. An anti-HuD monoclonal was biotinylated and combined with Streptavidin-ZAP (Cat. #IT-27); this conjugate was tested both in vitro and in vivo. Anti-HuD-SAP eliminated NCI-H69 and Neuro-2a cells at an EC50 of <0.5 μg/ml. 1 mg/kg of the conjugate injected directly into subcutaneous tumors generated in mice resulted in a temporary lack of tumor growth or regression of the tumor. The results demonstrate the potential of HuD as a therapeutic target for SCLC and neuroblastoma.

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

Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells.

Sims S, Bolinger B, Klenerman P (2015) Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells. Eur J Immunol 45:113-118. doi: 10.1002/eji.201445016

Summary: The standard CD8+ T-cell response to infection is a rapid proliferation followed by a reduction in number after the infection is cleared. Murine cytomegalovirus is an exception in that an infection generates a life-long latency with low-level sporadic replication. Immunodominant cells accumulate over time and stabilize at a high frequency. The authors examined a paradoxical boost following depletion of these cells with an M38 antibody attached to Streptavidin-ZAP (Cat. #IT-27). Mice were treated with 44 pM intraperitoneal injections. M38 is an epitope present on the effector CD8+ T cells. Following a significant depletion of cells, the population rebounded and reached a higher percentage of total CD8+ T-cells than before the depletion.

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

High-content analysis of antibody phage-display library selection outputs identifies tumor selective macropinocytosis-dependent rapidly internalizing antibodies.

Ha K, Bidlingmaier S, Zhang Y, Su Y, Liu B (2014) High-content analysis of antibody phage-display library selection outputs identifies tumor selective macropinocytosis-dependent rapidly internalizing antibodies. Mol Cell Proteomics 13:3320-3331. doi: 10.1074/mcp.M114.039768

Summary: Macropinocytosis, the internalization of large endocytic vesicles called macropinosomes, is upregulated in Ras-transformed cancers. To date, large-scale antibody generation strategies have not incorporated a selection method for antibodies. In this work the authors demonstrate screening and validation of the antibodies that utilize the macropinosome pathway. One method used was to biotinylate the antibodies and combine them with Streptavidin-ZAP (Cat. #IT-27) at a 1:1 ratio. The conjugate was applied to cells in a concentration curve starting at 200 nM in order to demonstrate internalization and cell killing.

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

Light-triggered, efficient cytosolic release of IM7-saporin targeting the putative cancer stem cell marker CD44 by photochemical internalization.

Bostad M, Kausberg M, Weyergang A, Olsen C, Berg K, Høgset A, Selbo P (2014) Light-triggered, efficient cytosolic release of IM7-saporin targeting the putative cancer stem cell marker CD44 by photochemical internalization. Mol Pharm 11:2764-2776. doi: 10.1021/mp500129t

Summary: CD44 is known as a common cancer stem cell (CSC) marker. Given that CSC's seem to have the ability to resist many therapeutic agents, the authors investigated the use of photochemical internalization (PCI) while targeting CD44-expressing CSC's. An immunotoxin was constructed by biotinylating a pan CD44 antibody and combining it with Streptavidin-ZAP (Cat. #IT-27) at a 4:1 biotinylated antibody to Streptavidin-ZAP molar ratio. Various cancer cell lines were incubated with the toxin at a concentration of 0.825 nM. The toxin showed specific cytotoxicity to CD44-expressing cell lines, demonstrating the efficacy of PCI in conjunction with targeted toxins to treat some cancers

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

Depletion of inflammatory dendritic cells with anti-CD209 conjugated to saporin toxin.

Alonso M, Gregorio J, Davidson M, Gonzalez J, Engleman E (2014) Depletion of inflammatory dendritic cells with anti-CD209 conjugated to saporin toxin. Immunol Res 58:374-377. doi: 10.1007/s12026-014-8511-6

Objective: To investigate a strategy that avoids monocyte intermediates to deplete inflammatory dendritic cells (DCs). Mice with an abundance of inflammatory DCs as a consequence of lipopolysaccharide exposure were treated with anti-CD209 antibody conjugated to saporin.

Summary: The results demonstrate depletion of CD209+ DCs. This strategy could prove useful for the targeted reduction of inflammatory DCs in disease.

Usage: Streptavidin-ZAP mixed with biotinylated Anti-CD209 was delivered i.v. to mice. Inflammatory DCs were markedly depleted.

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

The novel EpCAM-targeting monoclonal antibody 3-17I linked to saporin is highly cytotoxic after photochemical internalization in breast, pancreas and colon cancer cell lines.

Lund K, Bostad M, Skarpen E, Braunagel M, Krauss S, Duncan A, Hogset A, Selbo P (2014) The novel EpCAM-targeting monoclonal antibody 3-17I linked to saporin is highly cytotoxic after photochemical internalization in breast, pancreas and colon cancer cell lines. MAbs 6(4):1038-1050. doi: 10.4161/mabs.28207

Summary: The epithelial cell adhesion molecule (EpCAM) is an attractive diagnostic and therapeutic target for a wide range of human carcinomas. It has also been found on cancer stem cells, increasing the interest in targeting and eliminating cells that express it. The authors have created a monoclonal antibody that binds EpCAM, and use several assays to demonstrate the antibody's potential as an oncology tool. In one series of assays the biotinylated antibody was combined with streptavidin-ZAP (Cat. #IT-27), and in conjunction with photochemical internalization was shown to have specific cytotoxicity on several different cancer cell lines over a range of concentrations.

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

Immunoablation of cells expressing the NG2 chondroitin sulphate proteoglycan.

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. doi: 10.1111/joa.12141

Summary: In this work the authors use an antibody against the NG2-glia marker chondroitin sulphate proteoglycan (CSPG) along with Mab-ZAP (Cat. #IT-04) on cell lines and brain slices to eliminate cells expressing CSPG. The results demonstrate selective and effective killing, providing a method to study the function of these cells.

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

Deletion of naive T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance.

Hess SM, Young EF, Miller KR, Vincent BG, Buntzman AS, Collins EJ, Frelinger JA, Hess PR (2013) Deletion of naive T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance. Transpl Immunol 29(1-4):138-145. doi: 10.1016/j.trim.2013.10.005

Summary: The authors utilized biotinylated peptide-MHC class I tetramers with Streptavidin-ZAP (Cat. #IT-27) to selectively delete a specific population of alloreactive T cells in mice. Animals received iv 33-pmol injections of the toxic tetramer, and the data indicate that these toxic tetramers can prevent the induction of donor-specific responses that result in organ rejection.

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

Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties.

Bostad M, Berg K, Hogset A, Skarpen E, Stenmark H, Selbo PK (2013) Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties. J Control Release 168(3):317-326. doi: 10.1016/j.jconrel.2013.03.023

Summary: Targeted therapies for cancer can be trapped in the lysosome and compartmentalized away from the target. Photochemical internalization is a method to increase the efficacy of these compounds by releasing the therapeutic portion of the molecule from the endocytic vesicles to the cytosol by the use of light. The authors demonstrate this method on cells expressing the cancer stem cell marker CD133. Biotinylated antibodies against CD133 were combined with Streptavidin-ZAP (Cat. #IT-27) and applied to cell lines.

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

A novel model for evaluating therapies targeting human tumor vasculature and human cancer stem-like cells.

Burgos-Ojeda D, McLean K, Bai S, Pulaski H, Gong Y, Silva I, Skorecki K, Tzukerman M, Buckanovich RJ (2013) A novel model for evaluating therapies targeting human tumor vasculature and human cancer stem-like cells. Cancer Res 73(12):3555-3565. doi: 10.1158/0008-5472.CAN-12-2845

Objective: To evaluate tumor vascular markers (TVM) expression in a human embryonic stem cell–derived teratoma (hESCT) tumor model previously shown to have human vessels.

Summary: The model tested represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology.

Usage: In vitro - Anti-THY1-SAP (biotinylated Anti-THY1 mixed equimolar with Streptavidin-ZAP) was incubated with mesenchymal stem cells (MSC); resulting in statistically significant MSC death. In vivo - Anti-THY1-SAP or control (Rat IgG-SAP) was administered intravenously. Treated ovarian tumors showed delayed growth and significant reduction in central tumor viability.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Rat IgG-SAP (Cat. #IT-17)

Collagen-like cell-penetrating peptides

Yamazaki CM, Nakase I, Endo H, Kishimoto S, Mashiyama Y, Masuda R, Futaki S, Koide T (2013) Collagen-like cell-penetrating peptides. Angew Chem Int Ed Engl 52(21):5497-5500. doi: 10.1002/anie.201301266

Objective: To overcome drawbacks of cell-penetrating peptide (CPP)-conjugated forms, namely instability against attack by proteases, and binding to serum proteins that lowers the availability of the CPP to target cells.

Summary: Complexes were prepared by combining biotinylated CPPs with Streptavidin-ZAP to evaluate the impact of utilizing a rigid collagen-like triple-helical scaffold to improve CPP performance. There was low adsorption onto serum proteins and triple-helical CPPs are extremely stable in animal serum. Such unique properties are expected to be advantageous to long-term applications in cell culture systems and to in vivo drug delivery.

Usage: Biotinylated CPPs were interacted with Streptavidin-ZAP (SA-ZAP); IT-27: Streptavidin-ZAP

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

Photochemical internalization of CD133-targeting immunotoxins efficiently depletes sarcoma cells with stem-like properties and reduces tumorigenicity.

Stratford EW, Bostad M, Castro R, Skarpen E, Berg K, Hogset A, Myklebost O, Selbo PK (2013) Photochemical internalization of CD133-targeting immunotoxins efficiently depletes sarcoma cells with stem-like properties and reduces tumorigenicity. Biochim Biophys Acta 1830(8):4235-4243. doi: 10.1016/j.bbagen.2013.04.033

Summary: In this work the authors used photochemical internalization (PCI) to facilitate local cytosolic toxin release from various biotinylated-anti-CD133 antibodies coupled with Streptavidin-ZAP (Cat. #IT-27) in SW872 cells. This technique demonstrates potential for non-invasive sarcoma therapy.

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

Methods for evaluating cell-specific, cell-internalizing RNA aptamers.

Hernandez L, Flenker K, Hernandez F, Klingelhutz A, McNamara J, Giangrande P (2013) Methods for evaluating cell-specific, cell-internalizing RNA aptamers. Pharmaceuticals (Basel) 6:295-319. doi: 10.3390/ph6030295

Objective: Isolate aptamers that internalize upon binding to their cognate receptor on the cell surface

Summary: Among the methods used to characterize aptamers that internalize is a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Biotin-labeled A9g was conjugated to streptavidin-modified saporin (streptavidin-ZAP).  First, it was verified that conjugation of biotinylated aptamer to Streptavidin-ZAP (A9g-SAP) did not affect the inhibitory effect of the aptamer. Next, the effect was examined of A9g-SAP on PC3(PSMA+) and PC3(PSMA-) cells.  Cells were treated with varying amounts of aptamer-saporin conjugate for 72 h at 37°C and then an assay was performed to determine potential cytotoxicity of the conjugate.  Results confirm that A9g is internalized preferentially into target cells and that A9g is efficiently accessing the cytoplasm of target cells possibly through a mechanism of endosomal escape, resulting in inhibition of protein synthesis and ultimate cell-death.  FGF-SAP was used as a control.

Related Products: Streptavidin-ZAP (Cat. #IT-27), FGF-SAP (Cat. #IT-38)

Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type.

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. doi: 10.1186/1476-4598-12-11

Summary: The authors investigated a phenotypic antibody screening technique, in which antibodies are selected by function rather than target specificity. One facet of the screening procedure for hybridomas generated using a cancer cell line as antigen was the use of Mab-ZAP (Cat. #IT-04) to assess cell binding and internalization.

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

Targeted delivery of immunotoxin by antibody to ganglioside GD3: A novel drug delivery route for tumor cells.

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. doi: 10.1371/journal.pone.0055304

Summary: The authors used the mouse monoclonal antibody R24 against ganglioside G3 with Mab-ZAP (Cat. #IT-04) to test the viability of ganglioside G3 as a cancer therapy target. Varying concentrations of R24 were used on various cell lines with either 0.95 nM or 9.5 nM Mab-ZAP depending on the cell line.

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

Read the featured article in Targeting Trends.

Direct retino-raphe projection alters serotonergic tone and affective behavior.

Ren C, Luan L, Wui-Man Lau B, Huang X, Yang J, Zhou Y, Wu X, Gao J, Pickard GE, So KF, Pu M (2013) Direct retino-raphe projection alters serotonergic tone and affective behavior. Neuropsychopharmacology 38(7):1163-1175. doi: 10.1038/npp.2013.35

Summary: Although recent work has shown that some intrinsically photosensitive retinal ganglion cells (ipRGCs) are responsible for processing nonimage-forming visual functions, it is unclear whether the ipRGCs or conventional RGCs modulate affective behavior. The authors injected 2 μg of melanopsin-SAP (Cat. #IT-44) into each eye of gerbils, or biotinylated CTB monoclonal antibody coupled to Streptavidin-ZAP (Cat. #IT-27). The data suggest that retino-raphe signals modulate dorsal raphe nucleus serotonergic tone and affective behavior.

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

CD22 antigen is broadly expressed on lung cancer cells and is a target for antibody-based therapy.

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. doi: 10.1158/0008-5472.CAN-12-0173

Summary: The median overall survival of patients with advanced, unresectable, non-small cell lung cancer is 9-12 mos. A potential therapeutic target is CD22, a protein expressed on lung cancer cells. The authors examined the use of the monoclonal antibody HB22.7 as an antitumor agent. To assess internalization of the antibody, it was first incubated with 10 μg/ml Mab-ZAP (Cat. #IT-04) then applied to two different cancer cell lines in culture. Analysis of cell viability demonstrated that CD22 internalized when bound by the antibody-toxin complex, suggesting that targeting CD22 has therapeutic potential.

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

Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

Daniels-Wells TR, Helguera G, Rodriguez JA, Leoh LS, Erb MA, Diamante G, Casero D, Pellegrini M, Martinez-Maza O, Penichet ML (2013) Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1. Toxicol In Vitro 27(1):220-231. doi: 10.1016/j.tiv.2012.10.006

Summary: The antibody-avidin fusion protein ch128.1Av has been shown to target the human transferrin receptor 1 (TfR1) and kill malignant B cells by blocking the use of iron. Combination of this construct with a mono-biotinylated saporin custom conjugate produces an iron-independent toxicity to TfR1-expressing cells, even those that are resistant to ch128.1Av alone. The saporin-containing conjugate induces a transcriptional response consistent with oxidative stress and DNA damage. The data also show that the saporin conjugate is not toxic to human hematopoeietic stem cells.

Usage: An antibody-avidin fusion protein (ch128.1Av) was mixed with MonoBiotin-ZAP to make an immunotoxin that targets the human transferrin receptor 1 (TfR1).

Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP), Custom Conjugates

Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence.

Berstad MB, Weyergang A, Berg K (2012) Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence. Biochim Biophys Acta 1820(12):1849-1858. doi: 10.1016/j.bbagen.2012.08.027

Summary: A majority of patients develop acquired resistance to trastuzumab, the monoclonal antibody recognizing HER2, coupled to a toxin as a breast cancer therapeutic. One of the modes of resistance is that the therapeutic molecule is trapped inside an endocytic vesicle. PCI is a technique that facilitates cytosolic release of molecules in vesicles. The authors investigated the potency of biotinylated trastuzumab combined with streptavidin-ZAP (Cat. #IT-27) on several cell lines.

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

Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice

Rothenberg ME, Nusse Y, Kalisky T, Lee JJ, Dalerba P, Scheeren F, Lobo N, Kulkarni S, Sim S, Qian D, Beachy PA, Pasricha PJ, Quake SR, Clarke MF (2012) Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice. Gastroenterology 142:1195-1205.e1196. doi: 10.1053/j.gastro.2012.02.006

Objective: To investigate the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 stem cells.

Summary: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5+ stem cells.

Usage: For saporin experiments, small intestinal organoids with crypt buds and visible Paneth cells were passaged as single cells. Before embedding in Matrigel, dissociated cells were incubated with Anti-cKit-SAP (biotinylated-2B8 mixed with Streptavidin-ZAP). Treatment led to a marked decrease in organoid formation.

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

Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug.

Selbo PK, Weyergang A, Eng MS, Bostad M, Maelandsmo GM, Hogset A, Berg K (2012) Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug. J Control Release 159(2):197-203. doi: 10.1016/j.jconrel.2012.02.003

Summary: Many anti-cancer drugs are substrates of the ATP-binding cassette transporter ABCG2. Unfortunately ABCG2 is also thought to play an important role in multi-drug resistance and the protection of cancer stem cells against chemotherapeutics and photodynamic therapy. This paper examined whether photosensitizers used in photochemical internalization (PCI) are substrates for ABCG2. Streptavidin-ZAP (Cat. #IT-27) was combined with biotinylated EGF and applied to cells in culture; saporin (Cat. #PR-01) was used as a control. The data show that PCI with the EGF-saporin toxin did not utilize ABCG2 to enter cells.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Saporin (Cat. #PR-01)

Selective targeting of microglia by quantum dots.

Minami SS, Sun B, Popat K, Kauppinen T, Pleiss M, Zhou Y, Ward ME, Floreanig P, Mucke L, Desai T, Gan L ( 2012 ) Selective targeting of microglia by quantum dots. J Neuroinflammation 9(1):22 . doi: 10.1186/1742-2094-9-22

Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP)

Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells

Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang L-P, Gimotty PA, Gilks CB, Lal P, Zhang L, Coukos G (2011) Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature 475:226. doi: 10.1038/nature10169

Objective: To investigate whether a direct link between tumor hypoxia and tolerance occurs through the recruitment of regulatory cells.

Summary: Tumor hypoxia promotes the recruitment of regulatory T (Treg) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumor tolerance and angiogenesis.

Usage: In vivo depletion of CD4+ CD25+ cells was achieved by intraperitoneal administration of anti-CD25 or an immunotoxin consisting of anti-mouse CCR10 or anti-mouse CCR3 antibody conjugated at an equimolar ratio to Streptavidin–ZAP. Anti-CCR10–SAP depleted 90% of CCR101 or CCR31 cells. Anti-CCR10–SAP suppressed tumour growth and abrogated the effects of CCL28 overexpression, whereas anti-CCR3–SAP had no effect on tumor growth.

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

SIGLEC12, a human-specific segregating (pseudo)gene, encodes a signaling molecule expressed in prostate carcinomas.

Mitra N, Banda K, Altheide TK, 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. doi: 10.1074/jbc.M111.244152

Summary: Siglec 12 (sialic acid-binding immunoglobulin-like lectin 12) is a sugar molecule that has mutated in humans to be inactive, but is active in other primates. The human version is found on some macrophages, various epithelial cell surfaces, and some human carcinoma cell lines. Using Mab-ZAP (Cat. #IT-04) and monoclonal antibodies against Siglec 12, the researchers demonstrated binding and internalization in a prostate cancer cell line, indicating that Siglec 12 may be a target for some cancer therapies.

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

Sezary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-beta on the cell surface.

Chung JS, Shiue LH, Duvic M, Pandya A, Cruz PDJ, Ariizumi K (2011) Sezary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-beta on the cell surface. Blood 117(12):3382-3390. doi: 10.1182/blood-2010-08-302034

Summary: Syndecan-4 (SD-4) is a transmembrane heparan sulfate proteoglycan. The Sézary syndrome (SS) subset of cutaneous T-cell lymphoma overexpresses distinct heparan sulfate moieties, giving the authors a specific target for these cells. Biotinylated DC-HIL- Fc (the extracelluar domain of dendritic cell- associated heparan sulfate proteoglycan- integrin ligand fused to Fc of mouse IgG) was combined at a 1:1 molar ratio with streptavidin-ZAP (Cat. #IT-27). In vitro, this targeted toxin eliminated SS cells, preventing their proliferation and suggesting a method for SS treatment.

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

Human monoclonal antibodies to Sialyl-Lewisa (CA19.9) with potent CDC, ADCC, and antitumor activity.

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. doi: 10.1158/1078-0432.CCR-10-2640

Summary: In this work the authors investigated the use of a carbohydrate antigen, sialyl-Lewisa (CA19.9), as a target for cancer therapeutics. Human monoclonal antibodies were generated against CA19.9 and characterized using ELISA and flow cytometry. To assess internalization one antibody, 5B1, was combined with Hum-ZAP (Cat. #IT-22) and applied to CA19.9-expressing BxPC3 cells. The cytotoxicity of the 5B1-Hum- ZAP complex indicates that CA19.9 may be a target for cancer therapy.

Related Products: Hum-ZAP (Cat. #IT-22)

The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton.

Yang MY, Chaudhary A, Seaman S, Dunty J, Stevens J, Elzarrad MK, Frankel AE, St Croix B (2011) The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton. Biochim Biophys Acta 1813(1):39-49. doi: 10.1016/j.bbamcr.2010.11.013

Summary: Tumor endothelial marker 8 (TEM8) is a cell surface protein that is up-regulated on tumor blood vessels. Overexpression of this protein, however, produces a form that is not recognized by the SB5 monoclonal antibody used to bind TEM8. While cells expressing normal levels of TEM8 were killed by an application of biotinylated SB5 plus either 1 nM or 10 nM streptavidin-ZAP (Cat. #IT-27), cells overexpressing the protein did not bind the immunotoxin. Understanding the structural differences between the two forms of TEM8 will help in the design of therapeutic antibodies against these tumor cells.

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

Targeted delivery of saporin toxin by monoclonal antibody to the transcobalamin receptor, TCblR/CD320.

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. doi: 10.1158/1535-7163.MCT-10-0513

Summary: Vitamin B12 is necessary for cell proliferation. Cancer cells display an increased expression of TCb1R, the receptor that facilitates the intake of B12. In order to evaluate the potential of using immunotoxins to eliminate cancer cells expressing TCb1R the authors performed a series of in vitro experiments using their monoclonal antibodies plus Mab-ZAP (Cat. #IT-04). The results indicate that this is a viable therapeutic model that causes minimal peripheral damage.

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

Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity.

Kuroda K, Liu H, Kim S, Guo M, Navarro V, Bander NH (2010) Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity. Prostate 70(12):1286-1294. doi: 10.1002/pros.21164

Summary: Current treatments for prostate cancer are only moderately effective. In this work the authors examined the cytotoxic efficacy of an anti-prostate-specific membrane antigen (PMSA) antibody conjugated to saporin on PMSA-positive cell lines. hJ591, a humanized anti-PMSA antibody, was biotinylated and combined with streptavidin-ZAP (Cat. #IT-27). The hJ591-streptavidin-ZAP complex was specifically cytotoxic to PMSA-positive cell lines, and had anti-cancer activity in a xenograft model. This work demonstrates the anti-cancer potential of targeting PMSA.

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

Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate.

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.

Related Products: Mab-ZAP (Cat. #IT-04), Goat IgG-SAP (Cat. #IT-19)

Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice.

Vincent BG, Young EF, Buntzman AS, Stevens R, Kepler TB, Tisch RM, Frelinger JA, Hess PR (2010) Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice. J Immunol 184(8):4196-4204. doi: 10.4049/jimmunol.0903931

Summary: MHC class I tetramers have been used to identify antigen-specific cells. In this work the authors used a biotinylated tetramer in conjunction with streptavidin-ZAP (Cat. #IT-27) to eliminate a specific subset of reactive T cells associated with islets in vivo. NOD mice received three 4.36 µg intravenous injections of the tetramer/saporin complex over 12 days. The onset of type I diabetes in the treated mice was significantly delayed.

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

Depleting syndecan-4+ T lymphocytes using toxin-bearing dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand: A new opportunity for treating activated T cell-driven disease.

Akiyoshi H, Chung JS, Tomihari M, Cruz PD, Jr., Ariizumi K (2010) Depleting syndecan-4+ T lymphocytes using toxin-bearing dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand: A new opportunity for treating activated T cell-driven disease. J Immunol 184:3554-3561. doi: 10.4049/jimmunol.0903250

Summary: The dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) exclusively associates with syndecan-4 (SD-4), which is expressed on some activated T cells. The authors biotinylated DC-HIL and combined it with streptavidin-ZAP (Cat. #IT-27). This complex was then applied to resting or activated T cells in culture at a concentration of 10 µg/ml. Only activated T cells were bound and eliminated.

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

Read the featured article in Targeting Trends.

Photochemical internalization (PCI): a technology for drug delivery.

Berg K, Weyergang A, Prasmickaite L, Bonsted A, Hogset A, Strand MT, Wagner E, Selbo PK (2010) Photochemical internalization (PCI): a technology for drug delivery. (eds. Gomer C). In: Photodynamic Therapy. Methods in Molecular Biology. 635:133-145. Humana Press, Totowa, NJ. doi: 10.1007/978-1-60761-697-9_10

Summary: This review discusses photochemical internalization (PCI), which is a method used to overcome some of the intracellular barriers to introducing molecules into cancer cells. Some difficulties for such therapies include a low rate of release from endocytic vescicles and degradation of the therapeutic molecule by lysosomal enzymes. The use of streptavidin-ZAP (Cat. #IT-27) with a biotinylated anti-EGF receptor antibody is discussed.

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

See Also:

T-cell reconstitution without T-cell immunopathology in two models of T-cell-mediated tissue destruction.

Penaloza-MacMaster P, Masopust D, Ahmed R (2009) T-cell reconstitution without T-cell immunopathology in two models of T-cell-mediated tissue destruction. Immunology 128:164-171. doi: 10.1111/j.1365-2567.2009.03080.x

Summary: Although antigen-specific T cells are vital to adaptive immune responses, they also contribute to a variety of diseases. In this work the authors examined the possibility of selectively removing epitope-specific T cells while preserving immune function. Biotinylated MHC class I tetramers were combined with streptavidin-ZAP (Cat. #IT-27) and used in a mouse transferable T-cell-dependent neurological disease model. This technique resulted in a dramatic reduction in targeted antigen specific T cells with no observable bystander toxicity.

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

Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma.

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. doi: 10.1158/1078-0432.CCR-08-0499

Summary: Endosialin is an antigen expressed in many human cancer cell lines. As part of a wide-ranging study investigating clinical specimens, cell culture, and animal models, this group used Hum-ZAP (Cat. #IT-22) combined with a humanized anti-endosialin antibody in cell proliferation assays. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The anti-endosialin antibody and Hum-ZAP were incubated together in equimolar concentrations then applied to cells in culture. Various cancers, including synovial sarcoma, fibrosarcoma, and osteosarcoma among others, were found to express endosialin.

Related Products: Hum-ZAP (Cat. #IT-22), Mouse IgG-SAP (Cat. #IT-18)

Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells.

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. doi: 10.1593/tlo.08124

Summary: Tomoregulin is a type 1 transmembrane protein with a short cytoplasmic tail, and is found in the brain and prostate. After confirming cell surface localization by flow cytometry, and determining expression levels by whole-cell binding assays, the authors evaluated the use of tomoregulin as a target for immunotoxin therapy. Cells transfected with tomoregulin were treated with anti-tomoregulin + Mab-ZAP (IC50 = 160 pM; Cat. #IT-04) in vitro. The results demonstrate the potential for tomoregulin in prostate cancer treatment.

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

Targeting CUB domain-containing protein 1 with a monoclonal antibody inhibits metastasis in a prostate cancer model.

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. doi: 10.1158/0008-5472.CAN-07-1657

Summary: CUB domain-containing protein 1 (CDCP1) is an antigen expressed on several metastatic cancers, as well as on CD34+ and CD133+ myeloid leukemic blast cells. After demonstrating in vitro activity of the monoclonal antibody 25A11 with Mab-ZAP (Cat. #IT-04) and Hum-ZAP (Cat. #IT-22) the authors had a custom conjugation of 25A11 and saporin made for testing in mice. Goat-IgG-SAP (Cat. #IT-19) was used as a control for in vivo experiments, and saporin (Cat. #PR-01) was the control in vitro. The direct conjugate significantly inhibited tumor growth as well as metastasis in vivo.

Related Products: Mab-ZAP (Cat. #IT-04), Hum-ZAP (Cat. #IT-22), Goat IgG-SAP (Cat. #IT-19), Saporin (Cat #PR-01)

Scavenger receptor-A-targeted leukocyte depletion inhibits peritoneal ovarian tumor progression.

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. doi: 10.1158/0008-5472.CAN-06-4410

Summary: Vascular leukocytes (VLC) are immunosuppressive cells that facilitate tumor progression in ovarian cancer. One potential tumor therapy is to eliminate these cells. The authors determined that scavenger receptor-A is specifically expressed on VLCs. Mice were injected with tumor cells, as well as an anti-scavenger receptor-A antibody combined with Rat-ZAP (Cat. #IT-26). This was followed by additional treatment with the antibody-Rat-ZAP complex. Treatment with the immunotoxin eliminated VLCs, inhibited peritoneal tumor burden, and reduced ascites accumulation.

Related Products: Rat-ZAP (Cat. #IT-26)

Guanidinylated-Neomycin delivers large, bioactive cargo into cells through a heparan sulfate dependent pathway.

Elson-Schwab L, Garner OB, Schuksz M, Esko JD, Tor Y (2007) Guanidinylated-Neomycin delivers large, bioactive cargo into cells through a heparan sulfate dependent pathway. J Biol Chem 282(18):13585-13591. doi: 10.1074/jbc.M700463200

Summary: The uptake of high molecular weight drugs into cells is a stumbling block for some potential therapeutics. Using a neomycin derivative in which guanidinium groups have replaced the ammonium groups, the authors show heparan sulfate-dependent uptake of large molecules. The guanidine-neomycin was biotinylated, and incubated with streptavidin-ZAP (Cat #IT-27). This complex was effective in killing CHO cells in vitro, but was no more effective than streptavidin-ZAP alone on cells lacking heparan sulfate expression, demonstrating specificity.

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

Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin.

Hess PR, Barnes C, Woolard MD, Johnson MD, Cullen JM, Collins EJ, Frelinger JA (2007) Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin. Blood 109:3300-3307. doi: 10.1182/blood-2006-06-028001

Objective: To discover if pathogenic T cells could be selectively deleted.

Summary: A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact.

Usage: Streptavidin-SAP-coupled biotinylated tetramers were administered at low (22.2 pM) or high (66.6 pM) dose. Following the addition of Saporin Goat Polyclonal, affinity-purified FITC-labeled, T cells were subsequently incubated at either 37°C or 4°C, which permitted or prohibited endocytosis, respectively.

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

Read the featured article in Targeting Trends.

Targeted delivery and enhanced cytotoxicity of cetuximab-saporin by photochemical internalization in EGFR-positive cancer cells.

Yip WL, Weyergang A, Berg K, Tonnesen HH, Selbo PK (2007) Targeted delivery and enhanced cytotoxicity of cetuximab-saporin by photochemical internalization in EGFR-positive cancer cells. Mol Pharm 4(2):241-251. doi: 10.1021/mp060105u

Summary: Photochemical internalization (PCI) releases macromolecules from endocytic vesicles using photosensitizer activation by light. This technique allows the release of endocytosed molecules before degradation occurs in the lysosome. The authors demonstrate the proof-of-concept for this technique by combining biotinylated cetuximab (a chimeric monoclonal antibody to the EGFr) with streptavidin-ZAP (Cat. #IT-27). The conjugate was applied to three different human cancer cell lines, demonstrating enhanced specificity and toxicity against cells expressing the EGFr.

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

The nuclear DNA repair protein Ku70/80 is a tumor-associated antigen displaying rapid receptor mediated endocytosis.

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. doi: 10.1002/ijc.22212

Summary: The protein Ku70/80 is expressed in the nucleus of all cells. Tumor cell lines, however, have been shown to express Ku70/80 on the cell surface. In this study, the authors show that Ku70/80 is internalized into pancreatic carcinoma cells upon binding of the antibody INCA-X. INCA-X was combined with Mab-ZAP (Cat. #IT-04) and applied to several pancreatic carcinoma cell lines in vitro. Cell death in some of the treated lines demonstrates the potential of Ku70/80 as a therapeutic target.

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

High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells.

Collins BE, Blixt O, Han S, Duong B, Li H, Nathan JK, Bovin N, Paulson JC (2006) High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells. J Immunol 177(5):2994-3003. doi: 10.4049/jimmunol.177.5.2994

Objective: To demonstrate the dynamic equilibrium that exists between CD22 (Siglec-2) and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells.

Summary: The CD22 (Siglec-2) preferred ligand: sequence Siaa2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.

Usage: Cytotoxicity assay: BJAB lymphoma cell killing required both the targeting probe and the Streptavidin-ZAP, as no killing was observed in the absence of either.

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CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding.

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. doi: 10.1038/sj.bjc.6603222

Summary: Renal cell carcinoma (RCC) is usually resistant to chemotherapy. Using a proteomics approach, the authors found a potential target for immunotherapy in RCC. An antibody against CD70, a type II transmembrane receptor, was combined with Hum-ZAP (Cat. #IT-22). The complex was then added to an RCC-derived cell-line in vitro. The aCD70/Hum-ZAP complex demonstrated significant killing at several different concentrations. This work suggests that CD70 is a potential target antigen for RCC therapy.

Related Products: Hum-ZAP (Cat. #IT-22)

Myeloid precursors and acute myeloid leukemia cells express multiple CD33-related Siglecs.

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. doi: 10.1016/j.exphem.2006.03.003

Summary: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of cell surface receptors which bind to sialic acid. They are found mainly on leukocytes, and also on acute myeloid leukemia (AML) cells. The authors tested several anti-Siglec antibodies against U937 histiocytic lymphoma cells and THP-1 acute monocytic leukemia cells in vitro. When these antibodies were combined with Mab-ZAP (Cat. #IT-04), a second immunotoxin, the target cells were eliminated. The data suggest that Siglecs may be a viable target for AML therapy.

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

Evaluate Potential Targeting Molecules.

Kohls M (2006) Evaluate Potential Targeting Molecules. Nature Methods

Summary: Targeted toxins -- targeting agents conjugated to saporin -- are widely used to eliminate specific cell populations both in vitro and in vivo. For these molecules to be effective, it is vital that the targeting component of the conjugate specifically binds the cells of interest. A secondary conjugate, Streptavidin-ZAP, has been created by attaching the toxin saporin to streptavidin. The user can combine primary biotinylated material with Streptavidin-ZAP to quickly and economically screen potential targeting molecules for internalization and specificity. Once the appropriate targeting molecule is identified, a direct conjugation with saporin can be performed.

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Read the article.

Photochemically stimulated drug delivery increases the cytotoxicity and specificity of EGF-saporin.

Weyergang A, Selbo PK, Berg K (2006) Photochemically stimulated drug delivery increases the cytotoxicity and specificity of EGF-saporin. J Control Release 111(1-2):165-173. doi: 10.1016/j.jconrel.2005.12.002

Summary: In this study the authors investigated the use of photosensitizers located in endocytic vesicles that can be induced to release macromolecules upon activation by light. This process is called photochemical internalization, or PCI. Biotinylated EGF was combined with streptavidin-ZAP (Cat. #IT-27), and the compound was applied to various cell lines. The data shows that PCI increases the toxicity of EGF-saporin significantly in EGF receptor-expressing cell lines.

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

Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model.

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. doi: 10.1158/0008-5472.CAN-05-1221

Summary: The receptor protein tyrosine phosphatase ß (RPTPß) is overexpressed in astrocytomas, and is a potential target for tumor therapy. After testing antibodies against an extracellular domain of RPTPß in vitro with Mab-ZAP (Cat. #IT-04), two custom conjugates, 7E4B11-SAP and 7A9B5-SAP, were created by Advanced Targeting Systems. The authors tested the custom conjugates, using anti-DAT-SAP (Cat. #IT-25) as a positive control, and mouse IgG-SAP (Cat. #IT-18) as a negative control. The 7E4B11-SAP conjugate displayed significant antitumor activity in mice engrafted with U87 glioma cells.

Related Products: Mab-ZAP (Cat. #IT-04), Anti-DAT-SAP (Cat. #IT-25), Mouse IgG-SAP (Cat. #IT-18), Custom Conjugates

A cell-surface molecule selectively expressed on murine natural interferon-producing cells that blocks secretion of interferon-alpha.

Blasius A, Vermi W, Krug A, Facchetti F, Cella M, Colonna M (2004) A cell-surface molecule selectively expressed on murine natural interferon-producing cells that blocks secretion of interferon-alpha. Blood 103(11):4201-4206. doi: 10.1182/blood-2003-09-3108

Objective: To demonstrate the recruitment and function of natural interferon (IFN)-producing cells (IPCs) in murine infection models.

Summary: Incubation of IPCs with the antibody in vitro or administration of the antibody in vivo dramatically reduce secretion of IFN-α in response to deoxycytidylate-phosphatedeoxyguanylate (CpG) DNA without causing IPC depletion. Thus, the antibody identifies an IPC-specific surface molecule that, when engaged, inhibits IFN-α secretion.

Usage: Biotinylated 440c antibody mixed with Streptavidin-ZAP was applied to bone marrow-derived IPCs. Approximately 70% of CD11c+ B220hi 440c bright IPCs were depleted from culture within 36 hours.

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CEACAM6 as a novel target for indirect type 1 immunotoxin-based therapy in pancreatic adenocarcinoma.

Duxbury MS, Ito H, Ashley SW, Whang EE (2004) CEACAM6 as a novel target for indirect type 1 immunotoxin-based therapy in pancreatic adenocarcinoma. Biochem Biophys Res Commun 317(3):837-843. doi: 10.1016/j.bbrc.2004.03.128

Summary: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a cell-surface molecule that is overexpressed in a variety of human cancers. Here, the authors investigate the efficacy of a biotinylated antibody that recognizes CEACAM6 bound to streptavidin-ZAP (Cat. #IT-27) in elimination of tumor cells in vitro and in vivo. Treatment of cultured tumor cells induced significant specific cytotoxicity, while tumor growth was suppressed in a mouse xenograft model. These results indicate targeting of CEACAM6 may be a viable therapeutic strategy.

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

Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin.

Kohls MD, Lappi DA (2000) Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165. doi: 10.2144/00281pf01

Summary: Immunotoxins are useful tools for elimination of specific cell populations in vitro and in vivo for research and therapeutic applications. One of the factors limiting the use of immunotoxins is the selection of an appropriate antibody. Advanced Targeting Systems has created a reagent that allows researchers to select antibodies with the desired characteristics before an immuntoxin is made, purified, and assayed. Using a goat anti-murine IgG coupled to the ribosome-inactivating protein saporin, researchers can screen hundreds of antibodies in a time and cost-effective manner.

Related Products: Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), Hum-ZAP (Cat. #IT-22), Rat-ZAP (Cat. #IT-26), Anti-M-ZAP (Cat. #IT-30), Goat-ZAP (Cat. #IT-36)

Internalization of monoclonal antibodies selected for immunotoxin activity against small-cell lung cancer.

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. doi: 10.1089/hyb.1992.11.547

Related Products: Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), Hum-ZAP (Cat. #IT-22), , Rat-ZAP (Cat. #IT-26), Anti-M-ZAP (Cat. #IT-30)

Ribosome-inactivating proteins from plants: present status and future prospects.

Stirpe F, Barbieri L, Battelli MG, Soria M, Lappi DA (1992) Ribosome-inactivating proteins from plants: present status and future prospects. Bio/Technol 10:405-412. doi: 10.1038/nbt0492-405

Related Products: Saporin (Cat. #PR-01), Saporin Chicken Polyclonal, affinity-purified (Cat. #AB-17AP), Saporin Goat Polyclonal (Cat. #AB-15), MonoBiotin-ZAP (Cat. #BT-ZAP)

Selective killing of CD4+ and CD8+ cells with immunotoxins containing saporin.

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. doi: 10.1111/j.1365-3083.1989.tb01223.x

Related Products: Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), Hum-ZAP (Cat. #IT-22), Rat-ZAP (Cat. #IT-26), Anti-M-ZAP (Cat. #IT-30)

Rapid screening with indirect immunotoxin for monoclonal antibodies against human small cell lung cancer.

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.

Related Products: Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), Hum-ZAP (Cat. #IT-22), Rat-ZAP (Cat. #IT-26), Anti-M-ZAP (Cat. #IT-30)

Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies.

Lappi DA, Esch FS, Barbieri L, Stirpe F, Soria M (1985) Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies. Biochem Biophys Res Commun 129:934-942. doi: 10.1016/0006-291x(85)91981-3

Related Products: Saporin (Cat. #PR-01), Saporin Chicken Polyclonal, affinity-purified (Cat. #AB-17AP), Saporin Goat Polyclonal (Cat. #AB-15), MonoBiotin-ZAP (Cat. #BT-ZAP)

Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort) of Agrostemma githago L. (corn cockle) and of Asparagus officinalis (asparagus) and from the latex of Hura crepitans L. (sandbox tree).

Stirpe F, Gasperi-Campani A, Barbieri L, Falasca A, Abbondanza A, Stevens WA (1983) Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort) of Agrostemma githago L. (corn cockle) and of Asparagus officinalis (asparagus) and from the latex of Hura crepitans L. (sandbox tree). Biochem J 216:617-625. doi: 10.1042/bj2160617

Related Products: Saporin (Cat. #PR-01), Saporin Chicken Polyclonal, affinity-purified (Cat. #AB-17AP), Saporin Goat Polyclonal (Cat. #AB-15), MonoBiotin-ZAP (Cat. #BT-ZAP)