cancer-research

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 PMID: 909090

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)

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 PMID: 29771460

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:

• Kuroda K et al. Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity. Prostate 70(12):1286-1294, 2010.

Araldi D, Ferrari LF, Levine JD (2018) Role of GPCR (mu-opioid)-receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II). Pain 159(5):864-875. doi: 10.1097/j.pain.0000000000001155

Objective: To determine the the mechanisms mediating the induction of opioid-induced hyperalgesia and the prolongation of prostaglandinE2-induced hyperalgesia in type II hyperalgesic priming.

Summary: Understanding the mechanisms responsible for the induction of type II hyperalgesic priming, a form of neuroplasticity in the peripheral terminal of the primary afferent nociceptor, may provide useful information for the design of drugs with improved therapeutic profiles to treat neuroplasticity induced by chronic use of opioids.

Usage: SSP-SAP was prepared in saline (5 ng/mL), and 20 mL was injected intrathecally into rats, 14 days before nociceptive tests.

Related Products: SSP-SAP (Cat. #IT-11)

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)

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 PMID: 29313073

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)

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

Lund K, Olsen CE, Wong JJW, Olsen PA, Solberg NT, Høgset A, Krauss S, Selbo PK (2017) 5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin. J Exp Clin Cancer Res 36(1):185.. doi: 10.1186/s13046-017-0662-6

Objective: Investigate resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma.

Summary: Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and Anti-CD105-SAP was assessed using microscopy. MTS assay was used to investigate cytotoxic effects of photochemical internalization of Anti-CD105-SAP. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells using Anti-CD105-SAP in vitro. PCI-based targeting of CD105 may represent a potent anti-cancer strategy and should be further evaluated in preclinical models.

Usage: Cells were seeded (3000/well) in 96-well plates and allowed to attach overnight. The cells were incubated with the Anti-CD105-saporin (2.4 nM) or Saporin as a control (6.48 nM; Saporin was added in a molecular ratio of 2.7:1 to the immunotoxin) giving an equal ratio of Saporin to immunotoxin), with or without the photosensitizer TPCS2a (0.35 μg/ml) for 18 h.

Related Products: Anti-CD105-SAP (Cat. #IT-80)

Shramm PA, Ancheta L, Higgins D, Lappi DA (2017) A rapid, pH-sensitive screening method to detect internalization of cell surface markers for development of antibody-based pharmaceuticals to treat brain tumors. Neuroscience 2017 Abstracts 566.24 / H7. Society for Neuroscience, Washington, DC.

Summary: Some of the most potent treatments for cancers have been antibodies to cell surface proteins that cause tumor cell proliferation. Examples are cetuximab (antigen: EGFR) approved for colorectal cancer and Trastuzumab (ERBB2) for breast cancer. These antibodies have more than one effect on the cancer cell, but one of the most important is that, upon binding to the cell surface antigen, the complex is internalized by so-called antibody mediated internalization. As such, the mitogenic cell surface protein no longer plays a role in cancer cell division. Despite the blood brain barrier challenging systemic treatment for brain tumors, intracerebroventricular injection can produce similar results. For example, Gholamin et al., (Sci Transl Med 9:381, 2017) and Kang et al. (Sci Rep 6:34922, 2016) reported down-regulation of brain tumor mitogenic agents through antibody-mediated endocytosis. The quick and efficient screening of antibodies that internalize effectively is vital for determining suitability of an antibody as a therapeutic targeting agent. Here we describe a method for the efficient determination of internalization of cell surface molecules by antibodies using a pH-dependent fluorescent reporter cross-linked to a secondary antibody in a plate-based assay with visualization of internalization in hours. This conjugate is comprised of an affinity-purified monovalent secondary antibody against both the heavy and light chain of human or mouse IgG and is conjugated to a pH -dependent fluorescent reporter. The fluorescence from this reporter increases intensity as the pH of its surroundings becomes more acidic, as evident when exposed to the environment inside a cell (thereby providing evidence of internalization). A successful assay protocol has been developed to provide an EC50 by way of a fluorescence-detecting plate reader, which could be used to explore antibody candidates as therapeutics in a quick and reproducible manner.

Related Products: Fab-pHast human (Cat. #PH-01)

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See Also:

• Gholamin S et al. Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors. Sci Transl Med 9(381):eaaf2968, 2017.
• Kang BR et al. Cell surface GRP78 as a biomarker and target for suppressing glioma cells. Sci Rep 6:34922, 2016.
• Ancheta LR et al. Basigin-2 (EMMPRIN), a prognostic marker, is a dynamic portal of entry into cancer cells. Cancer Res 71(8):5218, 2011. Proceedings of the American Association for Cancer Research Annual Meeting, Orlando, FL
• Ancheta L et al. Method for screening neuronal tumor cell surface markers for high specificity and rapid internalization as potential oncologic treatments. Neuroscience 2017 Abstracts 612.11 / SS46, 2017. Society for Neuroscience, Washington, DC

Ancheta L, Shramm PA, Lappi DA (2017) Method for screening neuronal tumor cell surface markers for high specificity and rapid internalization as potential oncologic treatments. Neuroscience 2017 Abstracts 612.11 / SS46. Society for Neuroscience, Washington, DC.

Summary: Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules involved in the growth, progression, and spread of the tumor. These therapies are often cytostatic; they block tumor cell proliferation as opposed to chemotherapy that kills the cells. A primary approach to identify potential targets is the ability to compromise a ligand/receptor relationship that causes tumor cell proliferation. There are now many examples of the use of antibodies in tumor therapy to cause a breakdown in that relationship. In clinical use against brain tumors are antibodies to cell-surface EGFR, VEGFR, PDGFR, and c-kit. These work by down-regulation of the receptor by antibody-mediated internalization. It is crucial for development of a targeted therapy to have a method to determine the suitability of an antibody to cause internalization rapidly and completely. Here we describe a method for the efficient determination of internalization of cell surface molecules by antibodies: a cytotoxicity assay utilizing an antibody labeling method to streamline the process of multiple candidate screening. Cells are chosen that have significant levels of expression of the desired marker and the assay readout is definitive: cell death is demonstrated in 72 hours. This method is designed for the rapid screening of multiple antibodies for specificity and internalization in neuronal tumor cells to explore antibody candidates as therapeutics in a quick and reproducible manner.

Related Products: Fab-pHast human (Cat. #PH-01)

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)