cancer-research

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)

Yu X, Liu Z, Hou R, Nie Y, Chen R (2017) Nerve growth factor and its receptors on onset and diagnosis of ovarian cancer. Oncol Lett 14(3):2864-2868. doi: 10.3892/ol.2017.6527 PMID: 28928825

Objective: To investigate the effect of nerve growth factor (NGF) and its receptors on the onset and diagnosis of ovarian cancer.

Summary: NGF and its receptor can contribute to the occurrence of ovarian cancer, and the onset condition of ovarian cancer can be diagnosed through the detection of high or low expression of NGF and its receptors.

Usage: ELISA, WB, IHC (1:500)

Related Products: NGFR (mu p75) Rabbit Polyclonal, affinity-purified (Cat. #AB-N01AP)

Lamb YN (2017) Inotuzumab ozogamicin: First global approval. Drugs 77(14):1603-1610. doi: 10.1007/s40265-017-0802-5 PMID: 28819740

Summary: Inotuzumab ozogamicin, an anti-CD22 monoclonal antibody-calicheamicin conjugate, has received its first global approval as a promising treatment option. This novel therapy shows potential for addressing certain hematological malignancies and represents a significant advancement in the field of cancer treatment.

Casciello F, Al-Ejeh F, Kelly G, Brennan D, Ngiow S, Young A, Stoll T, Windloch K, Hill M, Smyth M, Gannon F, Lee J (2017) G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis. Proc Natl Acad Sci U S A 114:7077-7082. doi: 10.1073/pnas.1618706114 PMID: 28630300

Usage: Western blot

Related Products: Trans-4-Hydroxy-L-Proline Rabbit Polyclonal, Conjugated (Cat. #AB-T044)

Yao P, Ding Y, Han Z, Mu Y, Hong T, Zhu Y, Li H (2017) Suppression of asparaginyl endopeptidase attenuates breast cancer-induced bone pain through inhibition of neurotrophin receptors. Mol Pain 13:744806917708127. doi: 10.1177/1744806917708127 PMID: 28554249

Objective: To determine the functions and targeted suppression of asparaginyl endopeptidase (AEP) in a mouse model of breast cancer-induced bone pain.

Summary: Targeted suppression of AEP with specific small compounds significantly reduced bone pain while purified recombinant AEP proteins increased bone pain. AEP aggravates the development of breast cancer bone metastasis and bone pain by increasing the expression of neurotrophin receptors. AEP might be an effective target for treatment of breast cancer-induced bone pain.

Usage: Western blot

Related Products: NGFR (mu p75) Rabbit Polyclonal, affinity-purified (Cat. #AB-N01AP)

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

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

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

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

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

Havelin J, Imbert I, Sukhtankar D, Remeniuk B, Pelletier I, Gentry J, Okun A, Tiutan T, Porreca F, King T (2017) Mediation of movement-induced breakthrough cancer pain by IB4-binding nociceptors in rats. J Neurosci 37:5111-5122.. doi: 10.1523/JNEUROSCI.1212-16.2017

Objective: To define a novel preclinical measure of movement-induced breakthrough pain (BTP) that is observed in the presence of morphine controlling ongoing pain.

Summary: Novel compounds targeting IB4-binding nociceptors may improve pain management for cancer pain patients and other patient populations suffering from BTP that is inadequately treated by currently available medications.

Usage: To determine the effect of eliminating input from IB4-binding fibers, separate groups of rats received spinal administration of IB4-SAP or the control, Blank-SAP (3.2 mcg/20 mcl saline) followed by a 10 mcl flush of saline. Movement of an air bubble placed between drug solution and saline was used to monitor progress of the injection.

Related Products: IB4-SAP (Cat. #IT-10), Blank-SAP (Cat. #IT-21)

Burma NE, Bonin RP, Leduc-Pessah H, Baimel C, Cairncross ZF, Mousseau M, Shankara JV, Stemkowski PL, Baimoukhametova D, Bains JS, Antle MC, Zamponi GW, Cahill CM, Borgland SL, De Koninck Y, Trang T (2017) Blocking microglial pannexin-1 channels alleviates morphine withdrawal in rodents. Nat Med 23:355-360.. doi: 10.1038/nm.4281

Summary: The authors investigated the mechanisms underlying opiate withdrawal in rat. Depletion of spinal lumbar microglia by intrathecal injections of Mac-1–SAP (Cat. #IT-33; 20 mcg) decreased withdrawal behaviors and attenuated the severity of withdrawal without affecting morphine antinociception. Unconjugated Saporin (Cat. #PR-01; 20 mcg) was used as control and had no effect on spinal CD11b immunoreactivity or naloxone-induced morphine withdrawal.

Related Products: Mac-1-SAP rat (Cat. #IT-33), Saporin (Cat. #PR-01)