COMPOSITIONS AND METHODS RELATED TO xCT ANTIBODIES

A technology of antibodies and antibody fragments, applied in the fields of YFTTI, QTQNFKDAFSGRDSSITRLP, can solve the problems of SASP instability, off-target effects, low bioavailability, and insolubility

Pending Publication Date: 2020-05-22
AGILVAX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, SASP is unstable and insoluble under physiological conditions, has large off-target effects, low bioavailability, and requires high doses to inhibit xCT in vivo (Timmerman et al., Cancer Cell 2013, 24(4):450-65 ; Shitara et al., Gastric Cancer 2016; Linares et al., Expert Opin Drug Saf. 2011, 10(2):253-63; Robe et al., BMC Cancer 2009, 9:372)

Method used

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  • COMPOSITIONS AND METHODS RELATED TO xCT ANTIBODIES
  • COMPOSITIONS AND METHODS RELATED TO xCT ANTIBODIES
  • COMPOSITIONS AND METHODS RELATED TO xCT ANTIBODIES

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Experimental program
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Embodiment approach

[0071] In one embodiment, according to the present invention "K D " or " K D "values" are measured by radiolabeled antigen binding assays (RIA) performed with antibodies and Fab forms of antigen molecules, as described in the following assay, by titrating a series of unlabeled antigens with minimal concentrations of ( 125 1) Labeled antigen equilibrates Fab, then captures bound antigen with anti-Fab antibody-coated plates to measure solution binding affinity of Fab to antibody (Chen et al., (1999) J. Mol. Biol 293:865-881). To establish assay conditions, microtiter plates (Dynex) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then washed with 2 wt. BSA solution in PBS / vol% for two to five hours of blocking. In non-binding plates (Nunc#269620), 100pM or 26pM [ 125 I] Antigen is mixed with serial dilutions of Fab of interest. The Fab of interest is then incubated overnight. However, the incubation may be con...

Embodiment 1

[0146]To investigate whether antibodies induced by xCT VLPs affect xCT function and BCSC biology, IgG was purified from serum of control VLP or xCTVLP-treated mice. BCSC-enriched cells from the indicated sTNBC cell lines were incubated with purified antibodies, and 3D cultures were used to analyze BCSC self-renewal and proliferation, while the concentration of aldefluor-positive cells (a marker of BCSC phenotype) in spheroids was measured by FACS. Frequency and intercellular ROS levels (indicators of xCT function). As observed with other methods of xCT inactivation (siRNA, SASP treatment and DNA vaccination), xCT VLP-induced IgG antibodies reduced BCSC self-renewal, proliferation and increased ROS levels ( figure 1 ). The number of BCSCs was also significantly reduced in tumorspheres formed in cultures treated with xCT VLP-induced antibodies.

[0147] figure 1 showed that AX09 (an xCT epitope displayed on RNA phage VLP)-induced antibodies affects BCSC biology and inhibits...

Embodiment 2

[0207] Generation of monoclonal antibodies against xCT. Female BALB / c mice (Charles River Laboratory) were kept at the Center for Molecular Biotechnology, University of Turin, and were handled in accordance with the University Ethics Committee and European Guidelines 2010 / 63. Vaccination involved an intramuscular electroporation of the pVAX1-xCT DNA plasmid (encoding full-length mouse xCT protein), as previously described (Lanzardo et al., 2016). Starting on day 10 after DNA vaccination, mice were boosted 6 times per month with AX09-0M3 VLP (anti-third extracellular domain of human xCT protein; ECD3). Mice were bled before the first VLP vaccination and then two weeks after each vaccination, and serum was collected and stored at −20°C for subsequent analysis.

[0208] To test the functional effect of vaccination-induced antibodies, 4T1-derived tumorspheres enriched in xCT+ cancer stem cells (CSCs) were treated with medium alone, the xCT pharmacological inhibitor sulfasalazine ...

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Abstract

Certain embodiments are directed to therapeutic compositions having an xCT specific antibody.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 492,912, filed May 1, 2017, the entire contents of which are incorporated herein by reference. Background technique [0003] Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that lacks estrogen, progesterone, and HER2 receptors and accounts for 15% to 20% of all breast cancers in the United States. TNBC has a higher recurrence rate and poorer prognosis than other forms of breast cancer, and due to the lack of targetable surface receptors, TNBC is resistant to hormone and HER2-targeting therapies. The particularly aggressive features of TNBC may be due to the enrichment of cancer stem cells (CSCs), which possess unique biological properties necessary for the maintenance and spread of tumors, and which, through asymmetric division, can differentiate into cancer cells that make up the tumor mass ( Magee et al., Cancer...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/46C07K16/30
CPCC07K16/18C07K16/3015C07K2317/24C07K2317/622
Inventor 费德丽卡·佩里克莱约翰·奥罗克费德丽卡·卡瓦洛
Owner AGILVAX INC
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