Photosensitizing antibody-fluorophore conjugates

An antibody and antigen technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, drug combinations, etc., can solve problems such as side effects

Pending Publication Date: 2018-04-20
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, dose-limiting toxicity is ultimately related to the biodistribution and catabolism of the antibody conjugate
[0007] Conventional photodynamic therapy (PDT), which combines photosensitizers with the physical energy of non-ionizing light to kill cells, is less commonly used as a cancer therapy because current non-targeted photosensitizers are also absorbed by normal tissues, causing Serious side effects, although the excitation light itself is harmless in the near-infrared (NIR) range (Figure 9)

Method used

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  • Photosensitizing antibody-fluorophore conjugates
  • Photosensitizing antibody-fluorophore conjugates
  • Photosensitizing antibody-fluorophore conjugates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0191] Synthesis of IRDye 700-conjugated trastuzumab (anti-Her2)

[0192] This example describes the method used to conjugate the monoclonal antibody trastuzumab to IRye 700DX NHS Ester. Those skilled in the art will recognize that any antibody, such as any monoclonal antibody specific for a target cell surface protein, can be conjugated to the IRDye 700DX NHS Ester using similar methods.

[0193] The humanized HER2 antibody trastuzumab (Tra; Genentech, San Francisco, CA) (1 mg, 6.8 nmol) was mixed with IRDye 700DX NHS Ester (IR700; LI-COR Bioscience, Lincoln, NE) (66.8 μg, 34.2 nmol, 5mmol / L in DMSO) in 0.1mol / L Na 2 HPO 4 (pH 8.5) for 30-120 minutes at room temperature. Trastuzumab is a recombinant humanized monoclonal antibody (mAb) directed against the extracellular domain of the human epidermal growth factor receptor (EGFR) 2 (HER2) tyrosine kinase receptor. The mixture was purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ). Protein concentrat...

Embodiment 2

[0197] Selectively kills HER2+ cells

[0198] This example is described to demonstrate that the trastuzumab-IR700 compound described in Example 1 (referred to herein as Tra-IR700) can be used to selectively kill cells expressing HER2 (HER2+) while being negative for HER2 (HER2-) Cellular approach with minimal side effects.

[0199] NIH3T3 (3T3 / HER2+) cells transfected with HER2 gene were used for targeted photodynamic therapy (PDT). Balb / 3T3 cells expressing DsRed fluorescent protein but not expressing HER2 (Balb / 3T3 / DsRed) were used as a control. Incubate at 37°C in a humidified incubator in an atmosphere of 95% air and 5% carbon dioxide in tissue culture dishes in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin cell.

[0200] Fluorescence microscopy was performed with a BX51 or IX81 microscope (Olympus America, Melville, NY). Filters were set to detect IR700 and consisted of a 590-650 nm excitation filter and a 665-740 nm bandpass emissi...

Embodiment 3

[0205] Determining the radiation dose

[0206] To determine phototoxicity in response to different light exposure doses, use Fixable Green Dead Cell Stain Kit for flow cytometry of PDT-treated 3T3 / HER2+ cells. A LIVE / DEAD assay, which detects cells with damaged cell membranes, was performed within one hour of the treatment. As shown in Figure 1d, cell death in response to Tra-IR700-mediated PDT was light-dose-dependent after 1 h of PIT. Cells showed no significant phototoxicity in the absence of PIT or Tra-IR700.

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Abstract

The present disclosure relates to compositions and methods of killing cells. In particular examples, the method includes contacting a cell having a cell surface protein with a therapeutically effective amount of an antibody-IR700 molecule, wherein the antibody specifically binds to the cell surface protein, such as a tumor-specific antigen on the surface of a tumor cell. The cell is subsequently irradiated, such as at a wavelength of 660-740 nm at a dose of at least 1 J cm<-2>. The cell is also contacted with one or more therapeutic agents (such as an anti-cancer agent), for example about 0 to8 hours after irradiating the cell, thereby killing the cell. Also provided are methods of imaging cell killing in real time, using fluorescence lifetime imaging. Also provided are wearable devices that include an article of clothing, jewelry, or covering; and an NIR LED incorporated into the article, which can be used with the disclosed methods.

Description

[0001] This application is a divisional application of an invention patent application with an application date of June 27, 2012, an application number of 201280043973.2, and an invention title of "photosensitive antibody-fluorophore conjugate". [0002] Cross References to Related Applications [0003] This application is a continuation-in-part of U.S. Application No. 13 / 180,111, filed July 11, 2011, which claims priority to U.S. Provisional Application No. 61 / 363,079, filed July 9, 2010, both incorporated by reference way into this article. technical field [0004] The present application relates to antibody-IR700 conjugates and methods of using antibody-IR700 conjugates to kill cells specifically bound to the antibodies following irradiation with infrared (NIR) light. Also provided are devices incorporating NIR light emitting diodes (LEDs) that can also be used with the conjugates and methods of the present disclosure. Background technique [0005] In 2007, about 13% of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K41/00A61K47/68A61K49/00A61P35/00
CPCA61K41/0071A61K49/0032A61K49/0058C07K16/2863C07K16/3069C07K16/32C07K2317/24C07K2317/73A41D13/0002A41D2400/32A44C15/00A61K2039/505A61K47/6803A61K47/6869C07K16/30
Inventor 小林久隆P·库伊克M·博纳多
Owner UNITED STATES OF AMERICA
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