Abstract 2484: IR700-conjugated anti-CD44 monoclonal antibody for phototherapy theranostics of triple-negative breast cancer
Authors: Jin Jiefu , Krishnamachary Balaji , Kobayashi Hisataka , Bhujwalla Zaver 
Published: August 2015

CD44 transmembrane glycoproteins are cell adhesion molecules that play a role in cancer cell proliferation, migration, and invasion.1 CD44 expression is associated with stem-like breast cancer cells1 and is upregulated under hypoxia2 making it a significant target. Monoclonal antibody (mAb)-linked to photosensitizers have achieved targeted phototherapy.3 We developed a CD44 mAb-targeted photosensitizer complex for combined fluorescent detection and phototherapy (PT) of CD44 expressing triple negative breast cancer cells. The mAb conjugate is activated by near-infrared (NIR) light, and demonstrates phototoxic effects selectively in CD44 expressing breast cancer cells following cell membrane binding.

CD44 mAb was conjugated with a NIR phthalocyanine dye, IR700, to form CD44-IR700. MB-MDA-231 (CD44 positive), MCF-7 and BT-474 (CD44 negative) were selected for the cell studies. SDS-PAGE, UV spectroscopy and confocal fluorescence imaging were performed to determine purity, composition and cellular localization of the conjugate. Microscopic observation and cell viability tests were performed to study CD44-specific cell death. Tumor models were established by inoculating 2×106 MB-MDA-231 or BT-474 cells in the upper-right mammary fat pad of athymic female mice. In vivo fluorescence imaging was performed on a Li-Cor Pearl® Impulse scanner. Tumor bearing mice (n = 5 per group) injected with PBS, IR700, anti-CD44 mAb, IgG-IR700 and CD44-IR700 were monitored over a 3 week-period and sacrificed to harvest tumors. NIR light at a dose of 30 J/cm2 was applied on day 1 after injection.

CD44-IR700 demonstrated preferential binding to the cell membrane of MB-MDA-231 but not MCF-7 or BT-474 cells. CD44-specific cell death was initiated by CD44-IR700-mediated PT (&gt;90% of MB-MDA-231 cells were killed immediately, while 88% of MCF-7 cells and 90% of BT-474 cells were alive). Irradiation itself was harmless to cells and there was no cytotoxicity associated with IR700 and CD44 antibody or with CD44-IR700 in the absence of irradiation. In vivo fluorescence imaging verified the 3-fold increased retention of CD44-IR700 in MB-MDA-231 tumors compared to BT-474 tumors. CD44-IR700 injection together with a therapeutic dose of NIR light exposure resulted in significant growth delay in MB-MDA-231 tumors, but not BT-474 tumors. We did not observe tumor growth delay in separate groups of mice injected with PBS, IR700, anti-CD44 mAb, or IgG-IR700, irrespective of NIR light application. Histological studies confirmed a significant increase of necrosis in the treated MDA-MB-231 tumors.

This novel PT theranostic strategy provides a promising opportunity for treating CD44 expressing primary and metastatic triple negative breast cancer.

References: 1. Louderbough et al, Mol Can Res, 2011; 2. Krishnamachary et al, PloS one, 2012; 3. Mitsunaga et al, Nat Med, 2011.

Supported by NIH R01 CA136576 and P50 CA103175.

Citation Format: Jiefu Jin, Balaji Krishnamachary, Hisataka Kobayashi, Zaver Bhujwalla. IR700-conjugated anti-CD44 monoclonal antibody for phototherapy theranostics of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2484. doi:10.1158/1538-7445.AM2015-2484