Page 61 - D. Cancer biology

Development of Antibody Drug Conjugate Targeting c-Kit and
Validation of Effectiveness in Cancer

Jin-Ock Kim , and Sang Gyu Park 1,2
1
1 College of Pharmacy, Ajou University, Gyeonggi-do, 16499, Republic of Korea
2 NoveltyNobility, Gyeonggi-do, 13477, Republic of Korea
BACKGROUND METHODS

c-Kit is a member of the Type III receptor tyrosine kinase family. c-Kit is activated by Antibody Internalization Assay. GIST cells were stained with PE-labeled
stem cell factor (SCF), which plays a critical role in the differentiation, proliferation, NN2101 at 4℃. After washing, cells were incubated at 37℃ for 30minutes.
and survival of hematopoietic stem cells, melanocytes, and germ cells. Gain-of- And then cells were fixed and stained with APC-labeled LAMP-1 antibody,
function mutations resulting in constitutive c-Kit activation play a central pathogenic lysosomal marker.
role in gastrointestinal stromal tumors (GIST), subsets of melanoma, acute ADC Conjugation. Mixture of NN2101 and SMCC-DM1 (MedChemExpress)
myelogenous leukemia (AML), and acute systemic mastocytosis (ASM). was incubated in sodium phosphate buffer. NN2101-DM1 was separated
Imatinib which targets c-Kit, platelet‐derived growth factor receptor (PDGFR), and using PD-10 desalting column (GE Healthcare).
BCR‐ABL, was approved as a first‐line treatment and shown to induce a good Cytotoxicity Assay. Cells were plated onto 96-well plate (3000~8000
response in patients including leukemia, GIST, and systemic mastocytosis. Although cells/well) and incubated with NN2101 and NN2101-DM1 (up to 400 ug/ml) at
Imatinib is successful as therapeutics, some patients occurring c-Kit mutations within 37℃ for 3 or 4 days. Live cells were stained with Calcein AM (Invitrogen) or
2 to 5 years after starting therapy have been shown resistance to Imatinib. Of c-Kit Hoechst 33342 (Thermo Scientific) and counted using Celigo Imaging
mutations, activating mutation not only induces SCF-independent c-Kit activation but Cytometer (Nexcelom Bio-science) to determine the cell viability.
also is one of the causes of resistance to anti-tumor drug. In Vivo Studies in Tumor Xenograft model. Cancer cells mixed with
Here, we characterized a human anti-c-Kit antibody (NN2101) that blocks the SCF/c- Matrigel (Corning) were injected subcutaneously into right flank of 6-wk-old
Kit pathway following the high affinity (KD = 2.83 x 10 -12 M) against the CB17-SCID mice. When tumor approximately 200 mm in size, the mice were
3
immunoglobulin-like domain 2 of c-Kit. Also, we developed Antibody Drug Conjugate treated with NN2101, NN2101-DM1, IgG-DM1, and Imainib via i.V. or oral
(ADC) using SMCC-DM1 and examined its therapeutic efficacy in vitro and in vivo. administration. Tumor size were measured once in three days with a caliper.
RESULTS

A B C *
c-Kit Binding test

Fig3. Generation of anti-c-Kit
Antibody Drug Conjugate (ADC).
*DAR: Drug to Antibody Ratio

Imatinib-resistant tumor model

Fig1. FACS analysis for c-Kit expression
in GIST, Leukemia, and SCLC cell lines.

Imatinib-sensitive tumor model

Fig2. Internalization of NN2101 in cancer Fig4. In vitro cytotoxicity of anti-c-Kit ADC in c-Kit Fig5. In vivo efficacy of anti-c-Kit ADC in Imatinib-
resistant (A, B) and –sensitive (C, D) tumor xenograft
cells. positive and negative cell lines. model.
CONCLUSION REFERENCES Contact information
1. Characterization of NN2101 including cell 1. Abrams T, et al. Clinical cancer. 2018; 24(17):4297- Jin-Ock Kim
4308.
binding and internalization. Tel : +82-31-219-3491
2. Edris B, et al. PNAS. 2013; 110(9):3501-3506.
2. Generation of NN2101 ADC and charac- 3. Lebron MB, et al. Cancer biology & therapy. 2014; E-mail : kjo8909@ajou.ac.kr
15(9):1208-1218.
terization including DAR, in vitro and in vivo Sang Gyu Park
4. London CA, et al. Clinical cancer research. 2017;
efficacy. 23(10):2565-2574. Email : sgpark@ajou.ac.kr