Antibody-Drug Conjugate Targeting c-Kit

Antibody-Drug Conjugate Targeting c-Kit: Comparison

Please note this is a comparison between Version 3 by Rita Xu and Version 2 by Kwang-Hyeok Kim.

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC)

accounts for 15–25% of all lung cancers. It exhibits a rapid doubling time and a high degree of

invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study,

we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic

agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit

and specifically binds to SCLC cells expressing c-Kit with a binding affinity of KD = 5.5 10?9 M.

Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently

induced apoptosis in SCLC with an IC50 ranging from 158 pM to 4 nM. An in vivo assay using a

xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59%

(5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide

or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken

together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment.

c-Kit
small cell lung cancer
monoclonal antibody
antibody-drug conjugate

1. Introduction

Lung cancer is the leading cause of cancer-related deaths in the Western world and

is classified into two groups: small cell lung cancer (SCLC) and non-SCLC (NSCLC) ^[1].

SCLC, a neuroendocrine tumor, is distinguished from NSCLC by its rapid tumor growth,

high degree of invasiveness and early development of widespread metastases ^[2]. SCLC

is distinctly different from extrapulmonary small cell carcinoma with respect to disease

progression, prognosis, and etiology ^[3]. Without proper treatment, the life expectancy of

SCLC patients is less than four months. Although the five-year relative survival rate has

improved by 7% over the last few decades, it remains extremely poor ^[4].

A variety of molecular markers have been implicated in the pathogenesis and prognosis

of SCLC [5,6]^[5][6]. Paracrine or autocrine signal transduction pathways are widely used

to explain dysregulated SCLC growth ^[6]. In addition, tumor protein p53, retinoblastoma

protein, NOTCH, MYC, and phosphatidylinositol 3-kinase (PI3K) are aberrantly mutated

in SCLC; however, well-established etiological factors, such as EGFR mutations that occur

in NSCLC, have not been identified [7–10]^{[7][8][9][10]}. SCLC has a very aggressive course and is characterized

by genomic instability, increased vascularity, and a high metastatic potential ^[11].

Consequently, most SCLC patients already present with metastatic disease outside of the

chest, at the time of diagnosis, which results in premature death ^[12]. In addition, most

SCLC patients are current or former heavy smokers, which is associated with a high tumor

mutational burden, with C:G > A:T transversions being the most common type of base

substitutions [13,14]

^[13][14]

The c-Kit proto-oncogene encodes a transmembrane tyrosine kinase growth factor receptor

that belongs to the platelet-derived growth factor receptor (PDGFR) family [15,16]^[15][16]. Its ligand stem cell factor (SCF) is a hematopoietic growth factor that promotes the proliferation of

multiple hematopoietic stem cells [17,18]^[17][18]. In addition, c-Kit activity is dysregulated in

various cancers [19,20]^[19][20]. Previous studies reported that the expression of c-Kit containing

oncogenic mutations is either dysregulated and/or up-regulated in various cancers, which

results in SCF-independent c-Kit activation and an aggressive form of cancer ^[20]. Interestingly,

a variety of evidence indicates that SCLC cell lines and tumors express both the

c-Kit receptor and SCF mRNA, suggesting that these gene products constitute an autocrine

loop that mediates tumor cell survival and growth [21,22]^[21][22]. Although SCLC is significantly

correlated with smoking, it does not contain oncogenic c-Kit mutations. Immunohistochemical

staining showed that overexpression of c-Kit occurs in 70% of SCLC patients [23,24]^[23][24].

Imatinib, which was developed to target BCR-ABL, platelet-derived growth factor receptor

(PDGFR), and c-Kit, is currently used to treat chronic myeloid leukemia, acute lymphoid

leukemia, gastrointestinal stromal tumor (GIST), and hypereosinophilic syndrome [25–28]^{[25][26][27][28]}.

A variety of in vitro and in vivo studies demonstrated that imatinib exhibits therapeutic

efficacy against SCLC [29,30]^[29][30]. However, in phase 2 clinical trials, imatinib failed to exhibit

significant therapeutic efficacy as shown by a lack of objective responses [31–33]^[31][32][33]. Thus, an

alternative approach to target c-Kit in SCLC is needed. In th

2. 4C9 Antibody Specifically Binds to c-Kit

Firs study, we generated and

charactt, reserized 4C9, a human antibody targeting c-Kit. We developed an antibody-drug

conjugate (ADC) using DM1, a microtubule inrchibitor, coupled with N-succinimidyl-4-(Nmaleimidomethyl)

cyclohexane-1-carboxylate (SMCC) to generate 4C9-DM1, and then

evaluated its therapeutic efficacy in vitro and in vivo.

2. Results

2.1 4C9 Antibody Specifically Binds to c-Kit

First, we examined whether the 4C9 antibody specifically binds to c-Kit on cell surface.

FACS analysis revealed that 4C9 binds to various SCLC cell lines, including NCI-H526,

NCI-H1048, and NCI-H889, in a dose-dependent manner (Figure 1A). Interestingly, 4C9

antibody binding was saturated at 50 ng/mL in c-Kit-positive SCLC cell lines. In addition,

the expression of c-Kit was higher in NCI-H889 cells compared with that in NCI-H526 and

NCI-H1048 cells, which is consistent with a previous report ^[34]. However, 4C9 did not

show cross-reactivity with NCI-H446 and NCI-H2170 cells, which are c-Kit-negative SCLC

cell lines. WResearchers further examined the specific binding of 4C9 to c-Kit using siRNA knockdown

experiment. Western blot analysis showed that c-Kit siRNA efficiently decreased

protein expression in NCI-H1048 cells (Figure 1B). FACS analysis demonstrated that c-Kit

expression on the cell surface was also down-regulated by c-Kit siRNA. Taken together,

4C9 binds specifically to the extracellular domain of c-Kit on the cell surface.

Ijms 23 02264 g001 550

Figure

Figure 1. 1. Determination of specific binding of the 4C9 antibody to the surface of SCLC cells. (A)

SCLC cell lines were incubated with 4C9 antibody in a dose-dependent manner and analyzed by

FACS. (B) NCI-H1048 cells were transfected with control or c-Kit si-RNA and incubated for 72 h.

Binding of 4C9 (1 μg/mL) to NCI-H1048 cells was determined by FACS. The si-RNA-mediated

down-regulation of c-Kit protein expression was evaluated by Western blot analysis. Alpha-tubulin

was used as a loading control.

Next, wresearchers investigated whether the 4C9 antibody could inhibit the binding of SCF,

a ligand for c-Kit. Competitive enzyme-linked immunosorbent assay (ELISA) results

showed that the binding of 4C9 antibody to c-Kit was not affected by SCF, even at high

concentrations (Figure 2A), suggesting that 4C9 antibody binds to c-Kit independent of

SCF. In addition, wresearchers assessed whether 4C9 could inhibit SCF-mediated phosphorylation

of c-Kit. Using GIST-T1 cells, pretreatment with 4C9 antibody resulted in decreased c-

Kit phosphorylation in a dose-dependent manner; however, the phosphorylation levels

of the ERK and Akt, downstream molecules of c-Kit, were not changed by treatment

with 4C9 antibody (Figure 2B). Interestingly, total c-Kit levels decreased by 4C9 antibody

treatment (Figure 2B). Hence, a decrease in phosphorylated c-Kit levels may result from

decreased expression of c-Kit. A stability assessment of c-Kit indicated that the 4C9 antibody

dramatically decreased total c-Kit levels in a time-dependent manner in both GIST cell

lines and in some SCLC cell lines (Supplementary Figure S1), which may be associated

with ubiquitination-dependent degradation. Nevertheless, this needs further elucidation.

In contrast to GIST-T1, 4C9 did not reduce SCF-mediated c-Kit phosphorylation or c-Kit

stability in the NCI-H526 and NCI-H1048 cell lines (Figure 2C). Furthermore, the 4C9

antibody did not inhibit phosphorylation of ERK and Akt induced by SCF, which suggests

that the 4C9 antibody does not function as an antagonist of SCF/c-Kit signaling.

FigureFigure 2. 2. Characterization of the 4C9 antibody. (A) Human c-Kit (20 ng/well) was coated onto

96-well plates and the binding of the 4C9 antibody was investigated in the presence of human SCF at

the indicated concentrations. The results represent the mean ± SD of three independent experiments.

(B,C) GIST-T1, NCI-H526, or NCI-H1048 cells were treated with 4C9 at the indicated concentrations

in the presence or absence of SCF (100 ng/mL). The phosphorylation of c-Kit, Akt, and ERK was

assessed by Western blot analysis. In NCI-H1048 cells, phosphorylation of Y568/570 and Y823 by

SCF treatment was not detected. Alpha-tubulin was used as a loading control. The results represent

the mean ± SD of three independent experiments.

2.2. Generation and characterization of ADC (4C9-DM1)

3. Generation and Characterization of ADC (4C9-DM1)

Even though c-Kit is overexpressed in SCLC cell lines, naked antibodies cannot be

applied to treat SCLC because of the limited contribution of SCF/c-Kit signaling in the

pathogenesis of SCLC and failure of imatinib [32,33]. Therefore, the development of an

ADC would be a reasonable option to effectively treat SCLC. One important factor to

consider when creating an ADC is the internalization efficiency after the complex formation

of the antibody with the target molecule. Therefore, we first investigated whether the 4C9

antibody is internalized in SCLC cell lines. FACS analysis exhibited that the internalization

efficiency of the 4C9 antibody was 91% in NCI-H526, 76.6% in NCI-H1048, and 68.6%

in NCI-H889 cells (Figure 3A), which suggests that the 4C9 antibody could be used as

an efficient carrier for the specific delivery of toxin to treat SCLC. Next, we generated

an ADC using SMCC-DM1, which consists of a noncleavable linker and a microtubule

inhibitor. Because DM1 absorbs ultraviolet light at 252 nm [35], the absorbance of the naked

antibody (4C9) and ADC (4C9-DM1) at 252 nm was analyzed. The absorbance of 4C9-DM1

was higher than that of 4C9 at 252 nm (Figure 3B). The drug-antibody ratio (DAR) was

determined as described previously [36] and the DAR was approximately 2.16. SDS-PAGE

analysis exhibited that the conjugation of SMCC-DM1 to the 4C9 antibody resulted in a

slight size shift (Supplementary Figure S2). Since N-hydroxysuccinimide ester of the SMCC

linker reacts with primary amines of lysine residues in the antibody, the target binding

affinity of ADC may be affected. An ELISA demonstrated that the binding affinities of

4C9 and 4C9-DM1 to c-Kit were similar (Figure 3C). A quantitative analysis of the binding

affinity using surface plasmon resonance (SPR) indicated that the binding affinity of 4C9 to

human c-Kit was 5.5 × 10^-

Even though c-Kit is overexpressed in SCLC cell lines, naked antibodies cannot be applied to treat SCLC because of the limited contribution of SCF/c-Kit signaling in the pathogenesis of SCLC and failure of imatinib ^[32][33]. Therefore, the development of an ADC would be a reasonable option to effectively treat SCLC. One important factor to consider when creating an ADC is the internalization efficiency after the complex formation of the antibody with the target molecule. Therefore, researchers first investigated whether the 4C9 antibody is internalized in SCLC cell lines. FACS analysis exhibited that the internalization efficiency of the 4C9 antibody was 91% in NCI-H526, 76.6% in NCI-H1048, and 68.6% in NCI-H889 cells (Figure 3A), which suggests that the 4C9 antibody could be used as an efficient carrier for the specific delivery of toxin to treat SCLC. Next, researchers generated an ADC using SMCC-DM1, which consists of a noncleavable linker and a microtubule inhibitor. Because DM1 absorbs ultraviolet light at 252 nm ^[35], the absorbance of the naked antibody (4C9) and ADC (4C9-DM1) at 252 nm was analyzed. The absorbance of 4C9-DM1 was higher than that of 4C9 at 252 nm (Figure 3B). The drug-antibody ratio (DAR) was determined as described previously ^[36] and the DAR was approximately 2.16. SDS-PAGE analysis exhibited that the conjugation of SMCC-DM1 to the 4C9 antibody resulted in a slight size shift. Since N-hydroxysuccinimide ester of the SMCC linker reacts with primary amines of lysine residues in the antibody, the target binding affinity of ADC may be affected. An ELISA demonstrated that the binding affinities of 4C9 and 4C9-DM1 to c-Kit were similar (Figure 3C). A quantitative analysis of the binding affinity using surface plasmon resonance (SPR) indicated that the binding affinity of 4C9 to human c-Kit was 5.5 × 10⁻⁹ M (Ka = 2.27 × 10

⁴M^-

M⁻¹

s^-

s⁻¹

and Kd = 1.27 × 10^-

and Kd = 1.27 × 10⁻⁴

s^-

s⁻¹

), and 4C9-DM1 also showed similar binding affinity (5.46 × 10^-

), and 4C9-DM1 also showed similar binding affinity (5.46 × 10⁻⁹ M; Ka = 2.14 × 10

⁴M^-

M⁻¹

s^-

s⁻¹

and Kd = 1.16 × 10^-

and Kd = 1.16 × 10⁻⁴

s^-

s⁻¹

) (Figure 3D). Taken together, these results indicate that conjugation

using SMCC-DM1 to the 4C9 antibody did not affect the binding affinity of 4C9 antibody

for c-Kit.

) (Figure 3D). Taken together, these results indicate that conjugation using SMCC-DM1 to the 4C9 antibody did not affect the binding affinity of 4C9 antibody for c-Kit.

FigureFigure 3. 3. Characterization of 4C9-DM1. (A) The internalization of the 4C9 antibody into various

SCLC cell lines was determined using FACS analysis. SCLC cells were incubated with cycloheximide

(75 μg/mL) and blocked with Fc blocker for 10 min to inhibit Fc receptor-mediated internalization.

SCLC cells were incubated in the presence or absence of the 4C9 antibody at 4 '°C or 37 '°C for 1–4 h

and subjected to flow cytometry. The fluorescent signal of the 4C9/c-Kit complex on the cell surface

decreased after incubation at 37 '°C. (B) Optical absorbance of 4C9-DM1 compared with that of the

naked 4C9 antibody at 252 nm. The binding affinity of 4C9 and 4C9-DM1 to c-Kit was compared

using ELISA (C) and SPR (D).

2.3. 4C9-DM1 Exhibits Antitumor Activity In Vitro and In Vivo

4. 4C9-DM1 Exhibits Antitumor Activity In Vitro and In Vivo

Next, wresearchers analyzed the in vitro cytotoxicity using various SCLC cell lines (NCI-H526,

NCI-H889, NCI-H1048, NCI-H446, and NCI-H2170) and a breast cancer cell line (MDA-MB-

453). The c-Kit negative cell lines (NCI-H446, NCI-H2170, and MDA-MB-453) were

used as negative controls. 4C9-DM1 exhibited in vitro cytotoxicity against NCI-H526, NCI-H889,

and NCI-H1048 with half-maximal inhibitory concentration (IC₅₀) values ranging

from 158 pM to 4 nM. The in vitro cytotoxic activity of 4C9-DM1 against c-Kit-positive

cancer cell lines was 4- to >300-fold higher than that against c-Kit-negative cancer cell

lines (Figure 4A,B and Table 1). Interestingly, the cytotoxic activity of DM1 against the

c-Kit-positive cancer cells was 7- to >77 fold higher when applied as ADC rather than as

payload alone. By contrast, its cytotoxic activity was 2–5 times lower against c-Kit-negative

cancer cells (Table 1), suggesting the inclusion of a payload as an ADC may reduce off-target

toxicity. DM1 induces cell cycle arrest at the G2/M phase by inhibiting the assembly of

microtubules, resulting in apoptosis of actively dividing cells. Therefore, wresearchers analyzed the

effect of 4C9-DM1 on the cell cycle using NCI-H526, a c-Kit positive SCLC cell line, and

NCI-H446, a c-Kit negative SCLC cell line. As shown in Figure 4C, 4C9-DM1 significantly

increased the cell population in the G2/M phase in a time-dependent manner but 4C9

and IgG-DM1 did not. In addition, 4C9, 4C9-DM1, and IgG-DM1 did not alter the cell

population in the G2/M phase in c-Kit negative NCI-H446 cells, suggesting that cell cycle

arrest in NCI-H526 cells is specifically mediated by DM1 delivered by complex formation

with 4C9-DM1 and c-Kit.

Figure 4. 4C9-DM1 exhibits cytotoxicity against SCLC cells in vitro. (A,B) c-Kit positive or negative

SCLC cell lines were seeded into 96-well plates and incubated with 4C9 or 4C9-DM1 in a dose-dependent

manner for 3–5 days. Live cells were stained with Hoechst 33342 (10 μM) at 37 '°C for 30

min and quantitated using a Celigo Imaging Cytometer. Treatment with 4C9-DM1 decreased cell

viability in a dose-dependent manner. The results represent the mean ± standard error of the mean

of at least three independent experiments. (C) 4C9-DM1 induced cell cycle arrest at the G2/M phase.

SCLC cell lines were seeded into 96-well plates and incubated with 4C9 (1 μg/mL), IgG-DM1 (1

μg/mL), or 4C9-DM1 (1 μg/mL) for 24 and 48 h. Then, the cells were stained with propidium iodide

and analyzed using a Celigo Imaging Cytometer (** and *** vs. control; ^## and ^### vs. IgG-DM1). The

results represent the mean ± standard error of the mean of at least three independent experiments.

The results are presented as the mean ± standard error of the mean. The means were compared using

an unpaired Student’s two-sided t-test. ** p < 0.01, *** p < 0.001, ^## p < 0.01, ^### p < 0.001.

Table 1. IC₅₀ (nM) values of the tested materials.

c-Kit Expression	Tissue Type	Cell Line	4C9-DM1 *	SMCC-DM1
c-Kit positive	SCLC	NCI-H526	0.158	12.23
		NCI-H889	0.323	11.62
		NCI-H1048	4.08	30.45
c-Kit negative	SCLC	NCI-H446	16.58	6.97
	SCLC	NCI-H2170	35.5	20.17
	Breast cancer	MDA-MB-453	47.63	9.763

* Molar concentration was calculated as 180 kDa for the molecular weight of 4C9-DM1.

Based on the in vitro cytotoxicity assay, the in vivo efficacy of 4C9-DM1 was examined

using mouse models xenotransplanted with NCI-H526. Although IgG-DM1 did not

exert an effect, 4C9-DM1 significantly suppressed NCI-H526 tumor growth in a dose-dependent

manner (Figure 5A and Supplementary Figure S3A). Tumor growth inhibition

(TGI) rates of 4C9-DM1 at doses of 1, 3, and 5 mg/kg were 40%, 45%, and 59%, respectively,

compared with that of the vehicle control. The 4C9 antibody alone partially inhibited

tumor growth, but this effect was not statistically significant. Body weight losses due

to the administered materials were not observed (Figure 5A and Supplementary Figure

S3B), suggesting that there was no concern related to toxicity. Chemotherapy, including

etoposide, cisplatin, carboplatin, and lurbinectedin are used to treat SCLC patients as the

standard of care [37,38]^[37][38]. Although combination therapy using chemotherapeutic drugs is

effective, most SCLCs rapidly recur within 1 year ^[39]. Therefore, wresearchers determined whether the combination of 4C9-DM1 with chemotherapy could enhance the therapeutic efficacy

of SCLC. As shown in Figure 5B and Supplementary Figure S3C, 4C9-DM1, lurbinectedin,

and carboplatin/etoposide exhibited similar antitumor activities; the TGI rates of these

groups were 50% at day 18, compared with that of the vehicle control. Interestingly, the

combination of 4C9-DM1 with lurbinectedin or carboplatin/etoposide synergistically suppressed

tumor growth. The TGI rates of both groups were 85%, compared with that of the

vehicle control at day 18 with subsequent regrowth. The combinatorial treatment with

4C9-DM1 plus lurbinectedin induced body weight loss of approximately 10%; however,

body weight increased again after cessation of the treatment (Figure 5B and Supplementary

Figure S3D). This suggests that this combination may be used to treat SCLC with

manageable or mild toxicity.

Figure 5. 4C9−DM1 suppresses SCLC tumor growth in a xenograft mouse model. (A,B) Antitumor

activity of 4C9−DM1 was evaluated in an in vivo xenograft mouse model. NCI−H526 cancer cells

were implancted into immune-deficient mice as described in the Methods section. Mice with established

tumors were randomized into different treatment groups when the tumor volume reached ~

200 mm3 (n = 6). The animals were intravenously administered vehicle, 4C9, IgG−DM1, or

4C9−DM1. Carboplatin (60 mg/kg on days 1 and 11) and etoposide (3 mg/kg on days 1–5 and days

11–15) were intraperitoneally administered or combined with 4C9−DM1. Additionally, lurbinectedin

(0.08 mg/kg on days 1, 8, and 15) was intravenously administered or combined with

Figure 5. 4C9?DM1 suppresses SCLC tumor growth in a xenogray oft mouse model. (A,B) Antitumor

activity of 4C9-DM1 was evaluated in an in vivo xenograft mouse model. NCI-H526 cancer cells were

implanted into immune-deficient mice as described in the Methods section. Mice with established

tumors were randomized into different treatment groups when the tumor volume reached ~200

mm³ (n = 6). The animals were intravenously administered vehicle, 4C9, IgG-DM1, or 4C9-DM1.

Carboplatin (60 mg/kg on days 1 and 11) and etoposide (3 mg/kg on days 1–5 and days 11–15) were

intraperitoneally administered or combined with 4C9-DM1. Additionally, lurbinectedin (0.08 mg/kg

on days 1, 8, and 15) was intravenously administered or combined with 4C9-DM1 as indicated. Green

arrows indicate the administration of vehicle, IgG-DM1, 4C9, or 4C9-DM1, and blue and red arrows

indicate the administration of lurbinectedin and carboplatin, respectively (*, **, and *** vs. their

respective corresponding vehicle control; ^§ and ^§§§ vs. their respective corresponding 4C9-DM1

control; ^† vs. carboplatin/etoposide; ^‡ vs. lurbinectedin). The results are presented as the mean ±

standard error of the mean. The means were compared using an unpaired Student’s two-sided t-test.

* p < 0.05, ** p < 0.01, *** p < 0.001, ^† p < 0.05, ^‡ p < 0.01, ^§ p < 0.05, ^§§§ p < 0.001.

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