**4. Materials and Methods**

#### *4.1. Antibodies and Compounds*

A human version of ABL001 bispecific antibody (ABL001) was produced under Good Manufacturing Practices (GMP) regulation by Bi-Nex (Incheon, Korea), and a mouse version of ABL001 bispecific antibody (mABL001) was produced by ABL Bio Inc., R&D Center (Gyeonggi-do, Seongnam-si, Korea), as described in a previous report [23]. Paclitaxel and irinotecan HCl were purchased from Hanmi Pharmaceutical Co. Ltd. (Seoul, Korea).

#### *4.2. Cancer Cell Lines and Culture*

Human gastric cancer cell lines, MKN45 (KCLB No.80103) and SNU16 (KCLB No.00016), were purchased from KCLB (Korea Cell Line Bank, Seoul, Korea), and NUGC-3 (JCRB0822) was obtained from JCRB (JCRB Cell Bank, Ibaraki, Japan). Human colon cancer cell lines, Colo205 (CCL-222), WiDr (CCL-218), and SW48 (CCL-231) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). GAPF006 gastric cancer patientderived tissues and SW620 human colon cancer cell line (LIDE, Shanghai, China) were also used for in vivo mouse xenograft studies. DMEM/F12, RPMI-1640, Leibovitz's L-15, PBS, fetal bovine serum, 0.05% trypsin-EDTA, and antibiotic-antimycotic were purchased from Gibco (Carlsbad, CA, USA). Colo205, MKN45, SNU16, and NUGC-3 cells were cultured in RPMI-1640 culture medium containing 10% fetal bovine serum and antibiotic-antimycotic (1X). SW48 cells were cultured in DMEM/F12 culture medium containing 10% fetal bovine serum and antibiotic-antimycotic (1X). Colo205, MKN45, SNU16, NUGC-3, and SW48 cells were cultured in an incubator at 37 ◦C in a humidified atmosphere with 5% CO2 and 95% air. SW620 cells were cultured in Leibovitz's L-15 medium containing 10% fetal bovine serum in an incubator at 37 ◦C in free gas exchange with atmospheric air.

#### *4.3. Animals*

Eight-week-old female BALB/c nu/nu mice (Orient Bio Inc., Gyeonggi-do, Korea) were used for the efficacy tests in Colo205, WiDr, MKN45, and SNU16 xenograft models, eight-week-old female CB17 SCID (Envigo, Indianapolis, IN, USA) were used for the efficacy tests in the SW48 xenograft model, and eight-week-old female BALB/c nu/nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) were used for the efficacy tests in the SW620 xenograft model and human gastric PDX (Patient-Derived Xenograft) model (LIDE). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC), approval number: IACUC180067, approval date: 17 April 2018. Mice were maintained in a controlled environment (12 h light-dark cycle; temperature, 20–22 ◦C; 50–60% humidity), and ad libitum access to food and water.

### *4.4. Animal Studies*

To evaluate the in vivo efficacy of mABL001, MKN45, SNU16, and NUGC-3 human gastric cancer cells (5 × 106 cells/head) or Colo205, SW48, and WiDr human colon cancer cells (5 × 106 cells/head) were implanted in the flank of BALB/c nu/nu mice or CB17 SCID mice. When the tumors had grown to an average volume of 150–200 mm3, the mice were divided into homogenous groups (6–12 mice/group), and treated with an intraperitoneal injection mABL001 (1.25, 2, 3.25, or 6.5 mg/kg), or ABL001 (GAPF006 PDX model, 6.5 mg/kg) twice per week. To evaluate the in vivo efficacy of mABL001 with chemotherapy, tumor growth was measured after treatment with the mouse version, mABL001 in SW48 or SW620 human colorectal cancer xenograft models, with or without irinotecan

(20 or 40 mg/kg), respectively. BALB/c nu/nu mice were injected subcutaneously in the flank region with SW620 cells (5 × <sup>10</sup><sup>6</sup> cells/head) in 0.1 mL of HBSS or GAPF006 tumor tissue fragments (9 mm3, approximately 50–90 mg), and CB17 SCID mice were injected subcutaneously in the flank region with SW48 cells (5 × <sup>10</sup><sup>6</sup> cells/head). When the tumors had grown to an average volume of 150–200 mm3, the mice were divided into homogenous groups (7–10 mice/group). GAPF006 PDX model treated ABL001 (3.25 mg/kg) twice per week, and paclitaxel (15 mg/kg) was administered with an intraperitoneal injection once a week for three weeks. SW620 xenograft model treated mABL001 (2 mg/kg) twice per week, and irinotecan (40 mg/kg) were administered with an intraperitoneal injection once a week for three weeks.

Tumor size was measured twice per week using a caliper and then calculated using the formula, (length) × (width)<sup>2</sup> × 0.5. When the average tumor size of the control group reached 2000 mm3, the treatment was stopped, and the mice were sacrificed to measure the tumor weight, and immunofluorescence analysis was performed (SW620 xenograft model). The efficacy was expressed as tumor growth inhibition [%TGI (mean volume of treated tumors/mean volume of control tumors) × 100]. Some mice were perfused with 4% paraformaldehyde in PBS for further immunofluorescence analysis of tumors.

#### *4.5. Immunofluorescence Staining Analysis*

To investigate whether mABL001 affects tumor angiogenesis and tumor cell survival, SW620 tumor sections were analyzed by immunofluorescence staining. For immunofluorescence staining analysis, SW620 tumors were removed from mice after cardiac perfusion and then embedded in OCT solution (Cat#3801480; Leica, Wetzlar, Germany) to produce frozen tumor blocks. The frozen tumors were sectioned (4-μm; Leica CM3050S; Leica) and permeabilized with washing buffer (PBS containing 0.03% Triton X-100) for 10 min, then blocked with 5% normal goat serum (Cat#S-1000; Vector Laboratories, Burlingame, CA, USA) or horse serum (Cat#16050122; Gibco) in the washing buffer. Tumor vessels were stained with rat anti-mouse CD31 (1:100, Cat#553370; BD, Franklin Lakes, NJ, USA) and goat anti-mouse VEGFR-2 antibody (1:100, Cat#AF644; R&D Systems, Minneapolis, MN, USA), respectively. Apoptotic cells in the tumors were stained with rabbit anti-mouse/human activated caspase-3 antibody (1:200, Cat#AF835; R&D Systems). DLL4 levels were detected with goat anti-mouse DLL4 antibody (1:100, Cat#AF1389; R&D Systems), which is cross-reactive (about 50%) with human DLL4. After being washed three times, the sections were stained for each secondary antibody, Alexa-568-conjugated goat anti-rat IgG (1:250, Cat#A11077), donkey anti-goat IgG (1:250, Cat#A11057), Alexa-488-conjugated goat anti-rabbit IgG (1:500, Cat#A11008), or donkey anti-rat (1:500 or 1:250, Cat#A21208), all from Thermo Fisher Scientific (Waltham, MA, USA). Stained tumors were mounted with Vectashield (Vector Laboratories) containing DAPI (4 ,6-diamidino-2-phenylindole), and digital images of the tumors were captured using a Zeiss fluorescence microscope (Axio observer.7, Carl Zeiss, Oberkochen, Germany) with a camera (Axiocam, Carl Zeiss). Digital fluorescence images were analyzed using a Zeiss analysis software program (ZEN 2.6, Carl Zeiss).

### *4.6. Statistics*

Graph creation and statistical analysis were performed using GraphPad Prism (Graph-Pad software Inc., San Diego, CA, USA) version 8.4.3. Values were expressed as the means ± SEM. Normality of data was tested using the Shapiro–Wilk test or Anderson–Darling test. Comparison between two groups was performed using the Student's *t*-test. Multiple group comparisons were made parametric one-way ANOVA followed post hoc test (Tukey's test, *p* < 0.0001, *p* < 0.001, *p* < 0.01, *p* < 0.05 values were considered as significant). The nonparametric Kruskal–Wallis test was used for the other cases.

**Author Contributions:** Conceptualization, S.H.L. and W.-K.Y.; methodology, D.-H.Y. and Y.-S.L.; software, D.-H.Y. and Y.-S.L.; formal analysis, D.-H.Y., H.-B.L., S.L. and B.S.; investigation, E.H. and J.-H.A.; resources, I.R., D.K.; data curation, D.-H.Y. and J.-H.A.; writing—original draft preparation, D.-H.Y. and W.-K.Y.; writing—review and editing, Y.-S.C. and W.-K.Y.; visualization, Y.-S.L. and W.-K.Y.; supervision, W.-K.Y. and S.H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and all experimental protocols were approved by IACUC (approval number: IACUC180067; approved date: 17 April 2018).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** This work was supported by the National OncoVenture Program (No. HI11C1191).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**

