1. Supplemental Figure S1 KHK2805 KM8188 KM8188/113F(-f)
Fab region of KHK2805
Fab region of KM8188
113F(-f)-type Fc region
IgG1 Fc region
N-glycan at Asn297 with fucose
N-glycan at Asn297 without fucose
Supplemental Figure S1. Construct of KHK2805, KM8188 and KM8188/113F(-f). KHK2805, KM8188 and KM8188/113F(-f) are anti-FOLR1 humanized antibodies. Both KHK2805 and KM8188/113F(-f) have the 113F(-f)-type Fc region [11] for ADCC and CDC enhancement. KM8188 has a human IgG1 type Fc region with fucose on N-glycan at Asn297.

2. A B Supplemental Figure S2
Supplemental Figure S2. Confirmation of recombinant FOLR2 and FOLR3. Recombinant proteins (0.5 µg/mL FOLR1-Fc, FOLR2-Fc or FOLR3-Fc) were coated onto an ELISA plate. Various concentrations of anti-FOLR2-HRP (LS-C214284, LSBio, A) or anti-FOLR3-HRP (LS-C214296, LSBio, B) were reacted for 1 h, followed by TMB substrate reaction. After stopping the reaction, the A450 nm was measured. Each point represents the mean ± SD of triplicate experiments.

3. Supplemental Figure S3  + FOLR1-His
Supplemental Figure S3. IHC specificity of KM4193. A subcutaneously xenografted SKOV3 tumor was immunohistochemically stained by KM4193 with (+) or without (-) FOLR1-His as a competitor.

4. Supplemental Figure S4 IGROV1 Control CD46 CD55 CD59 OVMANA SKOV3
OVCAR3
Supplemental Figure S4. The complement regulatory protein expression on OC cells FITC-labeled antibodies for CD46, CD55 and CD59 (BD Biosciences) were used to stain OC cells at 4 C for 30 min. FITC-labeled mouse IgG1 (BD Biosciences) was used as a control antibody. The FITC intensity in the live-cell population was evaluated by FCM.

5. A B Supplemental Figure S5
Supplemental Figure S5. CDR-optimization caused higher affinity and CDC. (A) The relative affinity of rat/human chimeric KM4193 for FOLR1-His was evaluated by SPR. Humanized (CDR grafted) KM4193 and its CDR-modified versions are described as C101X, wherein C101 means cysteine positioning at 101 in the amino acid sequence of the heavy chain, and X means the substituted amino acid (T, F, I, Q, M, V, G, Y, S, D, A, W, R or P). Humanized KM4193 with C101T is KHK2805. (B) The CDC of anti-FOLR1 antibodies against IGROV1 cells were evaluated as described in the Materials and Methods section. Briefly, human sera were used as the source of complement at a final dilution of 1/6. After 2-h incubation at 37 C, the cell viability was measured. Each point represents the mean ± SD of triplicate experiments.

6. Supplemental Figure S6 FZ06 FZ44 FZ26 FZ12 FZ21 FZ32 FOLR1-PE
Mouse IgG1-PE
Not tested
Supplemental Figure S6. FOLR1(+) cancer cells in patient-derived ascites samples. The FOLR1(+) cancer cell population was detected in patient-derived ascites samples by FCM as described in the Materials and Methods section. FZ06, FZ44, FZ26, FZ12, FZ21 and FZ32 are the sample IDs. The histograms indicate the intensity of PE-conjugated anti-FOLR1 antibody (FOLR1-PE, A) or the control antibody (Mouse IgG1-PE, B) in the 7-AAD(-)FS(high)CD45(-)CD14(-) cell population. The number in each histogram indicates the percentage of FOLR1(+) cells in the population.

7. Supplemental Figure S7
IGROV1 cells
KHK2805
PBMCs ND
Supplemental Figure S7. IP-10 production in the culture supernatant of IGROV1 cell-KHK2805-PBMC coculture. The concentrations of IP-10 in the supernatant after 24-h coculture of IGROV1 cell-KHK2805-PBMC were measured by multi-spot electrochemiluminescence assays using the V-PLEX Human Cytokine 30-Plex Kit supplied by Meso Scale Discovery. +: cultured with IGROV1 cells, 0.01 or 1: cultured with 0.01 or 1 g/mL KHK2805, D1 or D2: cultured with PBMCs from donor 1 or 2 at a PBMC: IGROV1 cell ratio of 25: 1. The lower limit of quantification of IP-10 was pg/mL. ND: Not detected.

8. Supplemental Table S1
Supplemental Table S1. Ex vivo cytotoxic drug resistance profiling of malignant ascites cells.
Sample ID
FZ06
FZ32
FZ12
FZ26
FZ44
FZ21
Sex F
Age
59.9
48.1
50.4
62.4
62.5
75.5
Histology
Metastatic serous ovarian carcinoma
Ovarian adenocarcinoma
Ovarian serous carcinoma
Serous ovarian carcinoma
Metastatic ovarian carcinoma
CYCLOPHOSPHAMIDE R I S
CARBOPLATIN
CISPLATIN +GEMCITABINE
CISPLATIN
DOXIL
GEMCITABINE
TAXOL
TOPOTECAN
TAXOTERE
ETOPOSID
The six purchased malignant ascites cell samples and their profiles are listed. “R (Resistance, gray highlighted)” indicates a sample that exhibited no cell death in response to addition of compound, “I (Intermediate)” indicates some cell death, and “S (Sensitive)” indicates that most cells were killed upon treatment. These data were provided by Molecular Response.

9. Lymphocyte/Cancer cell ratio
Supplemental Table S2
Supplemental Table S2. An FCM analysis of the malignant ascites cells.
Patient No.
FZ06
FZ32
FZ12
FZ26
FZ44
FZ21
FOLR1 expression (MFI)
23106
15129
7615
5169
2594
1451
Lymphocyte/Cancer cell ratio
0.12
0.13
0.10
0.53
0.57
1.85
NK (% Lymphocyte)
7.4
10.6
10.3
10.4
15.8
19.8
T (% Lymphocyte)
33.9
46.3
25.5
34.4
67.3
38.5
The FOLR1 expression (mean fluorescent intensity, MFI) of cancer cells and the number of NK and T cells in the malignant ascites cells were evaluated by FCM as described in the Materials and Methods section. The numbers of NK and T cells are described as percentages relative to lymphocytes in the ascites.