[Supporting Information]

High-throughput cytotoxicity and antigen-binding

assay for screening small bispecific antibodies without

purification

Aruto Sugiyama, Mitsuo Umetsu*, Hikaru Nakazawa, Teppei Niide,

Ryutaro Asano, Takamitsu Hattori, and Izumi Kumagai

Table S1.List of fragments of variable region with affinity for CD3 or CD28 used to construct the diabody library used in the present study.

Fv, variable region; N.D., not determined.

Fv fragment with affinity for CD3 or CD28

Name Target Epitope Reference

L2K CD3 ε chain 1,2

OKT3 CD3 ε chain 3

UCHT1 CD3 ε chain 4,5

9.3 CD28 N.D. 6

Table S2.List of fragments of variable region with affinity for EGFR used to construct the diabody library used in the present study.

EGFR, epidermal growth factor receptor; Fv, variable region

Fv fragment with affinity for EGFR

Name Target Epitope Reference

7A7 EGFR domain III 7

175 EGFR domain III 8

225 EGFR domain III 9, 10

806 EGFR domain III 8

DL11EGFR domain III

11HER3 domain III

h-R3 EGFR domain vIII 12

425 EGFR domain III 13

528 EGFR domain III 14, 15

11F8 EGFR domain III 16

Table S3.Diabody library used in the present study.

Each diabody is referred to in the text by the number listed in this table. *, DL11 is an antibody with dual affinity for EGFR and HER3. EGFR, epidermal growth factor receptor; Fv, variable region.

　 Fv fragments with affinity for EGFR

7A7 175 225 806 DL11* h-R3 425 528 11F8

Fv fr

agm

ents

w

ith a

ffin

ity fo

r L2K (CD3)

OKT3 (CD3)

UCHT1 (CD3)

9.3 (CD28)

1

14

27

40

2

15

28

41

3

16

29

42

4

17

30

43

5

18

31

44

6

19

32

45

7

20

33

46

8

21

34

47

9

22

35

48

Fig. S1

Figure S1Schematic representation of the detection of diabody HL-31 by means of tag-sandwich enzyme-linked immunosorbent assay using anti-His tag antibody sc-8036 as the capture antibody and horseradish peroxidase-conjugated anti-C-myc tag antibody 9B11 as the detection diabody.

Fig. S2

Figure S2Western blotting analysis detected with HRP-conjugated anti-His tag antibody (sc-8036); Equal amounts of purified LH-21 and LH-21 in culture supernatant were loaded to Poly-Acrylamide Gel. The concentration of LH-21 in culture supernatant was determined by tag-sandwich ELISA, and each band density was analyzed by ImageJ software.

Fig. S3

Figure S3Comparisons of the sensitivity of the tag-sandwich enzyme-linked immunosorbent assay (A) for three HL-type diabodies and (B) for both structural configurations of the same diabodies. HL.

Fig. S4

Figure S4Cytotoxicity assay of unpurified diabodies against TFK-1 cells. Diabody concentrations were obtained (A) from Figure 3B and (B) from Figure 3C.

Fig. S5

Figure S5Surface plasmon resonance sensorgrams for the culture supernatant from Escherichia coli transformed with (A) a vacant vector or (B) a vector containing a gene fragment encoding diabody HL-21. The culture supernatants were diluted as indicated with fresh 2× YT medium, and the analysis was conducted by using a CM5 sensor chip coated with soluble epidermal growth factor receptor.

Fig. S6

Figure S6Surface plasmon resonance analysis of the HL and LH types of diabodies 21 and 22. A CM5 sensor chip coated with anti-C-myc antibody 9B11 was used for the analysis. KD, equilibrium dissociation constant.

Fig. S7

Figure S7Binding parameters estimated from the surface plasmon resonance sensorgrams of unpurified diabodies and of diabodies purified by mean of size-exclusion chromatography. kass, association rate constant; Rmax, maximum analyte binding capacity of the surface in resonance units (RU); KD, equilibrium dissociation constant; KA, equilibrium association constant; kdiss, dissociation rate constant.

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