Immunotoxins constructed with chimeric, short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

Figures & data

Figure 1 4–15% SDS-PAGE of murine and cRFB4 constructs under non-reducing (A) and reducing (B) conditions. Lane 1, cRFB4; lane 2, mcRFB4-P247W; lane 3, mcRFB4-I253A; lane 4, mcRFB4-H310A; lane 5, mcRFB4-H435A; lane 6, mcRFB4-AAA; lane 7, murine RFB4. This is one of three experiments.

Figure 2 Analysis of purified wild-type cRFB4 and His310Ala constructs using size-exclusion chromatography. (A) cRFB4; (B) mcRFB4-H310A. This is one of three experiments.

Figure 3 Autoradiograph of murine and chimeric RFB4 constructs on SDS gels. ¹²⁵I-labeled mAbs were incubated for 24 h with mouse serum at 37°C and then 4–15% SDS-PAGE was performed under non-reducing (A) or reducing (B) conditions. Lane 1, murine RFB4; lane 2, cRFB4; lane 3, mcRFB4-P247W; lane 4, mcRFB4-I253A; lane 5, mcRFB4-H310A; lane 6, mcRFB4-H435A; lane 7, mcRFB4-AAA. This is one of three experiments.

Figure 4 Analysis of the stability of cRFB4 constructs in mouse serum in vivo. (A) cRFB4: time 0 h; (B) cRFB4: time 24 h; (C) mcRFB4-H310A: time 0 h. (D) mcRFB4-H310A: time 24 h. Black line: measurement of radioactivity in cpm.; red line: measurement of the absorbance at 280 nm. IgM, immunoglobulin M; IgG, immunoglobulin G; Alb, albumin. This is one of two experiments.

Figure 5 4–15% SDS-PAGE of murine and chimeric RFB4 ITs under non-reducing conditions. Lane 1, RFB4-rRTA; lane 2, cRFB4-rRTA; lane 3, mcRFB4-P247W-rRTA; lane 4, mcRFB4-H310A-rRTA. This is one of three experiments.

Figure 6 Antigen-binding activity of the ITs vs. the corresponding cmAbs as determined by FACS. (A) (▴) cRFB4; (●) cRFB4-rRTA; (B) (▴) mcRFB4-P247W; (●) mcRFB4-P247W-rRTA; (C) (▴) mcRFB4-H310A; (●) mcRFB4-H310A-rRTA. This is one of three experiments.

Figure 7 PVL of anti-CD22 ITs in mice. Open column, RFB4-rRTA; Gray column, cRFB4-rRTA; Black column, mcRFB4-H310A-rRTA. Murine RFB4-rRTA and cRFB4-rRTA ITs showed similar toxicity regardless the doses (p > 0.26), while at the dose of 7.5 mg/kg, mcRFB4-H310A-rRTA is significantly less toxic as compared with both murine and chimeric RFB4 ITs (p < 0.04). This is one of two experiments.

Figure 8 Changes in the body weight of BALB/c mice after treatment with various doses of ITs. (A) 2.5 mg/kg; (B) 5 mg/kg; (C) 7.5 mg/kg; (■) RFB4-rRTA; (□) cRFB4-rRTA; (▴) mcRFB4-H310A-rRTA. This is one of two experiments.

Table 1 Pharmacokinetics of cRFB4 constructs in Swiss Webster micea

mAbs	T1/2 (H)b	AUC (H × ng/mL)	MRT (H)	FCR (day^−1)
cRFB4	263.7 ± 17.0	27,468 ± 1,627	375.6 ± 24.5	0.063 ± 0.004
mcRFB4-P247W	106.4 ± 4.2	9,640 ± 475	144.9 ± 6.2	0.156 ± 0.006
mcRFB4-I253A	58.1 ± 3.0	5,185 ± 104	72.9 ± 3.7	0.286 ± 0.015
mcRFB4-H310A	39.0 ± 0.9	4,280 ± 139	47.8 ± 1.2	0.425 ± 0.01
mcRFB4-H435A	59.7 ± 6.0	5,018 ± 510	72.2 ± 8.4	0.280 ± 0.03
mcRFB4-AAA	45.7 ± 1.0	4,228 ± 145	53.3 ± 0.5	0.362 ± 0.008

T_1/2, half life (beta); AUC, area under the curve; FCR, fractional catabolic rate; MRT, mean residence time.

a Groups of five mice were used; two experiments were performed.

b There are statistically significant differences between all pairs of mAbs (p < 0.001) with the exception of the mcRFB4-I253A vs. mcRFB4-H435A pair.

Table 2 Cytotoxicity of ITs on CD22⁺ Daudi tumor cellsa

ITs	IC50 (M)
RFB4-rRTA	1.8 × 10^−12
cRFB4-rRTA	1.6 × 10^−12
mcRFB4-P247W-rRTA	1.1 × 10^−12
mcRFB4-H310A-rRTA	1.2 × 10^−12

a Results of one experiment of three experiments performed.

Table 3 Pharmacokinetics of ITs and their corresponding mAbs in BALB/c micea

mAb or IT	T1/2 (H)b	AUC (H × ng/mL)	MRT (H)	FCR (day^−1)
cRFB4	340.7 ± 22.9	47,399 ± 2,369	490.5 ± 32.3	0.049 ± 0.003
mcRFB4-P247W	183.1 ± 6.5	22,759 ± 824	261.8 ± 9.4	0.091 ± 0.003
mcRFB4-H310A	60.5 ± 1.7	8,131 ± 280	84.6 ± 1.9	0.274 ± 0.008
cRFB4-rRTA	139.7 ± 9.2	12,848 ± 652	193.8 ± 13.0	0.119 ± 0.008
mcRFB4-P247W-rRTA	83.6 ± 6.4	6,160 ± 166	106.8 ± 8.0	0.199 ± 0.016
mcRFB4-H310A-rRTA	54.4 ± 3.1	4,010 ± 232	62.3 ± 4.4	0.305 ± 0.017

a Groups of five mice were used; two experiments were performed.

b There is a statistically significant difference between all pairs.