Mouse Anti-CEA Recombinant Antibody (clone H310A/H435Q); scFv Fragment (CAT#: HPAB-0348CQ)

Figure 1 Competition ELISA binding assay.

Plates were coated with recombinant NA-3 antigen. Increasing concentrations of wild-type, I253A, and H310A/H435Q unlabeled fragments were used to compete out the biotinylated intact, cT84.66 anti-CEA monoclonal antibody.

Kenanova, V., Olafsen, T., Crow, D. M., Sundaresan, G., Subbarayan, M., Carter, N. H., ... & Williams, L. E. (2005). Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti–carcinoembryonic antigen single-chain Fv-Fc antibody fragments. Cancer research, 65(2), 622-631.

Figure 2 Blood activity curves.

Dual biodistribution studies with ¹²⁵I- and ¹³¹I-labeled scFv-Fcs.

Kenanova, V., Olafsen, T., Crow, D. M., Sundaresan, G., Subbarayan, M., Carter, N. H., ... & Williams, L. E. (2005). Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti–carcinoembryonic antigen single-chain Fv-Fc antibody fragments. Cancer research, 65(2), 622-631.

Figure 3 Coronal microPET images of five constructs in representative individual athymic mice bearing LS174T xenografts (left shoulder) and C6 (right shoulder, except D) at three different time points.

Arrow, CEA-positive tumors. Following the imaging studies, tumors were dissected, weighed, and counted in a gamma counter. A, wild-type 42.5% ID/g, 185 mg LS174T tumor weight at 48 hours post injection; B, H435Q 10.6% ID/g, 64 mg at 123 hours; C, I253A 13.7% ID/g, 216 mg at 50 hours; D, H310A 10.6% ID/g, 174 mg at 53 hours; and E, H310A/H435Q 7.3% ID/g, 80 mg at 55 hours.

Kenanova, V., Olafsen, T., Crow, D. M., Sundaresan, G., Subbarayan, M., Carter, N. H., ... & Williams, L. E. (2005). Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti–carcinoembryonic antigen single-chain Fv-Fc antibody fragments. Cancer research, 65(2), 622-631.

Figure 4 A, ratios derived from the PET data comparing CEA-positive tumor-to-background levels of ¹²⁴I activity.

Early (3–4 hours), intermediate (16–18 hours), and late (48–90 hours) time points. B, ratios derived from the PET data comparing CEA-positive tumor-to-soft tissue levels of ¹²⁴I activity.

Kenanova, V., Olafsen, T., Crow, D. M., Sundaresan, G., Subbarayan, M., Carter, N. H., ... & Williams, L. E. (2005). Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti–carcinoembryonic antigen single-chain Fv-Fc antibody fragments. Cancer research, 65(2), 622-631.

Figure 5 ELISA measurements of pH-dependent binding of mFcRn and hFcRn to the scFv-Fc variants.

Binding responses of mFcRn to titrated amounts of WT, I253A, H310A, H435R, H435Q, and H310A/H435Q at pH 6.0 (A) and pH 7.4 (B), respectively. Binding of hFcRn to titrated amounts of WT, I253A, H310A, H435R, H435Q, and H310A/H435Q at pH 6.0 (C) and pH 7.4 (D). The numbers given represent the mean of triplicates.

Andersen, J. T., Foss, S., Kenanova, V. E., Olafsen, T., Leikfoss, I. S., Roopenian, D. C., ... & Sandlie, I. (2012). Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life. Journal of Biological Chemistry, 287(27), 22927-22937.

Figure 6 SPR binding profiles of anti-CEA scFv-Fc variants to immobilized mFcRn.

Representative sensorgram overlays obtained with 0.5 μm of the anti-CEA ScFv-Fc variants injected over a flow cell coupled with mFcRn (∼900 RU) at a flow rate of 50 μL/min (pH 6.0 and 7.4). A, WT; B, I253A; C, H310A; D, H435R; E, H435Q; and F, H310A/H435Q. The sensorgrams were zero-adjusted and reference cell data subtracted.

Andersen, J. T., Foss, S., Kenanova, V. E., Olafsen, T., Leikfoss, I. S., Roopenian, D. C., ... & Sandlie, I. (2012). Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life. Journal of Biological Chemistry, 287(27), 22927-22937.

Figure 7 Blood activity curves of ¹²³I-labeled scFv-Fc fragments in hFcRn transgenic mice.

Mice were injected with 96–130 μCi of ¹²³I-labeled scFv-Fc proteins (30 μg of labeled protein/mouse) under anesthesia. Blood samples were collected from the tail immediately after injection (0-h time point) and at time points 2, 4, 6, 12, 24, 48, and 72 h post-injection. Blood activity was determined using a Wallac WIZARD automatic gamma. The percentage of injected dose/g (% ID/g) was calculated. Each group represents four mice. Logarithmic scale, S.E. was used to measure variability.

Andersen, J. T., Foss, S., Kenanova, V. E., Olafsen, T., Leikfoss, I. S., Roopenian, D. C., ... & Sandlie, I. (2012). Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life. Journal of Biological Chemistry, 287(27), 22927-22937.

Figure 8 Blood clearance curves of ¹²³I-labeled scFv-Fc variants in WT BALB/c mice and hFcRn transgenic mice.

Blood samples were collected from the tail immediately after injection (0-h time point) and at time points 2, 4, 6, 12, 24, 48, and 72 h post-injection. Blood activity was determined using a Wallac WIZARD automatic gamma. The percentage of injected dose per gram (% ID/g) was calculated. Each group represents four mice. Logarithmic scale, S.E. was used to measure variability.

Andersen, J. T., Foss, S., Kenanova, V. E., Olafsen, T., Leikfoss, I. S., Roopenian, D. C., ... & Sandlie, I. (2012). Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life. Journal of Biological Chemistry, 287(27), 22927-22937.