Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry

Vladimir Tolmachev; Mikaela Friedman; Mattias Sandström; Tove L.J. Eriksson; Daniel Rosik; Monika Hodik; Stefan Ståhl; Fredrik Y. Frejd; Anna Orlova

doi:10.2967/jnumed.108.055525

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FIGURE 1.
Binding and affinity analysis of Z_EGFR:1907 monomer and dimer using surface plasmon resonance (Biacore) and flow cytometry. (A) Biacore sensorgrams obtained after injection of purified Z_EGFR:1907 monomer (▵) and Z_EGFR:1907 dimer (□) at 62.5 nM over sensor chip flow-cell surface containing amine-coupled EGFR-ECD. (B and C) Flow cytometric analysis and affinity measurements of EGFR-binding Affibody molecules to EGFR on A431 cells. Affibody molecules were fluorescently labeled site specifically to C-terminally introduced cysteine with Alexa Fluor 488. Equilibrium-binding curve for Z_EGFR:1907 monomer (B) and dimer (C). Data are average from 3 experiments and SD.
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FIGURE 2.
Cellular retention of radioactivity after interrupted incubation of EGFR-expressing A431 cells with EGFR-targeting Affibody molecules. Data are average value from 3 Petri dishes and SD. Error bars might not be seen because they are smaller than point symbols.
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FIGURE 3.
Tumor-to-organ ratios after injection of radiolabeled anti-EGFR Affibody molecules in BALB/c nu/nu mice bearing EGFR-expressing xenografts at 4 h after injection and injected dose of 3 μg (A), 24 h after injection and injected dose of 3 μg (B), 4 h after injection and injected dose of 50 μg (C), and 24 h after injection and injected dose of 50 μg (D). Data are average of 4 animals and SD.
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FIGURE 4.
Imaging of EGFR expression in A431 xenografts in BALB/c nude mice using ¹¹¹In-Bz-DTPA-Z₁₉₀₇ and ¹¹¹In-Bz-DTPA-(Z₁₉₀₇)₂. Planar γ-camera images were collected 24 h after administration of tracers. Arrows point to tumors (T) and kidneys (K).

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TABLE 1

Biodistribution of ¹¹¹In-Labeled Tracers (Injected Dose, 3 μg) 4 and 24 Hours After Injection in BALB/c nu/nu Mice Bearing EGFR-Expressing A431 Xenografts

	4 h after injection				24 h after injection
Organ or tissue	¹¹¹In-Bz-DTPA-Z_EGFR:1907	¹¹¹In-Bz-DTPA-Z_Taq	¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂	¹¹¹In-Bz-DTPA-(Z_Aβ)₂	¹¹¹In-Bz-DTPA-Z_EGFR:1907	¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂
Blood	1.5 ± 0.5^*	0.03 ± 0.01	1.5 ± 0.7	0.037 ± 0.002	0.07 ± 0.02^*	0.07 ± 0.02^†
Lung	1.3 ± 0.2^*^‡	0.25 ± 0.04	1.1 ± 0.2^†	0.15 ± 0.01	0.28 ± 0.04^*^‡	0.5 ± 0.2^†
Liver	5 ± 1^*^‡	1.4 ± 0.2	18 ± 5^†	0.6 ± 0.2	1.2 ± 0.3^*^‡	6 ± 1^†^‡
Spleen	0.7 ± 0.2^*	0.41 ± 0.06	1.1 ± 0.2^†	0.18 ± 0.03	0.33 ± 0.04^*	0.76 ± 0.05^†
Colon	1.2 ± 0.2^*	0.30 ± 0.05	1.6 ± 0.5	0.23 ± 0.05	0.44 ± 0.08^*	1.35 ± 0.09^†
Kidney	136 ± 11^*^‡	189 ± 19	73 ± 7^†	207 ± 35	102 ± 2^*	59 ± 7^†
Tumor	3.73 ± 0.08^*^‡	0.4 ± 0.2	1.57 ± 0.07	0.19 ± 0.07	2.0 ± 0.3^*^‡	1.23 ± 0.07^†
Muscle	0.29 ± 0.06^*	0.06 ± 0.01	0.24 ± 0.07	0.06 ± 0.04	0.09 ± 0.02^*	0.20 ± 0.05^†
Bone	0.5 ± 0.3	0.3 ± 0.2^§	0.7 ± 0.2	0.16 ± 0.10	0.3 ± 0.2^‡	0.6 ± 0.2^†
Brain	0.06 ± 0.02^*	0.004 ± 0.002	0.06 ± 0.03	NM	0.01 ± 0.01^‡	0.04 ± 0.03
GI tract^‖	3.6 ± 0.8^*^‡	1.0 ± 0.4	6.0 ± 0.7^†	1.0 ± 0.3	1.2 ± 0.2^*^‡	6 ± 1^†
Carcass^‖	13 ± 1^*	3 ± 1	12 ± 2^†	1.8 ± 0.3	4.9 ± 0.5^‡	9.6 ± 0.5^†

↵* Significant difference (P < 0.05, paired t test) between ¹¹¹In-Bz-DTPA-Z_EGFR:1907 and ¹²⁵I-PIB-Z_EGFR:1907.
↵† Significant difference (P < 0.05, paired t test) between ¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂ and ¹²⁵I-PIB-(Z_EGFR:1907)₂.
↵‡ Significant difference (P < 0.05, unpaired t test) between ¹¹¹In-Bz-DTPA-Z_EGFR:1907 and ¹¹¹In-Bz-DTPA- (Z_EGFR:1907)₂.
↵§ No significant difference (P > 0.05, unpaired t test) between ¹¹¹In-Bz-DTPA-Z_EGFR:1907 and ¹¹¹In-Bz-DTPA-Z_Taq.
↵‖ Data for gastrointestinal (GI) tract and carcass are presented as %IA per sample.
NM = nonmeasureable.
Data are presented as average %IA/g and SD (n = 4). Nonspecific Affibody molecules Z_Taq and (Z_Aβ)₂ have been included as negative control for ¹¹¹In-Bz-DTPA-Z_EGFR:1907 and ¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂, respectively.

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TABLE 2

Biodistribution of ¹²⁵I-Labeled Tracers (Injected Dose, 3 μg) 4 and 24 Hours After Injection in BALB/c nu/nu Mice Bearing EGFR-Expressing A431 Xenografts

	4 h after injection				24 h after injection
Blood or tissue	¹²⁵I-PIB-Z_EGFR:1907	¹²⁵I-PIB-Z_Taq	¹²⁵I-PIB-(Z_EGFR:1907)₂	¹²⁵I-PIB-(Z_Aβ)₂	¹²⁵I-PIB-Z_EGFR:1907	¹²⁵I-PIB-(Z_EGFR:1907)₂
Blood	1.2 ± 0.3^*	0.25 ± 0.02	1.1 ± 0.5	0.052 ± 0.003	0.04 ± 0.01^*	0.04 ± 0.01^†
Lung	0.9 ± 0.2^*	0.12 ± 0.03	1.0 ± 0.2^†	0.04 ± 0.01	0.05 ± 0.02^*	0.05 ± 0.02^†
Liver	2.1 ± 0.7^*^‡	0.21 ± 0.03	6 ± 2^†	0.05 ± 0.01	0.06 ± 0.01^*^‡	0.3 ± 0.2^†
Spleen	0.31 ± 0.08^*	0.11 ± 0.02	0.4 ± 0.2^†	0.065 ± 0.002	0.05 ± 0.02^*	0.04 ± 0.02^†
Colon	0.47 ± 0.10^*^‡	0.10 ± 0.07	0.73 ± 0.10	0.05 ± 0.02	0.02 ± 0.01^*	0.09 ± 0.10^†
Kidney	6.3 ± 0.6^*^‡	7 ± 2^§	10 ± 2^†	6 ± 2	NM	0.6 ± 0.2^†
Tumor	2.48 ± 0.10^*^‡	0.2 ± 0.1	1.9 ± 0.4	0.07 ± 0.05	0.26 ± 0.07^*^‡	0.46 ± 0.09^†
Muscle	0.16 ± 0.06^*	0.03 ± 0.01	0.17 ± 0.03	0.02 ± 0.01	0.02 ± 0.01^*	0.02 ± 0.02^†
Bone	0.34 ± 0.10	0.09 ± 0.03	0.32 ± 0.02	NM	0.14 ± 0.10	NM
Brain	0.04 ± 0.0^*	0.02 ± 0.02^§	0.05 ± 0.01	0.01 ± 0.01	0.01 ± 0.0^‡	0.010 ± 0.003
GI tract^‖	1.8 ± 0.5^*	0.8 ± 0.4	2.5 ± 0.2^†	0.5 ± 0.2	0.07 ± 0.01^*^‡	0.18 ± 0.09^†
Carcass^‖	7 ± 1^*	1.15 ± 0.09	8 ± 1^†	0.7 ± 0.2	0.6 ± 0.1	1.4 ± 0.2^†

↵* Significant difference (P < 0.05, paired t test) between ¹¹¹In-Bz-DTPA-Z_EGFR:1907 and ¹²⁵I-PIB-Z_EGFR:1907.
↵† Significant difference (P < 0.05, unpaired t test) between ¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂ and ¹²⁵I-PIB-(Z_EGFR:1907)₂.
↵‡ Significant difference (P < 0.05, unpaired t test) between ¹²⁵I-PIB-DTPA-Z_EGFR:1907 and ¹²⁵I-PIB-(Z_EGFR:1907)₂.
↵§ No significant difference (P > 0.05, unpaired t test) between¹²⁵I-PIB-Z_EGFR:1907 and ¹²⁵I-PIB-Z_Taq.
↵‖ Data for gastrointestinal (GI) tract and carcass are presented as % IA per sample.
NM = nonmeasurable.
Data are presented as average %IA/g and SD (n = 4). Nonspecific Affibody molecules Z_Taq and (Z_Aβ)₂ have been included as negative control for ¹²⁵I-PIB-Z_EGFR:1907 and ¹²⁵I-PIB-(Z_EGFR:1907)₂, respectively.

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TABLE 3

Biodistribution of ¹¹¹In-Bz-DTPA-Z_EGFR:1907 and ¹¹¹In-Bz DTPA-(Z_EGFR:1907)₂ (Injected Dose, 50 μg) 4 and 24 Hours After Injection in BALB/c nu/nu Mice Bearing EGFR-Expressing A431 Xenografts

	4 h after injection		24 h after injection
Organ or tissue	¹¹¹In-Bz-DTPA-Z_EGFR:1907	¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂	¹¹¹In-Bz-DTPA-Z_EGFR:1907	¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂
Blood	0.23 ± 0.05^*	0.18 ± 0.03^*	0.025 ± 0.006^*	0.038 ± 009^*
Lung	0.34 ± 0.03^*^†	0.68 ± 0.09^*	0.14 ± 0.03^*^†	0.33 ± 0.06
Liver	1.5 ± 0.2^*^†	6.9 ± 1.2^*	0.7 ± 0.1^*^†	3.6 ± 1.4
Spleen	0.34 ± 0.02^*^†	1.5 ± 0.2	0.29 ± 0.02^†	1.0 ± 0.3
Colon	0.45 ± 0.01^*^†	1.2 ± 0.2	0.22 ± 0.02^*^†	0.6 ± 0.1^*
Kidney	196 ± 4^*	169 ± 32^*	120 ± 10^*	118 ± 14^*
Tumor	3.35 ± 0.08^*^†	2.43 ± 0.35^*	2.39 ± 0.06^†	1.5 ± 0.3
Muscle	0.12 ± 0.05^*^†	0.24 ± 0.05	0.05 ± 0.02^†	0.13 ± 0.04
Bone	0.4 ± 0.1^*^†	0.9 ± 0.3	0.37 ± 0.08^†	0.7 ± 0.3
Brain	0.013 ± 0.004^*^†	0.020 ± 0.003^*	NM	0.010 ± 0.005^*
GI tract^‡	1.7 ± 0.3^*^†	3.5 ± 0.5^*	0.6 ± 0.1^*^†	1.4 ± 0.1^*
Carcass^‡	3.8 ± 0.4^*^†	9.0 ± 1.0	2.0 ± 0.3^*^†	5.8 ± 1.3^*

↵* Significant difference (P < 0.05, paired t test) between uptake after injection of 3 and 50 μg of tracer.
↵† Significant difference (P < 0.05, unpaired t test) between ¹¹¹In-Bz-DTPA-Z_EGFR:1907 and ¹¹¹In-Bz-DTPA-(Z_EGFR:1907)₂.
↵‡ Data for gastrointestinal (GI) tract and carcass are presented as %IA per sample.
NM = nonmeasurable.
Data are presented as average %IA/g and SD (n = 4).