Supplementary Material and Methods

Supplementary material and Methods

Animals C57BL/KalwRij mice were purchased from Harlan (Horst, the Netherlands). They were housed and treated following the conditions approved by the Ethical Committee for Animal Experiments of the Free University of Brussels (license no. LA1230281).

5TMM models 5T2MM and 5T33MM cells were isolated from diseased mice by flushing the BM out of the femurs and tibiae and crushing the vertebrae to release BM cells. BM mononuclear cells were purified by Lympholyte M (Cedarlane, Hornby, ON, Canada) gradient centrifugation at 1000g for 20min, generating enriched 5TMM cells. Monoclonal idiotype (5T2MMid and 5T33MMid) and anti-idiotype monoclonal antibody from these models were produced and purified as described before.

Dromedary immunization and immune Nanobody library construction A dromedary was subcutaneously injected with 200g purified 5T2MMid (in Gerbu-adjuvans) on days 0, 7, 14, 21, 28 and 35. On day 39, anti-coagulated blood was collected for the preparation of peripheral blood lymphocytes (PBLs). Total RNA from PBLs was used as template for cDNA synthesis. The variable domains of the heavy-chain-only antibodies were amplified in a two-step PCR. The amplified PCR products were ligated into the phagemid vector pHEN4. The ligated material was transformed into electro-competent E. coli TG1 cells hereby generating an immune Nanobody library of 1.5×107 transformants. Primers sequences and additional experimental details are described in depth elsewhere.3

Generation and production of anti-idiotypic Nanobodies Nanobodies were phage-displayed and used in biopannings, as generally described previously. 3 More in particular for the isolation of anti-idiotypic Nanobodies, several rounds of negative and positive selections were performed. Two methods for negative selections were chosen. In one method, negative selection occurred in solution whereby phages were incubated for 1.5h at room temperature (RT) with soluble purified 5T33MMid and total mouse IgG (100ug/ml each) before the mixture was added to microtiter plates containing immobilized 5T2MMid. In another method, 3 rounds of negative selections occurred on immobilized antigen, in which phages were incubated for 30min at RT in microtiter plates coated with 10µg of both purified 5T33MMid and total mouse IgG, after which non-bound phages were incubated with immobilized 5T2MMid. Positive selections were performed by phage-incubations in microtiter plates coated with 10 µg of purified 5T2MMid.

After positive selection, unbound phages were washed away and bound phages were eluted using 100mM triethylamine (pH=11.5). Eluted phages were reamplified for another round of positive and negative selections. Periplasmic extracts (PE) from randomly-chosen Nanobody-clones from each panning round were tested in ELISA for binding to 5T2MMid, 5T33MMid and total mouse IgG to select for potential anti-5T2MMid Nanobodies, and sequenced.

The sequences of all unique anti-5T2MMid Nanobodies and cAbBcII10, a control Nanobody targeting a bacterial enzyme,4 were re-cloned into expression vectors containing a hexahistidine-tag (His-tag) (pHen6c vector) or a His/Human influenza hemagglutinin (His/HA)-tag (pMECS vector). Nanobody R3B23 and cAbBcII10 were also re-cloned into a vector devoid of any tag (pHEN21 vector). The recombinant vectors were transformed into E. coli WK6 cells for Nanobody expression and generation of PE, as described before. 3 His-tagged and untagged Nanobodies were further purified by affinity chromatography on His-Select Nickel Affinity Gel or on protein A Sepharose beads (both from Sigma St Louis, MO, USA), respectively. Nanobodies were finally gelfiltrated using Superdex 75 16/60 columns (GE Healthcare Biosciences, Pittsburgh, PA, USA) in PBS.

Nanobody labeling with 99mTechnetium His-tagged Nanobodies were labeled with 99mTechnetium (99mTc) as previously described.3 Briefly, 99m [ Tc(H2O)3(CO)3] was synthesized using the Isolink labeling kit (Mallinckrodt Medical, Petten, The Netherlands) and attached to the Nanobodies through their His-tag. The 99mTc-labeled Nanobodies were purified using a NAP-5 column (GE Healthcare). The labeling yield was higher than 95% for all Nanobodies, as determined by instant thin-layer chromatography (iTLC).

Nanobody conjugation with 177Lutetium Untagged Nanobodies R3B23 and cAbBcII10 were conjugated with 177Lutetium (177Lu) as previously described.5 Briefly, bifunctional chelator 1B4M-DTPA was conjugated to the free -amino-groups of the Nanobodies. Nanobody-chelator complexes were purified using gelfiltration. Carrier-free 177Lu was obtained from ITG (Garching, Germany), with a specific activity of 3000GBq/mg. 4mCi (148MBq) 177Lu was added to a test vial containing Nanobody-DTPA and incubated for 30min at RT. The radiolabeled Nanobody solution was purified on a Nap-5 column and filtered. Quality control was performed using iTLC.

Pinhole SPECT/Micro-CT imaging and ex vivo biodistribution studies of 99mTc-labeled Nanobodies For each Nanobody healthy C57Bl/KaLwRij (n=6), 5T33MM diseased (n=6) or 5T2MM diseased mice (n=9) were intravenously (i.v.) injected with 1-2mCi (10 µg) 99mTc-labeled Nanobodies. At 1h post injection (p.i.) anesthetized mice were imaged using pinhole SPECT and micro-CT on separate devices, as described previously.6 30min after the imaging procedure, the mice were sacrificed, different organs and tissues were removed, weighed and the radioactivity was measured using an automated γ-counter (Cobra II Inspector 5033; Canberra Packard, Meriden, CT, USA). The amount of radioactivity uptake was calculated as percentage of injected activity per gram tissue or organ (%IA/g), corrected for decay.

Image analysis For visualisation and quantification of the images we used the AMIDE software (AMIDES’s medical Image Data Examiner). Based on CT-images, an ellipsoid region of interest (ROI) was drawn around the heart. Tracer uptake in heart, as a measurement of blood pool activity, is expressed as the counts in the tissue divided by the injected activity/cubic centimeter (%IA/cm³) ± standard error of the mean (SEM).

In vivo treatment with 177Lu-R3B23 Naïve mice were i.v. injected with 2x106 5T2MM cells/mice. Starting at week 1 after tumor inoculation, mice were divided into three groups. One group (n=3) received weekly i.v. saline injections, one group (n=6) received weekly i.v. 18.5Mbq 177Lu-cAbBCII10 and one group (n=6) was treated weekly i.v. with 18.5Mbq 177Lu-R3B23. Five weeks after treatment all mice were scanned by SPECT/micro-CT with 99mTc-R3B23 as described above. After 7 weeks of treatment, all mice were sacrificed and tumor load was analyzed by means of serum M-protein concentration and BM plasmocytosis: the first was quantified by electrophoresis and the latter by May-Grünwald-Giemsa staining of BM cytospin samples.

Statistics For statistical analysis the unpaired 2-tailed t-test was performed. P<0.05 was considered as statistically significant. Results are given as mean plus standard error of the mean (SEM). 1. Asosingh K, Radl J, Van Riet I, Van Camp B, Vanderkerken K. The 5TMM series: a useful in vivo mouse model of human multiple myeloma. The hematology journal : the official journal of the European Haematology Association / EHA 2000; 1(5): 351-6.

2. Vanderkerken K, De Raeve H, Goes E, Van Meirvenne S, Radl J, Van Riet I et al. Organ involvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells in the C57BL/KaLwRij mouse. British journal of cancer 1997; 76(4): 451-60.

3. Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S et al. Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circulation research 2012; 110(7): 927-37.

4. Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J et al. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrobial agents and chemotherapy 2001; 45(10): 2807-12.

5. D'Huyvetter M, Aerts A, Xavier C, Vaneycken I, Devoogdt N, Gijs M et al. Development of 177Lu-nanobodies for radioimmunotherapy of HER2-positive breast cancer: evaluation of different bifunctional chelators. Contrast media & molecular imaging 2012; 7(2): 254-64.

6. Put S, Schoonooghe S, Devoogdt N, Schurgers E, Avau A, Mitera T et al. SPECT Imaging of Joint Inflammation with Nanobodies Targeting the Macrophage Mannose Receptor in a Mouse Model for Rheumatoid Arthritis. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2013.