Supplementary Information 2 (doc 9926K)

Supplementary Information
A unique aptamer-drug conjugate for targeted therapy of multiple myeloma
Jianguo Wen,1 Wenjing Tao,2 Suyang Hao,1 Swaminathan P. Iyer, 3 Youli Zu1*
Supplementary Figure 1: CD38 is a specific biomarker for clinical diagnosis of MM cells. (A)
Marrow mononuclear cells of MM patients were stained with anti-CD38 antibody and analyzed by flow
cytometry. MM cells expressed the highest level of CD38, although weak expression was seen in other
cell populations, including a subset of activated T lymphocytes, monocytes, and blasts. (B) Tissue
immunohistochemical stains reveal that CD138 is less specific since it expresses in plasma cells as well
as normal epithelial cells.
Page 1 of 7
Supplementary Figure 2: Identification of MM cell-targeted and CD38-specific ssDNA aptamer
sequences. (A) For this purpose, we conducted a hybrid aptamer selection approach utilizing 15 rounds
of MM cell-based SELEX and 5 rounds of CD38 protein-based SELEX. The resultant ssDNA aptamer
pool was analyzed by next-generation sequencing with a total of 19,750,380 reads. (B) The top 20
aptamer sequences were analyzed using ClustalX software to generate a phylogenetic tree, and
clustered into three groups that shared a high degree of structural similarity (groups A, B, and C). Among
them, two dominant aptamer sequences, aptamer #1 and aptamer #2 were identified, accounting for
20% and 19% of total sequence reads, respectively (marked in red). (C) Aptamers in each group have
very similar sequences, such as the single-nucleotide differences seen between pairs #1 and #4, #2 and
8#, or #7 and #19.
Page 2 of 7
Supplementary Figure 3: Cell binding affinity and specificity of aptamer sequences. (A)
Representative aptamer sequences from each group, aptamers #1, #2, and #7, were synthesized and
conjugated with a Cy3 fluorochrome reporter. Synthetic aptamers were incubated with cultured MM cells
(MM1S) and CD38-negative control cells (HDLM2) and resultant cell binding was quantified by flow
cytometry. An anti-CD38 antibody was used as a standard control. Aptamer #1 showed the highest
binding affinity and specificity. (B) Predicted secondary structures of synthetic aptamers. (C) Affinity and
specificity of synthetic aptamer #1 was further confirmed in additional MM cells (RPMI8226 and NCIH929) and CD38-negative control cells (K299 and Jeko1).
Page 3 of 7
Supplementary Figure 4: The ssDNA aptamer sequence iss both MM cell-selective and CD38
protein-specific. (A) The CD38-specific aptamer #1 was truncated based on its functional structure
(#1S) and core sequence. Cell binding of aptamers was determined using MM cells (MM1S) and CD38negative cells (HDLM2), with anti-CD38 antibody as a standard control. The apparent dissociation
constants (Kd) were measured by flow cytometry. (B) Cell binding of synthetic aptamer #1S was
determined using MM cells (RPMI8226 and NCI-H929) and CD38-negative cells (K299 and Jeko1). (C)
Cell lysates of MM1S and HDLM2 were immunoprecipitated with aptamers #1 and #1S, and resultant
proteins were analyzed by western immunoblotting using anti-CD38 antibody. -actin was used as a
protein loading control. (D) RPMI8226 cells were transfected with CD38-specific siRNA to silence the
CD38 gene, or scrambled siRNA as a control (Scr siRNA). Down-regulation of cellular CD38 expression
was evaluated 48 hours post-transfection by flow cytometry with anti-CD38 antibody, and Aptamers #1
and #1S. (E) Selective cell binding and intracellular delivery of aptamer #1S occurred in a mixture of
MM1S cells (CD38+), but not HDLM2 cells (CD38-). All data are representative at least of three
independent experiments.
Page 4 of 7
Supplementary Figure 5: Synthetic ssDNA aptamers are biostable and selectively targeted MM
tumors in vivo. (A) ssDNA aptamer #1S and a control RNA aptamer were incubated in human serum
and their biostability was evaluated by gel electrophoresis of residual products at the indicated time
points. (B) Animal models bearing both a luciferase-tagged MM tumor (RPMI8226) and a CD38-negative
tumor (K299 lymphoma) were systemically administered with Cy5.5-labeled aptamer #1S through tail
veins. Tumor development was monitored by bioluminescence image scans and aptamer signals were
detected by fluorescence imaging with the Cy5.5 channel. All data are representative at least of three
independent experiments.
Page 5 of 7
Supplementary Figure 6: Formulation of ApDC with a CG-cargo sequence for high drug payload
and a pH-controlled structural switching mechanism for rapid drug release. (A) Schematic diagram
of ApDC formulation. (B) For drug loading, aptamer/CG-cargo structures were incubated with free DOX
at several molar ratios. Since free DOX is fluorescent (OD590) and its intercalated form is optically silent,
the DOX incorporation capacity of ApDC was determined by monitoring fluorescence resulting from
leftover unconjugated free DOX. Each aptamer/CG-cargo structure completely incorporated up to 5 DOX
molecules, a molar ratio of 1:5. (C) Synthetic aptamers alone and CG-cargo sequences alone were also
incubated with DOX and their drug loading capacities were evaluated. Changes in free DOX
fluorescence indicated that an intact aptamer/CG-cargo structure was indispensable for incorporating a
full DOX payload. (D) To confirm the presence of high DOX payload, ApDC was digested by DNase and
the released free DOX was monitored by fluorescence changes. (E) For stability assays, ApDC was
incubated at different temperatures and DOX release was monitored. (F) ApDC was stable in human
serum and carried more than 60% of DOX payload post-24 h incubation. (G) To test pH-controlled drug
release, ApDC was incubated in medium at pHs ranging from 7.4 to 1.0 for 30 min and released free
DOX in each reaction was monitored by fluorescence changes. Complete release of DOX payload from
ApDC was observed when the pH reached ≤5.0. All data are representative at least of three independent
experiments with mean ± SD.
Page 6 of 7
Supplementary Figure 7: ApDC selectively targets patient MM cells with little to no effects on
normal marrow cells. (A) Flow cytometry analysis revealed that MM cells in patient marrow specimens
could be distinguished by high levels of CD38 and CD138 expression. (B) To isolate primary MM cells,
patient marrow cells were stained with anti-CD138 antibody and primary CD138-positive MM cells and
CD138-negative background marrow cells were sorted (center diagram). Cell separation was confirmed
by flow cytometry analysis (scatter plots on both sides). (C) Primary MM cells isolated from individual
patients were treated with equal molar amounts of aptamers, free DOX, or ApDC and apoptosis was
determined by flow cytometry with Annexin V stain (left panel). Summary results from 4 patients are
shown as mean ± SD on the right. *P < 0.01.
Page 7 of 7