Targeted Polymersome Delivery of a Stapled Peptide for Drugging the Tumor Protein p53:BCL-2-Family Axis in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) remains a formidable diagnosis in need of new treatment paradigms. In this work, we elucidated an opportunity for therapeutic synergy in DLBCL by reactivating tumor protein p53 with a stapled peptide, ATSP-7041, thereby priming cells for apoptosis and enhancing their sensitivity to BCL-2 family modulation with a BH3-mimetic, ABT-263 (navitoclax). While this combination was highly effective at activating apoptosis in DLBCL in vitro, it was highly toxic in vivo, resulting in a prohibitively narrow therapeutic window. We, therefore, developed a targeted nanomedicine delivery platform to maintain the therapeutic potency of this combination while minimizing its toxicity via packaging and targeted delivery of a stapled peptide. We developed a CD19-targeted polymersome using block copolymers of poly(ethylene glycol) disulfide linked to poly(propylene sulfide) (PEG-SS-PPS) for ATSP-7041 delivery into DLBCL cells. Intracellular delivery was optimized in vitro and validated in vivo by using an aggressive human DLBCL xenograft model. Targeted delivery of ATSP-7041 unlocked the ability to systemically cotreat with ABT-263, resulting in delayed tumor growth, prolonged survival, and no overt toxicity. This work demonstrates a proof-of-concept for antigen-specific targeting of polymersome nanomedicines, targeted delivery of a stapled peptide in vivo, and synergistic dual intrinsic apoptotic therapy against DLBCL via direct p53 reactivation and BCL-2 family modulation.

are in labeled lanes, and unlabeled lanes contain samples irrelevant to this work.All gel samples were prepared with sodium azide (to quench DBCO:azide reactions) and NEM (to quench thiols and disulfide shuffling).Broad polymer smearing occurred in the presence of sodium azide in the gel samples.Gel samples were loaded such that, assuming 100% Fab conjugation, the Fab bands would be identical.This was calculated using the GPC-measured polymer concentration.

Figure S1 :
Figure S1: DLBCL cell death sensitivity to ATSP-7041.DLBCL cell lines were treated with ATSP-7041 or DMSO vehicle for 24 or 72 hr, and viability was measured.Data were normalized to untreated cells from the same time point, plotted as the mean of duplicates ± SEM, and fitted using nonlinear regression.EC50 is shown.Any absent error bars are smaller than the plotted point.

Figure S2 :
Figure S2: Changes in mRNA expression of the BCL-2 family in response to p53reactivation by ATSP-7041.DLBCL cell lines were treated with 1 M ATSP-7041 for 24 hr, and mRNA changes in the BCL-2 family were measured by qRT-PCR.Plotted values are the mean of the relative fold change of biological triplicates ± SEM, using GAPDH and B2M as housekeeping genes and DMSO-treated samples as reference controls.Changes were deemed significant via one-sample t-test (H0: CT = 0; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure S3 :
Figure S3: Cell death sensitivity to ABT-263 after priming with ATSP-7041.Cells were treated for 24 hr with ATSP-7041 or vehicle control.Pre-treated cells were then washed and plated with dose titrations of ABT-263, and viability was measured 24 hr later.Data were normalized to untreated cells from the same pre-treatment condition, plotted as the mean of duplicates ± SEM, and fitted using nonlinear regression.EC50 is shown.

Figure S4 :
Figure S4: LCMS analysis of purified ATSP-7041.Peptide purity was confirmed by LCMS, integrating the area under the A220 curve.Peptide purity was > 95% for each batch.ATSP-7041 eluted as two peaks due to the staple existing as two isomers, as previously reported.71

Figure S5 :
Figure S5: Characterization of PPS-PDS.(A) During PPS-SH polymerization, two peaks were visible by GPC with mobile phase DMF + 0.01 M LiBr at 50 C, and both peaks grew to larger MW with additional monomer.The larger MW polymer (leftmost peak) was presumably two PPS-SH polymers linked by a disulfide bond.(B) Reduction with TBP eliminated the higher MW peak, reducing disulfide polymers back into PPS-SH.The dispersity of reduced PPS-SH was 1.17.(C) 1 H NMR spectrum of PPS-PDS in CDCl3.

Figure S9 :
Figure S9: DNA coding sequences of CD19 and OspA Fabs and their protein translations.Fabs were designed with variable domains (Vκ and VH) for binding to either human CD19 (αCD19) or a xenoantigen control (αOspA).All Fabs shared the same constant domains (Cκ and CH).For αCD19-cys and αOspA-cys, the cysteine linker sequence was added to the Cterminal end of the CH domain.

Figure S10 :
Figure S10: Expression and binding validation of CD19 and OspA Fabs.(A) Fabs were designed using previously published sequences from antibodies that bind either human CD19 (CD19) or a xenoantigen control (OspA) as described in Materials and Methods. 73-76To enable site-specific conjugation to polymersomes, a flexible cysteine linker was encoded at the C-terminus of the heavy chain of each Fab to generate αCD19-cys and αOspA-cys.(B) Coomassie-stained SDS-PAGE of purified Fabs.Each Fab appears pure at the expected molecular weights.To confirm proper Fab formation, addition of DTT in the loading buffer reduces the interchain disulfide to generate polypeptides (heavy chain and light chain) that overlap at their expected molecular weights.(C) Flow cytometric measurement of Fab binding to a CD19 + DLBCL cell line, SU-DHL-5.Cells were stained with the indicated Fab, followed by an AF647-labeled αFab secondary antibody.αCD19 Fabs bind CD19 + DLBCL with or without the cysteine linker, and the control (αOspA) Fabs do not.

Figure S11 :
Figure S11: Full Coomassie-stained SDS-PAGE gels from Figure 4C.OspA-cys was reduced with varying amounts of TCEP, reacted with Sulfo-DBCO-PEG4-Maleimide, purified by desalting, and separated on SDS-PAGE gels.The gels were Coomassie-stained, and the bands in each lane were quantified using ImageJ.Unlabeled lanes contain samples irrelevant to this work.

Figure S13 :
Figure S13: Polymersome uptake into DLBCL cell lines depends upon CD19 cell surface expression and polymersome dose.DLBCL cell lines were treated with calcein-loaded polymersomes for 24 hr and then analyzed by flow cytometry.Both specific uptake/binding (CD19; purple) and non-specific uptake/binding (OspA; dark gray) were dose-dependent, but CD19-mediated uptake was significantly greater.The total polymer concentration in each treatment is indicated in μg/mL.

Figure S14 :
Figure S14: Full Coomassie-stained SDS-PAGE gel from Figure 4H.Bands from Figure 4Hare in labeled lanes, and unlabeled lanes contain samples irrelevant to this work.All gel samples were prepared with sodium azide (to quench DBCO:azide reactions) and NEM (to quench thiols and disulfide shuffling).Broad polymer smearing occurred in the presence of sodium azide in the gel samples.Gel samples were loaded such that, assuming 100% Fab conjugation, the Fab bands would be identical.This was calculated using the GPC-measured polymer concentration.

Figure S15 :
Figure S15: PEG-SS-PPS is non-toxic at high concentrations.OCI-Ly19 cells were treated with CD19-PSOMempty at varying concentrations of PEG-SS-PPS as high as 682 g/mL for 24 hr and 72 hr.No concentration was achieved that inhibited cell viability.Viability was measured using CellTiter-Glo 2.0, normalized to untreated cells, and plotted as the mean of duplicates ± SEM.

Figure S16 :
Figure S16: Representative gating strategy for flow cytometry of DLBCL cells from xenografts.After the treatment in Figure 5, (A) bone marrow and (B) subcutaneous tumors were isolated as single-cell suspensions and stained for flow cytometry.Debris was excluded via gating of FSC-A versus SSC-A.DLBCL cells (CD19 + CD20 + ) were distinguished from other cells in the tissue.The indicated live cell populations were used in Figure 5 to measure fluorescence from mice treated with vehicle or CD19-PSOMcalcein.

Figure S18 :
Figure S18: Polymersome delivery maintains the efficacy of ATSP-7041.OCI-Ly19 cells were treated with free ATSP-7041 (blue) or CD19-PSOMATSP-7041 (red) at varying concentrations of ATSP-7041 for 72 hr.Viability was normalized to untreated cells and plotted as the mean of duplicates ± SEM.