Supporting Information

Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive

Immunotherapy

Karlo Perica† β, Joan Glick Bieler λ, Christian Schütz β λ , Juan Carlos Varela β *, Jacqueline Douglass‡,

Andrew Skora‡, Yen Ling Chiu β *, Mathias Oelke β, Kenneth Kinzler‡, Shibin Zhou‡, Bert Vogelstein‡,

Jonathan P Schneck λ β †

†Department of Biomedical Engineering, β Ins�tute of Cell Engineering, * Sidney Kimmel Comprehensive

Cancer Center, λ Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA.

‡ Ludwig Cancer Research Center and Howard Hughes Medical Institute, Johns Hopkins School of

Medicine, Baltimore, MD, USA.

*Corresponding Author: Department of Pathology, Johns Hopkins School of Medicine, 733 N. Broadway,

MRB 639, Baltimore, Maryland 21205. jschnec1@jhmi.edu

Figure S1: Effect of Enrichment on Antigen Specific Frequency and Total Cells

Antigen specific frequency (left) and total antigen-specific cells obtained (right) seven days after culture

of a Positive (blue) or Positive + Negative (orange) E+E fraction, as described in Figure 3C, for the three

labeled antigens. Data shown are mean and standard deviation of at least three experiments. Figures

are not identical to those in Table because only experiments that included both a Positive and Positive +

Negative culture sample were included.

Figure S2. Micro-aAPC Are Not Effective For Antigen-Specific Enrichment

A) Binding of Micro- (top) and Nano- (bottom) aAPC to cognate pMEL (red) or non-cognate 2C (blue)

CD8+ T cells, characterized by fluorescent labeling of bound beads. No bead (grey) background is shown

as control. B) Micro-aAPC do not enrich cognate cells. Thy1.1+ pmel cells were incubated at a 1:1000

ratio with polyclonal, Thy1.2+ B6 splenocytes, and enrichment was attempted using Db-GP100

microparticles. Frequency of Thy1.1+ cells did not significantly increase after enrichment. C) Antigen-

specific cell frequency and percent of cells recovered, performed as in C with increasing amounts of

micro-aAPC.

Figure S3: Characterization of Expansion and Phenotype.

(A) Fold-expansion estimated by dilution of CFSE dye. Kb-Trp2 Enriched+Expanded cells diluted CFSE

below detectable limits (blue). To determine a lower limit of expansion, CFSE stained pmel cells were

activated with a moderate dose of Db-GP100 nano-aAPC (red); peaks represent two-fold dilution of CFSE

with each division. Numbers of divisions were derived from a proliferation model in FloJo (magenta,

with generation numbers in black). Thus, enriched+expanded cells underwent at least 8 divisions,

indicating greater than 1024 fold proliferation.

(B) Effector phenotype after enrichment and expansion. Seven days after stimulation againt Kb-Trp2

(top) or Kb-SIY (bottom), cells were stained with cognate dimer and gated on either antigen-specific cells

(red) or non-specific cells (blue). Activated upregulate CD44 and downregulate CD62L compared to Day

0 cells, consistent with an effector memory phenotype. Compared to non-cognate cells persisting in

culture on Day 7, they show higher expression of PD-1, IL7R, and KLRG1, differences which may be

attributed in part to the larger size and transiently enhanced transcriptional activity of stimulated cells.

Figure S4: T Cell Expansion with Bone-Marrow Derived DCs

(A) Bone marrow precursor cells activated for one week with IL-4, CD40L and show expression of

activated dendritic cells markers CD11b, MHC, and B7.1 (blue). Expression levels compared to isotype

control (red). (B) Trp2-specific T cells generated after one week of culture with Trp2-pulsed bone-

marrow derived DCs (left). Non-cognate SIINF (right) staining provided as control.

Figure S5: Expansion with Soluble MHC-Peptide

MHC must be conjugated to iron-dextran particles for effective expansion of the memory flu M1

response. M1-specific responses measured by tetramer staining after one week of expansion, without

enrichment. Soluble MHC-M1 as well as nano-aAPC bearing control MART1 peptide induced <2%

expansion after one week, whereas nano-aAPC bearing equivalent amounts of dimer induce up to 12%

antigen specific cells. MHC quantities expressed as volume of a 200 µg/ml solution. Results presented

are mean, SEM of three experiments.

MHC-peptide Protein (AA) Amino Acid Sequence

Balb/C mouse strain, CT26 tumor

Ld-A5-AH1 endogenous retroviral gp70, A5 variant (6-14)

SPSYAYHQF

Ld-SAF Mfsd12 major facilitator superfamily domain

SAFFSSFLM

Ld-FPS nucleotide diphosphate linked moiety

FPSENKHVY

C57BL/6J mouse strain, B16 tumor

Kb-TRP2 tyrosinase-related protein 2 (180-188)

SVYDFFVWL

Db-GP100 melanoma antigen (25-33) KVPRNQDWL

Kb-SIINF ovalbumin (257-264) SIINFEKL

Kb-LAY Tnpo3 Transportin 3 LAYLMKGL

Db-YTG Tubb3 Tubulin 3 YTGEAMDEM

Kb-VDW Kif18b Kinesin family member 18B VDWENVSPEL

Kb-RTF Cpsf3l Cleavage and polyadenylation specific factor 3-like

RTFANNPGPM

Db-IAM Fat1 FAT tumor suppressor homolog 1

IAMQNTTQL

Human

A2-NY-ESO1 cancer/testis antigen (157-165) SLLMWITQV

A2-MART1 melanA (26-35) ELAGIGILTV

Supplementary Table S1: Peptide antigens synthesized and used in this study to make nano-aAPC,

including both shared (shaded white) epitopes and neo-epitopes (shaded light grey).

Best Proteins for Peptides

(yellow=10mer)

Mutant Peptide

(includes

10mers)

Predicted

Affinity (nM)

Allele

Actn4 VTFQAFIDV 210 H-2-Kb

Atp11a QSLGFTYL 19 H-2-Kb

Cpsf3l RTFANNPGPM 2043 H-2-Db

Dag1 TTTTKKARV 2024 H-2-Kb

Ddb1 VLMINGEEV 153 H-2-Db

Ddx23 QTAMFTATM 112 H-2-Kb

Dpf2 LALPNNYCDV 318 H-2-Db

Eef2 ESFAFTADL 277 H-2-Kb

Fat1 IAMQNTTQL 5 H-2-Db

Fzd7 VAHVAAFL 87 H-2-Kb

Kif18b VDWENVSPEL 9066 H-2-Kb

Mthfd11 TILNCFHDV 1761 H-2-Kb

Orc2 VVPSFSAEI 39 H-2-Kb

Pbk AAVILRDAL 121 H-2-Db

Plod2 VWQIFENPV 111 H-2-Kb

S100a132510039O18 TVVCTFFTF 379 H-2-Kb

Sema3b VSAAQAERL 1487 H-2-Kb

Tm9sf3 AIYHHASRAI 191 H-2-Kb

Tnpo3 LAYLMKGL 69 H-2-Kb

Tubb3 YTGEAMDEM 991 H-2-Db

Wdr82 TNGSFIRLL 87 H-2-Kb

Supplementary Table S2: Candidate Neo-Epitopes

List of candidate peptide sequences containing neo-epitopes derived from B16 tumors, including MHC

affinity as predicted by NetMHC.

Manuscript Platform Ag-Specific Cells Fold Expansion

A2-MART1

Oelke et al.1 Micro-aAPC 7 Days: nd

28 Days: 30-60%

7 Days: nd

28 Days: ≈500x

Xu et al.2 Fast DC 6-7 Days: 2-11% 6-7 Days: Not

Described

Wӧlfl et al.3 ACE-CD8 10 Days: 20-50% 10 Days: >2000x

Current Nano-aAPC 14-38% 100x

A2-NY-ESO1

Butler et al.4 K562 aAPC 7 Days: <1%

600 Days: 10%

7 Days: <10x

400 Days: 1000x

Oelke et al. 1 Micro-aAPC 7 Days: nd

21 Days: 1.2%

7 Days: nd

28 Days: Not

Described

Chen et al.5 Peptide Pulsed

DC

7 days: Not Described

14 days: 0-0.04%

Not Described

Current Nano-aAPC 4-27% 1200x

Supplementary Table S3: Representative T Cell Expansion After One Week

Short-term expansion of A2-MART1 and A2-NY-ESO1 responses from healthy human donors. Where

one-week expansion was poor, highest reported expansion observed over several weeks is listed. nd:

not detectable.

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