Introduction Results

Key messages

F1 ONCOLOGY | 625 N FLAGLER DR, STE 625, WEST PALM BEACH FL 33401, 561-660-5049 | www.f1oncology.com

A High-Throughput Screening Strategy for the Identification of Novel Lymphoproliferative Elements

Laurence Jadin1, Hiba Shaban2, Anirban Kundu3, Benjamin Lopez1, Jianfang Hu1, Tiffany Lam1, Jaslem Valdez1, Tim Mayall1, Gregory Schreiber2, Scooter Willis2, Farzad Haerizadeh1, James Onuffer1, Gregory Frost2

1 F1 Oncology, San Diego CA, USA; 2 F1 Oncology, West Palm Beach FL, USA; 3 Exuma Biotechnology SEZC, Georgetown, Cayman Islands

ChimericAntigenReceptor (CAR) Tcell therapy is effectiveagainst certain leukemias andlymphomas andshows promise forotherincurable malignancies.Considerable challenges remain however to expand this platform technology beyond transplant-orientedhospitalcare.Centralizedmanufacturingof geneticallymodifiedTcells,lymphodepletingchemotherapyandpatientmanagementof current CAR-T therapies are associated with significant costs and treatment complexity. As a first step to reduce this treatmentcomplexity, the present study describes a high throughput combinatorial domain library screening method to identify syntheticlymphoproliferative elements (SLE) capable of driving in vivo expansion and survival of CAR-Tcells in a lymphoreplete host withoutthehomeostaticproliferationsignalsgeneratedbylymphodepletingchemotherapy.

barcoded SLE library

2.Experimental design

Human PBMCs were transduced with the library andcultured in vitro for several days. Expanded cells wereinjected into mice bearing xenograft tumors modified toexpress target antigens and compared to unmodifiedxenograft controls.The expansion rate of integratedcells wasmonitored weekly by quantitative PCR and after 21 days ofexposure, genomic DNA was isolated from blood, spleenandxenograft tumortissues.

1.Library design

DNA barcoded high-diversity semi-rationally designedlibraries of putative lymphoproliferative proteinsubdomains were assembled into a lentiviral vector co-expressing one of two different CARs. Each completeconstruct contained a dimerizing extracellular domain, atransmembrane domain, and an intracellular portioncomprised of two independent signaling domains, thatwere assembled into a library with a theoretical diversity ofalmost700,000uniquecombinations.

5. Assessment ofin vivo expansionand identificationof top candidates

Barcodes were quantified inblood, spleen and tumorusing NGS. In order to takethe variability of overallexpansion in each animalinto account, counts werescaled according to qPCR-measured lentivirus copynumbers, so that eachsample was representativeof one !g of original tissue.Associated constructs wereidentified by long readsequencing and topcandidates with consistentprevalence amongreplicates were selected forfurther characterization in asmall scale library.Comparison of samplesobtained from animalswhose xenograft tumorexpressed the targetantigen or not allowed us toevaluate CAR-mediatedsignalingdependency.

4. Library QC and characterization

PBMCs libraries were characterized for species richness as well as size and GC content bias after invitro expansion. Individualbarcodes were identifiedby Next-GenerationSequencingandcounted.Following artifact removal, observed and estimated species richness after in vitro expansion weredetermined based on the frequency and distribution of observed barcode species. Although notall possible combinations were obtained, the observed diversity in both libraries prior to injectionwas still above 100,000 individual species. Quantile plots of pre- and post-expansion distributionswere used to evaluate any potential selection bias during in vitro expansion in terms of constructsize or GC content. Little bias was observed with the exception of a slight under-representation ofvery largeconstructsafterexpansion.

Library 1 Library 2

6. Top candidatecompositionanalysis

Further analysis wasperformed to identifycommon key proteinsubdomains and pathwaysthat support selective in vivoexpansion and survival ofhuman lymphocytes in atumor-bearing mousemodel, dependently orindependently of a CAR-mediatedsignaling.Based on their overallperformanceinboth libraries,all parts were attributed ascore that was used to rankand select SLE candidates forfurthertesting.

Taken together,these results demonstrate that a high throughput combinatorial screening strategy with quantitative bioinformaticsis a viable method for identifying protein domain combinations capable of selectively driving human CAR-T cells in vivo. Thesesmall synthetic combinatorial protein domains may facilitate lymphodepleting chemotherapy-free regimens and lower CAR-T celldosesinthefuture.

7. In vivo and in vitro characterization of selected lymphoproliferative elements

3. Data Processing

Enriched barcodes were amplified using PCRand amplicons were subjected to HiSeq Next-Generation Sequencing. Barcode decodingwas achieved using PacBio long readsequencing analysis to align full-lengthconstruct sequences with barcodequantitation.

xenograft tumor Xenograft+antigentumor

Long read sequencing

PBMC + SLE + CARLibrary 1 = CD28-dLCKLibrary 2 = 4.1BB

Barcode sequencing Barcode sequencing

gDNA extraction

Amplicon prepand sequencing

CAR-T cell and LV copy number

quantification

Read normalization

QC, filteringBarcode identification

Data analysisCandidate

identification for small scale validation

Asmall scale library was built from top ranking candidates from both libraries 1 and 2 and tested for its ability to induce CAR-Tcellexpansion in a xenograft mouse model in the presence or absence of antigen, dependently or independently of CD28-dLCKCARsignaling.Significantexpansionwasobtainedinbothcases,andafewcandidateswithconsistentexpansionacrossreplicateswere identified for further characterization. Select SLEs were also tested for their ability to induce T cell expansion in vitro in theabsenceof cytokinesandantigen. Twoconstructsshowedconsistentexpansioninthreedifferentdonors.

Xenograft/no antigen Xenograft/antigen

Fig 1. Construction of the combinatorial synthetic lymphoproliferative element (SLE) library with self-correcting DNA barcode identifiers.

Fig 2. Experimental design and Next-Generation Sequencing data generation strategy. A combination of HiSeqand PacBio sequencing were used to allow high coverage barcode sequencing and identification of barcode-associated full length constructs of interest.

Fig 3. Next-generation sequencing data was processed using custom scripts and several R/Bioconductor packages (1, 2, 3, 4) to identify individual barcodes in reads and map them to their associated full length SLE construct sequence. qPCR was used to quantify overall expansion in tissues and normalize counts appropriately. Data analysis was performed using R (1).

Fig 6. Analysis of the subcomponents of the SLE library (A) Example count data where constructs containing different P3 parts were highlighted (colors). This data, collected in multiple tissues and across multiple replicates, was used to evaluate individual part performance. (B) FDR-adjusted p values for all parts in the presence of absence of antigen in library 1 vs

library2(left)and for overalltoprankingparts combining both libraries (right). (C) Pathway analysis and evaluation of the implication of specific signaling pathways in the performance of intracellular domains. ag, antigen.

Fig 4. (A) Histogram of the distribution of unique barcodes in the post-in vitro expansion, pre-injection samples of libraries 1 (left) and 2. (right) Species richness was above 100,000 for both libraries. (B) Size and GC content selection biases after in vitro initial expansion in libraries 1 (left) and 2 (right), showing minimal bias as compared with pre-injection samples.

A

B

References(1)www.R-project.org(2)www.bioconductor.org(3)BuschmannT,BystrykhLV(2013)BMCbioinformatics14(1),272.(4)M.Morganetal.(2009)Bioinformatics25:2607-2608.

3523

Methods and experimental design

• Thecurrent workdescribes ahigh-throughputmethod for screening lymphoproliferative elements in vivo fornext generationCAR-T therapies• Byquantification of selectivelyenrichedhuman PBMCswe can identifycombinatorial elementsthat promote CAR-T cell expansion and survival in an in vivo mouse model• Our platformis able to identify previouslypublishedlymphoproliferative elementsas well as new,potentcombinations of elementsthat are bothdependentorindependent of concomitant CAR signaling• This semi-rational screening strategy allows us tocontinue toidentify novel and potentiallystrongersynthetic lymphoproliferative peptidesin combinations that can be used in future CAR-T strategies

DNA

BarcodeDiversity 5 x 82 x 81 x 21

Total 697,410

STOPATG P1 P2 P3 P4

Extracellular TM Intracellular Intracellular

Blood−G05−CH

OBlood−G

06−CHO

Blood−G07−CH

OBlood−G

08−CHO

Blood−G09−CH

O

0 50 100 150 200

1

10

100

1000

1

10

100

1000

1

10

100

1000

1

10

100

1000

1

10

100

1000

Rank

Count

A BC

P3 P4

0.00

0.25

0.50

0.75

1.00

G32

G34

G35

G36

G39

G40

G43

G46

G47

G48

G49

G50

G33

G37

G38

G41

G42

G44

G45 G6

G8

G9

G10

G12

G18

G22

G23

G24 G7

G11

G13

G14

G15

G19

G21

G25

PBM

C

Prop

ortio

n of

sam

ple

E009−T006−S062−S053 (Junlz−CD4−CSF3R−CD79B)E008−T030−S057−S037 (Junlz−IFNAR2−CSF2RB−CD3D)E010−T021−S197−S049 (Junlz−EPOR−MyD88−CD28)E006−T023−S117−S080 (Junlz−FCER1G−IL6ST−ICOS)E007−T032−S064−S052 (Junlz−IFNGR2−CSF3R−CD79A)E008−T001−S121−S212 (Junlz−CD2−IL7RA−TNFRSF8)E006−T032−S197−S075 (Junlz−IFNGR2−MyD88−FCGR2C)

E009−T056−S104−S080 (Junlz−IL15RA−IL2RA−ICOS)E007−T005−S170−S076 (Junlz−CD3Z−IL31RA−FCGRA2)E008−T003−S138−S039 (Junlz−CD3E−IL12RB2−CD3G)E006−T006−S171−S215 (Junlz−CD4−IL31RA−TNFRSF18)E006−T056−S196−S212 (Junlz−IL15RA−MyD88−TNFRSF8)E006−T044−S186−S053 (Junlz−IL6R−MPL−CD79B)E008−T065−S069−S053 (Junlz−IL20RB−EPOR−CD79B)

E008−T038−S165−S052 (Junlz−IL2RA−IL23R−CD79A)E009−T062−S190−S074 (Junlz−IL18R1−MyD88−FCER1G)E008−T001−S064−S047 (Junlz−CD2−CSF3R−CD27)E006−T045−S072−S051 (Junlz−IL6ST−EPOR−CD40)EmptyE008−T031−S083−S212 (Junlz−IFNGR1−IFNAR2−TNFRSF8)E009−T032−S170−S074 (Junlz−IFNGR2−IL31RA−FCER1G)

E006−T038−S192−S039 (Junlz−IL2RA−MyD88−CD3G)E009−T010−S170−S074 (Junlz−CD28−IL31RA−FCER1G)E006−T064−S190−S080 (Junlz−IL20RA−MyD88−ICOS)E007−T039−S197−S080 (Junlz−IL2RB−MyD88−ICOS)E006−T006−S194−S211 (Junlz−CD4−MyD88−TNFRSF4)E008−T078−S190−S211 (Junlz−TNFRSF4−MyD88−TNFRSF4)E007−T054−S197−S212 (Junlz−IL13RA1−MyD88−TNFRSF8)

E010−T072−S192−S212 (Junlz−LEPR−MyD88−TNFRSF8)E009−T073−S062−S053 (Junlz−LIFR−CSF3R−CD79B)E010−T066−S197−S051 (Junlz−IL21R−MyD88−CD40)

Pre-injection

0.00

0.25

0.50

0.75

1.00

G32

G34

G35

G36

G39

G40

G43

G46

G47

G48

G49

G50

G33

G37

G38

G41

G42

G44

G45 G6

G8

G9

G10

G12

G18

G22

G23

G24 G7

G11

G13

G14

G15

G19

G21

G25

Prop

ortio

n of

sam

ple

E6−T4−IA12−IB11E2−T2−IA2−IB2E5−T5−IA5−IB5E4−T15−IA14−IB12E3−T8−IA6−IB7E2−T6−IA18−IB15E4−T8−IA5−IB8E6−T16−IA15−IB12E3−T3−IA3−IB3E2−T9−IA8−IB9E4−T4−IA4−IB4E4−T16−IA20−IB15E4−T12−IA11−IB11E2−T14−IA13−IB11E2−T7−IA7−IB7E6−T11−IA10−IB10

E2−T6−IA6−IB6E4−T20−IA17−IB14E1−T1−IA1−IB1E2−T22−IA19−IB15E6−T8−IA3−IB10E4−T7−IA9−IB9E6−T10−IA3−IB10E4−T18−IA10−IB12E3−T17−IA5−IB12E4−T4−IA16−IB13E2−T19−IA10−IB13E3−T24−IA5−IB15E5−T23−IA9−IB15E6−T13−IA12−IB11E5−T21−IA5−IB14

0

2000

4000

0 100 200Count

Frequency

0

500

1000

1500

2000

0 100 200Count

Frequency

A

B

C

Fig 5. (A) Species richness in multiple samples ascomparedwithpre-injection sample. (B) Correlation plots for all tested samples. (C) Contribution of top CAR-signaling independent constructs to the overall population in samples obtained from xenografts with no antigen (left panel) and of the top CAR-signaling dependent constructs to the overall population in samples obtained from xenograft overexpressing the antigen of interest (right panel) in libraries 1 and 2. xg, xenograft; ag, antigen.

A

agindpt

agdpt

AcknowledgementsWe would like to acknowledge Biao Chen, Quying Ding, Wenjie Yin, Feng Zhang, Wendy Zhang, Minghuan Zheng and Xun Zhuandtheirteamsfortheircontributionstothiswork.

1e+001e+011e+021e+031e+041e+05

pre−

inje

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nogr

aft b

lood−1

xeno

graf

t blo

od−2

xeno

graf

t blo

od−3

xeno

graf

t blo

od−4

xeno

graf

t blo

od−5

xeno

graf

t ant

igen−b

lood−1

xeno

graf

t ant

igen−b

lood−2

xeno

graf

t ant

igen−b

lood−3

xeno

graf

t ant

igen−b

lood−4

xeno

graf

t spl

enoc

ytes

_−1

xeno

graf

t spl

enoc

ytes

_−2

xeno

graf

t spl

enoc

ytes

_−3

xeno

graf

t spl

enoc

ytes

_−4

xeno

graf

t spl

enoc

ytes

_−5

xeno

graf

t spl

enoc

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_−6

xeno

graf

t ant

igen−s

plen

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es_−

1xe

nogr

aft a

ntig

en−s

plen

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es_−

2xe

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en−s

plen

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es_−

3xe

nogr

aft a

ntig

en−s

plen

ocyt

es_−

4xe

nogr

aft t

umor

_−1

xeno

graf

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or_−

2xe

nogr

aft t

umor

_−3

xeno

graf

t tum

or_−

4xe

nogr

aft t

umor

_−5

xeno

graf

t tum

or_−

6xe

nogr

aft a

ntig

en−t

umor

_−1

xeno

graf

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igen−t

umor

_−2

xeno

graf

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igen−t

umor

_−3B

arco

de d

iver

sity

1e+001e+011e+021e+031e+041e+05

pre−

inje

ctio

nxe

nogr

aft B

lood−1

xeno

graf

t Blo

od−2

xeno

graf

t Blo

od−3

xeno

graf

t Blo

od−4

xeno

graf

t Blo

od−5

xeno

graf

t Blo

od−a

ntig

en−1

xeno

graf

t Blo

od−a

ntig

en−2

xeno

graf

t Blo

od−a

ntig

en−3

xeno

graf

t Blo

od−a

ntig

en−4

xeno

graf

t Blo

od−a

ntig

en−5

xeno

graf

t spl

enoc

ytes−1

xeno

graf

t spl

enoc

ytes−2

xeno

graf

t spl

enoc

ytes−3

xeno

graf

t spl

enoc

ytes−4

xeno

graf

t spl

enoc

ytes−5

xeno

graf

t spl

enoc

ytes−a

ntig

en−1

xeno

graf

t spl

enoc

ytes−a

ntig

en−2

xeno

graf

t spl

enoc

ytes−a

ntig

en−3

xeno

graf

t spl

enoc

ytes−a

ntig

en−4

xeno

graf

t spl

enoc

ytes−a

ntig

en−5

xeno

graf

t tum

or−1

xeno

graf

t tum

or−2

xeno

graf

t tum

or−3

xeno

graf

t tum

or−4

xeno

graf

t tum

or−5

xeno

graf

t tum

or−a

ntig

en−1

xeno

graf

t tum

or−a

ntig

en−2

xeno

graf

t tum

or−a

ntig

en−3

xeno

graf

t tum

or−a

ntig

en−4

Bar

code

div

ersi

ty

preIV blood/xg blood/xg+ag spleen/xg spleen/xg+ag tumor/xg tumor/xg+ag

preIV blood/xg blood/xg+ag spleen/xg spleen/xg+ag tumor/xg tumor/xg+agba

rcod

e di

vers

ity

preIV blood/xg spleen/xg tumor/xg

preIV blood/xg+ag spleen/xg+ag tumor/xg+ag

preIV blood/xg spleen/xg tumor/xg

preIV blood/xg+ag spleen/xg+ag tumor/xg+ag

Library 1 Library 2

Library 1

Library 2

CAR-signaling independent

CAR-signaling independent

CAR-signaling dependent

CAR-signaling dependent

agindpt

agdpt

Observed diversity:127,634

Observed diversity:219,649

C

barc

ode

dive

rsity

p value

B

0

25

50

75

100

PBM

C_D

ay12

Bloo

d−G

05−C

HO

Bloo

d−G

06−C

HO

Bloo

d−G

07−C

HO

Bloo

d−G

08−C

HO

Bloo

d−G

09−C

HO

NPS

PL−G

05−C

HO

NPS

PL−G

06−C

HO

NPS

PL−G

07−C

HO

NPS

PL−G

08−C

HO

NPS

PL−G

09−C

HO

NPT

UM−G

05−C

HO

NPT

UM−G

06−C

HO

NPT

UM−G

07−C

HO

NPT

UM−G

08−C

HO

NPT

UM−G

09−C

HO

variable

Prop

ortio

n of

tota

l (%

)

E007−T080−S059−S080 (Junlz−TNFRSF9−CSF2RA−ICOS)

E008−T060−S190−S074 (Junlz−IL17RD−MyD88−FCER1G)

E007−T050−S190−S051 (Junlz−IL10RB−MyD88−CD40)

E007−T056−S170−S050 (Junlz−IL15RA−IL31RA−CD40)

E007−T054−S197−S212 (Junlz−IL13RA1−MyD88−TNFRSF8)

E010−T027−S117−S053 (Junlz−GHR−IL6ST−CD79B)

E009−T075−S165−S050 (Junlz−MPL−IL23R−CD40)

E006−T071−S165−S076 (Junlz−IL31RA−IL23R−FCGRA2)

E010−T055−S192−S051 (Junlz−IL13RA2−MyD88−CD40)

E010−T035−S190−S047 (Junlz−IL1RAP−MyD88−CD27)

E009−T073−S062−S053 (Junlz−LIFR−CSF3R−CD79B)

E006−T048−X001−S211 (Junlz−IL9R−Linker−TNFRSF4)

E008−T001−S064−S047 (Junlz−CD2−CSF3R−CD27)

E006−T047−S141−S050 (Junlz−IL7RA−IL13RA1−CD40)

E006−T064−S190−S080 (Junlz−IL20RA−MyD88−ICOS)

E008−T001−S121−S212 (Junlz−CD2−IL7RA−TNFRSF8)

E006−T023−S171−S211 (Junlz−FCER1G−IL31RA−TNFRSF4)

E006−T030−S129−S047 (Junlz−IFNAR2−IL10RA−CD27)

0

25

50

75

100

PBM

C_D

ay12

Bloo

d−G

17−C

HO−A

XL

Bloo

d−G

18−C

HO−A

XL

Bloo

d−G

19−C

HO−A

XL

Bloo

d−G

20−C

HO−A

XL

Bloo

d−G

21−C

HO−A

XL

NPS

PL−G

17−C

HO−A

XL

NPS

PL−G

18−C

HO−A

XL

NPS

PL−G

19−C

HO−A

XL

NPS

PL−G

20−C

HO−A

XL

NPS

PL−G

21−C

HO−A

XL

NPT

UM−G

17−C

HO−A

XL

NPT

UM−G

18−C

HO−A

XL

NPT

UM−G

19−C

HO−A

XL

NPT

UM−G

20−C

HO−A

XL

variable

Prop

ortio

n of

tota

l (%

)

E008−T032−S190−S213 (Junlz−IFNGR2−MyD88−TNFRSF9)

E006−T061−S190−S080 (Junlz−IL17RE−MyD88−ICOS)

E006−T069−S197−S053 (Junlz−IL27R−MyD88−CD79B)

E006−T070−S197−S037 (Junlz−IL27R−MyD88−CD3D)

E006−T073−X001−S074 (Junlz−LIFR−Linker−FCER1G)

E010−T077−S192−S074 (Junlz−PRLR−MyD88−FCER1G)

E007−T012−S054−S076 (Junlz−CD79A−CRLF2−FCGRA2)

E008−T004−S142−S212 (Junlz−CD3G−IL13RA2−TNFRSF8)

E009−T019−S194−S049 (Junlz−CSF3R−MyD88−CD28)

E010−T058−S121−S080 (Junlz−IL17RB−IL7RA−ICOS)

E008−T008−S194−S215 (Junlz−CD8RB−MyD88−TNFRSF18)

E009−T023−S190−S050 (Junlz−FCER1G−MyD88−CD40)

E007−T052−S102−S049 (Junlz−IL12RB1−IL1RL1−CD28)

E007−T052−S197−S075 (Junlz−IL12RB1−MyD88−FCGR2C)

E010−T032−S186−S050 (Junlz−IFNGR2−MPL−CD40)

E009−T028−S130−S212 (Junlz−ICOS−IL10RB−TNFRSF8)

E010−T012−S192−S214 (Junlz−CD79A−MyD88−TNFRSF14)

E006−T066−S109−X002 (Junlz−IL21R−IL3RA−STOP−PA)

0

25

50

75

100

PBM

C_D

ay12

G06

_CH

O−b

lood

G07

_CH

O−b

lood

G08

_CH

O−b

lood

G10

_CH

O−b

lood

G11

_CH

O−b

lood

G06

_CH

O−s

plen

ocyt

es_n

on_p

urifi

ed

G07

_CH

O−s

plen

ocyt

es_n

on_p

urifi

ed

G08

_CH

O−s

plen

ocyt

es_n

on_p

urifi

ed

G09

_CH

O−s

plen

ocyt

es_n

on_p

urifi

ed

G10

_CH

O−s

plen

ocyt

es_n

on_p

urifi

ed

G11

_CH

O−s

plen

ocyt

es_n

on_p

urifi

ed

G06

_CH

O−t

umor

_non

_pur

ified

G07

_CH

O−t

umor

_non

_pur

ified

G08

_CH

O−t

umor

_non

_pur

ified

G09

_CH

O−t

umor

_non

_pur

ified

G10

_CH

O−t

umor

_non

_pur

ified

G11

_CH

O−t

umor

_non

_pur

ified

variable

Prop

ortio

n of

tota

l (%

)

E006−T053−S186−S074 (Junlz−IL12RB2−MPL−FCER1G)

E009−T026−S100−S047 (Junlz−GHR−IL1RAP−CD27)

E006−T018−S141−S213 (Junlz−CSF2RB−IL13RA1−TNFRSF9)

E009−T062−S190−S074 (Junlz−IL18R1−MyD88−FCER1G)

E010−T042−S194−S050 (Junlz−IL4R−MyD88−CD40)

E009−T012−S154−X002 (Junlz−CD79A−IL18R1−STOP−PA)

E009−T020−S121−S037 (Junlz−CSF3R−IL7RA−CD3D)

E006−T044−S186−S053 (Junlz−IL6R−MPL−CD79B)

E007−T032−S064−S052 (Junlz−IFNGR2−CSF3R−CD79A)

E006−T057−S180−S051 (Junlz−IL17RA−LIFR−CD40)

E006−T023−S117−S080 (Junlz−FCER1G−IL6ST−ICOS)

E006−T006−S171−S215 (Junlz−CD4−IL31RA−TNFRSF18)

E009−T056−S104−S080 (Junlz−IL15RA−IL2RA−ICOS)

E006−T069−S177−S080 (Junlz−IL27R−LEPR−ICOS)

E008−T030−S057−S037 (Junlz−IFNAR2−CSF2RB−CD3D)

E006−T013−S196−S048 (Junlz−CD79B−MyD88−CD28)

E006−T032−S197−S075 (Junlz−IFNGR2−MyD88−FCGR2C)

0

25

50

75

100

PBM

C_D

ay12

G17

_CH

O−R

OR2−b

lood

G18

_CH

O−R

OR2−b

lood

G20

_CH

O−R

OR2−b

lood

G21

_CH

O−R

OR2−b

lood

G17

_CH

O−R

OR2−s

plen

ocyt

es_n

on_p

urifi

ed

G18

_CH

O−R

OR2−s

plen

ocyt

es_n

on_p

urifi

ed

G20

_CH

O−R

OR2−s

plen

ocyt

es_n

on_p

urifi

ed

G21

_CH

O−R

OR2−s

plen

ocyt

es_n

on_p

urifi

ed

G17

_CH

O−R

OR2−t

umor

_non

_pur

ified

G20

_CH

O−R

OR2−t

umor

_non

_pur

ified

G21

_CH

O−R

OR2−t

umor

_non

_pur

ified

variable

Prop

ortio

n of

tota

l (%

)

E008−T012−S157−S216 (Junlz−CD79A−IL20RB−TNFRSF18)

E009−T005−S142−S076 (Junlz−CD3Z−IL13RA2−FCGRA2)

E007−T021−S194−S211 (Junlz−EPOR−MyD88−TNFRSF4)

E006−T038−S154−X002 (Junlz−IL2RA−IL18R1−STOP−PA)

E010−T018−S165−S051 (Junlz−CSF2RB−IL23R−CD40)

E009−T032−X001−S211 (Junlz−IFNGR2−Linker−TNFRSF4)

E007−T048−S186−S053 (Junlz−IL9R−MPL−CD79B)

E006−T044−S062−S053 (Junlz−IL6R−CSF3R−CD79B)

E007−T029−S069−S076 (Junlz−IFNAR1−EPOR−FCGRA2)

E006−T049−S109−S074 (Junlz−IL10RA−IL3RA−FCER1G)

E009−T010−S177−S037 (Junlz−CD28−LEPR−CD3D)

E008−T033−S197−S216 (Junlz−IFNLR1−MyD88−TNFRSF18)

E006−T006−S062−S052 (Junlz−CD4−CSF3R−CD79A)

E007−T057−S195−S213 (Junlz−IL17RA−MyD88−TNFRSF9)

E006−T031−S109−S216 (Junlz−IFNGR1−IL3RA−TNFRSF18)

E006−T077−S129−X002 (Junlz−PRLR−IL10RA−STOP−PA)

●

●

● ●●

●

●●

●

●

● ●●

●

●●

●

●

● ●●

●

●●

●

●

● ●●

●

●

●

●

●

● ●●

●

●

●

●

●

● ●●

●

●●

●

●

● ●●

●●

●

●

●

● ●●

●

●

●

●

●

● ●●

●

● ●

Donor 1

Donor 2

Donor 3

0 5 10 15 20

0

20

40

60

0

20

40

60

0

20

40

60

Time (days)

Fold

exp

ansi

on

●

●

●

●

●

●

Construct 1

Construct 2

Construct 3

Construct 4

Construct 5

Construct 6

Construct 7

Construct 8

Construct 9

Construct 10

Construct 11

Construct 12

Construct 13

Construct 14

Construct 15

Construct 16

No SLE

Non transfected

Conclusions

0.00

0.25

0.50

0.75

1.00

G32

G34

G35

G36

G39

G40

G43

G46

G47

G48

G49

G50

G33

G37

G38

G41

G42

G44

G45 G6

G8

G9

G10

G12

G18

G22

G23

G24 G7

G11

G13

G14

G15

G19

G21

G25

PBM

C

Prop

ortio

n of

sam

ple E009−T006−S062−S053 (Junlz−CD4−CSF3R−CD79B)

E008−T030−S057−S037 (Junlz−IFNAR2−CSF2RB−CD3D)E010−T021−S197−S049 (Junlz−EPOR−MyD88−CD28)E006−T023−S117−S080 (Junlz−FCER1G−IL6ST−ICOS)E007−T032−S064−S052 (Junlz−IFNGR2−CSF3R−CD79A)E008−T001−S121−S212 (Junlz−CD2−IL7RA−TNFRSF8)E006−T032−S197−S075 (Junlz−IFNGR2−MyD88−FCGR2C)E009−T056−S104−S080 (Junlz−IL15RA−IL2RA−ICOS)E007−T005−S170−S076 (Junlz−CD3Z−IL31RA−FCGRA2)E008−T003−S138−S039 (Junlz−CD3E−IL12RB2−CD3G)E006−T006−S171−S215 (Junlz−CD4−IL31RA−TNFRSF18)E006−T056−S196−S212 (Junlz−IL15RA−MyD88−TNFRSF8)E006−T044−S186−S053 (Junlz−IL6R−MPL−CD79B)E008−T065−S069−S053 (Junlz−IL20RB−EPOR−CD79B)E008−T038−S165−S052 (Junlz−IL2RA−IL23R−CD79A)E009−T062−S190−S074 (Junlz−IL18R1−MyD88−FCER1G)

E008−T001−S064−S047 (Junlz−CD2−CSF3R−CD27)E006−T045−S072−S051 (Junlz−IL6ST−EPOR−CD40)EmptyE008−T031−S083−S212 (Junlz−IFNGR1−IFNAR2−TNFRSF8)E009−T032−S170−S074 (Junlz−IFNGR2−IL31RA−FCER1G)E006−T038−S192−S039 (Junlz−IL2RA−MyD88−CD3G)E009−T010−S170−S074 (Junlz−CD28−IL31RA−FCER1G)E006−T064−S190−S080 (Junlz−IL20RA−MyD88−ICOS)E007−T039−S197−S080 (Junlz−IL2RB−MyD88−ICOS)E006−T006−S194−S211 (Junlz−CD4−MyD88−TNFRSF4)E008−T078−S190−S211 (Junlz−TNFRSF4−MyD88−TNFRSF4)E007−T054−S197−S212 (Junlz−IL13RA1−MyD88−TNFRSF8)E010−T072−S192−S212 (Junlz−LEPR−MyD88−TNFRSF8)E009−T073−S062−S053 (Junlz−LIFR−CSF3R−CD79B)E010−T066−S197−S051 (Junlz−IL21R−MyD88−CD40)

Fig 7. (A) A small scale libraryusing 30 selected candidates induced enhancedin vitro expansion at 14 days post-injection as compared with the original libraries. (B) Identification of several SLE that persisted and/or expanded in a CAR/antigen signaling independent in multiple replicates (B) for further evaluation and characterization. (C) Antigen-independent cytokine-free in vitro expansion of PBMC transduced with a few selected constructs in three different healthy blood donors. Two constructs induced expansion in all tested donors.

p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.050

10

20

0 10 20 30 40 50Rank

−log

10(p

val

ue) Position

P1

P2

P3

P4

p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05

0

1

2

0 10 20 30 40 50Rank

−log

10(p

val

ue) Position

P1

P2

P3

P4

0

5

10

15

0 5 10 15−log10(p value), Library 1

−log

10(p

val

ue),

Libr

ary

2

Position

P1

P2

P3

P4

0

2

4

0 2 4−log10(p value), Library 1

−log

10(p

val

ue),

Libr

ary

2

Position

P1

P2

P3

P4

p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.05p = 0.050

10

20

0 10 20 30 40 50Rank

−log

10(p

val

ue) Position

P1

P2

P3

P4

Library 1 vs Library 2

Library 1 vs Library 2

CAR signal independent

Overall

Overall

CAR signal dependent

ctrl

diff

Interleukin.2.family.signalingInterleukin.15.signalingInterleukin.9.signalingSignal.TransductionSignaling.by.LeptinInterleukin.4.and.13.signalingPlatelet.Aggregation..Plug.Formation.Interleukin.17.signalingTranscriptional.regulationInterleukin.20.family.signalingInterleukin.35.SignallingIFNG.signalingIFNalpha.beta.signalingInterferon.SignalingInterleukin.18.signalingRAF.MAP.kinase.cascadeGPVI.mediated.activation.cascadeGPCR.IL3.IL5.GMCSF.signalingMAPK.signalingRUNX1.and.FOXP3.control.the.development.of.regulatory.T.lymphocytes..Tregs.HemostasisPlatelet.activation..signaling.and.aggregationDiseaseTLR.MYD88.TRAF.signalingAdaptive.Immune.SystemInnate.Immune.SystemPI3K.Akt.signalingInterleukin.1.family.signalingSurfactant.metabolismInterleukin.36.pathwayInterleukin.33.signalingSynapse.tyrosine.phosphatasesInterleukin.10.signalingRUNX1.regulates.transcription.of.genes.involved.in.interleukin.signalingInterleukin.6.family.signalingIL.6.type.cytokine.receptor.ligand.interactionsProlactin.GHR.signalingInterleukin.12.family.signalingMAPK.ERK.activationInterleukin.37.signalingImmune.SystemCytokine.Signaling.in.Immune.systemInterleukin.7.signalingSignaling.by.InterleukinsOther.interleukin.signaling

0.2

0.4

0.6

0.8

ctrl

diff

Adaptive.Immune.SystemFCERI.signalingCell.surface.interactions.at.the.vascular.wallGPVI.mediated.activation.cascadeHemostasisPlatelet.activation..signaling.and.aggregationClathrin.mediated.endocytosisNeutrophil.degranulationHIV.infectionCD28.dependent.signalingCostimulation.by.the.CD28.familyTCR.signaling.PD1.signalingAberrant.PI3K.Akt.signaling.in.cancerPI3K.Akt.signalingSignal.TransductionDiseaseBCR.signalingInnate.Immune.SystemFCGR.dependent.phagocytosisTNF.receptor.superfamily..TNFSF..members.mediating.non.canonical.NF.kB.pathwayCytokine.Signaling.in.Immune.systemTNFR2.non.canonical.NF.kB.pathwayTNFs.bind.their.physiological.receptorsImmunoregulatory.interactions.between.a.Lymphoid.and.a.non.Lymphoid.cellImmune.SystemTranscriptional.regulationRUNX1.and.FOXP3.control.the.development.of.regulatory.T.lymphocytes..Tregs.

0.2

0.4

0.6

0.8ctrl

diff

Adaptive.Immune.SystemFCERI.signalingCell.surface.interactions.at.the.vascular.wallGPVI.mediated.activation.cascadeHemostasisPlatelet.activation..signaling.and.aggregationClathrin.mediated.endocytosisNeutrophil.degranulationHIV.infectionCD28.dependent.signalingCostimulation.by.the.CD28.familyTCR.signaling.PD1.signalingAberrant.PI3K.Akt.signaling.in.cancerPI3K.Akt.signalingSignal.TransductionDiseaseBCR.signalingInnate.Immune.SystemFCGR.dependent.phagocytosisTNF.receptor.superfamily..TNFSF..members.mediating.non.canonical.NF.kB.pathwayCytokine.Signaling.in.Immune.systemTNFR2.non.canonical.NF.kB.pathwayTNFs.bind.their.physiological.receptorsImmunoregulatory.interactions.between.a.Lymphoid.and.a.non.Lymphoid.cellImmune.SystemTranscriptional.regulationRUNX1.and.FOXP3.control.the.development.of.regulatory.T.lymphocytes..Tregs.

0.2

0.4

0.6

0.8

0.00

0.25

0.50

0.75

1.00

G32

G34

G35

G36

G39

G40

G43

G46

G47

G48

G49

G50

G33

G37

G38

G41

G42

G44

G45 G6

G8

G9

G10

G12

G18

G22

G23

G24 G7

G11

G13

G14

G15

G19

G21

G25

PBM

C

Prop

ortio

n of

sam

ple E009−T006−S062−S053 (Junlz−CD4−CSF3R−CD79B)

E008−T030−S057−S037 (Junlz−IFNAR2−CSF2RB−CD3D)E010−T021−S197−S049 (Junlz−EPOR−MyD88−CD28)E006−T023−S117−S080 (Junlz−FCER1G−IL6ST−ICOS)E007−T032−S064−S052 (Junlz−IFNGR2−CSF3R−CD79A)E008−T001−S121−S212 (Junlz−CD2−IL7RA−TNFRSF8)E006−T032−S197−S075 (Junlz−IFNGR2−MyD88−FCGR2C)E009−T056−S104−S080 (Junlz−IL15RA−IL2RA−ICOS)E007−T005−S170−S076 (Junlz−CD3Z−IL31RA−FCGRA2)E008−T003−S138−S039 (Junlz−CD3E−IL12RB2−CD3G)E006−T006−S171−S215 (Junlz−CD4−IL31RA−TNFRSF18)E006−T056−S196−S212 (Junlz−IL15RA−MyD88−TNFRSF8)E006−T044−S186−S053 (Junlz−IL6R−MPL−CD79B)E008−T065−S069−S053 (Junlz−IL20RB−EPOR−CD79B)E008−T038−S165−S052 (Junlz−IL2RA−IL23R−CD79A)E009−T062−S190−S074 (Junlz−IL18R1−MyD88−FCER1G)

E008−T001−S064−S047 (Junlz−CD2−CSF3R−CD27)E006−T045−S072−S051 (Junlz−IL6ST−EPOR−CD40)EmptyE008−T031−S083−S212 (Junlz−IFNGR1−IFNAR2−TNFRSF8)E009−T032−S170−S074 (Junlz−IFNGR2−IL31RA−FCER1G)E006−T038−S192−S039 (Junlz−IL2RA−MyD88−CD3G)E009−T010−S170−S074 (Junlz−CD28−IL31RA−FCER1G)E006−T064−S190−S080 (Junlz−IL20RA−MyD88−ICOS)E007−T039−S197−S080 (Junlz−IL2RB−MyD88−ICOS)E006−T006−S194−S211 (Junlz−CD4−MyD88−TNFRSF4)E008−T078−S190−S211 (Junlz−TNFRSF4−MyD88−TNFRSF4)E007−T054−S197−S212 (Junlz−IL13RA1−MyD88−TNFRSF8)E010−T072−S192−S212 (Junlz−LEPR−MyD88−TNFRSF8)E009−T073−S062−S053 (Junlz−LIFR−CSF3R−CD79B)E010−T066−S197−S051 (Junlz−IL21R−MyD88−CD40)

0.00

0.25

0.50

0.75

1.00

G32

G34

G35

G36

G39

G40

G43

G46

G47

G48

G49

G50

G33

G37

G38

G41

G42

G44

G45 G6

G8

G9

G10

G12

G18

G22

G23

G24 G7

G11

G13

G14

G15

G19

G21

G25

PBM

C

Prop

ortio

n of

sam

ple E009−T006−S062−S053 (Junlz−CD4−CSF3R−CD79B)

E008−T030−S057−S037 (Junlz−IFNAR2−CSF2RB−CD3D)E010−T021−S197−S049 (Junlz−EPOR−MyD88−CD28)E006−T023−S117−S080 (Junlz−FCER1G−IL6ST−ICOS)E007−T032−S064−S052 (Junlz−IFNGR2−CSF3R−CD79A)E008−T001−S121−S212 (Junlz−CD2−IL7RA−TNFRSF8)E006−T032−S197−S075 (Junlz−IFNGR2−MyD88−FCGR2C)E009−T056−S104−S080 (Junlz−IL15RA−IL2RA−ICOS)E007−T005−S170−S076 (Junlz−CD3Z−IL31RA−FCGRA2)E008−T003−S138−S039 (Junlz−CD3E−IL12RB2−CD3G)E006−T006−S171−S215 (Junlz−CD4−IL31RA−TNFRSF18)E006−T056−S196−S212 (Junlz−IL15RA−MyD88−TNFRSF8)E006−T044−S186−S053 (Junlz−IL6R−MPL−CD79B)E008−T065−S069−S053 (Junlz−IL20RB−EPOR−CD79B)E008−T038−S165−S052 (Junlz−IL2RA−IL23R−CD79A)E009−T062−S190−S074 (Junlz−IL18R1−MyD88−FCER1G)

E008−T001−S064−S047 (Junlz−CD2−CSF3R−CD27)E006−T045−S072−S051 (Junlz−IL6ST−EPOR−CD40)EmptyE008−T031−S083−S212 (Junlz−IFNGR1−IFNAR2−TNFRSF8)E009−T032−S170−S074 (Junlz−IFNGR2−IL31RA−FCER1G)E006−T038−S192−S039 (Junlz−IL2RA−MyD88−CD3G)E009−T010−S170−S074 (Junlz−CD28−IL31RA−FCER1G)E006−T064−S190−S080 (Junlz−IL20RA−MyD88−ICOS)E007−T039−S197−S080 (Junlz−IL2RB−MyD88−ICOS)E006−T006−S194−S211 (Junlz−CD4−MyD88−TNFRSF4)E008−T078−S190−S211 (Junlz−TNFRSF4−MyD88−TNFRSF4)E007−T054−S197−S212 (Junlz−IL13RA1−MyD88−TNFRSF8)E010−T072−S192−S212 (Junlz−LEPR−MyD88−TNFRSF8)E009−T073−S062−S053 (Junlz−LIFR−CSF3R−CD79B)E010−T066−S197−S051 (Junlz−IL21R−MyD88−CD40)

0.00

0.25

0.50

0.75

1.00

G32

G34

G35

G36

G39

G40

G43

G46

G47

G48

G49

G50

G33

G37

G38

G41

G42

G44

G45 G6

G8

G9

G10

G12

G18

G22

G23

G24 G7

G11

G13

G14

G15

G19

G21

G25

PBM

C

Prop

ortio

n of

sam

ple E009−T006−S062−S053 (Junlz−CD4−CSF3R−CD79B)

E008−T030−S057−S037 (Junlz−IFNAR2−CSF2RB−CD3D)E010−T021−S197−S049 (Junlz−EPOR−MyD88−CD28)E006−T023−S117−S080 (Junlz−FCER1G−IL6ST−ICOS)E007−T032−S064−S052 (Junlz−IFNGR2−CSF3R−CD79A)E008−T001−S121−S212 (Junlz−CD2−IL7RA−TNFRSF8)E006−T032−S197−S075 (Junlz−IFNGR2−MyD88−FCGR2C)E009−T056−S104−S080 (Junlz−IL15RA−IL2RA−ICOS)E007−T005−S170−S076 (Junlz−CD3Z−IL31RA−FCGRA2)E008−T003−S138−S039 (Junlz−CD3E−IL12RB2−CD3G)E006−T006−S171−S215 (Junlz−CD4−IL31RA−TNFRSF18)E006−T056−S196−S212 (Junlz−IL15RA−MyD88−TNFRSF8)E006−T044−S186−S053 (Junlz−IL6R−MPL−CD79B)E008−T065−S069−S053 (Junlz−IL20RB−EPOR−CD79B)E008−T038−S165−S052 (Junlz−IL2RA−IL23R−CD79A)E009−T062−S190−S074 (Junlz−IL18R1−MyD88−FCER1G)

E008−T001−S064−S047 (Junlz−CD2−CSF3R−CD27)E006−T045−S072−S051 (Junlz−IL6ST−EPOR−CD40)EmptyE008−T031−S083−S212 (Junlz−IFNGR1−IFNAR2−TNFRSF8)E009−T032−S170−S074 (Junlz−IFNGR2−IL31RA−FCER1G)E006−T038−S192−S039 (Junlz−IL2RA−MyD88−CD3G)E009−T010−S170−S074 (Junlz−CD28−IL31RA−FCER1G)E006−T064−S190−S080 (Junlz−IL20RA−MyD88−ICOS)E007−T039−S197−S080 (Junlz−IL2RB−MyD88−ICOS)E006−T006−S194−S211 (Junlz−CD4−MyD88−TNFRSF4)E008−T078−S190−S211 (Junlz−TNFRSF4−MyD88−TNFRSF4)E007−T054−S197−S212 (Junlz−IL13RA1−MyD88−TNFRSF8)E010−T072−S192−S212 (Junlz−LEPR−MyD88−TNFRSF8)E009−T073−S062−S053 (Junlz−LIFR−CSF3R−CD79B)E010−T066−S197−S051 (Junlz−IL21R−MyD88−CD40)

0.00

0.25

0.50

0.75

1.00

G32

G34

G35

G36

G39

G40

G43

G46

G47

G48

G49

G50

G33

G37

G38

G41

G42

G44

G45

G6 G8 G9 G10

G12

G18

G22

G23

G24

G7 G11

G13

G14

G15

G19

G21

G25

PBM

C

Prop

ortio

n of s

ampl

e E009−T006−S062−S053 (Junlz−CD4−CSF3R−CD79B)E008−T030−S057−S037 (Junlz−IFNAR2−CSF2RB−CD3D)E010−T021−S197−S049 (Junlz−EPOR−MyD88−CD28)E006−T023−S117−S080 (Junlz−FCER1G−IL6ST−ICOS)E007−T032−S064−S052 (Junlz−IFNGR2−CSF3R−CD79A)E008−T001−S121−S212 (Junlz−CD2−IL7RA−TNFRSF8)E006−T032−S197−S075 (Junlz−IFNGR2−MyD88−FCGR2C)E009−T056−S104−S080 (Junlz−IL15RA−IL2RA−ICOS)E007−T005−S170−S076 (Junlz−CD3Z−IL31RA−FCGRA2)E008−T003−S138−S039 (Junlz−CD3E−IL12RB2−CD3G)E006−T006−S171−S215 (Junlz−CD4−IL31RA−TNFRSF18)E006−T056−S196−S212 (Junlz−IL15RA−MyD88−TNFRSF8)E006−T044−S186−S053 (Junlz−IL6R−MPL−CD79B)E008−T065−S069−S053 (Junlz−IL20RB−EPOR−CD79B)E008−T038−S165−S052 (Junlz−IL2RA−IL23R−CD79A)E009−T062−S190−S074 (Junlz−IL18R1−MyD88−FCER1G)

E008−T001−S064−S047 (Junlz−CD2−CSF3R−CD27)E006−T045−S072−S051 (Junlz−IL6ST−EPOR−CD40)EmptyE008−T031−S083−S212 (Junlz−IFNGR1−IFNAR2−TNFRSF8)E009−T032−S170−S074 (Junlz−IFNGR2−IL31RA−FCER1G)E006−T038−S192−S039 (Junlz−IL2RA−MyD88−CD3G)E009−T010−S170−S074 (Junlz−CD28−IL31RA−FCER1G)E006−T064−S190−S080 (Junlz−IL20RA−MyD88−ICOS)E007−T039−S197−S080 (Junlz−IL2RB−MyD88−ICOS)E006−T006−S194−S211 (Junlz−CD4−MyD88−TNFRSF4)E008−T078−S190−S211 (Junlz−TNFRSF4−MyD88−TNFRSF4)E007−T054−S197−S212 (Junlz−IL13RA1−MyD88−TNFRSF8)E010−T072−S192−S212 (Junlz−LEPR−MyD88−TNFRSF8)E009−T073−S062−S053 (Junlz−LIFR−CSF3R−CD79B)E010−T066−S197−S051 (Junlz−IL21R−MyD88−CD40)

Sample %Live %CAR+#CAR+

CellsTotal

%CD3+%CD3+ CD56-

%CD3+ CD56+ %CD4+ %CD8+

Construct 2 17.12% 41.24% 36137 97.12% 91.55% 5.57% 6.00% 85.02%Construct 10 27.34% 88.73% 95362 98.99% 88.33% 10.66% 14.07% 82.47%

No SLE 1.17% 89.72% 2330 79.35% 76.35% 3.00% 11.46% 71.37%Construct 2 14.17% 48.05% 32912 97.68% 78.64% 19.04% 20.61% 72.08%Construct 8 3.10% 80.25% 2048 94.09% 69.68% 24.41% 7.67% 87.11%

Construct 10 34.18% 86.27% 146733 98.25% 72.20% 26.05% 19.42% 76.83%No SLE 2.45% 82.62% 2696 78.56% 75.22% 3.34% 16.51% 64.13%

Construct 2 19.95% 17.05% 9655 95.46% 89.50% 5.96% 18.55% 72.82%Construct 10 45.72% 83.33% 100117 99.35% 98.45% 0.90% 35.64% 61.38%Donor 3

Phenotype Of Live Of CAR+

Donor 1

Donor 2

EmptySize

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

1000

1500

2000

2500

3000

1000 1500 2000 2500 3000size (nt), pre−expansion

size

(nt),

pos

t−ex

pans

ion

GC content

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●

0.40

0.45

0.50

0.55

0.60

0.65

0.40 0.45 0.50 0.55 0.60 0.65GC%, pre−expansion

GC

%, p

ost−

expa

nsio

n

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●

●

●

0.40

0.45

0.50

0.55

0.60

0.65

0.40 0.45 0.50 0.55 0.60 0.65GC%, pre−expansion

GC

%, p

ost−

expa

nsio

n

GC content

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●

●

1000

1500

2000

2500

3000

1000 1500 2000 2500 3000size (nt), pre−expansion

size

(nt),

pos

t−ex

pans

ion

Size

●● ●●

●

●

●

●

●

●

●

●0

3000

6000

9000

12000

8 10 12 14Time (days)

LV c

opie

s per

ug

gDN

A

●

●

●

●

●

●

Library 1/|Xenograft−antigen

Library 1/Xenograft−no antigen

Library 2/Xenograft−antigen

Library 2/Xenograft−no antigen

Validation library/Xenograft−antigen

Validation library/Xenograft−no antigen

Library 1/xg-ag

Library 1/xg

Library 2/xg-ag

Library 2 xg

Small scale library/xg-ag

Small scale library/xg−1

−0.8

−0.6

−0.4

−0.2

0

0.2

0.4

0.6

0.8

1

pre−injection

blood−xg−1

spleen−xg−1

tumor−xg−1

blood−xg−2

spleen−xg−2

tumor−xg−2

blood−xg−3

spleen−xg−3

tumor−xg−3

blood−xg−4

spleen−xg−4

tumor−xg−4

blood−xg−5

spleen−xg−5

tumor−xg−5

blood−xg−ag−1

spleen−xg−ag−1

tumor−xg−ag−1

blood−xg−ag−2

spleen−xg−ag−2

tumor−xg−ag−2

blood−xg−ag−3

spleen−xg−ag−3

tumor−xg−ag−3

blood−xg−ag−4

spleen−xg−ag−4

tumor−xg−ag−4

blood−xg−ag−5

spleen−xg−ag−5

pre−injectionblood−xg−1spleen−xg−1tumor−xg−1blood−xg−2spleen−xg−2tumor−xg−2blood−xg−3spleen−xg−3tumor−xg−3blood−xg−4spleen−xg−4tumor−xg−4blood−xg−5spleen−xg−5tumor−xg−5

blood−xg−ag−1spleen−xg−ag−1tumor−xg−ag−1blood−xg−ag−2spleen−xg−ag−2tumor−xg−ag−2blood−xg−ag−3spleen−xg−ag−3tumor−xg−ag−3blood−xg−ag−4spleen−xg−ag−4tumor−xg−ag−4blood−xg−ag−5spleen−xg−ag−5

−1

−0.8

−0.6

−0.4

−0.2

0

0.2

0.4

0.6

0.8

1

pre−injection

blood−xg−1

spleen−xg−1

tumor−xg−1

blood−xg−2

spleen−xg−2

tumor−xg−2

blood−xg−3

spleen−xg−3

tumor−xg−3

spleen−xg−4

tumor−xg−4

blood−xg−5

spleen−xg−5

tumor−xg−5

blood−xg−ag−1

spleen−xg−ag−1

tumor−xg−ag−1

blood−xg−ag−2

spleen−xg−ag−2

blood−xg−ag−3

spleen−xg−ag−3

tumor−xg−ag−3

blood−xg−ag−4

spleen−xg−ag−4

tumor−xg−ag−4

pre−injectionblood−xg−1spleen−xg−1tumor−xg−1blood−xg−2spleen−xg−2tumor−xg−2blood−xg−3spleen−xg−3tumor−xg−3spleen−xg−4tumor−xg−4blood−xg−5spleen−xg−5tumor−xg−5

blood−xg−ag−1spleen−xg−ag−1tumor−xg−ag−1blood−xg−ag−2spleen−xg−ag−2blood−xg−ag−3spleen−xg−ag−3tumor−xg−ag−3blood−xg−ag−4spleen−xg−ag−4tumor−xg−ag−4

Library 2

Library 1