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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
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 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
ytes
_−6
xeno
graf
t ant
igen−s
plen
ocyt
es_−
1xe
nogr
aft a
ntig
en−s
plen
ocyt
es_−
2xe
nogr
aft a
ntig
en−s
plen
ocyt
es_−
3xe
nogr
aft a
ntig
en−s
plen
ocyt
es_−
4xe
nogr
aft t
umor
_−1
xeno
graf
t tum
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
t ant
igen−t
umor
_−2
xeno
graf
t ant
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)
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●
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●
●
● ●●
●
●●
●
●
● ●●
●
●
●
●
●
● ●●
●
●
●
●
●
● ●●
●
●●
●
●
● ●●
●●
●
●
●
● ●●
●
●
●
●
●
● ●●
●
● ●
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