Cancer Immunology, Immunotherapy (submitted in 2014) Christian Krug et al.

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figure 1: agnistic anti-cd3 n PBMC PBMC frm healthy dnrs were expanded fr ten days using the agnistic anti-cd3 antibdy OKT3 and IL-2.

1 Cancer Immunlgy, Immuntherapy (submitted in 2014) Christian Krug et al. PBMC day 0 d 0 n anti-cd3 side scatter d 0 with anti-cd3 frward scatter PBMC day 10 d 10 n anti-cd3 side scatter d 10 with anti-cd3 frward scatter Supplementary figure 1: agnistic anti-cd3 n PBMC PBMC frm healthy dnrs were expanded fr ten days using the agnistic anti-cd3 antibdy OKT3 and IL-2. Cells, which were cultured withut the anti-cd3 antibdy, served as cntrl. PBMC were stained fr the murine anti-cd3 antibdy with a flurescence-labeled antibdy targeted against murine immunglbulin n day 0 and day 10 f culture. One representative f three experiments is depicted.

2 35 T2.A1 fresh 35 KATO III fresh relative lysis relative lysis CD3 CEA CD3 cn :1 20:1 6:1 2:1 effectr:target rati :1 20:1 6:1 2:1 effectr:target rati 35 T2.A1 thaw 35 KATO III thaw relative lysis relative lysis :1 20:1 6:1 2:1 effectr:target rati :1 20:1 6:1 2:1 effectr:target rati Supplementary figure 2: CAR-RNA-transfected T cells f healthy dnrs lyse antigen-bearing tumr cells PBMC frm healthy dnrs were expanded fr ten days using the agnistic anti-cd3 antibdy OKT3 and IL-2. The amplified T cells were electrprated with mrna encding a CEA-specific chimeric antigen receptr (CAR) r a CAR f irrelevant specificity (cn.). Cells were either used 6 h after electrpratin r crycnserved 2 h after electrpratin. Fresh and thawed T cells were assayed fr cytlytic activity in a standard 4 h chrmium 51 release assay, with T2.A1 (CEA - ) and KATO III (CEA + ) as target cells at the indicated ratis. The release f chrmium int the supernatant was determined and lysis was calculated. Mean values frm three independent experiments are depicted. Errr bars indicate standard errr f the mean.

3 Supplementary standard perating prcedure (SOP) 1: Expansin f T cells frm PBMC Equipment: cell centrifuge (e.g. Heraeus Multifuge 3 S-R; Cat.N ) tw cell culture incubatrs (e.g. Heraeus BBD 6220 CO2 Incubatr Cat.N ) vacuum pump and suctin system (e.g. Carl Rth bivac 106; Cat.N. KH49.1) Pipetby (e.g. Eppendrf easypet 3; Cat.N ) Cnsumables: X-viv 15 with Gentamycin and Phenl red (Lnza; Cat. N. BE04-418F) Prleukin IL-2, 18 x 10 6 IU / ml (Nvartis; ATC-cde L03AC01) Orthclne OKT-3, 1 g/l (Jannsen-Cilag; ATC-Cde L04AA02) cell factries (e.g. Nunc; Mat.n ) cell culture flasks T175, 600 ml (e.g. Nunc ) 50 ml cell culture tubes (e.g. Crning; Mat.N ) serlgical pipettes 5 ml (e.g. Crning; Mat.N. 4051) 10 ml (e.g. Crning; Mat.N. 4101) 25 ml (e.g. Crning; Mat.N. 4251) 50 ml (e.g. Crning; Mat.N. 4501) pipettes µl (e.g. Eppendrf Refence 2; Cat.N ) µl (e.g. Eppendrf Refence 2; Cat.N ) pipette filter tips 100 µl (e.g. Greiner filtertip 100 µl; Cat.N ) 1000 µl (e.g. MbP; Mat.N. 2079GIW) General remarks: all wrk n the pen prduct is perfrmed in a clean rm grade A (accrding t EU GMP classificatin) standard rm temperature: C; standard incubatr cnditins: 37 C; 5% CO 2 ; 95% relative humidity

4 C-applicable dcuments: SOP: generatin f peripheral bld mnnuclear cells (PBMC) SOP: determinatin f cell cncentratin SOP: FACS analyses f T cells SOP: electrpratin f T cells Starting material: peripheral bld mnnuclear cells (PBMC; 2 x 10 8 t 6 x 10 8 cells in 200 ml t 1000 ml) number f PBMC: x 10 6 vlume f PBMC: ml gently mix PBMC remve samples fr quality cntrl (with backup) 2 x 2 ml: sterility 2 x 1 ml: endtxin 2 x 1 ml: FACS-analysis fr expressin f CD3 (fr infrmatin nly) transfer all PBMC t 50 ml cell culture tubes; fill each tube with 50 ml cell suspensin transfer remains t an additinal 50 ml tube number f tubes: centrifuge tubes with settings as fllws: 10 minutes, rm temperature; 215 g; acceleratin: 9; deceleratin: 9 aspirate and discard supernatant resuspend each pellet in 5 ml X-viv 15 medium pl resuspended PBMC in T175 flask adjust cells t a cncentratin f 1.0x10 6 cells/ml with X-viv 15 medium: final cell vlume = Number f PBMC / 10 6 x ml = ml added vlume X-viv 15: ml - ( x 5 ml) = ml final cell vlume number f tubes add calculated vlume f X-viv 15 add a final cncentratin f 0.1 µg/ml Orthclne OKT-3 and 1.0x10 3 IU/ml IL-2 vlume Orthclne: ml / = 0.0 ml = µl final cell vlume add calculated vlume f Orthclne vlume IL-2: ml / = 0.0 ml = µl final cell vlume add calculated vlume f IL-2 mix cells thrughly but gently by inverting transfer cell suspensin t ne (vlume < 400 ml) r equally t 2 cell factries

5 vlume per cell factry: ml incubate cells at standard cnditins fr hurs start f incubatin: date:.. ; time: : end f incubatin: date:.. ; time: : length f incubatin: h add fresh IL-2 at a final cncentratin f 1.0x10 3 IU/ml vlume IL-2: ml / = 0.0 ml = µl vlume per cell factry add calculated vlume f IL-2 incubate cells at standard cnditins fr hurs start f incubatin: date:.. ; time: : end f incubatin: date:.. ; time: : length f incubatin: h transfer all cells t T175 flask resuspend cells remve cunting sample cunt cells accrding t SOP: determinatin f cell cncentratin cell cncentratin:. x 10 6 cells / ml cell cncentratin >= 2x10 5 cells/ml? n: abrt prductin yes: cntinue prtcl calculate ttal number f cells: ttal number f cells: ml x. x 10 6 cells / ml =. x 10 6 cell vlume cell cncentratin if ttal cell number is > 4.8 x 10 8 use 4 x 10 7 cells per factry, prepare 12 factries, discard excess: calculate cell vlume per factry: cell vlume per factry: 4 x 10 7 /. x 10 6 / ml = ml cell cncentratin transfer calculated cell vlume t factries adjust cell cncentratin t 2 x 10 5 / ml by adding X-viv 15 ad 200 ml per factry vlume X-viv 15: 200 ml - ml = ml cell vlume per factry

6 add calculated vlume f X-viv 15 add 11 µl IL-2 per factry if ttal number f cells is < 4.8 x 10 8 then divide cells int 1 t 12 cell factries: calculate number f cell factries (rund up): x 10 6 / 40 x 10 6 =. ~ cell factries ttal number f cells rund up calculate number f cells and vlume per factry: number f cells per factry: x 10 6 / = x 10 6 ttal number f cells number f factries vlume f cells per factry: ml / = ml ttal vlume f cells number f factries mix cells thrughly but gently by inverting transfer calculated vlume f cells t cell factries: adjust cell cncentratin t 2 x 10 5 cells per ml by adding X-viv 15: calculate ttal vlume per factry: ttal vlume per factry: x 10 6 / 0.2 x 10 6 = ml number f cells per factry X-viv 15 per factry: 200 ml - ml = ml vlume f cells per factry add fresh IL-2 at a final cncentratin f 1.0x10 3 IU/ml: vlume IL-2: ml / = 0.0 ml = µl ttal vlume per cell factry add calculated vlume f IL-2 incubate cells at standard cnditins fr hurs start f incubatin: date:.. ; time: : end f incubatin: date:.. ; time: : length f incubatin: h add same vlume f fresh IL-2 as calculated abve incubate cells at standard cnditins fr hurs start f incubatin: date:.. ; time: : end f incubatin: date:.. ; time: : length f incubatin: h

7 duble culture vlume by adding X-viv 15 (same vlume as ttal cell vlume per factry: vlume X-viv 15 added per factry: ml calculate new ttal vlume per factry: ml x 2 = ml vlume added add fresh IL-2 at a final cncentratin f 1.0x10 3 IU/ml: vlume IL-2: ml / = 0.0 ml = µl ttal vlume per cell factry add calculated vlume f IL-2 Incubate cells at standard cnditins fr hurs start f incubatin: date:.. ; time: : end f incubatin: date:.. ; time: : length f incubatin: h transfer all cells t 50 ml cell culture tubes; fill each tube with 50 ml cell suspensin transfer remains t an additinal 50 ml tube number f tubes: centrifuge tubes with settings as fllws: 10 minutes, rm temperature; 215 g; acceleratin: 9; deceleratin: 9 aspirate and discard supernatant resuspend each pellet in 5 ml X-viv 15 medium pl resuspended cells in T175 flask remve samples fr quality cntrl (with backup) 2 x 2 ml: sterility 2 x 1 ml: FACS-analysis f T cells resuspend cells remve cunting sample cunt cells accrding t SOP: determinatin f cell cncentratin cell cncentratin:. x 10 6 cells / ml calculate ttal number f cells: ttal number f cells:. x 10 6 cells / ml x 5 x = x 10 6 cell cncentratin number f tubes

8 Supplementary standard perating prcedure (SOP) 2: Electrpratin f activated T cells Equipment: cell centrifuge (e.g. Heraeus Multifuge 3 S-R; Cat.N ) tw cell culture incubatrs (e.g. Heraeus BBD 6220 CO2 Incubatr Cat.N ) vacuum pump and suctin system (e.g. Carl Rth bivac 106; Cat.N. KH49.1) Pipetby (e.g. Eppendrf easypet 3; Cat.N ) electrpratin device (e.g. Bi-Rad Gene Pulser Xcell, Eukarytic System; Mat.N ) refrigeratr 4 C 8 C Cnsumables: X-viv 15 with Gentamycin and Phenl red (Lnza; Cat. N. BE04-418F) RPMI 1640 (e.g. Lnza; Mat.N. BE F) OptiMEM withut phenl red (e.g. Invitrgen-Gibc; Mat.N ) Prleukin IL-2, 18 x 10 6 IU / ml (Nvartis; ATC-cde L03AC01) Orthclne OKT-3, 1 g/l (Jannsen-Cilag; ATC-Cde L04AA02) cell culture flasks T75, 200 ml (e.g. Nunc ) 50 ml cell culture tubes (e.g. Crning; Mat.N ) serlgical pipettes 5 ml (e.g. Crning; Mat.N. 4051) 10 ml (e.g. Crning; Mat.N. 4101) 25 ml (e.g. Crning; Mat.N. 4251) 50 ml (e.g. Crning; Mat.N. 4501) pipettes µl (e.g. Eppendrf Refence 2; Cat.N ) µl (e.g. Eppendrf Refence 2; Cat.N ) pipette filter tips 100 µl (e.g. Greiner filtertip 100 µl; Cat.N ) 1000 µl (e.g. MbP; Mat.N. 2079GIW) fine tip Pasteur pipette, 3 ml (e.g. VWR Cat. N )

9 electrpratin cuvettes (e.g. Peqlab; Mat.N ) CAR-mRNA in water, 1.5 µg/µl, aliquts à 90 µg, API-grade T cells generated accrding t SOP expansin f T cells frm PBMC General remarks: all wrk n the pen prduct is perfrmed in a clean rm grade A (accrding t EU GMP classificatin) standard rm temperature: C; standard incubatr cnditins: 37 C; 5% CO 2 ; 95% relative humidity C-applicable dcuments: SOP: expansin f T cells frm PBMC SOP: crycnservatin f T cells SOP: determinatin f cell cncentratin SOP: FACS analyses f T cells Starting material: T cells generated accrding t SOP expansin f T cells frm PBMC number f T cells: x 10 6 vlume f T cells: ml cncentratin f T cells: x 10 6 / ml Preparative wrk: remve RPMI 1640, OptiMEM, and X-viv 15 frm refrigeratr at least 30 min befre starting, t allw fr pre-warming t rm temperature time: : adjust electrpratin device t prtcl: square wave pulse prtcl: 500 V; 5 ms; 1 pulse; 4 mm gap cuvettes perfrm test pulse withut cuvette, nte vltage and drp: Vltage: V drp: % calculate number f cuvettes: if number f T cells is > 2.7 x 10 9 use 30 cuvettes if number f T cells is < 2.7 x 10 9 calculate number f cuvettes:

10 calculate number f cuvettes (rund up): x 10 6 / 90 x 10 6 =. ~ cuvettes number f T cells rund up transfer CAR-mRNA (ne aliqut per cuvette) frm deep freezer t refrigeratr t allw t thaw prepare T75 cell culture flasks in same number as cuvettes transfer 30 ml f X-viv 15 t each flask time at start f wrk: : if ttal cell number is < 2.7 x 10 9 use all cells, transfer all cells t 50 ml cell culture tubes; fill each tube with a maximum f 50 ml cell suspensin number f tubes: if ttal cell number is > 2.7 x 10 9 use a maximum f 2.7 x 10 9 cells calculate vlume cntaining 2.7 x 10 9 cells: vlume t be used: 2,700 x 10 6 / x 10 6 / ml = ml cncentratin f T cells gently but thrughly mix T cells by inverting transfer calculated vlume f cells t 50 ml cell culture tubes; fill each tube with a maximum f 50 ml cell suspensin number f tubes: discard rest f cells r crycnserve fr F&E accrding t SOP crycnservatin f T cells centrifuge tubes with settings as fllws: 10 minutes, rm temperature; 215 g; acceleratin: 9; deceleratin: 9 aspirate and discard supernatant resuspend each pellet in 10 ml RPMI 1640 pl cells in tw 50 ml cell culture tube centrifuge tubes with settings as fllws: 10 minutes, rm temperature; 215 xg; acceleratin: 9; deceleratin: 9 aspirate and discard supernatant resuspend each pellet in 20 ml OptiMEM pl cells in ne tube centrifuge tube with settings as fllws: 10 minutes, rm temperature; 215 xg; acceleratin: 9; deceleratin: 9 aspirate and discard supernatant calculate vlume t resuspend cells: vlume t resuspend cells: ( x 0.6 ml) ml =. ml

11 number f cuvettes add calculated vlume f OptiMEM and resuspend pellet remve CAR mrna frm refrigeratr, check if cmpletely thawed mix by tapping electrprate cells: A. transfer ne aliqut f mrna cmpletely t ne cuvette B. transfer 600 µl f cells t ne cuvette C. place cuvette in shck pd D. clse shck pd E. shck cells F. remve cuvette frm shck pd G. transfer cells frm cuvette t prepared T75 flask cntaining X-viv 15 medium with a fine tip Pasteur pipette H. repeat frm A fr all cuvettes Incubate cells at standard cnditins fr min start f incubatin: time: : end f incubatin: time: : length f incubatin: min remve samples fr quality cntrl (with backup) 2 x 2 ml: sterility 2 x 1 ml: endtxin 2 x 2 ml: FACS-analysis resuspend cells remve sample fr cunting crycnserve accrding t SOP: crycnservatin f T cells

12 Supplementary flw chart 1: Expansin f T cells frm PBMC time line X viv 15 OKT 3 IL 2 starting material: activated T cells adjust t 10 6 cells/ml in X viv 15 with 0.1 g/l OKT 3 with 1 IU/µl IL 2 QC sample: sterility, endtxin, percentage f CD3 + (FIO) day 0 IL 2 add 1 IU/µl f IL 2 day 2 QC sample: cunting yes cell cncentratin >2 x 10 5 / ml? n day 3 X viv 15 adjust t 2 x 10 5 cells / ml Abrt prductin IL 2 add 1 IU/µl f IL 2 day 5 X viv 15 IL 2 duble cell vlume with X viv 15 add 1 IU/µl f IL 2 day 7 Prduct: expanded T cells QC sample: cunting sterility, endtxin, FACS day 10 SOP: electrpratin f expanded T cells

13 Supplementary flw chart 2: Electrpratin f expanded T cells frm PBMC starting material: expanded T cells yes number f cell <2.7 x 10 9 n use all cells use 2.7 x 10 9 cells crycnserve excess crycnserved expanded T cells RPMI 1640 wash cells with RPMI 1640 OptiMEM wash cell with OptiMEM OptiMEM resuspend cells in OptiMEM at 1.5x10 8 /ml CAR mrna add 90 µg f mrna t each cuvette add 9x10 7 cells t each cuvette electrprate: 500 V, 5 ms, square wave pulse X viv 15 transfer cells t X viv 15 incubate cells fr min Prduct: CAR electrprated T cells QC sample: sterility, endtxin, FACS cunting SOP: crycnservatin f T cells

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