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THE MAIN US 20180193477A1TE A T HA N HAT ( 19) United States ( 12) Patent Application Publication ( 10 ) Pub . No. : US 2018 /0193477 A1 Ng et al. (43 ) Pub . Date : Jul. 12 , 2018 (54 ) DRUG -CONJUGATED BI- SPECIFIC A61K 39 /395 ( 2006 .01 ) ANTIGEN -BINDING CONSTRUCTS CO7K 16 / 46 (2006 .01 ) (52 ) U. S. CI. (71 ) Applicant: Zymeworks Inc ., Vancouver (CA ) CPC . .. A61K 47 /6803 ( 2017 . 08 ); A61K 2039 / 505 ( 2013 .01 ); A61P 35 / 02 ( 2018 .01 ) ; A61P 35 / 04 ( 72 ) Inventors : Gordon Yiu Kon Ng , Vancouver (CA ) ; (2018 .01 ) ; CO7K 16 / 2809 ( 2013 . 01 ); A61K Leonard G . Presta , San Francisco , CA 51/ 1042 ( 2013 .01 ); A61K 51/ 1027 (2013 . 01 ) ; (US ) ; Thomas Spreter Von A61K 51/ 103 ( 2013 .01 ) ; A61K 51 / 1051 Kreudenstein, Vancouver ( CA ) (2013 . 01 ) ; A61K 51/ 1072 (2013 .01 ); A61K 47/ 6817 ( 2017 .08 ); A61K 47/ 6811 ( 2017 .08 ); (21 ) Appl. No. : 15 /741 , 984 A61K 47 /6849 ( 2017 . 08 ) ; COZK 16 / 2803 ( 2013 .01 ) ; CO7K 16 / 32 (2013 .01 ) ; A61K ( 22 ) PCT Filed : Jul. 15, 2016 47 /6855 ( 2017 .08 ) ; A61K 47 /6869 ( 2017 . 08 ) ; CO7K 16 / 2863 ( 2013 .01 ) ; CO7K 16 / 28 ( 86 ) PCT No .: PCT/ CA2016 / 050839 ( 2013 .01 ) ; A61K 45 / 06 ( 2013 .01 ) ; A61K $ 371 (c ) ( 1 ) , 39 / 3955 ( 2013 . 01 ) ; A61K 39 /39558 (2013 .01 ) ; ( 2 ) Date : Jan . 4 , 2018 CO7K 16 /46 ( 2013 .01 ); CO7K 2317 / 56 (2013 . 01 ) ; CO7K 2317 /92 ( 2013 .01 ) ; CO7K Related U . S . Application Data 2317 / 73 (2013 .01 ) ; CO7K 2319 /00 ( 2013 .01 ) ; (60 ) Provisional application No . 62 /193 , 056 , filed on Jul. CO7K 2317 / 55 (2013 .01 ) ; CO7K 2317/ 622 15 , 2015 , provisional application No . 62/ 193 , 569, (2013 . 01 ) ; CO7K 2317 / 52 ( 2013 .01 ) ; CO7K 2317 /53 (2013 . 01 ); CO7K 2317 / 526 (2013 . 01 ); filed on Jul. 16 , 2015 . CO7K 2317 / 21 ( 2013 .01 ) ; CO7K 2317 /524 Publication Classification ( 2013 .01 ); CO7K 2317 /76 ( 2013 .01 ); CO7K (51 ) Int. CI. 2317/ 33 ( 2013 .01 ) ; C07K 2317 / 565 (2013 .01 ) ; A61K 47/ 68 ( 2006 . 01 ) A61P 35 /00 (2018 . 01 ) A61P 35 /00 ( 2006 .01 ) A61P 35 /02 ( 2006 . 01 ) (57 ) ABSTRACT A61P 35 /04 ( 2006 .01 ) Bispecific antigen -binding constructs e. g ., antibodies con CO7K 16 /28 ( 2006 . 01 ) jugated to drugs (ADCs ) , which bind CD3 and other cell A61K 51/ 10 ( 2006 . 01 ) surface target antigen such as tumor antigens e . g ., CD19 , CO7K 16 /32 ( 2006 . 01 ) CDH3 , HER2 , HER3 and EGFR antigens and methods of A61K 45 / 06 ( 2006 .01 ) use are disclosed . Patent Application Publication Jul. 12, 2018 Sheet 1 of 41 US 2018 / 0193477 A1

anti -target antigen anti -target antigen A antianti - CD3 anti -CD3

FIG.1A

jii. iv. anti- target antigen anti -target antigen

anti -CD3 anti -CD3 Patent Application Publication Jul. 12, 2018 Sheet 2 of 41 US 2018 / 0193477 A1

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SESSORS 1999DODOSOS 50L2 60H250 110

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CD19Sequences: 14 Kabat HD37 Kabat hVL2 Kabat HD37hVH2 hvH3 Kabat HD37 hVH2hvH3 FIG . 2 Patent Application Publication Jul, 12 , 2018 Sheet 4 of 41 US 2018/ 0193477 A1

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70 60 H? ? VHstabilizedsequences: VLstabilizedsequences: OLAA Anti-CD3sequences KabatVH KabatVH KabatVL KabatVL OKT3 hVH1 hVH2 OKT3 hVH1 hvH2 OKT3 HVL1 hVL2 ???3 HVL1 hVL1 FIG . 4 Patent Application Publication Jul. 12, 2018 Sheet 7 of 41 US 2018 / 0193477 A1

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102 TIL. concentration(nM) HumanPBMC 10-1100101

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1000 concentration(nM) 10100 JurkatCD3+Tcellline EC50(nM) 44.7 1.8 0.3 FIG.8

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Ramos Cells

o V6754 -DM1 (batch 1) b 12043 -DM1 (batch 1 ) + v12043 - DM4 A (3days) 8887Lý ITT1 * V6754 -DM1 (batch2 ) %Survivingfractions + v12043- DM1 (batch 2 ) etter V4371 -DM1 V6751- DM1 A V6249 - DM1 0H - 1 - - 3 - 2 - 1 0 1 2 3 4 LOG conc . (NM ) Jurkat Cells 1407 120 V6754- DM1 (batch 1 ) ART = 12043- DM1 (batch 1 ) + v12043 - DM4 (3days) + V6754 -DM1 (batch2 ) fractionsSurviving% + v12043- Dm1 (batch 2 ) V4371 -DM1 V6751 -DM1 + V6249 - DM1 04 - 3 - 2 - 1 0 1 2 3 LOG conc . (nM ) FIG.10 Raji Cells 1407 II 16754 -DM1 (batch 1) = v12043 -DM1 (batch 1 ) man teniment v12043 -DM4 (3days) + V6754 -DM1 (batch 2 ) %Survivingfractions - v12043 -DM1 (batch 2 ) * V4371 -DM1 + V6751 -DM1 * V6249 - DM1 OHA is 1 ó 1 ? LOG conc . (NM ) Patent Application Publication Jul. 12 , 2018 Sheet 14 of 41 US 2018 /0193477 A1

% CD20 + Raji cells

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CD8 + /CD69 + T cells 50 ,000 II 40, 000 FIG.11(cont'd) cellcount/well 30, 000

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CD20 + Raji B cells 70000

60000 ????? 50000 40000 cellcount/well 30000 20000 ??? ? ? ?? ? in preme . 10000 wwwwwww O woce 5nM 5nM 0.5nM 5nM PBMC-B8 + Raji 0 .0005nM 0.005nM 0.05nM 0.5nM 50nM 0.0005nM 0.005nM 0.05nM 0.5nM 50nM 0.0005nM 0.005nM 0.05nM 50nM v12043 V12043 V12043 SMCC -DM1 SPDB -DM4 CD8 + T cells 80 ,000 860 , 000 cellcount/well 40 ,000 8

5nM 0.5nM 5nM 5nM PBMC-B + Raji 0 .0005nM 0.005nM 0.050M 0.56M 50nM 0.0005nM 0.0050M 0.05nM 50nM 0.0005nM 0.0050M 0.050M 0.5nM 50nM v12043 V12043 V12043 ?????? SMCC??? - DM1 SPDB???? -DM4 CD8 + /CD69 + T cells 160 ,000 finishing F 140 , 000 120 ,000 toremindmeasurements 100 ,000 a wellcount/cell 80 ,000 .

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CD20 + B cells (Ramos ) 200 ,000 180 , 000 beeld 160 ,000 9140 ,000 XXX - - XXXXXXX .197 .04 .14 . . . . RHIA . . . . . Cellcountperwell 120 ,000 100 ,000 www.namaswamporecommanding 80 . 000 60 .000 tette 40 , 000 ww. SA

20 ,000 w

RamosNoTI PBMC-BNoTI V67540.5nM V67540.005nM V67540.05nM V67545nM V675450nM V6754-DM10.5nM V6754-DM15nM 5MDM10.6751- 5M.DMI46751- 18910.0005nM V8910.005nM V8910.05nM V4372/DM10.5nM V4372/DM15nM estaurantesdeartementet PBMC-BRamosNoTI V6754-DM10.005nM V6754-DM10.05nM V6754-DM150nM 005nMDM10.V6751- V6751-DM10.05nM V6751-DM150nM V4372/DM150nM

FIG.13

CD4 + T cells Ande 140 ,000 120 ,000 . VV.

. 100 ,000 www XXV.Ay 2 . 1 7 ...... 80 ,000 det Cell.countperwell . ???? WWWWWWWWWWW

60 ,000 . ie 40 ,000 . Love ervar 1. v.sawww wy. 20 , 000 N rares . W totorokritert NoiRamosti PBMC-BNOTI V67540.5nM V67545nM v8910.05nM V67540.006nM V67540.05nM V675450nM V6754-DM10.5nM v6751-01SnM V8910.005nM V4372/DM10.5nM t v675-DM15nM V4372/DM15nM PBMC-BRamosNoTIa V6754-DM10.005nM V6754-DM10.05nM V6754-DM15nM V6754-DM150nM V6751-DM10,005nM V6751-DM10.05nM V6751-DM150nM 0005nMV8910. V4372/DM150nM deforbehoor ???????????????????????????????????? WWWWWWWWWWWWWWWW Patent Application Publication Jul. 12 , 2018 Sheet 18 of 41 US 2018 /0193477 A1

PD - 1 + T cells

%oftotalTcells

. o68

. .

cccccccdwuwie www recede . ???w??riz??vwi??zzzzzzz?wz?w?????????? RamosNoTi PBMC-BNoTIO V67540.5nM V67540.005nM V67540.05nM V67545nM V675450nM V6754-DM10.5nM v6751-DM10.5M V8910.005nM V8910.05nM V4372/DM10.5nMCH V4372/DM150nMI PBMC-BRamosNoTI 005nMDM10.V6754- V6754-DM10.05nM V6754-DM15nM V6754-DM150nM V6751-DM10.005nM V6751-DM10.05nM v6751-DM15nM V6751-DM150nM V8910.0005nM V4372/DM15nM

P FIG.13(contd) Patent Application Publication Jul. 12 , 2018 Sheet 19 of 41 US 2018 /0193477 A1

CD20 + B cells (Raji )

200 , 000 giriririr...... INSS . . . Sirir: ...... rrrrSiriririr: : . . rir . . ririririririr i ririririr. . .. . ! ! iririririr i ririririririririririr iririririririririririr i ririririr iririririr iririririririririririrariririririricar : riririririr i riririririririr. . . 180 ,000 160 ,000 140 ,000 120 ,000 Cellcountperwell 100 , 000 80 ,000 TE 60 ,000 ere po

40 , 000 w 20 ,000 S RajiNoTI PBMC-BNoTI 05nMV67540. V67540.5nM V67545nM V8910.05nM PBMC-BRajiNoTI V67540.005nM V675450nM V6754-DM10.05nM V6754-DM10.50M V6754-DM15nM V6751-DM10.05nM v6751-DM10.5nM 46751-DM15nM V6751-DM150nM 0005nMV8910. V8910.005nM V4372/DM10.5nM V4372/DM15nM V6754-DM10.005nM V6754-DM150nM V6751-DM10.005nM 1 V4372/DM150nM

FIG.14 CD4 + T cells

180 ,000 2222222222 160, 000 140 , 000 120, 000 oorsienintersom Cellcountperwell 100 , 000 v 80, 000 60, 000 terjejer warzyn ???????????????????????? ? .wat 40, 000 members 20, 000 ??????? wasawarded .

NoTILPBMC-B TINoRaji V67540.5nM V67540.005nM V67540.05nM 5nMV6754 50nMV6754 V6754-DM10.5nM V6751-M5nM V8910.005nM V8910.05nM V4372/DM10.5nM PBMC-BRajiNoTI V6754-DM10.005nM 05nMDM10.V6754- V6754-DM15nM V6754-DM150nM V6751-DM10.005nM V6751-DM10.05nM v6761-DM105nM V6751-DM150nM V8910.0005nM 5nMDM1V4372/ V4372/DM150nM Patent Application Publication Jul. 12 , 2018 Sheet 20 of 41 US 2018 /0193477 A1

PD - 1 + T cells

. RERE PPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPP

2. E %oftotalTcellsopsporenproposamnapadowopcap C

ASTRA GREECEGREENERGETSKOG Wwwwwwwwwwwwwwwwww 8888888 IKAW

PBMC-BNoTIO RajiNoTI V67540.5nM V67540.005nM V67540.05nM V67545nM V675450nM V6754-DM10.5nM V6751-0,5M V8910.005nM 18910.05nM GREECE 1 PBMC-BRajiNoTI V6754-DM10.005nM V6754-DM10.05nM V6754-DM15nM V6754-DM150nM V6751-DM10.005nM V6751-DM10.05nM 5nMDM1v6751- V6751-DM150nM V8910.0005nM V4372/DM10.5nM V4372/DM15nM V4372/DM150nM ??????????????????????????????????????????????????????????????????????????? GREECE

FIG.14(cont'd) Patent Application Publication Jul. 12 , 2018 Sheet 21 of 41 US 2018 /0193477 A1

huB12-MCCDM1 DM1MCCV6751-+ DM1-huB12MCC- DM1MCC-Isotypestorey +V6751-MCCDM1 DM1MCC-Isotypeoen

4 4

3 T1 3 veytas RASANYA a XXXS* raabitual A LOGconcentration(nM) Th RamosCD19+Bcells K562CD19-/CD3cells ) H LOGconcentration(nM) -3210 -3210

WWUWARS TT 4- FTTTTT 140 20 ????? ? $ si fractions Surviving % fractions Surviving % FIG.15

4 4

3 3

AVX - N WANAWAKE ** * * * Vixens1 T Frente LOGconcentration(nM) HA LOGconcentration(nM) RajiCD19+Bcells Bo , JurkattCD3+cells -3210 -3210 www.beriwwdb TTTT

. . 100-) 140 120 ?????80 40 140 ??????120 100 80 0 fractions Surviving % fractions Surviving % Patent Application Publication Jul. 12 , 2018 Sheet 22 of 41 US 2018 /0193477 A1

nM 05. 0 (estimatedrange;n=4donors) nM 005. 0 >90% 20-40% 10-90%10-90% nM 0005 . 0 blinatumomab %Bcelldepletion nM 50 1997 harapan nM 5 nM 5 . 0 v15195 nM 05. 0 nM 005. 0 (estimatedrange;n=4donors) 16.FIG nM 50 BcelldepletionIC50 <0.5nM 0.05-5nM 0.5-5PM nM 5 Wuso v15195-MCCDM1 nM 05 . 0 nM 005. 0 ADC Isotype B - PBMC/ Raji only Raji PBMC-B/Rajicellculture v15195-MCCDM1 80000 0000 40000 20000 (72hincubation) v15195 blinatumomab cells + CD20 Patent Application Publication Jul. 12 , 2018 Sheet 23 of 41 US 2018 /0193477 A1

SUDHL-4 SUDHL-6 11-RS4 Nalm-6 Daudi

on wuos Wus FIG.17 5nM®.0 ^05nM .0 concentration

...... WWWWWWWWWW : : : : : 88878787872722M M MMMMM . Wusoo'o VACCESSSS Media uop?d?p 1185 g jabuer % Patent Application Publication Jul. 12 , 2018 Sheet 24 of 41 US 2018 /0193477 A1

tototo )05nM .0( blinatumomab )50nM (v15195 CD8+/CD25Tcells 11/1)50NM (DM1 -MCC -v15195 B-PBMC /Raji B-PBMC 30000 20000 counts cell total otted 1 ')05nM .0( blinatumomab V)50nM (v15195 CDB-HICDE8+TcellsCD8+/CD69Tcells FIG.18A )50nM (DM1 -MCC -v15195 B-PBMC /Raji B-PBMC carton30000 20000 10000 counts cell total trto

† )05nM .0( blinatumomab )50NM (v15195 CD8+Tcells † )50nM (DM1 -MCC -v15195 B-PBMC /Raji † B-PBMC 300002 20000 0000 counts cell total Patent Application Publication Jul. 12 , 2018 Sheet 25 of 41 US 2018 /0193477 A1

vblinatumomab(0.3nM) I16751(100nM) A66751(300nM) OKT3(0.3nM) •Media

I H )3nM. 0(OKT3 FIG.18B Proliferationindex Hot )3nM .0(blinatumomab HH1 )300nM (V6751 )100nM (V6751 - Media )SI ( Index Stimulation Patent Application Publication Jul. 12 , 2018 Sheet 26 of 41 US 2018 /0193477 A1

DM1-MCC -huBU12 CD8+/CD25 DM1-MCC -V6751 PBMC-B Media

wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww 'DM1 -MCC -huBU12 CD4+CD25 DM1Media-MCC -V6751 10000 8000 6000 4000 2000 count cell T FIG.19

DM1-MCC -huBU12 CD8+ DM1-MCC -V6751 PBMC-B Media DM1-MCC -huBU12 CD4+ DM1Media-MCC -V6751 400007 30000 10000 count cell T Patent Application Publication Jul. 12 , 2018 Sheet 27 of 41 US 2018 /0193477 A1

Raji/PBMC-B *V15195-MCCDM1 Av15195

tetto 5nM

1 CD8+/CD25Tcells 5nM.0 concentration HO 05nM.0 FIG.20A Raji+PBMC-Bculture(72hincubation,FACSanalysisofdifferentpopulations;5:1ETration=4donors) 30000 20000 10000 counts cell total

5nM

CD8+Tcells 5nM.0 K * concentration moto 05nM.0

30000 20000 10000 counts cell total Patent Application Publication Jul. 12 , 2018 Sheet 28 of 41 US 2018 /0193477 A1

28 /41 Dv15195-MCCDM1 ??Raji/PBMC-B Dv15195 50nM SnM 5nM . 0 05nM . 0 80de 50nM IL10 5nM v15195-DM1v15195 5nM . 0 Wuso ' o

OO Wuos50M ! FIG.20B 05nM .0 Raji+PBMC-Bculture(72hincubation,FACSanalysisofdifferentpopulations;5:1ETration=4donors) IL6 Ik 50nM v15195-DM1 K - SnM OCH - 5nM. 0 05nM. 0

50nM 5nMO OL - 5nM. 0 HU 05nM . 0 IFNg H- 50nM v15195-DM1

HIhreTTTTTTTTTTTTTTTTT H - 5M. 0 +444 05nM . 0 Patent Application Publication Jul. 12 , 2018 Sheet 29 of 41 US 2018 /0193477 A1

CD20 + Ramos B cells

140000 ?????? 120000

100000 . totalcellcountperwell .

80000 . 60000 .

40000 .

20000 . NoTI InM 1nM 0.1nM 10nM 0.1nM 10nM PBMConly 0.001nM 0.010M 100nM 100nM

unconjugated v15193 v15193 - VCPAB -MMAE

CD8 + T cells 7 ,000 . 00 6 ,000 . 00 dengannamankami 4949 5 ,000 . 00 sanan totalcellcountperwell 27 4 ,000 .00 man 3 ,000 .00 lamamenamatkanna 2 ,000 .00 1 , 000 . 00 withuania 0 .00 NoTI 10M 0.10M 1nM 10nM 0.1nM 10nM PBMConly 0.001nM 0.01nM 100nM 100nM HERRER unconjugated v15193 v15193 - VCPAB -MMAE

FIG .21 Patent Application Publication Jul. 12 , 2018 Sheet 30 of 41 US 2018 /0193477 A1

% T cells in peripheral blood

+ v12043 0 .3mg / kg v12043 0 . 1mg/ kg %CD45+/CD8Tcells - V12043- DM1 0. 3mg/ kg Dv12043- DM1 0 . 1mg/ kg v12043- DM4 0 . 3mg/ kg ooö 0 20 40 60 80 100 120 140 Hours after administration % B cells in peripheral blood

999999999999999999 NNNNNNNNNNNAABAwwwwwwwwwwwwww w AAAWAVAWANNYAWAWAAN9999999993939393 ?????????? ???????????????? ???????? ???????? ???????? ???????? ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? VnWVWZ

XYYAYAYA:W/VXx %CD45/CD20+Bcells FIG.22 ww w wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww ANAK

AWAK

üonö wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww W w wwwwwwwwwwwww 1

w YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY w ww YYYY

wwwwwto wwwwwwwwwwwwwwwwwwwwwwwwwwwwwww w w w wwwwwwwwwwwwwwwww Luxur 0 20 40 60 80 100 120 140 Hours after administration Patent Application Publication Jul. 12 , 2018 Sheet 31 of 41 US 2018 /0193477 A1

% CD3 + T cells in spleen 100

%ofhCD45+ 1 * w * *

Vehicle FIG.23 v15195-MCC -DM1 ,9mpk v15195 -MCC -DM1 ,3mpk V15195 -MCC -DM1 ,1v15195mpk -MCC -DM1 ,0.3v15760 mpk -MCC -DM1 ,9mg /kg

% CD3 + T cells in peripheral blood

H %ofhCD45+ Hofy. totoe. teft HH.

Vehicle v15195-MCC -DM1 ,9mpk v15195 -MCC -DM1 ,3mpk v15195 -MCC -DM1 ,1v15195mpk '-MCC -DM1 ,0. 3v15760 mpk '-MCC -DM1 ,9mg /kg Patent Application Publication Jul. 12 , 2018 Sheet 32 of 41 US 2018 /0193477 A1

Jurkat 40000 , - Synagis FIG.24 0000 - V2171 PHAbsignal(DAR+ sorption V15195 CellCountnormalized) 2000 ple torinosicherhe + V16371 itstech V13831

10000* typu 0 - aastane porno corpora mareP pentru paprm 0 .01 0 . 1 100 OM A431 150000 Synagis Internalizationassay(upto60nM) ANNEXT so V2171 plete 1PHAbsignal(DAR+ 100000 seulement v15195 com * V16371 CellCountnormalized) white v were 1 50000 distensilesiasticism deperiodistas en

0. 010 .1 RETO 100 OM

SKOV3 40000 Le Synagis 00 pit V2171 PHAbsignal(DAR+ angla V15195 CellCountnormalized) erilainen V16371 RSSde V13831 1

0 pm sem wanna us Let Lirik 0 .01 0 . 1 OL 00L Powe nM Patent Application Publication Jul. 12 , 2018 Sheet 33 of 41 US 2018 /0193477 A1

HCT- 116 20000 authon Synagis 15000 - V2171 - - V15195 PHAbsignal(DAR+ SARRERA audition v16371 CellCountnormalized) R - V13831 5000 RITOR bout A Internalizationassay(upto60nM) 0 -raamw T a ppeti nepagyroperty own transporta passerer espanyom 0 .01 1 0 . 1 10 100 nM

150000 Synagis en v2171 100000 poslodas - - v15195 PHAbsignal(DAR+ V16371 CellCountnormalized) V13831 00 WYPRIOR bg.www 0pm 0 .01 0 . 1 1 10 NM FIG.24(cont'd) Patent Application Publication Jul . 12 , 2018 Sheet 34 of 41 US 2018 / 0193477 Al

MCF7 cells continuous incubation ( 5 days) 125 ? ?? PPPIRRORNER , PIANO , Siam ?????????????? = = v6249 -SMCCDM1 ?????????? rern?????ease v13790 -SMCC - DM1 ????????sundation ? ?????eter v13792 -SMCC - DM1 ??????? rem v13831 -SMCC - DM1 %Survivingfractions(5days) ??

?????

? ? F - 3 - 2 1 - 0 1 2 3 4 LOG conc. ( nM )

Growthinhibition(noPBMCs) SKOV3 cells continuous incubation (5 days) 125

??? % 9e % % %% % % % er g y ,san ? Sessssssssssssssssssssssssee ???????? ?????????????? ???????????? ???????? ??????? tent _ v6249 - SMCC - DM1 Toner v13792 -SMCC -DM1 (5days) %Survivingfractions BeyNethods???Combie ?????????

???? ?????ers, exx°?? ? 1 ...... | LOG conc {nM ) FIG.25 Patent Application Publication Jul. 12 , 2018 Sheet 35 of 41 US 2018 /0193477 A1

JIMT- 1 cells continuous incubation ( 5 days )

PORT V6249SMCC - DM1 i version v13792 - SMCC -DM1 te velit esse v13831- SMCC -DM1 %Survivingfractions (5days)weBere Activere 7 3 LOG concó. (nM i? ) Growthinhibition(noPBMCs)

Jurkat cells continuous incubation ( 5 days ) 125

100 - este intrebarea me morationen e r at den eneste durant seu terme la mesa de lo nebo V6249 -SMCC -DM1 vendt V13790 -SMCC - DM1 Sarita V13792 - SMCC -DM1 %Survivingfractionsdays)5( Serendipity v13831 - SMCC -DM1

4 32. TOTTA LOG conc . (nM ) FIG.25(Cont'd) Patent Application Publication Jul. 12 , 2018 Sheet 36 of 41 US 2018 /0193477 A1

Co -culture assay ( E : T ratio of 2 : 1 , JIMT1 cells ) Her2 -CD3 (with PBMCs )

+ V13792 + V13792 -SMCC - DM1 LiveJIMT1cells * * * * * * * (%ofparentpopulation) * * * * * * *

0.000 0.005 0.050 0.500 5.000 50.000 Concentration (nM ) CDH3 -CD3 (with PBMCs )

+ v13831 * * * + V13831 -SMCC -DM1

* * * * LiveJIMT1cells (%ofparentpopulation) * * * * * * * * * * *

e 0.000 00..005 0.050 0.500 5.000 50.000 Concentration (nM ) FIG . 26 Patent Application Publication Jul. 12 , 2018 Sheet 37 of 41 US 2018 /0193477 A1

Co -culture assay ( E : T ratio of 2 : 1 , JIMT1 cells ) CD8 + cells CDH3 -CD3 400007 + V13831 I I + v13831- SMCC - DM1 AdjustedCounts(CD8+) 30000 20000 KAN 10000 205 206 o s son FIG.27 Concentration (nM ) CDH3 -CD3 40000 + v13831 30000 + v13831- SMCC -DM1 Adjustedcounts(CD8+CD25) 20000 . . JE10000 0.000 0.005 0.050 0.500 5.000 50.000 CDH3- CD3 20000 , + V13831 15000 v13831 - SMCC -DM1 A Adjustedcounts (CD8+CD694) 10000 E5000 Concentration (NM ) Patent Application Publication Jul. 12 , 2018 Sheet 38 of 41 US 2018 /0193477 A1

Co -culture assay ( E : T ratio of 2 : 1 , JIMT1 cells ) CD4 + cells CDH3- CD3 60000 + v13831 + v13831-- SMCC SMCC - - DM1OM AdjustedCounts(CD4+) I FIG.27(Cont'd) 200 WH1

Concentration ( M ) CDH3 -CD3 60000 + v13831 + v13831 - SMCC - DM1 40000 (CD4+CD25) P

Adjustedcounts . 20000 0 .005 BS B SO Concentration (nM ) CDH3 -CD3 30000 + v13831 + v13831 -SMCC -DM1 20000 Adjustedcounts (CD4+CD9) 10000 04 69 6 3 4 5 Concentration (nM ) Patent Application Publication Jul. 12 , 2018 Sheet 39 of 41 US 2018 /0193477 A1

Co -culture assay (E : T ratio of 2 : 1 , JIMT1 cells ) CD8 + cells Her2 -CD3 400007 + V13792 V13792 - SMCC -DM1 AdjustedCounts(CD8+) 30000 20000 FIG.27(Contd) 10000

0.005 Ons Concentration (nM ) Her2 -CD3 400007 with v13792 30000 To • V13792 -SMCC -DM1 1 Adjustedcounts(CD8+CD25) 20000 II 10000 ka hOCS 205 05 ? SO Concentration (nM ) Her2 - CD3 250007 20000 + v13792 + v13792 - SMCC - DM1 15000 (CD8+CD694) Adjustedcounts 10000 5000

0.05 * ** 0.005 Concentration (nM ) Patent Application PublicationPublication Jul. 12 , 2018 Sheet 40 of 41 US 2018 /0193477 A1

Co -culture assay ( E : T ratio of 2 : 1 , JIMT1 cells ) CD4 + cells andHer2 - CD3 are s omeone 60000 + V13792 • V13792 - SMCC -DM1 AdjustedCounts(CD4+) 40000

20000 FIG.27(Contd)

0.005DOS 0COS.05 SO Concentration (nM ) Her2- CD3 60000 * V13792 • v13792 - SMCC - DM1 00 H Adjustedcounts (CD4+CD25) 20000

0.000 0.005 0.050 0.500 5.000 50.000

Concentration (nM ) Her2 -CD3 30000 + v13792 V13792 - SMCC -DM1 2000 Adjustedcounts(CD4 +CD9) 10000

0.000 0.005 0.050 0.500 5.000 50.000 Concentration (nM ) Patent Application Publication Jul . 12 , 2018 Sheet 41 of 41 US 2018 /0193477 A1

5 :1 ( E : T ratio ) 80000 - 4 v13831 . * v13831 -SMCCDM1 Adjustedcounts(CTV+Live) 0000 - 40000 - T20000 - ? - Concentration (nM ) 1 : 5 ( E: T ratio ) 80000 - - v13831. 60000 - + v13831 - SMCC- DM1 Adjustedcounts(CTV+Live) - FIG.28 - 40000 - ??? -- - ?? ? ? ? IT1 Concentration (nM ) 1 : 50 (ET ratio ) 60000 - - v13831 4 v13831 - SMCC- DM1 AdjustedCounts(CTV+Live) 40000 HH + + +* ,> + 13831- SMCC - DM1 20000 -

0.005 0.05 . Concentration (nm ) US 2018 /0193477 A1 Jul. 12 , 2018

DRUG -CONJUGATED BI - SPECIFIC peptide construct that specifically binds a CD3 antigen ANTIGEN - BINDING CONSTRUCTS expressed on T cells ; and a second antigen -binding poly peptide construct that specifically binds a disease - associated SEQUENCE LISTING target antigen expressed on a target cell . The first and second [ 0001] Not applicable . antigen - binding polypeptides are operably linked ; and the antigen -binding construct is conjugated to a drug , optionally FIELD OF THE INVENTION to 2 different drugs. In some embodiments, the drug - conju [0002 ] The field of the invention is drug - conjugated bi gated antigen -binding construct displays higher killing specific antigen -binding constructs , e . g . , antibodies , com potency against target cells in vitro than a reference antigen prising a CD3 antigen - binding polypeptide construct, e . g . , a binding construct that is not conjugated to a drug . and does CD3 binding domain and a second antigen - binding poly not substantially deplete T cells when administered to a peptide construct, e . g ., a domain that binds a target antigen subject. The drug - conjugated antigen -binding construct expressed on a target cell, e . g . a tumor cell. comprises one or more drug molecules. The drug - conju gated antigen binding construct may comprise a heterodi BACKGROUND OF THE INVENTION meric Fc comprising a first Fc polypeptide linked to the first antigen -binding polypeptide construct with or without a first [0003 ] In the realm of therapeutic proteins , antibodies linker and a second Fc polypeptide linked to the second with their multivalent target binding features are excellent antigen - binding polypeptide construct with or without a scaffolds for the design of drug candidates . Advancing these second linker. In some embodiments , the target antigen is features further , designed bispecific antibodies and other CD19 . In some embodiments , the target antigen is CDH3. In fused multispecific therapeutics exhibit dual or multiple some embodiments , the target antigen is HER2 . In some target specificities and an opportunity to create drugs with embodiments , the target antigen is CDH3 . In some embodi novel modes of action . The development of such multivalent and multispecific therapeutic proteins with favorable phar ments , the target antigen is EGFR . In some embodiments the macokinetics and functional activity has been a challenge . target antigen is selected from Table LL . [0004 ] Bi- specific antibodies capable of targeting T cells [0008 ] One aspect of the present disclosure is a method of to tumor cells have been identified and tested for their killing target cells that express a target antigen on the cell efficacy in the treatment of cancers. Blinatumomab is an surface comprising contacting the target cells with an effec example of a bispecific anti -CD3 -CD19 antibody in a tive amount of a drug -conjugated antigen -binding construct format called BiTETM (Bi - specific T -cell Engager) that has in the presence of effector T cells , wherein the drug been identified for the treatment of B - cell diseases such as conjugated antigen -binding construct comprises a first anti relapsed B - cell non -Hodgkin lymphoma and chronic lym gen -binding polypeptide construct that specifically binds a phocytic leukemia (Baeuerle et al (2009 ) 12 :4941 -4944 ) and CD3 antigen expressed on the effector T cells , comprising a is FDA approved . T cell engagers directed against other first heavy chain variable ( VH ) region and a first light chain tumor -associated target antigens have also been made, and variable ( VL ) region ; a second antigen -binding polypeptide several have entered clinical trials : AMG110 /MT110 construct comprising a second VH region and a second VL EpCAM for lung cancer , gastric cancer and colorectal can region that specifically binds the target antigen ; and at least cer; AMG211/ MEDI565 CEA for gastrointestinal adenocar one drug conjugated to the antigen -binding construct; cinoma; and AMG 212 / BAY2010112 PSMA for prostate wherein the first and second antigen -binding polypeptide cancer ( see Suruadevara , C . M . et al , Oncoimmunology . constructs are operably linked ; and the target antigen is not 2015 June ; 4 (6 ) : e1008339 ) . CD3 ; and wherein ( a) the antigen -binding construct displays 10005 ] The BiTETM format is a bi - specific single chain higher affinity to the target antigen than to CD3 as measured antibody construct that links variable domains derived from by SPR or FACS analysis, and / or ( b ) the antigen -binding two different antibodies . Blinatumomab , is highly effica construct displays higher killing potency against target cells cious in B cell acute lymphocytic leukemia ( ALL ) with an bearing the target antigen than against T cells , as measured overall response rate of over 80 % , but despite the high in an in vitro assay . efficacy many patients relapse shortly after or during the [0009 ] Another aspect of the present disclosure is a treatment. In addition , T cell engagers have been shown to method of killing target cells that express a target antigen on be less effective in malignancies like chronic lymphocytic the cell surface in a subject, comprising administering to the leukemia (CLL ). There is a need for more efficacious and subject an effective amount of a drug -conjugated antigen durable T cell engager therapies. binding construct wherein the drug -conjugated antigen [ 0006 ] T cell engager antigen -binding constructs are binding construct comprises a first antigen -binding polypep described in the following: International application no . tide construct that specifically binds a CD3 antigen PCT /US2013 /050411 filed on Jul. 13 , 2013 and titled expressed on the T cells of the subject, comprising a first “ Bispecific Asymmetric Heterodimers Comprising Anti heavy chain variable (VH ) region and a first light chain CD3 Constructs ; " International application no . PCT/ variable (VL ) region ; a second antigen -binding polypeptide US2014 / 046436 filed on Jul. 11 , 2014 and titled “ Bispecific construct comprising a second VH region and a second VL CD3 and CD19 Antigen Binding Constructs; " PCT/ US2015 / region that specifically binds the target antigen ; and at least 011664 filed on Jan . 15 , 2015 and titled “ Bispecific CD3 and one drug conjugated to the antigen - binding construct; CD19 Antigen Binding Constructs ." wherein the first and second antigen -binding polypeptide constructs are operably linked ; and the target antigen is not SUMMARY OF THE INVENTION CD3 ; and wherein ( a ) the antigen -binding construct displays 10007 ] Described herein is a drug -conjugated antigen - higher affinity to the target antigen than to CD3 as measured binding construct comprising a first antigen - binding poly by SPR or FACS analysis ; and/ or (b ) the antigen - binding US 2018 /0193477 A1 Jul. 12 , 2018 construct displays higher killing potency against target cells BRIEF DESCRIPTION OF THE FIGURES bearing the target antigen than against T cells , as measured [0013 ] FIG . 1A depicts schematic representations of in an in vitro assay . designs of antigen -binding constructs conjugated to a drug , [ 0010 ] Another aspect of the present disclosure is a the drug being depicted by a “ star” . One binding domain of method of treating a disease , disorder or condition in a the antigen -binding constructs binds to a CD3 antigen , and subject in need thereof comprising administering to the the other binding domain binds to a “ target antigen " subject a therapeutically effective amount of a drug- conju expressed on a the cell surface of a target cell. Although gated antigen -binding construct wherein the drug -conju there is only one “ star ” , the construct may contain multiple drug molecules which can be the same, or different . FIG . gated antigen -binding construct comprises a first antigen 1A (i ) shows a representation of an exemplary antigen binding polypeptide construct that specifically binds a CD3 binding construct in which both of the antigen - binding antigen expressed on the T cells of the subject , comprising domains of the antigen - binding construct are scFvs , with the a first heavy chain variable ( VH ) region and a first light VH and VL regions of each scFv connected with a poly chain variable ( VL ) region ; a second antigen - binding poly peptide linker. Each scFv is also connected to one polypep peptide construct comprising a second VH region and a tide chain of a heterodimeric Fc with a hinge polypeptide second VL region that specifically binds the target antigen ; linker . The two polypeptide chains of the antigen -binding and at least one drug conjugated to the antigen - binding construct are covalently linked together via disulphide bonds construct ; wherein the first and second antigen - binding (depicted as thick solid lines lines ) . FIG . 1A ( ii ) depicts a polypeptide constructs are operably linked ; and the target representation of an exemplary antigen - binding construct antigen is not CD3 ; and wherein ( a ) the antigen - binding similar to 1A (i ) , except the CD3 binding domain is a Fab and construct displays higher affinity to the target antigen than to the target antigen binding domain are scFvs . FIG . 1A ( iii ) CD3 as measured by SPR or FACS analysis ; and /or (b ) the depicts a similar antigen - binding construct in which the CD3 antigen -binding construct displays higher killing potency binding domain is an scFv and the target antigen binding against target cells bearing the target antigen than against T domain is a Fab . FIG . 1A (iv ) depicts a similar antigen cells , as measured in an in vitro assay . binding construct in which the both the CD3 and target antigen binding domains are Fabs. [ 0011 ] Another aspect of the present disclosure is a com [0014 ] FIG . 1B depicts exemplary embodiments of anti position consisting of a drug -conjugated antigen -binding gen binding construct drug conjugates (ADCs ). FIG . 1B ( i ) construct comprising a first antigen -binding polypeptide shows a 4 - ( N -maleimidomethyl ) cyclohexane - 1 -carboxylate construct comprising a first VH region , and optionally a first (MCC ) - DM1 conjugate in which the linker- toxin is conju VL region , that specifically binds a CD3 antigen expressed gated via a lysine residue on the antigen binding construct ; on a T cell ; a second antigen -binding polypeptide construct FIG . 1B ( ii ) shows an N -succinimidyl - 4 -( 2 -pyridyldithio ) comprising a second VH region , and optionally a second VL butanoate (SPDB )- DM4 conjugate in which the linker- toxin region , that specifically binds a target antigen expressed on is conjugated via a lysine residue on the antigen binding a target cell ; and at least one drug conjugated to the construct ; FIG . 1B (iii ) shows a maleimidocaproyl -valine antigen - binding construct; wherein the first and second citrulline - p - aminobenzyloxycarbonyl (mc - Val - Cit -PABC ) antigen -binding polypeptide constructs are operably linked ; MMAE conjugate in which the linker - toxin is conjugated and wherein the target antigen is not CD3 ; and wherein the via a cysteine residue on the antigen binding construct . “ Ab ” target antigen is not CD3 ; and wherein the antigen -binding represents the antigen binding construct, which may be any construct displays higher affinity to the target antigen than to one of the designs shown in FIGS. 1A - 1D . “ n ” represents the CD3 as measured by SPR or FACS analysis . In some number of linker - toxin moieties conjugated to the antigen embodiments embodiment, the antigen - binding construct binding construct and is between 1 and 20 . displays higher killing potency against target cells bearing [0015 ] FIG . 2 depicts humanized CD19 VL and VH the target antigen than against T cells , as measured in an in sequences based on the mouse HD37 VL and VH sequences . vitro assay. Three humanized VL sequences have been provided : hVL2 , [0012 ] Another aspect of the present disclosure is an HVL2 ( D - E ) , and hVL2 ( D - S ) . HVL2 ( D - E ) contains a D to antigen - binding construct that binds to a CD3 epsilon sub E substitution in CDR L1, while hVL2 ( D - S ) contains a D unit comprising a first antigen binding polypeptide construct to S substitution in CDR L1 . Two humanized VH sequences comprising a VH region and a VL region wherein the VH have been provided : hVH2, and hVH3. The CDR sequences region comprises 3 CDRs comprising the amino acid are identified by boxes. The CDRs identified in this figure sequences of the VH CDRs of the humanized variant of are exemplary only. As is known in the art, the identification OKT 3 in Table Sl ; and the VL region comprises 3 CDRs of CDRsmay vary depending on the method used to identify comprising the amino acid sequences of the VL CDRs of the them . Alternate CDR definitions for the anti - CD19 VL and humanized variant of OKT3 in Table S1 . In one embodi VH sequences are shown in Table S1 . Modifications to ment, the construct comprises a VH region comprising an humanize these sequences with respect to the wild - type amino acid sequence selected from the amino acid sequence mouse HD37 antibody sequence are denoted by underlining . of hVH1 or hVH2 in FIG . 2 and an amino acid sequence that [0016 ] FIG . 3 depicts a table showing the number accord is at least 80 , 85 , 90 , 95 , 96 , 97 , 98 or 99 % identical to the ing to Kabat for the anti - CD19 VH and VL sequences, based amino acid sequence of hVH1 or hVH2 in FIG . 4 ; and the on the anti- CD19 HD37 antibody . VL region comprises an amino acid sequence selected from [0017 ] FIG . 4 depicts humanized CD3 VL and VH the amino acid sequence of hVL1 or hVL2 in FIG . 4 and an sequences based on the mouse OKT3 and teplizumab ( a amino acid sequence that is at least 80 , 85 , 90 , 95 , 96 , 97 , known humanized OKT3 ) sequences . Two VH sequences 98 or 99 % identical to the amino acid sequence of hVL1 or have been provided : hVH1 and hVH2. Two VL sequences hVL2 in FIG . 4 . have been provided : hVL1 and hVL2. The CDR sequences US 2018 /0193477 A1 Jul. 12 , 2018

arare identified by boxes. The CDRs identified in this figure CD19 antibody conjugated to DM1 huB12, and an isotype are exemplary only . As is known in the art , the identification non - specific IgG conjugated to DM1. of CDRsmay vary depending on the method used to identify [0029 ] FIG . 16 depicts the effects of an exemplary anti them . Alternate CDR definitions for the anti -CD3 VL and CD3 -CD19 antigen -binding construct v15195 , v15195 con VH sequences are shown in Table S1. Modifications to these jugated to DM1 and blinatumomab on Raji cells after 72 sequences with respect to the wild - type teplizumab antibody hours of culture . sequence are denoted by underlining (0030 ) FIG . 17 depicts the effect of v15195 conjugated to [0018 ] FIG . 5 depicts a table showing the number accord DM1 at various concentrations against various ALL and ing to Kabat for the anti -CD3 VH and VL sequences , based NHL cell lines : RS4 - 11 , Nalm - 6 , Daudi, SUDHL - 4 and on the anti -CD3 OKT3 antibody. SUDHL -6 . [0019 ] FIG . 6 depicts the SEC profile of a parentalmurine [0031 ] FIG . 18A depicts the effect of v15195 , v15195 anti - CD3- CD19 antigen - binding construct v6751 ( left ) and a conjugated to DM1 and blinatumomab in cultures of Raji humanized anti- CD3 - CD19 antigen - binding construct cells co - cultured with human PBMC on CD8 + T cells , v15192 ( right ), showing the greatly enhanced purity of CD8 + / CD69 + T cells and CD8 + / CD25 + T cells . FIG . 18B v15192 . depicts the proliferation observed in cultures of Raji cells [0020 ] FIG . 7 depicts a DSC thermogram of exemplary co -cultured with human PBMC - B cells with v6751 , blina humanized anti - CD3- CD19 antigen -binding constructs tumomab and OKT3 antibodies. compared to a parental murine anti - CD3 - CD19 antigen [0032 ] FIG . 19 depicts the effects of an exemplary anti binding construct, showing the increase in Tm ofthe human CD3 -CD19 antigen -binding construct v6751, V6751 conju ized variants. Variants marked as A and B represent different gated to DM1 and a control bivalentmono -specific antibody production batches of the same variant. anti -CD19 antibody , huBU12 , and huBU12 conjugated to [0021 ] FIG . 8 depicts the binding of a humanized anti DMI on various T cell subpopulations in cultures of CD3 -CD19 antigen -binding construct v15195 to (panel A ) Raji CD19 + B cells ; (panel B ) Jurkat CD3+ T cells . Panel PBMC - B cells : CD4 + . CD8 + , CD4 + / CD25 + and CD8 + / ( C ) depicts the percentage of T : B cell doublets detected CD25 + . when v15195 is incubated with human peripheral blood [0033 ] FIG . 20A depicts the effects of v15195 and v15195 mononuclear cells (PBMC ). T: B cell doublets were detected conjugated to DM1 on CD8 + and CD8 + /CD25 + T cells in as being both CD20 + and CD4 + or CD8 + . co - cultures of Raji cells with PBMC - B cells . FIG . 20B [0022 ] FIG . 9 depicts an exemplary UPLC - SEC profile of depicts the level of cytokines IFNg, IL6 and ?L10 in the an anti CD3- CD19 antigen -binding construct v12043 after cultures at 72 hours . conjugation to the toxin DM1 using an SMCC linker . [0034 ] FIG . 21 depicts the effects of exemplary anti -CD3 [0023 ] FIG . 10 depicts the results of an assay in which CD19 antigen -binding construct v15193 and v15193 con selected exemplary anti- CD3- CD19 variants that were con jugated to MMAE at various concentrations on CD8 + T cells jugated to DM1 or DM4 were tested at various concentra and target Ramos B cells in co -cultures with PBMC . tions for their ability to inhibit the growth of ( A ) Ramos B [ 0035 ] FIG . 22 depicts the effects of a single intravenous cells which express CD19 , ( B ) Jurkat T cells, which express administration to humanized mice of varying doses ranging CD3 and not CD19 , and ( C ) Raji B cells which express from 0 . 1 to 1 . 0 mg /kg of exemplary anti -CD3 -CD19 anti CD19 but not CD3 . gen -binding construct v12043 (without drug conjugation ) , [0024 ] FIG . 11 depicts the effects of various concentra v12043 - DM1 and v12043 - DM4 on B and T cells counts in tions of unconjugated variant anti -CD3 -CD19 variants humanized NSG mice over a 5 -day period after administra 12043 , v12043 -DM1 and v12043 - DM4 on ( A ) Raji cells , tion . (CD19 + ) ( B ) CD8 + T cells and ( C ) CD8 + /CD69 + T cells in [ 0036 ] FIG . 23 depicts the effects of a single intravenous 72 - hour cultures of Raji cells incubated with allogenic administration to humanized mice of varying doses ranging peripheral blood mononuclear cells that had been depleted from 0 . 3 to 9 . 0 mg/ kg of exemplary anti- CD3 - CD19 anti of B cells . gen -binding construct v15195 conjugated to DM1 on CD3 + [ 0025 ] FIG . 12 depicts the results of a second experiment T cells in spleen and peripheral blood at 8 days after conducted as in FIG . 11 . administration . 100261. FIG . 13 depicts the effects of various concentra [0037 ] FIG . 24 depicts the internalization into cells of tions of DM1- conjugated anti - CD3 - CD19 variants variants PHAb - labelled exemplary antigen -binding constructs anti 6754 and 6751 as well as DM1 conjugated control variants CD3 -CD19 v15195 , anti- CD3 - EGFR v16371 , and anti ( 1891, blinatumomab and v4372 bivalent monospecific anti CD3 -CDH3 v13831 and control antibodies ( v2171 UCHT1 CD19 antibody ) on ( A ) Ramos ( CD19 + ) target B cells, ( B ) anti - CD3 monospecific bivalent antibody ; and anti -RSV CD4 + T cells , and ( C ) PD - 1 + T cells in 72 -hour cultures of antibody, Synagis ) . Cell lines tested were Jurkat, A431 , Ramos cells incubated with allogenic peripheral blood SKOV3, HCT- 116 and JIMT1 . mononuclear cells that had been depleted of B cells . [ 0038 ] FIG . 25 depicts the direct cytotoxicity / growth inhi [0027 ] FIG . 14 depicts the results of a second experiment bition in the absence of T cells on target cells lines by conducted as in FIG . 13 . exemplary antigen - binding constructs anti- CD3 - CDH3 [ 0028 ] FIG . 15 . Depicts the cytotoxic effect at various v13831 , anti - CD3- HER2 v13792 , anti - CD3- HER3 v13790 concentrations of an exemplary anti -CD3 -CD19 antigen all conjugated to DM1 in comparison to an non - specific IgG binding construct v6751 conjugated to DM1 on Raji, an control v6249. Cell lines tested were MCF7, SKOV3 , ALL cell line , and Ramos , an NHL cell line , Jurkat, a T cell JIMT1 and Jurkat . line , and K562 , a cell line that expresses neither CD19 nor [0039 ] FIG . 26 depicts the effects of exemplary antigen CD3. Controls antibodies were a monspecific bivalent anti binding constructs anti- CD3 -CDH3 v13831, and anti - CD3 US 2018 /0193477 A1 Jul. 12 , 2018

HER2 v13792 and their DM1 conjugates at various concen benefit in the treatment of diseases such as cancer over trations on JIMT1 tumor target cells co - cultured with conventional T -cell engager therapeutics , none of which , to PBMCs. our knowledge , have incorporated a toxin or other drug . [0040 ] FIG . 27 depicts the T cell proliferation and activa Additionally the drug- conjugated bispecific antigen -binding tion of different T cell subpopulations in co - cultures of constructs that comprise antigen binding domains for CD3 JIMT1 tumor target cells and PBMC to which various and target antigens have potential in treating diseases other concentrations of DM1- conjugated or unconjugated anti than cancer , such as autoimmune or inflammatory diseases CD3 -CDH3 v13831 or anti -CD3 - HER2 v13792 were added . and diseases caused by intracellular pathogens, by combin The level of CD4 , CD4 + CD69, CD4 + CD25 , CD8, CD8 + ing the mechanisms of T cell - and drug -mediated killing . CD69, CD8 + CD25 positive T cells were evaluated for each [0045 ] Also described are pharmaceutical compositions construct. comprising the drug - conjugated antigen - binding constructs [0041 ] FIG . 28 depicts the effect of DM1- conjugated and and methods of treating a disease , disorder or condition e . g ., unconjugated anti- CD3 -CDH3 in co -cultures of JIMTtumor cancer, using the drug - conjugated antigen - binding con target cells and PBMC and different effector to target cell structs described herein . ratios. [0046 ] Described herein are drug - conjugated antigen binding constructs comprising a first antigen -binding poly DETAILED DESCRIPTION OF THE peptide construct that specifically binds a CD3 antigen INVENTION expressed on T cells , and a second antigen -binding poly [0042 ] Described herein are drug -conjugated bispecific peptide construct which and specifically binds a target antigen -binding constructs e . g . antibodies , often termed antigen , such as a tumor antigen expressed on the surface of antibody - drug conjugates or ADCs. Provided herein are tumor cells . The first and second antigen - binding polypep drug - conjugated antigen - binding constructs that bind to a tide constructs are operably linked, and the antigen - binding CD3 antigen expressed on T cells and to a second target construct is conjugated to a drug . The drug - conjugated antigen expressed on the surface of a target cell , for example antigen - binding construct displays higher killing potency a tumor cell , a cell responsible for autoimmunity or a cell against target cells bearing the target antigen in vitro than a infected with a pathogen . These drug -conjugated antigen reference antigen - binding construct that is not conjugated to binding constructs comprise a first antigen -binding domain a drug . that specifically binds to the CD3 antigen expressed on T [0047 ] The antigen -binding polypeptide constructs may cells , and a second antigen - binding domain that specifically have different formats . In some embodiments , the first and binds to another target antigen expressed on a the surface of second antigen - binding polypeptides each comprise a Fab or a target cell, and at least one drug molecule conjugated to the an scFv . In some embodiments the first antigen -binding antigen - binding construct. The first and second antigen polypeptide construct is a Fab and the second antigen binding domains may be operably linked to each other, or binding polypeptide is an scFv . In some embodiments the they may each be linked to a scaffold , such as an Fc domain , first antigen -binding polypeptide construct is a scFv and the as further described herein . second antigen -binding polypeptide is an scFv. In other [ 0043] Certain exemplary bispecific antigen -binding con embodiments , the first and second antigen -binding polypep structs used herein to produce ADCs have been shown tide constructs may both comprise Fabs or may both com elsewhere to be able to bridge CD3 -expressing T cells with prise scFvs . In certain embodiments , the CD3- binding poly CD19 -expressing B cells , with the formation of immuno peptide construct is an scFv and the target antigen - binding logical synapses . These antigen - binding constructs were construct is a Fab . able to mediate T cell- directed B cell depletion as measured [ 0048 ] In some embodiments , the drug - conjugated anti by in vitro and ex vivo assays , and as assessed in an in vivo gen -binding construct further comprises an heterodimeric model of disease . Fc , with a first Fc polypeptide linked to the first antigen [ 0044 ) In some embodiments described herein anti -CD3 binding polypeptide construct with or without a first linker target antigen drug - conjugated antigen -binding constructs and a second Fc polypeptide linked to the second antigen are shown to exhibit higher killing potency in depleting binding polypeptide construct with or without a second target tumor cells in vitro than the same antigen - binding linker . As described in detail below , in some embodiments , construct that does not comprise a drug . Unexpectedly, the heterodimeric Fc comprises a modified CH3 domain several exemplary CD3- target antigen drug - conjugated anti comprising asymmetric amino acid modifications that pro gen -binding constructs are shown herein to exhibit high mote the formation of the heterodimeric Fc and the killing potency against target antigen -expressing tumor cells dimerized CH3 domains having a melting temperature ( Tm ) in vitro while at the same time exhibiting low potency of about 68° C . or higher. In some embodiments, the against T cells . Additionally , in some embodiments , these asymmetric amino acid modifications are selected from ADCs are shown not to significantly deplete circulating T Table C below . cells in vivo in humanized mice when administered at doses [0049 ] In some embodiments , the second antigen -binding up to 3 mg/ kg . In view of the lack of impact on T cells , and polypeptide construct comprises the antigen -binding poly without being bound by theory , it appears that CD3 - target peptide construct specific for CD3 derived from an antibody antigen drug - conjugated antigen - binding constructs may selected from OKT3 ; TeplizumabTM (MGA031 , Eli Lilly ) ; exert their effect on target cells through two distinct mecha blinatumomab ; UCHT1 ; NI0401 ; visilizumab ; X35 - 3 , nisms: T cell -mediated killing , and toxin / small molecule VIT3 , BMA030 (BW264 /56 ) , CLB - T3 / 3 , CRIS7 , YTH12 . 5 , mediated killing resulting from internalization of the CD3 F111 - 409 , CLB - T3 . 4 . 2 , WT31 , WT32 , SPv - T3b , 11D8 , target antigen drug -conjugated antigen - binding constructs . XIII - 141, XIII -46 , XIII- 87 , 12F6 , T3 /RW2 - 8C8, T3 /RW2 Hence the anti - CD3 - target antigen drug - conjugated antigen 4B6 , OKT3D , M - T301, SP34 , SMC2 and F101. 01 ; or a binding constructs described herein may have an added humanized version thereof. Other CD3 binding moieties are US 2018 /0193477 A1 Jul. 12 , 2018 possible , and may be made by methods described herein . In Bi- Specific Antigen - Binding Constructs for Drug some embodiments , the antigen -binding polypeptide con Conjugation struct has the 6 CDRs of wild - type OKT3 , or the 6 CDRs of the stabilized variant of OKT3 , or a humanized variant of [0056 ] Provided herein are drug - conjugates of bispecific OKT3 in Table S1. antigen - binding constructs , e . g ., antibodies , that bind CD3 [0050 ] In some embodiments described herein , the target and a second antigen expressed on target cells . The antigen antigen ( cognate antigen — for the second antigen -binding binding construct itself comprises two antigen - binding poly polypeptide construct) is a B cell antigen . In some embodi peptide constructs , e . g. , antigen binding domains specifi ments , the target antigen is CD19 . Thus in some some cally binding either CD3 or the target antigen . In some embodiments wherein the tumor antigen is CD19 , the sec embodiments , the target antigen is associated with a tumor, ond antigen -binding polypeptide construct has the 6 CDRs for example CD19 , HER2 , HER3 , CDH3, or EGFR . In some of HD37 or the humanized variants of HD37 as shown in embodiments , the antigen - binding construct is derived from Table S1 . In some embodiments , the second antigen -binding known antibodies or antigen - binding constructs . As polypeptide construct comprises the antigen - binding poly described in more detail below , the antigen - binding poly peptide construct specific for CD19 derived from an anti peptide constructs may have the format of a Fab or an scFv body selected from the group consisting of 4G7 ; B4 ; B43 ; ( single chain Fv ) and may include an Fc . BU12; CLB - CD19 ; Leu- 12; SJ25 - C1; J4. 119, B43, SJ25C1, [0057 ] In some embodiments , first antigen - binding poly FMC63 ( IgG2a ) HD237 ( IgG2b ) , Mor -208 , MEDI- 551 , and peptide construct ( anti -CD3 ) may comprise a second scFv MDX - 1342 . comprising a second VL , a second scFv linker , and a second [0051 ] In other embodiments , the drug -conjugated antigen VH or it may comprise a Fab comprising a second VL and binding construct may be any of variants 6754 , 6751 , 1853 , a second VH . The second scFv may be selected from the 10151 , 6475 , 6749 , 10152 , 10153 , 6476 , 5850 , 5851 , 5852 , group consisting of the OKT3 scFv , a modified OKT3 scFv , 6325 , 1661, 1653 , 1662 , 1660 , 1666 , 1801, 6747 , 10149 , an OKT3 blocking antibody scFv , and a modified OKT3 10150 , 1380 or 12043, 151912 , 15193 , 15194 , 15195 , 17118 blocking antibody scFv , wherein the OKT3 blocking anti or 17119 . conjugated to a drug . body blocks by 50 % or greater the binding of OKT3 to the 10052 ] In many embodiments of drug -conjugated antigen epsilon subunit of the CD3 antigen . The second antigen binding construct having Fcs , there are modifications in the binding polypeptide construct may comprise the antigen CH2 domain to reduce or eliminate Fc gamma receptor binding polypeptide construct specific for CD3 derived from binding and thus they have no associated immune - cell an antibody selected from OKT3 ; TeplizumabTM (MGA031 , mediated effector activity . Eli Lilly ); Micromet , blinatumomab ; UCHT1 ; NI0401 ; [ 0053 ] In some embodiments of a drug - conjugated anti visilizumab ; X35 - 3 , VIT3 , BMA030 (BW264 / 56 ) , CLB - T3 / gen - binding construct , the affinity for the first antigen 3 , CRIS7 , YTH12 . 5 , F111 - 409 , CLB - T3 . 4 . 2 , WT31 , WT32 , binding polypeptide construct for CD3 is at least 2 , 5 , 10 , 15 SPV - T3b , 11D8 , XIII - 141 , XIII - 46 , XIII - 87 , 12F6 , T3 /RW2 or 20 - fold lower than and the affinity of the second antigen 8C8, T3 /RW2 - 4B6 , OKT3D , M - T301 , SMC2, F101. 01 or binding polypeptide construct for the target antigen , as SP34 . determined by SPR or FACS analysis . [ 0058 ] In some embodiments , for example , the second [0054 ] Also provided is a method of treating a disease , antigen -binding polypeptide construct (anti - CD19 ) may disorder or condition in a subject, the method comprising comprise an scFv comprising a first VL , a first scFv linker , administering an effective amount of the drug - conjugated and a first VH or it may comprise a Fab comprising a first antigen - binding construct of to the subject. In some embodi VL and a first VH . The first scFv may be selected from the ments , the cancer is a hematopoietic cancer, leukemia , a group consisting of an anti -CD19 antibody HD37 scFv , a lymphoma, a hematological cancer, a B -cell lymphoma, a modified HD37 scFv, an HD37 blocking antibody scFv, and non -Hodgkin ' s lymphoma , a cancer non - responsive to at a modified HD37 blocking antibody scFv , wherein the HD37 least one of a CD19 lytic antibody, a CD20 lytic antibody blocking antibody blocks by 50 % or greater the binding of and blinatumomab , a cancer cell regressive after treatment HD37 to the CD19 antigen . Alternatively , antigen -binding with blinatumomab , ALL , CLL , NHL , Mantle Cell Lym polypeptide constructs ( anti -CD19 ) may comprise the cor phoma, disseminated B cell diseases and metastases of the responding Fabs. The first antigen -binding polypeptide con brain , lung , liver, and /or bone . In some embodiments , the struct may comprise the antigen -binding polypeptide con tumor is a solid tumor. struct specific for CD19 derived from an antibody selected [ 0055 ] Also provided is a method of depleting target cells from the group consisting of 4G7; B4 ; B43; BU12 ; CLB in a subject comprising administering to the subject an CD19 ; Leu - 12 ; SJ25 - C1 ; J4 . 119 , B43 , SJ25C1, FMC63 effective amount of a drug - conjugated antigen -binding poly (IgG2a ) HD237 (IgG2b ) , Mor -208 , MEDI- 551, or MDX peptide construct comprising a first antigen -binding poly 1342 . peptide construct that monovalently and specifically binds to [0059 ] The heterodimeric Fc comprises first and second Fc a CD3 antigen expressed on T cells of the subject and a polypeptides each comprising a modified CH3 sequence second antigen -binding polypeptide construct that specifi capable of forming a dimerized CH3 domain , wherein each cally binds to an antigen expressed on the target cells , modified CH3 sequence comprises asymmetric amino acid wherein the first and second antigen - binding polypeptide modifications that promote formation of a heterodimeric Fc constructs are operably linked , and wherein the antigen and the dimerized CH3 domains have a melting temperature binding construct is conjugated to a drug . In some embodi (Tm ) of about 68° C . or higher . The first Fc polypeptide is ments , the tumor cells in the subject are depleted , but the T linked to the first antigen -binding polypeptide construct with cells are not substantially depleted . In some embodiments , a first hinge linker , and the second Fc polypeptide is linked the administration does not result in up - regulation of PD - 1 + to the second antigen -binding polypeptide construct with a ( inhibitory ) T cells in the subject . second hinge linker . In some embodiments , and as described US 2018 /0193477 A1 Jul. 12 , 2018 below , the CH2 domain of the Fc is modified to reduce or that correspond to the different classes of immunoglobulins eliminate binding of the drug - conjugated antigen -binding are called a , d , e, y , and u , respectively . constructs to Fc receptors . [ 0066 ] An exemplary immunoglobulin ( antibody ) struc 10060 ] The term “ antigen -binding construct” refers to any tural unit is composed of two pairs of polypeptide chains , agent , e . g ., polypeptide or polypeptide complex capable of each pair having one “ light” ( about 25 kD ) and one “ heavy ” binding to an antigen . In some aspects an antigen -binding chain (about 50 -70 kD ) . The N - terminal domain of each construct is a polypeptide that specifically binds to an chain defines a variable region of about 100 to 110 or more antigen of interest. An antigen -binding construct can be a amino acids primarily responsible for antigen recognition . monomer, dimer , multimer , a protein , a peptide, or a protein The terms variable light chain (VL ) and variable heavy or peptide complex ; an antibody, an antibody fragment, or an chain (VH ) refer to these light and heavy chain domains antigen -binding fragment thereof; an scFv and the like . An respectively . antigen - binding construct can be a polypeptide construct [0067 ] The IgG , heavy chain comprised of the VH , CHI, that is monospecific , bispecific , or multispecific . In some CH2 and CH3 domains respectively from the N to C -ter aspects , an antigen -binding construct can include , e . g ., one minus. The light chain is comprised of the VL and CL or more antigen -binding components ( e . g . , Fabs or scFvs ) domains from N to C terminus . The IgG heavy chain linked to one or more Fc . Further examples of antigen comprises a hinge between the CH1 and CH2 domains . binding constructs suitable for use in ADCs are described [0068 ] The term “ hypervariable region ” or “HVR ” , as below and provided in the Examples. used herein , refers to each of the regions of an antibody [0061 ] The term “ bispecific ” is intended to include any variable domain which are hypervariable in sequence and / or agent , e. g. , an antigen - binding construct, which has two form structurally defined loops (“ hypervariable loops " ) . antigen -binding moieties ( e . g . antigen -binding polypeptide Generally , native four- chain antibodies comprise six HVRs; constructs ) , each with a unique binding specificity . For three in the VH (H1 , H2, H3) , and three in the VL (L1 , L2 , example , a first antigen -binding moiety binds to an epitope L3 ) . HVRs generally comprise amino acid residues from the on a first antigen , and a second antigen -binding moiety binds hypervariable loops and / or from the complementarity deter to an epitope on a second antigen , where the first antigen is mining regions (CDRs ) , the latter being of highest sequence different from the second antigen . variability and /or involved in antigen recognition . With the [0062 ] For example , in some embodiments a bispecific exception of CDR1 in VH , CDRs generally comprise the agent may bind to , or interact with , ( a ) a cell surface target amino acid residues that form the hypervariable loops . molecule and ( b ) an Fc receptor on the surface of an effector Hypervariable regions (HVRs ) are also referred to as cell. In another embodiment , the agent may bind to , or " complementarity determining regions ” (CDRs ) , and these interact with ( a ) a first cell surface target molecule and ( b ) terms are used herein interchangeably in reference to por a second cell surface target molecule that is different from tions of the variable region that form the antigen - binding the first cells surface target molecule . In another embodi regions . This particular region has been described by Kabat ment, the agent may bind to and bridge two cells, i . e . interact et al. , U . S . Dept. of Health and Human Services, Sequences with ( a ) a first cell surface target molecule on a first call and of Proteins of Immunological Interest ( 1983 ) and by Chothia ( b ) a second cell surface target molecule on a second cell that et al. , J Mol Biot 196 : 901 - 917 ( 1987 ) , where the definitions is different from the first cell' s surface target molecule. include overlapping or subsets of amino acid residues when [0063 ] In some embodiments , the bispecific antigen compared against each other . Nevertheless , application of binding construct bridges CD3- expressing T cells with either definition to refer to a CDR of an antibody or variants CD19 - expressing B cells , with the formation of immuno thereof is intended to be within the scope of the term as logical synapses and / or mediation of T cell directed B cell defined and used herein . The exact residue numbers which depletion . encompass a particular CDR will vary depending on the [ 0064 ] A monospecific antigen -binding construct refers to sequence and size of the CDR . Those skilled in the art can an antigen - binding construct with a single binding specific routinely determine which residues comprise a particular ity . In other words, both antigen -binding moieties bind to the CDR given the variable region amino acid sequence of the same epitope on the same antigen . Examples of monospe antibody . cific antigen -binding constructs include the anti -CD19 anti [0069 ] The CDR regions of an antibody may be used to body HD37 and the anti - CD3 antibody OKT3 . construct a binding protein , including without limitation , an 0065 ] An antigen -binding construct can be an antibody or antibody, a scFv, a diabody , and the like . In a certain antigen - binding portion thereof. As used herein , an “ anti embodiment, the antigen -binding constructs described body ” or “ immunoglobulin ” refers to a polypeptide substan herein will comprise at least one or all the CDR regions from tially encoded by an immunoglobulin gene or immuno an antibody. CDR sequences may be used on an antibody globulin genes , or fragments thereof, which specifically bind backbone , or fragment thereof, and likewise may include and recognize an analyte ( e . g ., antigen ). The recognized humanized antibodies, or antibodies containing humanized immunoglobulin genes include the kappa , lambda , alpha , sequences . Methods of identifying CDR portions of an gamma, delta , epsilon and mu constant region genes , as well antibody are well known in the art. See , Shirai, H ., Kidera , as the myriad immunoglobulin variable region genes . Light A . , and Nakamura , H . , H3 - rules: Identification of CDR -H3 chains are classified as either kappa or lambda . The “ class” structures in antibodies , FEBS Lett ., 455 ( 1 ) : 188 - 197 , 1999 ; of an antibody or immunoglobulin refers to the type of and Almagro J C , Fransson , J. Front Biosci. 13 :1619 -33 constant domain or constant region possessed by its heavy (2008 ). chain . There are five major classes of antibodies: IgA , IgD , IgE , IgG , and IgM , and several of these may be further Antige- Binding Polypeptide Construct Format divided into subclasses (isotypes ) , e . g . , IgG , IgG2, IgGz, [0070 ] The bispecific antigen - binding construct com IgG4, IgA1, and IgA2. The heavy chain constant domains prises two antigen -binding polypeptide constructs , e . g. , anti US 2018 /0193477 A1 Jul. 12 , 2018 gen binding domains . The format of the antigen -binding position and /or length of the scFv linker polypeptide . Typi polypeptide construct determines the functional character cal peptide linkers comprise about 2 - 20 amino acids , and are istics of the bispecific antigen -binding construct . In one described herein or known in the art . Suitable , non - immu embodiment, the bi -specific antigen -binding construct has nogenic linker peptides include, for example , (GAS ), (SGA )n , an scFv - scFv format, i. e . both antigen -binding polypeptide (GAS )n , G (SG4 ) n, or G2 (SG2 ) n , linker peptides , wherein n is constructs are scFvs. In another embodiment the antigen generally a number between 1 and 10 , typically between 2 binding construct has an scFv -Fab format. In another and 4 . embodiment, both antigen - binding polypeptide constructs [0078 ] In some embodiments , the scFv linker is selected are Fabs. from Table below : 10071] The format " Single - chain Fy ” or “ scFv ” includes the VH and VL domains of an antibody, wherein these TABLE A domains are present in a single polypeptide chain . In some embodiments , the scFv polypeptide further comprises a scFv linker polypeptide sequences polypeptide linker between the VH and VL domains. For a CD19 review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113 , Rosenburg and Moore GGGGSGGGGSGGGGS eds. , Springer - Verlag , New York , pp . 269 -315 ( 1994 ) . [0072 ] Other antigen - binding polypeptide construct for CD3 mats include a Fab fragment or sdAb . GGGGSGGGGSGGGGS 10073 ] The “ Fab fragment” (also referred to as fragment antigen -binding ) contains the constant domain (CL ) of the SSTGGGGSGGGGSGGGGSDI light chain and the first constant domain (CHI ) of the heavy VEGGSGGSGGSGGSGGVD chain along with the variable domains VL and VH on the light and heavy chains respectively . The variable domains Generic linkers : comprise the complementarity determining loops (CDR , GGGGSGGGGSGGGGS also referred to as hypervariable region ) that are involved in antigen -binding . Fab ' fragments differ from Fab fragments GGGGSGGGGSGGGGSGGGGS by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cyste GSTSGGGSGGGSGGGGSS ines from the antibody hinge region . GSTSGSGKPGSGEGSTKG [0074 ] The “ Single domain antibodies ” or “ sdAb ” format is an individual immunoglobulin domain . Sdabs are fairly [ 0079 ] The scFv molecule may be optimized for protein stable and easy to express as fusion partner with the Fc chain expression and yield by including stabilizing disulfide of an antibody (Harmsen M M , De Haard H J ( 2007 ) . bridges between the heavy and light chain variable domains , “ Properties, production , and applications of camelid single for example as described in Reiter et al . ( Nat Biotechnol 14 , domain antibody fragments ” . Appl. Microbiol Biotechnol. 1239 - 1245 (1996 ) ). Hence, in one embodiment the T cell 77 ( 1 ) : 13 -22 ) . In some embodiments an antigen -binding activating bispecific antigen -binding molecule of the inven construct provided herein comprises an antigen -binding tion comprises a scFv molecule wherein an amino acid in the polypeptide construct that lacks a light chain , thus compris heavy chain variable domain and an amino acid in the light ing a single domain antibody. chain variable domain have been replaced by cysteine so scFv Format that a disulfide bridge can be formed between the heavy and [0075 ] The antigen -binding constructs described herein light chain variable domain . In a specific embodiment the are bi -specific , e . g . , they comprise two antigen - binding amino acid at position 44 of the light chain variable domain polypeptide constructs each capable of specific binding to a and the amino acid at position 100 of the heavy chain distinct antigen . In some embodiments , either or both anti variable domain have been replaced by cysteine (Kabat gen -binding polypeptide construct is in an scFv format. ( i . e . , numbering ). antigen - binding domains composed of a heavy chain vari [0080 ) As is known in the art, scFvs can also be stabilized able domain and a light chain variable domain , connected by mutation of CDR sequences, as described in [ Miller et al. , with a polypeptide linker) . In one embodiment said scFv are Protein Eng Des Set. 2010 July ; 23 ( 7 ) :549 -57 ; Igawa et al. , human . In another embodiment said scFv molecules are MAbs. 2011 May - June ; 3 ( 3 ): 243 -5 ; Perchiacca & Tessier , humanized . The scFvs are optimized for protein expression Annu Rev Chem Biomol Eng . 2012 ; 3 : 263 - 86 . ) . One or and yield by the modifications described below . more of the above noted modifications to the format and [0076 ] The scFv can be optimized by changing the order sequence of the scFv may be applied to scFvs of the of the variable domains VL and VH in the scFv . In some antigen -binding constructs . embodiments of an scFv in a antigen -binding construct [0081 ] Humanized CD19 VH and VL described herein , the C - terminus of the light chain variable [0082 ] In some embodiments , and in order to further region may be connected to the N -terminus of the heavy stabilize the antigen - binding constructs described herein , the chain variable region , or the C - terminus of the heavy chain wild - type sequences of the HD37 anti- CD19 antibody can variable region may be connected to the N -terminus of the be modified to generate humanized VH and VL polypeptide light chain variable region . sequences . Modifications to both the framework regions and [0077 ] The variable regions may be connected via a linker CDRs can be made in order to obtain VH and VL polypep peptide , or scFv linker, that allows the formation of a tide sequences to be used in the CD19 - binding scFvs and functional antigen - binding moiety . The scFv can be opti - Fabs of the antigen - binding constructs . In some embodi mized for protein expression and yield by changing com ments, the modifications are those depicted in FIG . 2 . In US 2018 /0193477 A1 Jul. 12 , 2018 some embodiments , the Tm of a humanized anti - CD19 [0087 ] In certain embodiments , an antigen -binding con binding domain is higher than than the Tm of an HD37 struct that binds to the antigen , or an antigen -binding mol binding domain . In some embodiments , the Tm of a human ecule comprising that antigen -binding moiety , has a disso ized anti -CD19 binding domain is at least 2 , at least 3 , at ciation constant ( K ) ) of < 1 uM , < 100 nM , < 10 nM , < 1 nM , least 4 , at least 5 , at least 6 , at least 7 , at least 8 , at least 10 < 0 . 1 nM , < 0 .01 nM , or < 0 .001 nM ( e . g . 10 - 8 M or less , e . g . degrees C . higher than than the Tm of an HD37 binding from 10 - 8 M to 10 " 13 M , e .g ., from 10 " 9 M to 10" 13 M ). domain . [0088 ] “ Affinity ” refers to the strength of the sum total of non - covalent interactions between a single binding site of a Humanized CD3 VH and VL molecule ( e . g . , a receptor ) and its binding partner ( e . g . , a [0083 ] In some embodiments , and in order to further ligand ) . Unless indicated otherwise , as used herein , " binding atabilize the antigen -binding constructs described herein the affinity ” refers to intrinsic binding affinity which reflects a wild - type sequences of the OKT3 anti -CDS3 antibody are 1 : 1 interaction between members of a binding pair ( e . g ., an modified to generate humanized VH and VL polypeptide antigen -binding moiety and an antigen , or a receptor and its sequences .Modifications to both the framework regions and ligand ) . The affinity of a molecule X for its partner Y can CDRs can be made in order to obtain VH and VL polypep generally be represented by the dissociation constant ( K ) ) , tide sequences to be used in the CD3 -binding scFvs and Fabs which is the ratio of dissociation and association rate con of the antigen - binding constructs . In some embodiments, the stants ( k and kon , respectively ) . Thus , equivalent affinities modifications are those depicted in FIG . 4 . In some embodi may comprise different rate constants, as long as the ratio of ments , the Tm of a humanized anti -CD19 scFv binding the rate constants remains the same. Affinity can be mea domain is higher than the Tm of an OKT3 or teplizumab sured by well established methods known in the art , includ binding domain . In some embodiments , the Tm of a human ing those described herein . A particular method for measur ized anti -CD19 scFv binding domain is at least 2 , at least 3 , ing affinity is Surface Plasmon Resonance (SPR ) , or whole at least 4 , at least 5 , at least 6 , at least 7 , at least 8 , at least cell binding assays with cells that express the antigen of 10 degrees C . higher than the Tm of an OKT3 or teplizumab interest. binding domain . [ 0089 ] “ Reduced binding” , for example reduced binding to an Fc receptor, refers to a decrease in affinity for the Antige- Binding Polypeptide Construct — Antigens respective interaction , as measured for example by SPR . For clarity the term includes also reduction of the affinity to zero 100841. The antigen -binding constructs described herein ( or below the detection limit of the analytic method ), i. e . specifically bind a CD3 antigen and a second target antigen . complete abolishment of the interaction . Conversely , [0085 ] As used herein , the term “ antigenic determinant” is “ increased binding” refers to an increase in binding affinity synonymous with “ antigen ” and “ epitope, ” and refers to a for the respective interaction . site ( e . g . a contiguous stretch of amino acids or a confor [0090 ] An “ activating T cell antigen ” as used herein refers mational configuration made up of different regions of to an antigenic determinant expressed on the surface of a T non - contiguous amino acids) on a polypeptide macromol lymphocyte , particularly a cytotoxic T lymphocyte , which is ecule to which an antigen - binding moiety binds , forming an capable of inducing T cell activation upon interaction with antigen - binding moiety - antigen complex . An epitope typi an antigen - binding molecule . Specifically , interaction of an cally includes at least 3 , and more usually , at least 5 or 8 - 10 antigen -binding molecule with an activating T cell antigen amino acids in a unique spatial conformation . The epitope may induce T cell activation by triggering the signaling may comprise amino acid residues directly involved in the cascade of the T cell receptor complex . In a particular binding and other amino acid residues, which are not directly involved in the binding , such as amino acid residues embodiment the activating T cell antigen is CD3. which are effectively blocked by the specifically antigen 10091 ] " T cell activation ” as used herein refers to one or binding peptide ; in other words, the amino acid residue is more cellular response of a T lymphocyte , particularly a within the footprint of the specifically antigen binding cytotoxic T lymphocyte , selected from : proliferation , differ peptide . Antibodies that recognize the same epitope can be entiation , cytokine secretion , cytotoxic effector molecule verified in a simple immunoassay showing the ability of one release , cytotoxic activity , and expression of activation antibody to block the binding of another antibody to a target markers. The T cell activating bispecific antigen -binding antigen . molecules of the invention are capable of inducing T cell [ 0086 ] “ Specifically binds ” , “ specific binding ” or “ selec activation . Suitable assays to measure T cell activation are tive binding ” means that the binding is selective for the known in the art described herein . antigen and can be discriminated from unwanted or non [ 0092 ] A “ target cell antigen ” or “ target antigen ” as used specific interactions. The ability of an antigen -binding con herein refers to an antigenic determinant presented on the struct to bind to a specific antigenic determinant can be surface of a target cell , for example a B cell in a tumor such measured either through an enzyme- linked immunosorbent as a cancer cell or a cell of the tumor stroma. Atumor antigen assay (ELISA ) or other techniques familiar to one of skill in is a target cell antigen expressed on a tumor cell . In some the art, e . g . surface plasmon resonance (SPR ) technique embodiments , a tumor antigen or may be overexpressed on (analyzed on a BIAcore instrument) (Liljeblad et al, Glyco tumor cells. As used herein , the terms " first” and “ second ” J 17 , 323 - 329 (2000 ) ) , and traditional binding assays (Hee with respect to antigen - binding moieties etc . , are used for ley, Endocr Res 28 , 217 - 229 ( 2002) ) . In one embodiment, convenience of distinguishing when there is more than one the extent of binding of an antigen - binding moiety to an of each type ofmoiety . Use of these terms is not intended to unrelated protein is less than about 10 % of the binding of the confer a specific order or orientation of the T cell activating antigen - binding construct to the antigen as measured , e . g ., bispecific antigen - binding molecule unless explicitly so by SPR . stated . US 2018 /0193477 A1 Jul. 12 , 2018

[0093 ] The term “ cross - species binding” or “ interspecies gen -binding construct comprises a CD3 antigen -binding binding " or " species cross- reactive ” as used herein means polypeptide construct which monovalently and specifically binding of a binding domain described herein to the same binds a CD3 antigen , the VH and VL regions of said CD3 target molecule in humans and other organisms for instance , antigen - binding polypeptide derived from the CD3 epsilon but not restricted to non - chimpanzee primates . Thus , “ cross specific antibody OKT3 . species binding " or " interspecies binding " is to be under [0098 ] In some embodiments , the binding affinity of the stood as an interspecies reactivity to the samemolecule “ X ” first antigen binding polypeptide construct specific for the ( i . e . the homolog ) expressed in different species , but not to epsilon subunit of CD3 is between about 1 nM to about 100 a molecule other than “ X ” . Cross -species specificity of a nM , or between about 20 nM to about 100 nM , or , e . g ., monoclonal antibody recognizing e . g . human CD3 epsilon , greater than 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , to a non -chimpanzee primate CD3 epsilon , e . g . macaque 80 , or greater than 90 nM . CD3 epsilon , can be determined , for instance, by FACS [0099 ] The epitope on the CD3 epsilon subunit to which analysis . The FACS analysis is carried out in a way that the the OKT3 antibody binds is identified by analysis of the respective monoclonal antibody is tested for binding to crystal structure of the OKT3 bound to CD3 epsilon (Kjer human and non -chimpanzee primate cells , e . g . macaque Nielsen L . et al ., ( 2004 ) Proc . Natl . Acad . Sci . USA 101 : cells , expressing said human and non - chimpanzee primate 7675 -7680 ). The polypeptide sequence of CD3 epsilon is CD3 epsilon antigens, respectively . An appropriate assay is provided in the Table below . shown in the following examples . The above -mentioned subject matter applies mutatis mutandis for the CD19 . The TABLE B FACS analysis is carried out in a way that the respective monoclonal antibody is tested for binding to human and CD3 Epsilon sequence non - chimpanzee primate cells , e . g . macaque cells, express Human T - cell MOSGTHWRVLGLCLLSVGVWGODGNEEMGGI ing said human and non - chimpanzee primate CD3 or CD19 surface TOTPYKVSISGTTVILTCPOYPGSEILWOHN antigens. glycoprotein DKNIGGDEDDKNIGSDEDHLSLKEFSELEOS CD3 epsilon GYYVCYPRGSKPEDANFYLYLRARVCENCME subunit , MDVMSVATIVIVDICITGGLLLLVYYWSKNR CD3 UniProt ID : KAKAKPVTRGAGAGGRORGONKERPPPVPNP [ 0094 ] The antigen -binding constructs described herein P07766 ( 207 DYEPIRKGQRDLYSGLNQRRI specifically bind a CD3 antigen . amino acids ) ( SEQ ID NO : 350 ) [0095 ] “ CD3 ” or “ CD3 complex ” as described herein is a complex of at least five membrane -bound polypeptides in [0100 ] Analysis of this structure indicates that the CDRs mature T -lymphocytes that are non -covalently associated of the OKT3 antibody , with respect to the sequence in Table with one another and with the T -cell receptor. The CD3 B , contact human CD3 epsilon at residues 56 -57 (SE ) , 68 - 70 complex includes the gamma, delta , epsilon , and zeta chains (GDE ) , and 101- 107 ( RGSKPED ) . The binding hotspots in ( also referred to as subunits ) . Non -human monoclonal anti these residues are underlined . These residues are considered bodies have been developed against some of these chains , as to be the epitope to which OKT3 binds. Accordingly , the exemplified by the murine antibodies OKT3 , SP34 , UCHT1 antigen -binding constructs described herein may comprise or 64 . 1 . ( See e . g . , June , et al. , J . Immunol. 136 : 3945 - 3952 an antigen - binding polypeptide construct that specifically ( 1986 ) ; Yang , et al. , J . Immunol. 137 : 1097 - 1100 (1986 ) ; and binds to this epitope . Hayward , et al. , Immunol. 64 :87 - 92 ( 1988 ) . Clustering of [0101 ] Provided herein are antigen - binding constructs CD3 on T cells , e . g . , by immobilized anti -CD3 -antibodies , comprising at least one CD3 binding polypeptide construct leads to T cell activation similar to the engagement of the T that binds to a CD3 complex on at least one CD3 expressing cell receptor but independent from its clone typical speci cell, where in the CD3 expressing cell is a T - cell . In certain ficity . Most anti -CD3 -antibodies recognize the CD38 -chain . embodiments , the CD3 expressing cell is a human cell. In 10096 ] In some embodiments , the anti -CD3 scFv or Fab is some embodiments , the CD3 expressing cell is a non an scFV or Fab of a known anti -CD3 antibody , or is derived human , mammalian cell . In some embodiments, the T cell is from , e. g. , is a modified version of the scFv or Fab of a a cytotoxic T cell . In some embodiments the T cell is a CD4 + known anti -CD3 antibody . Antibodies directed against or a CD8 + T cell . human CD3 which provide for variable regions (VH and [0102 ] In certain embodiments of the antigen -binding con VL ) to be employed in the bi -specific antigen - binding structs provided herein , the construct is capable of activating construct described herein are known in the art and include and redirecting cytotoxic activity of a T cell to a target cell OKT3 (ORTHOCLONE -OKT3TM (muromonab -CD3 ) . such as a B cell. In a particular embodiment , said redirection Additional anti- CD3 antibodies include “ OKT3 blocking is independent of MHC - mediated peptide antigen presenta antibodies ” that block by 50 % or greater the binding of tion by the target cell and and / or specificity of the T cell . OKT3 to the epsilon subunit of the CD3 antigen . Examples include but are not limited to TeplizumabTM (MGA031 , Eli Target Antigens Lilly ) ; UCHT1 ( Pollard et al. 1987 J Histochem Cytochem . 35 (11 ) : 1329 - 38 ) ; NI0401 (WO2007 / 033230 ) ; and visili CD19 zumab (US25834597 ) . [0103 ] B - cell antigen CD 19 (CD 19 , also known as B - cell [ 0097 ] In one embodiment, the bispecific antigen -binding surface antigen B4 , Leu - 12 ; Uniprot ID # P15391 ) is a construct comprises a CD3 antigen - binding polypeptide human pan - B -cell surface marker that is expressed from construct which monovalently and specifically binds a CD3 early stages of pre - B cell development through terminal antigen , where the CD3 antigen - binding polypeptide con differentiation into plasma cells . CD 19 promotes the pro struct is derived from OKT3 (ORTHOCLONE -OKT3TM liferation and survival of mature B cells . It associates in a (muromonab -CD3 ) . In one embodiment the bispecific anti - complex with CD21 on the cell surface . It also associates US 2018 /0193477 A1 Jul. 12 , 2018 with CD81 and Leu -13 and potentiates B cell receptor Immunol. 138, 2793 - 9) . Additional anti -CD19 antibodies (BCR ) signaling . Together with the BCR , CD 19 modulates include “ HD37 blocking antibodies” that block by 50 % or intrinsic and antigen receptor- induced signaling thresholds greater the binding of HD37 to the CD19 antigen . Examples critical for clonal expansion of B cells and humoral immu include but are not limited to HD237 ( IgG2b ) (Fourth nity . In collaboration with CD21 it links the adaptive and the International Workshop on Human Leukocyte Differentia innate immune system . Upon activation , the cytoplasmic tail tion Antigens, Vienna , Austria , 1989 ; and Pezzutto et al. , J . of CD 19 becomes phosphorylated which leads to binding Immunol. , 138 ( 9 ) : 2793 - 2799 ( 1987 ) ) ; 4G7 (Meecker ( 1984 ) by Src - family kinases and recruitment of PI - 3 kinase . It is Hybridoma 3 , 305 - 20 ) ; B4 (Freedman ( 1987 ) Blood 70 , also expressed on the vast majority of non -Hodgkin 's lym 418 - 27 ) ; B43 ( Bejcek ( 1995 ) Cancer Res . 55 , 2346 -51 ) and phoma (NHL ) cells as well as some leukemias . Mor -208 (Hammer (2012 ) Mabs4 : 5 , 571 -577 ) . [0104 ] Because of their critical role in regulating the [0107 ] In one embodiment said VH ( CD19 ) and immune system , disregulation of B cells is associated with VL (CD19 ) regions ( or parts , like CDRs, thereof ) are derived a variety of disorders . B - cell disorders , also referred to from the anti -CD19 antibody HD37 , provided by the HD37 herein as B - cell related diseases, are divided into excessive hybridoma (Pezzutto (1997 ) , J . Immunol. 138 , 2793 - 9 ) . or uncontrolled proliferation ( lymphomas , leukemias ) , and [0108 ] In some embodiments , the binding affinity of the defects of B - cell development/ immunoglobulin production second antigen - binding polypeptide construct for the target ( immunodeficiencies ). antigen is between about 0 . 1 nM to about 10 nM or less than The amino acid sequence of CD19 is as follows : 5 . 0 , 4 . 0 , 3 . 0 , 2 . 0 , 1 . 0 , 0 . 9 , 0 .09 , 0 . 9 , 0 . 7 , 0 . 6 , 0 . 5 , 0 . 4 , 0 . 3 , or less than 0 . 2 nM . In some embodiments , the binding affinity of the second antigen - binding polypeptide construct to MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGP CD19 on the surface of CD19 + target cells is in the range of 0 . 1 to 0 . 5 , 0 . 5 - 1 , 1 - 3 , 3 - 5 , 5 - 7 , 7 - 9 , 9 - 11 , 11 - 13 , 13 - 15 , TOOLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMG 15 - 17 , 17 - 19 or 19 -21 nM as measured by FACS analysis . GFYLCQPGPPSEKAWOPGWTVNVEGSGELFRWNVSDLGGLGCGLKN [0109 ] In certain embodiments , the antigen - binding poly peptide construct is an scFv or Fab construct that binds RSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSL CD19 on a B cell . In some embodiments the scFv or Fab SQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLEL construct is mammalian . In one embodiment said scFv or Fab construct is human . In another embodiment said scFv or KDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITA Fab construct is humanized . In yet another embodiment said scFv or Fab construct comprises at least one of human heavy RPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRK and light chain variable regions. RKRMTDPTRRFFKVTPPPGSGPONQYGNVLSLPTPTSGLGRAQRWA [0110 ] In certain embodiments , the antigen - binding poly peptide construct exhibits cross - species binding to a least AGLGGTAPSYGNPSSDVOADGALGSRSPPGVGPEEEEGEGYEEPDS one antigen expressed on the surface of a B cell. In some EEDSEFYENDSNLGQDOLSQDGSGYENPEDEPLGPEDEDSFSNAES embodiments , the antigen -binding polypeptide construct of an antigen - binding construct described herein bind to at least YENEDEELTOPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGI one of mammalian CD19 . In certain embodiments , the LYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGT CD19 antigen -binding polypeptide construct binds a human CD19 . WSTR . [0105 ] In some embodiments , the antigen -binding con CDH3 structs described herein include an antigen -binding polypep [0111 ] In some embodiments , a drug -conjugated antigen tide construct that binds to a CD19 antigen ( anti -CD19 scFv binding construct may have an antigen -binding polypeptide or Fab ) . construct directed against CDH3. CDH3 , also known as [ 0106 ] In some embodiments, the anti -CD19 scFv or Fab CADH3 ; cadherin 3 , type 1 or P - cadherin (Uniprot ID # is an scFv or Fab of a known anti- CD19 antibody, or is P22223 ) is a member of the cadherin family of cell adhesion derived from , e . g . , is a modified version of the scFv or Fab proteins that preferentially interact with themselves in a of a known anti- CD19 antibody . Antibodies directed against homophilic manner in cell -cell adhesion . CDH3 overexpres CD19 which provide for variable regions (VH and VL ) to be sion is associated with several types of cancer. In some employed in the bispecific antigen -binding construct embodiments , anti -CDH3 antibodies in Table KK are used described herein are known in the art and include HD37 , to derive antigen - binding polypeptide constructs specific for provided by the HD37 hybridoma (Pezzutto ( 1997 ) , J . CDH3 . TABLE KK Antibody Patent/ paper reference : anti -HER2 trastuzumab PCT /US1998 /026266 ; Baselga J. , et al, 1998 , Cancer Res. , 58 : 2825 - 31 pertuzumab PCT/ US 2005 / 025084; DeGrendele H ., 2003 , Clin Prostate Cancer , 2 : 143 - 5 ertumaxomab PCT/ EP2008/ 001551; Kiewe P , et al, 2006 , Clin . Cancer Res. , 12 : 3085 - 91 US 2018 /0193477 A1 Jul. 12 , 2018 11

TABLE KK -continued Antibody Patent/ paper reference : margetuximab PCT/ US2009 /038201 XMT- 1522 PCT/ US2015 /036431 MIL5 _ scFv Qiao C , et al. , 2013 , J. Biomol . Struct. Dyn ., 31 : 511 -21 702, 7F3 U . S . Pat . No . 14 , 511 ,604 anti- HER3 seribantumab PCT/ US2008 /002119 ; Schoeberl B . , et al. , 2009 , Sci Signal, 2 : ra31 patritumab PCT/ EP 2006 /01 2632; LoRusso P. , et al, 2013, Clin Cancer Res. , 19 : 3078 - 87 elgemtumab PCT/ EP2011/ 064407 ; Garrett J T ., et al. , 2013, Cancer Res . , 73: 6013 - 23 lumretuzumab PCT/ EP 2010 /070062 ; Mirschberger C . , et al. , 2013 , Cancer Res . , 73 : 5183 - 94 KTN3379 PCT/ US2012 /066038 ; Lee S . , et al. , 2015 , Proc Natl Acad Sci USA . , 112 : 13225 - 30 15D5 and 1D9 PCT/ US2011 /050322 REGN1400 PCT/ US2012 /056446 anti - EGFR cetuximab PCT/ US1996 /009847 ; Prewett M . , et al. , 1996 , J Immunother Emphasis Tumor Immunol , 19 : 419 - 27 panitumumab PCT /US2003 /015734 ; Yang X D , et al. , 2001 , Crit Rev Oncol Hematol. , 38 : 17 - 23 nimotuzumab PCT/ CA2012/ 050034 ; Spicer J. , 2005 , Curr. Opin . Mol. Ther ., 7 : 182 - 91 necitumumab PCT/ US2005 /009583 ; Lu D . , et al. , 2005 , J . Biol . Chem . , 280 : 19665 - 7 zalutumumab PCT/ US2002 /018748 ; Lammerts van Bueren J J . , et al. , 2008 , Proc . Natl. Acad . Sci. U . S . A ., 105 : 6109 - 14 matuzumab PCT/ EP2002 / 001687 ; Vanhoefer U . , et al. , 2004 , J. Clin . Oncol. , 22 : 175 - 84 imgatuzumab PCT/ IB 2006 / 000238 ; Gerdes C A . , et al. , 2013 , Clin Cancer Res. , 19 : 1126 - 38 depatuxizumab PCT/ US2007 /019988 ; Gan H K . , et al. , 2007 , J . Biol. Chem . , 282 : 2840 - 50 anti -CDH3 FF - 21101 PCT/ JP2010 /057694 Oncotherapy Clone # 6 PCT/ JP2007/ 054374 PF - 03732010 PCT/ IB 2006 /001053 ; 2010 , Zhang C C ., et al. , Clin . Cancer Res. , 16 : 5177 - 88 PCA062 PCT/ IB2015 / 058801 PF - 06671008 PCT/ IB2015 /054829

HER2 , HER3 and EGFR Other Target Antigens [0112 ] HER2, HER3 and EGFR are a HER receptors . A [0113 ] In some embodiments , the drug - conjugated anti “ HER receptor" is a receptor protein tyrosine kinase which gen - binding construct comprises a second antigen -binding belongs to the human epidermal growth factor receptor polypeptide construct that is specific for one of the target (HER ) family and includes EGFR , HER2 , HER3 and HER4 antigens provided in Table LL . In some embodiments , the receptors . A HER receptor will generally comprise an extra target antigen is a pathogen - derived antigen . In an embodi cellular domain , which may bind an HER ligand ; a lipophilic ment, the target antigen is a viral antigen . In some embodi transmembrane domain ; a conserved intracellular tyrosine ments , the target antigen is a fungal antigen . In some kinase domain ; and a carboxyl- terminal signaling domain embodiments , the target antigen is bacterial. In some harboring several tyrosine residues which can be phospho embodiments , the target antigen is a parasite antigen . In rylated . HER2, HER3 and EGFR are overexpressed in some embodiments the target antigen is associated with a numerous types of cancer. In some embodiments , the anti hematological cancer . In some embodiments , the target HER2, anti- HER3 and anti - EGFR antibodies in Table KK antigen is expressed on a solid tumor. In some embodiments , are used to derive antigen - binding polypeptide constructs . the target antigen is associated with an autoimmune disease . TABLE LL Target Antigens Viral targets Family GenusGenus Virus Retroviridae Lentivirus human immunodeficiency virus Papillomaviridae Many Human papilloma virus Paramyxoviridae Pneumovirus Human respiratory syncytial virus US 2018 /0193477 A1 12 Jul. 12 , 2018

TABLE LL - continued Target Antigens Viral targets Filoviridae Ebolavirus Ebola virus Coronaviridae Betacoronavirus SARS coronavirus Orthomyxoviridae Influenza A , B , C Influenza Hepadnaviridae Orthohepadnavirus Hepatitis B virus Flaviviridae Hepacivirus Hepatitis C virus Flaviviridae Flavivirus Zika virus Flaviviridae Flavivirus Dengue virus Flaviviridae Flavivirus West Nile Virus Herpesviridae Simplexvirus Herpes simplex virus Herpesviridae Lymphocryptovirus Epstein - Barr Virus Herpesviridae Varicellovirus Varicella -Zoster virus Herpesviridae Cytomegalovirus Cytomegalovirus Bacterial/ fungal Targets Family Genus Species Brucellaceae Brucella B . melitensis Chlamydiaceae Chlamydia C . trachomatis Chlamydiaceae Chlamydophila C . pneumoniae Clostridiaceae Clostridium C . difficile Coxiellaceae Coxiella C . burnetii Legionellaceae Legionella L . pneumophila (many more ) Listeriaceae Listeria L . monocytogenes Mycobacteriaceae Mycobacterium M . tuberculosis , M . leprae Neisseriaceae Neisseria N . gonorrhoeae , N . meningitidis Rickettsiaceae Rickettsia Numerous species in three groups: Spotted fever ( R . rickettsii ) Typhus ( R . prowazekii ) and Scrub - typhus ( Orientia tsutsugamushi) Enterobacteriaceae Salmonella S . bongori , S . enterica Enterobacteriaceae Shigella S . boydii , S . dysenteriae , S . flexneri, S . sonnei Enterobacteriaceae Yersinia Y. pestis , Y. pseudotuberculosis Tremellaceae Cryptococcus C . neoformans Trichocomaceae Aspergillus Aspergillus spp Parasitic Targets Family Genus Species Cryptosporidiidae Cryptosporidium C . parvum Plasmodium Plasmodium P . falciparum , P . vivax , P . ovale , and P . malariae Trypanosomatidae Leishmania L . donovani ( ~ 20 species infect humans ) Sarcocystidae Toxoplasma T . gondii Trypanosomatidae Trypanosoma T . cruzi , T . brucei Human targets Gene ID Uniprot ID Disease Association Cancer Hemooncology CD8 P10966 T cell activation ? CD19 P15391 B - cell malignancies , autoimmune disease CD20 P11836 Chronic Lymphocytic Leukemia , Non - Hodgkin ' s Lymphoma, Rheumatoid Arthritis CD22 P20273 Non - Hodgkin ' s Lymphoma, B - cell malignancies CD30 P28908 Anaplastic Large Cell Lymphoma Hematologic malignancies Hodgkin Lymphoma CD33 P20138 Acute myeloid leukemia CD37 P11049 Acute myeloid leukemia Chronic Lymphocytic Leukemia Non Hodgkin ' s Lymphoma CD38 P28907 Hematologic malignancies , Multiple Myeloma CD44v6 P16070 Squamous cell carcinoma, Hematologic malignancies CD74 PO4233 Chronic Lymphocytic Leukemia Multiple Myeloma US 2018 /0193477 A1 Jul. 12 , 2018

TABLE LL -continued Target Antigens Viral targets CD79b P40259 Non - Hodgkin 's Lymphoma, Systemic lupus erythematosus CD133 043490 Acute lymphoblastic leukemia Acute myeloid leukemia CD138 P18827 Multiple Myeloma IL -3Ra P26951 Acute myeloid leukemia , Hodgkin Lymphoma ???? Q02223 B -cell malignancies, Multiple Myeloma CLEC12A Q5QGZ9 Acute myeloid leukemia FLT3 P36888 Acute myeloid leukemia ROR 1 001973 Chronic Lymphocytic Leukemia , B cell malignancies Solid tumor CD70 (CD27L ) P32970 Renal Cell Carcinoma, Autoimmune Diseases, Cancer, Inflammatory Diseases CD117 P10721 Inflammatory Diseases , Cancer , Acute myeloid leukemia CD56 P13591 Multiple Myeloma, Solid Tumors CD98 P08195 head and neck squamous cell carcinoma cells with stem cell properties Notch 1 P46531 solid tumors - broad indications Notch 2 004721 solid tumors - broad indications Notch 3 Q9UM47 solid tumors - broad indications Notch 4 Q99466 solid tumors - broad indications DL44 Q9NR61 solid tumors - broad indications PSMA Q04609 Prostate Cancer PSA P07288 Prostate Cancer PSCA 043653 Prostate Cancer STEAP1 Q9UHES Prostate Cancer, Multiple Others CEACAM4 075871 Colorectal Cancer CEACAM5 P06731 Colorectal Cancer , Pancreatic Cancer , Gastric Cancer alpha - V integrin P06756 melanoma, glioma , ovarian , and breast cancer EphA2 P29317 solid tumors Ephalo Q5JZY3 Breast Cancer Ep??? P16422 solid tumors Cadherin - 19 J3KTP3 Melanoma P - cadherin P22223 solid tumors Nectin - 4 Q96NY8 Metastatic Urothelial Cancer Glypican 3 P51654 Liver Cancer EGFR / EGFRvIII P00533 solid tumors - broad indications VEGFR P17948 endothelial cell - solid tumor HER2 /neu PO4626 Breast Cancer , Head and Neck Cancer , ovarian , prostate Her3 P21860 Solid tumors IGF1R P08069 solid tumors and hematological malignancies C -MET P08581 Solid tumors folate receptor alpha P15328 Ovarian Cancer folate receptor beta P14207 Acute myeloid leukemia , Ovarian Cancer Endothelin B receptor P24530 Melanoma TF ( Tissue Factor ) P13726 Pancreatic Cancer, Acute Lung Injury, Inflammatory Diseases MSLN Q13421 Mesothelioma, Breast Cancer , Ovarian cancer ENPP3 014638 Liver Cancer, Renal Cell Carcinoma TPBG Q13641 Non -Small Cell Lung cancer, Renal Cell Carcinoma FAP Q12884 Stromal Targeting, Colorectal Cancer HMW -MAA QOUVK1 Melanoma, Breast Cancer A33 099795 Colorectal Cancer B7 - H3 Q5ZPR3 Solid tumors B7 - H4 Q7Z7D3 Solid tumors GPNMB Q14956 Breast Cancer , Melanoma , CFC1B POCG36 Solid tumors TACSTD ( Trop2) P09758 Breast Cancer , Gastric Cancer, Pancreatic Cancer US 2018 /0193477 A1 Jul. 12 , 2018 14

TABLE LL - continued Target Antigens Viral targets TAG -72 Q9XVS1 Prostate , Breast , Colon , Lung , and Pancreatic cancers TIM - 3 Q8TDOO Immune Checkpoint , Cancer , Autoimmunity , Inflammation Guanylyl cyclase C P25092 Pancreatic Cancer (GCC ) /GUCY2C SLC44A4 Q53GD3 Pancreatic Cancer, Prostate Cancer SLC34A2 095436 Non - Small Cell Lung cancer , Ovarian Cancer SLC39A6 Q13433 Breast Cancer CanAg (a glycoform of P15941 Breast Cancer MUC1 ) Mucin 16 (CA125 ) Q8WX17 Epithelial Ovarian Cancer, Breast Cancer CAIX Q16790 Renal Cell Carcinoma RAAG12 N - linked carbohydrate Adenocarcinoma epitope Sialyl Lewisa carbohydrate epitope Gastrointestinal cancers Lewis Y (Le ( y ) ) antigen carbohydrate epitope Gastrointestinal cancers Autoimmune disease / Inflammation CD19 see above autoimmune disease CD20 see above Rheumatoid Arthritis CD70 (CD27L ) see above Autoimmune Diseases , Cancer, Inflammatory Diseases CD79b see above Systemic lupus erythematosus IL - 5Ra Q01344 Asthma, Chronic obstructive pulmonary disease IL -23R Q9NPF7 Inflammatory Diseases , Autoimmune Diseases , Cancer TF ( Tissue Factor) see above Acute Lung Injury , Inflammatory Diseases TIM - 3 see above Autoimmunity, Inflammation Viral infections - human targets TSG101 099816 HIV , Herpes , Influenza , Ebola WNV E 091KZ4 West Nile Virus CD81 P60033 HCV (entry ) CD4 PO1730 HIV CXCR4 P61073 HIV CCR5 P51681 HIV Integrin al P20701 HIV

Scaffolds Fc of Antigen - Binding Constructs . [0114 ] In some embodiments , the antigen - binding con [0116 ] Fc polypeptides make excellent scaffolds for anti structs described herein comprise a scaffold . A scaffold may gen -binding polypeptide constructs . Certain antigen - binding be a peptide , polypeptide , polymer , nanoparticle or other constructs described herein comprise an Fc , e. g ., a dimeric chemical entity . In embodiments where the scaffold is an Fc Fc. In some embodiments , the Fc is a a heterodimeric Fc or dimeric Fc, the antigen - binding polypeptide construct ( s ) comprising first and second Fc polypeptides each compris ing a modified CH3 sequence, wherein each modified CH3 of the antigen - binding construct may be linked to either the sequence comprises asymmetric amino acid modifications N - or C - terminus of the scaffold . A dimeric Fc can be that promote the formation of a heterodimeric Fc and the homodimeric or heterodimeric . dimerized CH3 domains have a melting temperature ( Tm ) of [0115 ] In embodiments where the scaffold is a peptide or about 68° C . or higher, and wherein the first Fc polypeptide polypeptide, the antigen - binding construct or antigen -bind is linked to the first antigen - binding polypeptide construct , ing polypeptide construct may be linked to the scaffold by with a first hinge linker, and the second Fc polypeptide is genetic fusion with or without polypeptide linkers . In other linked to the second antigen -binding polypeptide construct embodiments , where the scaffold is a polymer or nanopar with a second hinge linker. ticle , the antigen - binding construct may be linked to the 101171. The term “ Fc domain ” or “ Fc region ” herein is used scaffold by chemical conjugation . In some embodiments, the to define a C -terminal region of an immunoglobulin heavy scaffold is an albumin polypeptide or split albumin poly chain that contains at least a portion of the constant region . peptide. The use of split albumin polypeptides as scaffolds The term includes native sequence Fc regions and variant Fc for antigen -binding polypeptide constructs is fully described regions . Unless otherwise specified herein , numbering of in PCT/ CA2012 /050131 , PCT/ US2013 /050408 and PCT / amino acid residues in the Fc region or constant region is US2013 /050411 all of which are hereby incorporated by according to the EU numbering system , also called the EU reference in their entirety. index , as described in Kabat et al , Sequences of Proteins of US 2018 /0193477 A1 Jul. 12 , 2018 15

Immunological Interest , 5th Ed . Public Health Service , to 447 of the full - length human IgG1 heavy chain . Amino National Institutes of Health , Bethesda, Md. , 1991 . An “ Fc acids 231 - 238 are also referred to as the lower hinge . The polypeptide” of a dimeric Fc as used herein refers to one of CH3 sequence comprises amino acid 341 - 447 of the full the two polypeptides forming the dimeric Fc domain , i .e . a length human IgG1 heavy chain . polypeptide comprising C - terminal constant regions of an [0126 ] Typically an Fc can include two contiguous heavy immunoglobulin heavy chain , capable of stable self - asso chain sequences ( A and B ) that are capable of dimerizing. ciation . For example , an Fc polypeptide of a dimeric IgG Fc With respect to the antigen binding constructs described comprises an IgG CH2 and an IgG CH3 constant domain herein , in some embodiments the first scFv is linked to chain sequence . A of the heterodimeric Fc and the second scFv is linked to 10118 ] An Fc domain comprises either a CH3 domain or a chain B of the heterodimeric Fc . in some embodiments the CH3 and a CH2 domain . The CH3 domain comprises two second scFv is linked to chain A of the heterodimeric Fc and CH3 sequences , one from each of the two Fc polypeptides the first scFv is linked to chain B of the heterodimeric Fc . of the dimeric Fc . The CH2 domain comprises two CH2 0127 ] In some aspects, one or both sequences of an Fc sequences, one from each of the two Fc polypeptides of the include one or more mutations or modifications at the dimeric Fc. following locations: L351, F405 , Y407, T366 , K392 , T394 , [ 0119 ] In some aspects , the Fc comprises at least one or T350 , 5400 , and / or N390 , using EU numbering . In some two CH3 sequences . In some aspects, the Fc is coupled , with aspects , an Fc includes a mutant sequence shown in Table X . or without one or more linkers , to a first antigen - binding construct and / or a second antigen -binding construct . In In some aspects , an Fc includes the mutations of Variant 1 some aspects , the Fc is a human Fc. In some aspects , the Fc A - B . In some aspects , an Fc includes the mutations of is a human IgG or IgG1 Fc . In some aspects , the Fc is a Variant 2 A - B . In someaspects , an Fc includes the mutations heterodimeric Fc . In some aspects , the Fc comprises at least of Variant 3 A - B . In some aspects, an Fc includes the one or two CH2 sequences . mutations of Variant 4 A - B . In some aspects , an Fc includes [0120 ] In some aspects , the Fc comprises one or more the mutations of Variant 5 A - B . modifications in at least one of the CH3 sequences. In some TABLE C aspects , the Fc comprises one or more modifications in at least one of the CH2 sequences. In some aspects , an Fc is a IgG1 Fc sequence and variants single polypeptide . In some aspects , an Fc is multiple Human IgG1 FC APELLGGPSVFLFPPKPKDTLMIS peptides, e . g . , two polypeptides . sequence 231 - 447 RTPEVTCVVDVSHEDPEVKFNWY [0121 ] In some aspects , the Fc is an Fc described in patent ( EU - numbering ) VDGVEVHNAKTKPREEQYNSTYRV applications PCT/ CA2011 / 001238 , filed Nov. 4 , 2011 or VSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYT PCT/ CA2012 /050780 , filed Nov. 2 , 2012 , the entire disclo LPPSRDELTKNOVSLTCLVKGFYP sure of each of which is hereby incorporated by reference in SDIAVEWESNGOPENNYKTTPPVL its entirety for all purposes . DSDGSFFLYSKL TVDKSRWOOGNV [0122 ] Modified CH3 Domains FSCSVMHEALHNHYTOKSLSLSPG [ 0123 ] In some aspects , the antigen -binding construct K ( SEQ ID NO : 361 ) described herein comprises a heterodimeric Fc comprising a Variant modified CH3 domain that has been asymmetrically modi IgGi Fc fied . The heterodimeric Fc can comprise two heavy chain sequence constant domain polypeptides: a first Fc polypeptide and a ( 231 - 447 ) Chain Mutations second Fc polypeptide, which can be used interchangeably A L351Y _ F405A _ Y407V provided that Fc comprises one first Fc polypeptide and one second Fc polypeptide. Generally , the first Fc polypeptide B T366L _ K3 92M _ T394W comprises a first CH3 sequence and the second Fc polypep A tide comprises a second CH3 sequence . L351Y _ F405A _ Y407V [0124 ] Two CH3 sequences that comprise one or more B T366L _ K392L _ T394W amino acid modifications introduced in an asymmetric fash ion generally results in a heterodimeric Fc , rather than a A T350V _ L351Y _ F405A _ Y407V homodimer, when the two CH3 sequences dimerize . As used B T350V _ T366L _ K392 L _ T394W herein , “ asymmetric amino acid modifications” refers to any modification where an amino acid at a specific position on Hamm A T350V _ L351Y _ F405A _ Y407V a first CH3 sequence is different from the amino acid on a B T350V _ T366L _ K392M _ T394W second CH3 sequence at the same position , and the first and sesecond CH3 sequence preferentially pair to form a heterodi u A T350V _ L351Y _ S400E _ F405A _ Y407V

mer , rather than a homodimer. This heterodimerization can u be a result of modification of only one of the two amino B T350V _ T366L _ N390R _ K392M _ T394W acids at the same respective amino acid position on each sequence ; or modification of both amino acids on each [0128 ] The first and second CH3 sequences can comprise sequence at the same respective position on each of the first amino acid mutations as described herein , with reference to and second CH3 sequences. The first and second CH3 amino acids 231 to 447 of the full -length human IgG1 heavy sequence of a heterodimeric Fc can comprise one or more chain . In one embodiment, the heterodimeric Fc comprises than one asymmetric amino acid modification . a modified CH3 domain with a first CH3 sequence having [ 0125 ] Table C provides the amino acid sequence of the amino acid modifications at positions F405 and Y407 , and a human IgG1 Fc sequence , corresponding to amino acids 231 second CH3 sequence having amino acid modifications at US 2018 /0193477 A1 Jul. 12 , 2018 16 position T394 . In one embodiment, the heterodimeric Fc which the heterodimeric CH3 domain has a stability that is comprises a modified CH3 domain with a first CH3 comparable to a wild - type homodimeric CH3 domain . In an sequence having one or more amino acid modifications embodiment, the one or more asymmetric amino acid modi selected from L351Y , F405A , and Y407V , and the second fications promote the formation of a heterodimeric Fc CH3 sequence having one or more amino acid modifications domain in which the heterodimeric Fc domain has a stability selected from T366L , T3661, K392L , K392M , and T394WW . that is comparable to a wild -type homodimeric Fc domain . [ 0129 ] In one embodiment, a heterodimeric Fc comprises In an embodiment, the one or more asymmetric amino acid a modified CH3 domain with a first CH3 sequence having modifications promote the formation of a heterodimeric Fc amino acid modifications at positions L351, F405 and Y407 , domain in which the heterodimeric Fc domain has a stability and a second CH3 sequence having amino acid modifica observed via the melting temperature ( Tm ) in a differential tions at positions T366 , K392 , and T394 , and one of the first scanning calorimetry study , and where the melting tempera or second CH3 sequences further comprising amino acid ture is within 4° C . of that observed for the corresponding modifications at position Q347 , and the other CH3 sequence symmetric wild - type homodimeric Fc domain . In some further comprising amino acid modification at position aspects , the Fc comprises one or more modifications in at K360. In another embodiment, a heterodimeric Fc comprises least one of the CH3 sequences that promote the formation a modified CH3 domain with a first CH3 sequence having of a heterodimeric Fc with stability comparable to a wild amino acid modifications at positions L351, F405 and Y407 , type homodimeric Fc . and a second CH3 sequence having amino acid modifica [0134 ] In one embodiment, the stability of the CH3 tions at position T366 , K392 , and T394 , one of the first or domain can be assessed by measuring the melting tempera second CH3 sequences further comprising amino acid modi ture of the CH3 domain , for example by differential scan fications at position Q347 , and the other CH3 sequence ning calorimetry (DSC ) . Thus , in a further embodiment, the further comprising amino acid modification at position CH3 domain has a melting temperature of about 68° C . or K360 , and one or both of said CH3 sequences further higher. In another embodiment, the CH3 domain has a comprise the amino acid modification T350V . melting temperature of about 70° C . or higher. In another [0130 ] In one embodiment, a heterodimeric Fc comprises embodiment, the CH3 domain has a melting temperature of a modified CH3 domain with a first CH3 sequence having about 72° C . or higher. In another embodiment, the CH3 amino acid modifications at positions L351 , F405 and Y407 , domain has a melting temperature of about 73° C . or higher . and a second CH3 sequence having amino acid modifica In another embodiment, the CH3 domain has a melting tions at positions T366 , K392 , and T394 and one of said first temperature of about 75° C . or higher. In another embodi and second CH3 sequences further comprising amino acid ment, the CH3 domain has a melting temperature of about modification of D399R or D399K and the other CH3 78° C . or higher . In some aspects , the dimerized CH3 sequence comprising one or more of T411E , T411D , K409E , sequences have a melting temperature ( Tm ) of about 68 , 69 , K409D , K392E and K392D . In another embodiment, a 70 , 71, 72 , 73 , 74 , 75 , 76 , 77, 77 .5 , 78 , 79, 80 , 81 , 82, 83 , heterodimeric Fc comprises a modified CH3 domain with a 84, or 85° C . or higher . first CH3 sequence having amino acid modifications at 10135 ] In some embodiments , a heterodimeric Fc com positions L351 , F405 and Y407 , and a second CH3 sequence prising modified CH3 sequences can be formed with a purity having amino acid modifications at positions T366 , K392 , of at least about 75 % as compared to homodimeric Fc in the and T394 , one of said first and second CH3 sequences expressed product . In another embodiment, the heterodi further comprises amino acid modification of D399R or meric Fc is formed with a purity greater than about 80 % . In D399K and the other CH3 sequence comprising one or more another embodiment, the heterodimeric Fc is formed with a of T411E , T411D , K409E , K409D , K392E and K392D , and purity greater than about 85 % . In another embodiment, the one or both of said CH3 sequences further comprise the heterodimeric Fc is formed with a purity greater than about amino acid modification T350V. 90 % . In another embodiment , the heterodimeric Fc is 0131 ] In one embodiment, a heterodimeric Fc comprises formed with a purity greater than about 95 % . In another a modified CH3 domain with a first CH3 sequence having embodiment, the heterodimeric Fc is formed with a purity amino acid modifications at positions L351 , F405 and Y407 , greater than about 97 % . In some aspects, the Fc is a and a second CH3 sequence having amino acid modifica heterodimer formed with a purity greater than about 75 , 76 , tions at positions T366 , K392 , and T394 , wherein one or 77 , 78 , 79 , 80 , 81, 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91, 92, both of said CH3 sequences further comprise the amino acid 93, 94, 95 , 96 , 97 , 98, or 99 % when expressed . In some modification of T350V . aspects , the Fc is a heterodimer formed with a purity greater [0132 ] In one embodiment, a heterodimeric Fc comprises than about 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83, 84 , 85 , 86 , 87 , a modified CH3 domain comprising the following amino 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , or 99 % when acid modifications , where “ A ” represents the amino acid expressed via a single cell . modifications to the first CH3 sequence , and “ B ” represents [ 0136 ] Additional methods for modifying monomeric Fc the amino acid modifications to the second CH3 sequence : polypeptides to promote heterodimeric Fc formation are A : L351Y _ F405A _ Y407V , B : T366L _ K392M _ T394W , known in the art. For example , see International Patent A :L351Y F405A Y407V , B : T366L K392L _ T394W , Publication No. WO 96 /027011 (knobs into holes ), in A : T350V _ L351Y _ F405A _ Y407V , B : T350V _ T366L Gunasekaran et al. (Gunasekaran K . et al. ( 2010 ) J Biot K392L _ T394W , A : T350V _ L351Y _ F405A _ Y407V , Chem . 285 , 19637 - 46 , electrostatic design to achieve selec B : T350V _ T366L _ K392M _ T394W , A : T350V _ L351Y _ tive heterodimerization ), in Davis et al. (Davis , J H . et al . S400E _ F405A _ Y407V , and /or B : T350V _ T366L _N390R __ (2010 ) Prot Eng Des Set; 23 ( 4 ): 195 - 202 , strand exchange K392M _ T394W . engineered domain ( SEED ) technology ) , and in Labrijn et al [0133 ] The one or more asymmetric amino acid modifi - [Efficient generation of stable bispecific IgG1 by controlled cations can promote the formation of a heterodimeric Fc in Fab - arm exchange . Labrijn A F , Meesters JI, de Goeij B E , US 2018 /0193477 A1 Jul. 12 , 2018 17 van den Bremer E T , Neijssen J , van Kampen MD, each of the two Fc polypeptides of the dimeric Fc . Typically , Strumane K , Verploegen S , Kundu A , Cramer M J , van the modifications to the CH2 domain are symmetric and are Berkel P H , van de Winkel J G , Schuurman J , Parren P W . thus the same on both CH2 sequences of the Fc polypep Proc Natl Acad Sci USA . 2013 Mar. 26 ; 110 ( 13 ) :5145 - 50 . tides. However, asymmetric mutations are also possible in [0137 ] CH2 Domains the presence of mutations on the CH3 domain that enhance [ 0138 ] As indicated above, in some embodiments , the Fc heterodimerization . In one embodiment, the CH2 domain of the antigen -binding construct comprises a CH2 domain in comprises modifications to reduce FcyR or Clq binding addition to a CH3 domain . As an example , the amino acid and / or effector function . sequence of the CH2 domain of an IgG1 Fc is identified as amino acids 239- 340 of the sequence shown in Table A . The Modifications to Reduce Effector Function : CH2 domain of the Fc binds to Fc receptors and complement [0144 ] Fc modifications reducing FcyR and /or comple and is thus involved in mediating effector cell functions . ment binding and /or effector function are known in the art . [0139 ] The terms “ Fc receptor” and “ FcR ” are used to Recent publications describe strategies that have been used describe a receptor that binds to the Fc region of an antibody, to engineer antibodies with reduced or silenced effector and includes Fc gamma receptors ( FcyRs ) and the neonatal activity ( see Strohl, WR ( 2009 ) , Curr Opin Biotech 20 : 685 receptor FcRn . 691 , and Strohl , W R and Strohl L M , “ Antibody Fc [ 0140 ) Generally , an FcyR is one which binds an IgG engineering for optimal antibody performance ” In Thera antibody ( a gamma receptor ) and includes receptors of the peutic Antibody Engineering, Cambridge : Woodhead Pub FcyRI, FcyRII , and FcyRIII subclasses in humans, including lishing (2012 ) , pp 225 -249 ) . These strategies include reduc allelic variants and alternatively spliced forms of these tion of effector function through modification of receptors. FcyRII receptors include FcyRIIA (an “ activating glycosylation , use of IgG2/ IgG4 scaffolds, or the introduc receptor” ) and FcyRIIB (an “ inhibiting receptor” ), which tion of mutations in the hinge or CH2 regions of the Fc . For have similar amino acid sequences that differ primarily in example , US Patent Publication No . 2011 /0212087 ( Strohl) , the cytoplasmic domains thereof. Immunoglobulins of other International Patent Publication No . WO 2006 / 105338 (Xe isotypes can also be bound by certain FcRs ( see , e . g . , ncor ), US Patent Publication No . 2012 /0225058 (Xencor ), Janeway et al. , Immuno Biology : the immune system in US Patent Publication No . 2012 / 0251531 (Genentech ) , and health and disease, (Elsevier Science Ltd . , NY ) (4th ed . , Strop et al ( 2012 ) J . Mol. Biol. 420 : 204 - 219 ) describe 1999 ) ) . Activating receptor FcyRIIA contains an immuno specific modifications to reduce FcyR or complement bind receptor tyrosine -based activation motif (ITAM ) in its cyto ing to the Fc . plasmic domain . Inhibiting receptor FcYRIIB contains an [0145 ] Specific , non - limiting examples of known symmet immunoreceptor tyrosine -based inhibition motif (ITIM ) in ric amino acid modifications to reduce FcyR or complement its cytoplasmic domain reviewed in Daeron , Annu . Rev . binding to the Fc include those identified in the following Immunol. 15 : 203 - 234 ( 1997 ) ). FcRs are reviewed in table : Ravetch and Kinet , Annu . Rev . Immunol 9 : 457 - 92 ( 1991 ); Capel et al. , Immunomethods 4 : 25 - 34 ( 1994 ) ; and de Haas TABLE D et al ., J . Lab . Clin . Med . 126 : 330 -41 ( 1995 ) . Other FcyRs, including those to be identified in the future , are encom modifications to reduce FcYR or complement binding to the Fc passed by the term “ FcR ” herein . An FcyR are also found in other organisms, including but not limited to mice , rats , Company Mutations rabbits , and monkeys . Mouse FcyRs include but are not GSK N297A limited to FcyRI (CD64 ) , FcyRII (CD32 ), FcyRIII (CD 16 ) , Ortho Biotech L234A / L235A Protein Design labs IGG2 V234A /G237A and FcyRIII - 2 (CD 16 - 2 ) . FcyRs are expressed by effector Wellcome Labs IGG4 L235A /G237A / E318A cells such as NK cells or B cells . GSK IGG4 S228P / L236E [ 0141] Complement activation requires binding of the Alexion IGG2/ IgG4 combination complement protein Clq to antigen - antibody complexes . Merck IGG2 H268Q /V309L / A330S / A331S Bristol- Myers C220S / C226S/ C229S/ P238S Residues in the CH2 domain of the Fc are involved in the Seattle Genetics C226S/ C229S /E3233P /L235V /L235A interaction between Clq and the Fc . Amgen E . coli production , non glycosylated [ 0142 ] Some of the antigen -binding constructs described Medimune L234F /L235E /P331S herein are able to bind FcRn . As is known in the art , binding Trubion Hinge mutant, possibly C226S /P230S to FcRn recycles endocytosed antibody from the endosome back to the bloodstream (Raghavan et al. , 1996 , Annu Rev [014 ] In one embodiment , the Fc comprises at least one Cell Dev Biol 12 : 181 - 220 ; Ghetie et al. , 2000 , Annu Rev amino acid modification identified in the above table . In Immunol 18 : 739 - 766 ) . This process , coupled with preclu another embodiment the Fc comprises amino acid modifi sion of kidney filtration due to the large size of the full cation of at least one of L234 , L235 , or D265 . In another length molecule , results in favorable antibody serum half embodiment, the Fc comprises amino acid modification at lives ranging from one to three weeks . Binding of Fc to L234 , L235 and D265 . In another embodiment, the Fc FcRn also plays a key role in antibody transport . FcRn is comprises the amino acid modifications L234A , L235A and responsible for the transfer of maternal IgGs to the fetus D265S . (Guyer et al. , J . Immunol. 117 :587 ( 1976 ) ; and Kim et al. , [0147 ] In some embodiments the Fc comprises one or J . Immunol . 24 :249 ( 1994 ) ). Binding of the FcRn to IgG more asymmetric amino acid modifications in the lower involves residues in the CH2 and CH3 domains of the Fc . hinge region of the Fc as described in International Patent 10143] Modifications in the CH2 domain can affect the Application No. PCT /CA2014 /050507 . Examples of such binding of FcRs to the Fc . As indicated above , the CH2 asymmetric amino acid modifications that reduce Feyr domain of the Fc comprises two CH2 sequences, one on binding are shown in Table E : US 2018 /0193477 A1 Jul. 12 , 2018

TABLE E TABLE F - continued Asymmetric mutations that reduce Feyr binding Hinge linker polypeptide sequences ( SEO ID NOS : ) Chain A Chain B SEQ L234D /L235E L234K / L235K ID NO : E233A / L234D /L235E E233A / L234R /L235R L234D /L235E E233K / L234R / L235R 1003 Hinge - 3 GTCPPCP E233A / L234K / L235A E233K / L234A /L235K 1004 Hinge - 3 GGCACATGCCCTCCATGTCCA Hinge Linkers [ 0148 ] In the antigen -binding constructs described herein , Dissociation Constant (KD ) and Maximal Binding (Bmax ) the first Fc polypeptide is linked to the first antigen -binding [ 0150 ] In some embodiments , an antigen -binding con polypeptide construct with a first hinge linker , and the struct is described by functional characteristics including but second Fc polypeptide is linked to the second antigen not limited to a dissociation constant and a maximal binding . binding polypeptide construct with a second hinge linker. 10151] The term " dissociation constant ( K ) ) ” as used Examples of hinge linker sequences are well -known to one herein , is intended to refer to the equilibrium dissociation of skill in the art and can be used in the antigen - binding constant of a particular ligand -protein interaction . As used constructs described herein . Alternatively, modified versions herein , ligand - protein interactions refer to , but are not lim of known hinge linkers can be used . ited to protein -protein interactions or antibody - antigen inter [ 0149 ] The hinge linker polypeptides are selected such actions. The Ky measures the propensity of two proteins that they maintain or optimize the functional activity of the ( e . g . AB ) to dissociate reversibly into smaller components antigen - binding construct . Suitable linker polypeptides ( A + B ), and is define as the ratio of the rate of dissociation , include IgG hinge regions such as, for example those from also called the " off- rate ( k . ) ” , to the association rate , or IgG , IgG2, or IgG4, including the upper hinge sequences “ on -rate (kon ) " . Thus, K , equals kokon and is expressed as and core hinge sequences. The amino acid residues corre a molar concentration (M ). It follows that the smaller the sponding to the upper and core hinge sequences vary Kp , the stronger the affinity of binding . Therefore , a Ky of depending on the IgG type , as is known in the art and one 1 mM indicates weak binding affinity compared to a K , of of skill in the art would readily be able to identify such 1 nM . Ky values for antigen -binding constructs can be sequences for a given IgG type . Modified versions of these determined using methods well established in the art . One exemplary linkers can also be used . For example , modifi method for determining the Ky of an antigen -binding con cations to improve the stability of the IgG4 hinge are known struct is by using surface plasmon resonance ( SPR ), typi in the art (see for example , Labrijn et al . ( 2009 ) Nature cally using a biosensor system such as a Biacore® system . Biotechnology 27 , 767 -771 ) . Examples of hinge linker Isothermal titration calorimetry (ITC ) is anothermethod that sequences are found in the following Table . In some can be used to determine . embodiments , the drug - conjugated antigen -binding con 10152 ] The term “ Bmax ” , or maximal binding , refers to structs described herein have modifications to the hinge the maximum antigen -binding construct binding level on the region to modify or optimize potency of the construct. cells at saturating concentrations of antigen -binding con struct. This parameter can be reported in the arbitrary unit TABLE F MFI for relative comparison , or converted into an absolute value corresponding to the number of antigen -binding con Hinge linker polypeptide sequences structs bound to the cell with the use of a standard curve . ( SEO ID NOS : ) [0153 ] The binding characteristics of an antigen - binding SEO construct can be determined by various techniques . One of ID NO : which is the measurement of binding to target cells express 995 IqG1 ???scDKTHTCPPCP ing the antigen by flow cytometry (FACS , Fluorescence activated cell sorting ) . Typically , in such an experiment, the 996 IgG1 GAGCCCAAGAGCTGTGATAAGACCC target cells expressing the antigen of interest are incubated ACAccTGccc?cccTGTcCA with antigen -binding constructs at different concentrations , 997 V1661 AAEPKSSDKTHTCPPCP washed , incubated with a secondary agent for detecting the antigen -binding construct, washed , and analyzed in the flow 998 v1661 GCAGCCGAACCCAAATCCTCTGATA cytometer to measure themedian fluorescent intensity (MFI ) AGACCCACACATGCcc??CATGTcc representing the strength of detection signal on the cells , which in turn is related to the number of antigen - binding 999 Hinge - 1 ???ssDKTHTCPPCP constructs bound to the cells . The antigen -binding construct 1000 Hinge - 1 GAGCCTAAAAGCTCCGACAAGACCC concentration vs . MFI data is then fitted into a saturation ??????ccccACCTTGTccG binding equation to yield two key binding parameters , Bmax and apparent Kp . 1001 Hinge - 2 DKTHTCPPCP [0154 ] Apparent Kp, or apparent equilibrium dissociation 1002 Hinge - 2 GACAAGACCCACACATGCCCACCTT constant , represents the antigen - binding construct concen GTcCG tration at which half maximal cell binding is observed . Evidently , the smaller the K , value , the smaller antigen binding construct concentration is required to reach maxi US 2018 /0193477 A1 Jul. 12 , 2018 mum cell binding and thus the higher is the affinity of the an amino acid sequence comprising the VL of the antigen antigen - binding construct. The apparent Ky is dependent on binding polypeptide construct and a second vector compris the conditions of the cell binding experiment, such as ing a nucleic acid that encodes an amino acid sequence different receptor levels expressed on the cells and incuba comprising the VH of the antigen - binding polypeptide con tion conditions, and thus the apparent K , is generally struct . In one embodiment, the host cell is eukaryotic , e. g . a different from the K , values determined from cell -free Chinese Hamster Ovary (CHO ) cell , or human embryonic molecular experiments such as SPR and ITC . However, kidney (HEK ) cell , or lymphoid cell ( e . g . , YO, NSO , Sp20 there is generally good agreement between the different cell ) . In one embodiment, a method of making an antigen methods. binding construct is provided , wherein the method com [ 0155 ] In some embodiments of a drug- conjugated anti prises culturing a host cell comprising nucleic acid encoding gen - binding construct described herein , one antigen - binding the antigen -binding construct , as provided above , under polypeptide construct has a higher affinity for its cognate conditions suitable for expression of the antigen -binding antigen than the other . In most embodiments of a drug construct, and optionally recovering the antigen -binding conjugated antigen - binding construct, the first antigen -bind construct from the host cell (or host cell culture medium ). ing polypeptide construct has a lower affinity for CD3 than [0159 ] For recombinant production of the antigen -binding the second antigen -binding polypeptide construct has for the construct , a nucleic acid encoding an antigen -binding con target antigen . In some embodiments , the construct has at struct, e . g ., as described above , is isolated and inserted into least 2 , at least 5 , at least 10 , at least 20 , at least 30 , at least one or more vectors for further cloning and / or expression in 40 , at least 50 , at least 60 , at least 70 , at least 80 , at least 90 a host cell . Such nucleic acid may be readily isolated and or at least 100 - fold lower affinity for the CD3 antigen than sequenced using conventional procedures ( e . g . , by using for the target antigen , as measured by SPR ; and / or has an an oligonucleotide probes that are capable of binding specifi affinity of less than 10 nM for target cells bearing the target cally to genes encoding the heavy and light chains of the antigen and an affinity in the range of 10 nM - 500 nM for T antigen -binding construct) . cells as measured by FACS . [0156 ] In many embodiments , the affinity for CD3 will be [0160 ] Suitable host cells for cloning or expression of lower than the affinity for the target antigen . In one embodi antigen -binding construct -encoding vectors include pro ment of a CD3 -CD19 drug - conjugated antigen -binding con karyotic or eukaryotic cells described herein . struct, the affinity for CD3 is lower than the affinity for [0161 ] A “ recombinant host cell” or “ host cell ” refers to a CD19 . In further embodiments , the affinity for CD3 is at cell that includes an exogenous polynucleotide , regardless of least 2 , 5 , 10 , 15 or 20 - fold lower than the affinity for CD19 . the method used for insertion , for example , direct uptake , In one specific embodiment, the affinity of a CD3- CD19 transduction , f -mating , or other methods known in the art to drug -conjugated antigen -binding construct is 2 nM for create recombinant host cells . The exogenous polynucle CD19 and 30 nM for CD3 . Affinities may be determined by otide may be maintained as a nonintegrated vector, for SPR . In some embodiments the affinity of the second example , a plasmid , or alternatively , may be integrated into antigen - binding polypeptide construct for CD19 antigen the host genome. expressed on a B cell is in the range of about 0 . 5 - 1 , 1 - 3 , 3 - 5 , [0162 ] As used herein , the term “ eukaryote ” refers to 5 - 7 , 7 - 9 , 9 - 11, 11 - 13 , 13 - 15 , 15 - 17 , 17 - 19 or 19 - 21 nM , and organisms belonging to the phylogenetic domain Eucarya the affinity of the first antigen -binding polypeptide construct such as animals ( including but not limited to , mammals , for CD3 expressed on a T cell is in the range of about 5 - 10 , insects , reptiles , birds , etc . ), ciliates, plants ( including but 10 - 15 , 15 - 20 , 20 - 15 , 25 - 30 , 30 - 35 , 35 - 40 , 40 - 50 , 50 - 55 , not limited to , monocots, dicots, algae , etc . ) , fungi, yeasts , 55 - 60 , 60 - 70 , 70 -80 , 80 - 90 or 90 - 100 nm , as determined by flagellates, microsporidia , protists , etc . FACS analysis . [0163 ] As used herein , the term “ prokaryote ” refers to prokaryotic organisms. For example , a non - eukaryotic Methods of Preparation of Antigen -Binding Constructs organism can belong to the Eubacteria ( including but not [0157 ] Antigen -binding constructs described herein may limited to , Escherichia coli , Thermus thermophilus , Bacillus be produced using recombinant methods and compositions , stearothermophilus, Pseudomonas fluorescens, Pseudomo e .g ., as described in U .S . Pat. No . 4 ,816 , 567 . nas aeruginosa , Pseudomonas putida , etc . ) phylogenetic [ 0158 ] In one embodiment, an isolated nucleic acid encod domain , or the Archaea ( including but not limited to , Metha ing an antigen -binding construct described herein is pro nococcus jannaschii , Methanobacterium thermoautotrophi vided . Such nucleic acid may encode an amino acid cum , Halobacterium such as Haloferax volcanii and sequence comprising the VL and /or an amino acid sequence Halobacterium species NRC - 1 , Archaeoglobus fulgidus, comprising the VH of the antigen -binding construct ( e . g ., Pyrococcus furiosus, Pyrococcus horikoshii , Aeuropyrum the light and / or heavy chains of the antigen -binding con pernix , etc .) phylogenetic domain . struct ) . In a further embodiment, one or more vectors ( e . g ., [0164 ] For example , antigen - binding constructs may be expression vectors ) comprising such nucleic acid are pro produced in bacteria , in particular when glycosylation and vided . In one embodiment, the nucleic acid is provided in a Fc effector function are not needed . For expression of multicistronic vector. In a further embodiment, a host cell antigen - binding construct fragments and polypeptides in comprising such nucleic acid is provided . In one such bacteria, see , e . g . , U . S . Pat . Nos. 5 ,648 ,237 , 5 , 789 ,199 , and embodiment, a host cell comprises ( e .g . , has been trans 5 ,840 ,523 . (See also Charlton , Methods in Molecular Biol formed with ): (1 ) a vector comprising a nucleic acid that ogy , Vol. 248 ( B . K . C . Lo , ed . , Humana Press, Totowa , N . J ., encodes an amino acid sequence comprising the VL of the 2003 ) , pp . 245 - 254 , describing expression of antibody frag antigen - binding construct and an amino acid sequence com ments in E . coli. ) After expression , the antigen - binding prising the VH of the antigen -binding polypeptide construct, construct may be isolated from the bacterial cell paste in a or (2 ) a first vector comprising a nucleic acid that encodes soluble fraction and can be further purified . US 2018 /0193477 A1 Jul. 12 , 2018 20

[0165 ] In addition to prokaryotes, eukaryotic microbes ods also include electrophoretic , immunological, precipita such as filamentous fungi or yeast are suitable cloning or tion , dialysis , and chromatofocusing techniques . Ultrafiltra expression hosts for antigen -binding construct - encoding tion and diafiltration techniques , in conjunction with protein vectors, including fungi and yeast strains whose glycosy concentration , are also useful. As is well known in the art , lation pathways have been “ humanized , ” resulting in the a variety of natural proteins bind Fc and antibodies, and production of an antigen -binding construct with a partially these proteins can find use in the present invention for or fully human glycosylation pattern . See Gerngross, Nat. purification of antigen - binding constructs . For example , the Biotech . 22 : 1409 - 1414 ( 2004 ) , and Li et al. , Nat. Biotech . bacterial proteins A and G bind to the Fc region . Likewise , 24 :210 - 215 ( 2006 ). the bacterial protein L binds to the Fab region of some [ 0166 ] Suitable host cells for the expression of glycosy antibodies . Purification can often be enabled by a particular lated antigen -binding constructs are also derived from mul fusion partner. For example , antibodies may be purified ticellular organisms ( invertebrates and vertebrates ). using glutathione resin if a GST fusion is employed , Ni+ 2 Examples of invertebrate cells include plant and insect cells . affinity chromatography if a His -tag is employed , or immo Numerous baculoviral strains have been identified which bilized anti - flag antibody if a flag - tag is used . For general may be used in conjunction with insect cells , particularly for guidance in suitable purification techniques , see , e . g . incor transfection of Spodoptera frugiperda cells. porated entirely by reference Protein Purification : Principles 0167] Plant cell cultures can also be utilized as hosts . See , and Practice, 3rd Ed . , Scopes , Springer - Verlag , NY, 1994 , e . g ., U . S . Pat. Nos. 5 ,959 , 177 , 6 ,040 ,498 , 6 ,420 ,548 , 7 ,125 , incorporated entirely by reference . The degree of purifica 978 , and 6 ,417 , 429 (describing PLANTIBODIESTM tech tion necessary will vary depending on the use of the antigen nology for producing antigen - binding constructs in trans binding constructs . In some instances no purification is genic plants ) . necessary . 10168 ] Vertebrate cells may also be used as hosts . For [0171 ] In certain embodiments the antigen -binding con example , mammalian cell lines that are adapted to grow in structs are purified using Anion Exchange Chromatography suspension may be useful . Other examples of useful mam including, but not limited to , chromatography on Q - sephar malian host cell lines are monkey kidney CV1 line trans ose , DEAE sepharose, poros HQ , poros DEAF , Toyopearl Q , formed by SV40 (COS - 7 ) ; human embryonic kidney line Toyopearl QAE , Toyopearl DEAE , Resource / Source Q and ( 293 or 293 cells as described , e . g . , in Graham et al ., J . Gen DEAE , Fractogel Q and DEAE columns. Virol. 36 : 59 ( 1977 ) ); baby hamster kidney cells (BHK ) ; [ 0172 ] In specific embodiments the proteins described mouse sertoli cells ( TM4 cells as described , e. g ., in Mather, herein are purified using Cation Exchange Chromatography Biol. Reprod. 23 : 243 -251 (1980 ) ) ; monkey kidney cells including , but not limited to , SP - sepharose , CM sepharose , (CV1 ) ; African green monkey kidney cells (VERO - 76 ) ; poros HS , poros CM , Toyopearl SP, Toyopearl CM , human cervical carcinoma cells (HELA ) ; canine kidney Resource / Source S and CM , Fractogel S and CM columns cells (MDCK ; buffalo rat liver cells ( BRL 3A ) ; human lung and their equivalents and comparables . cells (W138 ); human liver cells (Hep G2) ; mouse mammary [ 0173 ] In addition , antigen - binding constructs described tumor (MMT 060562 ) ; TRI cells , as described , e . g . , in herein can be chemically synthesized using techniques Mather et al. , Annals N . Y . Acad . Sci. 383 : 44 - 68 ( 1982 ) ; known in the art ( e . g . , see Creighton , 1983 , Proteins : Struc MRC 5 cells ; and FS4 cells . Other useful mammalian host tures and Molecular Principles, W . H . Freeman & Co . , N . Y cell lines include Chinese hamster ovary (CHO ) cells , and Hunkapiller et al. , Nature , 310 : 105 - 111 ( 1984 )) . For including DHFR - CHO cells (Urlaub et al . , Proc. Natl. example , a polypeptide corresponding to a fragment of a Acad . Sci. USA 77 :4216 ( 1980 )) ; and myeloma cell lines polypeptide can be synthesized by use of a peptide synthe such as YO , NSO and Sp2/ 0 . For a review of certain mam sizer . Furthermore , if desired , nonclassical amino acids or malian host cell lines suitable for antigen -binding construct chemical amino acid analogs can be introduced as a substi production , see , e . g ., Yazaki and Wu , Methods in Molecular tution or addition into the polypeptide sequence . Non Biology, Vol. 248 (B . K . C . Lo , ed ., Humana Press, Totowa, classical amino acids include , but are not limited to , to the N . J .) , pp . 255 - 268 (2003 ) . D - isomers of the common amino acids, 2 , 4diaminobutyric [0169 ] In one embodiment, the antigen - binding constructs acid , alpha - amino isobutyric acid , 4aminobutyric acid , Abu , described herein are produced in stable mammalian cells , by 2 -amino butyric acid , g - Abu , e - Ahx , 6amino hexanoic acid , a method comprising : transfecting at least one stable mam Aib , 2 -amino isobutyric acid , 3 - amino propionic acid , orni malian cell with : nucleic acid encoding the antigen -binding thine, norleucine, norvaline, hydroxyproline , sarcosine, cit construct, in a predetermined ratio ; and expressing the rulline , homocitrulline , cysteic acid , t -butylglycine , t -buty nucleic acid in the at least one mammalian cell. In some lalanine, phenylglycine , cyclohexylalanine , ]- alanine , embodiments , the predetermined ratio of nucleic acid is fluoro - amino acids, designer amino acids such as ] -methyl determined in transient transfection experiments to deter amino acids, C ]- methyl amino acids, N ]- methyl amino mine the relative ratio of input nucleic acids that results in acids, and amino acid analogs in general. Furthermore , the the highest percentage of the antigen -binding construct in amino acid can be D (dextrorotary ) or L (levorotary ). the expressed product. [0174 ] In some embodiments , the antigen - binding con [0170 ] If required , the antigen - binding constructs can be structs described herein are substantially purified . The term purified or isolated after expression . Proteins may be iso " substantially purified ” refers to a construct described lated or purified in a variety of ways known to those skilled herein , or variant thereof that may be substantially or in the art . Standard purification methods include chromato essentially free of components that normally accompany or graphic techniques , including ion exchange , hydrophobic interact with the protein as found in its naturally occurring interaction , affinity , sizing or gel filtration , and reversed environment, i . e . a native cell, or host cell in the case of phase , carried out at atmospheric pressure or at high pressure recombinantly produced antigen -binding construct that in using systems such as FPLC and HPLC . Purification meth certain embodiments , is substantially free of cellular mate US 2018 /0193477 A1 Jul. 12 , 2018 rial includes preparations of protein having less than about modifications of N - linked or O - linked carbohydrate chains , 30 % , less than about 25 % , less than about 20 % , less than and addition or deletion of an N - terminal methionine residue about 15 % , less than about 10 % , less than about 5 % , less as a result of procaryotic host cell expression . The antigen than about 4 % , less than about 3 % , less than about 2 % , or binding constructs described herein are modified with a less than about 1 % (by dry weight) of contaminating protein . detectable label, such as an enzymatic , fluorescent, isotopic When the antigen - binding construct or variant thereof is or affinity label to allow for detection and isolation of the recombinantly produced by the host cells , the protein in protein . In certain embodiments, examples of suitable certain embodiments is present at about 30 % , about 25 % , enzyme labels include horseradish peroxidase , alkaline about 20 % , about 15 % , about 10 % , about 5 % , about 4 % , phosphatase , beta - galactosidase , or acetylcholinesterase ; about 3 % , about 2 % , or about 1 % or less of the dry weight examples of suitable prosthetic group complexes include of the cells . When the antigen -binding construct or variant streptavidin biotin and avidin /biotin , examples of suitable thereof is recombinantly produced by the host cells, the fluorescent materials include umbelliferone , fluorescein , protein , in certain embodiments, is present in the culture fluorescein isothiocyanate , rhodamine, dichlorotriazinylam medium at about 5 g / L , about 4 g / L , about 3 g / L , about 2 ine fluorescein , dansyl chloride or phycoerythrin ; an g / L , about 1 g / L , about 750 mg/ L , about 500 mg/ L , about example of a luminescent material includes luminol ; 250 mg/ L , about 100 mg/ L , about 50 mg/ L , about 10 mg/ L , examples of bioluminescent materials include luciferase , or about 1 mg/ L or less of the dry weight of the cells . In luciferin , and aequorin ; and examples of suitable radioactive certain embodiments , a “ substantially purified ” antigen material include iodine, carbon , sulfur, tritium , indium , binding construct produced by the methods described technetium , thallium , gallium , palladium , molybdenum , herein , has a purity level of at least about 30 % , at least about xenon , fluorine . 35 % , at least about 40 % , at least about 45 % , at least about 50 % , at least about 55 % , at least about 60 % , at least about [0181 ] In some embodiments , antigen -binding constructs 65 % , at least about 70 % , specifically , a purity level of at described herein are attached to macrocyclic chelators that least about 75 % , 80 % , 85 % , and more specifically , a purity associate with radiometal ions. level of at least about 90 % , a purity level of at least about [0182 ] In some embodiments , the antigen -binding con 95 % , a purity level of at least about 99 % or greater as structs described herein are modified by either natural pro determined by appropriate methods such as SDS/ PAGE cesses, such as post - translational processing , or by chemical analysis , RP -HPLC , SEC , and capillary electrophoresis . modification techniques which are well known in the art. In [ 0175 ] Post - Translational Modifications: certain embodiments , the same type of modification may be [0176 ] In certain embodiments antigen - binding constructs present in the same or varying degrees at several sites in a described herein are differentially modified during or after given polypeptide . In certain embodiments , polypeptides translation . from antigen -binding constructs described herein are [0177 ] The term “modified ," as used herein refers to any branched , for example, as a result of ubiquitination , and in changes made to a given polypeptide, such as changes to the some embodiments are cyclic , with or without branching . length of the polypeptide , the amino acid sequence , chemi Cyclic , branched , and branched cyclic polypeptides are a cal structure , co - translational modification , or post - transla result from posttranslation natural processes or made by tionalmodification of a polypeptide . The form “ (modified ) ” . synthetic methods . Modifications include acetylation , acy term means that the polypeptides being discussed are option lation , ADP - ribosylation , amidation , covalent attachment of ally modified , that is , the polypeptides under discussion can flavin , covalent attachment of a heme moiety , covalent be modified or unmodified . attachment of a nucleotide or nucleotide derivative , covalent [0178 ] The term “ post -translationally modified ” refers to attachment of a lipid or lipid derivative , covalent attachment any modification of a natural or non - natural amino acid that of phosphotidylinositol, cross - linking , cyclization , disulfide occurs to such an amino acid after it has been incorporated bond formation , demethylation , formation of covalent cross into a polypeptide chain . The term encompasses, by way of links, formation of cysteine , formation of pyroglutamate , example only , co - translational in vivo modifications , co formylation , gamma- carboxylation , glycosylation , GPI translational in vitro modifications ( such as in a cell - free anchor formation , hydroxylation , iodination , methylation , translation system ) , post - translational in vivo modifications , myristylation , oxidation , pegylation , proteolytic processing , and post - translational in vitro modifications . phosphorylation , prenylation , racemization , selenoylation , 0179 ]. In some embodiments , the modification is at least sulfation , transfer- RNA mediated addition of amino acids to one of : glycosylation , acetylation , phosphorylation , amida proteins such as arginylation , and ubiquitination . (See , for tion , derivatization by known protecting/ blocking groups, instance , PROTEINS STRUCTURE AND MOLECU proteolytic cleavage and linkage to an antibody molecule or LAR PROPERTIES , 2nd Ed . , T . E . Creighton , W . H . Free antigen - binding construct or other cellular ligand . In some man and Company, New York ( 1993 ) ; POST- TRANSLA embodiments , the antigen - binding construct is chemically TIONAL COVALENT MODIFICATION OF PROTEINS, modified by known techniques, including but not limited , to B . C . Johnson , Ed . , Academic Press , New York , pgs . 1 - 12 specific chemical cleavage by cyanogen bromide , trypsin , ( 1983 ) ; Seifter et al ., Meth . Enzymol . 182 :626 -646 ( 1990 ) ; chymotrypsin , papain , V8 protease , NaBH ; acetylation , Rattan et al. , Ann . N . Y . Acad . Sci. 663: 48 -62 (1992 )) . formylation , oxidation , reduction ; and metabolic synthesis [0183 ] In certain embodiments , antigen -binding con in the presence of tunicamycin . structs described herein are attached to solid supports, which [0180 ] Additional post - translational modifications of anti are particularly useful for immunoassays or purification of gen - binding constructs described herein include, for polypeptides that are bound by , that bind to , or associate example , N - linked or O - linked carbohydrate chains , pro with proteins described herein . Such solid supports include, cessing of N - terminal or C -terminal ends ) , attachment of but are not limited to , glass , cellulose , polyacrylamide , chemical moieties to the amino acid backbone , chemical nylon , polystyrene, polyvinyl chloride or polypropylene . US 2018 /0193477 A1 Jul. 12 , 2018

Functional Activity of Drug - Conjugated Antigen - Binding Jurkat T cells . In some embodiments the target antigen is Constructs and Assays to Measure Function CDH3 and the construct is internalized at least 10 -50 - fold [0184 ] The antigen - binding constructs described herein more into target cells than Jurkat T cells . In some embodi can be assayed for functional activity ( e . g . , biological activ ments the target antigen is CD19 and the construct is ity ) using or routinely modifying assays known in the art , as internalized at least 10 - 50 - fold more into target cells than well as assays described herein . Jurkat T cells . [ 0185 ] Methods of testing the biological activity of the [0188 ] The impact of the drug -conjugated antigen -binding antigen - binding constructs and drug - conjugated antigen constructs on T cells can also be assessed by culturing T binding constructs described herein can be measured by cells from human blood ( PBMC ) with the constructs, with or various assays as described in the Examples. Such methods without target cells bearing the target antigen , and analyzing include in vitro assays measuring T cell -mediated killing of the resulting T cell subpopulations in the culture using FACS target cells bearing the target antigen that is specifically to detect T cell surface markers PD1, CD4, CD , CD25 , bound by the second antigen -binding polypeptide construct. CD69 and CD45 . In some embodiments , the assay is carried For example , the killing of target cells bearing the relevant out as in Examples 14 , 15 or 16 . In some embodiments , the target antigens can be measured in cultures comprising construct does not increase the number of inhibitory PD1 + human whole blood , PBMCs, or PBMCs from which the B cells . In some embodiments, an anti- CD3- CD19 - drug con cells have been removed , referred to herein as “ PBMC - B ) as jugate causes less activation of inhibitory (PD - 1 + ) T cells a source of effector T cells . Such assays may also be carried than blinatumomab . out using purified T cell cultures . This type of assay detects [0189 ] In some embodiments , the drug -conjugated anti both T -cell mediated killing of target cells bearing the target gen -binding constructs display killing of Raji or Ramos antigen and any killing that occurs through internalization of tumor B cell with high potency , and killing of Jurkat tumor the drug - conjugated construct by the target cells . Thus in T cells with low potency. In some embodiments , the potency some embodiments described herein , the killing potency of of the drug - conjugated antigen -binding constructs is at least a drug - conjugated antigen -binding construct such as an 1 . 5 , 2 , 4 , 6 , 8 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 or more - fold anti - CD3 - CD19 , CD3 - CDH3 , CD3- HER2 , CD3 -HER3 OR higher on B cells than on T cells . In a specific embodiment, CD3- EGFR against a target cell bearing the target antigen is the potency of a CD3 - CD19 antigen - binding construct on observed to be higher than the reference unconjugated Ramos B cells is about 0 .5 nM , and the potency on T cells construct . In some embodiments , the drug - conjugated anti is about 23 nM . In some embodiments , the potency on target gen -binding constructs described herein display increased cells is at least 2 - fold higher than on T cells . In another Raji or Ramos tumor B cell killing compared to a reference embodiment, the potency on target cells is at least 4 - fold unconjugated antigen -binding construct having the same higher than on T cells . In another embodiment, the potency CDRs and binding affinity . on target cells is at least 6 - fold higher than on T cells . In [0186 ] The direct cytoxicity of a drug - conjugated antigen another embodiment, the potency on target cells is at least binding construct may be determined by culturing the con 8 - fold higher than on T cells . In another embodiment, the struct with a target cell bearing the target antigen to which potency on target cells is at least 10 - fold higher than on T the second antigen -binding polypeptide construct is cells . In another embodiment , the potency on target cells is directed . In some embodiments described herein , the target at least 15 - fold higher than on T cells . antigen is CD19 , HER2 , HER3, EGFR or CDH3, and the [0190 ] In some embodiments , the drug - conjugated anti cytoxicity is determined by culturing the construct with gen -binding construct have a cleavable linker and do not target cells bearing the relevant target antigen , for example , reduce the number of T cells in an assay compared to a as described in Example 22 . In this type of assay , it is reference construct with a non - cleavable linker or a refer possible to assess whether cell killing by means of internal ence construct that is not conjugated to a drug . ization of the drug , in the absence of any T -cell mediated [0191 ] The impact of the drug - conjugated antigen - binding killing . constructs on T cells can also be evaluated in vivo , as was [ 0187] The impact of the drug -conjugated antigen -binding done in Examples 18 and 19 . In some embodiments , the construct on T cells can be measured in several ways . The drug - conjugated antigen - binding constructs did not reduce internalization of the construct into T cells can be measured the number of circulating T cells or the number of splenic T by coupling a dye or other detectable agent to the construct , cells in a humanized NSG mouse when administered at and culturing it with T cells and monitoring the amount of doses ranging from 0 . 1 mg/ kg to 3 . 0 mg/ kg . In some dye that accumulates in the T cell , for example , as described embodiments the construct tested was an anti -CD3 -CD19 in Example 21, in which Jurkat T cells were used . This can drug conjugate . In some embodiments , the drug - conjugated be compared in the same experiment with the internalization antigen - binding construct does not substantially impact the of the construct into target cells bearing the target antigen . In some embodiments described herein , the internalization level of CD45 + /CD8 + T cells in the peripheral blood of of the drug - conjugated construct into T cells is lower than humanized NSG mice over a 5 -day period . into target cells bearing the target antigen . In some embodi [0192 ] In some embodiments , anti - CD3 - CD19 antigen ments , the internalization into T cells is at least 10 , 20 , 30 , binding constructs described herein are capable of synapse 40 , 50 , 60 , 70 , 80 , 90 or 100 - fold lower than internalization formation and bridging between CD19 + Raji B - cells and into target cells bearing target antigen . In some embodi Jurkat T - cells as assayed by FACS and/ or microscopy . In ments , the T cell is a Jurkat cell. In some embodiments the some embodiments , the drug -conjugated antigen -binding target antigen is EGFR and the construct is internalized at constructs described herein display less activation of inhibi least 10 - 50 - fold more into target cells than T cells . In some tory (PD - 1 + ) T cells than blinatumomab . embodiments the target antigen is EGFR and the construct [0193 ] In certain embodiments , the assays are those is internalized at least 10 -50 -fold more into target cells than described in the examples below . US 2018 /0193477 A1 Jul. 12 , 2018

[0194 ] In some embodiments , the functional characteris by means well -known in the art, such as, for example , tics of the bispecific antigen -binding constructs described reducing and non -reducing gel chromatography, protein herein are compared to those of a reference antigen -binding affinity chromatography, and affinity blotting . See generally , construct. The identity of the reference antigen -binding Phizicky et al. ,Microbiol . Rev. 59: 94 - 123 ( 1995 ) . In another construct depends on the functional characteristic being embodiment, the ability of physiological correlates of a measured or the distinction being made . For example , when antigen -binding construct protein to bind to a substrate ( s ) of comparing the functional characteristics of exemplary anti antigen -binding polypeptide constructs of the antigen -bind CD3 -CD19 bispecific antigen -binding constructs , the ref ing constructs described herein can be routinely assayed erence antigen -binding construct may be the anti CD19 using techniques known in the art. antibody HD37 and / or the anti CD3 antibodies OKT3 or teplizumab . In other embodiment , the reference antigen Antigen -Binding Construct Drug Conjugates (ADCs ) binding construct is a construct described herein , e . g . , 1891 [0198 ] In many embodiments provided herein antigen (blinatumomab ) or bivalent anti -CD19 ( v4371 ) . In some binding constructs are conjugated to a drug , e . g. , a toxin , a embodiments , the reference antigen - binding construct is the chemotherapeutic agent , a small molecule therapeutic , an same variant without a conjugated drug, for example, com immune modulator e . g . a cytokine, or a radioisotope . paring v12043 with v12043 conjugated to DM1 with an Numerousmethods of preparing ADCs (antibody drug con SMCC linker. jugates or antigen binding construct drug conjugates ) are [ 0195 ] The degree to which an antibody blocks binding to known in the art and are described in U . S . Pat. No . 8 ,624 , a reference antibody , for example , OKT3 or HD37 can be 003 (pot method ), U . S . Pat. No . 8 , 163 , 888 (one - step ) , and assessed using a competition assay in which the test anti U .S . Pat . No . 5 ,208 , 020 ( two - step method ) for example . body is able to inhibit or block specific binding of the OKT3 0199 ) In some embodiments , the drug is selected from a or HD37 antibody (reference antibody ) to its target antigen maytansine , auristatin , calicheamicin , or derivative thereof. ( see , e . g ., Junghans et al. , Cancer Res. 50 : 1495 , 1990 ; In other embodiments , the drug is a maytansine selected Fendly et al. Cancer Research 50 : 1550 - 1558 ; U . S . Pat. No . from DM1 and DM4. Further examples are described below . 6 , 949, 245 for examples of assays) . A test antibody competes [0200 ] In certain embodiments , the antigen binding con with a reference antibody if an excess of a test antibody ( e . g . , struct is conjugated to a drug via a linker. The linker may be at least 2x , 5x , 10x , 20x , or 100x ) inhibits or blocks binding cleavable or non - cleavable . Non - limiting examples of link of the reference antibody by , e . g ., at least 50 % , 60 % , 70 % , ers are described below . 75 % , 80 % , 85 % , 90 % , 95 % , or 99 % as measured in a [0201 ] In some embodiments , one molecule of drug is competitive binding assay . Test antibodies identified by conjugated to an antigen - binding construct, but in others , competition assay (blocking antibodies ) include those bind multiple drug molecules may be conjugated to the same ing to the same epitope as the reference antibody and antigen - binding construct . The drug - to - antigen binding con antibodies binding to an adjacent epitope sufficiently proxi struct ratio (DAR ) can be , e . g ., in the range of 1 . 0 to 6 . 0 , or mal to the epitope bound by the reference antibody for steric 3 . 0 to 5 . 0 , or 2 . 0 to 4 . 0 . In some embodiments described hindrance to occur. herein , the DAR ranges from 2 . 2 to 3 . 5 . In some embodi [ 01961. For example , in one embodiment where one is ments , the DAR is 1 . 0 , 2 . 0 , 3 . 0 , 4 . 0 , 5 . 0 , 6 . 0 , 7 . 0 or 8 . 0 . assaying for the ability of a antigen - binding construct [0202 ] In certain embodiments , the ADCs have the general described herein to bind an antigen or to compete with formula 1 : another polypeptide for binding to an antigen , or bind to an Fc receptor and / or anti- albumin antibody , various immuno A - ( L - ( D )m ) . ( I) assays known in the art can be used , including but not where A is an antigen binding construct as described herein ; limited to , competitive and non - competitive assay systems L is a linker ; Dis a drug ; m is an integer between 1 and about using techniques such as radioimmunoassays , ELISA ( en 10 , and n is an integer between 1 and about 20 . In certain zyme linked immunosorbent assay ) , " sandwich ” immuno embodiments , m is between about 1 and about 5 , or between assays , immunoradiometric assays, gel diffusion precipita 1 and 2 . In some embodiments , m is 1 . In some embodi tion reactions , immunodiffusion assays, in situ ments , n is between 1 and 10 , for example , between 1 and immunoassays (using colloidal gold , enzyme or radioisotope 8 , between 2 and 8 , between 2 and 6 , or between 2 and 4 . In labels , for example ) , western blots , precipitation reactions, some embodiments , L may be absent. agglutination assays ( e . g. , gel agglutination assays , hemag glutination assays ), complement fixation assays , immuno Drugs fluorescence assays, protein A assays, and immunoelectro [ 0203 ] The drug moiety of the ADCs may be a compound phoresis assays , etc . In one embodiment, antibody binding is or moiety having a cytostatic or cytotoxic effect. In some detected by detecting a label on the primary antibody. In embodiments the antigen - binding construct is conjugated to another embodiment, the primary antibody is detected by a cytotoxic agent. detecting binding of a secondary antibody or reagent to the [0204 ] The term “ cytotoxic agent” as used herein refers to primary antibody . In a further embodiment, the secondary a substance that inhibits or prevents the function of cells antibody is labeled . Many means are known in the art for and / or causes destruction of cells . The term is intended to detecting binding in an immunoassay and are within the include radioactive isotopes ( e . g . 211 At, 1311 , 1251, 90Y , scope of the present invention . 186Re, 188Re, 153Sm , 212Bi, 32P and 177Ln ), chemotherapeu [0197 ] In certain embodiments , where a binding partner tic agents , and toxins such as small molecule toxins or (e .g ., a receptor or a ligand ) is identified for an antigen enzymatically active toxins of bacterial, fungal, plant or binding domain comprised by a antigen -binding construct animal origin , including fragments and /or variants thereof. described herein , binding to that binding partner by an One skilled in the art will appreciate that some of these antigen - binding construct described herein is assayed , e . g . , categories of drugs overlap and are thus not intended to be US 2018 /0193477 A1 Jul. 12 , 2018 24. mutually exclusive . For example , toxins may also be con where Y is ( CR2) m - , each R is independently H or sidered as chemotherapeutic agents in the sense that they are C1- C6 alkyl, m is 1 , 2 or 3 , and www indicates the point of chemical compounds that may be used to treat cancer. In some embodiments, the drug is an analogue or derivative of attachment to linker L ( see U . S . Pat. No . 5 ,208 ,020 , a naturally occurring toxin . Examples of such naturally RE39151, WO 2007/ 056550 and Widdison et al ., (2006 ) J. occurring toxins include , but are not limited to ,maytansines , Med . Chem . , 49 :4392 - 4408 ) . auristatins , dolastatins, tubulysins , hemiasterlins, cali [0208 ] In some embodiments , the drug included in the cheamicins, duocarmycins, pyrrolobenzodiazapenes , ama ADC is a maytansinoid having the general formula ( II ) in toxins, camptothecins, Pseudomonas exotoxin (PE ) , diph which Y is – CH2CH2 – CH2CH2CH (CH3 ) - or theria toxin (DT ) , deglycosylated ricin A ( dgA ) and gelonin . _ CH , CH , C (CH ) , . All stereoisomers of the maytansine In some embodiments , the drug is an analogue or derivative of a naturally occurring toxin having a peptidyl scaffold . drug moiety are contemplated for the ADCs described Non - limiting examples of such toxins include auristatins , herein , i. e . any combination of R and S configurations at the dolastatins , tubulysins , hemiasterlins and amatoxins . chiral carbons . [ 0205 ) In certain embodiments , the drug comprised by the [0209 ] In some embodiments , the drug included in the ADCs is a toxin , or a toxin derivative or analogue, where the ADC is a maytansinoid having the following stereochemis toxin , derivative or analogue is a microtubule disrupting try ( general formula ( IIA ) ) : agent or a DNA modifying agent. Examples of toxins that are microtubule disrupting agents include , but are not lim ited to , maytansines , auristatins , dolastatins , tubulysins , ( IIA ) hemiasterlins , and analogues and derivatives thereof. Examples of toxins that are DNA modifying agents include , but are not limited to , calicheamicins and other enediyne Y — S antibiotics, duocarmycins, pyrrolobenzodiazapenes, ama toxins, camptothecins, and analogues and derivatives thereof. Maytansines ci H3C [0206 ] As indicated above, in some embodiments the drug III. is a maytansine or maytansine analogue or derivative (“ may tansinoid ” ) . Exemplary maytansinoids include DM1 (mer H3CO ** 111111 tansine , emtansine , N . - deacetyl- N _ ' - ( 3 -mercapto - 1 - oxopro pyl) maytansine ), DM3 ( N , '- deacetyl - N , '- ( 4 -mercapto - 1 oxopentyl) maytansine ) , and DM4 (ravtansine , soravtansine , N2 -deacetyl - N , '- ( 4 -methyl - 4 -mercapto - 1 - oxopentyl )may tansine ) (see U . S . Patent Publication No . US 2009 / 0202536 ). Other examples of naturally occurring , synthetic and semi-synthetic maytansinoids are described in Cassady et al. , ( 2004 ) Chem . Pharm . Bull . 52 ( 1 ): 1 - 26 , and in U . S . CHHO Pat . Nos. 4 , 256 , 746 ; 4 , 361, 650 ; 4 ,307 ,016 ; 4 ,294 ,757 ; 4 , 424 ,219 ; 4 ,331 , 598 ; 4 , 364, 866 ; 4 , 313 , 946 ; 4 ,315 , 929 ; 4 , 362 ,663 ; 4 , 322 , 348 and 4 , 371 ,533 . Many positions on where Y is as defined above for general formula ( II ). maytansine compounds are known to be useful as the [ 0210 ] In some embodiments , the drug included in the linkage position , depending upon the type of link . For ADC is a maytansinoid having the general formula (II ) or example, for forming an ester linkage, the C - 3 position (IIA ) in which Y is CH ,CH , — ( e . g . DM1) , having a hydroxyl group , the C - 14 position modified with CH2CH2CH ( CH3) - ( e . g . DM3) or — CH _ CH _ C ( CH3 ) hydroxymethyl, the C - 15 position modified with a hydroxyl 2 - ( e . g . DM4 ) . In some embodiments , the drug included in group and the C - 20 position having a hydroxyl group are all the ADC is a maytansinoid having the general formula ( II ) suitable . [0207 ] In certain embodiments , the drug included in the or ( IIA ) in which Y is CH CH — ( e. g. DM1) or ADC is a maytansinoid having the general formula ( II) : - CH2CH2C ( CH3) 2 - ( e . g . DM4) . Dolastatins and Auristatins maiE [0211 ] In some embodiments , the drug is a dolastatin or an HzCY — S auristatin , such as auristatin E ( also known in the art as a derivative of dolastatin - 10 ) or auristatin F , or an analogue or derivative thereof. The auristatin can be , for example , an ester formed between auristatin E and a keto acid . For H3C example , auristatin E can be reacted with paraacetyl benzoic Ci acid or benzoylvaleric acid to produce auristatin EB ( AEB ) and auristatin EVB ( AEVB ), respectively . Other typical H3CO auristatins include auristatin F phenylenediamine ( AFP ) , monomethylauristatin F (MMAF ), and monomethylaurista tin E (MMAE ) . The synthesis and structure of exemplary auristatins are described in U .S . Pat . Nos . 6 , 884 , 869 ; 7 , 098 , 308 ; 7 ,256 , 257 ; 7 ,423 , 116 ; 7 ,498 , 298 and 7 , 745 , 394 , each HO NH of which is incorporated by reference herein in its entirety CH30 and for all purposes . 10212 ] The dolastatin or auristatin may be conjugated to the antigen binding construct via the amino ( N ) - terminus or US 2018 /0193477 A1 Jul. 12 , 2018 25

the carboxy ( C ) - terminus of the drug molecule . In some C3- C , cycloalkyl , aryl , carboxamide , carboxyl, cyano , embodiments , the drug is an auristatin or analogue or C - C6 haloalkyl, C , -C6 haloalkoxy, halo , hydroxyl, nitro , derivative thereof and is conjugated to the antigen binding thio and thio - C1- C , alkyl; construct via the N - terminus of the drug molecule . X is C ( O )NHCH (CH _ R ) , or X is absent ; Examples of auristatin analogues suitable for conjugation R * is selected from aryl, heteroaryl and C3- C , cycloalkyl, via the N - terminus of the drug molecule include those each optionally substituted with one substituent selected described in U . S . Pat. Nos. 7 , 498 , 298 and 7 ,659 , 241 . from amino and hydroxyl, and 10213 ] In some embodiments , the drug is MMAE or R4 and RS are each independently H or C , - C , alkyl. MMAF. In some embodiments , the drug is MMAE or (0216 ) In the context of general formula ( III) , the term MMAF and is conjugated to the antigen binding construct “ aryl” refers to a radical derived from a 6 - to 12 -membered via the N -terminus of the drug molecule as shown below , mono - or bicyclic hydrocarbon ring system in which at least where www indicates the point of attachment to linker L : one ring is aromatic ; the term “ aryl -alkyl ” refers to an alkyl

Afrika MMAE

OH hogy MMAF

[0214 ] In some embodiments , the drug is an auristatin or group substituted with one aryl substituent; the term analogue or derivative thereof and is conjugated to the " cycloalkyl- alkyl” refers to an alkyl group substituted with antigen binding construct via the C - terminus of the drug one cycloalkyl substituent; the term " heteroaryl” refers to a molecule . Examples of auristatin analogues suitable for radical derived from a 6 - to 12 -membered mono - or bicyclic conjugation via the C -terminus of the drug molecule include ring system wherein at least one ring atom is a heteroatom , those described in International Patent Publication Nos. WO such as O , N or S , and at least one ring is aromatic ; the term 2002 /088172 and WO 2016 / 041082 . " heteroaryl -alkyl ” refers to an alkyl group substituted with [0215 ] In some embodiments , the drug is an auristatin of one heteroaryl substituent; the term " heterocyclyl” refers to general formula (III ) : a radical derived from a 3 - to 12 -membered mono - or bicyclic non -aromatic ring system wherein at least one ring atom is a heteroatom such as O , N or S ; the term “ alkoxy ( 111 ) carbonyl” refers to C ( O ) O -alkyl ; the term “ alkylamino " refers to — NH -alkyl ; the term “ amino - alkyl” refers to an alkyl group substituted with one amino substituent ; the term Rx “ amino -aryl ” refers to an aryl group substituted with one N amino substituent; the term “ amino -cycloalkyl ” refers to a cycloalkyl group substituted with one amino substituent; the Rs l Ì term " carboxamide” refers to C (O )NH2 ; the term “ haloalkyl ” refers to an alkyl group substituted with one or more halo substituents ; the term “ haloalkoxy ” refers to HN - S- RP O - haloalkyl, and the term “ thio -alkyl ” refers to - S -alkyl . OO [0217 ] In certain embodiments , the drug is an auristatin of general formula ( III ) and is conjugated to the antigen wherein : binding moiety via the R group . R² is selected from C2- C6 alkyl, aryl , aryl- C , -C alkyl, C4 - C , cycloalkyl , C3 - C , cycloalkyl- C , - C . alkyl, heteroaryl, Tubulysins heteroaryl- C / -C . alkyl and heterocyclyl, each optionally substituted with one or more substituents selected from [0218 ] In some embodiments , the drug is a tubulysin . C - C6 alkoxy , C1 -C6 alkoxycarbonyl , C7 - C , alkyl, C1- C6 Naturally occurring tubulysins include, for example , tubuly alkylamino , amino , amino - C -Co alkyl , amino - aryl, amino sins A , B , C , D , E , F , G , H , I, U , V , W and Z : US 2018 /0193477 A1 Jul. 12 , 2018

[ 0222 ] Additional examples of hemiasterlin analogues are described in International Patent Publication No . WO 2014 / 144871 . [0223 ]. In certain embodiments , the drug is a hemiasterlin analogue or derivative having general formula (IV ) : -Z ?Z 7 Š CO2H (IV ) R18 O Tubulysin A : R ' = Ac; R2 = CH2OC (O )CH2CH (CH3 ) 2 ; 1 R26R26 R3 — OH =O Tubulysin B : R = Ac; R2 = CH2OC (O )CH2CH2CH2 ; R27 =O R = OH Tubulysin C : R = Ac; R2 = CH ,OC (O )CH CHz; R3— OH R17 R16 Tubulysin D : R = Ac; R2 = CH2OC (O )CH2CH (CH3 ) 2 ; R = H wherein : Tubulysin E : R = Ac; R2 = CH2OC ( O )CH2CH2CH2 ; R20 is selected from optionally substituted alkyl, optionally R - H substituted alkylamino , optionally substituted cycloalkyl, Tubulysin F : R ' = Ac ; R2 = CH2OC ( O )CH2CH3 ; R = H optionally substituted aryl, optionally substituted heterocy Tubulysin G : R = Ac ; R2 = CH , OC ( O ) CH = C (CH3 ) 2 ; clyl and optionally substituted heteroaryl; R _ OH R27 is selected from optionally substituted alkyl, optionally Tubulysin H : R = Ac; R2= CH , OC ( O )CH2 ; R = H substituted alkylamino , optionally substituted cycloalkyl, Tubulysin I: R = Ac ; R2 = CH OC (O ) CH3; R = OH optionally substituted aryl, optionally substituted heterocy Tubulysin U : R - Ac ; R = R ? = H clyl and optionally substituted heteroaryl; Tubulysin V : R = R ? — R = H R16 and R17 are each independently H or Cl- , alkyl , and Tubulysin W : R = H ; R2 = CH2OC ( O )CH2CH2CH2 ; Reis Ci , alkyl or — SH . R > OH 0224 ) In the context of general formula ( IV ) , the term Tubulysin X : R = Ac; R = H ; R = OH “ alkyl ” refers to a straight or branched chain substituent Tubulysin Z : R = R2 = H ; R = OH consisting solely of carbon and hydrogen atoms, which is [ 0219 ] Therapeutically useful analogues and derivatives saturated or unsaturated and has from one to 12 carbon of tubulysins have also been described (see , for example , atoms; the term “ alkylamino ” refers to a substituent of the International Patent Publication No . WO 2014 / 126836 and formula — NHR , or — NR , R , , where each R , is indepen U . S . Patent Publication No. US 2016 /0130299 ) . dently an alkyl substituent containing one to 12 carbon [0220 ] The tubulysin or tubulysin analogue or derivative atoms; the term " cycloalkyl ” refers to a stable non - aromatic may be conjugated to the antigen binding construct through monocyclic or polycyclic hydrocarbon substituent consist a free hydroxyl group , or it may be modified to include an ing solely of carbon and hydrogen atoms, which may include amine group that can be used for conjugation as described in fused or bridged ring systems, having from 3 to 10 carbon U .S . Patent Publication US 2016 /0130299 . atoms; the term “ aryl” refers to a hydrocarbon ring substitu ent comprising hydrogen , 6 to 18 carbon atoms and at least Hemiasterlins one aromatic ring ; the term " heterocyclyl” refers to a stable 3 - to 18 -membered non - aromatic ring substituent which [ 0221 ] In some embodiments , the drug is a hemiasterlin or consists of 2 to 12 carbon atoms and from one to 6 analogue or derivative thereof. Various analogues and heteroatoms selected from N , O and S , and the term " het derivatives of hemiasterlin having anti -mitotic activity have eroaryl” refers to a 5 - to 14 -membered ring system substitu been described (see , for example , International Patent Pub ent comprising hydrogen atoms , one to 13 carbon atoms, one lication Nos. WO 1996 /33211 and WO 2004 / 026293 ). U . S . to 6 heteroatoms selected from N , O and S , and at least one Pat. No . 7 ,579 , 323 describes an analogue of hemiasterlin , aromatic ring . referred to as HTI- 286 , that possesses potent anti -mitotic [0225 ] In certain embodiments , the drug is a hemiasterlin activity and which has been assessed in clinical trials for the of general formula ( IV ) and is conjugated to the antigen treatment of cancer. In certain embodiments , the drug is binding construct via the R26 substituent . In some embodi HTI- 286 or a derivative thereof: ments , the drug is a hemiasterlin of general formula (IV ) and is conjugated to the antigen binding construct via the Roy substituent. Calicheamicins [0226 ] In some embodiments , the drug is a calicheamycin ?? or analogue or derivative thereof. Various analogues and HN . derivatives of calicheamycin suitable for conjugation to an antigen binding construct have been described ( see , for HTI -286 example , International Patent Publication No . WO 2015 / 063680 , U . S . Pat . Nos. 5, 773 ,001 ; 5 ,714 ,586 and 5 ,770 , 701) . US 2018 /0193477 A1 Jul. 12 , 2018 27

Duocarmycins Chemotherapeutic Agents [0227 ] In some embodiments , the drug is a duocarmycin [0231 ] In some embodiments the antigen binding con or analogue or derivative thereof. Naturally - occurring duo struct is conjugated to a chemotherapeutic agent. Examples carmycins include , for example , duocarmycins A , B1, B2 , include but are not limited to cisplatin and Lapatinib . A C1, C2 , D and SA , as well as CC - 1065 . Various analogues " chemotherapeutic agent” is a chemical compound useful in and derivatives of duocarmycins have been described , the treatment of cancer. including adozelesin , bizelesin and centanamycin . Other [0232 ] Examples of chemotherapeutic agents include analogues and derivatives are described in U . S . Pat . Nos. alkylating agents such as thiotepa and cyclosphosphamide 4 ,912 , 227 ; 5 ,070 ,092 ; 5 , 084 , 468 ; 5 , 332 ,837 ; 5 ,641 , 780 ; (CYTOXANTM ) ; alkyl sulfonates such as busulfan , impro 5 ,739 , 350 and 8 , 889, 868. Various groups on the duocarmy sulfan and piposulfan ; aziridines such as benzodopa , carbo cin molecule may be modified to allow for conjugation to an quone, meturedopa , and uredopa ; ethylenimines and meth antigen binding construct. A non - limiting example is pro ylamelamines including altretamine , triethylenemelamine, vided in Elgersma et al ., ( 2015 ) Mol. Pharmaceutics, triethylenephosphoramide , triethylenethiophosphaoramide 12 : 1813 - 1835 . and trimethylolomelamine ; nitrogen mustards such as chlorambucil , chlornaphazine , cholophosphamide, estra Pyrrolobenzodiazapenes mustine , ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan , novembichin , phenester [0228 ] In some embodiments , the drug is a pyrrolobenzo ine , prednimustine, trofosfamide , uracil mustard ; nitrosureas diazapene (PBD ) or an analogue or derivative thereof, such such as carmustine , chlorozotocin , fotemustine , lomustine , as a PBD dimer. Various PBD dimers have been described nimustine , ranimustine; antibiotics such as aclacinomysins , including , for example , those described in U . S . Pat . Nos . actinomycin , authramycin , azaserine , bleomycins, cactino 6 ,884 ,799 ; 7, 049 , 311 ; 7 ,511 , 032 ; 7 ,528 , 126 ; 7 ,557 ,099 and mycin , calicheamicin , carabicin , carminomycin , carzinophi 9 ,056 , 914 . In some embodiments , the drug is a PBD dimer lin , chromomycins, dactinomycin , daunorubicin , detorubi or an analogue or derivative thereof. The PBD dimer struc cin , 6 - diazo - 5 - oxo - L - norleucine , doxorubicin , epirubicin , ture is believed to improve the fit at the binding site of DNA . esorubicin , idarubicin , marcellomycin , mitomycins, myco PBD dimers may be conjugated to the antigen binding phenolic acid , nogalamycin , olivomycins, peplomycin , pot construct through one of a number of potential linkage sites firomycin , puromycin , quelamycin , rodorubicin , streptoni on the PBD dimer , such as the five -membered pyrrolo ring , grin , streptozocin , tubercidin , ubenimex , zinostatin , the tether between the PBD units , the N10 - C11 imine group zorubicin ; anti- metabolites such as methotrexate and 5 - fluo or the C2 position (see , for example , International Patent rouracil ( 5 - FU ) ; folic acid analogues such as denopterin , Publication Nos . WO 2007 /085930 , WO 2009 /016516 , WO methotrexate , pteropterin , trimetrexate ; purine analogs such 2011 / 130598 , WO 2011 / 130613 and WO 2011/ 130616 ; U . S . as fludarabine , 6 -mercaptopurine , thiamiprine , thioguanine ; Patent Publication No . US 2011 /0256157 ). pyrimidine analogs such as ancitabine , azacitidine, 6 - azau ridine , carmofur , cytarabine , dideoxyuridine , doxifluridine , Amatoxins enocitabine , floxuridine , 5 -FU ; androgens such as caluster [ 0229 ]. In some embodiments, the drug is an amatoxin , one , dromostanolone propionate , epitiostanol, mepitiostane , such as a - Amanitin , B - Amanitin , y - Amanitin or e - Amanitin , testolactone ; anti - adrenals such as aminoglutethimide , mito or an analogue or derivative thereof. In some embodiments , tane , trilostane ; folic acid replenisher such as frolinic acid ; the drug is a -Amanitin or an analogue or derivative thereof. aceglatone ; aldophosphamide glycoside ; aminolevulinic Amatoxins are cyclic peptides composed of eight amino acid ; amsacrine ; bestrabucil ; bisantrene ; edatraxate ; defo acids and thus present a number of potential sites for famine ; demecolcine ; diaziquone; elfornithine ; elliptinium conjugation . Various amatoxins and analogues thereof have acetate ; etoglucid ; gallium nitrate ; hydroxyurea ; lentinan ; been described (see , for example , European Patent No . EP lonidamine ; mitoguazone; mitoxantrone ; mopidamol; nitra 1 859 811 , U . S . Pat. No . 9 ,233 , 173 and International Patent crine ; pentostatin ; phenamet ; pirarubicin ; podophyllinic Publication No. WO 2014 /043403 ) . acid ; 2 - ethylhydrazide ; procarbazine ; PSK7; razoxane ; sizo firan ; spirogermanium ; tenuazonic acid ; triaziquone ; 2 ', 2 ' , Camptothecins 2 - trichlorotriethylamine ; urethan ; vindesine ; dacarbazine ; mannomustine ; mitobronitol; mitolactol; pipobroman ; gacy [ 0230 ] In some embodiments , the drug is a camptothecin tosine ; arabinoside (“ Ara - C " ) ; cyclophosphamide ; taxanes , (CPT ) or analogue or derivative thereof , such as irinotecan e . g . paclitaxel ( TAXOL® , Bristol- Myers Squibb Oncology , (CPT - 11 ) , SN - 38 ( 7 - ethyl- 10 -hydroxy - camptothecin ), Princeton , N . J . ) and doxetaxel ( TAXOTERE? , Rhone - Pou 10 -hydroxy camptothecin , topotecan , lurtotecan , 9 -amin lenc Rorer , Antony, France ) ; chlorambucil ; gemcitabine ; ocamptothecin or 9 - nitrocamptothecin . Other examples of 6 -thioguanine ; platinum analogs such as cisplatin and car CPT analogues and derivatives include 7 -butyl - 10 - amino boplatin ; vinblastine; platinum ; etoposide (VP - 16 ) ; ifosf camptothecin and 7 -butyl - 9- amino - 10 , 11- methylenedioxy amide; mitomycin C ; mitoxantrone ; vincristine ; vinorelbine ; camptothecin (see U . S . Patent Publication No . US 2005 / navelbine ; novantrone ; teniposide ; daunomycin ; aminop 0209263 ) and aniline containing derivatives of these terin ; xeloda ; ibandronate ; CPT- 11 ; topoisomerase inhibitor compounds as described in Burke et al. , ( 2009) , Bioconj. RFS 2000 ; difluoromethylornithine (DMFO ) ; retinoic acid ; Chem . 20 (6 ): 1242 - 1250 . Conjugation of camptothecin and esperamicins; capecitabine; and pharmaceutically accept its analogues or derivatives to the antigen binding construct able salts , acids or derivatives of any of the above. Also may be achieved via modification of various groups in the included in this definition are anti- hormonal agents that act drug molecule . Non - limiting examples are provided in to regulate or inhibit hormone action on tumors such as Burke et al. , ( 2009 ) , Bioconj. Chem . 20 ( 6 ) : 1242 - 1250 and anti- estrogens including for example tamoxifen , raloxifene , Sharkey et al ., (2012 ) Mol. Cancer Ther. 11 :224 -234 . aromatase inhibiting 4 ( 5 ) - imidazoles, 4 -hydroxytamoxifen , US 2018 /0193477 A1 Jul. 12 , 2018 trioxifene , keoxifene , LY117018 , onapristone , and tore - reagent, isothiocyanates , aldehydes and acid anhydrides mifene ( Fareston ) ; and anti - androgens such as flutamide , such as diethylenetriaminepentaacetic anhydride (DTPA ) . nilutamide , bicalutamide , leuprolide , and goserelin ; and Other examples include succinimido - 1 , 1 , 3 , 3 - tetra -methyl pharmaceutically acceptable salts , acids or derivatives of uronium tetrafluoroborate ( TSTU ) and benzotriazol- 1 - yl any of the above . oxytripyrrolidinophosphonium hexafluorophosphate (Py [0233 ] In some embodiments , the chemotherapeutic agent BOP ) . Non - limiting examples of functional groups capable is an anthracycline , such as doxorubicin , epirubicin , idaru of reacting with an electrophilic group on the antigen bicin , daunorubicin ( also known as daunomycin ) , nemoru binding construct or drug ( such as an aldehyde or ketone bicin or an analogue or derivative thereof . Various groups carbonyl group ) include hydrazide , oxime, amino , hydra within the anthracycline molecule may be modified for zine , thiosemicarbazone , hydrazine carboxylate and arylhy conjugation to the antigen binding construct. For example , drazide . derivatization of daunorubicin and doxorubicin for conju [0237 ] In certain embodiments , a linker that includes a gation to antibodies has been described (see , for example , functional group that allows for bridging of two interchain Kratz et al. , ( 2006 ) Current Med . Chem . 13: 477 -523 ; U . S . cysteines on the antibody binding construct may be used , Pat. No . 6 ,630 , 579 ) . such as a ThioBridgeTM linker (Badescu et al . , ( 2014 ) Bioconjug . Chem . 25 : 1124 - 1136 ), a dithiomaleimide (DTM ) Linkers linker ( Behrens et al ., 2015 , Mol. Pharm . 12 :3986 - 3998 ) , a [ 0234 ] In some embodiments , the drug is linked to the dithioaryl( TCEP )pyridazinedione based linker (Lee et al. , antigen binding construct , e . g . , antibody , by a linker. Linkers ( 2016 ) Chem . Sci. 7 :799 - 802 ) or a dibromopyridazinedione are bifunctional or multifunctional moieties capable of link based linker (Maruani et al. , (2015 ) Nat. Commun . 6 :6645 ) . ing one or more drugs to the antigen binding construct . In [0238 ] A variety of linkers for linking drugs to antibodies some embodiments , the linker may be bifunctional (or and other antigen binding constructs are known in the art, monovalent ) such that it links a single drug to a single site including hydrazone -, disulfide - and peptide -based linkers. on the antigen binding construct . In some embodiments , the [0239 ] Suitable linkers typically are more chemically linker may be multifunctional ( or polyvalent) such that it stable to conditions outside the cell than to conditions inside links more than one drug to a single site on the antigen the cell , although less stable linkers may be contemplated in binding construct . Multifunctional linkers may also be used certain situations, such as when the drug is selective or to link one drug to more than one site on the antigen binding targeted and has a low toxicity to normal cells . Suitable construct in some embodiments . linkers include , for example , cleavable and non - cleavable [0235 ] Attachment of a linker to an antibody or other linkers . A cleavable linker is typically susceptible to cleav antigen binding construct can be accomplished in a variety age under intracellular conditions , for example , through of ways, such as through surface lysines , reductive - coupling lysosomal processes. Examples include linkers that are to oxidized carbohydrates , and through cysteine residues protease -sensitive , acid - sensitive or reduction - sensitive . liberated by reducing interchain disulfide linkages . Alterna Non - cleavable linkers by contrast, rely on the degradation of tively , attachment of a linker to an antigen binding construct the antibody in the cell , which typically results in the release may be achieved by modification of the antigen binding of an amino acid - linker - cytotoxin moiety . construct to include additional cysteine residues (see , for [0240 ] Suitable cleavable linkers include , for example , example , U . S . Pat. Nos. 7 , 521, 541 ; 8 , 455 ,622 and 9 ,000 , peptide - containing linkers cleavable by an intracellular pro 130 ) or non - natural amino acids that provide reactive tease , such as lysosomal protease or an endosomal protease . handles, such as selenomethionine , p -acetylphenylalanine , In exemplary embodiments, the linker can be a dipeptide formylglycine or p -azidomethyl - L -phenylalanine ( see , for containing linker , such as a valine -citrulline ( Val - Cit ) or a example , Hofer et al. , ( 2009 ) Biochemistry 48: 12047 - 12057 ; phenylalanine - lysine (Phe - Lys ) linker. Other examples of Axup et al. , ( 2012 ) PNAS 109 : 16101 - 16106 ; Wu et al. , suitable dipeptides for inclusion in the linkers include Val ( 2009 ) PNAS 106 :3000 - 3005 ; Zimmerman et al. , (2014 ) Lys, Ala - Lys, Phe -Lys , Val- Cit , Phe - Cit , Leu -Cit , Ile - Cit , Bioconj. Chem . 25 : 351 -361 ), to allow for site - specific con Trp - Cit , Phe - Arg, Ala - Phe, Val- Ala , Met - Lys , Asn - Lys , Ile jugation . Pro , Ile- Val, Asp - Val, His - Val, Met- (D )Lys , Asn - (D ) Lys, [ 0236 ] The linkers include a functional group capable of Val - ( D ) Asp , NorVal- ( D ) Asp , Ala - ( D )Asp , Mez Lys- Pro , reacting with the target group or groups on the antigen PhenylGly - ( D )Lys , Met - ( D )Lys , Asn - ( D ) Lys, Pro - ( D ) Lys binding construct and one ormore functional groups capable and Met - ( D )Lys . Linkers may also include longer peptide of reacting with a target group on the drug . Suitable func sequences in some embodiments , such as the tripeptides tional groups are known in the art and include those Met - Cit - Val, Gly - Cit - Val, ( D ) Phe - Phe -Lys or ( D ) Ala -Phe described , for example , in Bioconjugate Techniques (G . T. Lys, or the tetrapeptides Gly - Phe -Leu -Gly or Ala -Leu - Ala Hermanson , 2013 , Academic Press ). Non -limiting examples Leu . of functional groups for reacting with free cysteines or thiols [0241 ] Additional suitable cleavable linkers include dis include maleimide, haloacetamide, haloacetyl, activated ulfide - containing linkers . Examples of disulfide - containing esters such as succinimide esters , 4 -nitrophenyl esters , pen linkers include, but are not limited to , N -succinimydyl - 4 tafluorophenyl esters, tetrafluorophenyl esters , anhydrides , ( 2 -pyridyldithio ) butanoate (SPBD ) and N - succinimydyl- 4 acid chlorides, sulfonyl chlorides, isocyanates, and isothio ( 2 -pyridyldithio ) - 2 - sulfo butanoate ( sulfo - SPBD ) . Disul cyanates . Also useful in this context are “ self -stabilizing ” fide- containing linkers may optionally include additional maleimides as described in Lyon et al. , ( 2014 ) Nat. Biotech groups to provide steric hindrance adjacent to the disulfide nol. 32 : 1059- 1062. Non - limiting examples of functional bond in order to improve the extracellular stability of the groups for reacting with surface lysines and amines include linker, for example , inclusion of a geminal dimethyl group . activated esters such as N -hydroxysuccinamide (NHS ) Other suitable linkers include linkers hydrolyzable at a esters or sulfo -NHS esters, imido esters such as Traut' s specific pH or within a pH range , such as hydrazone linkers . US 2018 /0193477 A1 Jul. 12 , 2018

Linkers comprising combinations of these functionalities [ 0247 ] In some embodiments , in general formula (VI ) : may also be useful, for example , linkers comprising both a hydrazone and a disulfide are known in the art . Z is [ 0242 ] A further example of a cleavable linker is a linker comprising a B - glucuronide, which is cleavable by B - glu [0248 ] curonidase , an enzyme present in lysosomes and tumor interstitium ( see , for example , De Graaf et al. , (2002 ) Curr. Pharm . Des. 8 : 1391 - 1403 ) . [ 0243 ] Cleavable linkers may optionally further comprise one or more additional functionalities such as self- immola tive and self - elimination groups , stretchers or hydrophilic moieties . [ 0244 ] Self - immolative and self - elimination groups that find use in linkers include , for example , p - aminobenzyloxy carbonyl (PABC ) and p -aminobenzyl ether (PABE ) groups , [0249 ] In some embodiments , in general formula (VI ) : and methylated ethylene diamine (MED ) . Other examples of self - immolative groups include, but are not limited to , Str is aromatic compounds that are electronically similar to the [0250 ] PABC or PABE group such as heterocyclic derivatives, for example 2 - aminoimidazol- 5 -methanol derivatives as described in U . S . Pat. No . 7 , 375 , 078 . Other examples include groups that undergo cyclization upon amide bond hydrolysis , such as substituted and unsubstituted 4 -amin - (CH2 ) 2 - 0 — ; - (CH2CH2O ) , — C — obutyric acid amides (Rodrigues et al. (1995 ) Chemistry Biology 2 :223 - 227) and 2 - aminophenylpropionic acid — ( CH2) p — ( CH2CH20 )? — * — ; amides ( Amsberry, et al. ( 1990 ) J Org. Chem . 55 :5867 5877 ). Self - immolative/ self- elimination groups, alone or in combination are often included in peptide -based linkers, but - (CH2CH20 ), — (CH2 ) , - — ; may also be included in other types of linkers. In some R or embodiments , the linker may include one or more self Z- immolative and self - elimination groups , for example , a — ( CH2) p — C — N — ( CH2) , — Ö — PABC group , a PABE group , or a combination of a PABC or 0 7R - PABE group and an MED . - ( CH2) p — C — N — ( CH2CH20 ) q - C [0245 ] Stretchers that find use in linkers for ADCs include, for example , alkylene groups and stretchers based on ali phatic acids , diacids, amines or diamines, such as diglyco wherein late, malonate , caproate and caproamide . Other stretchers Ris H or C , - C6 alkyl; include , for example, glycine based stretchers and polyeth p is an integer between 2 and 10 , and ylene glycol (PEG ) or monomethoxy polyethylene glycol q is an integer between 1 and 10 . (MPEG ) stretchers. PEG and mPEG stretchers also function [0251 ] In some embodiments , in general formula (VI ) : as hydrophilic moieties and may be particularly useful with hydrophobic drugs , although their use in linkers with other Str is drugs is also contemplated in some embodiments . [0246 ] In certain embodiments , the linker included in the [ 0252 ] ADCs of the present disclosure are peptide -based linkers of

general formula (VI ) : O O = — (CH2 ) , — Ö — or - (CH2CH20 )? — Ö — ( VI) c. . . ahol 2 + Str In AAIFAA2 ImtXtoD wherein p and q are as defined above. wherein : In some embodiments , in general formula (VI ) : Z is a functional group capable of reacting with the target Str is group on the antigen binding construct; Str is a stretcher; [0253 ] AA , and AA , are each independently an amino acid , wherein AA1- [ AA2] m forms a protease cleavage site ; X is a self- immolative group ; D is a drug ; — (CH2 ) , — C — or - (CH2CH20 )? — C — n is 0 or 1 ; m is 1 , 2 or 3 , and wherein p is an integer between 2 and 6 , and o is 0 , 1 or 2 . q is an integer between 2 and 8 . US 2018 /0193477 A1 Jul. 12 , 2018 30

[ 0254 ] In some embodiments , in general formula (VI ) : [0263 ] In some embodiments , the linker is a disulfide AA - [AA ] m is selected from Val- Lys , Ala - Lys, Phe -Lys , containing linker and the ADC has general formula (VII ) : Val- Cit , Phe - Cit, Leu -Cit , Ile - Cit , Trp -Cit , Phe - Arg , Ala Phe, Val - Ala , Met- Lys , Asn -Lys , Ile -Pro , Ile - Val, Asp -Val , (VII ) His - Val, Met- (D ) Lys, Asn -( D ) Lys, Val- ( D )Asp , NorVal- ( D ) RR Asp , Ala- (D )Asp , Mez Lys- Pro , PhenylGly- ( D ) Lys, Met -( D ) A Lys, Asn - ( D ) Lys, Pro -( D ) Lys, Met - ( D )Lys , Met - Cit - Val, D Gly - Cit - Val , ( D )Phe - Phe -Lys , (D ) Ala -Phe -Lys , Gly -Phe Leu -Gly and Ala -Leu - Ala - Leu . RR [0255 ] In some embodiments , in general formula ( VI) : wherein : m is 1 (i . e . AA -[ AA2 ] m is a dipeptide ). A is the antigen binding construct; [0256 ] In some embodiments , in general formula (VI ) : D is the drug; Yis — (CH2 ) . — or — (CH2CH2O ) , — , wherein p and q are AA ,- [ AA ] m is a dipeptide selected from Val- Lys, Ala - Lys, each independently an integer between 1 and 10 ; Phe- Lys , Val- Cit , Phe - Cit, Leu - Cit , Ile - Cit and Trp - Cit . each R is independently H or C , -C . alkyl; [ 0257] In some embodiments , in general formula (VI ) : n is 1 , 2 or 3 , and each X is independently selected from p -aminobenzyloxy wherein carbonyl (PABC ) , p -aminobenzyl ether (PABE ) and meth ylated ethylene diamine (MED ) . [0258 ] In some embodiments , in general formula ( VI) : n is 1 . [ 0259 ] In some embodiments , in general formula (VI ) : represents an amide bond formed between the linker and the E - amino group of a surface lysine on the antigen binding o is 1 or 2 . construct . [0260 ] In some embodiments , in general formula (VI ) : [0264 ] In some embodiments in general formula (VII ) : p and q are each independently an integer between 1 and 4 . [0265 ] In some embodiments in general formula ( VII) : Z is Y is – (CH ) n - and p is an integer between 1 and 4 . [0266 ] In some embodiments in general formula ( VII ) : [ 0261] each R is independently H or Me. [0267 ] In some embodiments in general formula ( VII ) : n is 1 or 2 [0268 ] In some embodiments in general formula ( VII) : Y is — (CH ) , and p is an integer between 1 and 4 ; each R is independently H or Me, and n is 1 or 2 . [ 0269 ] Examples of commonly used cleavable linkers that may find use in the ADCs of the present disclosure in some embodiments include, but are not limited to , linkers com prising SPBD , sulfo -SPBD , hydrazone , Val- Cit , maleido caproyl MC( or mc) , mc- Val- Cit , mc- Val - Cit - PABC , Phe Str is Lys, mc- Phe - Lys or mc- Phe -Lys - PABC . [0270 ] Various non -cleavable linkers are known in the art for linking drugs to targeting moieties and may be useful in [0262 ] the ADCs of the present disclosure . Examples of non cleavable linkers include linkers having an N - succinimidyl ester or N - sulfosuccinimidyl ester moiety for reaction with the cell binding agent, as well as a maleimido - or haloacetyl — ( CH2) p — Ö — or — ( CH2CH20 )? — C — based moiety for reaction with the drug , or vice versa . An example of such a non - cleavable linker is based on sulfos uccinimidyl- 4 - [ N -maleimidomethyl ] cyclohexane - 1 -car wherein p is an integer between 2 and 6 , and q is an integer boxylate ( sulfo -SMCC ) . Sulfo - SMCC conjugation typically between 2 and 8 ; occurs via a maleimide group which reacts with sulfhydryls ( thiols , — SH ) on the drug moiety , while the sulfo - NHS ester mis 1 and AA - [ AA ] m is a dipeptide selected from Val- Lys , is reactive toward primary amines ( as found in lysine and the Ala -Lys , Phe -Lys , Val - Cit, Phe - Cit, Leu -Cit , Ile -Cit and protein or peptide N - terminus ) . Other non - limiting examples Trp - Cit ; of such linkers include those based on N - succinimidyl each X is independently selected from p - aminobenzyloxy 4 - (maleimidomethyl ) cyclohexanecarboxylate ( SMCC ) , carbonyl (PABC ) , p - aminobenzyl ether (PABE ) and meth N -succinimidyl - 4 - (N -maleimidomethyl ) -cyclohexane - 1 ylated ethylene diamine (MED ). carboxy - ( 6 -amidocaproate ) (“ long chain ” SMCC or LC SMCC ) , K -maleimidoundecanoic acid N -succinimidyl ester n is 1 , and (KMUA ), y -maleimidobutyric acid N - succinimidyl ester o is 1 or 2 . (GMBS ) , e -maleimidocaproic acid N -hydroxysuccinimide US 2018 /0193477 A1 Jul. 12 , 2018

ester ( EMCS ) , m - maleimidobenzoyl- N -hydroxysuccinim - R ' is selected from optionally substituted alkyl , optionally ide ester (MBS ), N - la -maleimidoacetoxy )- succinimide substituted alkylamino , optionally substituted cycloalkyl, ester (AMAS ) , succinimidyl- 6 - ( ß -maleimidopropionamido ) optionally substituted aryl, optionally substituted heterocy hexanoate (SMPH ) , N -succinimidyl 4 - ( p -maleimidophe clyl, optionally substituted heteroaryl , COR27 — nyl) - butyrate (SMPB ), and N -( p -maleimidophenyl ) isocya CSR27 — , - OR27 — and — NHR27 — , wherein each R27 nate (PMPI ) . Other examples include those comprising a is independently selected from optionally substituted alkyl , haloacetyl- based functional group such as N - succinimidyl - optionally substituted alkylamino , optionally substituted 4 - iodoacetyl) -aminobenzoate (SIAB ) , N - succinimidyl cycloalkyl , optionally substituted aryl, optionally substituted iodoacetate (SIA ) , N -succinimidyl bromoacetate (SBA ) and heterocyclyl and optionally substituted heteroaryl; N -succinimidyl 3 -( bromoacetamido )propionate ( SBAP ). each AA is independently an amino acid , wherein ( AA ) ' [0271 ] Other examples of non -cleavable linkers include (AA ) , taken together comprise an amino acid sequence maleimidocarboxylic acids, such as maleimidocaproyl capable of facilitating cleavage of the JPB ; (MC ) . x is an integer from 0 to 25 ; [ 0272] In certain embodiments , the antigen binding con L ' is a remaining portion of the linker or is absent; struct is conjugated to the drug via a sulphonamide -contain A is the antigen binding construct . ing linker as described in International Patent Publication [0274 ] In some embodiments , the antigen -binding con No . WO 2015 /095953 . In some embodiments , the antigen struct is coupled to the drug via a cleavable linker e . g . a binding construct is conjugated to the drug via a linker SPBD linker or a maleimidocaproyl- valine -citrulline - p having general formula (VIII ) : aminobenzyloxycarbonyl (mc - Val - Cit- PABC ) linker. In some embodiments , the antigen - binding construct is coupled to the drug via a non -cleavable linker e . g . a MCC linker (VIII ) formed using SMCC or sulfo -SMCC . [ 0275 ] Selection of an appropriate linker for a given ADC L3 — A may be readily made by the skilled person having knowl edge of the art and taking into account relevant factors , such ZH as the site of attachment to the antigen binding construct, any structural constraints of the drug and the hydrophobicity wherein : of the drug ( see, for example , review in Nolting , Chapter 5 , R is selected from optionally substituted alkyl , optionally Antibody - Drug Conjugates : Methods in Molecular Biology, substituted alkylamino , optionally substituted cycloalkyl, 2013 , Ducry (Ed .) , Springer) . A number of specific linker optionally substituted aryl, optionally substituted heterocy toxin combinations have been described and may be used clyl, optionally substituted heteroaryl , COR27 — with the antigen binding constructs described herein to - CSR 7 — , - OR27 — and — NHR27 — , wherein each R27 prepare ADCs in certain embodiments . Examples include , is independently selected from optionally substituted alkyl, but are not limited to , cleavable peptide -based linkers with optionally substituted alkylamino , optionally substituted auristatins such as MMAE and MMAF , camptothecins such cycloalkyl, optionally substituted aryl, optionally substituted as SN - 38 , duocarmycins and PBD dimers ; non - cleavable heterocyclyl and optionally substituted heteroaryl ; MC -based linkers with auristatins MMAF and MMAE ; acid - labile hydrazone -based linkers with calicheamicins and P3 is the drug or a portion of the drug ; doxorubicin ; disulfide -based linkers with maytansinoids L is a linker or a portion of a linker , and such as DM1 and DM4, and bis -maleimido - trioxyethylene A is the antigen binding construct . glycol (BMPEO ) - based linkers with maytansinoid DM1 [0273 ] In some embodiments , the antigen binding con ( see , for example , Peters & Brown, ( 2015 ) Biosci . Rep . struct is conjugated to the drug via a linker having general e00225 ; Dosio et al . , ( 2014 ) Recent Patents on Anti - Cancer formula (IX ) : Drug Discovery 9 : 35 - 65 ; US Patent Publication No . US 2015 /0374847 ) . ( IX ) Preparation of ADCs i _ r - N - 1 - A [ 0276 ] The ADC may be prepared by one of several routes known in the art , employing organic chemistry reactions , conditions , and reagents known to those skilled in the art (see , for example , Bioconjugate Techniques (G . T . Herman wherein - L - A has the structure : son , 2013 , Academic Press ) . For example , conjugation may be achieved by ( 1 ) reaction of a nucleophilic group or an electrophilic group of an antibody with a bivalent linker reagent, to form antibody -linker intermediate Ab -L , via a covalent bond , followed by reaction with an activated drug (AA ) moiety D ; or (2 ) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug - linker intermediate D -L , via a covalent bond , followed by reaction with the nucleophilic group or an wherein : electrophilic group of an antibody. Conjugation methods ( 1) P3 is a remaining portion of the drug ; and ( 2 ) may be employed with a variety of antibodies , drug the - NH - group bonded to R ' forms a peptide bond ( the moieties , and linkers to prepare the ADCs described here . junction peptide bond or JPB ) with (AA )' ; Various prepared linkers , linker components and toxins are US 2018 /0193477 A1 Jul. 12 , 2018 32 commercially available or may be prepared using standard starch , glucose , lactose, sucrose , gelatin , malt, rice , flour, synthetic organic chemistry techniques (see , for example , chalk , silica gel , sodium stearate , glycerol monostearate , March ' s Advanced Organic Chemistry (Smith & March , talc , sodium chloride, dried skim milk , glycerol, propylene , 2006 , Sixth Ed ., Wiley) ; Toki et al. , ( 2002 ) J. Org. Chem . glycol, water, ethanol and the like. The composition , if 67 : 1866 - 1872 ; Frisch et al. , ( 1997 ) Bioconj. Chem . 7 : 180 desired , can also contain minor amounts of wetting or 186 ; Bioconjugate Techniques (G . T . Hermanson , 2013 , emulsifying agents , or pH buffering agents . These compo Academic Press ) ) . In addition , a number of pre - formed sitions can take the form of solutions, suspensions, emul drug - linkers suitable for reaction with a selected antigen sion , tablets , pills, capsules , powders , sustained - release for binding construct are also available commercially , for mulations and the like . The composition can be formulated example , linker - toxins comprising DM1, DM4, MMAE , as a suppository , with traditional binders and carriers such as MMAF or Duocarmycin SA are available from Creative triglycerides . Oral formulation can include standard carriers BioLabs (Shirley , N . Y . ) . such as pharmaceutical grades of mannitol, lactose , starch , [0277 ] Several specific examples ofmethods of preparing magnesium stearate , sodium saccharine , cellulose , magne ADCs are known in the art and are described in U . S . Pat . No . sium carbonate , etc . Examples of suitable pharmaceutical 8 ,624 , 003 ( pot method ) , U . S . Pat. No . 8 , 163 , 888 ( one - step ) , carriers are described in “ Remington ' s Pharmaceutical Sci and U . S . Pat . No. 5 , 208 ,020 ( two - step method ) . Other ences ” by E . W . Martin . Such compositions will contain a methods are known in the art and include those described in therapeutically effective amount of the compound , prefer Antibody - Drug Conjugates : Methods in Molecular Biology , ably in purified form , together with a suitable amount of 2013 , Ducry ( Ed . ) , Springer. In addition , various antibody carrier so as to provide the form for proper administration to drug conjugation services are available commercially from the patient . The formulation should suit themode of admin companies such as Lonza Inc. ( Allendale , N . J . ) , Abzena istration . PLC (Cambridge , UK ) , ADC Biotechnology ( St. Asaph , [ 0281 ] In certain embodiments , the composition compris UK ) , Baxter BioPharma Solutions (Baxter Healthcare Cor ing the construct is formulated in accordance with routine poration , Deerfield , Ill. ) and Piramel Pharma Solutions procedures as a pharmaceutical composition adapted for (Grangemouth , UK ) . intravenous administration to human beings . Typically , com [ 0278 ] The average number of drugs conjugated to the positions for intravenous administration are solutions in antigen binding construct (drug - to - antibody ratio or DAR ) sterile isotonic aqueous buffer . Where necessary, the com may be determined by standard techniques such as UV /VIS position may also include a solubilizing agent and a local spectroscopic analysis , ELISA - based techniques, chroma anaesthetic such as lignocaine to ease pain at the site of the tography techniques such as hydrophobic interaction chro injection . Generally , the ingredients are supplied either matography (HIC ) , UV -MALDI mass spectrometry (MS ) separately or mixed together in unit dosage form , for and MALDI- TOF MS. In addition , distribution of drug example , as a dry lyophilized powder or water free concen linked forms ( for example , the fraction of antigen binding trate in a hermetically sealed container such as an ampoule constructs containing zero , one , two , three , etc . drugs ) may or sachette indicating the quantity of active agent. Where the also optionally be analyzed . Various techniques are known composition is to be administered by infusion , it can be in the art to measure such distribution , including MS ( with dispensed with an infusion bottle containing sterile pharma or without an accompanying chromatographic separation ceutical grade water or saline . Where the composition is step ) , hydrophobic interaction chromatography, reverse administered by injection , an ampoule of sterile water for phase HPLC or iso - electric focusing gel electrophoresis injection or saline can be provided so that the ingredients ( IEF ) (see , for example , Wakankar et al. , ( 2011) mAbs may be mixed prior to administration . 3 : 161- 172 ) . [0282 ] In certain embodiments , the compositions described herein are formulated as neutral or salt forms. Pharmaceutical Compositions Pharmaceutically acceptable salts include those formed with [0279 ] Also provided herein are pharmaceutical composi anions such as those derived from hydrochloric , phosphoric , tions comprising a drug -conjugated antigen -binding con acetic , oxalic , tartaric acids, etc . , and those formed with struct described herein . Pharmaceutical compositions com cations such as those derived from sodium , potassium , prise the construct and a pharmaceutically acceptable ammonium , calcium , ferric hydroxide isopropylamine , tri carrier. ethylamine , 2 - ethylamino ethanol, histidine , procaine , etc . [ 0280 ] The term “ pharmaceutically acceptable ” means approved by a regulatory agency of the Federal or a state Methods of Treatment government or listed in the U . S . Pharmacopeia or other generally recognized pharmacopeia for use in animals , and [0283 ] Also described herein are methods of treating a more particularly in humans . The term “ carrier” refers to a disease or disorder comprising administering to a subject in diluent , adjuvant , excipient, or vehicle with which the thera which such treatment, prevention or amelioration is desired , peutic is administered . Such pharmaceutical carriers can be an antigen -binding construct described herein , in an amount sterile liquids, such as water and oils , including those of effective to treat , prevent or ameliorate the disease or petroleum , animal, vegetable or synthetic origin , such as disorder . peanut oil , soybean oil , mineral oil, sesame oil and the like . [0284 ] Disorder and disease are used interchangeably and In some aspects , the carrier is a man -made carrier not found refer to any condition that would benefit from treatment with in nature . Water can be used as a carrier when the pharma an antigen - binding construct or method described herein . ceutical composition is administered intravenously . Saline This includes chronic and acute disorders or diseases includ solutions and aqueous dextrose and glycerol solutions can ing those pathological conditions which predispose the also be employed as liquid carriers , particularly for inject mammal to the disorder in question . In some embodiments , able solutions. Suitable pharmaceutical excipients include the disorder is cancer. US 2018 /0193477 A1 Jul. 12 , 2018 33

[0285 ] The term “ subject” refers to an animal which is the fungi that express antigens on the cell surface of an infected object of treatment, observation or experiment. An animal host. Targets useful in these constructs are found in Table may be a human , a non - human primate , a companion animal LL . ( e . g ., dogs, cats , and the like ), farm animal (e . g ., cows, [0291 ] In some embodiments , the drug - conjugated anti sheep , pigs , horses , and the like ) or a laboratory animal ( e . g ., gen -binding constructs do not substantially deplete the T rats, mice, guinea pigs , and the like ) . cells of a subject to which the construct is administered . As 0286 ]. The term “ mammal” as used herein includes but is used herein “ substantially deplete ” T cells means reduce the not limited to humans, non -human primates, canines , number of T cells to a number that is less than about 75 % , felines , murines, bovines , equines , and porcines . less than about 50 % , or less than about 25 percent of the [0287 ] “ Treatment” refers to clinical intervention in an pre - administration number. attempt to alter the natural course of the individual or cell [0292 ] In certain embodiments is provided a method for being treated , and can be performed either for prophylaxis or the prevention , treatment or amelioration of cancer, said during the course of clinical pathology . Desirable effects of method comprising administering to a subject in need of treatment include preventing occurrence or recurrence of such prevention , treatment or amelioration a pharmaceutical disease , alleviation of symptoms, diminishing of any direct composition comprising an antigen - binding construct or indirect pathological consequences of the disease, pre described herein . venting metastasis , decreasing the rate of disease progres [0293 ] In certain embodiments is a method of treating sion , amelioration or palliation of the disease state, and cancer in a mammal in need thereof, comprising adminis remission or improved prognosis . In some embodiments , tering to the mammal a composition comprising an effective antigen - binding constructs described herein are used to amountof the pharmaceutical composition described herein , delay development of a disease or disorder. In one embodi optionally in combination with other pharmaceutically ment, antigen - binding constructs and methods described active molecules . In certain embodiments , the cancer is a herein effect tumor regression . In one embodiment, antigen lymphoma or leukemia . binding constructs and methods described herein effect [0294 ] In some embodiments , the cancer is a lymphoma or inhibition of tumor/ cancer growth . leukemia or a B cell malignancy , or a cancer that expresses [0288 ] Desirable effects of treatment include, but are not CD19 , or non - Hodgkin ' s lymphoma (NHL ) or mantle cell limited to , preventing occurrence or recurrence of disease , lymphoma (MCL ) or acute lymphoblastic leukemia (ALL ) alleviation of symptoms, diminishment of any direct or or chronic lymphocytic leukemia (CLL ) or rituximab - or indirect pathological consequences of the disease , prevent CHOP (CytoxanTM /AdriamycinTM vincristine/ prednisone ing metastasis , decreasing the rate of disease progression , therapy ) - resistant B cell cancer , or a blinatumomab - resistant amelioration or palliation of the disease state , and remission or refractory B cell cancer . In some embodiments , the cancer or improved prognosis . In some embodiments , construct is a solid tumor. In some embodiments , the cancer is a constructs described herein are used to delay development of non - inflammatory tumor that is not easily infiltrated with a disease or to slow the progression of a disease . lymphocytes. [ 0289] The term “ effective amount” as used herein refers [0295 ] In a further aspect , the antigen -binding constructs to that amount of construct being administered , which will and drug - conjugated antigen -binding constructs described accomplish the goal of the recited method , e . g ., relieve to herein are for use in the manufacture or preparation of a some extent one or more of the symptoms of the disease , medicament. In one embodiment, the medicament is for condition or disorder being treated . The amount of the treatment of cancer . In certain embodiments , the medica composition described herein which will be effective in the ment is for the treatment of lymphoma or leukemia . In other treatment, inhibition and prevention of a disease or disorder embodiments , the medicament is for the treatment of cancer associated with aberrant expression and / or activity of a described above . In another embodiment, the medicament is therapeutic protein can be determined by standard clinical for use in a method of treating cancer comprising adminis techniques. In addition , in vitro assays may optionally be tering to patient having cancer, an effective amount of the employed to help identify optimal dosage ranges . The pre medicament. cise dose to be employed in the formulation will also depend [0296 ] In certain embodiments , the methods and uses on the route of administration , and the seriousness of the described herein further comprise administering to the disease or disorder, and should be decided according to the patient an effective amount of at least one additional thera judgment of the practitioner and each patient' s circum peutic agent, e . g ., cytotoxic agents , chemotherapeutic stances . Effective doses are extrapolated from dose - response agents , cytokines , growth inhibitory agents , kinase inhibi curves derived from in vitro or animal model test systems. tors, anti - angiogenic agents , cardioprotectants , immunos timulatory agents , immunosuppressive agents , protein tyro Therapeutic Uses : sine kinase (PTK ) inhibitors , other antibodies , Fc fusions, or [ 0290 ] In an aspect , the antigen -binding constructs and immunoglobulins , or other therapeutic agents . drug -conjugated antigen -binding constructs described 10297 ] In certain embodiments , the additional therapeutic herein are used in antibody - based therapies which involve agent is for preventing and / or treating cancer . Such combi administering the antigen - binding constructs , or nucleic nation therapy encompasses combined administration acids encoding antigen -binding constructs to a patient for (where two or more therapeutic agents are included in the treating one or more diseases , disorders , or conditions. Such same or separate formulations ) , and separate administration , disorders , diseases and conditions may include, but are not in which case , administration of the antigen - binding con limited to , cancer ( hematological, solid tumor or metastatic ) , struct described herein can occur prior to , simultaneously , autoimmune diseases , inflammatory diseases, and diseases and / or following , administration of the additional therapeu caused by pathogen such as viruses , bacteria , parasites or tic agent and / or adjuvant. US 2018 /0193477 A1 Jul. 12 , 2018 34

[ 0298 ] The antigen - binding constructs and drug - conju - administering a protein , including an antibody , of the inven gated antigen -binding constructs described herein may be tion , care must be taken to use materials to which the protein administered alone or in combination with other types of does not absorb . treatments ( e. g ., radiation therapy , chemotherapy, hormonal 10305 ]. In another embodiment, the antigen - binding con therapy , immunotherapy and anti - tumor agents ) . structs or composition can be delivered in a vesicle , in [0299 ] Demonstration of Therapeutic or Prophylactic particular a liposome ( see Langer, Science 249: 1527 - 1533 Activity : (1990 ) ; Treat et al. , in Liposomes in the Therapy of Infec tious Disease and Cancer, Lopez -Berestein and Fidler ( eds . ) , [0300 ] The drug - conjugated antigen -binding constructs or Liss , New York , pp . 353 - 365 ( 1989 ) ; Lopez -Berestein , ibid . , pharmaceutical compositions described herein are tested in pp . 317 - 327 ; see generally ibid .) vitro , and then in vivo for the desired therapeutic or pro [0306 ] In yet another embodiment, the antigen -binding phylactic activity , prior to use in humans. For example , in constructs or composition can be delivered in a controlled vitro assays to demonstrate the therapeutic or prophylactic release system . In one embodiment, a pump may be used utility of a compound or pharmaceutical composition ( see Langer , supra ; Sefton , CRC Crit. Ref. Biomed . Eng . include , the effect of a compound on a cell line or a patient 14 : 201 ( 1987 ) ; Buchwald et al. , Surgery 88 : 507 ( 1980 ) ; tissue sample . The effect of the compound or composition on Saudek et al. , N . Engl. J. Med . 321 :574 ( 1989 )) . In another the cell line and / or tissue sample can be determined utilizing embodiment, polymeric materials can be used (see Medical techniques known to those of skill in the art including , but Applications of Controlled Release , Langer and Wise ( eds . ) , not limited to , rosette formation assays and cell lysis assays . CRC Pres . , Boca Raton , Fla . ( 1974 ) ; Controlled Drug Bio [ 0301 ] Therapeutic /Prophylactic Administration and availability , Drug Product Design and Performance , Smolen Composition : and Ball ( eds. ) , Wiley , New York ( 1984 ) ; Ranger and Peppas, J . , Macromol. Sci. Rev . Macromol. Chem . 23 :61 0302 ] Provided are methods of treatment, inhibition and ( 1983 ); see also Levy et al. , Science 228 : 190 ( 1985 ) ; During prophylaxis by administration to a subject of an effective et al. , Ann Neurol. 25 :351 (1989 ) ; Howard et al. , J . Neuro amount of an antigen -binding construct or pharmaceutical surg . 71: 105 (1989 ) ) . In yet another embodiment, a con composition described herein . In an embodiment, the anti trolled release system can be placed in proximity of the gen -binding construct is substantially purified ( e. g ., substan therapeutic target , e . g . , the brain , thus requiring only a tially free from substances that limit its effect or produce fraction of the systemic dose ( see , e . g ., Goodson , in Medical undesired side - effects ) . In certain embodiments , the subject Applications of Controlled Release , supra , vol. 2 , pp . 115 is an animal, including but not limited to animals such as 138 ( 1984 )) . cows, pigs , horses , chickens, cats , dogs, etc . , and in certain [0307 ] Other controlled release systems are discussed in embodiments, a mammal, and most preferably human . the review by Langer (Science 249: 1527 - 1533 ( 1990 ) ) . [0303 ] Various delivery systems are known and can be used to administer an antigen -binding construct formulation Kits and Articles of Manufacture described herein , e. g ., encapsulation in liposomes, micropar [ 0308 ] Also described herein are kits comprising one or ticles, microcapsules, recombinant cells capable of express more antigen - binding constructs described herein . Indi ing the antigen -binding constructs , receptor -mediated endo vidual components of the kit would be packaged in separate cytosis (see , e . g . , Wu and Wu , J. Biol. Chem . 262: 4429 - 4432 containers and , associated with such containers , can be a ( 1987 ) ) , construction of a nucleic acid as part of a retroviral notice in the form prescribed by a governmental agency or other vector, etc . Methods of introduction include but are regulating the manufacture , use or sale of pharmaceuticals or not limited to intradermal, intramuscular, intraperitoneal, biological products , which notice reflects approval by the intravenous , subcutaneous, intranasal, epidural, and oral agency of manufacture, use or sale . The kit may optionally routes . The antigen -binding constructs may be administered contain instructions or directions outlining the method of use by any convenient route , for example by infusion or bolus or administration regimen for the antigen -binding construct , injection , by absorption through epithelial or mucocutane sometimes referred to as a " package insert ” . ous linings ( e . g ., oral mucosa , rectal and intestinal mucosa , [0309 . When one or more components of the kit are etc . ) and may be administered together with other therapeu provided as solutions , for example an aqueous solution , or a tic agents . Administration can be systemic or local. Suitable sterile aqueous solution , the container means may itself be routes of administration include intraventricular and intrath an inhalant, syringe , pipette , eye dropper, or other such like ecal injection ; intraventricular injection may be facilitated apparatus, from which the solution may be administered to by an intraventricular catheter, for example , attached to a a subject or applied to and mixed with the other components reservoir , such as an Ommaya reservoir. Pulmonary admin of the kit . istration can also be employed , e . g . , by use of an inhaler or [0310 ] The components of the kit may also be provided in nebulizer , and formulation with an aerosolizing agent. dried or lyophilized form and the kit can additionally contain [ 0304 ] In a specific embodiment , it is desirable to admin a suitable solvent for reconstitution of the lyophilized com ister the antigen -binding constructs, or compositions ponents . Irrespective of the number or type of containers , the described herein locally to the area in need of treatment; this kits described herein also may comprise an instrument for may be achieved by , for example , and not by way of assisting with the administration of the composition to a limitation , local infusion during surgery , topical application , patient. Such an instrument may be an inhalant, nasal spray e . g . , in conjunction with a wound dressing after surgery , by device, syringe , pipette , forceps, measured spoon , eye drop injection , by means of a catheter , by means of a suppository, per or similar medically approved delivery vehicle . or by means of an implant, said implant being of a porous , [0311 ] In another aspect described herein , an article of non - porous , or gelatinous material, including membranes , manufacture containing materials useful for the treatment, such as sialastic membranes, or fibers . Preferably , when prevention and /or diagnosis of the disorders described above US 2018 /0193477 A1 Jul. 12 , 2018 35

is provided . The article of manufacture comprises a con glutamine , glutamic acid , glycine , histidine , isoleucine , leu tainer and a label or package insert on or associated with the cine, lysine , methionine, phenylalanine , praline , serine , container . Suitable containers include , for example , bottles, threonine , tryptophan , tyrosine , and valine ) and pyrrolysine vials , syringes , IV solution bags , etc . The containers may be and selenocysteine . Amino acid analogs refers to com formed from a variety of materials such as glass or plastic . pounds that have the same basic chemical structure as a The container holds a composition which is by itself or naturally occurring amino acid , i. e ., an a carbon that is combined with another composition effective for treating , bound to a hydrogen , a carboxyl group , an amino group , and preventing and / or diagnosing the condition and may have a an R group , such as, homoserine, norleucine, methionine sterile access port (for example the container may be an sulfoxide, methionine methyl sulfonium . Such analogs have intravenous solution bag or a vial having a stopper pierce modified R groups ( such as , norleucine ) or modified peptide able by a hypodermic injection needle ) . At least one active backbones, but retain the samebasic chemical structure as a agent in the composition is a T cell activating antigen naturally occurring amino acid . Reference to an amino acid binding construct described herein . The label or package includes , for example , naturally occurring proteogenic insert indicates that the composition is used for treating the L - amino acids ; D - amino acids, chemically modified amino condition of choice . Moreover , the article of manufacture acids such as amino acid variants and derivatives , naturally may comprise ( a ) a first container with a composition occurring non - proteogenic amino acids such as B - alanine , contained therein , wherein the composition comprises an ornithine , etc . , and chemically synthesized compounds hav antigen - binding construct described herein ; and ( b ) a second ing properties known in the art to be characteristic of amino container with a composition contained therein , wherein the acids . Examples of non - naturally occurring amino acids composition comprises a further cytotoxic or otherwise include , but are not limited to , a -methyl amino acids (e . g . therapeutic agent. The article ofmanufacture in this embodi a -methyl alanine ) , D - amino acids , histidine - like amino ment described herein may further comprise a package insert acids ( e . g . , 2 - amino -histidine , B -hydroxy -histidine , homo indicating that the compositions can be used to treat a histidine ) , amino acids having an extra methylene in the side particular condition . Alternatively , or additionally , the article chain ( “ homo " amino acids ) , and amino acids in which a of manufacture may further comprise a second ( or third ) carboxylic acid functional group in the side chain is replaced container comprising a pharmaceutically -acceptable buffer , with a sulfonic acid group ( e . g . , cysteic acid ). The incorpo such as bacteriostatic water for injection (BWFI ) , phos ration of non - natural amino acids, including synthetic non phate -buffered saline , Ringer ' s solution and dextrose solu native amino acids, substituted amino acids , or one or more tion . It may further include other materials desirable from a D - amino acids into the proteins of the present invention may commercial and user standpoint, including other buffers , be advantageous in a number of different ways . D - amino diluents , filters , needles, and syringes . acid -containing peptides, etc ., exhibit increased stability in vitro or in vivo compared to L - amino acid - containing coun Polypeptides and Polynucleotides terparts . Thus, the construction of peptides , etc ., incorporat [0312 ] The antigen -binding constructs described herein ing D -amino acids can be particularly useful when greater comprise at least one polypeptide . Also described are poly intracellular stability is desired or required . More specifi nucleotides encoding the polypeptides described herein . The cally, D - peptides, etc ., are resistant to endogenous pepti polypeptides and polynucleotides are typically isolated . dases and proteases , thereby providing improved bioavail [ 0313 ] As used herein , “ isolated ” means an agent ( e .g ., a ability of the molecule , and prolonged lifetimes in vivo polypeptide or polynucleotide ) that has been identified and when such properties are desirable . Additionally , D - pep separated and / or recovered from a component of its natural tides , etc . , cannot be processed efficiently for major histo cell culture environment. Contaminant components of its compatibility complex class II - restricted presentation to T natural environment are materials that would interfere with helper cells , and are therefore , less likely to induce humoral diagnostic or therapeutic uses for the antigen -binding con immune responses in the whole organism . struct , and may include enzymes, hormones , and other [0316 ] Amino acids may be referred to herein by either proteinaceous or non - proteinaceous solutes . Isolated also their commonly known three letter symbols or by the refers to an agent that has been synthetically produced , e . g . , one - letter symbols recommended by the IUPAC - IUB Bio via human intervention . chemical Nomenclature Commission . Nucleotides, likewise , [0314 ] The terms " polypeptide, ” “ peptide” and “ protein " may be referred to by their commonly accepted single - letter are used interchangeably herein to refer to a polymer of codes. amino acid residues . That is , a description directed to a [03171 Also described herein are polynucleotides encod polypeptide applies equally to a description of a peptide and ing polypeptides of the antigen -binding constructs. The term a description of a protein , and vice versa . The terms apply " polynucleotide” or “ nucleotide sequence ” is intended to to naturally occurring amino acid polymers as well as amino indicate a consecutive stretch of two or more nucleotide acid polymers in which one or more amino acid residues is molecules. The nucleotide sequence may be of genomic , a non -naturally encoded amino acid . As used herein , the cDNA , RNA , semisynthetic or synthetic origin , or any terms encompass amino acid chains of any length , including combination thereof. full length proteins , wherein the amino acid residues are [0318 ] The term “ nucleic acid ” refers to deoxyribonucle linked by covalent peptide bonds . otides, deoxyribonucleosides, ribonucleosides, or ribonucle [ 0315 ] The term “ amino acid ” refers to naturally occurring otides and polymers thereof in either single - or double and non -naturally occurring amino acids, as well as amino stranded form . Unless specifically limited , the term acid analogs and amino acid mimetics that function in a encompasses nucleic acids containing known analogues of manner similar to the naturally occurring amino acids . natural nucleotides which have similar binding properties as Naturally encoded amino acids are the 20 common amino the reference nucleic acid and are metabolized in a manner acids ( alanine, arginine, asparagine, aspartic acid , cysteine , similar to naturally occurring nucleotides. Unless specifi US 2018 /0193477 A1 Jul. 12 , 2018 36 cally limited otherwise , the term also refers to oligonucle ton , Proteins: Structures and Molecular Properties ( W H otide analogs including PNA (peptidonucleic acid ), analogs Freeman & Co . ; 2nd edition ( December 1993 ) of DNA used in antisense technology ( phosphorothioates, [0322 ] The terms “ identical ” or percent “ identity ,” in the phosphoroamidates, and the like ) . Unless otherwise indi context of two or more nucleic acids or polypeptide cated , a particular nucleic acid sequence also implicitly sequences , refer to two or more sequences or subsequences encompasses conservatively modified variants thereof (in that are the same. Sequences are " substantially identical” if cluding but not limited to , degenerate codon substitutions ) they have a percentage of amino acid residues or nucleotides and complementary sequences as well as the sequence that are the same ( i . e ., about 60 % identity , about 65 % , about explicitly indicated . Specifically , degenerate codon substi 70 % , about 75 % , about 80 % , about 85 % , about 90 % , or tutions may be achieved by generating sequences in which about 95 % identity over a specified region ), when compared the third position of one or more selected (or all) codons is and aligned for maximum correspondence over a compari substituted with mixed -base and /or deoxyinosine residues son window , or designated region as measured using one of (Batzer et al. , Nucleic Acid Res . 19 : 5081 ( 1991 ); Ohtsuka et the following sequence comparison algorithms ( or other al. , J. Biol. Chem . 260 : 2605 - 2608 ( 1985 ); Rossolini et al ., algorithms available to persons of ordinary skill in the art ) Mol. Cell. Probes 8 :91 - 98 ( 1994 ) ) . or by manual alignment and visual inspection . This defini [ 0319] " Conservatively modified variants ” applies to both tion also refers to the complement of a test sequence . The amino acid and nucleic acid sequences. With respect to identity can exist over a region that is at least about 50 amino particular nucleic acid sequences , “ conservatively modified acids or nucleotides in length , or over a region that is 75 - 100 variants " refers to those nucleic acids which encode identi amino acids or nucleotides in length , or, where not specified , cal or essentially identical amino acid sequences , or where across the entire sequence of a polynucleotide or polypep the nucleic acid does not encode an amino acid sequence, to tide . A polynucleotide encoding a polypeptide of the present essentially identical sequences . Because of the degeneracy invention , including homologs from species other than of the genetic code , a large number of functionally identical human , may be obtained by a process comprising the steps nucleic acids encode any given protein . For instance , the of screening a library under stringent hybridization condi codons GCA , GCC , GCG and GCU all encode the amino tions with a labeled probe having a polynucleotide sequence acid alanine . Thus , at every position where an alanine is described herein or a fragment thereof, and isolating full specified by a codon , the codon can be altered to any of the length cDNA and genomic clones containing said polynucle corresponding codons described without altering the otide sequence . Such hybridization techniques are well encoded polypeptide . Such nucleic acid variations are known to the skilled artisan . " silent variations, ” which are one species of conservatively [0323 ] For sequence comparison , typically one sequence modified variations . Every nucleic acid sequence herein acts as a reference sequence , to which test sequences are which encodes a polypeptide also describes every possible compared . When using a sequence comparison algorithm , silent variation of the nucleic acid . One of ordinary skill in test and reference sequences are entered into a computer, the art will recognize that each codon in a nucleic acid subsequence coordinates are designated , if necessary , and ( except AUG , which is ordinarily the only codon for methio sequence algorithm program parameters are designated . nine , and TGG , which is ordinarily the only codon for Default program parameters can be used , or alternative tryptophan ) can be modified to yield a functionally identical parameters can be designated . The sequence comparison molecule . Accordingly , each silent variation of a nucleic algorithm then calculates the percent sequence identities for acid which encodes a polypeptide is implicit in each the test sequences relative to the reference sequence , based described sequence . on the program parameters . [0320 ] As to amino acid sequences, one of ordinary skill [ 0324 ] A " comparison window ” , as used herein , includes in the art will recognize that individual substitutions, dele reference to a segment of any one of the number of con tions or additions to a nucleic acid , peptide , polypeptide , or tiguous positions selected from the group consisting of from protein sequence which alters, adds or deletes a single amino 20 to 600 , usually about 50 to about 200 , more usually about acid or a small percentage of amino acids in the encoded 100 to about 150 in which a sequence may be compared to sequence is a “ conservatively modified variant” where the a reference sequence of the same number of contiguous alteration results in the deletion of an amino acid , addition positions after the two sequences are optimally aligned . of an amino acid , or substitution of an amino acid with a Methods of alignment of sequences for comparison are chemically similar amino acid . Conservative substitution known to those of ordinary skill in the art . Optimal align tables providing functionally similar amino acids are known ment of sequences for comparison can be conducted , includ to those of ordinary skill in the art . Such conservatively ing but not limited to , by the local homology algorithm of modified variants are in addition to and do not exclude Smith and Waterman ( 1970 ) Adv. Appl. Math . 2 :482c , by the polymorphic variants , interspecies homologs , and alleles homology alignment algorithm of Needleman and Wunsch described herein . (1970 ) J . Mol. Biol. 48 :443 , by the search for similarity [ 0321] Conservative substitution tables providing func method of Pearson and Lipman ( 1988 ) Proc. Nat' l . Acad . tionally similar amino acids are known to those of ordinary Sci. USA 85 : 2444 , by computerized implementations of skill in the art . The following eight groups each contain these algorithms (GAP , BESTFIT , FASTA , and TFASTA in amino acids that are conservative substitutions for one the Wisconsin Genetics Software Package , Genetics Com another: 1 ) Alanine ( A ) , Glycine ( G ) ; 2 ) Aspartic acid ( D ) , puter Group , 575 Science Dr. , Madison , Wis . ) , or by manual Glutamic acid ( E ); 3) Asparagine (N ) , Glutamine ( Q ); 4 ) alignment and visual inspection ( see , e . g ., Ausubel et al. , Arginine ( R ) , Lysine ( K ); 5 ) Isoleucine ( I ) , Leucine ( L ), Current Protocols in Molecular Biology ( 1995 supplement ) ) . Methionine ( M ), Valine ( V ) ; 6 ) Phenylalanine ( F ) , Tyrosine [ 0325 ] One example of an algorithm that is suitable for ( Y ) , Tryptophan ( W ) ; 7 ) Serine ( S ) , Threonine ( T ) ; and determining percent sequence identity and sequence simi T0139 8 ) Cysteine ( C ) , Methionine ( M ) (see , e . g . , Creigh - larity are the BLAST and BLAST 2 . 0 algorithms, which are US 2018 /0193477 A1 Jul. 12 , 2018 37 described in Altschul et al. ( 1997 ) Nuc . Acids Res. 25 :3389 example , a recombinant polynucleotide encoding a polypep 3402 , and Altschul et al. ( 1990 ) J . Mol. Biol. 215 : 403 - 410 , tide contained in a vector is considered isolated . Further respectively . Software for performing BLAST analyses is examples of an isolated polynucleotide include recombinant publicly available through the National Center for Biotech polynucleotides maintained in heterologous host cells or nology Information available at the World Wide Web at purified (partially or substantially ) polynucleotides in solu ncbi. nlm .nih .gov . The BLAST algorithm parameters W , T , tion . An isolated polynucleotide includes a polynucleotide and X determine the sensitivity and speed of the alignment. molecule contained in cells that ordinarily contain the poly The BLASTN program ( for nucleotide sequences ) uses as nucleotide molecule , but the polynucleotide molecule is defaults a wordlength ( W ) of 11, an expectation ( E ) or 10 , present extrachromosomally or at a chromosomal location M = 5 , N = - 4 and a comparison of both strands. For amino that is different from its natural chromosomal location . acid sequences , the BLASTP program uses as defaults a Isolated RNA molecules include in vivo or in vitro RNA wordlength of 3 , and expectation ( E ) of 10 , and the BLO transcripts , as well as positive and negative strand forms, SUM62 scoring matrix ( see Henikoff and Henikoff ( 1992 ) and double -stranded forms. Isolated polynucleotides or Proc . Natl. Acad. Sci. USA 89: 10915) alignments ( B ) of 50 , nucleic acids described herein , further include such mol expectation ( E ) of 10 , M = 5 , N = - 4 , and a comparison of both ecules produced synthetically , e . g. , via PCR or chemical strands . The BLAST algorithm is typically performed with synthesis . In addition , a polynucleotide or a nucleic acid , in the “ low complexity ” filter turned off . certain embodiments , include a regulatory element such as [0326 ] The BLAST algorithm also performs a statistical a promoter , ribosome binding site , or a transcription termi analysis of the similarity between two sequences ( see , e . g ., nator . Karlin and Altschul ( 1993 ) Proc . Natl. Acad . Sci. USA [0331 ] The term “ polymerase chain reaction ” or “ PCR ” 90 : 5873 - 5787 ) . One measure of similarity provided by the generally refers to a method for amplification of a desired BLAST algorithm is the smallest sum probability (P (N ) ), nucleotide sequence in vitro , as described , for example, in which provides an indication of the probability by which a U .S . Pat . No . 4 ,683 , 195 . In general, the PCR method match between two nucleotide or amino acid sequences involves repeated cycles of primer extension synthesis , would occur by chance . For example , a nucleic acid is using oligonucleotide primers capable of hybridising pref considered similar to a reference sequence if the smallest erentially to a template nucleic acid . sum probability in a comparison of the test nucleic acid to [0332 ] By a nucleic acid or polynucleotide having a the reference nucleic acid is less than about 0 . 2 , or less than nucleotide sequence at least , for example , 95 % " identical” to about 0 .01 , or less than about 0 .001 . a reference nucleotide sequence of the present invention , it [ 0327] The phrase " selectively ( or specifically ) hybridizes is intended that the nucleotide sequence of the polynucle to ” refers to the binding , duplexing , or hybridizing of a otide is identical to the reference sequence except that the molecule only to a particular nucleotide sequence under polynucleotide sequence may include up to five point muta stringent hybridization conditions when that sequence is tions per each 100 nucleotides of the reference nucleotide present in a complex mixture ( including but not limited to , sequence . In other words, to obtain a polynucleotide having total cellular or library DNA or RNA ). a nucleotide sequence at least 95 % identical to a reference [0328 ] The phrase " stringent hybridization conditions” nucleotide sequence , up to 5 % of the nucleotides in the refers to hybridization of sequences of DNA , RNA , or other reference sequence may be deleted or substituted with nucleic acids, or combinations thereof under conditions of another nucleotide , or a number of nucleotides up to 5 % of low ionic strength and high temperature as is known in the the total nucleotides in the reference sequence may be art . Typically, under stringent conditions a probe will hybrid inserted into the reference sequence . These alterations of the ize to its target subsequence in a complex mixture ofnucleic reference sequence may occur at the 5 ' or 3 ' terminal acid ( including but not limited to , total cellular or library positions of the reference nucleotide sequence or anywhere DNA or RNA ) but does not hybridize to other sequences in between those terminal positions , interspersed either indi the complex mixture . Stringent conditions are sequence - vidually among residues in the reference sequence or in one dependent and will be different in different circumstances . or more contiguous groups within the reference sequence . Longer sequences hybridize specifically at higher tempera As a practical matter, whether any particular polynucleotide tures . An extensive guide to the hybridization of nucleic sequence is at least 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % acids is found in Tijssen , Laboratory Techniques in Bio or 99 % identical to a nucleotide sequence of the present chemistry and Molecular Biology - Hybridization with invention can be determined conventionally using known Nucleic Probes , “ Overview of principles of hybridization computer programs, such as the ones discussed above for and the strategy of nucleic acid assays” ( 1993 ). polypeptides ( e . g . ALIGN - 2 ) . [ 0329 ] As used herein , the terms " engineer , engineered , [0333 ] A derivative, or a variant of a polypeptide is said to engineering ” , are considered to include any manipulation of share “ homology” or be “ homologous” with the peptide if the peptide backbone or the post - translational modifications the amino acid sequences of the derivative or variant has at of a naturally occurring or recombinant polypeptide or least 50 % identity with a 100 amino acid sequence from the fragment thereof. Engineering includes modifications of the original peptide . In certain embodiments, the derivative or amino acid sequence, of the glycosylation pattern , or of the variant is at least 75 % the same as that of either the peptide side chain group of individual amino acids, as well as or a fragment of the peptide having the same number of combinations of these approaches . The engineered proteins amino acid residues as the derivative . In certain embodi are expressed and produced by standard molecular biology ments , the derivative or variant is at least 85 % the same as techniques. that of either the peptide or a fragment of the peptide having [ 0330 ] By “ isolated nucleic acid molecule or polynucle the same number of amino acid residues as the derivative . In otide” is intended a nucleic acid molecule , DNA or RNA , certain embodiments , the amino acid sequence of the deriva which has been removed from its native environment. For tive is at least 90 % the same as the peptide or a fragment of US 2018 /0193477 A1 Jul. 12 , 2018 38

the peptide having the same number of amino acid residues described herein are disclosed by the present application to as the derivative . In some embodiments , the amino acid the same extent as if each single chain polypeptide or sequence of the derivative is at least 95 % the same as the heterodimer were set forth individually . Thus, selection of peptide or a fragment of the peptide having the same number particular components to form individual single chain poly of amino acid residues as the derivative . In certain embodi peptides or heterodimers is within the scope of the present ments , the derivative or variant is at least 99 % the same as disclosure that of either the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative . [ 0340 ] The section headings used herein are for organiza [0334 ] The term “ modified ,” as used herein refers to any tional purposes only and are not to be construed as limiting changes made to a given polypeptide, such as changes to the the subject matter described . length of the polypeptide , the amino acid sequence , chemi [0341 ] It is to be understood that the methods and com cal structure , co - translational modification , or post - transla tional modification of a polypeptide. The form “ (modified )" positions described herein are not limited to the particular term means that the polypeptides being discussed are option methodology, protocols , cell lines, constructs , and reagents ally modified , that is , the polypeptides under discussion can described herein and as such may vary . It is also to be be modified or unmodified . understood that the terminology used herein is for the [ 0335 ]. In someaspects , an antigen - binding construct com purpose of describing particular embodiments only , and is prises an amino acids sequence that is at least 80 , 85 , 90 , 91 , not intended to limit the scope of the methods and compo 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , or 100 % identical to a relevant sitions described herein , which will be limited only by the amino acid sequence or fragment thereof set forth in the appended claims. Table ( s ) or accession number( s ) disclosed herein . In some [ 0342 ] All documents , or portions of documents , cited in aspects , an isolated antigen - binding construct comprises an the application including , but not limited to , patents , patent amino acids sequence encoded by a polynucleotide that is at applications , articles , books, manuals , and treatises are least 80 , 85 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , or 100 % hereby expressly incorporated by reference in their entirety identical to a relevant nucleotide sequence or fragment for any purpose . All publications and patents mentioned thereof set forth in Table ( s ) or accession number ( s ) disclosed herein . herein are incorporated herein by reference in their entirety [ 0336 ] Unless defined otherwise , all technical and scien for the purpose of describing and disclosing , for example , tific terms used herein have the same meaning as is com the constructs and methodologies that are described in the monly understood by one of skill in the art to which the publications, which might be used in connection with the claimed subject matter belongs . In the event that there are a methods, compositions and compounds described herein . plurality of definitions for terms herein , those in this section The publications discussed herein are provided solely for prevail. Where reference is made to a URL or other such their disclosure prior to the filing date of the present appli identifier or address, it is understood that such identifiers can cation . Nothing herein is to be construed as an admission change and particular information on the internet can come that the inventors described herein are not entitled to ante and go , but equivalent information can be found by search date such disclosure by virtue of prior invention or for any ing the internet. Reference thereto evidences the availability other reason . and public dissemination of such information . Terms under stood by those in the art of antibody technology are each EXAMPLES given the meaning acquired in the art , unless expressly defined differently herein . [0343 ] The following specific and non -limiting examples [0337 ] It is to be understood that the general description are to be construed as merely illustrative , and do not limit the and following detailed description are exemplary and explanatory only and are not restrictive of any subject matter present disclosure in any way whatsoever. Without further claimed . elaboration , it is believed that one skilled in the art can , [0338 ] In this application , the use of the singular includes based on the description herein , utilize the present disclosure the plural unless specifically stated otherwise . to its fullest extent. All publications cited herein are hereby [ 0339 ] In the present description , any concentration range , incorporated by reference in their entirety. Where reference percentage range , ratio range , or integer range is to be is made to a URL or other such identifier or address , it is understood to include the value of any integer within the understood that such identifiers can change and particular recited range and , when appropriate , fractions thereof ( such information on the internet can come and go , but equivalent as one tenth and one hundredth of an integer ) , unless information can be found by searching the internet . Refer otherwise indicated . As used herein , " about” means + 10 % of ence thereto evidences the availability and public dissemi the indicated range , value , sequence , or structure , unless nation of such information . otherwise indicated . It should be understood that the terms [0344 ] Exemplary bispecific anti - CD3 - CD19 , anti -CD3 “ a ” and “ an ” as used herein refer to “ one or more ” of the CDH3 , anti -CD3 -HER2 , anti -CD3 -HER3 and anti -CD3 enumerated components unless otherwise indicated or dic EGFR antigen -binding constructs were made as described tated by its context. The use of the alternative ( e . g ., " or” ) below . An exemplary schematic representation of these type should be understood to mean either one , both , or any of constructs is shown in FIGS . 1A - D . All formats are based combination thereof of the alternatives . As used herein , the on the heterodimeric Fc constructed by known mutations in terms “ include ” and “ comprise ” are used synonymously . In the CH3 domain (Von Kreudenstein et al. , MAbs. 2013 addition , it should be understood that the individual single 5 (5 ) :646 - 54 ) . Exemplary constructs were conjugated to chain polypeptides or immunoglobulin constructs derived drugs to make ADCs using exemplary drugs DM1, DM4 and from various combinations of the structures and substituents MMAE . US 2018 /0193477 A1 Jul. 12 , 2018 39

Example 1 . Description , Expression and [ 0353 ] (VLVH SS ) or (VHVL SS ) indicates disulfide Purification of Bi- Specific Anti -CD19 - CD3 stabilized scFv utilizing the published positions VH 44 Antigen - Binding Constructs Useful for ADCs in and VL 100 , according to the Kabat numbering system , Dual scFv Format to introduce a disulphide link between the VH and VL of the scFv [Reiter et al. , Nat . Biotechnol . 14 : 1239 [0345 ) Bispecific antibodies against CD3 and CD19 were 1245 (1996 ) ]. designed , expressed and characterized as described in PCT / [ 0354 ] (CDR C -> S ) - indicates a mutation in the H3 US2015 /011664 . Briefly , the genes encoding the antibody CDR of OKT3 as referenced below heavy and light chains were constructed via gene synthesis [0355 ] (VHVL linker) - indicates VH and VL con using codons optimized for human /mammalian expression . nected by the linker SSTGGGGSGGGGSGGGGSDI. The scFv -Fc sequences were generated from a known anti [0356 ] Fc numbering is according to EU index as in Kabat CD3 and CD19 scFv BiTETM antibody (Kipriyanov et . al. , referring to the numbering of the EU antibody ( Edelman et 1998 , Int. J Cancer: 77 , 763- 772 ) , anti - CD3 monoclonal al. , 1969, Proc Natl Acad Sci USA 63 :78 - 85 ) ; Fab or antibody OKT3 (Drug Bank reference : DB00075 ) . The dual variable domain numbering is according to Kabat (Kabat scFv variants made are described in Table 1 . and Wu , 1991; Kabat et al, Sequences of proteins of immu nological interest . 5th Edition — US Department of Health TABLE 1 and Human Services , NIH publication no . 91 - 3242 , p 647 Dual scFv variants ( 1991 ) ) . [0357 ) The variants described in Table 1 include variant Variant Chain A Chain B Fc 875 , a preliminary design , which was used as a starting point 873 aCD19 _ HD37 scFv aCD3 Het Fc 1 to generate antigen -binding constructs with improved yield (blinatumomab ) scFv and biophysical properties. The modifications include sta 875 aCD19 _ HD37 scFv ACD3 _ OKT3 scFv Het Fc 1 1661 aCD19 _ HD37 scFv aCD3 _ OKT3 scFv Het Fc 2 ; bilization of the scFv by VLVH disulfide engineering and /or FcYR KO 2 adding stabilizing CDR mutations . All variants include a 1653 aCD19 _ HD37 scFv ACD3 _ OKT3 scFv Het Fc 2 heterodimeric Fc (Het Fc 1 or Het Fc 2 ) and can be (CDR C - > S ) expressed with or without mutations in the CH2 domain 1662 aCD19 _ HD37 scFv ACD3 _ OKT3 scFv Het Fc 2 ; ( FcYR KO 1 or FcYR KO 2 ) to abolish Fc effector activity . (CDR C - > S ) FcyR KO 2 1660 aCD3 _ OKT3 scFv aCD19 _ HD37 scFv Het Fc 2 Variants including this modification to the Fc are referred to ( VHVL linker ) as having an Fc knockout or Fc KO . 1666 aCD3 _ OKT3 scFv aCD19 _ HD37 scFv Het Fc 2 ; [ 0358 ] Variants 875 , 1661, 1653 , 1662 , 1660 , 1666 , 1801, (VHVL linker ) FcYR KO 2 and 1380 are initial designs of the CD3- CD19 antigen 1801 aCD19 _ HD37 scFv ACD3_ OKT3 scFv Het Fc 2 ( VLVH SS ) binding constructs developed , while variants 6747 , 10149 , N1 aCD19 _ HD37 scFv ACD3 _ OKT3 scFv Het Fc 2 ; and 12043 exemplify designs that include modifications ( VLVH SS ) FcYR KO 2 designed to further improve yield and biophysical properties 6747 aCD19 _ HD37 scFv ACD3 _ OKT3 scFv Het Fc 2 (VLVH SS ) (VLVH SS ) of the CD3 -CD19 antigen - binding constructs (see Example 10149 aCD19 _ HD37 scFv ACD3 _ OKT3 scFv Het Fc 2 ; 3 - 4 for additional details ) . Variants N1, N3 and N10 have (VLVH SS ) (VLVH SS ) FcYR KO 2 also been designed and the biophysical and functional N3 aCD19 _ HD37 scFv ACD3 _ OKT3 scFv Het Fc 2 characteristics of these variants can be predicted from the (VLVH SS ) (CDR C - > S ) ( VLVH data provided herein . SS) 10150 aCD19 _ HD37 scFv aCD3 _ OKT3 scFv Het Fc 2 ; [0359 ] The VHVL disulfide engineering strategy for both ( VLVH SS ) (CDR C - > S ) (VLVH FcyR KO 2 the CD3 and CD19 scFvs utilized the published positions SS ) VH 44 and VL 100 , according to the Kabat numbering 1380 aCD19 _ HD37 scFv ACD3 _ BiTE scFv Het Fc 2 ; FcYR KO 1 system , to introduce a disulphide link between the VH and N10 aCD19 _ HD37 scFv, ACD3 _ OKT3 scFv Het Fc 2 VL of the scFv [Reiter et al ., Nat. Biotechnol. 14 : 1239 - 1245 humanized (VLVH SS ) (VLVH SS ) ( 1996 ) ] . The mutation of C to S in the H3 CDR of aCD3 OKT3 scFv was generated as described in Kipryanov et al. , in Protein Engineering 10 : 445 -453 ( 1997 ) . [0346 ] Het Fc 1 = Chain A : L351Y _ F405A _ Y407V ; [0360 ] The final gene products were sub - cloned into the Chain B : 1366L _ K392M _ T394W (EU numbering sys mammalian expression vector PTT5 (NRC - BRI, Canada ) tem for IgG1 Fc ) and expressed in CHO cells (Durocher , Y . , Perret, S . & [0347 ] Het Fc 2 = Chain A : T350V _ L351Y _ F405A _ Kamen , A . High -level and high - throughput recombinant Y407V ; Chain B : T350V _ T366L _ K392L T394W protein production by transient transfection of suspension [0348 ] FcyR KO 1 = Chain A : L234A _ L235A ; Chain B : growing CHO cells . Nucleic acids research 30 , E9 (2002 ) ) . L234A _ L235A [0361 ] The CHO cells were transfected in exponential [0349 ] FcyR KO 2 = Chain A : D265S _ L234A _ L235A ; growth phase (1 . 5 to 2 million cells /mL ) with aqueous 1 Chain B : D265S _ L234A _ L235A mg/mL 25 kDa polyethylenimine (PEI , Polysciences ) at a [0350 ) aCD19 _ HD37 scFv - N - to C - terminal order of EI: DNA ratio of 2 . 5 : 1 . (Raymond C . et al . A simplified variable regions is VL /VH unless otherwise indicated polyethylenimine -mediated transfection process for large [ 0351 ] ACD3 _ OKT3 scFv - N - to C - terminal order of scale and high -throughput applications. Methods. 55 ( 1 ) : 44 variable regions is VL /VH unless otherwise indicated . 51 ( 2011 ) ) . In order to determine the optimal concentration The VLVH are connected by a (GGGGS ) z linker . range for forming heterodimers, the DNA was transfected in [0352 ] QCD3 _ BiTE scFv - N - to C - terminal order of optimal DNA ratios of the heavy chain A (HC - A ) , and heavy variable regions is VH /VL and linker and composition chain B (HC - B ) that allow for heterodimer formation ( e .g . is identical to blinatumomab . HC - A /HC - B / ratios = 50 :50 % ) . Transfected cells were har US 2018 /0193477 A1 Jul. 12 , 2018 40 vested after 5 - 6 days with the culture medium collected after TABLE S1 - continued centrifugation at 4000 rpm and clarified using a 0 . 45 um filter . CDR sequences CD3 and CD19 antigen binding constructs [ 0362] The clarified culture medium was loaded onto a MabSelect Sure (GE Healthcare ) protein - A column and Antigen binding constructs CDR sequence washed with 10 column volumes of PBS buffer at pH 7 .2 . The antibody was eluted with 10 column volumes of citrate H2 : INPSRGYT buffer at pH 3 . 6 with the pooled fractions containing the antibody neutralized with TRIS at pH 11 . The protein was H3 : ARYYDDHYSLDY desalted using an Econo - Pac 10DG column (Bio - Rad ) . Humanized VARIANT of OKT3 ( CD3 binding short 10363] In some cases , the protein was further purified by gel filtration , 3 . 5 mg of the antibody mixture was concen L1 : SSVSY trated to 1 . 5 mL and loaded onto a Superdex 200 HiLoad L2 : DTS 16 /600 200 pg column (GE Healthcare ) via an AKTA Express FPLC at a flow -rate of 1 mL /min . PBS buffer at pH L3 : OOWSSNP 7 . 4 was used at a flow -rate of 1 mL /min . Fractions corre H1 : GYTFTRYT sponding to the purified antibody were collected , concen trated to ~ 1 mg/ mL and stored at - 80° C . H2 : INPSRGYT [ 0364 ] An additional purification step using , protein L H3 : ARYYDDHYSLDY chromatography after protein a purification could be carried out by the method as follows. Capto L resin was equilibrated Humanized VARIANT of OKT3 ( CD3 binding ) long with PBS and the variant was added to the resin and L1: SASSSVSYMN incubated at RT for 30 min . The resin was washed with PBS , and bound protein was eluted with 0 . 5 ml 0 . 1 M Glycine , pH L2 : DTSKLAS L3 : QQWSSNPFT [ 0365 ] The purity and yield of the final product was estimated by LC MS/ and UPLC -SEC as described in detail Hl : GYTFTRYTMH in PCT /US2015 /011664 . All variants were expressed and H2 : YINPSRGYTN purified to > 95 % heterodimer purity without contaminating homodimers . H3 : YYDDHYSLDY [ 0366 ] The clones that correspond to each bispecific HD37 ( CD19 binding ) short anti- CD3- CD19 antigen - binding construct are shown in Table XX ( at the end of the Examples ), and the correspond L1 : QSVDYDGDSYL ing sequence composition of each clone is shown in Table L2 : DAS YY. The CDR sequences used in the variants are shown in Table S1. L3 : QOSTEDPWT H1 : GYAFSSYW TABLE S1 H2 : IWPGDGDT CDR sequences CD3 and CD19 antiqen binding constructs H3 : RETTTVGRYYYAMDY Antigen binding constructs CDR sequence Humanized VARIANT of HD37 ( CD19 binding ) short Wild - type OKT3 ( CD3 binding ) L1 : QSVDYEGDSYL L1 : SSVSY L2 : DAS L2 : DTS L3: QOSTEDPWT L3 : QQWSSNP H1 : GYAFSSYW Hl : GYTFTRYT H2 : IWPGDGDT H2 : INPSRGYT H3 : ARYYDDHYCLDY H3 : RETTTVGRYYYAMDY Stabilized VARIANT of OKT3 ( CD3 binding ) Humanized VARIANT of HD37 ( CD19 binding ) short L1 : SSVSY L1 : QSVDYSGDSYL L2 : DTS L2 : DAS L3 : QQWSSNP L3 : QOSTEDPWT H1 : GYTFTRYT H1 : GYAFSSYW US 2018 /0193477 A1 Jul. 12 , 2018 41

TABLE S1 - continued TABLE S1 - continued CDR sequences CD3 and CD19 CDR sequences CD3 and CD19 antigen binding constructs antigen binding constructs Antigen binding constructs CDR sequence Antigen binding constructs CDR sequence H2 : IWPGDGDT Humanized VARIANT of HD37 ( CD19 binding ) long H3 : RETTTVGRYYYAMDY L1 : RASQSVDYSGDSYL HD37 (CD19 binding ) long L2 : DASNLVS L1 : KASOSVDYDGDSYL L3 : QOSTEDPWT H1 : GYAFSSYWMN L2 : DASNLVS H2 : QIWPGDGD TN L3 : QOSTEDPWT H3 : RETTTVGRYYYAMDY H1 : GYAFSSYWMN H2 : QIWPGDGD TN Example 2 : Description , Expression and Purification of Exemplary Bi- Specific H3 : RETTTVGRYYYAMDY Antigen - Binding Anti - CD3- CD19 Constructs in a Hybrid Heterodimer Fc Format or in Full -Size Humanized VARIANT of HD37 ( CD19 binding ) long Antibody Format [ 0367 ] Additional bispecific anti -CD3 - CD19 antigen L1 : RASQSVDYEGDSYL binding constructs 1853, 6754, 10151, 6750 , 6751, 6475 , L2 : DASNLVS 6749, 10152 , 10153, and 6518 were prepared as described in Example 1 . These constructs are based on the same antigen L3 : QOSTEDPWT binding domains as variant 875 but have been engineered for H1 : GYAFSSYWMN improved yield and biophysical properties . The modifica tions include changing one or both scFvs to the equivalent H2 : QIWPGDGDTN Fab format and /or stabilization of the scFv by VL - VH H3 : RETTTVGRYYYAMDY disulfide engineering and stabilizing CDR mutations . The details of the variant compositions are shown in Table 2 . TABLE 2 Summary of Variants and Composition

Variant # WT FC (FcgR knock -out ) * Chain 1 Chain 2

Dual scFv 875 ( 1661) ACD3 _OKT3 scFv aCD19 _ HD37 scFv heterodimer 873 ACD3 _blinatumomab scFv aCD19 _ HD37 scFv Fc variants 1653 ACD3 _ OKT3 scFv (CDR C - > S ) aCD19 _ HD37 scFv Hybrid 1853 (6754 ) ACD3 _ Teplizumab Fab ACD19 _HD37 scFv heterodimer N5 ( 10151) aCD3 _ Teplizumab Fab aCD19 _HD37 scFv (VHVL SS ) Fc variants 6750 (6751 ) ACD3 _OKT3 scFv ACD19 _HD37 Fab 6475 (6749 ) CD3 _ OKT3 scFv (CDR C - > S ) ACD19 _ HD37 Fab N7 (10152 ) ACD3 _ OKT3 scFv (VLVH SS ) aCD19 _ HD37 Fab N11 ( 10153) aCD3 _OKT3 scFv (CDR C - > S ) aCD19 _HD37 Fab ( VLVH SS ) 6476 aCD3 _ blinatumomab scFv ACD3 _ HD37 Fab Full size mAb 6518 (N12 ) aCD3 _ Teplizumab Fab ACD19 _HD37 Fab

* All variants have the following CH3 mutations : Chain 1 : T350V L351 Y F405A _ Y407V ; Chain 2 : T350V _ T366L K392L _ T394W Variants in brackets refer to the equivalent Fc knockout variant that include the additional mutations D265S _ L234A _ L235A on both heavy chains . This abolishes binding of the Fc to FcyRs. US 2018 /0193477 A1 Jul. 12 , 2018

[0368 ] The anti- CD19 scFv and anti -CD3 scFv sequences approach is based on the humanization and stabilization were generated as described above . The anti -CD19 Fab method as described by Ewert et al. , ( Ewert et al. , Methods (HD37 Fab ) is a chimeric Fab using the HD37 VH and VL 34 (2004 ) 184 - 199 ) and in addition includes detailed analy sequences fused to human IgG1 CH and CL sequences sis of the VH /VL three dimensional structures to identifying respectively . The scFv or VH -CH domains are fused to one potential VH /VL framework positions responsible for the chain of the heterodimeric Fc . The anti -CD3 Fab (tepizumab low stability . Further, the framework and CDR sequences Fab ) was generated from the known sequence of humanized were analyzed for potential sites of post -translational modi OKT3 antibody teplizumab (Eli Lilly ). The VH - CH domain fications, including de - amidation , aspartate isomerization was fused to one chain of the heterodimeric Fc . and protease cleavage . [ 0369 ] The scFv disulfide engineering strategy (VHVL [0379 ] The engineered humanized anti -CD3 and anti SS ) for both the anti - CD3 and anti - CD19 scFvs utilized the CD19 VL and VH sequences and the sequence alignment to published positions VH 44 and VL 100 , according to the the known parental murine antibodies HD37 and OKT3 and Kabat numbering system , to introduce a disulphide link the humanized teplizumab are shown in FIGS. 2 and 4 between the VH and VL of the scFv [Reiter et al. , Nat. respectively . Critical positions identified by the structure Biotechnol. 14 : 1239 - 1245 ( 1996 ) ] . guided humanization and stabilization approach are under [ 0370 ] The following variants contain a mutation to the lined and highlighted in bold in FIGS. 2 and 4 . The engi anti - CD3 scFv to improve stability and yield , as reported neered humanized sequences indicated hVH /HVL were used previously [Kipriyanov et al. , Prot . Eng . 10 ( 4 ) :445 -453 for construction of the bispecific variants as described in ( 1997) ] . v1653, 66475 and v10153 have an anti -CD3 Example 4 . (OKT3 ) with Cysteine to Serine mutation at position 100A of the VH CDR3. Example 4 . Expression and Purification of [0371 ] Details of the cloning, expression and character Bi- Specific Anti -CD19 -CD3 Antigen - Binding ization of hybrid and full sized anti -CD3 -CD19 antigen Constructs with Improved Yield and Biophysical binding constructs are provided in PCT/US2014 /046436 . Properties [0372 ] The clones that correspond to each bispecific [0380 ] Bispecific anti -CD3 - CD19 antibodies designed for anti -CD3 -CD19 and antigen - binding construct are shown in improved yield and biophysical stability were constructed as Table XX , and the corresponding sequence composition of described in Table 3 and Example 3 . Variant v10149 and each clone is shown in Table YY . V6751 are initial murine dual scFv heterodimer Fc and [0373 ] Controls hybrid heterodimer Fc designs of the CD3 - CD19 antigen 0374 ) v891 has a polypeptide sequence that is identical to binding constructs ( see Example 1 and Example 2 for further description ) . Variants v12043 and v15192 - v15195 exem blinatumomab ( BiTETM ) and includes an anti- CD3 scFv and plify humanized designs that include variable domain frame anti -CD19 scFv (50 kDa ) . work and CDR modifications designed to further improve [ 0375 ] Variant 4371 is a bivalent monospecific anti - CD19 yield and biophysical properties of the CD3 and CD19 antibody (used in Seattle Genetics ' anti -CD19 antibody antigen -binding constructs . drug conjugate known as SGN - 19A denintuzumab mafodo [0381 ] The anti -CD19 murine HD37 scFv has been tin . ) described in Example 1 and the Fab anti- CD19 murine [0376 ] In some experiments , polyclonal human IgG is HD37 is a chimeric Fab using the HD37 VH and VL used as a control and is referred to as v6249 . sequences fused to human IgG1 CH and CL sequences respectively. The humanized HD37 Fab is a Fab composed Example 3 . Humanization and Stabilization of of the humanized HD37 VH and VL sequences hVH2 and Anti- CD3 and Anti -CD19 Antibodies hVL2 ( D - E ) ( FIG . 2 ) fused to human IgG1 CH and CL [0377 ] The known murine and humanized anti- CD3 and sequences respectively . The humanized HD37 scFv is com CD19 antibodies BiTETM antibody (Kipriyanov et. al. , 1998 , posed of the humanized HD37 VH and VL sequences hVH2 Int. J Cancer : 77 ,763 - 772 ) , anti -CD3 monoclonal antibody and hVL2 ( D - E ) (FIG . 2 ) and has the identical VH /VL OKT3 (Drug Bank reference : DB00075 ), anti -CD3 mono orientation and linker as described for v10149 above . The clonal antibody teplizumab (Drug Bank reference : murine anti -CD3 scFv is identical to the scFv in the parental DB00075 ) and anti - CD19 monoclonal antibody HD37 (Kip variant v875 ( Table 1) and the humanized anti -CD3 scFvs riyanov et. al ., 1998 , Int. J Cancer : 77, 763 -772 ; Pezzutto , A . were generated from the engineered VH and VL sequences et al. , 1986 , Leukocyte Typing II . Vol. 2 . Springer - Verlag . as described in FIG . 4 and Table 3 . Heidelberg New York . P . 391 . ) exhibit low production yield [0382 ] (VLVH SS ) indicates disulfide stabilized scFv and biophysical stability . utilizing the published positions VH 44 and VL 100 , [ 0378 ] To improve the yield and biophysical properties of according to the Kabat numbering system , to introduce the HD37 and OKT3 based antibodies we used a structure a disulphide link between the VH and VL of the scFv guided approach for humanization and stabilization . This FReiter et al ., Nat. Biotechnol. 14 : 1239 - 1245 ( 1996 ) ]. TABLE 3 Summary of Variants and Composition Bispecific Anti -CD19 chain on Anti - CD3 chain on variant # heavy chain A heavy chain B VH /VL mutations for improved stability v10149 murine HD37 scFv murine OKT3 scFv Original murine HD37 and OKT3 VH /VL sequences (VLVH SS ) (VLVH SS ) US 2018 /0193477 A1 Jul. 12 , 2018 43

TABLE 3 -continued Summary of Variants and Composition Bispecific Anti- CD19 chain on Anti- CD3 chain on variant # heavy chain A heavy chain B VH /VL mutations for improved stability v12043 murine HD37 scFv murine OKT3 scFv Original murine OKT3 VH / VL sequences (VLVH SS ) (VLVH SS ) VHVL framework mutations for HD37 CDR mutation Asp - >Glu at position 28 of HD37 VL V6751 murine HD37 Fab murine OKT3 scFv Original murine HD37 and OKT3 VH /VL sequences v15192 humanized HD37 Fab humanized OKT3 scFv VHVL framework mutations for HD37 and OKT3 (hVH1 /HVL1 ) CDR mutation Cys - > Ser at position 100A of OKT3 VH CDR mutation Asp - > Glu at position 28 of HD37 VL v15193 humanized HD37 Fab humanized OKT3 scFv VHVL framework mutations for HD37 and OKT3 (hVH1 / HVL2 ) CDR mutation Cys - > Ser at position 100A of OKT3 VH CDR mutation Asp - >Glu at position 28 of HD37 VL v15194 humanized HD37 Fab humanized OKT3 scFv VHVL framework mutations for HD37 and OKT3 (hVH2 /HVL1 ) CDR mutation Cys - > Ser at position 100A of OKT3 VH CDR mutation Asp - Glu at position 28 of HD37 VL v15195 humanized HD37 Fab humanized OKT3 scFv VHVL framework mutations for HD37 and OKT3 (hVH2 /HVL2 ) CDR mutation Cys - > Ser at position 100A of OKT3 VH CDR mutation Asp - >Glu at position 28 of HD37 VL v 17119 humanized OKT3 scFv humanized HD37 Fab VHVL framework mutations for HD37 and OKT3 (hVH2 / HVL2 ) CDR mutation Cys - > Ser at position 100A of OKT3 VH CDR mutation Asp - >Glu at position 28 of HD37 VL

[ 0383] The humanized Fab and scFv sequences are fused [ 0389 ] The bispecifc antibodies were purified by Protein A to the heterodimer Fc chains as described for the parental affinity chromatography and subsequent gel filtration , as murine variants in Examples 1 and 2 . All variants have the described in Example 1 . FIG . 6 and Table 4 show the results following CH2 mutations : Heavy chain A : T350V _ L351Y _ of the preparative SEC purification and the final post puri F405A _ Y407V ; Heavy chain B : T350V _ T366L _K392L _ fication yield of the initial parental murine variant v6751 and T394W . The respective heavy chain CH3 mutations can the engineered humanized variants . either be on the anti- CD19 chain or the anti -CD3 chain . All [0390 ] The initial murine variant v6751 shows close to variants further comprise the additional mutations D265S 50 % high molecular aggregates after protein A purification L234A _ L235A on both heavy chains to abolish binding of and a low overall yield , while the engineered variants show the Fc to FcyRs. minimal aggregates and 2 - 3 - fold increased yield . The final 10384 ) Fc numbering is according to EU index as in Kabat post purification yield is comparable to positive control referring to the numbering of the EU antibody (Edelman et commercial antibodies. al. , 1969, Proc Natl Acad Sci USA 63 : 78 -85 ) ; Fab or variable domain numbering is according to Kabat ( Kabat TABLE 4 and Wu , 1991 ; Kabat et al, Sequences of proteins of immu nological interest. 5th Edition US Department of Health Production yield of humanized variants and Human Services , NIH publication no . 91- 3242 , p 647 Purification Post PA / SEC yield ( 1991) ) . Sample Name process (mg / L ) [0385 ] The murine HD37 and OKT3 sequences were v10149 PA / SEC 2 . 5 humanized and further modified for improved yield and v12043 PA / SEC 5 . 2 stability by the following changes : i ) the humanized anti V6751 PA / SEC 9. 2 v15192 PA / SEC 25 . 2 CD3 scFvs can utilize the published Cysteine to Serine v15193 PA / SEC 27 . 8 mutation at position 100A of the VH CDR3 [Kipriyanov et v15194 PA / SEC 29 .6 al. , Prot. Eng . 10 ( 4 ) :445 -453 (1997 ) ] and the variants v15195 PA / SEC 21. 2 v15192 -v15195 and v17119 ( Table 3 ) contain the Serine mutation for improved stability , ii ) the sequence of the humanized anti - CD19 CDR was modified at position 28 to Example 5 . Thermal Stability of Engineered eliminate a potential Aspartate isomeration site that could impact antigen binding , iii ) specific VHVL framework posi Bi- Specific Anti - CD19 -CD3 Antigen - Binding tions were identified to potentially impact stability and yield Constructs (Example 3 ); these positions are underlined and highlighted [0391 ] The thermal stability of the stability engineered in bold in FIGS. 2 and 4 . bispecific anti- CD19 - CD3 constructs in comparison to the murine parental variants was assessed by differential scan [ 0386 ] The clones that correspond to each bispecific ning calorimetry (DSC ) . anti - CD3 -CD19 and antigen - binding construct are shown in [0392 ] All DSC experiments were carried out using a GE Table XX , and the corresponding sequence composition of VP - Capillary instrument. The proteins were buffer - ex each clone is shown in Table YY . changed into PBS (pH 7 . 4 ) and diluted to 0 . 3 to 0 . 7 mg/ mL [0387 ] Include Table with 0 . 137 mL loaded into the sample cell and measured [0388 ] The bispecific antibodies against CD3 and CD19 with a scan rate of 1° C . /min from 20 to 100° C . Data was were designed , expressed and characterized as described in analyzed using the Origin software (GE Healthcare )with the PCT /US2015 /011664 and in Examples 1 and 2 . PBS buffer background subtracted US 2018 /0193477 A1 Jul. 12 , 2018 44

[0393 ] Table 5 shows a list of the estimated melting [0399 ] All SPR binding experiments were carried out temperatures ( Tm ) for the individual anti -CD3 and anti - using a BioRad ProteOn XPR36 instrument at 25° C . with CD19 Fab and scFvs of the parental murine vs. the stability 10 mM HEPES, 150 mM NaC1, 3 . 4 mM EDTA , and 0 .05 % engineered humanized constructs . Tween 20 at pH 7 . 4 . Recombinant CD3epsilon / delta Fc fusion protein (Sino Biological ; http :/ /www . sinobiological. TABLE 5 com /CD3D -CD3 -Delta - Protein -g - 10182 .html ) was cap tured on anti - Fc capture sensorchips. Purified antibodies A : Thermal stability of engineered anti - CD19 binding domains were indirectly captured on the sensorchip by binding the Anti- CD19 binding domain Tm (DSC ) recombinant CD3 fusion protein when injected at 25 uL /min for 240s (resulting in approx. 500 RUS) following a buffer mHD37 scFv 53° mHD37 Fab 65° injection to establish a stable baseline . Resultant K , values hHD37 Fab (hVH2 / HVL2 ( D - E ) ) - 72°( * ) were determined from binding isotherms using the Equilib rium Fit model with reported values as the mean of three B : Thermal stability of engineered anti - CD3 binding domains independent runs . [0400 ] Table 6 summarizes the results of the SPR binding Anti- CD3 binding domain Tm (DSC ) of the engineered humanized bispecific constructs v15192 OKT3 scFv 63° v15195 . All engineered constructs bind to CD19 and CD3 Teplizumab Fab 660 antigens equivalent to the parental murine construct v6751 . Teplizumab scFv ~ 62° ( * ) The stability engineered humanized constructs have equiva hOKT3 scFv (hVH2 /HVL2 ) ~ 72° ( * ) lent binding to CD3 antigen compared to the parental v6751 . ( * ) the DSC was measured on variants in IgG format; due to the overlap of CH2, Fab and scFv transitions with similar melting temperatures the specific Tm could only be estimated (see FIG . 7 ) TABLE 6 [0394 ] The anti- CD19 and anti- CD3 Fabs and scFvs were SPR binding of engineered anti- CD19 constructed as described above (Examples 1 and 4 ) , CD3 variants to recombinant CD3 expressed as bispecific or monospecific Fc constructs and Sample the purified constructs were measured by DSC as described . capture (RU ) KD (M ) Rmax (RU ) FIG . 7 illustrates a representative DSC thermogram of V6751 959. 93 1 . 41E - 07 178 . 72 selected engineered variants vs. the parentalmurine control. v15193 988 .48 3 .70E - 07 174 . 01 v15194 975 .92 3 .49E - 07 179 . 12 The melting transitions of the individual domains as sum v15195 1032 .89 4 . 27E - 07 marized in Table 5 were estimated by comparison of the 192. 69 engineered vs . the parental murine DSC thermograms. [0395 ] The results in Table 5 and FIG . 7 show that the humanized constructs with engineered variable domains Example 7 : Whole Cell Binding to CD19 + Raji have increased stability compared to their murine parental Tumor Cells and CD3 + Jurkat T Cells and Human constructs . The final stabilized hybrid variants v15192 PBMCs v15195 have thermal melting temperatures of over 72° C ., comparable to Fabs of commercial IgG antibodies. [0401 ] The ability of the humanized bispecific anti - CD19 [ 0396 ] As illustrated in Table 4 and 5 , the structure guided CD3 constructs to bind to CD3 - and CD19 - expressing cells stability engineering yields a significant improvement in was assessed via whole cell FACS binding analysis as expression and thermal stability . Further, comparison to the described previously (PCT / US2015 /011664 ) . The binding to humanized Teplizumab shows that the improvement in yield CD19 + Raji B cells (ATCC : CCL - 86 ; Pulvertaft , Lancet and stability is independent of the sequence humanization , 1964 ) and CD3 + Jurkat cells (Weiss , J Immunol 1984 ) and but is most likely due to specific changes to VH /VL posi the apparent binding affinities of variant v15195 are shown tions that we have identified by our structure guided in FIGS . 8A and B . approach as critical for the Fab / scFv stability . [0402 ] The bispecific anti -CD19 -CD3 constructs (exem [0397 ] In conclusion , our structure guided humanization plified by v15195 in FIG . 8 ) bound human Raji CD19 + NHL and stabilization approach has identified new humanized B cells with high affinity ( apparent affinity of 2 . 4 nM ) and OKT3 and HD37 VH /VL sequences with significantly human CD3 + Jurkat T cells with low affinity (apparent improved yield and stability . In difference to the known affinity of 44 . 7 nM ) . murine and humanized HD37 and OKT3 scFvs , which [0403 ] The ability of bispecific T cell engagers to crosslink exhibit low expression and stability , our engineered variants T cells and target B cells is a prerequisite for activity . show yield and stability comparable to commercial IgG and Therefore , in addition to assessing binding to isolated B and thus allow the development as therapeutic antibodies . T cell lines as shown in FIGS . 8A and B we tested the ability of the bispecific anti -CD19 -CD3 constructs to crosslink Example 6 . Antigen Binding of Engineered autologous B and T cells in human PBMC . Freshly isolated Bi- Specific Anti - CD19 - CD3 Antigen -Binding human and PBMCs were incubated with v15195 on ice and the percentage of B cell: T cell doublets were analyzed by Constructs FACS to determine the concentration dependent ability of [ 0398 ] To determine whether the engineered bispecific crosslinking B and T cells. The percent T :B doublets were constructs v15192 -v15195 bind to CD19 and CD3 antigens defined as FSC - W -high cells within the CD20 + SSClow equivalent to the parental murine construct v6751, the population . Greater than 75 % of the identified doublets were binding affinity to CD19 and CD3 was measured by SPR and CD4 + or CD8 + , suggesting that they had formed doublets whole cell FACS as described below . with T cells . US 2018 /0193477 A1 Jul. 12 , 2018 45

[0404 ] As illustrated in FIG . 8C , the analysis of B : T cell TABLE 7 doublets in human PBMC demonstrated that v15195 cross links B and T cells in human PBMC in a concentration Conjugation of bispecific anti- CD3 - CD19 variants dependent manner and at concentrations below 1 nM . Variants Conjugate DAR % purity YieldYield [ 0405 ] Together , this data shows that the bispecific anti 12043 SMCC - DM1 91 CD19 -CD3 constructs preferentially binds B cell and cross 12043 SPBD - DM4 ??? 6754 SMCC - DM1 links B and T cells at concentrations below 2 nM , while 6751 SMCC - DM1 ? 97 binding to isolated T cells at significantly lower concentra 4372 SMCC -DM1 ? 90 tions of above 40 nM . This preferential binding of B cells 15195 SMCC -DM1 ? NNNNNNNONU and crosslinking of B and T cells at low concentrations , while only binding isolated T cells at low concentrations, [0408 ] FIG . 9 shows an exemplary UPLC - SEC profile of allows for development of bispecific drug - conjugates that v12043 - SMCC -DM1 after conjugation . Other variants will preferentially bind B cells and activate T cells without behaved similarly . impacting isolated T cells . Example 9 . Impact of Bispecific Format on In Vitro Activity of Anti -CD3 -CD19 Antigen - Binding Example 8 : Drug Conjugation of Bi- Specific Drug -Conjugates Against B and T Tumor Cell Anti -CD19 - CD3 Antigen -Binding Constructs Lines [0409 ] To test the cytotoxicity and potency of anti - CD3 [0406 ] A schematic of exemplary anti- CD3 - CD19 anti CD19 conjugates on target B cells and T cells , selected gen -binding construct drug conjugate is shown in FIG . 1 . bispecific anti -CD3 -CD19 conjugates with identical CDRs, Anti -CD3 -CD19 antigen - binding constructs were conju but differing antigen binding format of scFv or Fab were gated to either DM1 using the non - cleavable linker SMCC tested in a growth inhibition assay using B and T tumor cell or to DM4 using the cleavable linker SPBD as described lines . The selected variants in FIG . 10 and Table 8 have below . Variants were conjugated to either DM1 or DM4 previously been shown to have similar binding affinities to using a one - step procedure . The starting protein sample was CD19 and CD3 . The affinity of all selected variants to CD19 first exchanged into a buffer composed of 50 mM potassium is ~ 2 nM , the affinity to CD3epsilon is ~ 40 nM . phosphate pH 6 . 5 , 50 mM NaCl and 2 mM EDTA using a [0410 ] The extent of cytotoxicity was measured in cell PD - 10 column, and adjusted to a protein concentration of cultures of CD19 + Raji or Ramos (ATCC : CRL - 1596 ; 2 - 10 mg/ ml . A 10 mM solution of SMCC -DM1 (Levena Klein , Intervirology 1975 ) non -Hodgkin lymphoma (NHL ) Biopharma US, San Diego , Calif .) or SPBD - DM4 (Levena target B cell lines and CD3 + Jurkat T cell line in comparison Biopharma US, San Diego , Calif. ) dissolved in dimethylac to non - specific IgG SMCC - DM1 conjugate ( v6249 ) and etamide (DMA ) was then added to 7 .5 molar equivalents of monospecific anti -CD19 antibody huBU12 conjugated to the protein sample . DMA was further added to a final SMCC -DM1 as positive control ( v4371) . The monospecific concentration of 10 % v / v and the sample was mixed briefly. anti -CD19 antibody huBU12 is currently being evaluated as The reaction mixture was incubated at 25° C . overnight with a MC -MMAF drug conjugate (denintuzumab mafodotin ) in mixing . The product was then exchanged into a buffer Phase I and Phase II clinical trials in NHL and B - ALL composed of 20 mM sodium succinate pH 5 . 0 using a PD - 10 (Gerber , Blood 2009 ; Albertson TM , Proceeding : AACR column, and the protein concentration and drug - to -antibody Annual Meeting 2014 ) . Potential off -target cytotoxicity of ratio (DAR ) were calculated based on the absorbance at 252 the SMCC -DM1 conjugates was measured against the target and 280 nm . The buffer was adjusted to a final composition cell line , K562 ( ATCC : CCL - 243 ) which does not express of 20 mM sodium succinate , 6 % w /v trehalose and 0 .02 % CD19 or CD3. The selected antibodies were diluted in media w / v polysorbate 20 , pH 5 . 0 . High performance liquid chro and added to the target Raji , Ramos , Jurkat or K562 cells in triplicate and incubated for 24 hr. Cells were washed , media matography - size exclusion chromatography (HPLC -SEC ) replaced and cell survival was evaluated after a 3 day was performed to determine the purity of the ADC , using the incubation at 37° C . Cell viability was measured using Tosoh G3000 - SWXL column ( 7 . 8 mmx30 cm ), in 100 mM Sulforhodamine B with absorbance read at 510 and 540 nm sodium phosphate, 300 mM sodium chloride, pH 7 . 0 , at a following standard procedures . Data was normalized to flow rate of 1 ml/min . untreated control and analysis was performed in GraphPad [0407 ] SMCC - DM1 conjugates of v12043 , v6754 , 6751 , prism . 15195 and 4372 had a yield of over 70 % , a purity of > 85 % [0411 ] All anti - CD3 -CD19 conjugates showed no off and a drug / antibody ratio (DAR ) of 2 . 2 - 3 . 5 as summarized target activity against the cell line K562 which does not in Table 7. The SPBD -DM4 conjugate of v12043 had a yield express CD19 or CD3, similar to the non - specific IgG of 70 % and a purity of 82 % . SMCC -DM1 control v6249 (data not shown ) . TABLE 8 Cytotoxicity of selected anti -CD3 - CD19 variants on B and T cells Ramos Jurkat Therapeutic IC50 IC50 Window (IC50 Rank Variant Format Linker -toxin (nM ) (nM ) Jurkat /Ramos ) 1 6751 aCD3 (scFv ). SMCC - DM1 0 .4562 23 .39 51. 3 ACD19 (Fab ) US 2018 /0193477 A1 Jul. 12 , 2018 46

TABLE 8 - continued Cytotoxicity of selected anti - CD3 - CD19 variants on B and T cells Ramos Jurkat Therapeutic IC50 IC50 Window ( IC50 Rank Variant Format Linker- toxin (NM ) (nM ) Jurkat /Ramos ) - 12043 aCD3 ( scFv ) SMCC - DM1 2 . 982 26 .55 8 . 9 aCD19 ( scFv ) (batch 2 ) -3 12043 aCD3 ( scFv ) . SPBD - DM4 0 . 2885 2 . 121 7 . 4 aCD19 ( scFv ) A 12043 aCD3 ( scFv ) . SMCC -DM1 0 .5399 2 . 496 4 .6 aCD19 ( scFv ) (batch 1 )

oC 4371 aCD19 control SMCC - DM1 6 .025 24 . 33 4 . 0 6249 Non -specific SMCC - DM1 37 . 14 134 . 7 3 . 6 IgG control x 6754 aCD3 (Fab ) - SMCC -DM1 3 .974 0 . 1891 0 . 0 ACD19 ( scFv ) (batch 1 ) x 6754 aCD3 (Fab )- SMCC -DM1 6 .251 0 .1672 O ACD19 ( scFv ) (batch 2 )

10412 ]. The growth inhibition results , as illustrated in FIG . T cells as described in Example 10 and also in internaliza 10 and Table 8 , show unexpectedly, that the cytotoxictovic tion assays with tumor and T cell lines (Example 21) . activity on target B cells of all monovalent anti- CD19 bispecific antibodies is comparable to or better than the Example 10 . In Vitro Efficacy of Exemplary bivalent monospecific anti -CD19 -SMCC -DM1 control Anti -CD3 -CD19 Antigen - Binding Construct V4371 . The conjugated variant 6751 has a potency of 0 .45 Drug - Conjugates in Primary Human Blood Samples nM on Ramos B cells and the bivalent monospecific positive control v4371 conjugate has a potency of ~ 6 nM . 104171 To further test the preferential killing of target B [0413 ] The growth inhibition results suggest in addition an cells without affecting T cells and T cell activity , the selected unexpected difference between the different hybrid and dual variants were tested in primary blood cultures with alloge scFv anti -CD3 - CD19 -MCC - DM1 conjugates. Variant6751 - neic Ramos and Raji lymphoma cell lines . This assay MCC - DM1 (with the anti -CD3 in scFv format and the reflects the cytotoxic activity of the anti -CD3 -CD19 conju anti - CD19 in Fab format) is highly active towards B cells gates towards the allogeneic target B cells mediated by the with EC50 of 0 . 45 nM , while having very low activity on T cell redirected activity of the bispecific , and also the Jurkat T cells , similar to the non - specific controls v4371 and conjugated drug delivered by internalization of the antigen V6249 . In contrast , the variant v6754 (with the anti -CD3 in binding construct by the target B cells . To measure the effect Fab format and the anti -CD19 in scFv format ) has similar of the conjugates on the T cell population the T cell activity , potency on target B cells and T cells . A potential therapeutic activation and proliferation were analyzed . As relevant window of killing target B cell without impacting T cells markers for total T cell counts CD4 and CD8 have been was calculated as shown in Table 8 . The data suggest a measured whereas T cell activation of the CD8 and CD4 T therapeutic window and killing of target B cell without cells was measured by the established early and late T cell impacting the T cells for the variant6751 and 12043, but not activation markers CD69 and CD25 , respectively. 6754 . [0418 ] In addition , the T cell exhaustion marker PD - 1 was measured to evaluate the potential effect on T cell inhibition [0414 ] These results show that unexpectedly , a bispecific and exhaustion . PD -1 (Programmed cell Death protein 1 ) T cell engager drug conjugate can be developed to prefer functions as an immune checkpoint and plays an important entially bind and kill target B cells , while not impacting the role in down regulating the immune system by preventing T cells . As result of to the preferential binding and activity , the activation of T -cells and promoting T cell apoptosis , the bispecific T cell engager drug conjugate has the potential while reducing apoptosis in regulatory T cells ( suppressor T to have a dual mechanism of action of: i ) T cell redirected cells ) (Francisco L M , Sage P T , Sharpe A H ( July 2010 ) B cell killing and ii ) B cell killing through internalization of Immunological Reviews 236 : 219 -42 ) the conjugated toxin payload . [0419 ] Human blood ( 120 - 140 mL ) for individual studies [0415 ] Further, the data suggests the preferential behav was collected from donors and PBMC were freshly isolated . iour is dependent on one or all of the following character PBMCswere further processed to derive the subpopulations istics of the bispecific : i ) monospecific targeting of the CD3 without autologous B cells ( PBMC - B ) . Resting PBMCs antigen , ii ) low affinity binding to the CD3 antigen , iii ) were used as effector cells and Raji or Ramos human B cells format and geometry of the bispecific . In conclusion , the as target cells and the ratio of T cells to allogeneic Raji or results allow the identification of the bispecific format Ramos B cells was adjusted to an E : T ratio of 5 : 1. B cell and ( including Fab vs scFv and hybrid vs. dual scFv or full size T cell populations , at day 0 , were determined by FACS . Ig bispecific and Ig isotype and hinge ) and CD3e affinity as Exclusive B cell markers included CD19 and CD20 . T cell the critical parameters that have to be optimized for the populations were measured by CD3, CD4 and CD8 and T development of bispecifc CD3 T cell engager drug conju cell activation and potential exhaustion was measured by gates . CD69 , CD25 and PD1, respectively as described above . [0416 ] This conclusion and the ranking of variants with Quadruplicate wells were plated for each control and experi different format is confirmed in activity assays with primary mental condition and co - cultures were incubated in 5 % US 2018 /0193477 A1 Jul. 12 , 2018 47

CO2, 37° C . and stopped at 72 hours . T and B cells were [0423 ] The clinical and preclinical experience of Blinatu assessed for their respective proportions in the culture by momab indicate that the T cell redirected response is highly FACS . The collected culture cells were stained for CD45 , donor dependent and can be limited by mechanisms of T cell CD20 and 7 - AAD FACS detection . FACS analysis was immunosuppression (Kohnke , 2015 ) . As illustrated above carried out by InCyte / Flow Jo as follows: A Guava 8HT flow all tested bispecifics ( including blinatumomab ) induced up cytometer was used for analysis of cell subpopulations. regulation of PD1 and in some donors the unconjugated Between 5 ,000 events for FSC /SSC and compensation bispecific were ineffective in depleting the target B cells . In wells , and 30 , 000 events for experimental wells were ana contrast, the conjugated bispecific T cell engager showed lyzed by cytometry . A threshold was set to skip debris and activity in these cultures , suggesting that the dual mecha RBCs. All B cells were confirmed to be double positive for nism of action can potentially overcome limited efficacy in CD19 and CD20 at Day 0 , which allowed for monitoring of patients with high T cell immunosuppression . CD20 as appropriate B cell marker. In a control experiment Raji and Ramos cell cultures without PBMC were incubated Example 11. Cytotoxicity of Bispecific with the variants analyzed for B cell cytotoxicity after 72 Anti- CD19 -CD3 - SMCC -DM1 Drug Conjugates hrs . Against ALL , NHL Tumor Cell Lines Grown in [0420 ] FIGS. 11 and 12 show the results of an n = 2 repeat Culture without T Effector Cells with two individual PBMC donors and allogeneic Raji B cells . The non - conjugated variant v12043 has a potency of [0424 ] To test the cytotoxicity and potency of the human < 0 . 05 nM on the Raji target B cells for both donors and ized bispecific anti- CD3 -CD19 variants with improved bio induces T cell proliferation and activation with similar physical properties ( see Example 3 - 5) , selected variants potency. The non -conjugated variant v12043 is able to were conjugated to SMCC - DM1 as described in Example 8 . deplete - 50 % of target B cells by the T cell redirected All SMCC -DM1 conjugates of v6751, v15192 , v15193 , mechanism . In contrast, the drug conjugates show an v15194 , v15195 had comparable yield of over 70 % , purity equivalent T cell mediated B cell depletion at concentrations of > 90 % and a drug /antibody ratio (DAR ) of 3 . 1 - 3 . 5 . below 0 . 5 nM , but in addition are able to further deplete the [0425 ] The extent of cytotoxicity was measured in cell target Raji B cells at concentrations above 0 .5 nM . Unex cultures of different CD19 + non -Hodgkin lymphoma (NHL ) pectedly , the conjugates show only at the highest concen and acute lymphocytic leukemia ( ALL ) tumor B cell lines in tration of 50 nM an impact on the T cell proliferation , but do comparison to non - specific IgG SMCC -DM1 conjugate not have an impact at lower concentrations . This is in line ( Isotype DM1) and monospecific anti- CD19 antibody with the data presented in FIG . 10 and Table 8 . huBU12 conjugated to auristatin as positive control. The [0421 ] FIGS. 13 and 14 show the results of a separate monospecific anti -CD19 antibody huBU12 is currently repeat experiment of the hybrid variants v6751 and v6754 being evaluated as a MC -MMAF drug conjugate (denintu with Raji and Ramos target B cells using fresh PBMCs from zumab mafodotin ) in Phase I and Phase II clinical trials in the same donor as in FIG . 12 . The activity of the anti - CD3 NHL and B - ALL (Gerber , Blood 2009 ; AlbertsonTM , Pro CD19 variants is compared to the positive controls blinatu ceeding: AACR Annual Meeting 2014 ) . momab and the anti -CD19 monospecific conjugate . The 10426 ] The impact on T cells is tested on CD3 + Jurkat T results in FIGS. 13 and 14 show a similar additional activity cells. Potential off -target cytotoxicity of the SMCC -DM1 of the conjugates on the target B cell depletion at higher conjugates was measured against the target cell line , K562 concentrations compared to the non - conjugated v6751 and which does not express CD19 or CD3 . The experiment was V6754 . As suggested by the growth inhibition assay in FIG . conducted as described in detail in Example 9 . 10 , the assay confirms the unexpected difference of v6751 and v6754 conjugates on the T cell , with v6754 conjugate 10427 ] FIG . 15 illustrates the results for a selected subset having no impact on the T cells while mediating potent of target cell lines and Table 9 summarizes the results in killing of the target B cells . comparison to the anti -CD19 antibody positive control. [0422 ] In addition to the B cell depletion and T cell counts TABLE 9 measured in the previous experiments ( FIGS . 11 and 12 ), the up - regulation of PD - 1 was measured . PD - 1 plays an impor Cytotoxicity of MCC -DM1 drug conjugates against ALL , tant role in down regulating the immune system by prevent NHL tumor cell lines grown in culture without T cells ing the activation of T - cells and promoting T cell apoptosis v15195 -MCC - huBU12 -MCC ( Francisco L M , Sage P T , Sharpe A H ( July 2010 ) Immu DM1 DM1 nological Reviews 236 : 219 -42 ) . PD - 1 up - regulation has Target cell line ( IC50 nM ) ( IC50 nM ) been speculated to be a mechanism of resistance to T cell redirected therapies [ Junttila et al. , Cancer Res ( 2014 ) ALL ( CD19 + , CD3- ) 5561- 71 ; Kohnke, 2015 ]. As shown in FIG . 14 with PBMC Nalm - 6 (ATCC : CRL - 3273) 0 . 7 * and allogeneic Raji cells all variants , including the positive RS4 ; 11 ( ATCC : CRL - 1873 ) < 5 * ûuü * control blinatumomab induced up -regulation of PD - 1 in DLBCL (CD19 + , CD3 - ) > 80 % of T cells and no significant B cell depletion of the SUDHL - 4 ( ATCC : CRL - 2957 ) 1 . 2 4 . 7 non - conjugated variants . The conjugated variants were able SUDHL - 6 ( ATCC : CRL - 2959 ) 1 . 8 < 5 * to deplete the Raji B cells at higher concentrations , but not Burkitt (CD19 + , CD3- ) the non -conjugated variants . In addition , the anti -CD3 Raji * * 1 .5 6 . 9 CD19 conjugates in comparison to the non - conjugated com Ramos * * 0 . 4 6 . 0 parators show a lower % of PD - 1 expressing T cells at higher Daudi ( ATCC : CCL - 213 ) 2 . 1 5 * concentrations. T US 2018 /0193477 A1 Jul. 12 , 2018 48

TABLE 9 -continued be mediated by the T cell redirected activity of the bispecific , but also by the conjugated drug delivered by internalization Cytotoxicity of MCC - DM1 drug conjugates against ALL , of the antigen -binding construct by the target B cells . NHL tumor cell lines grown in culture without T cells 10434 ] Further , the results show the benefit of a dual v15195 -MCC huBU12 -MCC mechanism as the T cell mediated activity of both the DM1 DM1 unconjugated v15195 and the positive control Blinatu Target cell line ( IC50 nM ) (IC50 nM ) momab at efficacious concentrations is highly donor depen T - cell leukemia ( CD19 - , CD3 + ) dent and not sufficient to kill > 90 % of the target B cells in this assay. Jurkat 24 . 3 23 . 4 AML (CD19 + , CD3 - ) Example 13 . Cytotoxicity of Bispecific K562 ( ATCC : CCL - 243 ) Greater Greater Anti -CD19 -CD3 -SMCC - DM1 Drug Conjugate than 50 nM than 50 nM Against Tumor Cell Lines Grown in Culture with T Cells [0428 ] For the results indicated with * only a 5 point [0435 ] To further test the activity of the bispecific anti concentration curve was measured and the Kd could not be CD19 -CD3 - SMCC -DM1 drug conjugates , the extent of fitted with confidence . The results of e . g . < 5 indicates that at cytotoxicity was measured in co - cultures of different CD19 + the concentration of 5 nM over 50 % of cells were depleted . non - Hodgkin lymphoma (NHL ) or acute lymphocytic leu The results indicated with * * refer to data collected for the kemia (ALL ) tumor B cell lines and primary T cells . murine v6751 - SMCC -DM1 conjugate . [0436 ] The variant v15195 -MCC -DM1 was tested in pri [0429 ] As shown in Table 9 , the bispecific anti -CD3 - CD19 mary blood cultures with allogeneic NHL or ALL cell line . drug conjugates show potent killing of NHL and ALL tumor The experimental set - up was conducted as described above B cells lines while not significantly impacting the growth of in Example 12 . the Jurkat T cells . All anti -CD3 - CD19 conjugates showed no [0437 ] FIG . 17 shows potent killing of different NHL and off- target activity against the cell line K562 , which does not ALL target B cell lines by the bispecific anti -CD19 -CD3 express CD19 or CD3, similar to the non - specific IgG SMCC -DM1 drug conjugate and confirms preferential kill SMCC -DM1 control (data not shown ) . ing of target B cells without impacting T cells . The T cell 10430 ] In addition , the potency was comparable or greater counts are not impacted up to the highest tested concentra than the positive control huBU12 -MCC - DM1 and v15195 tion of 50 nM (data not shown ). MCC -DM1 exhibited a wide range of target cell cytotoxic killing across human cancer cell lines. Example 14 . T Cell Activation and Proliferation Effects of Bispecific Anti -CD19 -CD3 -SMCC - DM1 Example 12 . Cytotoxicity of Bispecific Drug Conjugate in Comparison to Blinatumomab Unconjugated Anti- CD19 -CD3 and Bispecific and OKT3 Antibodies Anti -CD19 - CD3 - SMCC -DM1 Drug Conjugates [0438 ] The clinical dosing of the commercial therapeutic Against Tumor Cell Lines Grown in Culture with T antibody Blinatumomab is limited by toxicities that are Cells thought to be T cell mediated and associated with the extent [0431 ] The target B cell cytotoxic activity of the SMCC of T cell proliferation and activation ( Chatenoud , 1986 ; DM1 conjugated and unconjugated variant v15195 was Abramowicz , 1989 ; Goebeler , 2011 ; Bargou , 2008 ; Topp , further evaluated in comparison to the approved therapeutic 2011 ; Klinger, 2010 ; International Patent Publication No. antibody Blinatumomab . The bispecific variant v15195 was WO2011051307A1; GoebelerMEJ Clin Oncol 2016 ; Topp , specifically chosen because of the over 100 fold lower T cell Lancet Oncol 2015 ) redirected potency compared to Blinatumomab . This lower [0439 ] To evaluate the potential therapeutic index of T cell mediated potency is sufficient to mediate target B cell v15195 , the ability of v15195 to induce T cell activation and killing in vitro and in vivo , while resulting in lower T cell proliferation was assessed in co - cultures of Raji cancer B activation and proliferation compared to Blinatumomab at cells and human PBMC and compared to the in vitro activity 1000 fold lower concentration (see Example 14 ) . Impor of Blinatumomab at a concentration equivalent to the clini tantly , the lower potency yields compatible potencies for the cally tolerated exposure . ( The maximum tolerated doe T cell redirected and DM1 mediated cytotoxicity and (MTD ) of Blinatumomab in Phase 1 r / r - NHL trial is 60 enables a dual mechanism of action . ug/ m2 /day (Goebeler MEJ Clin Oncol 2016 ) ; for compari [ 0432 ] The cytotoxic activity of the bispecific anti- CD3 son to a safe dose and exposure of Blinatumomab , a con CD19 conjugates was measured in comparison to the non centration of 0 . 05 nM Blinatumomab or equivalent to the conjugated anti - CD3- CD19 variant and the positive control exposure at the dose of 40 ug /m2 / day was chosen ). BlinatumomabTM (blinatumomab , BiTETM ) . To measure the [0440 ] The co -culture experiment was conducted as fol effect of the conjugates on the T cell population the T cell lows: On Day 0 , blood was collected from each of 4 donors activity , activation and proliferation were further analyzed and PBMCs were freshly isolated . PBMCs were further as described in Example 13 . The assay was performed with processed to derive the subpopulation of PBMC without B n = 4 primary blood donors and the experimental set - up was cells (PBMC - B ) . Resting PBMC - B were used as effector conducted as described above in Examples 11 - 14 . cells and Raji human B cells as target cells and the ratio of [0433 ] As illustrated in FIG . 16 , comparison of the activity T cells to allogeneic Raji cells was adjusted to an E : T ratio of the unconjugated v15195 and the DM1 conjugated of 5 : 1 . The mixtures were incubated together with the v15195 confirms that the cytotoxic activity of the anti -CD3 antibody constructs for 3 days , after which the collected CD19 conjugates towards the allogeneic target B cells can primary cells were stained for CD4 , CD , CD69, CD25 US 2018 /0193477 A1 Jul. 12 , 2018 49

FACS detection . FACS analysis of the different populations PBMC cultures were incubated in 5 % CO2, 37° C . and was carried out by InCyte / FlowJo as follows: Between stopped at 72 hours . T cell populations were assessed by 5 ,000 event for FSC /SSC and compensation wells , and FACS . The cell pellets were resuspended in various antibody 30 ,000 events for experimental wells were analyzed by cocktails for flow cytometry analysis . A Guava 8HT flow cytometry. A threshold was set to skip debris and RBCs. cytometer was used for analysis of cell subpopulations. As [ 0441] FIG . 18A illustrates the results from n = 4 donors negative controls an anti -CD19 bivalent monospecific anti after 72 h incubation for the CD8 + T cell populations . The body (huB12 ; see Example 11 ) and untreated cultures were analysis shows the total CD8 + T cell counts , which is an used . The results are shown in FIG . 19 . indirect measure of the induced T cell proliferation and also [0448 ] The results indicate that v15195 does not activate the extend of T cell activation , measured by the early and T cells in cultures of PBMC lacking B cells , but activates T late T cell activation markers CD69 and CD25 ( see Example cells in presence of target B cells . Variant v15195 shows 10 ) , respectively . strictly target dependent T cell activation . [ 0442 ] The results show that at efficacious concentrations of 100 - 1000 fold above the in vitro EC50 ( see FIG . 16 and Example 16 . T Cell Activation , Proliferation and Example 12 ), v15195 induced lower T cell proliferation and Cytokine Release of Bispecific activation than clinically tolerated concentrations of Blina tumomab . Anti -CD19 - CD3 -SMCC - DM1 Drug Conjugate 10443 ]. In addition to the FACS based analysis of induced v15195 , in Comparison to Unconjugated v15195 T cell proliferation and activation , the v15195 induced T cell [0449 ] The ability of the bispecific SMCC -DM1 conju proliferation was evaluated in a thymidine cell proliferation gated and un - conjugated constructs to induce T cell prolif assay in PBMC cultures , as depicted in FIG . 18B . The eration and activation was assessed in two different assays as thymidine based assay presents a differentmeasure of T cell described below . The assay was performed with n = 4 primary proliferation and activation as it is a measure of the total blood donors and the experimental set - up was conducted induced proliferation potential in the PBMC culture . The identical to Examples 12 and 14 . thymidine based analysis provides a complementary mea [0450 ] FACS Analysis of T Cell Proliferation / Activation sures to the FACS based method described above . In the in Raji /PBMC - B Cultures : assay illustrated in FIG . 18B , v6751, the original parental [0451 ] On Day 0 , blood was collected from each of 4 murine variant to v15195 was used ( see Table 3 ) . donors and PBMCs were freshly isolated . PBMCs were [0444 ] The thymidine cell proliferation assay in PBMC further processed to derive the subpopulation of PBMC cultures was conducted as follows: On Day 1 , blood was without B cells (PBMC - B ) . Resting PBMC - B were used as collected from each of 3 donors and PBMCs were freshly effector cells and Raji human B cells as target cells and the isolated . The test items were prepared for a final concentra ratio of T cells to allogeneic Raji cells was adjusted to an E : T tion of 0 . 3 and 100 nM and combined with the PBMCs, ratio of 5 : 1 . The mixtures were incubated together with the plated at 250 ,000 cells/ well . The mixtures were incubated for 3 days , after which tritiated thymidine was added to the antibody constructs for 3 days , after which the collected cell containing wells for a final of 0 . 5 uCi thymidine /well ; primary cells were stained for CD4, CD , CD69, CD25 the plates were incubated for an additional 18 hours , after FACS detection . which the plates were frozen . Total incubation time was 4 [0452 ] Cytokine Analysis of Raji /PBMC -B Co -Culture days . The plates were filtered and counted (CPMs ) using a Supernatant : B - counter. From the averages , a Stimulation Index ( SI) was [0453 ] Raji co - culture experiments were set- up as calculated as follows and the data was tabulated : average described above and levels of IFN - Y , IL - 6 and IL - 10 were CPM of test item /average CPM of media only . The average assessed by luminex after 3 days of incubation . E : T ratio in human PBMC collected from healthy donors [0454 ] The results of the FACS based analysis of T cell was ~ 10 : 1 CD3 + T cells to CD19 + B cells . proliferation and activation in PBMC - B / Raji co - cultures is [0445 ] As illustrated in FIG . 18B , the comparison to shown in FIG . 20A . Cytokine production is presented in Blinatumomab and OKT3 demonstrated lower total cell FIG . 20B . proliferation in cultures of v6751 even at 1000 fold higher [0455 ] The results in FIGS . 20A and 20B illustrate that concentrations . This suggests that the bispecific CD19 - CD3 conjugation of v15195 with SMCC -DM1 enhanced T cell drug conjugate does not impact T cell at to the highest proliferation and activation at low concentrations , compared evaluated concentration and further that the therapeutic to unconjugated v15195 . In addition , SMCC - DM1 conju index is potentially higher than Blinatumomab . gated v15195 enhanced production of the pro -inflammatory cytokines, IFN - y and IL - 6 compared to unconjugated Example 15: Target B Cell -Dependence of T Cell v15195 . While SMCC -DM1 conjugated v15195 only Activation by Bi- Specific Heterodimer Variants in induced a modest increase in the anti - inflammatory cytokine Human PBMC IL - 10 , the unconjugated form caused a dose -dependent [0446 ] The dependence of T -cell activation by the exem increase . plary anti -CD19 -CD3 - SMCC - DM1 bispecific variant [0456 ] The difference in T cell activation and particularly v15195 on target B cells was determined in human PBMCs. cytokine profile is an unexpected and relevant result , since The experiment was carried out as described below . for example IL10 release has been associated with T cell [ 04471 Human blood ( 120 - 140 mL ) was collected from suppressive mechanisms ( e . g . regulatory T cell expansion ) donors and PBMC were freshly isolated from donors . which limit the efficacy of T cell engagers . The bispecific T PBMCs were further processed to derive the subpopulation cell engager drug conjugate could therefore potentially be of PBMC without B cells (PBMC - B ) . Quadruplicate wells less susceptible to T cell suppressive mechanisms. In addi were plated for each control and experimental condition and tion the increased release of the pro - inflammatory cytokine US 2018 /0193477 A1 Jul. 12 , 2018 50

INFy, which is a key regulator for T cell and macrophage [0461 ] Following conjugation , excess linker - toxin solu activation , has the potential to synergize and enhance the T tion was removed by centrifugation of the resin at 14800 cell redirected activity. rpm for 2 minutes . The resin was then washed three times 04571. This difference in T cell activation and cytokine with PBS containing 5 % ( v / v ) DMA to remove any residual release is dose dependent and correlates with the dose linker- toxin , followed by three washes with PBS (pH 7 . 4 ) to response of DM1 mediated target cell depletion , which remove any remaining DMA co -solvent . The antibody -drug suggests that the effect is mediated by the activity of the drug conjugate was released from the resin by incubating the conjugate . As previously reported , DM1 and DM1- ADCs resin for 15 minutes in Release buffer ( ADC Biotechnology can mediate immunogenic cell death and are highly syner Ltd . ) followed by centrifugation at 14800 rpm for 2 minutes. gistic with immunomodulatory agents , like anti -PD1 and The filtrate was then desalted by G25 gel permeation chro anti - CTLA4 (Mueller et al . , Science Transl Med 2015 ) . The matography (GE Healthcare IllustraTM NAPTM - 5 column ) results suggest that addition of a toxin like DM1 has the into a buffer containing 10 mM sodium acetate , 9 % sucrose potential to improve the efficacy of the anti- CD19 - CD3 (pH 5 .0 ) , followed by filtration through a sterile 0 .22 um bispecific , by inducing immunogenic /pro - inflammatory cell PES membrane . death (Mueller et al ., Science Transl Med 2015 ). [ 0462 ] Purity of the final antibody - drug conjugate was assessed by high performance liquid chromatography - size Example 17 . Cytotoxicity of Bispecific exclusion chromatography (HPLC -SEC ) on a TSKgel Anti -CD19 - CD3 Conjugated to MMAE Against G3000SWXL 7 .8 mmx30 cm , 5 um column ( TOSOH Bio Tumor Cell Lines Grown in Culture with T Cells science LLC ) in 10 % IPA , 0 .2M potassium phosphate , [ 0458 ] To further test the preferential killing of target B 0 .25M potassium chloride , pH 6 .95 at a flow rate of 0 . 5 cells without affecting T cells , v15193 was conjugated to the mL/ min . The drug - to -antibody ratio (DAR ) of the antibody toxin MMAE using a cleavable linker (maleimidocaproyl drug conjugate was determined using hydrophobic interac valine - citrulline - p -aminobenzyloxycarbonyl (mc - vc tion chromatography (HIC ) HPLC on a Butyl -NPR 4 . 6 PABC ) ) . The cytotoxic activity of the bispecific anti -CD3 mmx3 .5 cm , 2 . 5 um column ( TOSOH Bioscience LLC ) run CD19 conjugates was measured in comparison to the non at 0 . 8 mL /min with a 12 -minute linear gradient of A – 1 .5M conjugated anti - CD3- CD19 variant in primary blood (NH2 ) , SO4, 25 mM NaPi, pH 6 . 95 and B - 25 mM NaPi, pH cultures with allogeneic Ramos lymphoma cell lines . Pos 6 . 95 , 25 % IPA . sible cytotoxicity towards T cells as a result of drug conju 0463 ] The final yield of the v15193 -mc - vc - PABC gation using a cleavable linker was also assessed . MMAE conjugate was 39 % , with a purity of > 98 % and an [0459 ] To prepare the antibody drug conjugate , the anti average DAR of 3 . 7 . body was first bound to a “ Lock -Release " resin ( ADCRO1 , [0464 ] To measure the effect of the conjugate on the T cell ADC Biotechnology Ltd . ), a proprietary resin for immobi population , the T cell activity , activation and proliferation lization of antibodies for conjugation , at 1 mg/ 100 uL resin were further analyzed as described in Example 14 . The assay loading . Antibody disulfide binds were then reduced by was performed with n = l primary blood donors and the addition of a solution of tris( carboxyethyl) phosphine experimental set- up was conducted as described above in ( TCEP ) in PBS (pH7 . 4 ) with 2 mM EDTA to the bound Example 12 . antibody at 6 molar equivalents of TCEP to bound antibody 10465 ] As illustrated in FIG . 21 , comparison of the activity and incubation of the mixture at 20° C . for 120 minutes with of the unconjugated and the MMAE conjugated bispecific continuous mixing. Excess TCEP was removed by washing variant (v15193 vs . v15193 -mc - vc - PABC -MMAE (x3 ) with PBS (pH7 . 4 ). ( “* v15193 -VC -MMAE ” ) ) confirms that the cytotoxic activity [0460 ] For conjugation , a solution of mc- vc -PABC of the anti - CD3 - CD19 conjugate towards the allogeneic MMAE ( ADC Biotechnology Ltd . , see structure below ) ( 10 target B cells can be mediated by the T cell redirected mM stock in dimethyl acetamide (DMA ) ) equivalent to 6 activity of the bispecific , but also by the conjugated drug molar equivalents of linker - toxin to bound antibody was first delivered by internalization of the antigen - binding construct prepared in PBS containing 5 % ( v / v ) DMA . This linker by the target B cells . toxin solution was added to the bound antibody and the [ 0466 ] Further , the results show unexpectedly that mixture incubated at 20° C . for 60 minutes with continuous v15193 -mc -vc -PABC -MMAE had little to no effect on T shaking. cell counts suggesting that bispecific T cell engager drug

mc - vc - PABC -MMAE :

OMe 0 IZ Meo NH OH NH NH2 US 2018 /0193477 A1 Jul. 12 , 2018 51 conjugates can be developed both with non - cleavable linkers Example 19. In Vivo Response of Anti -CD3 - CD19 (as described above ), and with cleavable linkers such as Antigen - Binding Construct Drug - Conjugates in mc - Val- Cit - PABC . Hu ( CD34 + )NSG Mice [ 0473 ] To further evaluate the impact of anti- CD3- CD19 Example 18 . In Vivo Response to Exemplary conjugates on the T cells and T cell activation at higher Anti -CD3 - CD19 Antigen - Binding Construct doses and maximum exposures of over 300 nM , v15195 Drug - Conjugates in Humanized Hu( CD34 + )NSG MCC - DM1 was analyzed in an in vivo study in humanized Mice mice . The in vivo activation and redistribution of autologous [ 0467] To further evaluate the impact of anti- CD3 - CD19 T cells was measured in humanized (CD34 + ) NSG -SGM3 conjugates on the T cell population and activity , selected (NSG strain : NOD . Cg - Prkdescid 112rgim1Wjl Tg ( CMV - IL3 , variants were analyzed in an in vivo study in humanized CSF2 ,KITLG ) 1Eav /MloySzv ; Jackson laboratory ) mice mice . The in vivo B cell depletion and activation and ( E : T < 1 : 5 ) after a single dose IV injection at 9 mg/ kg , 3 redistribution of autologous T cells was measured in human mg/ kg , 1 mg/ kg , and 0 .3 mg/ kg . As control, a variant with ized ( CD34 + ) NSG mice (E : T - 1 : 5 ) after a single dose IV only the anti -CD19 Fab connected to the heterodimeric Fc , injection of v12043 SMCC -DM1 and SPDB - DM4 conju but lacking the anti -CD3 scFv (v15760 ) was used . The gates in comparison to the non - conjugated v12043 . monospecific anti -CD19 control variant v15760 was conju 10468] . For humanization of mice , 2 week -old NSG (NOD gated to SMCC - DM1 as described in Example 8 , with a scid gamma, NOD .Cg -Prkdcscid 112rgtmlWji/ SzJ ) mice purity of > 90 % and DAR of 3 . 5 . were injected with human (CD34 + ) HSC from human fetal [ 0474 ] Humanized ( CD34 + ) mice present a good model liver ( Jackson Laboratory. Humanized (CD34 + ) NSG mice system to evaluate human T cell activation , redistribution develop human T cell and B cell linages within 12 weeks . and expansion in a mouse model, whereas the proliferation Average T cell to B cell ratio in humanized (CD34 + ) NSG and maturation of human B cells is partially deficient in these models ( Ito et al. , Cellular & Molecular Immunology is ~ 1 : 5 to 1 : 1 . 2012 ; 9 : 208 -214 ; Brehm et al ., Curr Opin Endocrinol [0469 ] Humanized (hCD34 + ) NSG were dosed with 1 Diabetes Obes. 2010 ; 17 ( 2 ) : 120 - 125 ) . We therefore don ' t intravenous ( IV ) bolus injection at day ( ( at 0 . 3 and 0 . 1 expect to see a significant effect of the conjugated drug DM1 mg/ kg doses) and the autologous circulating B and T cell on the human B cells, whereas the human T cell activation populations were analysed at 48 h post injection and at day and proliferation is well established in these models and the 5 upon termination , similar to previously described ( PCT/ main aim of this study is to assess the impact of the US2015 /011664 ) . The T cell and B cell populations were anti - CD3 -CD19 bispecific drug conjugate on the T cell analyzed by FACS . The specific B and T cell markers activation and expansion . analyzed were human CD45 , CD20 , CD4, CD8 and CD69 , [0475 ] The study was conducted as previously described as described above in Example 10 . in Example 18 and PCT /US2015 /011664 . Briefly , human [0470 ] The in vivo serum exposure of the variants , as ized (hCD34 + ) NSG - SGM3 mice were purchased from shown in Table 5 , was estimated from previous data in NSG Jackson laboratory . The bispecific anti -CD3 -CD19 ADC , mice ( see PCT/ US2015 /011664 ) . v15195 -MCC -DM1 was dosed with 1 intravenous (IV ) bolus injection at day 0 ( at 0 . 3 , 1 , 3 and 9 mg/ kg doses ) and TABLE 10 the autologous circulating B and T cell populations in peripheral blood and isolated spleen were analysed at day 8 Estimated serum exposure for v12043 upon termination , similar to previously described (PCT / Serum conc . 0 . 3 mg/kg US2015 /011664 ) . The T cell and B cell populations were 0 . 5 h > 30 nM analyzed by FACS . The specific B and T cell markers 24 h 10 nM analyzed were human CD45 , CD20 , CD4, CD8 and CD69, 48 h 6nM as described above in Example 10 and 18 . 72 h 3 nM [0476 ] FIG . 23 shows the total CD3 + T cell counts in 120 h 1 nM peripheral blood and isolated spleen at day 8 post injection of the single dose . The results show that only at the selected [0471 ] FIG . 22 shows the impact on the B and T cell highest dose of 9 mg/ kg a significant effect on the T cells in counts in circulation after single dose injection of v12043 circulation and in the spleen was observed . No impact on T and the v12043 SMCC - DM1 and SPBD - DM4 conjugates . cell counts and activation was observed at 3 mg /kg and All variants and dose levels were effective in depletion the lower doses . circulating B cells (CD20 + B cells ) and no significant [0477 ] The in vivo serum exposure of v15195 , as shown difference on T cell counts and T cell activation , as measured in Table 11 , was estimated from previous data of the by CD69 expression on CD4 and CD8 positive T cells , was unconjugated variants in NSG mice ( PCT/ US2015 / 011664 ) . observed between the groups. [0478 ]. The estimated serum exposure in Table 11 suggests [ 0472 ) The estimation of the serum exposure in Table 10 that the bispecific T cell engager -MCC - DM1 conjugates can suggests a Cmax of close to 50 nM , which was the maximum be dosed up to at least 3 mg /kg and an associated Cmax of concentration used in the in vitro assay in primary blood > 300 nM , without long term impact on the autologous T cultures. The in vivo single dose study confirms that at even cells . This is an exposure and Cmax similar to other SMCC at the highest dose of 0 . 3 mg/ kg and a Cmax close to 50 nM , DM1 antibody frug conjugates in clinical development and the anti -CD3 - CD19 conjugates have no negative impact on thus allows the development of T cell engager drug conju T cells . Further, the conjugates do not reduce the T cell gates at dose levels that are standard for other -MCC -DM1 activation and T cell redirected activity on the B cells . ADCs in development (Jumbe at al. , J Pharmacokinet Phar US 2018 /0193477 A1 Jul. 12 , 2018 52 macodyn (2010 ) 37 :221 -242 ; Lu et al. , Cancer Chemother TABLE 12 Pharmacol (2014 ) 74 :399 -410 ). Summary of Variants and Composition TABLE 11 Bispecific variant # Anti - tumor antigen chain Anti -CD3 chain Estimated serum exposure v13831 ACDH3_ Clone # 6 Fab BiTEx - 12C scFv ( VL / VH ) Serum conc . 9 mg/kg 3 mg/ kg 1 mg/ kg 0. 3 mg/ kg v13792 AHER2 _ ( trastuzumab ) Fab BiTEx - I2C scFv ( VL /VH ) v13790 aHER3 _ Mab205 Fab BiTEx - 12C scFv ( VL /VH ) 0 . 5 h > 900 nM > 300 nM > 30 nM > 30 nM v16371 AEGFR _ ( cetuximab ) Fab BiTEx - 12C scFv (VL /VH ) 24 h 300 nM 100 nM 10 nM 10 nM 48 h 180 nM 60 nM 6nM 6 nM All variants have the following CH3 mutations: Heavy chain A : T350V _ L351Y _ F405A _ 72 h 90 nM 30 nM 3 nM 3 nM Y407V , Heavy chain B : T350V _ T366L _ K392L _ T394W . Chain A or B can be either on 120 h 30 nM 10 nM 1 nM 1 nM the anti- CD3 or the anti- tumor antigen chain . [0481 ] Fc numbering is according to EU index as in Kabat referring to the numbering of the EU antibody (Edelman et al. , 1969, Proc Natl Acad Sci USA 63 :78 - 85 ) ; Fab or Example 20 . Expression and Purification of variable domain numbering is according to Kabat (Kabat Bi- Specific Anti- Tumor -CD3 Antigen - Binding and Wu, 1991 ; Kabat et al, Sequences of proteins of immu Constructs for Solid Tumor Indications nological interest . 5th Edition — US Department of Health and Human Services , NIH publication no . 91- 3242 , p 647 [0479 ] Bispecific antibodies against CD3 and CDH3 , (1991 ) ) . HER2 , HER3 or EGFR were designed , expressed and char [0482 ] The clones that correspond to each bispecific acterized as described in PCT/ US2015 /011664 . Briefly , the anti - tumor - CD3 and antigen -binding construct are shown in genes encoding the antibody heavy and light chains were Table XX , and the corresponding sequence composition of constructed via gene synthesis using codons optimized for each clone is shown in Table YY . human /mammalian expression . The scFv - Fc sequences were [0483 ] The bispecific antibodies against CD3 and CDH3, generated from a known anti- CD3 scFv BiTETM antibody HER2, HER3 or EGFR were designed , expressed and char (Kipriyanov et . al ., 1998 , Int. J Cancer: 77 , 763 - 772 ) and acterized as described in PCT /US2015 /011664 and in anti - CD3 monoclonal antibody OKT3 (Drug Bank refer Examples 1 and 2 . ence: DB00075 ). The CDH3 Fab sequences were generated [0484 ] The bispecifc antibodies were purified by Protein A from a known anti -CDH3 monoclonal antibody (PCT / affinity chromatography and subsequent gel filtration , as JP2009/ 007333 ) . The HER2 Fab sequences were generated described in Example 1 . from trastuzumab ( PCT /US1998 /026266 , Baselga J et al. , 1998 , Cancer Res : 58 ,2825 -31 ) and the HER3 Fab Example 21. In Vitro Internalization of Bispecific sequences were generated from a known anti -HER3 mono Anti - Tumor -CD3 PHAb - Conjugates on Solid clonal antibody ( PCT / EP2010 / 070062, Mirschberger C , et Tumor Cell Lines and Jurkat T Cell Line al. , 2013 , Cancer Res : 73 , 5183 - 94 ) . EGFR sequences were [0485 ] Conjugation of a pHAb dye to antibodies is a generated from cetuximab (PCT /US1996 /009847 , Prewett method used to assess internalization of a given antibody by M et al. , 1996 , J Immunother Emphasis Tumor Immunol: a cell . The dyes only fluoresce under low pH conditions such 19 ,419 - 27 ) . The Fab - scFv variants made are described in as those found the the endosome/ lysosome , indicating inter Table 12 . nalization of the antibody . Exemplary bispecific anti -tumor 10480 ] The humanized anti -CD3 OKT3 scFv was gener CD3 antigen -binding construct pHAb conjugates were made ated identical to anti - CD3 scFv of the murine OKT3 variants as follows. Variants were conjugated to pHAb Amine Reac v875 or the humanized OKT3 variant v15195 , described tive Dye as per the manufacture ' s protocol for in - solution above. The anti- CD3 BiTEX -IC2 scFv was generated from antibody conjugation (Promega ). the VH and VH sequences as described in (US 20110275787 [0486 ] The starting protein sample was first exchanged in A1 ) , which is cross - reactive with non - chimpanzee primate 10 mM sodium bicarbonate buffer (pH 8 .5 ) using a desalting CD3. The humanized OTK3 scFv or the BiTEx - IC2 scFv column . A 10 mg/ ml solution of PHAb Amine Reactive Dye were fused to one chain of the heterodimeric Fc . The dissolved in a 1 : 1 DMSO -water mix was then added at a 20 anti -CDH3 monoclonal , Clone # 6 Fab is a chimeric Fab molar excess to the antibody sample . The reaction mixture using the murine Clone # 6 VH and VL sequences fused to was incubated for 60 minutes with mixing . Unreacted dye human IgG1 CH and CL sequences respectively . The anti was removed using a desalting column. The antibody con HER2 monoclonal Fab consists of the humanized VH and centration and DAR were calculated after measuring absor VL sequences of trastuzumab fused to human IgG1 CH and bance at 280 nm and 532 nm . High performance liquid CL sequences , respectively . The Fab of the anti -HER3 chromatography- size exclusion chromatography (HPLC monoclonal is a fusion of the humanized VH and VL SEC ) was performed to determine the purity of the conju sequences of lumretuzumab (PCT / EP2010 /070062 ; Mirsch gates , using the Superdex 200 column ( 8 . 6 5x150 mm ) , in berger C . , et al. , 2013 , Cancer Res. , 73 : 5183 - 94 ) to human D - PBS + 0 .01 % Polysorbate 20 , at a flow rate of 0 .25 ml/min . IgG1 CH and CL sequences , respectively . The anti - EGFR [0487 ] PHAb conjugates of v13831, v16371 had a yield of monoclonal, cetuximab Fab is a chimeric Fab using the over 60 % , a purity by HPLC -SEC of > 90 % and a drug murine cetuximab VH and VL sequences fused to human antibody ratio (DAR ) of 1. 5 - 3 . 3 . IgG1 CH and CL sequences , respectively . In all cases, the [ 0488 ] The extent of internalization was measured in VH -CH domains of the antibodies are fused to the second several tumor cell lines, SKOV3 (ATCC : HTB 77 ), A431 chain of the heterodimeric Fc . (ATCC : CRL - 1555 ) , HCT- 116 (ATCC : CCL - 247 ) and US 2018 /0193477 A1 Jul. 12 , 2018 53

JIMT1 (AddexBio # C0006005 ) , in comparison to non - spe can be transferred to different tumor targets . In addition , the cific IgG PHAb conjugate (v15195 ) and an anti - CD3 PHAL low impact on T cells is not specific to a particular epitope conjugated mAb , v2171 (UCHT1 , Beverley PC and Callard on CD3e, but likely rather dependent on the format and R E . , 1981, Eur J Immunol ., 11: 329 -34 ; PCT/ US1993 / geometry of the bispecific , as described in Example 9 . 007832 ). The anti - RSV antibody , Synagis (PCT / US1991 / 002668 ) , was used as a negative control. The impact on T Example 22 . Cytotoxicity of Bispecific cells is tested on CD3 + Jurkat T cell. The selected antibodies Anti - Tumor -CD3 - SMCC - DM1 Drug Conjugates were diluted in media and added to the target cells in Against Breast, Ovarian Tumor Cell Lines Grown triplicate and incubated for 1 hr. Cells were washed , media in Culture without T Cells replaced and antibody internalization was evaluated using ImageExpress following standard procedures . Data was [0492 ] To test the cytotoxicity and potency of the bispe normalized to untreated control and analysis was performed cific anti - tumor- CD3 variants , selected variants were con in GraphPad prism . jugated to SMCC - DM1 as described in Example 8 . All [0489 ] The internalization results , as illustrated in FIG . 24 SMCC -DM1 conjugates of v13831 , v13792 and v13790 had and Table 13 show that pHAb conjugated v13831 and comparable yield of over 70 % , purity of > 90 % and a v16371 are rapidly internalized by SKOV3, A431, HCT- 116 drug / antibody ratio (DAR ) of 3 . 1 - 3 . 5 . and JIMT1 cells , with Kd values in the nM range and [0493 ] The extent of cytotoxicity was measured in cell internalization of the variants is dependent upon expression cultures of different breast (MCF7 (ATCC : HTB -22 ) and of the target antigen . Unexpectedly , these variants are poorly JIMT1 ) and ovarian ( SKOV3 ) tumor cell lines in compari internalized by the Jurkat T cell line . Furthermore , the son to non -specific IgG SMCC -DM1 conjugate ( Isotype bivalent anti -CD3 PHAb conjugated mAb ( v2171 , UCHT1) DM1) . The impact on T cells is tested on CD3 + Jurkat T was internalized by Jurkat cells at close to 2 nM , while the cells . The experiment was conducted as described in detail bispecifics based on two different anti - CD3 scFvs , which in Example 9 . target slightly different CD3e epitopes (OKT3 and xBiTE ) , showed low internalization into Jurkat T cells ( 72 nM and [0494 ] FIG . 25 illustrates the results for a selected subset > 100 nM , respectively ). of target cell lines and Table 14 summarizes the results in [0490 ] Therefore, the data suggests that the bispecific comparison to the IgG - DM1 control. tumor- CD3 antibodies are preferentially internalized by [0495 ] The results of the cytotoxicity study, as illustrated tumor cells compared to CD3+ cells making it less likely that in FIG . 25 and Table 14 show that the DM1 conjugated ADC versions of these bispecifics would exhibit toxicity variants , v13831, v13792 , and v13790 , exhibit potent killing towards the T cells being engaged by the CD3 arm . of breast and ovarian tumor cell lines but do not significantly TABLE 13 Differential internalization of pHAb conjugated bispecific tumor- CD3 variants by tumor cell lines EC50 (NM )

v13831 v16371 v15195 v2171 bispecific bispecific bispecific aCD3 control Target cell line ( CDH3- BITEx) ( EGFR - BiTEx ) ( aCD19 -hOKT3 ) (hUCHT1 ) Solid Tumor HCT- 116 3 .92 > 100 ( EGFR - /CDH3 + ) A431 (EGFR + /CDH3 + ) 4 . 15 2 .46 JIMT1 < 10 * 2 .90 (EGFR + /CDH3 +) SKOV3 > 100 2 .05 (EGFR + /CDH37 ) T -cell leukemia Jurkat (CD19 - , CD3 + ) > 100 > 100 71. 95 13. 2 * Estimated EC50 ; accurate fitting of the parameters was not possible due to increased background levels at antibody concentrations greater than 100 nM ( see FIG . 24 for comparison ). [ 0491] The results illustrate further, that the concept of a impact the growth of the Jurkat T cells . This preferential bispecific T cell engager ADC with dual functionality , as killing of tumor cell lines is similar to the preferential established in Examples 1 - 14 for CD19 -CD3 bispecifics can internalization of the variants by tumor cell lines presented be expanded to different solid tumor antigens and solid in Example 22 . The non - specific variant, v6249 -SMCC tumor targeting bispecific T cell engagers . The preferential DM1, does not exhibit any significant killing of tumor cell tumor targeting without impacting the T cells is not specific lines or Jurkat T cells until concentrations greater than 100 to the CD19 antigen , and the concept for the design of nM are used . This highlights the role target specificity plays bispecific T cell engager ADCs, as described in Example 9 , in the activity of the anti - tumor -CD3 bispecific antibodies. US 2018 /0193477 A1 Jul. 12 , 2018 54

TABLE 14 Cytotoxicity of SMCC -DM1 drug conjugates against breast , ovarian tumor cell lines grown in culture without T cells EC50 (nM ) v13831 - SMCC - v13792 -SMCC - v13790 - SMCC - v6249 -SMCC DM1 DM1 DM1 DM1 Target cell line ( CDH3- BITEx ) ( HER2- BITEx ) ( HER3- BITEx ) ( hIgG) Breast cancer MCF7 3 . 1 12 . 6 3 . 6 50 . 2 JIMT1 1 . 4 3 . 4 34 . 1 Ovarian cancer SKOV3 0 . 1 21. 5 T - cell leukemia ( CD19 - , CD3 + ) Jurkat 12 . 2 16 . 7 15 . 3 53 . 7

[ 0496 ] Therefore, the data suggests that, like the CD19 - enhanced tumor cell killing compared to the unconjugated CD3 bispecifics , the bispecific anti- tumor- CD3 drug conju versionversions of these variants at concentrations greater than 0 .05 gates preferentially kill tumor cells compared to CD3 + cells . nM . This is likely a result of drug delivery to the target tumor This further supports the conclusion that bispecific ADCS cells through internalization of the antigen -binding con would would possess potent anti- tumor activity while exhib struct. iting little to no toxicity towards the T cells being engaged [0500 ] Further, the results show the benefit of a dual by the CD3 arm . mechanism as the T cell mediated activity of the unconju Example 23 . Cytotoxicity of Unconjugated and gated variants is highly donor dependent and not sufficient SMCC - DM1 Conjugated Anti - Tumor - CD3 to kill > 90 % of the target tumor cells in this assay. Bispecifics Against Tumor Cell Lines Grown in Example 24 . T Cell Proliferation and Activation of Culture with T Cells Bispecific Anti - Tumor - CD3- SMCC -DM1 Drug [0497 ] To further test the preferential killing of target Conjugates Compared to Unconjugated tumor cells without effecting T cells and T cell activity, Anti - Tumor -CD3 Bispecifics selected variants were tested in primary blood cultures with allogeneic JIMT1 cell line. The cytotoxic activity of the [ 0501 ] The ability of the SMCC -DM1 conjugated bispe bispecific anti- CD3 - tumor conjugates was measured in com cifics , anti -CDH3 -CD3 and anti -HER2 -CD3 , and their par parison to the non - conjugated anti -CD3 -tumor variant. To ent unconjugated constructs to induce T cell activation and measure the effect of the conjugates on the T cell population proliferation was assessed as described below . the T cell activity , activation and proliferation were further [0502 ] On Day 0 , blood was collected from each of 4 analyzed as described in Example 23 . The assay was per donors and PBMCs were freshly isolated . Resting PBMC formed with n = 1 primary blood donors and the experimental were used as effector cells and JIMT1 cells as target cells set- up was conducted as described above in Example 5 with and the ratio of T cells to allogeneic JIMT1 cells was minor modifications described below . adjusted to an E : T ratio of 2 : 1 . The mixtures were incubated [0498 ] Specifically , on day 0 JIMT1 cells were first together with the antibody constructs for 4 days, after which labeled with CellTracer violet ( a live / dead stain ) . Following the collected primary cells were stained for CD4, CD8 , the labeling of the JIMT1 target cells , PBMCs were isolated CD69, and CD25 . FACS analysis of the different popula for use as effector cells . Rested PBMCs were mixed with the tions was carried outby InCyte/ Flow Jo as follows: Between labeled JIMT1 cells such that the ratio of T cells to alloge 5 ,000 event for FSC / SSC and compensation wells , and neic JIMT1 cells was adjusted to an E : T ratio of 2 : 1 . The 30 ,000 events for experimental wells were analyzed by mixtures were incubated together with the antibody con cytometry. A threshold was set to skip debris and RBCs. structs for 4 days , after which the JIMT1 cells were collected [0503 ] The results of the FACS based analysis of T cell and viability was assessed though FACS analysis of CTV proliferation and activation in PBMC / JIMT1 co - cultures is levels . This was carried out by InCyte / Flow Jo as follows: shown in FIG . 27 . The results illustrate that at efficacious Between 5 , 000 event for FSC /SSC and compensation wells , concentrations, the SMCC -DM1 conjugated variants induce and 30 ,000 events for experimental wells were analyzed by a modest increase in the total CD8 + and CD4 + T cell cytometry . A threshold was set to skip debris and RBCs. populations compared to the unconjugated parent variants . [ 0499 ] As illustrated in FIG . 26 , both the unconjugated Total CD8 + and CD4 + T cell counts is an indirect measure and SMCC -DM1 conjugated forms of the variants , v13831 of induced T cell proliferation suggesting that conjugation of and v13792 exhibited potent cytotoxic activity towards a toxin to an anti - tumor- CD3 bispecific can enhance T cell JIMT1 cells . The results show that this cytotoxic activity proliferation induced by the unconjugated bispecific . Simi towards the allogeneic target tumor cells can be mediated by larly , a modest increase in CD25 + and CD697 (early and late the T cell redirected activity of the bispecific . Interestingly , stage T cell activation markers , respectively ) T cells is also SMCC -DM1 conjugation of variants v13831 and v13792 observed in when the co -cultures are stimulated with the US 2018 /0193477 A1 Jul. 12 , 2018 55

SMCC -DM1 conjugated variants compared to the unconju especially when T effector cell concentrations are low . This gated variants , suggesting increased activation of the T cells . may be of particular importance in indications where T cell infiltration of tumors is low . Example 25 . T Cell Proliferation and Activation of Bispecific Anti - Tumor -CD3 - SMCC -DM1 Drug Conjugates Compared to Unconjugated TABLE XX Anti - Tumor -CD3 Bispecifics Range of High , H1 (Heavy H2 (Heavy L1 ( Light L3 ( Light Intermediate and Low Effector to Target Ratios Variant Chain 1 Chain 2 Chain 1 Chain 2 Number Clone No. ) Clone No. ) Clone No. ) Clone No. ) 105041 To further delineate the role of each mechanism of action of the bispecific anti -tumor -CD3 - SMCC -DM1 drug 873 1064 1065 875 1064 1067 conjugates, T cell redirected killing and killing through 1661 2183 2176 internalization of the conjugated toxin payload , the cyto 1653 1842 2167 toxic activity of the anti -CD3 - CDH3 conjugate , v13831 1662 2183 2177 SMCC -DM1 was measured in comparison to its non -con 1660 2174 2175 jugated parent v13831 . The variants were tested in primary 1666 2184 2185 blood cultures with allogeneic JIMT1 cell line at three 1801 1842 2228 6747 5243 2227 different E : T ratios . The assay was performed with n = 1 10149 6692 6689 primary blood donors . 10150 6692 6690 0505 ] on day 0 JIMT1 cells were first labeled with 1380 1844 1890 CellTracer violet (a live/ dead stain ). Following the labeling 12043 7239 6689 of the JIMT1 target cells , PBMCs were isolated for use as 1853 2304 2175 effector cells . Rested PBMCs were mixed with the labeled 6754 5239 2185 2309 JIMT1 cells such that the ratio of T cells to allogeneic JIMT1 10151 5239 6691 2309 cells was adjusted to an E : T ratios of 5 : 1, 1 : 5 and 1: 50 . The 6750 5241 5238 2310 6751 5242 2176 2310 mixtures were incubated together with the antibody con 6475 2305 2171 2310 structs for 4 days, after which the JIMT1 cells were collected 6749 5242 2177 2310 and viability was assessed though FACS analysis of CTV 10152 5242 6689 2310 levels. This was carried out by InCyte /FlowJo as follows: 10153 5242 6690 2310 Between 5 ,000 event for FSC / SSC and compensation wells , 6476 2305 2170 2310 and 30 ,000 events for experimental wells were analyzed by 6518 2304 2304 2309 2309 cytometry . A threshold was set to skip debris and RBCs. 891 1109 [0506 ] The results presented in FIG . 28 , suggest that at 4372 3344 3345 3346 3346 higher E : T ratios ( eg . 5 : 1 ) , the cytotoxic activity of the 15192 9288 9284 9289 bispecific anti -CDH3 -CD3 antibody, v13831 towards the 15193 9288 9285 9289 15194 9288 9286 9289 allogeneic target tumor cells can be mediated by the T cell 15195 9288 9287 9289 redirected activity of the bispecific . Thus , as the number of 17119 11176 11177 11175 T effector cells decrease , as is the case in the 1 : 5 and 1 : 50 17118 11178 11179 11175 E : T ratios , tumor cell killing is nearly lost . Unlike the 13831 8074 3320 8071 unconjugated variant, v13831 - SMCC - DM1 was effective 13792 1015 3320 even at the lower E : T ratios ( 1 : 5 and 1 : 50 ) as a result of its 13790 3320 3299 second mode of action , drug delivery to the target tumor 16371 3537 3320 3357 cells through internalization of the antigen - binding con 873 1064 1065 struct. Therefore , the data further supports the benefit of a 17119 11176 11177 11175 dual mechanism of action as the T cell mediated activity of 17118 11178 11179 11175 the unconjugated variants is highly donor dependent and not sufficient to kill > 90 % of the target tumor cells in this assay, US 2018 /0193477 A1 Jul. 12 , 2018 95

S27-Y31 T96088- D49-S51 01-N106 1 -1 -1 -1 -1 -1 - ad e T 0122 S240

TABLEYY WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV CAGATCGTCCTGACACAGAGC?CAGCTATCATGTCAGCAAccccccccGAGAAAGTCACAATGACT?????AGCCAGc????cTGTGAGCTACATGAACTGGTATCAGCA Sequence MHEALHNHYTOKSLSLSPGK AAG SSVSY TCCTCTGTGAGCTAC QOWSSNPFT CAGCAGTGGAGTTCAAACCCATTCACT DTS GACACATCC GTTLTVSS GGCCAGGGGACTACCCTGACAGTGAGCTCC Descrip clonetion 2176Full 2176Full 2176VL 2176VL 2176L1 2176L1 2176L3 2176L3 2176L2 2176L2 2176VH 2176VH SEQ ID No. 1.OIVLTOSPAIMSASPGEKVTMTCSASSSVSYMNWYOOKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCOOWSSNPFTFGSGTKLEINGGGGSGG GGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDK5SSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAAEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVSVLTVLHQD GAAAAGCGGAAcc?cccccAAGAGATGGATCTACGACACATCCAAGCTGuccTCTGGAGTGccTGCTCACTICAGGGGCAdcGGCTCTGGGAccAGTTATTCACTG??? ????ccGGCATGGAGGCCGAAGATGccGcTAccTACTATTGCCAGCAGTGGAGTICAAAccCATICACTTTTGGATCTGGCACCAAGCTGGAAATTAATGGCGGAGGAG GCTCCGGAGGAGGAGGGTCTGGAGGAGGAGGAAGTCAGGTGCAGCTGCAGCAGTCCGGAGCAGAGCTGGCTCGACCAGGAGCTAGTGTGAAAATGTCCTGTAAGGC AAGCGGCTACACCTTCACACGGTATACCATGCATTGGGTGAAACAGAGACCCGGGCAGGGACTGGAATGGATCGGGTACATTAATCCTAGCCGAGGATACACAAACTAC AACCAGAAGTTTAAAGACAAGGCCACTCTGACCACAGATAAGAGCTCCTCTACCGCTTATATGCAGCTGAGTTCACTGACATCTGAGGACAGTGCAGTGTACTATTGCGC CAGGTACTATGACGATCACTACTGTCTGGATTATTGGGGCCAGGGGACTACCCTGACAGTGAGCTCCGCAGCCGAACCTAAATSTAGTGACAAGACTCATACCTGCCCCC ?TTGTCCAGCACCAGAGGCTGCAGGAGGACCTIccGTGTTccTGTTTccAccCAAACCAAAGGATACTCTGATGATCTcccGGACACCTGAAGTCACTTGCGTGGTCGTG AGCGTGTCTCACGAGGACCCCGAAGTCAAGTTTAACTGGTACGTGGACGGCGTCGAGGTGCATAATGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACATATC GCGTCGTGTCTGTCCTGACTGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAATGCAAGGTGAGCAACAAGGCACTGCCTGCCCCAATCGAGAAGACAATTA GCAAAGCAAAGGGGCAGCCCCGAGAACCTCAGGTCTACGTGCTGCCTCCATCTCGGGACGAGCTGACTAAAAACCAGGTCAGTCTGCTGTGTCTGGTGAAGGGCTTCTA ?cCAAGCGATATTGCTGTGGAGTGGGAATccAATGGGCAGcccGAAAACAATTA???GACTIGGcccccTGTccTGGACTCAGATGGGAGCTIc?????GTATAGTAAACTGACCGTGGACAAGTCACGGTGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCATGAGGCCCTGCACAATCATTACACCCAGAAATCTCTGAGTCTGTCACCCGGC 3.OIVLTOSPAIMSASPGEKVTMTCSASSSVSYMNWYOOKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCOOWSSNPFTFGSGTKLEIN CAGATCGTccTGACACAGAGC?CAGCTATCATGTCAGCAAGccccGGCGAGAAAGTCACAATGACTTGCTCAcccAGc?ccTCTGTGAGCTACATGAACTGGTATCAGCA4.GAAAAGCGGAACCTcccccAAGAGATGGATCTACGACACATCCAAGCTGGCCTCTGGAGTGCCTGCTCACTICAGGGGCAGCGGCTCTGGGACCAGTTATTCACTG??? ATTTCCGGCATGGAGGCCGAAGATGCCGCTACCTACTATTGCCAGCAGTGGAGTTCAAACCCATTCACTTTTGGATCTGGCACCAAGCTGGAAATTAAT i 6. o o 10. QVOLOQSGAELARPGASVKMSCKASGYTFTRYTMHWVKORPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDK5SSTAYMOLSSLTSEDSAVYYCARYYDDHYCLDYWGQ11. CAGGTGCAGCTGCAGCAGTccGGAGCAGAGCTGGCTCGAccAGGAGCTAGTGTGAAAATGTccTGTAAGGCAAGCGGCTACAccTTCACACGGTATACCATGCATTGG GTGAAACAGAGACCCGGGCAGGGACTGGAATGGATCGGGTACATTAATCCTAGCCGAGGATACACAAACTACAACCAGAAGTTTAAAGACAAGGCCACTCTGACCACAGATAAGAGCTccTC?AccGc???T?TGCAGCTGAGTTCACTG????cTGAGGACAGTGCAGTGTACTATTGCGccAGGTACTATGACGATCACTACTGTCTGGATTATTGG US 2018 /0193477 A1 Jul. 12 , 2018 57

G147 T154 -1 A218 Y229 -1 1172 T179 -1 A258 K367 1- G368 G473 1- -1 -1

TABLEYY-continued CAGGTACTATGACGATCACTACTCCCTGGATTATTGGGGCCAGGGGACTACCCTGACAGTGAGCTCCGCAGCCGAACCTAAATCTAGTGACAAGACTCATACCTGCCCCC Sequence GYTFTRYT GGCTACACCTTCACACGGTATACC ARYYDDHYCLDY GCCAGGTACTATGACGATCACTACTGTCTGGATTAT INPSRGYT ATTAATCCTAGCCGAGGATACACA AAAG MHEALHNHYTOKSLSLSPGK Descrip clonetion 2176H1 2176H1 2176H3 2176H3 2176H2 2176H2 2176CH2 2176CH2 2176CH3 2176CH3 2177Full 2177Full SEO ID No. 13. 14. 15. 16. 17. 18. APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK19. GCACCAGAGGCTGCAGGAGG?????ccGTGTTccTGTTTcCAcccAAACCAAAGGATACTCTGATGATCTcccGGACAccTGAAGTCACTTGCGTGGTCGTGAGCGTGTC20. TCACGAGGACCCCGAAGTCAAGTTTAACTGGTACGTGGACGGCGTCGAGGTGCATAATGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACATATCGCGTCGT GTCTGTCCTGACTGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAATGCAAGGTGAGCAACAAGGCACTGCCTGCCCCAATCGAGAAGACAATTAGCAAAGC 21.GQPREPOVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPG 22.GGGCAGccccGAGAAccTCAGGTC?AcGTGCTGcc?ccATc?cGGGACGAGCTGACTAAAAAccAGGTCAGTCTGCTGTGTCTGGTGAAGGGC??????ccAAGCGATA TTGCTGTGGAGTGGGAATCCAATGGGCAGCCCGAAAACAATTACCTGACTTGGCCCCCTGTCCTGGACTCAGATGGGAGCTTCTTTCTGTATAGTAAACTGACCGTGGAC AAGTCACGGTGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCATGAGGCCCTGCACAATCATTACACCCAGAAATCTCTGAGTCTGTCACCCGGC 23.QIVLTOSPAIMSASPGEKVTMTCSASSSVSYMNWYOQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCOOWSSNPFTFGSGTKLEINGGGGSGG GGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKORPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDK5SSTAYMOLSSLTSEDSAVYYCARYYDDHY SLDYWGOGTTLTVSSAAEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHOD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOVYVLPPSRDELTKNOVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV 24.CAGATCGTCCTGACACAGAGCCCAGCTATCATGTCAGCAAGCCCCGGCGAGAAAGTCACAATGACTTGCTCAGCCAGCTCCTCTGTGAGCTACATGAACTGGTATCAGCA GAAAAGCGGAACCTCCCCCAAGAGATGGATCTACGACACATCCAAGCTGGCCTCTGGAGTGCCTGCTCACTTCAGGGGCAGCGGCTCTGGGACCAGTTATTCACTGACA ATTTCCGGCATGGAGGCCGAAGATGCCGCTACCTACTATTGCCAGCAGTGGAGTTCAAACCCATTCACTTTTGGATCTGGCACCAAGCTGGAAATTAATGGCGGAGGAG GCTCCGGAGGAGGAGGGTCTGGAGGAGGAGGAAGTCAGGTGCAGCTGCAGCAGAGCGGAGCAGAGCTGGCTCGACCAGGAGCTAGTGTGAAAATGTCCTGTAAGGC AAGCGGCTACACCTTCACACGGTATACCATGCATTGGGTGAAACAGAGACCCGGGCAGGGACTGGAATGGATCGGGTACATTAATCCTTCCCGAGGATACACAAACTAC AACCAGAAGTTTAAAGACAAGGCCACTCTGACCACAGATAAGAGCTCCTCTACCGCTTATATGCAGCTGAGTTCACTGACATCTGAGGACAGTGCAGTGTACTATTGCGC ??????CAGCACCAGAGGCTGCAGGAGGACCTAGCGTGTTccTGTTTCCAC?CAAACCAAAGG?????cTGATGATC??ccGGACACCTGAAGTCACTTGTGTGGTCGTG US 2018 /0193477 A1 Jul. 12 , 2018 58

N10601- S27-Y31 088-T96 D49-551 -1 -1 -1 0122 S240 -1 G147 T154 -1 A218 Y229 -1 1172 T179 -1 A258

TABLEYY-continued

Sequence AAG SSVSY TCCTCTGTGAGCTAC QOWSSNPFT CAGCAGTGGAGTTCAAACCCATTRACT DTS GACACATCC GTTLTVSS GGCCAGGGGACTACCCTGACAGTGAGCTCC GYTFTRYT GGCTACACCTTCACACGGTATACC ARYYDDHYSLDY GCCAGGTACTATGACIGATCACTACTcccTGGATTAT INPSRGYT ATTAATCCTTCCCGAGGATACACA DescripDescrip 55 | clonetion 2177VL 2177VL 2177L1 2177L1 2177L3 2177L3 2177L2 2177L2 2177VH 2177VH 2177H1 2177H1 2177H3 2177H3 2177H2 2177H2 2177CH2 SEQID ID No. AGCGTGTCTCACGAGGACCCCGAAGTCAAGTTTAACTGGTACGTGGACGGCGTCGAGGTGCATAATGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACATATC GCGTCGTGTCTGTCCTGACTGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAATGCAAGGTGAGCAACAAGGCACTGCCTGCCCCAATCGAGAAGACAATTA GCAAAGCAAAGGGGCAGCCCCGAGAACCTCAGGTCTACGTGCTGCCTCCATCTCGGGACGAGCTGACTAAAAACCAGGTCAGTCTGCTGTGTCTGGTGAAGGGCTTCTA TCCAAGCGATATTGCTGTGGAGTGGGAATCCAATGGGCAGCCCGAAAACAATTACCTGACTTGGCCCCCTGTCCTGGACTCAGATGGGAGCTTCTTTCTGTATAGTAAAC TGACCGTGGACAAGTCACGGTGGCAGCAGGGAAACGTCTTTAGCTGTTCCGTGATGCATGAGGCCCTGCACAATCATTACACCCAGAAATCTCTGAGTCTGTCACCCGGC 25.QIVLTOSPAIMSASPGEKVTMTCSASSSVSYMNWYQOKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN 26.CAGATCGTCCTGACACAGAGCCCAGCTATCATGTCAGCAAGCCCCGGCGAGAAAGTCACAATGACTTGCTCAGCCAGCTCCTCTGTGAGCTACATGAACTGGTATCACA GAAAAGCGGAACCTCCCCCAAGAGATGGATCTACGACACATCCAAGCTGGCCTCTGGAGTGCCTGCTCACTTCAGGGGCAGCGGCTCTGGGACCAGTTATTCACTGACA ATTTCCGGCATGGAGGCCGAAGATGCCGCTACCTACTATTGCCAGCAGTGGAGTTCAAACCCATTCACTTTTGGATCTGGCACCAAGCTGGAAATTAAT 27. 28. 29. 30. 31. 32. 33.QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKORPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDK5SSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQble 34.CAGGTGCAGCTGCAGCAGAGCGGAGCAGAGCTGGCTCGACCAGGAGCTAGTGTGAAAATGTCCTGTAAGGCAAGCGGCTACACCTTCACACGGTATACCATGCATTGG GTGAAACAGAGACCCGGGCAGGGACTGGAATGGATCGGGTACATTAATCCTTCCCGAGGATACACAAACTACAACCAGAAGTTTAAAACAAGGCCACTCTGACCACA GATAAGAGCTCCTCTACCGCTTATATGCAGCTGAGTTCACTGACATCTGAGGACAGTGCAGTGTACTATTGCGCCAGGTACTATGACGATCACTACTCCCTGGATTATTGG 35. 36. 37. 38. 39. 40. APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEOYNSTYRVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAK41.