In Vivo Characterization Platform

Our integrated suite of in vitro assays allows for a comprehensive evaluation of various therapeutic targets and modalities, including TCEs, ADCs and autoimmune & inflammation molecules.

Efficient In Vitro Evaluation Platform for ADC, TCE, and Autoimmune & Inflammation Molecules Wenyuan Cao, Jianxin Mao, Jianchang Cao, Jiansheng Wu

WuXi Biologics, NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)

In early stages of drug development, the challenge lies in screening and identifying the most promising molecules from vast amounts of accumulating data. This poster presents our efficient, high-throughput in vitro evaluation methods, developed based on literatures, patents, and extensive project experience. Fig 1. One-Stop, Integrated In Vitro Platform for Therapeutic Antibody and ADC Evaluation Introduction Abstract The development of therapeutic antibodies and antibody-drug conjugates (ADCs) involves high costs and extensive R&D cycles. An efficient in vitro evaluation and screening strategy can significantly mitigate these challenges. WuXi Biologics’ in vitro platform provides efficient solutions for biologics evaluation, including internalization, cytotoxicity & bystander killing, and plasma stability assays for ADCs; T cell killing, activation and cytokine release assays for T cell engagers (TCEs); and ligand competitive binding, reporter gene and proliferation assays for autoimmune & inflammation molecules. Moreover, 500+ custom cell lines have been generated for bioassays. This one-stop, integrated in vitro platform efficiently streamlines therapeutic development.

Case Study 3: In Vitro Characterization of TCEs Fig 4. Reporter Gene Assay, T Cell Killing, Cytokine Production and T Cell Activation Assay to Characterize Molecular Functions of TCEs

6$7,1($1"8$-$"&99+.":68&;<"=)5>?!>1,35>73("@'1$$-)-5"A,1"!"#$%%"&'()*+(),-

!"#$%%"&#'()&'(*+",-.

!"#$ !"#%

!" #$

!" #%

!" #&

!" "

!" &

!" %

!" $

!"#$%&'(

!"#$%%"4)%%)-5

!"#$%%"&'%%'()"*+"!,- +."#$%% !//0'

=;; =?;

Molecular Assays

Cellular Assays • Reporter gene assay (30+) • Proliferation assay • Internalization assay • Apoptosis assay

Immune Assays • Mixed Lymphocyte Reaction • Ag-specific T cell proliferation • T/ ɣδ T/ Treg/ NK expansion/activation assay • ɣδ T/ NK degranulation assay • T/NK cytotoxicity assay • T/NK exhaustion and activation assay • APC activation assay

?; @; A; B;

!"# !"$ %&'()*+,-'.(/'0

• Binding assay • Competition binding assay • Epitope binning assay • Dimerization assay • Receptor occupancy

>?; ;

• Cell cycle analysis • Cell migration assay • Agglutination assay

;<;;;=

;<;=

=;;

=;;;;

/12"3456

! 23+4'5'2367'.89:.;;<=>'<>'23?&'23@&'23A'('0.BB; !"#$%%"&'()*+(),-"

#.(,/)-$"01,23'(),-

Biochemical Assays

Fc Eector Function

! !"#$%&'(#"$)&'!*$+&'!*$,-&'./01

• Treg suppression assay • Cytokine release assay

!"#$%&'()&**+,-%,-%!"./%0%!&11

• Enzymatic assay • Phosphorylation assay

• ADCC assay • CDC assay • ADCP assay

!"#$ ! %&'(')*'%+,-./&%0'(( ,1123 4%56708

422

!"# !"$ %&'()*+,-./,0

!"# !"$ %&'()*+,-'.(/'0

23222224 232224 2324

422

?<?????@ ?<???@

?<?@

@??

Fig 2. Various Methods for Protein and Cellular Binding Analysis Case Study 1: Binding and Competition Assays

78*9-:;

19*%+:78

Fig 5. High-Throughput Protein-Based and Cellular Functional Screening Methods, and Proliferation & Immune Assays for Evaluating Autoimmune and Inflammation Molecules Case Study 4: Autoimmune & Inflammation Molecules

.&"/(0$1-$12

!"#$%&'()*+,%-'

! !"#$%&'()*+%, ! -./012 ! 0!3 ! 4%55()*+%, ! 4%552-./01

! 6#"2,?*5(B?'8$&#'*52:#5%8?5%+2C%D=DE2)&+@%8&B&82*'$&)#,&%+F ! ;>"%%2B#":*$+ ! 4%552G24%55 ! !"#$%&'2G2!"#$%&' ! 4%552G2!"#$%&'

0,+)1-,1'2,%,'344$5'60237

81)9":,1$-")%';'<**=%,'344$54

!"#$%&'&'"()*'&+),'-.(/ ! <%;=>)="#0;=?0)8+?"#)'%*>@(,'A+,6"''*%*; ! B0)#%//0*'+(*)(;#*%,)1+,06=+(,+ .C> DEFGD3.C>HFG @$#69'"+C'@"%9%I0/(@ !"#$%&'()*+,-"-".,'/"%&"%#

! :'$$0$("+&0*6)%#*+ ,6"''*%*; ! .*&$(//()%#*+,%;*($%*;

! !"#$%&'"()%#*+(,,(- ! .//0*'+(,,(-,1+2345+6'$$+(6)%7()%#*8+ 6-)#9%*'+"'$'(,'+(,,(-8+ !"#$%

! 65#7289$#:%$"9 ! 63-; ! <&=>($>"#?=>@?$2:#,%+2*"%2*A*&5*)5%

+,-#./0120134.!"##.5.!"##6.78!9

5627 ("(8%99%:&';% ,'-.(/)6,":<%= 5647)%99%:&';% ,'-.(/)6,":<%=

410

, -,,,, .,,,, /,,,, 0,,,, 1,,,, 2,,,,

&'!" ( $'!" ( ('!" ( )'!" ( !'!" *

Case study 1: HTP intact mass analysis for ID !"#$"#%&'#&(&)*+',&-.//&/"#.&01&2345 :966 &!

60+7/ 60+78 !"#$%#!'()#*+ *9*!"#$%#!'()#*6

213

+,! 78/9:;<-=/09>/? 5@AB0C-/0?: .<??-/0?:

:666

308 30:

!"# $%&'()*+,&-'.&/

210

!"#$%&!'()#*+

8966

,-(./*$01#23)*41"#'15 !"#$%&!'()#*6

>?"&@$% !"(&=",

8666

013

966

!" #$ !" #% !" #& !" #! !" " !" ! "

010

67666867668 6768 678 8 86 866 8666 6

!"!!!# !"!!# !"!# !"#

#!!

0102 012

200

,(,,,- ,(,-

-,, -,,,,

+70*:";<

56?)A(BC !"#$%&'(

!"#$%&'()'*+ !"#$%&'(

+,-./0-1023 !"#$%&'(

30;&<#=>

!*+,-,.,/*&/0&!"#$%#1232*12*.&4255&36/5,0267.,/*

!"#$%&'()

",87*1&4/932.,.,/*&7::7;

3$2,45,#%&2,6&+(78#**1$129()&/+$6/*(:*//(;$1*'($1(<=>(?*//

!"##$%&'"$(&)*+,"

!"##-.$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!"##-/$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!"##-0

Fig 6. Cell Line Development for Bioassays, Including Target Protein Overexpression, Reporter Gene Cell Lines/Pools, and Target Gene Knockout Cell Lines Case Study 5: Cell Line Development for Bioassays

&!!!

%!!!!

#$!!!

%!!!

!"##$% !"##$& !"##$'

$!!!!

#!!!!

$!!!

#!!!!

$!!!

#!!!

! !""#$%&'()*$%+,,$,-.+'$/+.+01(+2$3)0$4-)1''15'$

!"!!!# !"!#

#!! #!!!!

!"!!!# !"!#

#!! #!!!!

!"!!!# !"!#

#!! #!!!!

&'()*+,-

%&'()*+,

'()*+,-.

!"#$%&'2%+%'7+)89):&'3%44'5*+%

1%/)#&%#'2%+%'3%44'5*+%6())4

!"#$%&'(#)&%*+',-%#%./#%00*)+

!"##-1$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!"##-2$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!"##-3

!"##$( !"##$) !"##$*

'!!

#!!! #$!! %!!! %$!!

&'# &'$

+,(

%!!!

()*# ()*$ +,-./ ,0123456718291: +,-.# ,0123456718291:

&!!

!"#$%&'()&

!"#$%&'()*

$!!!

%!!

#!!!

$!!

! $!!

0(12/& 3/(45

!"!!!# !"!#

#!! #!!!!

!"!!!# !"!#

#!! #!!!!

!"!!!# !"!#

#!! #!!!!

;&'0<68)

&'()*+,-

()*+,-./

0(12/& 3/(45

6257802%6%(90'0:;'0<<7(2%(2%=>?1@

*('+&,-.&//

Case Study 2: In Vitro Characterization of ADCs Fig 3. Internalization, Cytotoxicity and Bystander Killing, and Serum/Plasma Stability Assay for High-Throughput Screening and Functional Characterization of ADCs

#$!

!"# %&' ()*+,-./0#

#!!

-.%/0,,

12(/3(45%/0,,

!"#$%"&'()&#(*"+,"&'-.(.

7$%89:;'&.9&+7#&<('(#-

/-#*#*0(1(#-+2+3-.#&"4$%+5(''("6

!"!#

!"#

A1?BCD,4/D+"& 54)(' )(E0, FG HBHA

%,12&345-6,78

! !"#$%&'(# ?3#%#%@868#3'<00/3

! !"#$%&'()'*+,%&%-./0"&'

Random Recombination, Flp-In™ System, Lentiviral System, and CRISPR Knockout

Serum/Plasma: human, cyno, mouse and rat

#!! )*+,-./001213,+4,567+$88*9

Antigen

Conclusions WuXi Biologics has developed a comprehensive in vitro evaluation platform to facilitate the therapeutic development. For ADCs: Internalization, cytotoxicity & bystander killing, and plasma stability assays For TCEs: T cell killing, activation, and cytokine release assays For autoimmune & inflammation molecules: Ligand competitive binding, reporter gene, proliferation and immune assays • • • Acknowledgments We would like to express our sincere gratitude to the scientists in the CRO Services Department at WuXi Biologics for their invaluable support in developing in vitro platforms. Additionally, we extend our appreciation to the legal, public relations, and marketing departments at WuXi Biologics for their assistance with patent filings and the preparation and production of written materials. WuXi Biologics is a global leading Contract Research, Development and Manufacturing Organization (CRDMO) o ffe ring end-to-end solutions to enable partners to discover, develop and manufacture biologics from concept to commercialization. About WuXi Biologics For more information see us at wuxibiologics.com

*+#"5A3B# *+%"5A3B# *+C"5A3B# A-;>4D0"5A3B# 567+$88*9,;1E4

Internalization

Late endosome /Iysosome

Incubation at 37°C for 0/1/4/7/14/21 days

Target cell

$%!

!"!!# !"!# !"#

#!!

:;1.",<18=

: anti-Fc F(ab)2-pHrodo

: Antibody or ADC

)*+,+-././0121.-

! !"#$%&';)'<68&'=>"96$89:

#!!

Antigen

)8#"9:;<#$55)= )8>"9:;<#$55)= )8%"9:;<#$55)= :?/.-@A"9:;<#$55)=

Antigen affinity puriflcation DAR value detection

Released payload

Acid quench

$%!

Target cell

!"!!# !"!# !"#

#!!

Results

! !"#$%&#'()&'*%')+(,"-./0(*1/20(-2")'( ./3(4.$ ! 54''6&.1#2.3(4'#'.)'(./3(34"76$26 ./$%8231(4.$%2(9:;<=(*./(8'(.))'))'3(81( >?6!@ ! ;774'7.$%2/(*./(8'('A.#".$'3(81(@B?6 CD>?(E%$,(F#"24')*'/*'(3'$'*$%2/ ! ;/$%7'/(8%/3%/7(.*$%A%$1(981(B>G@;H5;?@=0( $2$.#(;:?(I(./$%8231(J"./$%F%*.$%2/(981( B>G@;H>?6!@=(*./(8'($')$'3($2('A.#".$'( ;:?()$.8%#%$1

+/32",4356

! !"#$%&'1)'230%0%4"'5,/6789:

! .30#/9&",'A8BB89:

!"#$%&

'$()*$+,-.)%

#$! %&'()()*+,+('%)-%./0+"12,%34567894:

!"# $%&'()*+,-./01 "23 $%&'()*+,-./01 !"# $%&'()*+,456781 "23 $%&'()*+,456781

#!!

!"!!!# !"!#

#!! #!!!!

&)-,;-(</(+)-3-=: !"#$%&'#()

To discuss this p oster : PS _B D @w u x i b iolo g ics . com

Page 1