Target Protein Antibody Antibody Batch to

selection & Expression Discovey characterisation batch QC

Protein Design and QC

Antibody development and characterisation have a great impact on antibody specificity, performance and consistency

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Advantages and constraints of polyclonal antibodies

Advantages • Relatively fast to produce • Recognize multiple epitopes in a single target • Useful for certain applications; IP

Constrains • Recognize multiple epitopes in a single target • Cross-reactivity with homologous proteins • Low sensitivity and specificity • Batch-to-batch variability • Initial discovery and production is in vivo

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High Nov I epitopes

Affinity

Low

Low High

Specificity

Compared to other common antibody platforms, rabbit monoclonal antibodies have the highest affinity and specificity

Advantages and constraints of hybridoma-derived monoclonal antibodies

Monoclonal (hybridoma) antibody

Advantages • Recognize a single epitope • High specificity • No or low batch-to-batch variability

Constrains • Vulnerable to stability issues and loss of epitope recognition • Unable to produce against toxic antigens • Antibody discovery phase is in vivo

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Recombinant antibodies produced through a recombinant DNA platform A) Recombinant monoclonal antibody development - in vivo approach

NGS~~ L.., I

Isolation of -------. /VH

coo•--~circulating B cells

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~~~ )- " ' ,IJ_ Hybridoma~ -II}derived 11 ~ Isolation and 00

immortalization of B cells Monoclonal .. antibody

B) Recombinant monoclonal antibody development Recombinantin vitro approach

Phage M13 monoclonal AxioMx technologyCoa1 PIO'"n

1 antibodyPhage Display /

Recombinant antibodies Tal1t:>e<' (~eop.e$)

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(?700 cop.cs)

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Recombinant monoclonal antibody

development by phage display

Recombinant affinity reagents can be tagged and used as traditional antibodies, or can be recombined back to Fc fusions, Fabs, IgGs, etc.

Advantages and constraints of phage

display recombinant antibodies

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Performance of Polyclonal vs recombinant monoclonal antibodies to histone modifications by ChIP

Polyclonal H3K4me3

ab8580 Lot-A ab8580 Lot-B

20 17.73 20

113 37 15

8 62 :: lCl .61 1· l~ 6,.65 1' 6,.78

0.97 1.12 0.93 0.91 1.03 1.07

0 - - - 0 - -9.Q~ R\f ,SJ'(- {$~ :1,.r? R~'f- 9.~ R\f {$~ :1,.r? R~,SJ'(­cl'~~ ~eff 0"' ~ ~ ~ 4f

Variation in signal among batches

RabMAb® Recombinant monoclonal H3K4me3

ab213224 Lot-A ab213224 Lot-B

20 20 15.60 14.63

15 15 9.35 9.33

10 lO

5 50. 97 1.04 0. 99 0.97 1.11 0.93I II - - - I ll - - ­0 0

-ts~ Cf() ~'(- ~~ ~ ~r?~'(-9.~ R..:f #'(- R -f$ ~9.<J QO ~ 0"' ~ ,. $' .# 0"' ~-t:' ~eff

There is clear consistency of Ch IP data between lots A and B.

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RabMAb rabbit monoclonal antibodies

NGS antibody selection Phage display antibody

discovery

Target selection & Protein

Protein Design Expression and QC

Antibody development

Antibody characterisation

Batch to batch QC

Recombinant monoclonal

Antibody generation

Antibody development and characterisation have a great impact on antibody specificity, performance and consistency

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Extensive antibody characterisation and validation is key to antibody specificity and performance

Label free Homogeneous HT assay ELISA Off-rate ranking development and

screening Colored product~

donor acceptor

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Substrate ,..,,/

WB Flow cytometryICC IHC I 2 J

250k0a ­

150k0a ­

100k0a ­

75k0a ­

50kDa ­

37k0a ­

25k0a ­20k0• ­

15k01 ­

10k0a ­

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Recombinant anti-BCMA RabMAb® perform in multiple applications

IP 2 input +

250 kDa 250 kDa 150 kDa 150 kDa

100 kDa 100 kDa

75 kDa 75 kDa

u 50 kDa Q. +- lgG heavy chain 50 kDa j 37 kDa ICC"*- 37 kDa <l E O> :'.! ~ 25 kDa ~ 25 kDa ~20kDa

~ 20 kDa ~15 kDa ~ § 15kDa

.c

81o koa - "' 1!-~ 10 kDa 0 " 0 0u

BCMA- A lexa Fluor©488 (530/30BP)

Flow Cytometry

2 3

Western blot lmmunoprecipitation

Ab245940 anti-BCMAMM cell line U266B1

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Enhanced validation level - extended toIHC validation on multispecies and multi-tissue microarray

relevant tumour tissue

NSCLC TMA

Mouse cerebrum

mg;d'l.!,!I RabMAb

Recombinant Anti-Uteroglobin antibody [EPR19846) (ab213203) Recombinant anti- PD-L1 (CAL 1 O ab237726

Tailored approach to verify antibody specificity

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HAP1 WT

HAP1- Vimentin KO

HAP1 WT Isotype Ctrl

HAP1- vimentin KO Isotype Ctrl

GAPDH

WT KO WT KO WT KO WT KO

Beta Ill Tubulin

WT KO Helo HEK293

Impact of antibody format on specificity and consistency

Rabbit polyclonal Anti-beta Ill Tubulin Recombinant Anti-beta Ill Tubulin antibody antibody (ab 18207) [EPRl 9591] [ab215037)

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260 kDa -

160 kDa -125 kDa -

90kDa -

70kDa -

50kDa -

38kDa -

30kDa -

25kDa -

15kDa -

itJ·!Bi$ Ant i-BACEl antibody [EPRl9523]

Host: Rabbit

Clonallty: Monoclonal (EPR19523)

260 kDa

160 kDa 125 kDa

90 kDa

70 kDa

050 kDa -Q.

~ 38 kDa - <( ...

030 kDa - N

s25 kDa.c

"' ~ 15 kDa u

l'.@M

Top cited clones do not always perform the best

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Combining recombinant antibody technologies and high validation standards to develop the best reagents

lmmunohistochemical analysis of paraffin-embedded lmmunofluorescent analysis of Neuro-2a (Mouse Human astrocytoma tissue labelling Pan Trkwith abl81560 neuroblastoma cells) cells labelling Pan Trkwith abl81560

Pan-Trk immunohistochemistry is an efficient and reliable screen for NTRK fusions

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Antibody discovery driven by a broad range of platforms & screening approaches

Rapid RabMAb® BCell Cloning NGS Phage display

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Over 18,000 . /-_tl Recombinant~ ~ recombinant

rabbit • * ,#.RabMAbs monoclonal -HJ

antibody -II · Botch - botch consistency

Best-in-class antibody discovery platforms supports generation of highest performance recombinant antibodies

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Questions?