Antigens & Antibodies II Polyclonal antibodies vs Monoclonal ...
Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies...
Transcript of Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies...
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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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
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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?