recombinant monoclonal antibodies: an indispensable ... · 1/21/2020 · monoclonal antibodies...
TRANSCRIPT
![Page 1: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/1.jpg)
![Page 2: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/2.jpg)
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
abeam
![Page 3: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/3.jpg)
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
abeam
![Page 4: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/4.jpg)
High Nov I epitopes
Affinity
Low
Low High
Specificity
Compared to other common antibody platforms, rabbit monoclonal antibodies have the highest affinity and specificity
![Page 5: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/5.jpg)
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
abeam
![Page 6: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/6.jpg)
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
""'YJ
~~~ )- " ' ,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$)
abeam
(?700 cop.cs)
![Page 7: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/7.jpg)
• •
• • • •
• •
![Page 8: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/8.jpg)
Recombinant monoclonal antibody
development by phage display
Recombinant affinity reagents can be tagged and used as traditional antibodies, or can be recombined back to Fc fusions, Fabs, IgGs, etc.
![Page 9: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/9.jpg)
Advantages and constraints of phage
display recombinant antibodies
• •
• • • •
• •
•
![Page 10: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/10.jpg)
Performance of Polyclonal vs recombinant monoclonal antibodies to histone modifications by ChIP
Polyclonal H3K4me3
ab8580 Lot-A ab8580 Lot-B
20 17.73 20
113 37 15
8 62 :: lCl .61 1· l~ 6,.65 1' 6,.78
0.97 1.12 0.93 0.91 1.03 1.07
0 - - - 0 - -9.Q~ R\f ,SJ'(- {$~ :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.
abeam
![Page 11: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/11.jpg)
•
RabMAb rabbit monoclonal antibodies
NGS antibody selection Phage display antibody
discovery
![Page 12: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/12.jpg)
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
abeam
![Page 13: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/13.jpg)
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
,__
Substrate ,..,,/
WB Flow cytometryICC IHC I 2 J
250k0a
150k0a
100k0a
75k0a
50kDa
37k0a
25k0a 20k0•
15k01
10k0a
abeam
![Page 14: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/14.jpg)
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
abeam
![Page 15: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/15.jpg)
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
abeam
![Page 16: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/16.jpg)
HAP1 WT
HAP1- Vimentin KO
HAP1 WT Isotype Ctrl
HAP1- vimentin KO Isotype Ctrl
![Page 17: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/17.jpg)
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)
abeam
![Page 18: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/18.jpg)
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
abeam
![Page 19: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/19.jpg)
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
abeam
![Page 20: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/20.jpg)
Antibody discovery driven by a broad range of platforms & screening approaches
Rapid RabMAb® BCell Cloning NGS Phage display
~l...,LUJI
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
abeam 20
![Page 21: Recombinant Monoclonal Antibodies: An Indispensable ... · 1/21/2020 · monoclonal antibodies Monoclonal (hybridoma) antibody Advantages • Recognize a single epitope • High](https://reader034.vdocuments.net/reader034/viewer/2022052218/5f0a50937e708231d42b0cbd/html5/thumbnails/21.jpg)
Questions?