Giulio Russo Recombinant mono- and multi-clonal antibodies as higher quality substitutes for animal sera in diagnostics 15.10.2020 | Antibody Webinar Series - PETA International Science Consortium
www.abcalis.com Phage display mAbs in therapy
9 animal free phage display-derived therapeutic mAbs approved 53 animal free phage display-derived therapeutic mAbs in clinical trials (Aug. 2020)
(Atezolizumab, Avelumab, Belimumab, Necitumumab, Ramucirumab, Guselkumab, Lanadelumab, Emapalumab, Raxibacumab)
Shim H. Curr Pharm Des. 2016, 22:6538-59 | Alfaleh et al., 2020, doi: 10.3389/fimmu.2020.01986
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 2 Rec Abs advantages for therapy
animal free generation antigen binding regions from human naïve libraries fully human IgG antibodies
sequence defined reagent unlimited reproducibility and scalable batch size
Annu. Rev. Med. 67, 165–83 (2016) customizable selection process constant epitope-/conformation-specific regions neutralizing antibodies
Fc improvable after selection
tunable stability/affinity Brinkmann and Kontermann, 2017, mAbs
molecular engineering
scFv, Fab, IgG, bispecifics, CAR-T, By Bioconjugator - Own work, CC BY-SA 4.0, site-specific conjugation (ADC) IgG https://commons.wikimedia.org/w/index.php?curid=58772304
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 3 Rec Abs advantages for diagnostics and research
animal free generation antigen binding regions from human naïve libraries
sequence defined reagent
customizable selection process constant Also beneficial for regions non-therapeutic
Fc improvable after selection applications?
molecular engineering IgG
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 4 Rec Abs advantages for diagnostics and research
animal free generation antigen binding regions from human naïve libraries reduce animal farming
sequence defined reagent unlimited reproducibility and known composition customizable selection process constant Also beneficial for negative selection of regions unwanted cross-reactivities non-therapeutic
Fc improvable after selection applications? tunable stability/affinity YES molecular engineering free choice of format/Fc-part IgG
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 5 Rec Abs advantages for diagnostics and research
animal free generation antigen binding regions from human naïve libraries Fc species origin: reduce animal farming human sequence defined reagent unlimited reproducibility and known composition mouse customizable selection process constant negative selection of regions unwanted cross-reactivities
Fc rabbit improvable after selection tunable stability/affinity …or many others molecular engineering free choice of format/Fc-part IgG (…added by DNA cloning from known genes)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 6 Why non-therapeutic recAbs are not so common?
• conservatism: animal derived Ab generation established for >40 years
• unawareness of today’s technology and existing high quality examples
• unawareness of the various additional opportunities of recAbs
• unawareness of existing commercial sources
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 7 Mission
Abcalis aims at offering a concrete alternative to animal-based diagnostic reagents introducing thoroughly characterized, animal- free, sustainable and sequence-defined recombinant Mono- and Multi-clonal antibodies generated with the phage display technology.
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 8 Problem: animal derived antibodies
More than 5000 Abs from 51 different providers tested via Western Blot and IHC on tissue microassays.
Less than 50% showed a staining pattern that is consistent with literature or bioinformatics data!
Berglund, L. et al., 2008, Cell Proteomics
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 9 Animal polyclonals
today's products: contains IgG with OTHER specificities Blood
?
?
Customer problems:
- limited amount COSTS for individual batch validation & QC - batch to batch variation RISK of unspecific reactions - non-reproducible NO product continuity animal sera - undefined composition
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 10 Recombinant MULTICLONALS
Abcalis recAb: today's sequence products: defined recAb's
- batch to batch variation ✓ animal-free - NO batch to batch variation - undefined composition ✓ unlimited batch size - FULLY DEFINED composition -✓nonreproducible-reproducible - ANY TARGET possible - limited= Product amount continuity animal sera - SCALABLE production
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 11 Technology
determination of the enzyme/dye final antibody mix conjugation
the world’s antibody antibody addition of species gene repertoire phage display Fc-part of choice MULTICLONAL (IgG mix)
validation in final assay
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 12 When is multi-clonality determinant?
HRP Rabbit anti-human Abs binding to coated hIgG Anti-human antibodies: Rab rec. Monoclonal IgG (SH1844-E3) Rabbit Polyclonal 1 goat anti-rabbit Rabbit Polyclonal 2 Rabbit Polyclonal 3
Rabbit anti-human
hIgG
polystyrene plate well
Indirect ELISA set-up
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 13 When is multi-clonality determinant?
Human IgG detection in serum sample
HRP HRP
Sandwich ELISA set-up
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 14 When is multi-clonality determinant?
Human IgG detection in serum sample
HRP HRP HRP
Sandwich ELISA set-up
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 15 MULTICLONAL assembly
System: Forté-Bio Octet Qke (BLI) RecAbs binding to sensor immobilized hIgG Antigen Loading: 9 µg/ml hIgG1 for 120 s Ab concentration: 50 nM
5 mAb mix
3 mAb mix
1 mAb
https://www.creative-biolabs.com/Octet-system.html
Biolayer interferometry molecular interactions and kinetics measurement
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 16 MULTICLONAL assembly
Captured
Antigen Saturating concentrations (human IgG Ab 1 2x saturating concentration anti-myc tag)
Immobilized Antigen (scFv-myc)
Capillary electrophoresis immunoassay set-up
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 17 MULTICLONAL assembly
Captured
Antigen Saturating concentrations (human IgG 2x saturating concentration anti-myc tag) 2x saturating concentration
Immobilized Antigen (scFv-myc) Ab 2
Capillary electrophoresis immunoassay set-up
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 18 MULTICLONAL assembly
Captured
Antigen Saturating concentrations (human IgG Ab 1 2x saturating concentration anti-myc tag) 2x saturating concentration each at saturating concentration
Immobilized Antigen MULTICLONAL (scFv-myc) Ab 2
Capillary electrophoresis immunoassay set-up
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 19 MULTICLONAL species specificity
mAb 9
mAb 4 MULTICLONAL
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 20 MULTICLONAL subclasses specificity
mAb 6
mAb 7 MULTICLONAL
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 21 Conclusion
https://abcalis.com/multiclonals/
Recombinant MULTICLONAL antibodies are a concrete alternative to animal derived polyclonal antibodies!
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 22 Rec Abs advantages for diagnostics and research
animal free generation antigen binding regions from human naïve libraries reduce animal farming
sequence defined reagent IMMORTAL unlimited reproducibility and known composition CUSTOMIZABLE customizable selection process constant negative selection of regions unwanted reactivities
Fc Encourages improvable after selection tunable stability/affinity thorough
molecular engineering characterization free choice of Fc-part IgG
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 23 Rec Abs advantages for diagnostics and research
animal free generation antigen binding regions from human naïve libraries reduce animal farming
sequence defined reagent IMMORTAL unlimited reproducibility and known composition CUSTOMIZABLE customizable selection process constant negative selection of regions unwanted reactivities
Fc Encourages improvable after selection tunable stability/affinity thorough
molecular engineering characterization free choice of Fc-part IgG
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 24 Rec Abs advantages for diagnostics and research
animal free generation antigen binding regions from human naïve libraries reduce animal farming
sequence defined reagent IMMORTAL unlimited reproducibility and known composition CUSTOMIZABLE customizable selection process constant negative selection of regions unwanted reactivities
Fc Encourages improvable after selection tunable stability/affinity thorough
molecular engineering characterization free choice of Fc-part IgG
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 25 COVID-19 Diagnostics
>100 antibodies tested against SARS-CoV-2
Applications:
• Positive control in serological test instead of patient sera
• Virus detection in rapid First tests antigen tests ongoing!
www.abcam.com/content/structural-and-functional-mechanism-of-sars-cov-2-cell-entry
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 26 anti-S1 SARS-CoV-2 clone ABC68 validation
the world’s antibody antibody addition of species gene repertoire phage display Fc-part of choice validation in final assay
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 27 Binding in ELISA
Sandwich ELISA Indirect ELISA
HRP
HRP Binding to anti-human Fc S1 mutants antibody anti-mouse Fc antibody
Specificity profile Human SARS-CoV-2 Fc-tag (different hcov) S1 SARS-CoV-2 Ab mouse IgG
SARS-CoV-2 Ab Storage stability mouse IgG SARS-CoV-2 and reproducibility S1 polystyrene plate well polystyrene plate well
ABC54 ABC75 ABC54 ABC75 Binding to PFA fixed antigen (IF) ABC63 ABC76 ABC63 ABC76 ABC68 ABC82 ABC68 ABC82
Binding to denatured antigen (WB)
Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 28 Denatured antigen binding
Western Blot
ABC68 Anti-human Fc Lane: 01- 02- 03- 04- 05- 06- 07- 08- 09- 10- 11- 12- 13- 14- 15- 16- 17- CTR+ Binding to S1 mutants
Specificity profile (different hcov) Antigen preparation: Non-reducing conditions Storage stability SDS-PAGE protein separation and reproducibility SARS-CoV-2 Ag. loading: 0.5 µg/lane S1-hFc (monomer > 100 kDa) Binding to PFA fixed antigen (IF) Detection: Rec. mIgG anti-SARS-CoV-2 S1 -> 1 µg/mL Binding to denatured antigen (WB) anti-mouse-AP conjugated
Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 29 Antigen binding after fixation
Calu-3 cells infected with SARS-CoV-2 (strain Zagreb) Calu-3 cells uninfected Immunofluorescence anti-SARS-CoV-2 S1 staining ABC68 (mIgG) 20 µg/mL
Binding to Note: S1 mutants cell fixation in 6% PFA 1h RT DAPI
Specificity profile (different hcov)
Storage stability and reproducibility anti-dsRNA J2 Ab (mIgG) Binding to PFA fixed antigen (IF) 20 µg/mL
DAPI Binding to denatured antigen (WB)
Binding to soluble or plate-coated antigen (ELISA)
Data: Ulfert Rand, Luka Cicin-Sain, HZI
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 30 Antigen binding after fixation
Calu-3 cells infected with SARS-CoV-2 (strain Zagreb) Calu-3 cells uninfected Immunofluorescence anti-SARS-CoV-2 S1 staining ABC68 (mIgG) 20 µg/mL
Binding to Note: S1 mutants cell fixation in 6% PFA 1h RT DAPI
Specificity profile (different hcov)
Storage stability and reproducibility anti-dsRNA J2 Ab (mIgG) Binding to PFA fixed antigen (IF) 20 µg/mL
DAPI Binding to denatured antigen (WB)
Data: Ulfert Rand, Luka Cicin-Sain, HZI Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 31 Antibody stability
HPLC-SEC (size exclusion chromatography) mIgG ABC68 binding to S1-hFc after storage
ABC68
Binding to S1 mutants Monomeric peak Specificity profile (different hcov)
Storage stability and reproducibility
Binding to PFA fixed antigen (IF) Separation column: AdvanceBio SEC 300 A 2 7 µm 7 8 x 150 mm, PL 1180 3301 Storage in phosphate buffer saline (PBS) pH 7,4 Binding to denatured antigen (WB) without stabilizers or protectants.
Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 32 Batch-to-batch consistency
mIgG ABC68 binding to S1-hFc Production ID:
Binding to S1 mutants
Specificity profile Transient production in (different hcov) mammalian and insect cells!
Storage stability and reproducibility Average signal
Binding to PFA fixed antigen (IF)
Binding to denatured antigen (WB)
Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 33 Human coronaviruses cross-reactivity profile
Binding to S1 mutants
Specificity profile (different hcov)
Storage stability and reproducibility Same genus Binding to PFA fixed antigen (IF) and same lineage
Binding to denatured antigen (WB)
Jaimes et al., 2020, J Mol Biol. Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 34 Human coronaviruses cross-reactivity profile
ABC68
Binding to S1 mutants
Specificity profile (different hcov)
Storage stability and reproducibility
Binding to PFA fixed antigen (IF)
Binding to denatured antigen (WB)
Jaimes et al., 2020, J Mol Biol. Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 35 SARS-CoV-2 mutants detection
SARS-CoV-2 identified mutations
(15 Oct. 2020)
Rakha, A., Rasheed, H., Batool, Z., Akram, J., Adnan, A., & Du, J. (2020). COVID-19 Variants Database: A repository for Human SARS-CoV-2 Polymorphism Data. bioRxiv. http://covid-19.dnageography.com/ Binding to S1 mutants ABC68 Specificity profile (different hcov)
Storage stability and reproducibility
Binding to PFA fixed antigen (IF)
Binding to denatured antigen (WB)
Binding to soluble or plate-coated antigen (ELISA)
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 36 Rec Abs advantages for diagnostics and research
animal free generation antigen binding regions from human naïve libraries reduce animal farming
sequence defined reagent unlimited reproducibility and known composition IMMORTAL customizable selection process constant negative selection of regions CUSTOMIZABLE unwanted reactivities SUSTAINABLE Fc improvable after selection tunable stability/affinity SPECIFIC
molecular engineering free choice of Fc-part IgG
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 37 Dept. Biotechnology
Thanks to all the
Luka Cicin-Sain, HZI Ulfert Rand cooperators and funders!
15.10.2020 | Antibody Webinar Series - PETA International Science Consortium – Russo G. - Slide 38