VL-Methods-InVitro-AntibodyDiscovery.pdf

In-Vitro Antibody Discovery

1. IN VITRO DISCOVERY OF ANTIBODIES AND ANTIBODY-FRAGMENTS
Creating Building Blocks for Targeted Therapy
Dr. Andreas Stengl

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This is us…
https://bioimaging.bio.lmu.de/research/research-group-stengl/index.html

3. Cancer Therapy
Why is treating/curing cancer so challenging?
•Only few differences between a patients cancer cells and healthy cells
èChallenging to distinguish and influence selectively
•A population of cancer cells constantly evolves
èHeterogeniety within the population
èThe cancer cell population can adapt to a selection pressure, e.g. treatment
•cancer cells are altered cells from the patient’s own body
èPatient to patient variation

4. Cancer Therapy
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https://www.facingourrisk.org/info/risk-management-and-treatment/cancer-treatment/by-treatment-type/targeted-therapy/overview

5. Targeted Therapy
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https://www.facingourrisk.org/info/risk-management-and-treatment/cancer-treatment/by-treatment-type/targeted-therapy/overview
Advantages:
•Higher efficacy
•Fewer side effects
•Paves the way for personalized medicine
Concept:
Use distinctive characterisitic(s)/targetable marker(s) to selectivly manipulate the desired sub-pupulation of cells (e.g. cancer cells)
Challenges:
•Identification of suitable markers
•Resistance/evasion
•Fewer patients/drug -> less economic

6. Targeted Therapy
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Hanahan & Weinberg, 2011, Cell
healthy cell cancer cell
Hallmarks of cancer

7. healthy cell cancer cell
Hallmarks of cancer
Targeted Therapy
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Hanahan & Weinberg, 2011, Cell

8. Targeted Therapy –Molecular Markers
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Cancer cells can be distinguished from healthy cells by certain (molecular) markers:
•Mutations
•Epigentic marks
•Changes in protein expression levels (eg. cell surface receptors)
•Receptor localisation and activity
•Cell-physiological properties
•Microenvironment
•…
extracellular markerintracellular marker

9. Targeted Therapy –Our activities
Targeted
Degradation
Triggered
Apoptosis
DeliveryConjugation
ADCsMultispecificsAbs CAR-T Cells
Extracellular TargetingIntracellularTargeting
Discovery
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10. Antibodies as Targeting Building Blocks
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Antibody Discovery Strategies
Key advantages
•Amenable to many antibody formats
•Animals not needed for discovery or manufacturing
•Selection of fully human antibodies feasible
•Antibodies can be selected at defined conditions
•Antigens need not be immunogenic
•Greater control over specificity and target epitopes
Key disadvantages
•Access to well-validated display libraries is difficult and expensive
•High-quality antigen preparations are required for selection
•Often requires in vitroaffinity maturation
•Know-how and infrastructure needed for selection and screening
•Developability issues must be actively addressed during early discovery
Adapted from Laustsen, Andreas H. et al. Trends in Biotechnology, Volume 39, Issue 12,
1263 - 1273 , 2021

12. Synthetic, Naïve Libraries -Process
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Library Design
Adapted from www.proteogenix.science
Yang Zhang (2023) Evolution of phage display libraries for therapeutic antibody discovery, mAbs, 15:1, 2023
(antibody)
format
Display
technology
library
type
Scaffold
Diversity
Genotype-Phenotype
Linkage

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Display Technologies
https://pipebio.com/blog/antibody-display-technologies
Display technologyLibrary size (unique clones)References
Phage display1011–12 Schwimmer etal. 2013; Tiller
etal. 2013
Bacterial display1011 Bessette etal. 2004
Yeast surface
display109 Benatuil etal. 2010; Rosowski
etal. 2018
Mammalian display107–9 Parthiban etal. 2019;
Waldmeieretal. 2016
Ribosome display1012–15 Lipovsek and Plückthun
2004; Plückthun 2012
Valldorf, Bernhard, …, Zielonka, Stefan. "Antibody display technologies: selecting the cream of the crop" Biological Chemistry, vol. 403, no. 5-6, 2022, pp. 455-477. https://doi.org/10.1515/hsz-2020-0377
Coupling of Genotype (Sequence) and Phenotype (Binding properties)
èEnables an enrichment process that favors defined properties (e.g. strong binding, specificitiy)
BUT ALSO co-enriches the encoding sequences (identitiy)

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Phage Display and Mammalian Display
Valldorf, Bernhard, …, Zielonka, Stefan. "Antibody display technologies: selecting the cream of the crop" Biological Chemistry, vol. 403, no. 5-6, 2022, pp. 455-477. https://doi.org/10.1515/hsz-2020-0377
Phage DisplayMammalian Display
Key advantages
Large max. library size èsuitable for naïve libraries
Robust and simple host system (E. coli)
Most frequently used èProtocols and literature available
Key disadvantages
Not feasible for full-length IgGs
Risk of enriching binders against blocking reagents or plastic
Avidity effects are challenging to avoid
Phage-handling requires experienced and well-trained scientists
Key advantages
Selection of candidates in the final format (for IgGs)
èLower risk of changes in (binding) characteristic after reformatting
Expression system during selections can be identical to production system
èPTMs and expression behaviour close to final product
Key disadvantages
Drastically lower max. library size ènot very suitable for naïve libraries
Less robust host cells (longer incubation times, more laborious handling)
“one candidate per cell” is challenging to achieve

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Universal, Naïve Libraries
www.proteogenix.science
Yang Zhang (2023) Evolution of phage display libraries for therapeutic antibody discovery, mAbs, 15:1, 2023
Key advantages
•Precise control over sequence and diversity
•Library characteristics can be designed (e.g. removal/exclusion
of developability liabilities)
•Artificial binder formats can be used
•Animals not needed for discovery or manufacturing
•Selection of fully human antibodies feasible
•Not limited to immunogenic antigens (e.g. self or toxic antigens)
Key disadvantages
•Scaffold diversity is typically lower
•Synthetic DNA libraries are custom products èthus costly
•Choices must be made èexpert knowledge needed

17. sdAb CDR Design
PDB ID: 70AP, 6M0J
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FW1CDR1FW2CDR2FW3 CDR3FW4
•Restrict AA usage
•Remove developability liabilites
•Remove PTM-motives
•Remove unwanted restriction sites
•Optimize Codon usage
VHH repertoire analysis
CDR3
Library Diversity

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Synthetic, Naïve Libraries and Phage Display
Yang Zhang (2023) Evolution of phage display libraries for therapeutic antibody discovery, mAbs, 15:1, 2023

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Appendix
Key advantagesKey drawbacks
Isolated from animal plasmaOnly limited know-how and simple
infrastructure neededAntigens need to be immunogenic
Benefits from in vivoaffinity maturationAntibodies remain in original species format
DNA/RNA immunization possible
Batch-to-batch variation
Animals needed for discovery and continuous
manufacture
Low abundance of relevant antibodies
Undefined/uncharacterized composition
Antigens may lose 3D structure when
combined with denaturing adjuvants
Generated by hybridoma cellsBenefits from in vivoaffinity maturationAnimals needed for discovery
Established infrastructure in many
laboratories
Antibodies remain in original format and
humanization/deimmunizationrequired for
therapeutic use
DNA/RNA immunization possible
Reproducibility and quality challenges if
hybridoma cell lines are used for continuous
production
Preserves information on the nature and
dynamics of the antibody response
Limited control over specificity and targeted
epitope
Antigens may lose 3D structure when
combined with denaturing adjuvants
Reclonedfrom animal B cellsBenefits from in vivoaffinity maturationAnimals needed for discovery
DNA/RNA immunization possibleAntigens need to be immunogenic
Preserves information on the nature and
dynamics of the antibody response
Limited control over specificity and targeted
epitope
Know-how and infrastructure needed for
screening and recloning
Variable domainsremain in original format
and humanization/deimmunization required
for therapeutic use
Antigens may lose 3D structure when
combined with denaturing adjuvants
Advantages and drawbacks of different antibody discovery strategies
Reclonedfrom human B cellsFully human format (no humanization or
deimmunization required)B cell material not easily obtainable
DNA/RNA immunization possibleKnow-how and infrastructure needed for
screening and recloning
Preserves information on the nature and
dynamics of the antibody response
Ethical challenges in many cases (including GDPR
considerations)
Reclonedfrom B cells from transgenic animalsFully human format (no humanization or
deimmunization required)Transgenic animals needed for discovery
Benefits from in vivoaffinity maturationAccess to transgenic technology is difficult and
expensive
DNA/RNA immunization possibleLimited control over specificity and targeted
epitope
Preserves information on the nature and
dynamics of the antibody response
Know-how and infrastructure needed for
screening and recloning
Antigens may lose 3D structure when combined
with denaturing adjuvants
Display technologies involving non-animal-derived
recombinant antibody repertoiresAmenable to many antibody formatsAccess to well-validated display libraries is
difficult and expensive
Animals not needed for discovery or
manufacturing
High-quality antigen preparations are required for
selection
Antibodies can be selected at defined conditionsOften requires in vitroaffinity maturation
Antigens need not be immunogenicKnow-how and infrastructure needed for
selection and screening
Greater control over specificity and target
epitopes
Developability issues must be actively addressed
during early discovery
Cloned from immunized animals and display
selected
DNA/RNA immunization possibleAnimals needed for discovery
Benefits from in vivoaffinity maturationAntigens need to be immunogenic
Preserves information on the nature and
dynamics of the antibody response
Requires creation of a dedicated library for each
antigen
Limited antibody repertoire cloning effortSequence and epitope diversity could be limited
due to the biases in the animal immune response
Antibodies can be selected at defined conditions
Know-how and infrastructure needed for
selection and screening
Variable domains remain in original format and
humanization/deimmunization required for
therapeutic use
Antigens may lose 3D structure when combined
with denaturing adjuvants
Laustsen, Andreas H. et al. Trends in Biotechnology, Volume 39, Issue 12, 1263 - 1273 , 2021