Humanization by CDR-grafting consists in transferring parental (commonly rodent) complementarity determining regions (CDR) into human framework (FR) regions. Parental antibody specificity and affinity are conserved thanks to the preservation of residues implicated in antigen binding. BIOTEM uses a validated and modernized version of the classical CDR-grafting technology.

1. Our Commitment makes the Difference!
Humanization by CDR-grafting
v3

2. BIOTEM: Company Presentation
Our Commitment makes the Difference!
 Contract Research Organization (C.R.O.) in immunotechnology
 Creation in 1980
 High qualified staff (38 employees including 7 PhD and 9 engineers)
 2000 m2 / 21,500 ft² facility
(Auvergne Rhône-Alpes Region – France)

3. Our Mission: To Provide High Added Value Services in Immunotechnology
 Expertise and know-how (35+ years)
 Exclusive technologies
 Contract with Guaranteed Results
 100 % fee-for-services
Client’s Worldwide Presence
Clients
• Big-Pharma
• Diagnostics companies
• Biotech companies
• Academic laboratories
• Hospitals & NGO
 Framework agreements
 No Claims
 No royalties
 No follow-up rights
Our Commitment makes the Difference!
Research Tax Credit accreditation (CIR)

4. BIOTEM: Activity Overview & Applications
Our Commitment makes the Difference!
Mouse & Rat Monoclonal Antibodies
Hybridoma / Phage Display
Rabbit Monoclonal Antibodies
Phage Display
Custom Species Antibodies
Phage Display (Veterinary Applications)
Polyclonal Antibodies
Several species – Custom protocols
Immunoassays
ELISA Development
Lateral Flow Development
R&D and
Diagnostic Antibodies

5. BIOTEM: Activity Overview & Applications
Our Commitment makes the Difference!
Ultimate Humanization®
Germinalization from macaque antibodies
Fully Human Antibodies
Phage Display from immune human donors
Humanized Antibodies
CDR-Grafting from rodent antibodies
Chimeric Antibodies
Custom Species Antibodies
Phage Display (Veterinary Applications)
Antibody Characterization
Sequencing, Affinity determination, Physicochemical analyses, Stability studies,…
Antibody Production & Modification
Hybridoma Production, Affinity maturation, CHO platform,…
Therapeutic
Antibodies
Additional
Services

6. Guaranteed results
Quality Management
BIOTEM is certified ISO 9001:2008 (Quality Management Systems)
and ISO 13485:2012 (IVD Medical Device Services)
• Development process management (R&D, Diagnostic, Therapeutic)
• Carrying out quality controls on delivered products (Antibodies and Immunoassays)
• Complying with ethics and regulations
• Traceability and Risk analysis
• Respecting delivery deadlines
• Ensuring optimal project management
• Ensuring the safety of production means and devices
Success rate (2014 – 2015)
• 269 Projects carried out
• 258 projects delivered as per order
 96% success rate
Very satisfied customers
Satisfied customers
Global satisfaction
Little satisfied or dissatisfied customers
Our Commitment makes the Difference!

7. Humanization: Technology Overview
Our Commitment makes the Difference!
Parental
Antibody
VL
VH
Antigen

8. Chimeric
Antibody
VL
VH
Humanization: Technology Overview
Our Commitment makes the Difference!
Parental
Antibody
VL
VH
Antigen
Chimerization
• VH & VL Sequencing
• Choice & validation of human
constant regions

9. Chimeric
Antibody
VL
VH
Humanization: Technology Overview
Our Commitment makes the Difference!
Parental
Antibody
VL
VH
Antigen
Chimerization
• VH & VL Sequencing
• Choice & validation of human
constant regions
CDR grafting (2 cycles)
Grafting parental residues
involved in antigen recognition on
human variable frameworks
Humanized
Antibody

10. Chimeric
Antibody
VL
VH
Humanization: Technology Overview
Our Commitment makes the Difference!
Parental
Antibody
VL
VH
Antigen
Chimerization
• VH & VL Sequencing
• Choice & validation of human
constant regions
CDR grafting (2 cycles)
Grafting parental residues
involved in antigen recognition on
human variable frameworks
Sequence optimization
• Tolerance (Immunogenicity)
• Manufacturability
• Drugability
• Affinity (potentially)
Humanized
Antibody
Optimized Lead
Antibody

11. Chimeric
Antibody
VL
VH
Humanization: Technology Overview
Our Commitment makes the Difference!
Parental
Antibody
VL
VH
Antigen
Chimerization
• VH & VL Sequencing
• Choice & validation of human
constant regions
CDR grafting (2 cycles)
Grafting parental residues
involved in antigen recognition on
human variable frameworks
Sequence optimization
• Tolerance (Immunogenicity)
• Manufacturability
• Drugability
• Affinity (potentially)
Humanized
Antibody
Optimized Lead
Antibody
CHO platform for recombinant antibody production
(small & large scale productions)

12. Chimeric
Antibody
VL
VH
Humanization: Technology Overview
Our Commitment makes the Difference!
Parental
Antibody
VL
VH
Antigen
Chimerization
• VH & VL Sequencing
• Choice & validation of human
constant regions
CDR grafting (2 cycles)
Grafting parental residues
involved in antigen recognition on
human variable frameworks
Sequence optimization
• Tolerance (Immunogenicity)
• Manufacturability
• Drugability
• Affinity (potentially)
Humanized
Antibody
Optimized Lead
Antibody
CHO platform for recombinant antibody production
(small & large scale productions)
QC QC QCQuality Controls
 Affinity (Blitz or Biacore)
 Protein analysis (Polyacrylamide Gel Electrophoresis)
 Endotoxin analysis
 Aggregation and oligomerization (analytical ultra-centrifugation, dynamic light scattering and SEC-HPLC)
 Stability (differential scanning calorimetry & circular dichroism)
 Isoelectric point determination (capillary or bidimentional gel isoelectrofocussing)
 Other quality controls upon request

13. Murine Parental Antibody
Humanization: In silico analysis 1/8
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
Our Commitment makes the Difference!

14. Murine Parental Antibody
CDR1 CDR2 CDR3
Humanization: In silico analysis 2/8
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
 CDR identification + Other information
Our Commitment makes the Difference!

15. Murine Parental Antibody
CDR2 CDR3
Humanization: In silico analysis 3/8
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
 CDR identification + Other information
 Classification of each position according to 3 levels
CDR1
Our Commitment makes the Difference!
Critical Position : involved in antigen-antibody recognition

16. Murine Parental Antibody
CDR2 CDR3
Humanization: In silico analysis 4/8
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
 CDR identification + Other information
 Classification of each position according to 3 levels
CDR1
Our Commitment makes the Difference!
Critical Position : involved in antigen-antibody recognition
Potential Contribution Position : potentially involved in antigen-antibody recognition

17. Neutral Position : not involved in antigen-antibody recognition
Critical Position : involved in antigen-antibody recognition
Murine Parental Antibody
CDR2 CDR3
Humanization: In silico analysis 5/8
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
 CDR identification + Other information
 Classification of each position according to 3 levels
Potential Contribution Position : potentially involved in antigen-antibody recognition
CDR1
Our Commitment makes the Difference!

18. Neutral Position
Critical Position
Murine Parental Antibody
CDR2 CDR3
Humanization: In silico analysis 6/8
Potential Contribution Position
Human Germline
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
 CDR identification + Other information
 Classification of each position according to 3 levels
 Identification of most appropriate Human acceptor sequences (VH & VL)
CDR1
Our Commitment makes the Difference!

19. Neutral Position
Critical Position
Murine Parental Antibody
CDR2 CDR3
Humanization: In silico analysis 7/8
Potential Contribution Position
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
 CDR identification + Other information
 Classification of each position according to 3 levels
 Identification of most appropriate Human acceptor sequences (VH & VL)
 Identification of problematic positions / Optimal substitution suggestions
Problematic position
Human Germline
CDR1
Our Commitment makes the Difference!

20. Neutral Position
Critical Position
Murine Parental Antibody
CDR2 CDR3
Humanization: In silico analysis 8/8
Potential Contribution Position
Problematic position
HUMANIZATION STRATEGY
(Report)
In silico analysis
- Primary sequence analysis
- 3D molecular modeling (option)
- IMGT blast (Human & Murine Germlines)
 CDR identification + Other information
 Classification of each position according to 3 levels
 Identification of most appropriate Human acceptor sequences (VH & VL)
 Identification of problematic positions / Optimal substitution suggestions
Human Germline
CDR1
Our Commitment makes the Difference!

21. Humanization: CDR grafting – 1st cycle 1/4
Murine Parental Antibody
CDR2 CDR3
Human Germline
Neutral Position
Critical Position
Potential Contribution Position
Problematic position
CDR1
1st Cycle of humanization
Goal: Validation of optimal Human frameworks (VH/VL pairing) with 100 % affinity retained
Our Commitment makes the Difference!

22. Humanized (1st Cycle)
1st Cycle of humanization
Goal: Validation of optimal Human frameworks (VH/VL pairing) with 100 % affinity retained
 Critical and potential contribution residues are maintained as in parental antibody
Humanization: CDR grafting – 1st cycle 2/4
Neutral Position
Critical Position
Potential Contribution Position
Problematic position
Murine Parental Antibody
CDR2 CDR3
Human Germline
CDR1
Our Commitment makes the Difference!

23. Humanized (1st Cycle)
Humanization: CDR grafting – 1st cycle 3/4
Neutral Position
Critical Position
Potential Contribution Position
Problematic position
Murine Parental Antibody
CDR2 CDR3
Human Germline
1st Cycle of humanization
Goal: Validation of optimal Human frameworks (VH/VL pairing) with 100 % affinity retained
 Critical and potential contribution residues are maintained as in parental antibody
 Neutral positions are all switched to Human germline residues
CDR1
Our Commitment makes the Difference!

24. Humanized (1st Cycle)
Humanization: CDR grafting – 1st cycle 4/4
Neutral Position
Critical Position
Potential Contribution Position
Problematic position
Murine Parental Antibody
CDR2 CDR3
Human Germline
1st Cycle of humanization
Goal: Validation of optimal Human frameworks (VH/VL pairing) with 100 % affinity retained
 Critical and potential contribution residues are maintained as in parental antibody
 Neutral positions are all switched to Human germline residues
 Production of variants in CHO
 Evaluation & Quality controls
CDR1
Our Commitment makes the Difference!

25. Humanization: CDR grafting – 2nd cycle 1/3
Neutral Position
Critical Position
Potential Contribution Position
Problematic position
Humanized (1st Cycle)
Murine Parental Antibody
CDR2 CDR3
Human Germline
2nd Cycle of humanization
Goal: improving further the Germinality Index (GI)
CDR1
Our Commitment makes the Difference!

26. Humanized (2nd Cycle)
Humanization: CDR grafting – 2nd cycle 2/3
Neutral Position
Critical Position
Potential Contribution Position
Problematic position
Humanized (1st Cycle)
Murine Parental Antibody
CDR2 CDR3
Human Germline
2nd Cycle of humanization
Goal: improving further the Germinality Index (GI)
 Residues with potential contribution are mutated to Human germline
CDR1
Our Commitment makes the Difference!

27. Humanized (2nd Cycle)
Humanization: CDR grafting – 2nd cycle 3/3
Neutral Position
Critical Position
Potential Contribution Position
Problematic position
Humanized (1st Cycle)
Murine Parental Antibody
CDR2 CDR3
Human Germline
2nd Cycle of humanization
Goal: improving further the Germinality Index (GI)
 Residues with potential contribution are mutated to Human germline
 Production of variants in CHO
 Evaluation & Quality controls
 Only substitutions that do not affect affinity are conserved
CDR1
Our Commitment makes the Difference!

28. Sequence optimization
- Antibody tolerance in humans by addressing the physico-chemical properties (heterogeneity, fragmentation,
aggregation)
- Manufacturability
- Drugability
- Affinity (potentially)
Humanized (2nd Cycle)
Humanization: Sequence optimization 1/2
Problematic position
Our Commitment makes the Difference!

29. Sequence optimization
- Antibody tolerance in humans by addressing the physico-chemical properties (heterogeneity, fragmentation,
aggregation)
- Manufacturability
- Drugability
- Affinity (potentially)
 Substitutions of amino-acid prone to post-translational modifications (PTM) and/or amino-acid combinations
that may lead to antibody denaturation (deamination, isomerization, N-glycosylation, oxidation,…)
 Production of variants in CHO
 Evaluation & Quality controls
Humanized (2nd Cycle)
Humanization: Sequence optimization 2/2
Problematic position
Humanized (sequence optimization)
Substitution
Our Commitment makes the Difference!

30. Chimeric
Antibody
VL
VH
Humanization: Technology Overview
Our Commitment makes the Difference!
Parental
Antibody
VL
VH
Antigen
Chimerisation
• VH & VL Sequencing
• Choice & validation of human
constant regions
CDR grafting (2 cycles)
Grafting parental residues involved
in antigen recognition on human
variable frameworks
Sequence optimisation
• Tolerance (Immunogenicity)
• Manufacturability
• Drugability
• Affinity (potentially)
Humanized
Antibody
Optimized Lead
Antibody
 No Claims
 No royalties
 No follow-up rights
100 % fee-for-service
BIOTEM does not claim any intellectual properties or any other right
on the developed antibodies and guarantees the entire freedom to
operate for the CLIENT

31. Antibody Humanization : Testimonials
“We worked with BIOTEM for the in silico humanization of our antibodies by
CDR grafting and were very satisfied with the quality of the reports as well as by the
applied price. The amino acid modifications proposed by BIOTEM for the humanization
and the optimisation of the antibodies were well explained and prioritized, which
rendered the testing of the humanization variants more efficient. I particularly enjoyed
the ease of interaction with the specialists and their very professional advice.”
C. Mary – Ose Immunotherapeutics
“In the context of one of our maturation projects, we have contacted BIOTEM
for the humanization by CDR-grafting of our murine monoclonal antibody. BIOTEM’s
dedicated project team has always taken in consideration all our requests and
constraints while maintaining the high quality of their services and providing specific
advice during every stage of the project. The obtained results are perfectly in line with
our expectations.”
Y. Campion – Technologytransfer office - SATT Nord
Tech Transfer Accelerator Network

32. info@biotem.fr www.biotem-antibody.com