Aim of the Study Identification of individual responds of Rheumatoid Arthritis (RA) patients to the anti TNFa- therapy with Adalimumab, to support the rheumatologist with diagnostic data for a rational dose decision. Technology The recoveryELISA© allows the detection of three parameter in one assay. The concentration of the Free Target (ng/mL), Free Drug (µg/mL) and the Neutralization Rate (%) of the drug in patients can be monitored. Results The concentration of free Adalimumab and TNFa- neutralization rate allowed a separation of the cohort in three groups. Supra-therapeutic patients 18% showed high level of free Adalimumab and high TNFa- neutralization values. All patients in this group exceed the therapeutic window (5.0- 10.0 µg/mL) of free Adalimumab and no free TNFa was detected. The patients in the therapeutic window 47% showed balanced TNFa- neutralization values, indicating that these patients received an ideal medication dose. 35% of the cohort had low concentrations of free Adalimumab, low TNFa- neutralization values and free Antigen was found in the patients sera. Conclusion Up to now the dose decision for RA patients is mainly based on less specific parameters such as Erythrocyte Sedimentation Rate (ESR) or C- Reactive Protein (CRP) concentration. With more diagnostic data, a detailed view of the individual patient’s drug dose can be monitored. In particular patients with high concentrations of free target and low TNFa- neutralization could improve the disease pathology with an additional administration of Adalimumab. Subsequently extra dose of Adalimumab could be avoided for patients with high TNFa- neutralization values. Outlook The recoveryELISA™ technology can be customized for therapeutic antibodies in preclinical phase. IL6/Tocilizumab, IL12, IL23/Ustekinumab and IL-17A/ Secukinumab will be our next recoveryELISA© development goals.

1. Biologics&Biosimilars Congress 2017
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Identify the drug dose for each patient individually
Challenge: Detect free antigen and free drug concentration during therapy
I´m so different, so why is the
medication dose the same?

2. Classical ELISA
TAB
Antigen
Biologics&Biosimilars Congress 2017
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Challenge
1st AB
2nd AB HRP

3. Classical ELISA
2nd AB HRP
TAB
Antigen
1st AB
Biologics&Biosimilars Congress 2017
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Challenge

4. Classical ELISA
2nd AB HRP
TAB
Antigen
1st AB
Biologics&Biosimilars Congress 2017
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Challenge

5. TAB Antigen
Additive Library
Antigen, TAB
Antigen/ TAB complex
Stabilization
Matrix adjustments
Plate Coating
Streptavidin
Poly Streptavidin
Biotin …
Resources
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Conjugate 1
Linker lengths …
Conjugate 2
HRP
Poly HRP …
Development platform

6. TAB
TAB- poly HRP
Capture AB
Antigen
60µl Serum
BioTeZ Algorithm
Non linear regression,
Michaelis-Menten &
Law of mass action
96 well plate&reader
Solution
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Source: Strohner, Pavel, Stephanie Korn, Roland Buhl, and Gunther Becher. “The Recovery-
ELISA--a Novel Assay Technique to Monitor Therapy with Humanized Antibodies: The
Example of Omalizumab.” Journal of Immunoassay & Immunochemistry 34, no. 1 (2013): 83–
93. doi:10.1080/15321819.2012.683501.
The recoveryELISA® (CE marked IVD) is a combination of a sandwich ELISA and a competition ELISA

7. Solution/Pipetting scheme
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Antigen
CALIBRATORS
Topping up Samples with Antigen
CALIBRATORS
TAB
CALIBRATORS

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Free Target
concentration
as indicator of
patients drug
need.
Neutralization rate
gives information's
about the ability of the
drug to neutralize free
target antigens and
allows prediction of
dose variations effects.
A low neutralization
rate can serve as
indicator for anti-drug
antibodies (ADAs).
Solution
Free Drug concentration
shows individual drug
dose to meet the
therapeutic window to
avoid over and under
treatment.
Administered drug dose
and measured free drug
show individual drug
absorption and drug
metabolism.

9. Therapeutic free Drug Monitoring (TDM)
Therapeutic free Antigen Monitoring (TAM)
Therapeutic Drug/Antigen Monitoring
Solution Summary
Biologics&Biosimilars Congress 2017
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10. ~ 1 % of the world population are affected
Adalimumab/TNFa in RA
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Source: *www.healthline.com **Sacks,Luo and
Helmick, Arthritis Care Res
Observational clinical study cohort of 17
patients treated with adalimumab dose
(40mg) every 14 day or according to the
rheumatologist every 7 days (BOOST)
*
**

11. Adalimumab/TNFa in RA
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12. DAS28/Boost in RA
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13. Consequences RA Study
* Key findings towards optimising adalimumabtreatment: the
concentration–effectcurve Pouw et al Ann Rheum Dis. 2015
Mar;74(3):513-8. doi: 10.1136/annrheumdis-2013-204172.
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*

14. Consequences RA Study
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Patients Brand et al Laine et al BioTeZ
Cohort Size (N) 17 1137 50
Supra-Therapeutic 18% 22% n.d.
Therapeutic Window 47% 42% n.d.
Sub-Therapeutic 35% 36% n.d.
Laine J. et al Cost-effectiveness of routine measuring of serum drug concentrations in
treatment of RA patients with TNF-α blockers Targets and Therapy
Brand et al Improved dose decision in TNFa-inhibiting therapy with comprehensive
diagnostics data Under review at The Journal of Rheumatology

15. Treatment following existing
guideline DAS28 as main
disease indicator
Follow up study on RA patients with
Horizon2020 & Innovationsfond proposals
Open label treatment
including diagnostic data
and real world evidence
RA Cohort
Consequences RA Study
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N=260 N=260

16. Omalizumab HRP
aIgE
IgE
Examines the underlying mechanisms of Fast vs. Slow responders to first injection of
Omalizumab chronic spontaneous urticaria (CSU) patients.
IgE/Omalizumab in CSU
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Source: Gericke et al J Allergy Clin Immunol. 2016 Nov 9. doi:
10.1016/j.jaci.2016.07.047. Serum autoreactivity predicts time to
response to omalizumab therapy in chronic spontaneous urticaria.

17. PCSK9/Evolocumab and Alirocumab in high LDL
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Source: PCSK9 Inhibitors Pradeep Natarajan, Sekar Kathiresan
DOI: http://dx.doi.org/10.1016/j.cell.2016.05.016

18. Alirocumab- HRP
Evolocumab
PCSK9
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PCSK9/Evolocumab and Alirocumab in high LDL
Evolocumab- HRP
Alirocumab
PCSK9

19. Future Project IL6 and sIL6R
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Chalaris et al Interleukin-6 Trans-Signalingand Colonic Cancer Associated
with InflammatoryBowel Disease
Article in Digestive Diseases 30(5):492-9 · October 2012
DOI: 10.1159/000341698

20. Preclinical recoveryELISA application
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In Vivo Drug stability tests
Drug/Antigen binding identification
In early biological development stages
Preclinical phases
60µl Serum ≈100µl blood are needed
Source: https://en.wikipedia.org/wiki/Laboratory_rat#/media/File:Wistar_rat.jpg

21. Feasibility Package
recoveryELISA Development
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6 3 2
Additive,Linker&Coating Library
Optimization in buffer system
We need: 2mg TAB,
2mg Capture AB &
0.3mg Antigen
Matrix Free Package
Sandwich in Serum, Plasma &
Matrix Adjustment
We need: Patient samples
Algorithm
Adjustment
Ready to use kits
More Services: Stability&cross reactivity tests, scale up and production
For each TAB/Antigen
recoveryELISA
individually
Weeks
We deliver
Sample measurement
as a service and deliver the
recoveryELISA results.
We need: patients samples
Single kit components
e.g. Poly HRP TAB or Plates

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For assistance and more information's please contact
Dr. Janko Brand j.brand@biotez.de
Come for a chat at booth # 5
Find us next to the poster # 13
Thank you for your attention!