![Page 1: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/1.jpg)
Detection of Immune Complex Formation in Non-Clinical Studies and
Implications for Clinical Risk Assessment
Steven J Swanson, Ph.D.
Executive Director, Medical Sciences/Clinical Immunology [email protected]
![Page 2: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/2.jpg)
Immune Complex Related Pathology
§ Have been observed in non clinical studies § Risk factors for acute effects include:
– mAb therapeutics (due to their large MW) – IV administration (due to large amount of drug
entering the circulation within a short time)
![Page 3: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/3.jpg)
Circulating Immune Complex Assays
§ Traditional assays have not met the need § Variable results
§ Lack of specificity
§ Immune complexes comprise a diverse population and may have variable stability
§ New methods are evolving
![Page 4: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/4.jpg)
Hypersensi)vity reac)ons (HSRs)
§ Inappropriate or damaging immune and inflammatory response that is harmful to the host
§ Reac)ons ini)ated by Ab and drug-‐ADA complexes are referred to as “Immediate” Hypersensi)vity and manifest in minutes to hours aDer “an)body drug”
§ Types • Type I – Immediate type; IgE mediated • Type II – IgG or IgM an7body-‐mediated cell cytotoxicity (ADCC) or complement-‐mediated lysis of cells (CDC)
• Type III – IgG mediated immune complex reac)ons resul)ng in forma)on, deposi)on and complement ac)va)on with local )ssue destruc)on
• Type IV – Delayed-‐type; Th cell mediated
![Page 5: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/5.jpg)
Forma)on and clearance of immune complex
Immune Complex"+"
Small / Intermediate" soluble complexes"
Large insoluble"complex"
Clearance by mø in liver and spleen"
C3"
binding to "CR1 on RBCs"
Phagocyte Removal
Compliments of Patsy Giclas,PhD
![Page 6: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/6.jpg)
CICs are transferred from RBCs to liver macrophages
Macrophage
CR4
CR3
C3b
CR1 Factor F
Complement Components
Immune Complex C4 C2
C1s C1r
C1q
C3b
![Page 7: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/7.jpg)
Saturated IC clearance or large complexes can contribute to pathology
Immune Complex"+"
Small soluble"complex"
Large insoluble"complex"
Immune Complex!Disease!
Clearance by mø in liver and spleen"
C3"
binding to "CR1 on RBCs"
Overload"Immune Complex!
Disease!
C3/C4 depletion"
![Page 8: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/8.jpg)
Immune complexes and vasculi7s
Complement
![Page 9: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/9.jpg)
Non-‐Clinical Observa)on When Drug Was Cleared Between Doses § NHPs administered mul)ple injec)ons of IgG1/IgG2 human an)bodies
§ Not a Group effect; individual animals
§ Effects noted a short period aDer dosing (minutes to ~2 days)
§ In IV dosed groups (vs SC) at lower (eg 10-‐50 mg/kg) as opposed to higher doses (i.e., 300 mg/kg)
§ Poten)al clinical findings post-‐dose: – Vomi)ng, difficulty breathing, weakness/lethargy, death – Prominent bleeding or bruising at injec)on site – Petechial hemorrhages
§ Clinical pathology findings: – Ac)vated platelets +/-‐ change in platelet counts – Decreased neutrophils and monocytes
§ Affected animal(s) had: – High ADAs – Below Quan)fiable Limits (BQL) drug prior to next dose
NOTE: Example only, does not always occur
![Page 10: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/10.jpg)
ADA/drug Ab complex complicates CI ADA detec)on and PKDM drug Ab detec)on
Low Dose Mid-dose High-dose
ADA +; PK +/- ADA -; PK + ADA +/-; PK +/- Reliability Factor:
![Page 11: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/11.jpg)
Immunoassay for huAb drug induced-‐immune complexes in NHP
CIC Assay Standard Curve using 1:1 Positive Control
0.01 0.1 1 10 1000
50
100
150
Conc. ug/mL
S/N
![Page 12: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/12.jpg)
CIC Immunoassay Valida)on Parameters
§ Sample with S/N > 3.35 = CIC Posi)ve
§ Sample dilu)on: 1:20 ; 1:400 and 1:8000
§ Quan)ta)on based on a 1:1 CIC standard – Dynamic range : 0.250-‐10 mcg/ml CIC – Precision : 25%
§ Sensi)vity: 142 ng/ml
§ Quan)fiable Limit: 250 ng/ml
§ Drug tolerance @ QL: 1 mcg/ml IgG2
![Page 13: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/13.jpg)
1
10
100
1000
1
10
100
1000
10000
100000
0 10 20 30 40 50 60
Immun
e Co
mplex
S/N
Drug / ADA
ng
/mL
Day
Drug
ADA
Immune Complex
Dynamics of Detecting ADA & CIC in Presence of High Serum Drug levels
Note: These are representative data for demonstration purposes
![Page 14: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/14.jpg)
Case Study 1: Immune Complex in Non-Human Primate § Single animal presented on Day 23 of 28 day study with:
– Lymphadenopathy – Inflammatory leukogram – Decreased serum drug concentrations – Early euthanasia
§ Other differentials considered: Infectious (TB or atypical Mycobacteria, protozoal—T. cruzi), test-article related effect
§ Additional data collected: ADA, CIC, special stains for infectious organisms
§ WOE for immune complex-mediated etiology – No evidence of infection – Single animal affected – CIC detected when symptomatic, and decreasing drug concentrations – Histopathology
• Chronic active inflammation aorta-coronary artery branch point • Pyogranulomatous lymphadenitis
![Page 15: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/15.jpg)
Case Study 1 (IgG2 mAb): Altera)on in PK Levels and Associated Pathology in an ADA Nega)ve Animal
CIC +
Day 22 – SC- 25 mg/kg 2X/week
(Vasculitis observed)
![Page 16: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/16.jpg)
Case Study 2 (IgG2 mAb) : Clinical and Anatomic Pathology Finding of an IC Mediated Hypersensi)vity
l Clinical signs: l Decreased activity; decreased use of left hindlimb
l Ecchymosis/petechiation was observed on all 4 limbs l Timing:
l Occurred 24 hrs after dose administration
l Occurred after 23rd dose (ie, late in dosing phase) in an individual animal l CI / TK:
l Binding ADAs observed D57; increasing levels of Abs at D113, D141, D156 l Positive immune complexes (CIC) on D156
l Decreased bioactivity (D57, D113) and serum concentration (D57 through D156)
l Histopathology: l Multifocal vasculitis / thrombosis of small vessels in skin and GI serosa
![Page 17: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/17.jpg)
Day
Serum Concentration1 Bioactive Drug Level Antibody Positive Animals
in Group 4 Immune Complex
Assay
Animal X
Grp 4 Mean Animal X Grp 4
Mean Animal
X Animal
Y Animal
Z Animal
X Grp 2 - 4
(µg/mL) (µg/mL) (Signal/Noise Ratio) (µg/mL)
57 172 934 149 945 9.08 Neg Neg
85 < 2 1010
113 < 2 978 BQL 958 5739 1.17 Neg
141 < 2 968 8046 1.32 1.48 Neg
156 < 2 3077 Neg Neg > 32
162 1040 1.43 1.56 Neg
183 9822 1061 1.51 1.45 Blank squares indicate blood sample not collected 1 Pre-dose, 2 7-d post-dose
Serum concentration: 5x to 500x decreased D57 through D156 Bioactivity: 6x to 1000x decreased D57 and D113
ADA: S/N 3000x to 8000x increased D113 through D156 CIC: Positive D156
Case Study 2: Data
Evidence of Immune Complex Forma)on in Animal X
![Page 18: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/18.jpg)
Impact to the Programs
§ Clinical and pathologic changes consistent with immune complex secondary to ADA forma)on
§ Consequences of IC forma)on were not a direct TA-‐related effect – ADA forma)on in NHP not clinically relevant
§ There was no impact to either program – Did not impact the NOAEL or safety margins – No impact on )melines or clinical trials progress
![Page 19: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/19.jpg)
“Triggers” for CIC Assay § Unusual PK/PD/pathology findings in animals that test ADA nega)ve (Case Study 1)
§ Post-‐dose clinical signs (e.g. fain7ng, weakness, etc) or clinical/anatomic pathology findings in animals ADA+ on study (Case Study 2) – IV dosing – Predose (trough) drug low or BQL – Robust ADA
§ To test the asser7on that immune complexes are the cause of the pathology findings in ADA+ animals (Case Study 2)
![Page 20: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/20.jpg)
ADAs Rarely Cause Safety Issues § Our challenge
– Determine why some human IgGs cause ADA-‐mediated toxicity • Animals with IC-‐related effects are CIC+ • Not all CIC+ animals have adverse events
– What is the role of route, dose, infusion rates, an)body vs an)gen excess, molecule characteris)cs, etc.?
– How does CIC size correlate with adverse effects/pathology?
– What does CIC composi)on tell us? • Detect C3b on CIC • Cyno IgG subclass • Other serum proteins
![Page 21: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/21.jpg)
Conclusion
§ A validated CIC assay provides direct evidence of circula)ng immune complexes (human IgG drugs/cyno IgG ADAs)
§ In Case Study 1, CIC results were cri)cal in explaining the altera)on in PK levels and associated pathology in an animal that was ADA nega)ve by tradi)onal methods
§ In Case Study 2, CIC results supported the clinical and anatomic pathology findings of an immune-‐complex mediated type III hypersensi)vity reac)on
![Page 22: Detection of Immune Complex Formation in Non-Clinical ... · PDF fileDetection of Immune Complex Formation in Non-Clinical Studies and Implications for Clinical Risk Assessment](https://reader031.vdocuments.net/reader031/viewer/2022030404/5a79cf557f8b9a9e0c8d1751/html5/thumbnails/22.jpg)
Acknowledgements
§ Clinical Immunology – Dan Mytych – Dohan Weeraratne – Jill Miller – Mike Moxness – Rocio Lopez – Naren Chirmule
§ Compara)ve Biology & Safety Sciences – Nancy Everds – Ka)e Sprugel – Jeanine Bussiere – Jon Werner