# Presentation: 862015

##  UNWANTED IMMUNOGENICITY    Implications for Follow-on Biologicals       Robin Thorpe PhD.,FRCPath.

## Unwanted Immunogenicity 

**Induce antibodies**

- Proteins

- Patients

- Alter
- Pharmaco-
- kinetics

-  Neutralise biological
- effects and compromise
- further therapy e.g. Factor
- VIII, IFN2a, GM-CSF

-  Cross-react with native
- protein and induce adverse
- Symptoms e.g. Epo, MGDF

- No effect

##   Examples - Immunogenic Proteins

- <date/time>

-   Examples - Immunogenic Proteins

| Protein Category  | Protein | Type & Producer cells  | Binding antibodies | Neutralizing antibodies | Clinical consequences  |
| --- | --- | --- | --- | --- | --- |
| Non human proteins | Calcitonin | Natural | Yes  | Yes  | Loss of efficacy |
|   | Insulin | Natural | Yes  | Yes  | Loss of efficacy uncommon |
|   | Glucocerebosidase1 | Natural | Yes | Yes | Loss of efficacy  |
| Human proteins | Factor VIII 2 | Natural | Yes | Yes | Loss of efficacy |
|   | Follicle stimulating Hormone3 | Natural | No | No | - |
|   | IFN-α2a | rDNA  | Yes | Yes | Loss of efficacy  |
|   | GM-CSF | rDNA
rDNA | Yes
Yes | Yes
No | Loss of efficacy 
No loss of efficacy |
| Homologous to native proteins |  |  |  |  |  |
|  | G-CSF |                                               rDNA | No |            No | No loss of efficacy |
|   | IFN-β | rDNA | Yes | Yes | Loss of efficacy   |
|  | Epo |  rDNA | Yes | Yes | Cross reacted with endogenous protein and caused adverse effects. |
|   | IL-25 | rDNA | Yes | Yes | Loss of efficacy associated with both types of antibodies. |
| Sequence variants | IFN-β | rDNA | Yes | Yes | Loss of efficacy  |
|   | IFN-α Con 1 | rDNA | Yes | No | Loss of efficacy not reported |
| Chemically Modified | Pegylated MGDF | rDNA | Yes | Yes | Cross reacted with endogenous protein and caused adverse effects. |
|  Hybrid molecules | GM-CSF/IL-3 hybrid (PIXY 321) | rDNA | Yes | Yes | Clinical efficacy abrogated  |
|   | TNFR2-Ig | rDNA | Yes | No | No correlation with clinical responses or adverse effects. |

## Immunogenicity Problem

- Different classes of Protein Therapeutics can be Immunogenic
- Animal derived proteins - insulin, calcitonin
- Human derived proteins - growth hormone, Factor VIII
- Human recombinant proteins - Sequence variants,   Chemical modifications,  Fusion proteins, Hybrid molecules
- Antibody Therapeutics –
-         -   Murine, Chimeric, Humanized
-         -   Fully Human antibodies

## Immunogenicity Of Proteins Spectrum

- Some biotherapeutics appear not to be immunogenic e.g. G-CSF, IFN-.
- Other proteins e.g. IL-2, GM-CSF, IFN-, IFN-are immunogenic - induced antibodies can impact on clinical responses
- Some proteins e.g. MGDF, Epo are immunogenic –induced antibodies can cross-react with endogenous protein and cause severe adverse effects.

## Immunogenicity Testing

-    Testing for antibody responses is essential for ensuring :
-  Clinical safety of a biological therapeutic.
-  Product Comparability

## Immunogenicity Testing - How?

-                  Binding characteristics
- Binding Assays – ELISA
- Radioimmunoprecipitation Assays
- Surface Plasmon resonance - Biacore
- Immunoblotting
-                 Neutralising characteristics
- Bioassay

## Choice Of Antibody Assay

- Each assay has its own advantages and disadvantages. Some of these may be related to the nature of the sample, nature of the antigen etc.

## Immunogenicity Testing

- A complement of assays is necessary for assessing immunogenicity.

## Antibody Characterization

- ELISA

-    SPR

- No correlation between ELISA and neutralising abs.

- Good correlation between SPR and neutralising abs – not absolute.

## Incidence of GM-CSF antibodies in CRC patients given GM-CSF combination therapy

## Effect of GM-CSF antibodies on GM-CSF induced expansion of leukocytes

## Characteristics of GM-CSF antibodies

- 6,14 - strong ELISA, weak SPR, no
-                    neutralization
-    10 - strong binding ELISA/SPR,
-                    no neutralization
-      7 - strong binding ELISA/SPR,
-                    strong neutralization
-      2 - strong binding ELISA/SPR,
-                    moderate neutralization
-     11- strong binding ELISA/SPR,
-                     weak neutralization
-                    ***Kinetics is variable***

- () – indicates month at which antibodies became detectable;
- nd – not detectable

## Antibodies induced in patients receiving GM-CSF therapy

**All possibilities found, i.e;**
**(i)****	****Antibodies which ****bind and neutralize**** GMCSF**
**(ii)****	****Antibodies which ****bind, but **_**do not**_** neutralize**** GMCSF**
**(iii)****	****Antibodies against ****non-product related proteins**** e.g. *****E. Coli *****derived proteins (expression system related proteins)**
**(iv)****	****Antibodies against ****product and non-product**** ****related proteins**
**(v)****	****No antibodies**

## BIACORE characterization of anti-EPO Abs

** **

** **

** **

** **

** **

** **

** **

** **

**BIACORE characterization of anti-EPO Abs**

***  RU = Response Unit**
**   ****Positive control antibody had a dissociation rate of –0.8 RU/min**

- Data – courtesy of Steve Swanson, Amgen

## Immunogenicity Testing

**Monitoring for antibodies  - how long?**
**    ****Product related, dependent on schedule, nature of**
**    ****disease etc**
**          ****- assessment of sequential samples may need to be**
**             ****considered**
**          ****- post-marketing surveillance too !!! **

## Assessment of Immunogenicity - Preclinical 

-  Prediction Of Immunogenicity – identifying T cell epitopes, use of computer algorithms.
-  Animal models using:
-      - Lower mammals
-      - Other mammals e.g. dogs
-      - Non - human primates
-      - Immune tolerant transgenic mice

## Issues with using animals for assessment of immunogenicity

- Human proteins - lower mammals
-    - usually immunogenic in animal species; data often does not correlate with antibody response in the clinic.
-    - ***Useful for determining relative immunogenicity e.g. routes of administration, formulation etc***
- Homologous proteins - animals** **
-    - immune system different to human,
-    - protein preparation different to human version
***   ******- Benefits of this approach in predicting immunogenicity in humans is limited***
**    *****   ***

## Relative Immunogenicity of IFN2a

- Braun et al (1997) : Pharm. Res _14_, 1472 -1478

-     **Frequency**

**Route**

## Issues with using animals for assessment of immunogenicity

- Human proteins may not necessarily be active (fully) in animal species
- Preclinical (tox) studies often carried out in non-human primates - rhesus, cynos, chimps
**  ‘*****How predictive are these studies for assessment of immunogenicity in humans?’***

## Utility of primate models in predicting immunogenicity in humans?

- TPO - neutralizing TPO antibodies were detected in both monkeys and humans, these cross-reacted with endogenous TPO and caused thrombocytopenia.
-     Human GH and lys-pro Insulin - weakly  immunogenic in primates and also in humans (Zwickl et al., 1991, 1995).
- In other cases, particularly for those products where amino acid sequence is not well conserved across species (e.g., IFNs, GM-CSF) antibody induction is frequent in animals but lower in human patients.
-      An anti-IgE product - highly immunogenic in Cynos (30/31) as opposed to patients (1/10) by aerosol route; no abs detected with iv/sc delivery of product in both species (Bussiere et al., 1997; Sweeney et al., 2002).
***   ******Evidence to predict immune response from studies in primates is very limited. Dependent on protein/product.***

## Immunogenicity Profile of GM-CSF:      Human and Primate

|    
   Species |    Dose (g)
and frequency of
GM-CSF  |  
        Type of 
       Treatment | 
Treatment
Cycles | 
  Binding
Antibodies
  | 
Neutralizing Antibodies
  |
| --- | --- | --- | --- | --- | --- |
| 
Human | 
      250 x 5 |        
Product C + vaccine | 
4
(biweekly) | 
10/30 | 
1/30  |
|  
Rhesus
Macaques  | 
250 x 4  | 
Product C + vaccine  |           
         4
    (weekly)* |    
   7/8            | 
     7/8  |
| 
Rhesus
Macaques 
 | 
250 x 4  | 
Product C 
alone |           
         4
    (weekly)* |    
   7/8            | 
     7/8  |

## Preclinical Studies

- Pre-clinical studies may not necessarily be
-  indicative of the immunogenicity in man.

##  Immunogenicity of proteins - Factors

**Molecular structure - novel epitopes, glycosylation, aggregation, degradation, oxidation, deamidation**
**Product impurities**
**Formulation**
**Dose, route, frequency of administration and duration of therapy**
**Immune Status of the Patients, Disease**
**Immunomodulatory properties of the protein**

## Regulatory Agency Views

- EMEA - NfG on Comparability of Medicinal Products Containing Biotechnology-derived Proteins as Drug  Substance. Annex on non-clinical and clinical considerations (July’02; Jan’ 03)
-   ‘Immunogenicity must always be addressed by clinical data, unless clinically relevant immunogenicity can be excluded by other means’
- ICH S6 Guidance  -
-    ‘Antibody response should be characterized  ........and their appearance correlated with any pharmacological and/or  toxicological changes’

## Comparability

**    ****Extrapolation of published data generated using a particular product to another can be misleading and invalid.**

## Comparability

**Conclusions** on immunogenicity of products
- obtained by **comparing data** from **different**
**studies** using different products are usually
-                             **invalid**.

## Comparative Immunogenicity

**    ****Compares immunogenicity of products ;**
**    ****Studies need to be designed to demonstrate whether the immunogenicity of the products is the same or significantly different. **
**    **
**    ****This may affect the design of the studies &                     their interpretation.**
**    ****The consequences of immunogenicity also must be compared.                                                    **

## Immunogenicity Studies -  Biogenerics

**The immunogenicity of the marketed product**
**does not influence the need for comparative **
**immunogenicity studies.**
**However, if the imunogenicity profiles of marketed &**
**biogeneric products are significantly different, they can be**
**considered DISSIMILAR**
**        **

## Acknowledgements

- Chris Bird
- Paula Dilger
- Meenu Wadhwa
- Rose Gaines Das

- Hakan Mellstedt
- Eric Small
- Meg von Mehren
- Francois Verdier
- Steve Swanson