challenges of immunogenicity testing for fusion … · •michael benson, ba •elizabeth zavala...

CHALLENGES OF IMMUNOGENICITY TESTING FOR FUSION PROTEIN BIOTHERAPEUTICSJessica St. Charles, PhD, Director Laboratory Sciences – Immunochemistry/ImmunologyCharles River Laboratories - MattawanEBF Barcelona November 2018

EVERY STEP OF THE WAY

EVERY STEP OF THE WAY2

OUTLINE

Overview of Fusion Proteins

Challenges | Impact of Immunogenicity

Solutions to the Challenges | Impact of Immunogenicity

Case Study: Fusion Protein ABC

Points for Consideration

OVERVIEW OF FUSION PROTEINSIntroduction

3 EVERY STEP OF THE WAY

Recombinant Technology:• Combine two or more genes encoding for different parent proteins • Results in a single protein with functional properties derived from

both parent proteinsPurpose: • Stability and delivery • Target engagement/specificity• Toxicity (cell killing)Fc-Fusion Proteins – most common type

1980 20001990 2010

Humira®1st fully human

therapeutic mAb

Humulin®1st recombinant protein

for Human use

Orthoclone OKT3®1st therapeutic mAb

(murine)

Enbrel®1st Fusion Protein

Removab®1st multiple

function antibody

OVERVIEW OF FUSION PROTEINSCont.


Advantages:• Simplifies manufacture and drug delivery

• Manufacture 1 vs. 2 proteins• 1 vs. 2 Biodistribution profile

• New functionalities created

• Increased half-life• Increased target specificity

• Therapeutic benefits: • Reduced side effects

• Longer dosing intervals• Improved activity

Disadvantages:• Difficulties with manufacturing:

• Unrelated proteins being put together = noncompatible partners

• Could cause aggregation, misfolding of one or both domains

• Formulation issues:

• Conflicting stability requirements

• Immunogenicity:

• Formation of novel epitopes at the fusion junction (even if all human partners)

CHALLENGES


EMA• “Non-analogue therapeutic proteins like fusion proteins may contain neo-epitopes due to the

introduction of foreign peptide sequences, e.g. in linkages/junctions.”• “Fusion proteins composed of a foreign and self-protein, as well as chimeric proteins, may be of

concern, especially because of the potential of the foreign moiety to provoke an immune response to the self-protein (epitope spreading).”

FDA• “For some therapeutic protein products, the sponsor may need to investigate the ADA to specific

epitopes to which immune responses are specifically generated. For example, determination of epitope specificity is recommended for some fusion molecules because the region where the two molecules join may form a neoantigen, and immune responses to this region may arise.”

• “Because of epitope spreading, immune responses to other parts of the molecule may ensue, leading to the generation of antibodies to the therapeutic protein product or its endogenous counterpart”

Impact of Immunogenicity – Regulatory Guidance

SOLUTIONS TO THE CHALLENGES


EMA• Strategy 1: Multiple assays may be required for measuring immune responses to the variable epitopes

within a fusion protein• Strategy 2: Utilize the competitive inhibition principle in the confirmatory assay to identify the specificity

of the ADA

FDA• Strategy: Multiple assays to measure immune responses to different domains on multiple functional

domain proteins

Impact of Immunogenicity

CASE STUDY: FUSION PROTEIN ABC


CASE STUDY: FUSION PROTEIN ABCBackground

N

N

N

N

Protein AC

C

C

C

C

B

A

CBA

Protein B

Protein C

Fusion Protein ABC


CASE STUDY: FUSION PROTEIN ABCImmunogenicity Assessment Approach

Immunogenicity: Screen and Confirm Assay for Fusion Protein ABC

Sample Confirm Positive for anti-Fusion Protein ABC?

STOPNO

YES

1) Perform Titer2) Perform Competitive Inhibition Assay for ADA Characterization with Proteins A, B, C and ABC

Sample shows inhibition with Proteins A, B, or C?

Neutralizing Assay for Characterized ADA

(anti-A, B, or C)

Neutralizing Assay for Fusion Protein ABC

YESNO

CASE STUDY: FUSION PROTEIN ABCImmunogenicity Assay for Fusion Protein ABC - Screening


Homogenous Bridging Format • Protein ABC – Biotin• Protein ABC – SulfoTag• PC: Antibody that binds to Protein ABC

Assay Parameters:• Sensitivity: ~90 ng/mL• Drug tolerance:

• 146 µg/mL at PCH (20µg/mL)• 0.779 µg/mL at PCL (250ng/mL)

• Intra-/inter-assay precision within 20% CV

CASE STUDY: FUSION PROTEIN ABCImmunogenicity Assay for Fusion Protein ABC – Competitive Inhibition Assay


Confirm Protein A Confirm Protein B Confirm Protein C Confirm Protein ABC


CASE STUDY: FUSION PROTEIN ABCCharacterization of ADA with Competitive Inhibition Assay

• PC Approach:• Option 1: Single antibody that binds to all 4 components• Option 2: Cocktail of multiple antibodies that bind to all 4

components• Results:

• Multiple PCs were assessed using both PC options• Homogenous bridging format utilized from initial ADA

assay• Could NOT identify a suitable PC utilizing either option above

• PC1 – reactive with proteins B and C• PC2 – reactive with protein A

• New Approach: Step-wise assay format (new cap and dAb)• This would allow interchanging of the PCs in the format• Results: Same species PCs for each fusion protein component

could not be identified



• Move forward with 4 separate assays for each component• Utilize Nab cell-based assays specific to characterized ADA

anti-A ADA

anti-B ADAanti-C ADA

anti-ABC ADA

protein A

protein Bprotein C

protein ABC

Unbound and washed away

Addition of detection





Immunogenicity: Screen and Confirm Assay for Fusion Protein ABC

Sample Confirm Positive for anti-Fusion Protein ABC?

STOPNO

YES

1) Perform Titer2) Perform Competitive Inhibition Assay for ADA Characterization with Proteins A, B, C and ABC



(anti-A, B, or C)


YESNO


CASE STUDY: FUSION PROTEIN ABCImmunogenicity Assessment Approach - #2

Immunogenicity Three Tier Assay with Fusion Protein ABC

Sample Titer Positive for anti-Fusion Protein ABC?

STOPNO

YES


Sample shows NAb positive for Protein ABC?

Neutralizing Assay for anti-A, B, or C

Sample Positive for one of the Protein Components (A, B, C)

Conclude ADA Characterization is anti-ABC

YESNO

STOPNO

YES

Conclude ADA Characterization is anti-A, B, or C


POINTS TO CONSIDERSummary and Conclusions

Increasing number of fusion proteins in development

• Opportunity to introduce improved stability and delivery mechanisms with better target engagement and reduced toxicity

• Multi-functional fusion proteins also being developed

Fusion proteins introduce multiple advantages and disadvantages

• Advantages include simplifying manufacturing and drug delivery, availability of new functionalities and therapeutic benefits

• Disadvantages include difficulties within manufacturing and formulation along with the potential for increased immunogenicity

Immunogenicity challenges and solutions

• Challenges – formation of neo-epitopes and epitope spreading• Solutions – increased specificity testing with multiple assays to multiple epitopes or utilize competitive inhibition

assays

Does increased specificity testing require better availability of PCs within the market?

ACKNOWLEDGMENTS


• Rachel Sterling, MS• Michael Benson, BA• Elizabeth Zavala• Neha Ainapure• Brad Reinhart, PhD• Arkadeep Sinha, PhD• Elaina Breznau, PhD• Amy Smith, BA• Roger Hayes, PhD

Thank You…

QUESTIONS??

EVERY STEP OF THE WAY 18

EXTRA SLIDES


OVERVIEW OF FUSION PROTEINSDrug Market


History:• 1982 – first recombinant protein for human use

• Humulin® – human insulin• 1986 – first therapeutic mAb (murine)

• Orthoclone® (OKT3)• 1998 – first fusion protein

• Enbrel® - still best selling fusion protein to date• 2002 – first therapeutic fully human mAb

• Humira®• 2009 – first multispecific antibody

• Removab® (tri-functional)• 1998-2013: 8 fusion proteins reach market

Schmidt S. R, 2013


Screening Assay with Fusion Protein ABC

Sample Screens Potential Positive?

Confirmatory Assay with Fusion Protein ABC

Sample Confirms Positive?

Titer Assay with Fusion Protein ABC

Sample is Titer Positive?

STOP

STOP

STOP

NO

YES

NOYES

NO YES

Competitive Inhibition Assay for ADA Characterization with Proteins A, B, C and ABC



(anti-A, B, or C)


YESNO


EXTRA SLIDES




• PC Approach:• Option 1: Single antibody that binds to all 4 components• Option 2: Cocktail of multiple antibodies that bind to all 4

components• Results:

• Multiple PCs were assessed using both PC options• Homogenous bridging format utilized from initial ADA

assay• Could NOT identify a suitable PC utilizing either option above

• PC1 – reactive with proteins B and C• PC2 – reactive with protein A

• New Approach: Step-wise assay format (new cap and dAb)• This would allow interchanging of the PCs in the format• Results: Same species PCs for each fusion protein component

could not be identified• Move forward with 4 separate assays for each component• Utilize Nab cell-based assays specific to characterized ADA



anti-A ADA

anti-B ADA

anti-C ADA

anti-ABC ADA

protein A

protein B

protein C

protein ABC





challenges of immunogenicity testing for fusion … · •michael benson, ba •elizabeth zavala...

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