Bioanalytical Community

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

Hi John,

My position is that we don't need multiple NAb assays since the competitive LBA (cLBA) can account for NAbs for all modes of action.  Since the first step in each MOA (payload mediated death, ADCC, complement mediated lysis) is binding of target by ADC, the cLBA is more practical than having to set up multiple functional assays and should suffice.  Although one can argue that the cell-based killing assay also begins with a target binding by ADC and in theory accounts for all MOAs, the lower complexity of a cLBA assay trumps having to set up a more challenging cell-based assay. 

Uma.

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

Hi John,

My position is that we don't need multiple NAb assays since the competitive LBA (cLBA) can account for NAbs for all modes of action.  Since the first step in each MOA (payload mediated death, ADCC, complement mediated lysis) is binding of target by ADC, the cLBA is more practical than having to set up multiple functional assays and should suffice.  Although one can argue that the cell-based killing assay also begins with a target binding by ADC and in theory accounts for all MOAs, the lower complexity of a cLBA assay trumps having to set up a more challenging cell-based assay. 

Uma.

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

Hi Erik,

Thanks for your thoughts on this. I have yet to come across evidence on inhibition of internalization post binding.  If the community can confirm these observations, it would be greatly appreciated.  After internalization, it is my understanding from the literature that linkers get denatured to release the payload, and with them any ADA that are bound.  So post-internalization events are less of a concern to me.  Open to further thoughts and evidence to the contrary.

Good to touch base with you again after the acid denaturation of ADA discussion.

Thanks,

Uma.

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

Hi John,

My position is that we don't need multiple NAb assays since the competitive LBA (cLBA) can account for NAbs for all modes of action.  Since the first step in each MOA (payload mediated death, ADCC, complement mediated lysis) is binding of target by ADC, the cLBA is more practical than having to set up multiple functional assays and should suffice.  Although one can argue that the cell-based killing assay also begins with a target binding by ADC and in theory accounts for all MOAs, the lower complexity of a cLBA assay trumps having to set up a more challenging cell-based assay. 

Uma.

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

Hi John,

My position is that we don't need multiple NAb assays since the competitive LBA (cLBA) can account for NAbs for all modes of action.  Since the first step in each MOA (payload mediated death, ADCC, complement mediated lysis) is binding of target by ADC, the cLBA is more practical than having to set up multiple functional assays and should suffice.  Although one can argue that the cell-based killing assay also begins with a target binding by ADC and in theory accounts for all MOAs, the lower complexity of a cLBA assay trumps having to set up a more challenging cell-based assay. 

Uma.

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

Hi John,

My position is that we don't need multiple NAb assays since the competitive LBA (cLBA) can account for NAbs for all modes of action.  Since the first step in each MOA (payload mediated death, ADCC, complement mediated lysis) is binding of target by ADC, the cLBA is more practical than having to set up multiple functional assays and should suffice.  Although one can argue that the cell-based killing assay also begins with a target binding by ADC and in theory accounts for all MOAs, the lower complexity of a cLBA assay trumps having to set up a more challenging cell-based assay. 

Uma.

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

Hi John,

My position is that we don't need multiple NAb assays since the competitive LBA (cLBA) can account for NAbs for all modes of action.  Since the first step in each MOA (payload mediated death, ADCC, complement mediated lysis) is binding of target by ADC, the cLBA is more practical than having to set up multiple functional assays and should suffice.  Although one can argue that the cell-based killing assay also begins with a target binding by ADC and in theory accounts for all MOAs, the lower complexity of a cLBA assay trumps having to set up a more challenging cell-based assay. 

Uma.

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences

For the first question "1️⃣ For ADCs, is a cell-based NAb assay considered a requirement or would a competitive ligand-binding assay (LBA) be acceptable to demonstrate neutralizing of binding for the antibody component?" in my opinion, a cell-based assay is not necessary for NAb characterization for ADCs, and a competitive LBA assay should suffice for the following reason:

The first critical step in the MOA of an ADC involves binding to the target molecule on the cell surface prior to internalization, followed by release of payload and cell death via payload action.  Without the first step, the rest cannot happen (I am ignoring non-target dependent passive uptake of ADCs and any FcR bound internalization into the target cell). 

Since an LBA assay will identify NAbs that inhibit engagement of ADC with the target, it will suffice for NAb characterization.  ADAs that bind the non-target engaging parts of the ADC (e.g. anti-linker/payload ADA) are going to be denatured in the endosomal compartment; everything but the payload is destroyed there.  Thus, the concern that such ADA are not accounted for in the LBA NAb assay is negated.

Additionally, there are advantages to an LBA NAb assay for ADCs where the Fc portion retains functional activity (e.g., ADCC and complement mediated lysis of target cell). The assay will account for the first critical step of target binding for ADCC and CML as well.  The cell-based killing assay only accounts for payload mediated MOA and not ADCC or complement mediated lysis.  So, it would be an incomplete assessment of functional activity. 

Uma Kavita, Ph.D.

Senior Director, Oncology Integrated Bioanalysis Strategy

AstraZeneca

R&D | Clinical Pharmacology & Safety Sciences