Antibodies for bioanalytical and patient drug level monitoring assays for ipilimumab

Recombinant monoclonal anti-idiotypic antibodies, highly specific for the immune checkpoint inhibitor drug ipilimumab (Yervoy).

Anti-Ipilimumab Inhibitory Antibodies

These antibodies inhibit the binding of the drug ipilimumab to its target, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and therefore detect free drug. An antibody pair is ideal for the development of a pharmacokinetic (PK) bridging ELISA. Three antibodies of high, medium, and low affinity are suitable as a surrogate positive control for an anti-drug antibody (ADA) assay.

Catalog Numbers: HCA327, HCA328, HCA329, HCA330

Anti-Ipilimumab Drug/Target Complex Antibody

This drug-target complex binder specifically recognizes the ipilimumab/CTLA-4 complex. It detects ipilimumab only when it is bound to CTLA-4; it does not bind to free ipilimumab or free CTLA-4. It can be used as the detection antibody in a PK antigen capture assay, as an alternative format to the bridging ELISA.

Catalog Number: HCA331​​

These recombinant, monoclonal antibodies are generated using Human Combinatorial Antibody Library (HuCAL®) technology and CysDisplay®, a proprietary method of phage display, and use guided selection methods to obtain highly targeted reagents. The result is highly specific and high affinity antibodies, ideal for development of PK assays. HuCAL antibodies are fully human and, when in full length IgG format, can be used as a surrogate positive control or calibrator in an ADA assay. The in vitro production of recombinant antibodies results in a consistent, secure supply of these critical reagents throughout preclinical development and clinical trials.

More information about anti-idiotypic antibody binding types and their properties

Anti-biotherapeutic antibody quality control and characterization


Antibodies Specific to Ipilimumab

Catalog #

Clone

Antibody Specificity

Binding Type

Format

Affinity
KD, nM

Assay Development Recommendations

HCA330

AbD34433

Ipilimumab

Inhibitory

Fab-FSx21

0.3

PK bridging ELISA capture antibody

HCA329

AbD34429ia

Ipilimumab

Inhibitory

Human IgG1

12

PK bridging ELISA detection antibody

ADA assay

HCA329P

AbD34429ia

Ipilimumab

Inhibitory

Human IgG1 HRP labeled

12

PK bridging ELISA detection antibody

HCA327

AbD34283ia

Ipilimumab

Inhibitory

Human IgG1

162

ADA assay

HCA328

AbD34428ia

Ipilimumab

Inhibitory

Human IgG1

0.3

ADA assay

HCA331​​

AbD34294

Ipilimumab/CTLA-4

Complex-specific

Fab-FSx21

2522

PK antigen capture assay

1 F=DYKDDDDK-tag Sx2=StrepX-StrepX-tag
2 Affinity measured in the monovalent Fab format


Pharmacokinetic Assay

Schematic image of PK Bridging ELISA. Anti-idiotypic capture antibody, Fab format (purple), monoclonal antibody drug (gold), anti-idiotypic detection antibody, Ig format (blue), labeled with HRP.

Schematic image of PK Bridging ELISA. Anti-idiotypic capture antibody, Fab format (purple), monoclonal antibody drug (gold), anti-idiotypic detection antibody, Ig format (blue), labeled with HRP.

Fig. 1. Ipilimumab PK ELISA bridging format using antibodies HCA330 and HCA329P.

Fig. 1. Ipilimumab PK ELISA bridging format using antibodies HCA330 and HCA329P.


Measurement of free drug with Human Anti-Ipilimumab Antibodies

In Figure 1, Human Anti-Ipilimumab Antibody clone AbD34433 (HCA330) was coated on a microtiter plate at 1 µg/ml and left overnight. Washing and blocking was performed with PBST + 5% BSA. PBST with 10% human serum, spiked with increasing concentrations of ipilimumab was added. Detection was performed using HRP conjugated Human Anti-Ipilimumab Antibody, clone AbD34429ia (HCA329P) diluted to 2 µg/ml in HISPEC Assay Diluent (BUF049A), and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: Ipilimumab PK bridging ELISA protocol HCA330 and HCA329P


Schematic image of PK Bridging ELISA with type 2 antibodies

Schematic image of PK antigen capture format ELISA. Drug target (red), monoclonal antibody drug (gold), drug target complex detection antibody, Fab format (purple), anti-tag detection antibody labeled with HRP (gray).

Fig. 2. Ipilimumab PK ELISA antigen capture format using antibody HCA331.

Fig. 2. Ipilimumab PK ELISA antigen capture format using antibody HCA331.


In Figure 2, a microtiter plate was coated overnight with recombinant human CTLA-4 at a concentration of 5 µg/ml. After washing and blocking with PBST + 5% BSA, 10% human serum was added spiked with increasing concentrations of ipilimumab. Detection was performed using Human Anti-Ipilimumab Antibody, clone AbD34294 (HCA331) at a concentration of 2 µg/ml, followed by an HRP conjugated anti-DYKDDDDK tag antibody in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: Ipilimumab PK antigen capture ELISA protocol HCA331


Anti-Drug Antibody Assay – Bridging ELISA 

Schematic image of fully human anti-idiotypic antibody in anti-drug antibody (ADA) assay

Schematic image of ADA bridging assay. Monoclonal antibody drug as capture antibody and detection antibody labeled with HRP (gold), fully human anti-idiotypic antibody, Ig format (blue).

Fig. 3. ADA bridging ELISA using antibodies HCA327, HCA328, and HCA329.

Fig. 3. ADA bridging ELISA using antibodies HCA327, HCA328, and HCA329.


Immunogenicity assay using Human Anti-Ipilimumab Antibodies as the calibration standard

In Figure 3, ipilimumab was coated at 1 µg/ml on a microtiter plate overnight. After washing and blocking with PBST + 5% BSA, PBST with 10% human serum was added, spiked with increasing concentrations of Human Anti-Ipilimumab Antibody, clone AbD34283ia (HCA327, black), clone AbD34428ia (HCA328, red), or clone AbD34429ia (HCA329, blue). Detection was performed using HRP conjugated ipilimumab at 2 µg/ml in HISPEC Assay Diluent (BUF049A), and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: Ipilimumab ADA bridging ELISA using antibodies HCA327, HCA328, and HCA329


Inhibition ELISA

Fig. 4. Demonstration of the inhibitory property of antibody HCA329.

Fig. 4. Demonstration of the inhibitory property of antibody HCA329.


In figure 4, recombinant human CTLA-4 was coated on a microtiter plate at 1 µg/ml overnight. After washing and blocking with PBST + 5% BSA, HRP conjugated ipilimumab was added (0.3 µg/ml), spiked with increasing concentrations of Human Anti-Ipilimumab Antibody, clone AbD34429 (HCA329) in the monovalent Fab format. Free HRP conjugated ipilimumab, still capable of binding to the human CTLA-4 coated plate, was detected using QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.


Demonstration of Antibody Specificity

Fig. 5. Specificity of Human Anti-Ipilimumab Drug/Target Complex Antibody HC331.

Fig. 5. Specificity of Human Anti-Ipilimumab Drug/Target Complex Antibody HC331.

In Figure 5, a microtiter plate was coated overnight with various antigens at a concentration of 5 µg/ml. After washing and blocking with PBST + 5% BSA, detection was performed using Human Anti-Ipilimumab Antibody, clone AbD34294 (HCA331) at a concentration of 2 μg/ml in HISPEC Assay Diluent (BUF049A), followed by an HRP conjugated anti-DYKDDDDK tag antibody and QuantaBlu Fluorogenic Peroxidase Substrate.


Licensed use: For in vitro research purposes and for commercial applications for the provision of in vitro testing services to support preclinical and clinical drug development. Any re-sale in any form or any other commercial application needs a written agreement from Bio-Rad.