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Single-chain antibody-based immunotoxins targeting Her2/neu: design optimization and impact of affinity on antitumor
Yu Cao1, James D Marks, Qian Huang
1Immunopharmacology and Targeted Therapy Laboratory, Departmentof Experimental Therapeutics, University of Texas MD AndersonCancer Center, Houston, Texas 77054, USA.
Abstract:
Recombinant immunotoxins, consisting of single-chain variable fragments (scFv) genetically fused to polypeptide toxins, represent potentially effective candidates for cancer therapeutics. We evaluated the affinity of various anti-Her2/neu scFv fused to recombinant gelonin (rGel) and its effect on antitumor efficacy and off-target toxicity. A series of rGel-based immunotoxins were created from the human anti-Her2/neu scFv C6.5 and various affinity mutants (designated ML3-9, MH3-B1, and B1D3) with affinities ranging from 10(-8) to 10(-11) mol/L. Against Her2/neu-overexpressing tumor cells, immunotoxins with increasing affinity displayed improved internalization and enhanced autophagic cytotoxicity. Targeting indices were highest for the highest affinity B1D3/rGel construct. However, the addition of free Her2/neu extracellular domain (ECD) significantly reduced the cytotoxicity of B1D3/rGel because of immune complex formation. In contrast, ECD addition had little impact on the lower affinity constructs in vitro. In vivo studies against established BT474 M1 xenografts showed growth suppression by all immunotoxins. Surprisingly, therapy with the B1D3-rGel induced significant liver toxicity because of immune complex formation with shed Her2/neu antigen in circulation. The MH3-B1/rGel construct with intermediate affinity showed effective tumor growth inhibition without inducing hepatotoxicity or complex formation. These findings show that while high-affinity constructs can be potent antitumor agents, they may also be associated with mistargeting through the facile formation of complexes with soluble antigen leading to significant off-target toxicity. Constructs composed of intermediate-affinity antibodies are also potent agents that are more resistant to immune complex formation. Therefore, affinity is an exceptionally important consideration when evaluating the design and efficacy of targeted therapeutics.
Insights
Optimizing recombinant immunotoxin design for cancer therapy is crucial. While high-affinity anti-Her2/neu agents show potent antitumor effects, intermediate-affinity versions offer better safety by avoiding off-target toxicity from immune complex formation.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Recombinant immunotoxins (scFv-toxin fusions) are promising cancer therapeutics.
- Targeting Her2/neu is a validated strategy for breast cancer treatment.
Purpose of the Study:
- To evaluate the impact of anti-Her2/neu single-chain variable fragment (scFv) affinity on immunotoxin efficacy and toxicity.
- To determine optimal affinity for potent antitumor activity with minimal off-target effects.
Main Methods:
- Constructed recombinant immunotoxins by fusing anti-Her2/neu scFv variants (C6.5, ML3-9, MH3-B1, B1D3) with varying affinities to recombinant gelonin (rGel).
- Assessed in vitro cytotoxicity, internalization, and targeting indices against Her2/neu-overexpressing cells.
- Evaluated in vivo antitumor efficacy and toxicity in BT474 M1 xenograft models.
Main Results:
- Higher affinity immunotoxins (e.g., B1D3/rGel) demonstrated enhanced cellular uptake and cytotoxicity but formed immune complexes with soluble Her2/neu, causing liver toxicity.
- Intermediate-affinity immunotoxin (MH3-B1/rGel) effectively inhibited tumor growth without significant off-target toxicity or immune complex formation.
- Lower affinity constructs showed reduced efficacy but were less prone to immune complex-mediated toxicity.
Conclusions:
- Immunotoxin affinity is a critical factor influencing both therapeutic efficacy and safety.
- Intermediate-affinity antibodies offer a favorable balance, providing potent antitumor activity while mitigating off-target toxicity associated with high-affinity interactions and immune complex formation.
- Rational design of immunotoxin affinity is essential for developing safe and effective targeted cancer therapies.
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