HOL3 Antibody
Description
Knobs-into-Holes (KIH) Antibody Platform
The KIH technology enables heterodimerization of antibody heavy chains by introducing complementary mutations ("knob" and "hole") in their CH3 domains . This approach is widely used to produce bispecific antibodies (BsAbs) with asymmetric Fc regions. Key features include:
Glycosylation and Effector Function
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Fc Glycosylation: Mammalian-expressed KIH antibodies retain Fc glycosylation, enabling effector functions like antibody-dependent cell-mediated cytotoxicity (ADCC) .
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Afucosylation Impact: Removing fucose from one CH2 domain (asymmetric afucosylation) enhances ADCC potency to levels comparable to fully afucosylated antibodies .
Table 1: ADCC Activity of Asymmetric KIH Antibodies
| Heterodimer | Fucosylation State | ADCC EC50 (ng/mL) | FcγRIIIa Binding (EC50) |
|---|---|---|---|
| H3 (CHO) | Fully fucosylated | 8.3 | Reduced affinity |
| H6 | Fully afucosylated | 1.43 | High affinity |
| H4/H5 | Hemi-afucosylated | 1.5–2.1 | Equivalent to H6 |
Data derived from anti-CD20 KIH antibodies .
Immunogenicity Risk
KIH mutations (e.g., Y407V) show minimal immunogenicity risk in preclinical assessments:
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MAPPs Analysis: T-cell epitopes overlapping KIH mutations (e.g., Y407V) were detected in ≤2/10 donors, suggesting low immunogenic potential .
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Benchmark Comparison: KIH antibodies exhibit ADA (anti-drug antibody) rates comparable to low-risk benchmarks like bevacizumab .
Therapeutic Bispecific Antibodies
KIH-engineered BsAbs have been used to target:
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CD19 × CD3: Blinatumomab (FDA-approved for acute lymphoblastic leukemia) .
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Tumor Antigens × Immune Checkpoints: Preclinical candidates targeting PD-L1, CTLA4, and CD47 .
Research Tools
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Immunoprecipitation/Blotting: Bispecific KIH antibodies paired with hapten-conjugated reagents reduce background noise in assays .
Stability and Challenges
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Thermal Stability: KIH mutations may reduce Tm by 5–10°C compared to wild-type Fc .
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Aggregation Risk: Hole homodimers (e.g., Y407V mutants) show increased aggregation in transient expression systems .
Future Directions
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
FAQs for Researchers on Knobs-Into-Holes (KIH) CH3 Antibody Engineering
Advanced Research Questions
How do glycoform asymmetries in KIH heterodimers influence FcγRIIIa binding?
KIH technology enables systematic study of asymmetric glycosylation. Experiments show that hemi-afucosylation (one chain afucosylated) suffices for maximal ADCC enhancement, while sialylation has negligible impact .
Methodological Insight:
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Produce H-L fragments separately in CHO (fucosylated) and Fut8KO (afucosylated) cells, then assemble heterodimers .
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Assess FcγRIIIa binding via ELISA and ADCC using NK cell lines or PBMCs .
Contradiction Analysis:
Earlier studies suggested sialylation reduces ADCC, but KIH experiments show no statistical difference in EC50 between sialylated (10.5 ng/mL) and desialylated (8.3 ng/mL) heterodimers . This highlights context-dependent effects of glycosylation.
How can researchers resolve low ADCC activity in transiently expressed KIH antibodies?
Transient expression often yields heterodimers with atypical glycosylation (e.g., hyper-sialylation) due to incomplete Fc domain folding.
Optimization Strategies:
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Use stable cell lines to improve folding and reduce aberrant glycosylation .
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Introduce Fut8KO to eliminate core fucose, boosting FcγRIIIa affinity .
What are the pitfalls in interpreting FcγR binding data for KIH antibodies?
FcγRIIIa binds asymmetrically to the Fc homodimer, complicating data interpretation for KIH heterodimers.
Methodological Insight:
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Use asymmetric FcγRIIIa-binding assays (e.g., SPR with monovalent receptors) to isolate contributions from each chain .
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Compare binding kinetics of hemi-afucosylated (e.g., H4, H5) vs. fully afucosylated (H6) heterodimers to confirm redundancy in fucose removal .
Data Reconciliation:
While afucosylation is known to enhance ADCC, KIH studies reveal that 50% afucosylation (one chain) achieves ~90% of maximal activity, challenging assumptions that both chains must be modified .
How can phage display improve KIH antibody stability?
Phage libraries screen for CH3 mutations that enhance heterodimer stability without disrupting FcγR binding.
