Overview of Knobs-in-holes Bispecific Antibody

Backgrounds of Knobs-in-holes Bispecific Antibody

Bispecific antibodies (bsAbs) are a class of engineered antibodies that can simultaneously bind to two different antigens or epitopes, thus expanding the therapeutic scope and potential of conventional monoclonal antibodies (mAbs). BsAbs have been widely explored for various applications, such as cancer immunotherapy, inflammatory diseases, and infectious diseases. BsAbs can exert novel modes of action that are not achievable by mAbs, such as recruiting effector cells to enhance tumor killing, blocking redundant signaling pathways to overcome resistance, differentiating diseased cells from normal cells for improved safety and efficacy, and provoking synergistic effects by co-targeting two antigens. However, bsAbs also pose significant challenges in terms of design, production, and quality control, due to the complexity and diversity of their molecular architectures.

Fig.1 Classical Molecular Platform of Bsabs and Representative Antibodies (Ma, 2021)

Among the numerous formats of bsAbs that have been developed, knobs-into-holes (KIHs) technology is one of the most widely used and well-validated methods for generating bispecific IgG antibodies. KIHs technology involves engineering the CH3 domains of two distinct heavy chains to create either a "knob" or a "hole" in each chain to promote heterodimerization. The knob is formed by introducing a bulky amino acid (Tyr) at position 366 (EU numbering), while the hole is formed by replacing a bulky amino acid (Tyr) at position 407 with a smaller one (Thr). The knob fits into the hole of the opposite chain, resulting in a stable and specific heterodimeric Fc region. The light chains are paired with their cognate heavy chains by using either common light chains or crossover mutations at the CH1-CL interface. KIHs technology enables the production of bispecific IgG antibodies with similar properties and behaviors as natural IgG antibodies, such as long half-life, Fc-mediated effector functions, and low immunogenicity.

Structural Features of Knobs-in-holes Bispecific Antibody

Knobs-in-holes (KIHs) bispecific antibody is a type of bispecific IgG antibody that has a heterodimeric Fc region and two different Fab arms. The Fc region is composed of two distinct heavy chains that are engineered to form a stable and specific heterodimer by introducing a "knob" and a "hole" mutation in the CH3 domains. The knob is a bulky amino acid (Tyr) at position 366 (EU numbering) that fits into the hole, which is a smaller amino acid (Thr) at position 407, of the opposite chain. The light chains are paired with their cognate heavy chains by using either common light chains or crossover mutations at the CH1-CL interface. The Fab arms can bind to two different antigens or epitopes, thus conferring dual specificity to the KIHs bispecific antibody.

The structure of KIHs bispecific antibody can be characterized by various methods, such as Western blotting, liquid chromatography-mass spectrometry (LC-MS), size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and X-ray crystallography. These methods can provide information on the molecular weight, purity, assembly, stability, and conformation of the KIHs bispecific antibody. For example, Western blotting can confirm the presence of knob and hole mutations by using specific antibodies against them. LC-MS can identify the amino acid sequences and post-translational modifications of the KIHs bispecific antibody. SEC can separate and quantify the monomeric and dimeric forms of the KIHs bispecific antibody. AUC can measure the sedimentation coefficient and molecular shape of the KIHs bispecific antibody. X-ray crystallography can reveal the three-dimensional structure and interactions of the KIHs bispecific antibody.

The structure of KIHs bispecific antibody is similar to that of natural IgG antibody, except for the knob and hole mutations in the CH3 domains and the possible differences in the Fab arms. Therefore, KIHs bispecific antibody can retain most of the properties and behaviors of natural IgG antibody, such as long half-life, Fc-mediated effector functions, and low immunogenicity. However, some structural features of KIHs bispecific antibody may also affect its functionality, stability, potency, and immunogenicity. For example, the knob and hole mutations may introduce steric hindrance or electrostatic repulsion between the CH3 domains, resulting in reduced thermal stability or increased aggregation tendency. The Fab arms may have different affinities or avidities for their respective antigens, resulting in different binding kinetics or stoichiometries. The Fab arms may also interfere with each other or with the Fc region, resulting in altered antigen binding or Fc receptor binding. Therefore, careful structural and functional characterization of KIHs bispecific antibody is essential for its optimal design and production.

Generation Methods of Knobs-in-holes Bispecific Antibody

Knobs-in-holes (KIHs) bispecific antibody can be generated by various methods, such as cell-based expression, cell-free expression, and chemical conjugation. Each method has its own advantages and disadvantages in terms of efficiency, scalability, quality, and cost.

Cell-based expression is the most common method for producing KIHs bispecific antibody. It involves co-transfecting two plasmids encoding the knob and hole heavy chains and their respective light chains into mammalian cells, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. The cells are then cultured and harvested, and the KIHs bispecific antibody is purified by affinity chromatography. Cell-based expression can produce high-quality and homogeneous KIHs bispecific antibody with natural glycosylation and folding. However, cell-based expression also faces some challenges, such as low expression yield, high production cost, complex optimization of plasmid ratio and culture conditions, and potential contamination or instability of the product.

Cell-free expression is an emerging method for producing KIHs bispecific antibody. It involves using a cell-free system, such as Xpress CF, to synthesize the knob and hole heavy chains and their respective light chains in vitro from DNA templates. The cell-free system can be either a lysate-based system or a reconstituted system that contains the essential components for protein synthesis. The cell-free system can also be supplemented with prefabricated knob or hole to improve the assembly and yield of KIHs bispecific antibody. Cell-free expression can produce KIHs bispecific antibody rapidly and efficiently without the need for cell culture and maintenance. Cell-free expression can also allow for high manipulability and flexibility of the system to optimize the plasmid ratio, reaction conditions, and construct design. However, cell-free expression may also have some limitations, such as low scalability, high variability, unnatural glycosylation and folding, and potential degradation or aggregation of the product.

Chemical conjugation is another method for producing KIHs bispecific antibody. It involves using chemical cross-linkers to covalently link two monospecific antibodies or Fab fragments that have been separately produced by cell-based or cell-free expression. Chemical conjugation can produce KIHs bispecific antibody with precise stoichiometry and orientation of the two Fab arms. However, chemical conjugation may also introduce unwanted modifications or impurities to the KIHs bispecific antibody, such as heterogeneity, immunogenicity, toxicity, or reduced activity.

Therefore, different methods for generating KIHs bispecific antibody have their own pros and cons, and the choice of method depends on the specific needs and goals of each project.

Clinical Data of Knobs-in-holes Bispecific Antibody

Knobs-in-holes (KIHs) bispecific antibody has shown promising clinical results in various indications, especially in oncology. Several KIHs bispecific antibodies have been approved or are under clinical development for different types of cancers, such as hematological malignancies and solid tumors.

The first and so far the only approved KIHs bispecific antibody is blinatumomab (Blincyto), which targets CD19 and CD3 antigens. Blinatumomab was approved by the US Food and Drug Administration (FDA) in 2014 for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). It was later expanded to include patients with minimal residual disease (MRD)-positive B-cell precursor ALL in 2018 and patients with newly diagnosed B-cell precursor ALL in 2020. Blinatumomab works by engaging T-cells to kill CD19-positive B-cells, which are malignant in ALL.

The approval of blinatumomab was based on several clinical trials that demonstrated its efficacy and safety in different settings of ALL. In a phase II trial (BLAST) involving 116 patients with MRD-positive B-cell precursor ALL, blinatumomab achieved a complete MRD response rate of 78% and a median relapse-free survival of 23.6 months. In a phase III trial (TOWER) involving 405 patients with relapsed or refractory B-cell precursor ALL, blinatumomab improved the median overall survival from 4 months to 7.7 months compared with standard chemotherapy. In another phase III trial (ALCANTARA) involving 267 patients with relapsed or refractory Philadelphia chromosome-positive (Ph+) B-cell precursor ALL, blinatumomab achieved a complete remission or complete remission with partial hematological recovery rate of 36% compared with 17% for standard chemotherapy. In a phase III trial (Children's Oncology Group AALL1331) involving 208 children and young adults with high-risk first relapse B-cell precursor ALL, blinatumomab improved the event-free survival from 32% to 54% compared with standard chemotherapy.

The most common adverse events associated with blinatumomab are cytokine release syndrome (CRS), neurologic toxicity, infections, pyrexia, headache, and neutropenia. CRS is a systemic inflammatory response that can cause fever, hypotension, hypoxia, and organ dysfunction. Neurologic toxicity can manifest as seizures, encephalopathy, tremor, aphasia, and other symptoms. The management of these adverse events includes dose interruption or discontinuation, supportive care, and administration of corticosteroids or tocilizumab.

Several other KIHs bispecific antibodies are currently under clinical development for various types of cancers.

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