Overview of Quadroma (or Hybrid-Hybridoma) Technology

Bispecific antibodies (BsAbs) are artificially designed antibodies that have two different antigen-binding sites, which can simultaneously recognize two different antigens or two different epitopes on the same antigen, thereby achieving multiple functions, such as redirecting effector cells to tumor cells, blocking two signal pathways, enhancing targeted drug delivery, etc. Quadroma technology is a method of producing BsAbs based on the fusion of two different hybridoma cell lines, also known as hybrid-hybridoma technology, which is one of the earliest BsAb production technologies. Quadroma technology was first developed by Milstein et al. in 1983 to produce BsAbs with anti-insulin and anti-nerve growth factor specificity. Quadroma technology can produce BsAbs with a complete IgG structure that have high stability and affinity but also have drawbacks such as low efficiency, high heterogeneity, heavy chain and light chain mismatch, etc. Therefore, many other types of BsAb production technologies have emerged, such as genetic engineering technology, chemical conjugation technology, and small molecule BsAb technology.

The Principle and Steps of Quadroma Technology

Quadroma technology is based on the somatic fusion of two different hybridoma cell lines that produce monoclonal antibodies with different specificities. Hybridoma cell lines are derived from the fusion of B cells and myeloma cells, which can secrete monoclonal antibodies against a single antigen or epitope. To produce BsAbs, two hybridoma cell lines from different species (usually mouse and rat) are fused together to form quadroma cells, which can express heavy and light chains from both parent cell lines. These chains randomly assemble in the cell to form different types of immunoglobulin molecules. Among these molecules, only a small fraction are the desired BsAbs, which have two different antigen-binding regions from the parent antibodies. The proportion of BsAbs is about 6.25%. The other types of molecules include the parent antibodies, monovalent antibodies with only heavy or light chains, and bivalent antibodies with mismatched heavy and light chains. These molecules need to be removed through purification steps. To obtain BsAbs, the quadroma cells are cloned and expanded, and their culture supernatants are screened for the presence of BsAbs by ELISA or other methods. The quadroma cells that produce BsAbs are stabilized and cultured on a large scale, and the BsAbs are collected and purified.

Fig.1 Generation of bispecific antibodies by quadroma technology (Kroesen,1998)

The Characteristics of Quadroma Technology

Quadroma technology has some advantages over other BsAb production technologies. For example, it can produce BsAbs with a complete IgG structure, which have high stability and affinity, and can trigger Fc-mediated effector functions such as ADCC and CDC. This makes them suitable for therapeutic applications that require a long half-life and potent cytotoxicity. In addition, it does not require genetic engineering or chemical conjugation, which simplifies the production process and reduces the risk of immunogenicity or toxicity. This also avoids the need for complex cloning, expression, and purification systems that are often associated with other BsAb production technologies.

However, Quadroma technology also has some drawbacks that limit its application. First, it has low efficiency because only a small fraction of the molecules are the desired BsAbs, and the other types of molecules need to be removed by purification steps, which increases the cost and time. The yield of BsAbs from Quadroma technology is usually less than 1 mg/L, compared with more than 10 mg/L from other technologies. Second, it has high heterogeneity because it uses hybridoma cell lines from two different species, which may cause immune rejection in the human body. The use of mouse and rat antibodies also limits the diversity of antigen targets that can be recognized by BsAbs. Third, it has a heavy chain and light chain mismatch problem because quadroma cells express heavy and light chains from both parent cell lines, which randomly assemble in the cell and may form mismatched bivalent antibodies that affect the function and specificity of BsAbs. This problem can be partially solved by using hybridoma cell lines from the same species or by using affinity chromatography to separate the desired BsAbs.

The Applications of Quadroma Technology

Quadroma technology has been used to produce various BsAbs with different functions, especially in the field of cancer immunotherapy. BsAbs produced by Quadroma technology can redirect immune cells to tumor cells, block tumor-associated antigens or receptors, or deliver cytotoxic agents to tumor sites.

Table 1. Examples of BsAbs produced by Quadroma technology

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