Difficulties in preparing bispecific antibodies
The random combination of four different polypeptide chains will produce more than 10 different products, and only one product is the desired BsAb, so a large amount of by-products will be produced in the preparation of antibodies. These by-products generally include homodimers, antibodies lacking one light chain, half antibodies, heavy chain polymers, light chain polymers, free heavy chains, and light chains. These by-products are not common in the preparation of standard monoclonal antibodies, and therefore pose a great challenge for the downstream process of BsAb preparation.
Although genetic engineering technology can summarize the generation of heterodimers, it is still difficult to completely eliminate homodimerization. Commonly used protein A chromatography is difficult to separate these dimers from the target BsAb. For BsAbs whose parent antibodies have large differences in isoelectric point or hydrophobicity, they can be separated by methods such as ion exchange, hydrophobic exchange, and hydroxyapatite. However, for BsAbs with small differences, engineering transformation is required, such as isoelectric point engineering, affinity engineering, and scFv-Fc fusion.
Important advantages of bispecific antibodies
BsAb can simultaneously bind dual targets, block dual signaling pathways, play unique or overlapping functions, and effectively prevent drug resistance. Receptor tyrosine kinases (RTKs) are a class of enzyme-linked receptors that play an important regulatory role in cell proliferation. The abnormally high expression of RTKs on the surface of tumor cells leads to malignant proliferation of tumor cells, so it is also an important target for tumor treatment.
Single-target monoclonal antibodies against RTKs have been widely used in tumor therapy. However, tumor cells can perform immune escape by switching signaling pathways or by activating intracellular signals through HER family members or homo/heterodimers between different members. BsAb drugs can block two or more RTKs or their ligands at the same time, which can reduce the escape of tumor cells and improve the therapeutic effect.
BsAb has greater specificity, targeting and reduced off-target toxicity. The two antigen binding arms bind different antigens, which effectively enhances the binding specificity and targeting of the antibody to cancer cells and reduces off-target side effects.
BsAb can also effectively reduce the cost of treatment. Compared with monoclonal antibodies, BsAb has stronger tissue penetration rate, tumor cell killing efficiency and clinical indications, and the dose can be reduced to a minimum of 1/2000 of the original, significantly reducing the treatment costs.
Dozens of bispecific antibodies are currently in clinical development, and more bispecific antibody molecules are in the preclinical research stage, and most of the bispecific antibodies that have entered the clinical stage are used in the field of cancer treatment. From the target point of view, most projects targeting PD-1/PD-L1, CTLA-4 and HER2, such as PD-1/CD47, PD-1/IL-10, PD-1/VEGF, CD47/ VEGF, etc.
There are multiple BsAbs that are conducting clinical trials for the treatment of tumors, and their structures mostly contain CD3 antigen binding sites (used to recruit T cells to the vicinity of tumor cells), and another binding site includes CD19, CD20, CD33, CD123, HER2, CEA, ganglioside GD2, PSMA, gpA33, etc. In addition, there are some BsAbs: HER2/HER3, IL1α/IL1β, IL13/IL17, IL17A/IL17F and CD30/CD16A.
As a forward-looking technology, it faces many challenges in industrialization, such as solving mismatches and purification, improving the instability of downstream processes, ensuring the stability of double antibodies and balancing the expression of two antibodies. With the advancement of technology, I believe that in the future, more and better strategies can be used to optimize various BsAbs, so that they have more powerful efficacy and lower side effects, and bring gospel to cancer patients.