Development of Human-Derived Antibody Gene Mice and Their Application in the Treatment of Diseases

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One of the earliest attempts to develop antibody drugs for the treatment of infectious diseases was made using the animal-derived serum. The hybridoma technology was successfully established in 1975, and it contributed to the research regarding therapeutic antibodies. In 1986, the first therapeutic mouse-derived antibody drug was approved for clinical use.

Therapeutic antibodies comprise leading drugs for treating tumors and other human-related disorders. The development and improvement in genome editing technology is a revolutionary evolution in the field of therapeutic antibody drugs, which are clinically important for the treatment of several diseases, particularly the ones related to oncology.

l What Historical Stages Have Therapeutic Antibody Development Gone Through? What Milestones Have Been Achieved in the Treatment of Diseases?

Here are the key stages and milestones in the development of therapeutic antibody development:

  1. In 1975, the hybridoma technology made the unlimited production of monoclonal antibodies possible.
  1. In 1986, the FDA approved the mouse-derived anti-CD3 antibody or Orthoclone OKT3 for treating acute organ transplant rejection. Owing to high toxicity and shorter half-life, the murine antibody was withdrawn from the market.
  1. In 1994, anti-GPIIB/IIIa antigen-binding fragment antibody (Fab) was approved for the treatment of platelet coagulation inhibition and associated cardiovascular diseases. Anti-CD20 was the first chimeric antibody approved for the treatment of non-Hodgkin’s lymphoma.
  1. In 1997, the humanized anti-IL-2 receptor antibody was approved for the prevention of organ transplant rejection. In 1998, the humanized anti-HER2 antibody or Herceptin was approved for the treatment of metastatic breast cancer and gastroesophageal junction adenocarcinoma.
  1. In 2002, the first fully human antibody, the anti-TNF-α antibody, was approved for the treatment of rheumatoid arthritis using phage display technology. The use of these antibodies now includes ulcerative colitis, inflammatory bowel disease, ankylosing spondylitis, and psoriasis.
  1. In 2011, the immune checkpoint-related molecules included Yervoy, the first fully human antibody which targets immune checkpoint CTLA-4 in 2011.
  1. In 2014, Opdivo, a fully human anti-PD1 antibody, and Keytruda, a humanized anti-PD1 antibody, were used for the treatment of melanoma, non-small lung cancer, head and neck cancer, kidney cancer, and Hodgkin’s lymphoma. Therapeutic antibodies are amongst the best-selling drugs.
  1. To date, approximately 80 antibodies have been approved by FDA for clinical use, 30 of which are therapeutic oncologic antibodies. The pharmaceutical market is valued at approximately $115.2 billion, with sales expected to reach $300 billion by the year 2025.
l Why Do We Need to Develop Human-Derived Antibodies? What are the Technologies and Strategies for Human-Derived Antibody Development?

The disadvantages of murine-derived antibodies include human anti-mouse antibody (HAMA) reactions which increase the clearance of antibodies and elicit allergic reactions.

The developmental strategies of human-derived antibodies have been derived. The first fully human antibody was screened using the phage display technology. The human antibodies were developed using mouse models with human antibody genomes. The human-derived antibodies were obtained using human hybridoma cells and associated B lymphocytes. The chimeric antibody technology was used for the development of humanized antibodies. The chimeric antibody retained the specificity of the antibody binding antigen. The complementary decision region grafting technique (CDR) comprises antibodies of human origin will only a small binding antigen decision region sequence in mouse-derived antibody.

In contrast to chimeric antibodies, antibodies derived from CDR transfer technology are less immunogenic and have formed the basis for the treatment of oncology and autoimmune diseases. With 10% murine antibody sequences, these humanized antibodies carry the risk of immune rejection and hypersensitivity reactions.  

The humanized antibody development technology involves protein platforms and the formation of the recombinant peptide. Antibody fragments are the first human-derived antibodies using this technology. The phage display technology obtains the antibody directly and does not involve in vivo immune responses. The human antibody gene mouse model was established in the early 1990s and has facilitated associated clinical applications. In the initial stages, this platform is relatively slow. After acquiring initial antibodies, the antibodies demonstrated improved affinity and efficacy. Drugs developed using this technology provide better evaluation for indicators of drug generating properties.

l How Do the Basic Structures of Therapeutic Antibodies Interrelate with Clinical Disease Treatment?

IgG is the most prevalent therapeutic antibody comprising heavy and light chains. The heavy chain backbone forms a fragment crystallization region (Fc), whereas the two arms of the antibody are attributed to antigen-binding domains (Fab). IgG binds to the corresponding agent and demonstrates therapeutic effects, including antibody-dependent cytotoxic response (ADCC) and complement-mediated cytotoxic response (CDC). The most suitable subtype of IgG therapeutic antibodies is IgG1, followed by IgG4 and IgG2. The therapeutic antibodies approved for clinical use may comprise intact antibodies, Fc fusion proteins, and Fab antibody fragments. The antibody fragments are time- and cost-efficient as well as have better infiltration for tumors. However, these antibodies lack the Fc region, which influences the stability, circulation, and therapeutic efficacy.

Large molecule antibody drugs have high target specificity and target toxicity. The route of administration is intravenous or subcutaneous, and the drugs are absorbed by the lymphatic system. The human-derived antibodies are produced using the mammalian cell expression system. Using this system, the antibodies can be produced in greater quantities and undergo post-translational modification. Most of the antibodies are produced using Chinese hamster ovary (CHO) cells. Other sources include mouse myeloma cells, human embryonic kidney cells, and E. coli.

I. What are the Strategies and Methods for Establishing Human Antibody Gene Mouse Models? What is the Current Progress of the Clinical Application of these Human Antibodies?

The mouse immune system is employed to produce diverse combinations of the human antibodies via natural recombination and somatic hypermutation of the human antibody genes in the mouse models. The gene mouse technology does not require humanization, has greater antibody combination diversity, and optimized antibody maturation and cloning. However, the large genomic region of the Ig gene imposes a great challenge to the development of the human antibody gene in the mouse. The idea of the development of transgenic mice and the production of human antibodies was proposed in 1985. The antibody heavy chain gene vector was constructed in 1989, followed by the production of human antibodies by transgenic mice in 1993. The HuMab mouse technology platform was developed in 1994 and was used to express the heavy and light chain genes of the human antibody. The yeast artificial chromosome (YAC) vectors were used for constructing the XenoMouse model for expressing human-derived antibody genes. However, both the XenoMouse and HuMab mouse models lacked constant and variable region genes influencing the effectiveness of antibody production.

The human antibody variable regions were inserted into the mouse genome upstream to the constant regions using Cre/loxP and bacterial artificial chromosome (BAC) techniques, developing the KyMouse model with high affinity and high-frequency somatic mutagenesis. The VelocImmune mouse model was constructed using large fragments of human antibody genes and a microinjection method to introduce the BAC vector into mouse cells to achieve target replacement while retaining a constant region of mouse antibody genes.

The human antibody gene mouse technology platform has been used by seven major biopharmaceutical companies for developing therapeutic antibodies. The platforms include XenoMouse, HuMab, and VelocImmune mouse models. Anti-CTLA-4 and anti-PD-1 antibodies have been developed using HuMab mouse models and were initially used for melanoma treatment. Using the same model, anti-IL-23 and anti-IL-12 antibodies were developed for the treatment of Crohn’s ileitis and psoriasis, respectively. Using the XenoMouse platform, seven therapeutic human antibodies have been approved, including an anti-EGFR antibody for the treatment of metastatic colon cancer and an anti-IL-17 antibody for psoriasis. Similarly, VelocImmune mouse technology is employed for the production of four human antibody drugs such as anti-IL-4 and anti-IL-6 receptor antibodies.  

II. What are the Future Trends of Human Antibody R&D?

Therapeutic antibody drugs have been used for the treatment of autoimmune, infectious, and oncologic diseases. The therapeutic antibodies are divided into naked antibody drugs and drugs that elevate the therapeutic value of antibodies. Naked antibodies are directly used for the treatment of diseases. On the contrary, the second type of antibody-drug further modifies and processes the antibodies, including antibody-chemical drug couples, immunoliposomes, and antibody-immune cytokine binding. Biantigen antibody development enables the antibodies to recognize different antigens simultaneously. For instance, antibodies against CD3 and CD19 are employed for the treatment of B-cell acute lymphoblastic leukemia.

Newer trends in research and development include antibody Fc engineering, chimeric antigen receptor (CAR) T-cell therapy, and the production of human antibodies from B-cell isolation and screening. Even though the production of antibodies using single B-cell technology has not yet been approved by FDA, therapeutic antibodies serve as a powerful tool for diagnostic and pharmacokinetic applications.    

III. What is the Current Progress in the Development of Human Antibodies against NCCV?

Scientists have applied human antibody gene mice and single B-cell isolation screening for the development of antibodies against neoplastic pneumonia. The first anti-neo-coronavirus antibody to enter the drug trial is Eli Lilly’s neutralizing antibody. Regeneron and Harbour BioMed are among the first companies to develop anti-neo-coronavirus antibodies which target the stinger S protein. The side effects of human-derived antibodies include the development of anti-drug antibodies, impaired drug clearance, and severe paralysis.

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