HOPE Monoclonal Antibodies: Genetically Engineered Monoclonal Antibodies via Mass Spectrometry or Protein Design analysis of antibodies of Recovered patients. by Dr. Michellie Hernandez, MD is licensed under CC BY-SA 4.0
Neutralizing antibodies are the antibodies of recovered patients that best bind to the antigen’s epitope and found to be the most efficient in animal models. The following is a method in the development of monoclonal antibodies by discovering and utilizing the best neutralizing antibodies of recovered patients of any disease or tumor that produces antibodies. These selected neutralizing antibodies can be used as potential guides towards the development of genetically engineered monoclonal antibodies.
For years different methods to create genetically engineered monoclonal antibodies have been attempted in animal models, but found to be too expensive and time consuming for mass production. The following is an attempt to lower the cost in the development process of genetically engineered monoclonal antibodies by suggesting a few innovative experiments inspired by published research papers like Barderas, R., Benito-Peña, E. The 2018 Nobel Prize in Chemistry: phage display of peptides and antibodies. (Barderas et al. 2019)
I like to call the genetically engineered monoclonal antibodies (GE-mAb) produced by my method, HOPE Monoclonal Antibodies (HOPE mAb). The following steps in development of HOPE mAb are specific for COVID19, although these steps can be followed for other diseases or tumors that produce antibodies in recovered patients, thus can be used as a guideline for genetic engineered monoclonal antibodies development.
The process in summary is done with the help of mass spectrometry or Protein Design decoding both the mRNA sequence of the Fab component of an effective antibody against SARS-COV2 from recovered COVID19 patient and the mRNA sequence of the constant region of a fully human monoclonal antibody. This will be followed by uniting the two mRNA sequences to form the mRNA of a full monoclonal antibody specific against SARS-COV2.
Parts of the following steps require permission of US patents.
1. Conduct antibody study in recovered COVID19 patients to collect serum samples to test for effectiveness of each sampled antibody to bind to SARSCOV-2 spike protein with neutralization tests. Essays with nonpathogenic remnant of SARSCOV-2 containing spike proteins can be used to test for the efficacy in binding of the antibodies to the spike protein in order to reduce the costs and need of BSL3 labs. One can also confirm with tandem mass spectrometry analysis to select the best antibody with the most cross binding between antibody and spike protein. Select the most effective antibody specific against SARSCOV-2 in the study.
2. Via HPLC and mass spectrometry or Protein Design decode the mRNA sequence of the Fab component (the binding site of the antibody to the antigen) of the most effective antibody against SARS-COV-2 selected in step 1. If protein design is used, computational models with machine learning can make the Fab component of the antibody into a linear protein structure, decode the amino acid sequence, decode the codons then decode the mRNA sequence.
3. Obtain the mRNA sequence of the constant region of a fully humane monoclonal antibody (mumab) via HPLC and mass spectrometry or Protein Design. Same computational model algorithms as above.
4. Unite both mRNA sequences obtained in steps 2 and 3 so that the union of the two encodes for a complete fully human monoclonal antibody (mumab) effective against SARS-CoV2.
5. Obtain the mRNA sequence of an In Vitro transcribed mRNA (IVT mRNA) encoding the united mRNA sequence. Per Schlake, T., Thess, A., Thran, M. et al. mRNA prepared by in vitro transcription (IVT) is increasingly appreciated as a drug substance for delivery of recombinant proteins. (Schlake et al. 2018)
6. To make IVT mRNA production even more cost-effective one can test mass production of IVT mRNA with recombinant DNA technology. Synthesize a synthetic DNA sequence that upon transcription will transcribe the mRNA sequence in Step 5 (IVT mRNA without the delivery system). The synthetic DNA is inserted into a plasmid and with the use of recombinant DNA technology in E. Coli or yeast culture, clones of IVT mRNA could be reproduced. If non-immunogenic delivery system for IVT mRNA are proven to be safe and effective in animal models, IVT mRNA vectors can be used as delivery of mAb in humans similar to how IVT mRNA is being tested to be used as passive immunity vaccines. But instead of delivering the antigen of SARS-COV2, the IVT mRNA will deliver the mAb to the plasma cells in humans.
Additional methods of making mAb production more cost-effective using protein design are:
In Vitro mAb production in yeast culture:
Add a promotor to IVT mRNA in step 5 and after cloning the IVT mRNA in step 6, stimulate transcription of pDNA encoding the IVT mRNA and stimulate translation of IVT mRNA within the yeast culture for mass mAb production within the yeast culture. Provide a medium rich in amino acids necessary for the mAb production. This process is similar to the process of producing foreign protein synthesis in yeast cultures by use of plasmid with encoding DNA as done in past experiments. (Ridder et al. 1995 and Griffiths et al. 2000)
Use of transgenic animal models:
Synthesize synthetic DNA which transcribed encodes the united mRNA and inseminate in ovum lamb and proceed to select transgenic progeny to have HOPE monoclonal antibodies secreted in the progeny lamb’s milk. (Griffiths et al. 2000)
Use of animal models:
Synthesize synthetic DNA which transcribed encodes the united mRNA and insert in vitro to hybridoma and later inseminate in animal models. One can also test if in vitro production of mAb can be done without the use of animal models. (Van Hoecke et al. 2019)
In either of these options used, you must follow with the extraction of mAb and the quality testing of the pure mAb. Animal Testing for safety and efficacy of HOPE monoclonal antibodies followed by human trials.
Hypothesis: The efficacy of the HOPE monoclonal antibodies should prove to be the same as the selected antibody from step 1 if the protein design computational models decoded the mRNA correctly. If efficacy proves to be less than the selected antibody review the protein design computations in decoding the mRNA sequence or the purification mechanisms of the monoclonal antibody. This method should also prove to be a more cost effective way of developing genetically engineered mAb in the future for a number of diseases or tumors that produce antibodies in recovered patients.
Discussion: Worldwide distribution of a safe and effective vaccine to achieve herd immunity can last years to accomplish. In the meantime HOPE monoclonal antibodies can be developed and used to reduce mortality due to COVID19 as well as treat immunocompromised individuals in which the vaccine might prove to be ineffective.
Ideally an IVT mRNA vector encoding the united mRNA sequence of the mAb, can deliver to plasma cells in humans similar to how passive immunity vaccines are being developed to deliver the mRNA sequence of a known antigen. This process will bypass any animal models required for the mAb development and reduce the costs of mAb development in the future. Per an article by Van Hoecke, L., Roose, K, immunogenicity of IVT mRNA is still a problem for use of IVT mRNA in humans. (Van Hoecke et al. 2019)
Thus I suggest until a non-immunogenic IVT mRNA delivery system can be found, one can stimulate promotors in IVT mRNA to mass produce mAb production in vitro in hybridoma, or yeast or plant cultures instead in order to bypass the costs and limitations of using animal models.
NOTE: A few steps in the method process are subject to US patents.
Acknowledgements: I will like to thank Dr. Mary Ruebush who encouraged me to continue in the beginning stages of my research.
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