Hope Monoclonal Antibodies as potential treatment for COVID-19

mhg20
Posted September 20, 2020 from Dominican Republic

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

Abstract:

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.

Article:

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. 1

 

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. 2

 

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.

 

 

Other methods of making mAb production more cost-effective using protein design are:

vIn 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. 3, 4

 

vUse 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. 4

 

vUse 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. 5

 

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. 4

 

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.

 

Created by: Dr. Michellie Hernández, MD

 

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

 

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.

 

References:

 

1.Barderas, R., Benito-Peña, E. The 2018 Nobel Prize in Chemistry: phage display of peptides and antibodies. Anal Bioanal Chem 411, 2475–2479 (2019). https://doi.org/10.1007/s00216-019-01714-4

 

2.Schlake, T., Thess, A., Thran, M. et al. mRNA as novel technology for passive immunotherapy. Cell. Mol. Life Sci. 76, 301–328 (2019). https://doi.org/10.1007/s00018-018-2935-4

 

3.Ridder, R., Schmitz, R., Legay, F. et al. Generation of Rabbit Monoclonal Antibody Fragments from a Combinatorial Phage Display Library and Their Production in the Yeast Pichia pastoris. Nat Biotechnol 13, 255–260 (1995). https://doi.org/10.1038/nbt0395-255

 

  1. Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000. Recombinant DNA technology in eukaryotes. Available from: https://www.ncbi.nlm.nih.gov/books/NBK22002/

 

  1. Van Hoecke, L., Roose, K. How mRNA therapeutics are entering the monoclonal antibody field. J Transl Med 17, 54 (2019). https://doi.org/10.1186/s12967-019-1804-8

 

  1. Pardi, N., Secreto, A., Shan, X. et al. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge. Nat Commun 8, 14630 (2017). https://doi.org/10.1038/ncomms14630

 

7.M POTTER, ... P CHANG, in Artificial Cells, Cell Engineering and Therapy, 2007  https://www.sciencedirect.com/topics/medicine-and-dentistry/hybridoma

 

8.Norbert Pardi1, Michael J. Hogan1, Frederick W. Porter2 and Drew Weissman1, mRNA vaccines — a new era in vaccinology.  NATURE REVIEWS | DRUG DISCOVERY VOLUME 17 | APRIL 2018 https://www.nature.com/articles/nrd.2017.243.pdf?origin=ppub

 

9.Goodwin, S., McPherson, J. & McCombie, W. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet 17, 333–351 (2016). https://doi.org/10.1038/nrg.2016.49

 

10.Kuhlman B, Bradley P. Advances in protein structure prediction and design. Nat Rev Mol Cell Biol. 2019 Nov;20(11):681-697. doi: 10.1038/s41580-019-0163-x. Epub 2019 Aug 15. PMID: 31417196; PMCID: PMC7032036. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032036/

 

11.Stothard, Paul.  "The Sequence Manipulation Suite: JavaScript Programs for Analyzing and Formatting Protein and DNA Sequences." , 28(6), pp. 1102–1104

     https://www.future-science.com/doi/pdf/10.2144/00286ir01

 

12.Nicholas Callahan, Jennifer Tullman, Zvi Kelman, John Marino, Strategies for Development of a Next-Generation Protein Sequencing Platform, Trends in Biochemical Sciences, Volume 45, Issue 1, 2020, Pages 76-89, ISSN 0968-0004, https://doi.org/10.1016/j.tibs.2019.09.005

 

13.Lorimer, D., Raymond, A., Walchli, J. et al. Gene Composer: database software for protein construct design, codon engineering, and gene synthesis. BMC Biotechnol 9, 36 (2009). https://doi.org/10.1186/1472-6750-9-36

 

14.Tian, J., Yan, Y., Yue, Q. et al. Predicting synonymous codon usage and optimizing the heterologous gene for expression in E. coli . Sci Rep 7, 9926 (2017). https://doi.org/10.1038/s41598-017-10546-0

 

15.Jones, P., Dear, P., Foote, J. et al. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321, 522–525 (1986). https://doi.org/10.1038/321522a0

 

16.Traggiai, E., Becker, S., Subbarao, K. et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med 10, 871–875 (2004). https://doi.org/10.1038/nm1080

 

17.Reff ME , Carner K , Chambers KS et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20 . Blood 1994 ; 83 : 435 .  https://www.researchgate.net/profile/Ronald_Raab2/publication/15692056_Depletion_of_B_cells_in_vivo_by_a_chimeric_mouse_human_monoclonal_antibody_to_CD20/links/00b7d53c9089b8c37b000000/Depletion-of-B-cells-in-vivo-by-a-chimeric-mouse-human-monoclonal-antibody-to-CD20.pdf

 

18.Neil S. Lipman, Lynn R. Jackson, Laura J. Trudel, Frances Weis-Garcia, Monoclonal Versus Polyclonal Antibodies: Distinguishing Characteristics, Applications, and Information Resources, ILAR Journal, Volume 46, Issue 3, 2005, Pages 258–268, https://doi.org/10.1093/ilar.46.3.258

 

 

 

                                                                                                                                            

This story was submitted in response to Dispatches from the COVID-19 Pandemic.

Comments 5

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Tamarack Verrall
Sep 20
Sep 20

Thank you for the information on the work you have been doing on a way to protect people from covid. We depend on scientists like you to be searching for safe ways to solve this pandemic. Good luck with your research.

mhg20
Sep 20
Sep 20

Thank-you! I had posted it in my LinkedIn and I have submitted it to Lancet for peer review, but I am awaiting if it will be accepted for publication and for a possible fee waiver since I am not affiliated with an university and have been working on the research on my own.

Nini Mappo
Sep 22
Sep 22

It is informative to get into the other side of Covid, the data world. Good on you for all that you are doing in the fight to keep our communities safe. I hope that you are safe yourself, so that you can look after others better.

Hello, Dr. Michellie,

Thank you for sharing this hypothesis you drafted. Very informative. Please update us on any progress of this research.

mhg20
Sep 22
Sep 22

Thanks Karen, I will let you know if it gets published in any journals for peer-review. I have submitted it to 3 so far and one came back saying no. So I'll see if the other two are willing to publish it as a commentary since I do not have an academic research background and I'm only a physician it wouldn't classify as an article.