The HOPE Method monoclonal antibodies and bio proteins: A Game-Changer in Antibody Engineering and Pathway for material science to transition towards bio proteins mass production.

The HOPE Method by Dr. Michellie Hernandez edited illustration of Deb Bose (Hernandez et al., 2023)

Written and edited by Michellie Hernandez, MD, with the help of ChatGPT (revised ChatGPT content along with personal content by the author)

Published: 10/04/2024

Edited: 10/11/2024

Abstract

The HOPE Method is a six-step process of mAbs production from antibodies collected from blood samples of human donors with a potent adaptive immune response against a pathogen or target of interest. Dr. Michellie Hernandez ideated the process in 2020 (Hernandez 2020), but named it the HOPE method in 2023 (Hernandez et al. 2023). This process may also be used to either identify the genes of bio proteins for current recurrent DNA technology or an innovative method to mass produce bio proteins without knowing the protein's genetic information.

Steps of the HOPE method

1. Obtain blood samples from recovered patient populations with the pathogen or neoantigen of interest. For purposes of bio proteins, a purified sample will be obtained.

2. Computational analysis of protein (selected antibody or bio protein in Step 1) structure with HPLC mass spectrometer and cryogenic electron microscopy (cryo-EM).This data can be entered into trained computational models to complete de novo peptide sequencing to predict the protein's linear amino acid sequence, accomplishing further decoding and prediction of the mRNA sequence with algorithms analyzing the codon table. To reverse engineer antibodies, the selection of the best antibody should be the efficiency of the best FAB region and the best FC region, thus can potentially be two different antibodies or one antibody that has both potent FAB and FC region. Computational analysis of the selected antibody’s FAB (the antibody's binding site to the antigen) and FC region should be done separately.

3. Decode the mRNA sequence of the FAB and Fc region (in case of antibody reverse engineering) from the predicted linear amino acid sequence in the computational models in Step 2; using reverse translation and codon engineering computational models. In natural translation, the mRNA sequence codes for an amino acid with three nucleic acids combined called the codon. In reverse translation or back translation on some articles, the mRNA is predicted from linear amino acid sequence data based on algorithms from trained computational models that have analyzed the codon chart and numerous natural translations of mRNA sequence to its known amino acid sequences, thus creating an algorithm to decode mRNA from linear amino acid sequences. This same process of decoding mRNA from the predicted linear amino acid sequence can also be done to reverse engineer bio proteins.

4. In the case of antibody reverse engineering, unite the mRNA sequences of the FAB and FC region obtained in step 3 so that the union of the two encodes a complete, fully human monoclonal antibody (mab) effective against the target of interest. Obtain an In Vitro transcribed mRNA (IVT mRNA) encoding the combined mRNA sequence (Hernandez et al., 2023).

5. Synthesize a synthetic DNA sequence that, upon transcription, will transcribe the mRNA sequence in Step 5 (IVT mRNA without the delivery system) via reverse transcription and genetic engineering. In case of bio protein reverse engineering, steps 4 and 5 provides an innovative approach to mass produce bioproteins as current recombinant biomanufacturing of bio proteins uses the genes of proteins, instead the HOPE method the DNA sequence obtained in Step 5, coding for a IVT mRNA for the bio protein of interest to mass produce.

6. The synthetic DNA is inserted into a plasmid, and with the use of recombinant DNA technology in E. Coli or yeast culture (Ridder et al., 1995), clones of IVT mRNA could be reproduced (Hernandez et al., 2023).”

Proteins from every living species on Earth follow the same transcription process, albeit the units of ribosomes may differ between species, the transcription process of the central dogma that converts mRNA to codons to amino acid sequences is followed by all. Thus is the reason why decoding mRNA sequence is key for their bio manufacturing. The HOPE method ideation process did not follow a methodology, but was an interdisciplinary approach to take advantage of the amazing selection process seen in potent human adaptive immune system to bind to its target. Upon its ideation, I became aware that it can also be applied to bio protein mass production.

The research proposal enhances the modern recombinant monoclonal antibody production by integrating AI, bioengineering and the bio utilization of the by product of potent adaptive immune system. Although not considered Biomimicry since the conceptualization was not achieved through the Biomimicry methodology and because it involves bio utilization of species, I like to consider the HOPE method as nature inspired.

Biomimicry combines “Ethos,” which is about reconnecting with and respecting nature, and “Emulation,” which means incorporating nature’s forms, processes, or systems into your designs at different levels—macro, micro, or nano. To evaluate these designs, we use nature’s patterns, known as life’s principles, which ensure balance and sustainability. This approach cultivates an appreciation for how nature has evolved, allowing every living being to exist in harmony with the Earth and its environment.

I am currently ideating version 2 of the HOPE method which might follow more Biomimicry life principles and utilize less bio utilization. The HOPE method version 2 would take some steps of the HOPE method to develop a molecular imprinting the FAB region of antibodies with nanoparticles. I am working on more details on how to do that, since it requires more data to train the AI that perhaps is still not available. This will substitute the monoclonal antibodies for nanoparticles that can target the same targets of the antibody to reduce costs in targeted diagnostics and therapeutics. I still believe the HOPE method was inspired by nature, since the ideation of the HOPE method was inspired on how the natural world already targets its immune response and takes advantage of the potent targeted antibodies already made from the selection process of adaptive immune system.

The HOPE method in comparison to traditional and modern methods of biomanufacturing

The main differences between the HOPE method with traditional and modern methods of monoclonal antibody (mAb) production are as follows:

1. Source of Antibodies:

   • Traditional Method: Antibodies are derived from hybridomas, which are created by fusing B cells from immunized mice with myeloma cells (HeLa cells).

   • Modern Method: Antibodies are produced using recombinant DNA technology, where the genes encoding the antibody are isolated and inserted into expression vectors for production in various cell types.

   • HOPE Method: Selection of the best antibody from population studies with pathogen of interest or personalized studies detecting antibodies specific against neoantigens of oncologic patients. Upon computational analysis and confirmation of efficient binding capacity of FAB region and immune response of FC region.

2. Production Process:

   • Traditional Method: Involves the use of hybridoma technology, which requires the fusion of B cells with myeloma cells (HeLa cells), followed by the selection of a single clone that produces the desired antibody. The antibodies are then produced in vivo by injecting mice with hybridoma cells or in vitro through cell culture.

   • Modern Method: Utilizes genetic engineering techniques to clone the genes for the antibody's heavy and light chains into expression vectors. These vectors are then introduced into suitable host cells, such as yeast, bacteria, insect cells, or mammalian cells, for large-scale production.

   • HOPE Method: Instead of incorporating the gene of the antibody into a plasmid, which would be much longer since it would include the introns and well as the exons, synthetic DNA coding for the IVT mRNA of the antibody would be incorporate onto the plasmid.

3. Cost and Time Efficiency:

   • Traditional Method: The process is expensive and time-consuming, requiring the maintenance of mice models, cell fusion, and screening for the desired antibody-producing clone.

   • Modern Method: Recombinant DNA technology offers a more cost-effective and time-efficient approach, as it eliminates the need for animal models and allows for direct production in cell culture systems.

   • HOPE Method: Cost-effectiveness is of yet to be determined, but once the process would reduce the time in trail and error of the efficacy of the mAb if proper computational predictions are made decoding the mRNA sequence of the selected best antibody in step 1 of the process.

4. Scalability and Consistency:

   • Traditional Method: Scaling up production can be challenging and may result in variability in antibody quality due to differences in animal health and culture conditions.

   • Modern Method: Offers better scalability and consistency in antibody production, as it relies on controlled cell culture conditions and can be automated to a greater extent.

   • HOPE Method: Scalability is similar to modern recombinant technology, but should be enhanced due to the smaller DNA sequences added to the plasmid facilitating the recombinant DNA technology production in bacteria or yeast.

5. Post-Translational Modifications:

   • Traditional Method: The in vivo production method may result in antibodies with post-translational modifications that differ from human antibodies, potentially leading to immunogenicity issues.

   • Modern Method: Recombinant production systems can be engineered to perform post-translational modifications more similar to human cells, reducing the risk of immunogenicity.

   • HOPE Method: Determination of post-translational modifications is to be determined as more data must be collected to determine its regulation of post-translational modifications. The mRNA encodes only exons that translates to the linear amino acid sequence of the protein, scientists must collect data from microRNA and other factors that are key in the regulation of post-translational modifications in antibodies production to determine if a common post-translational modification regulation exists within the translation of antibodies.

     
     

6. Therapeutic Applications:

   • Traditional Method: The antibodies produced are often murine or chimeric, which may limit their therapeutic use due to immunogenicity.

   • Modern Method: Allows for the production of fully human or humanized antibodies, which are more suitable for therapeutic applications as they are less likely to elicit an immune response in humans.

   • HOPE Method: The potential applications of the HOPE Method are vast and span multiple fields:

     • Infectious Diseases: The method's ability to identify and produce NAbs and bNAbs from recovered patients could lead to the development of highly effective treatments for viral infections like COVID-19. By targeting epitopes with a low mutation history, the HOPE Method could stay ahead of the antigenic drift of pathogens. Potent antibodies specific against extracellular microbial or parasitic pathogens that are not considered neutralizing antibodies since its due to a nonviral infection, can also be used in the process of developing diagnostics and therapeutics of nonviral infectious diseases.

     • Cancer Therapy: Personalized medicine takes a significant leap forward with the HOPE Method's capability to produce mAbs specific to tumor neoantigens. This could lead to more targeted and effective treatments for cancer patients via antibody drug conjugates or personalized diagnostic immunoassays, potentially preventing metastasis and altering the tumor microenvironment.

     • Rapid Diagnostics: The method can facilitate the creation of rapid diagnostic tests for various diseases, including arising pandemics. By identifying antibodies that bind most tightly to specific antigens, the HOPE Method can enhance the sensitivity and specificity of diagnostic assays.

     • Vaccine Development: By identifying the most effective NAbs or potent antibodies specific against nonviral pathogens, the HOPE Method can guide the development of mRNA vaccines. As these potent antibodies bind to epitopes, scientists can determine the best epitopes to focus the vaccine development towards. Incorporating the mRNA sequence of the epitopes recognized by these antibodies into vaccine designs could elicit a strong immune response, by incrementing the chances that the patient's immune system will generate a similar antibody response.

     • Material Science: The HOPE Method's computational models can also be applied to the production of bioproteins, offering a sustainable alternative to plastics and potentially impacting climate change innovations. The method can be used to decode the mRNA of a protein whose gene is not known and once that is known proceed in one of two ways.

       • Scientists can identify the gene within the species genome by looking for the same split exon sequences in the DNA (remember DNA contains introns and exons thus why the mRNA which only codes for the genes exons would appear to be split up within the DNA as well as DNA will have a missing hydroxyl group within their nucleic acids and DNA has Thymine while RNA has Uracil). Using AI algorithms identify where similar DNA sequences appear in the genome and test for the protein's gene within that region.

       • Scientists can also try a different approach in recombinant DNA technology, by not using the protein's gene within the plasmid and using synthetic DNA instead coding for IVT mRNA that would direct the bacterial or yeast ribosomes to produce the bio protein of interest. Which to my knowledge has not been attempted as of yet.0

   • Precision Medicine: The method's focus on personalized antibody production could pave the way for a new era in precision medicine, where treatments are tailored to the individual's unique immune response.

Conclusion

In summary, the HOPE method attempts to enhance modern methods of mAb production offering significant advantages over traditional hybridoma technology in terms of cost, time, scalability, consistency, and therapeutic applicability. Modern methods of mass producing recombinant bio proteins require prior knowledge of the gene of the bio protein of interest,

the HOPE method provides a way to facilitate identifying the gene of the bio protein within the species genome to mass produce with modern recombinant technology or a new innovative version of recombinant technology utilizing synthetic DNA sequencing encoding IVT mRNA of the bio protein of interest within the plasmid.

As the method matures and undergoes rigorous testing for safety and efficacy, it could become a cornerstone of global health initiatives, offering hope to patients and communities affected by a wide range of diseases. As well as a stepping stone in the process of mass producing bio proteins.

References

 Hernandez M, Bose D (2023) The HOPE Method: Reverse Engineering Antibodies of recovered Patients and Bioproteins. J Appl Microb Res. Vol: 6 Issu: 1 (09-20). https://www.innovationinfo.org/articles/JAMBR/JAMBR-164.pdf

Hernandez M, Bose D (2022) The HOPE method: reverse engineering antibodies of recovered patients and bioproteins. ARPHA Preprints. https://doi.org/10.3897/arphapreprints.e95037 

Hernandez M (2021) Preprint: HOPE Monoclonal Antibodies: Genetically engineered monoclonal antibodies via mass spectrometry or cryogenic electron microscopy followed by protein design analysis of antibodies of recovered patients. DOI:10.13140/RG.2.2.31167.02727

Hernandez M (2020) 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. https://www.worldpulse.org/story/hope-monoclonal-antibodies-as-potential-treatment-for-covid19-18496

Funding: Independent funding was provided by the MD Biomimicry pro bono project on The HOPE method for monoclonal antibodies and bio proteins.

Author contributions: Dr. Michellie Hernandez (MH) and Artificial Intelligence

Conceptualization: MH, ChatGPT, and PopAI

Methodology: MH

Investigation: MH

Visualization: MH

Funding acquisition: MH

Project administration: MH

Supervision: MH

Writing – original draft: MH, ChatGPT, and PopAI

Writing – review & editing: MH and Grammarly

Competing interests: The author denies any competing interests.