Generating Antibodies Against P. aeruginosa
Introduction: Antibiotic resistant bacterial infections pose a significant threat to human health. Pseudomonas aeruginosa, a gram-negative pathogen, was recently recognized as a serious threat by the Center for Disease Control (CDC), and ranked the second most critical drug resistant bacteria in need of new treatments by the World Health Organization (WHO). Despite the need for new treatments, new antibiotics approvals have decreased to fewer than one approval per year, creating an urgent need for new agents to treat pan resistant organisms. Monoclonal antibodies engineered to target bacterial cells, and conjugated with small molecule drugs (antibody-drug-conjugate) have unique pharmacodynamics and mechanisms of bactericidal action, making antibody-drug-conjugates ideal drug candidates for the treatment of infectious diseases. We hypothesize than an ADC targeting resistant P. aeruginosa will be an effective bactericidal agent by delivering a potent antibacterial drug selectively to P. aeruginosa. The purpose of this project is to identify antibodies with high binding affinity to P. aeruginosa that can then be subsequently developed into antibody-drug-conjugates to treat resistant P. aeruginosa.
Materials and Methods: We conducted a high-throughput screen of a single-domain (VHH) antibody library using antibody phage display following published protocols to identify antibodies with high affinity binding against the P. aeruginosa exopolysaccharide Psl. Recovered clones were subsequently sequenced. Recovered sequences were then cloned into expression vectors and antibodies were expressed in CHO cell culture. Binding of the resulting anti-Psl mAbs was verified by ELISA assay and in cell culture against P. aeruginosa.
Results and Discussion: Expected results- Using antibody phage display we identified three clones that demonstrated high affinity binding to our antigen of interest. Sequences for each clone were recovered and submitted to the public domain. Anticipated: Following cloning and transfection, expression of the subsequent antibody demonstrated selective binding to P. aeruginosa. This project demonstrates the ability to use an antibody phage library to screen for and select sequences that bind to a known antigen.
Conclusions: This project addresses the urgent need for the development of new last line therapies. Development of this class of drugs could reduce deaths associated with resistant infections, decrease adverse drug reactions caused by off target toxicities, reduce hospital burden and overall healthcare cost, and potentially offer highly effective prophylactic agents for patients at risk of hospital acquired resistant infections.Published in College of Pharmacy, Virtual Poster Session Spring 2020
Jeremy, nicely presented poster. Can you clarify for me whether the figures are hypothetical/anticipated results? How long will it take to reach the point of having the outcomes?
Thank you for the question Dr. Keefe. As of now, the figures are hypothetical. Repeat scale up of phage, binding, elution, and infection steps resulted in similar phage titers and increased bacterial colony counts. The third and final round of selection resulted in ~10^9 colonies indicating the selection of binders.
After three rounds of selection individual colonies were isolated we attempted producing soluble fragments in a 96 well format. I had some issues with the methods in this step (difficulties producing soluble fragments). So we decided to send a few clones for sequencing to see if the sequences aligned with what was expected. This step is currently pending. Ideally, we would have sequenced clones that bound to antigen on the ELISA, but since we were having difficulties with expression this seemed like a logical step. Further work will include analyzing the sequences, repeat ELISA, and possibly moving forward with cloning the known sequence into
an expression vector, and subsequent transient transfection into CHO cell culture to express an antibody. This antibody would then need to be further developed by the medicinal chemists in the laboratory to conjugate a small molecule drug to it. So, at this point this study we are still very “high” up on the biopharmaceutical pipeline, meaning that extensive in-vitro testing and successes would need to be obtained before moving to animal models, and ultimately to in-human testing to demonstrate this therapeutic as a successful option. As for a timeline, if things went well I would say maybe 6 months (depending on COVID lab closures of course).
This is so relevant, especially now. Thanks.
Interesting concept. Do you ever look at what epitope the Ab is recognizing? Another question, do you expect “drug resistance” to occur with these Abs, or is the epitope a large region?
Thank you for the question Dr. Lim. We are focusing on a specific antigen- Exopolysaccharide PSI, an outer cell surface membrane protein expressed by all strains of P. aeruginosa, which has been shown to be a promising target with demonstrated bactericidal action by human antibodies in other studies. See the references below. As for resistance, one hypothesis is conjugating multiple small molecules (drug efflux pump inhibitors) etc, to decrease the development of resistance.
10. Ali SO, Yu XQ, Robbie GJ, et al. Phase 1 study of MEDI3902, an investigational anti-Pseudomonas aeruginosa PcrV and Psl bispecific human monoclonal antibody, in healthy adults. Clin Microbiol Infect. 2018.
11. DiGiandomenico A, Warrener P, Hamilton M, et al. Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening. The Journal of experimental medicine. 2012;209(7):1273-1287.
12. Le HN, Quetz JS, Tran VG, et al. MEDI3902 Correlates of Protection against Severe Pseudomonas aeruginosa Pneumonia in a Rabbit Acute Pneumonia Model. Antimicrob Agents Chemother. 2018;62(5).
13. Periasamy S, Nair HA, Lee KW, et al. Pseudomonas aeruginosa PAO1 exopolysaccharides are important for mixed species biofilm community development and stress tolerance. Frontiers in microbiology. 2015;6:851.
Very interesting study. We used phage display quite a bit in the past. What are your next steps? We usually quantified affinity using SPR.
Thank you for the comment Dr. Barrows. The next steps will involve repeating the soluble fragment ELISA, sequencing, and then converting each of the anti-Psl VHH to heavy chain antibodies (VHH-Fc) by sequence and ligation independent cloning (SLIC) into pVITRO1 to replace the first constant heavy domain (CH1) and by removing the light chain sequences of trastuzumab (pVITRO1-trastuzumab-IgG1/, Addgene). The resulting expression vector, pVITRO1-antiPseudomonas, will be sequence verified and then transfected into ExpiCHO-S cells. The expressed antibodies will be purified by affinity chromatography using a Protein A resin using a FPLC. Binding of the resulting anti-Psl mAbs will be verified by ELISA assay and in cell culture against P. aeruginosa (alot of successful steps would need to occur for this to all happen, but this is the ideal scenario).
Jeremy this study has so many amazing implications for patients battling chronic wounds. I saw a few patients during clinical rotations that had MDR Pseudomonas in a chronic pressure wound and new therapies would greatly increase their healing! Hopefully this technology is the future 🙂
Thank you Taylor!
Hi Jeremy, interesting project. To bad that solubility got in the way. Is the intent to look at the complementarity-determining region (CDR) sequences and clone them into a VHH framework?
Thank you Dr. Herron, yes the intent is to look at the CDR sequences and clone them into a vector for expression in CHO culture.
Jeremy, great poster! It’s rough when experiments don’t go right the first time, but I’m sure it was a valuable experience for you. Very interesting topic and I hope it can continue forward!
Thank you Gabe!
The topic of passive immunity is an important one in the battle of these seriously resistant GNB, especially in patients with immune compromise. Your work represents an important building block for studies in mice, etc. and ultimately humans and looks to be truly exciting!! Productive work, Jeremy. Thank you for sharing this potentially important data!! Good Luck!!
Thank you Dr. Orlando!
Nice job! This is an interesting projects with lots of clinical implications. I look forward to seeing the next steops.
Thank you Dr. Tyler!
Jeremy, this brings awareness to the importance of antibody studies. It seems the next few years are going to be landmark times for antibody studies in bacterial and viral infectious disease. good job!
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