Cancer immunotherapy holds enormous potential in combating malignant tumors. Messenger ribonucleic acid (mRNA) has emerged as a widely studied tool in recent years, functioning within the cytoplasm and eliminating the risk of unintentional insertion or mutation that was associated with plasmid DNA (pDNA) in the past. mRNA possesses the ability to encode tumor antigens (TAs), immune cell receptor cytokines, and antibodies. However, due to the inherent instability of mRNA's structure, there is a need to develop effective delivery systems. Lipid nanoparticles (LNPs) have become a crucial medium for mRNA delivery in cancer immunotherapy, offering protection to mRNA and enhancing intracellular delivery efficiency.
LNPs for mRNA Delivery
LNPs can serve as drug delivery systems for encapsulating small molecules, nucleic acids, small interfering RNA (siRNA), and mRNA. They are composed of ionizable lipids, helper lipids, cholesterol, polyethylene glycol (PEG) lipids, and mRNA. The characteristic feature of ionizable lipids is their pH sensitivity. Due to their head groups' ability to change charge, they can transition from a positively charged state at low pH to a neutral state at physiological pH. This property allows them to form "mRNA ionizable cationic lipid" complexes that stabilize and protect mRNA in a pH-dependent manner. Another key component, PEG-lipids, prolongs circulation and enhances stability by preventing macrophage activation and phagocytosis. The choice of PEG-lipids depends on the molar mass of PEG and lipid length, which can impact targeted delivery and cellular uptake efficiency. Helper lipids and cholesterol play crucial roles in the formation of LNPs, controlling LNP fluidity or rigidity. Particularly, cholesterol can influence effective LNP delivery and distribution and, through specific modifications, can selectively target certain cell types.
Tumor Immunotherapy Strategies Based on mRNA-LNPs
LNP-based cancer immunotherapy uses the following four main strategies.
(1) Activating the immune response by encoding TAs.
(2) Expressing antigen receptors, such as CAR (Chimeric Antigen Receptor) or T cell receptor (TCR).
(3) Encoding adjuvants to stimulate the immune system.
(4) Encoding immune-related proteins, such as cytokines and antibodies.
Cancer Antigen Presentation
TA is a protein expressed in cancer cells, recognized as foreign by the immune system, and presented to T cells and B cells by antigen-presenting cells (APCs). This can induce a potent anti-cancer immune response. TA can exist in various forms, including full-length proteins, antigenic peptides, or pDNA encoding specific cancer antigens. Some studies have suggested that personalized neoantigens used in dendritic cell (DC) vaccines can have effective anti-tumor effects. In clinical trials, the feasibility of inducing an immune response in malignant melanoma using autologous tumor mRNA has been evaluated. DCs transfected with mRNA encoding TAs have become an effective cancer treatment strategy, showing long-term survival rates in clinical trials for brain cancer, prostate cancer, renal cell carcinoma, and melanoma. Notably, 50% of metastatic melanoma patients receiving DC vaccines alone or in combination with Interleukin-2 (IL-2) achieved long-term survival without severe adverse reactions. Given these anti-tumor effects, Oberli and colleagues optimized LNPs and demonstrated the intracellular delivery of mRNA-encoded TAs to APCs, promoting cytotoxic CD8+ T cell responses in a melanoma mouse model. In another study, in an OVA-bearing mouse model, LNP-OVA mRNA and C16-R848 effectively inhibited tumor growth. Sasaki and colleagues reported a method for optimizing LNPs using a microfluidic device to select the appropriate size and lipid composition. They delivered E.G7-OVA mRNA using A-11-LNP and compared it with two other LNP formulations, showing that A-11-LNP exhibited superior gene expression activity and maturation in DCs and demonstrated a significant anti-tumor therapeutic effect in the E.G7-OVA tumor model.
In another study, Chen and colleagues reported a lymph node-targeting mRNA vaccine based on LNPs, named 113-O12B, for cancer immunotherapy. In an in vivo model with B16F10-OVA, the targeted delivery of mRNA to the lymph nodes triggered a robust CD8+ T cell response to the encoded full-length OVA.
CAR Engineered Immune Cells
Engineered immune cell therapy has the potential to specifically target cancer cells for cancer treatment. CARs encoded by mRNA can be delivered to immune cells either in vitro or in vivo, where they are expressed on the cell surface to target cancer cells. Several clinical and preclinical studies have demonstrated the potential of mRNA-encoded CARs in cancer immunotherapy. Billingsley and colleagues have shown the feasibility of generating CAR-T cell therapies based on mRNA delivery platforms by developing ionizable lipids and optimizing LNP libraries with various combinations. LNPs encapsulating mRNA encoding CD19 CAR expressed CD19 CAR at levels similar to or higher than electroporation. CARs generated using this approach can also be applied to NK cell therapy, although viral vectors are currently more widely used, even though this method has greater potential.
One significant limiting factor of mRNA-based CARs is their relatively short duration of expression, which may restrict their therapeutic effectiveness. To overcome this challenge, researchers are exploring strategies to enhance mRNA stability and prolong CAR expression. These strategies include optimizing mRNA sequences and structures, optimizing LNP formulations to enhance intracellular delivery and release, with the aim of enabling sustained or repeated administration of mRNA-encoded CARs.
Adjuvants
Immunogenic adjuvants play a role in modulating antigen recognition, upregulating co-stimulatory molecules, and inducing cytokine signaling. Toll-like receptors (TLRs) recognize conserved structures present in various pathogens and trigger innate immune responses, especially the production of Type I interferons (IFNs). Because TLR agonists can bridge innate and adaptive immune responses, they hold great promise as adjuvants in cancer therapy. Stimulator of Interferon Genes (STING) is a protein crucial in the innate immune response, and its activation triggers signaling cascades involving transcription factors like interferon regulatory factor 3 (IRF-3) and CD8+ T cell immune signals. Due to its central role in immune responses, STING has become a promising avenue for developing cancer immunotherapy.
In one study, combining LNP-encapsulated mRNA vaccines with an adjuvant (STING-V155M mutant of Interferon Gene Stimulator) enhanced immune responses in preclinical models and clinical research. This adjuvant was initially discovered in a patient with STING-associated vasculopathy of infancy (SAVI) and was found to significantly increase CD8+ T cells, enhance the immunogenic response to vaccines, and activate Type I interferon pathway via nuclear factor kappa B (NF-κB) and interferon-stimulated response elements (ISRE). When used in combination with an mRNA vaccine targeting human papillomavirus (HPV) cancer proteins, STING-V155M reduced tumor growth and improved survival in vaccinated mice, indicating the potential of mRNA-encoded adjuvants in cancer immunotherapy.
Cytokines: Encoding Immune-Related Proteins
The IL-1 and IL-12 families collaborate to elicit anti-inflammatory and anti-tumor immune responses. The IL-1 family (comprising IL-1, IL-18, IL-33, IL-36, IL-37, and IL-38) participates in early immune responses following antigen invasion. IL-36, in particular, is associated with a favorable prognosis in cancer and stimulates APCs and T cells. The IL-12 family of cytokines serves as a bridge between innate and adaptive immunity, with IL-23 being a member that regulates immune responses and exhibits anti-tumor effects. Hewitt and colleagues designed an mRNA-LNP delivery system encoding OX40, IL-36, and IL-23 for mono- and combination therapy against tumors. Delivery of the three mRNAs (OX40, IL-36, and IL-23) via LNPs effectively activated DCs and T cells, significantly enhancing anti-cancer effects compared to individual mRNA treatments. These strategies triggered both innate and adaptive immune responses, re-attacking tumors, and effectively preventing tumor recurrence. In another study, the use of mRNA-LNPs encoding cytokines for anti-cancer therapy was demonstrated. These cytokines (IL-12, IL-27, and GM-CSF) acted synergistically, increasing T cell survival in the tumor microenvironment (TME) and promoting memory T cells through IFN-γ and IL-10. This combination had a significant tumor-suppressive effect in a melanoma model without toxicity. The combination of IL-12 and IL-27 attracted B cells, macrophages, CD4+/CD8+ T cells, and NK cells, demonstrating the potential of mRNA-LNP delivery systems for multiple cytokines to mobilize immune cells and provide effective therapy.
Antibodies: Encoding Immune-Related Proteins
Antibody therapy has shown remarkable efficacy in clinical settings, but it still has certain limitations. Stability issues, the complexity of large-scale manufacturing, and treatment costs can hinder its widespread application. One approach to address the limitations and challenges of conventional antibodies is to deliver mRNA encoding antibodies for in vivo antibody production. HER2 antibody (i.e., trastuzumab) is a prime example of targeted cancer treatment. When trastuzumab binds to HER2 on cancer cells, it exerts its anticancer effects by blocking cancer cell proliferation and survival pathways. Building on this mechanism, Rybakova and colleagues utilized LNP delivery to express trastuzumab-encoding mRNA. When injected into mice, the concentration of trastuzumab antibody expressed in the serum gradually increased until it disappeared after 7 days. This suggests that mRNA-LNP delivery encoding antibodies can serve as an alternative to traditional antibody therapy.
Sahin's team designed mRNA encoding RiboMABs targeting CD3 and Claudin6 (CLDN6), one of the tumor-associated antigens (TAAs). After injecting CD3×CLDN6-RiboMAB LNPs into mice, they monitored the concentration of these mRNA-derived antibodies in the serum over time. The mRNA gradually decreased within 144 hours, while the antibody proteins rapidly disappeared after 6 hours of dosing. This study demonstrates that low-dose mRNA-based on LNP can be repeatedly administered and replicated, allowing for continuous antibody production. This overcomes the limitations of the short half-life of antibody therapy and showcases its potential clinical applicability.
In another study by Thran and colleagues, they explored the use of rituximab (a CD20 antibody used for targeted lymphoma therapy) and investigated the utility of chemically modified mRNA in passive immunity. They designed LNP-encapsulated mRNA encoding rituximab and evaluated its anti-tumor effects. In an in vivo lymphoma model, the group treated with LNP mRNA therapy showed a higher tumor suppression rate and survival rate compared to the group treated with recombinant rituximab antibody therapy. Furthermore, a single injection of mRNA-LNP was sufficient to achieve rapid, stable, and sustained serum antibody titers, providing preventive and therapeutic protection against deadly rabies infections or botulinum toxin poisoning. These findings suggest that mRNA-LNP encoding antibodies offer better delivery and therapeutic efficacy compared to their recombinant protein counterparts.
Conclusion and Future Outlook
In summary, mRNA-based therapeutic strategies have garnered significant attention in recent years due to their simplicity of manufacture and the ability to encode proteins without genomic mutations. However, the instability of mRNA structures necessitates effective carriers or delivery systems to enhance intracellular uptake. With the emergence of nanoparticle-targeted delivery technologies, LNPs have become an innovative delivery platform capable of improving mRNA stability and intracellular delivery efficiency, making them highly promising candidates for cancer immunotherapy. mRNA encoding cancer antigens, CARs, adjuvants, cytokines, and antibodies has demonstrated potential in reducing tumor growth, further underscoring the potential of LNP-facilitated mRNA-based cancer immunotherapy. As research and development in this field continue to advance, we can anticipate the emergence of more effective, personalized, and safer treatment approaches. These advancements hold the promise of improving treatment outcomes for cancer patients, mitigating adverse effects of therapy, and enhancing overall quality of life. Ultimately, these iterative improvements will continue to elevate the efficacy of treatment and provide new hope and opportunities in cancer therapy based on mRNA.
References
1. Han, J.; et al. Lipid nanoparticle-based mRNA Delivery Systems For Cancer Immunotherapy. Nano Convergence. 2023, 10(36).
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Compared with viral vectors, non-viral delivery vectors are easier to scale up and have the ability to solve the most critical safety issues. Thanks to the LNP-mRNA vaccine that came to the fore during the COVID-19 pandemic, lipid nanoparticles (LNP) are undoubtedly the most advanced and widely studied non-viral vectors at present. Typically, LNP is composed of 4 components: ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Each component determines the stability, transfection efficiency, and safety of LNP. It plays a vital role. To design a drug carrier based on lipid nanoparticles, we first need to clarify the basic principles behind LNP design.
Physicochemical Characteristics to Consider When Designing LNP
Formulation: Molar Ratio, Weight Ratio and N:P
The lipid molar ratio determines the composition of the lipid particles and affects their size, polydispersity, and efficacy. However, an important feature that distinguishes LNPs from lipoplexes is that their structure cannot be predicted simply from the molar composition of the formulation. LNP needs to be characterized by cryo-electron microscopy and other characterization methods to characterize the LNP structure under different formulas.
Changes in the content of different components will not only affect the morphology of LNP, but also affect the encapsulation efficacy. Studies have found that when the ICL molar ratio increases, the cholesterol content decreases proportionally, which will lead to a decrease in the encapsulation efficiency of loaded nucleic acids. Cholesterol is important for the interaction between ICL and nucleic acids, so cholesterol deficiency results in reduced encapsulation efficiency. In addition, if the formulation contains a higher proportion of PEG lipids (>2.5 mol%), the encapsulation efficiency will be significantly reduced. At the same time, due to the commonly found human anti-PEG antibody response, lower PEG lipid ratios are often considered in formulation design, or are gradually replaced by analogues.
Fatty Acid Dissociation Constant (pKa)
The pKa of ionizable cationic lipids (ICL) determines the ionization behavior and surface charge of LNPs, and further affects the stability and toxicity of LNPs. Traditional permanently charged cationic lipids such as DOTAP (trimethyl-2, 3-dioleoyloxypropyl ammonium bromide), which were used in earlier studies for nucleic acid delivery, easily interact with negatively charged serum proteins in the body. The occurrence of aggregation will lead to rapid clearance of LNP by the mononuclear phagocyte system, seriously shortening the half-life of LNP in vivo. And this aggregation effect increases the risk of toxic side effects, leading to red blood cell membrane damage and hemolysis.
Ideally, at acidic pH (i.e., the endosomal environment), the head group of the ICL should become positively charged again to facilitate binding to negatively charged lipids exposed on the endosomal membrane. This interaction causes the lipid carrier to form an inverted hexagonal structure, further promoting the destruction of the proendosomal membrane and delivering therapeutic nucleic acids into the cytoplasm. For example, studies have found that LNP pKa values of 6.2-6.5 and 6.6-6.9 are beneficial for in vivo hepatic delivery of siRNA and intramuscular injection of mRNA vaccines, respectively.
Physicochemical Properties
The physicochemical properties of LNPs, including size, surface charge (zeta potential), and surface modifications, have a direct impact on the efficacy, pharmacokinetics, and biodistribution of LNPs. LNP size is critical to overall drug targeting and circulation longevity. Studies have shown that smaller particles are more likely to evade clearance by mononuclear phagocyte machinery and generally have a longer circulating half-life. In addition, particles smaller than 100 nm can easily penetrate target tissues through porous endothelial cells. In addition to different particle sizes caused by formula changes, different LNP preparation methods can also be used, such as extrusion to achieve smaller and more uniform particle sizes.
The Main Components and Principles of LNP
Ionizable Cationic Lipids (ICLs)
Ionizable cationic lipids are key components in LNP formulations and have an amphiphilic structure with a positively charged hydrophilic amine head linked to a long lipophilic tail. The structure of ICLs can be divided into amine headgroups (Headgroups), hydrophobic tail groups (Tails) and internal linking segments (Linkers).
Depending on the number of amine groups in the head group, cationic lipids can be divided into monoamine-based or polyamine-based lipids. The most famous DLin-MC3-DMA (MC3), SM-102 and ALC-0315 are all monoamine-based lipids and are the only three ionizable cationic lipids approved by the FDA for RNA delivery. However, these three ionizable cationic lipids are not biodegradable, and accumulation in the body will produce potential cytotoxicity. Researchers often focus on tuning the structure of lipid tails to enhance potency or confer specific functions by changing the number of tails, designing linear or branched structures, and introducing unsaturated or biodegradable bonds. For example, the unsaturated tail of MC3 promotes endosomal escape of siRNA, and the ester bond of L319 can accelerate the degradation of intracellular lipids.
Phospholipids
Phospholipids are accessory lipids that assist lipid nanoparticles in self-assembly and endosomal escape. In preclinical research and clinical applications, commonly used phospholipids are DSPC and DOPE. DOPE is a phosphoethanolamine with two unsaturated chains (C18). Due to its unsaturated nature, DOPE has clotogenic characteristics and therefore promotes the fusion of LNPs with endosomal membranes by promoting a non-bilayer inverted hexagonal structure. In contrast, DSPC is a saturated amphipathic lipid. It has a neutral overall charge and consists of a quaternary amine and a negatively charged phosphate group connecting two saturated chains (C18). Due to the saturation characteristics, DPSC has a cylindrical shape and therefore does not exhibit membrane instability properties but tends to form a stable bilayer structure. The two mRNA COVID-19 vaccines already on the market, Moderna and BioNTech/Pfizer, both use DPSC.
Cholesterol
Cholesterol helps increase the stability of LNPs and assists in cell membrane fusion. Optimizing the structure of cholesterol can also improve the delivery efficiency of LNPs and give LNPs specific functions. Some studies have screened β-sitosterol LNP (eLNP) among natural cholesterol analogs, which significantly improves transfection efficiency. By analyzing the SAR of this cholesterol analog, they found that eLNP has a polyhedral structure and is composed of different surface lipids. This may facilitate endosomal escape and mRNA release.
PEG Lipids
Pegylated (PEG) lipids are mainly used to reduce nanoparticle aggregation, reduce phagocytosis by mononuclear phagocytes, and prolong systemic circulation time. However, PEG lipids can also hinder target cell interaction and endosomal escape, thereby reducing transfection efficiency. By optimizing the PEGylation chemistry and the density of PEG on the LNP surface, the pharmacokinetics and pharmacodynamics of LNPs can be altered.
Research data shows that the shorter the PEG lipid carbon chain, the faster the desorption rate. The length of the dialkyl chain and PEGylated lipid concentration/molecular weight significantly affect the pharmacokinetics, pharmacodynamics, and biodistribution of LNPs in vivo. PEG that is too short (<1 kDa) cannot prevent the interaction of nanoparticles with serum proteins and therefore cannot effectively extend circulation time. However, very long PEG (>5 kDa) or high molar ratio of PEG (>15 mol%) will lead to reduced membrane permeability, thereby severely reducing cellular uptake. Therefore, PEG molecules with medium molecular weight (∼2 kDa) are usually used for LNP modification at a proportion of <5%.
Stability and Storage of LNPs
Stability studies are critical in helping to determine the shelf life and optimal storage conditions of the final product. The stability of LNP-nucleic acid formulations depends on processing parameters such as temperature, humidity, and light, as well as the properties of the nucleic acid and lipid excipients. It is known that commercially available mRNA LNP vaccines are frozen due to lack of long-term stability in solutions at 2-8°C. The limited shelf life under refrigerated conditions is mainly attributed to the chemical degradation of the mRNA and the reactive properties of nucleotide and lipid impurities in solution.
Methods to improve LNP-RNA stability include formulation strategies such as the addition of buffers, surfactants, and other excipients. In addition, the use of effective process controls and the use of appropriate cryoprotectants (sucrose, trehalose or mannitol) for freezing or lyophilization are often used. Although this process is known to affect LNP size and encapsulation, these properties are not the only determinants of LNP performance. Furthermore, the choice of excipients in solution (e.g., buffers and sugars) is crucial for this method, indicating that the solution medium has a strong influence on the formation and colloidal stability of LNPs upon reconstitution. The colloidal stability of LNPs will be affected by their lipid composition, suggesting that efficient lipid mixing and interaction with RNA cargo is necessary to obtain a more stable product.
References
Cárdenas, M. et al. Review of structural design guiding the development of lipid nanoparticles for nucleic acid delivery. COCIS. 2023, 66: 101705.
Mendonça, M.C.P. et al. Design of lipid-based nanoparticles for delivery of therapeutic nucleic acids. Drug Discov Today. 2023, 28(3): 103505.
Lu, J. et al. Regulatory perspective for quality evaluation of lipid nanoparticle-based mRNA vaccines in China. Biologicals. 2023, 84: 101700.
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Why RNA production by fermentation
RNA is a vital biomolecule with numerous biological functions, and its importance has led to a growing demand for RNA-based therapeutics and diagnostic tools. However, producing RNA at a large scale has been a challenge, and traditional methods such as chemical synthesis are time-consuming and expensive. In recent years, advances in biotechnology have led to the development of new methods for large-scale RNA production, including fermentation-based approaches.
What is RNA production by fermentation
Large-scale RNA Production Through Fermentation
Fermentation is a process that involves the use of microorganisms to convert substrates into a desired products. Fermentation has been widely used for the production of antibiotics, proteins, enzymes, peptides and organic acids. More recently, fermentation has been explored as a method for producing RNA on a large scale. The basic principle of RNA fermentation is to use microorganisms to produce RNA through transcription, the process of copying DNA into RNA. The microbial cells are engineered to produce high levels of RNA, followed by RNA harvesting and purification.
Approaches to RNA fermentation
The different approaches to RNA fermentation are depending on the type of RNA being produced and the specific application.
Using genetically engineered bacterial strains
This approach uses bacterial strains that have been genetically engineered to overproduce RNA. These bacteria can be grown in large fermenters and are capable of producing large amounts of RNA in a relatively short period of time. Several strains can be optimized for specific types of RNA, such as messenger RNA (mRNA) or small interfering RNA (siRNA) with different structural and functional properties.
Using cell-free systems
This approach is derived from bacterial cells but do not contain the intact cells themselves. Cell-free systems are capable of producing high levels of RNA in a short period of time, making them useful for rapid prototyping and testing of RNA-based therapeutics and diagnostics. These techniques can also be used to create RNA that has undergone modifications, such chemically modified RNA or RNA with certain sequences or structures.
Using genetically engineered yeast
In this method, yeast is used as a host to produce RNA. Large amounts of RNA can be produced by yeast, as has been demonstrated. Yeast cells can synthesize more complicated RNA molecules because they are eukaryotic, like human cells. Moreover, a promising technique involves employing RNA viruses that infect yeast cells to create RNA. However as yeast-based systems are still in their infancy, further study is required to optimize the production process and raise the output and caliber of RNA produced.
Advantages of fermentation-based approaches for RNA production
Scalability of RNA production through fermentation
Fermentation can be easily scaled up to produce large quantities of RNA, making it an attractive option for commercial production. In addition, fermentation can be carried out in a controlled environment, allowing for precise control of the fermentation conditions and the quality of the RNA produced.
Sustainability of RNA production through fermentation
Fermentation is a more sustainable and environmentally friendly method compared to traditional methods, as it uses renewable resources such as carbohydrates as the substrate for RNA production. It also reduces the amount of waste generated during the production process, as the microbial cells can be recycled or used for other purposes.
Cost-effectiveness of RNA production through fermentation
Fermentation is more cost-effective than chemical synthesis, particularly for large-scale production. Chemical synthesis requires expensive reagents and equipment, and the process is time-consuming and requires a high degree of expertise. In contrast, fermentation-based approaches are simpler and more efficient, and easier to scale up.
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In recent years, PROteolysis TArgeting Chimeras (PROTAC), which utilizes the cell's own degradation mechanisms to eliminate specific disease-related proteins, has emerged as one of the most promising methods. Apart from PROTAC, various targeted protein degradation (TPD) strategies are emerging, involving molecular glue, Autophagy-Targeting Chimera (AUTEC), Autophagosome Tethering Compound (ATTEC), and Autophagy Targeting Chimera (AUTOTAC). These compounds are generally bifunctional small molecules, consisting of two parts: one part contains an E3 recruitment ligand to activate the ubiquitination cascade, and the other part contains a head that targets the protein of interest (POI), assembled using an appropriate linker. Once inside the body, a POI-PROTAC-E3 ternary complex is formed, inducing POI ubiquitination and subsequent degradation through the ubiquitin-proteasome system (UPS). In many cases, their relatively large molecular weight (usually > 1000 Da) may affect oral bioavailability, solubility, and/or in vivo pharmacokinetic properties (not conforming to the Lipinski's Rule of Five). Additionally, certain intracellular proteins may be resistant to such molecules because they might not be substrates for proteasomal clearance. To address these issues and complement PROTAC, antibodies, with their high selectivity and affinity for any immunogenic target (in principle) and their successful commercialization, have been extensively applied to lysosome-based degradation strategies. Depending on their composition, this class of molecules includes antibody-based PROTAC (AbTAC/PROTAB), Lysosome-Targeting Chimeras (LYTAC), GlueBody Targeting Chimera (GlueTAC), Cytokine Receptor-Targeting Chimeras (KineTACs), and Signal-Mediated Lysosome-Targeting Chimeras (SignalTACs). These technologies not only significantly expand the scope of TPD but also offer new insights into antibody drug discovery.
Signal-Mediated Lysosome-Targeting Chimera (SignalTAC)
Membrane protein degradation technology relies on specific cell surface lysosome-targeting receptors to exert its effects. However, the expression of these internalizing receptors varies depending on tissue and cell type, which poses significant limitations to their further application. The internalization of lysosome-targeting receptors is a complex process mediated by signals within the receptor's cytoplasmic domain. Most signals are based on double leucine or tyrosine-based peptide sequences, which are recognized by components of the clathrin coat to ensure the precise transport of proteins to lysosomes through clathrin-mediated endocytosis. Among these, the tyrosine-based lysosome sorting signal NPXY (Asn-Pro-X-Tyr, where X represents any amino acid) in the low-density lipoprotein receptor (LDLR) can induce the internalization and lysosomal transport of anti-EGFR nanobodies. Therefore, based on this research, Professor Xiaoqing Cai's research group at Sun Yat-sen University proposed that membrane protein complexes with lysosome sorting signals could form SignalTACs for protein degradation. After binding to membrane proteins, SignalTACs dissociate from the membrane protein in the acidic endosomal compartment and enter lysosomes for degradation.
The Cation-Independent Mannose-6-Phosphate Receptor (CI-M6PR) is a single transmembrane glycoprotein that mediates the internalization of extracellular ligands and the intracellular sorting of newly synthesized lysosomal enzymes. Its efficacy does not depend on any lysosome shuttle receptor. Therefore, SignalTAC technology was developed using CI-M6PR's inherent lysosome sorting signal. The internalization and sorting of CI-M6PR are guided by a signal based on double leucine (composed of a cluster of acidic amino acids and subsequent double leucine). Based on this information, a series of IgG/Nb-based SignalTACs was constructed, where the CI-M6PR sorting signal was fused to the C-terminus of IgG antibodies using heavy chains, light chains, or both. Among these, Nb1 showed significantly enhanced cell fluorescence compared to the parental 5F7. Additionally, SignalTACs containing the RRRRK sequence, based on Trastuzumab (Tz) modification (Ab6), exhibited the highest internalization capability. Structure-activity relationship studies indicated that the double leucine-based sequence DEDLL (Asp7-Ile11) is critical for its function. Moreover, truncation of 13 residues at P1 of the sequence was found to synergistically enhance Nb1 internalization.
The use of IgG-type SignalTACs to treat HER2+ cell lines (SKBR3, BT474, and SKOV3) showed that SignalTACs Ab1-Ab6 induced degradation in all three cell lines. Further investigation of the degradation effects of Ab6 revealed that the maximum protein degradation occurred after treating SKBR3 cells with a concentration of 100 nM for 48 hours. Mechanistic studies indicated that clathrin-mediated endocytosis is a key pathway for SignalTAC-induced degradation. However, further research is needed to fully understand the molecular mechanism of SignalTAC-induced degradation. In addition to causing degradation of the target protein HER2, SignalTAC also downregulates downstream signaling (PI3K/AKT/mTOR) and promotes apoptosis and inhibits the proliferation of HER2-driven cancer cells.
Cytokine Receptor-Targeting Chimeras (KineTACs)
Due to limitations in lysosome delivery targeting chimeras, such as low modularity, high development difficulty, poor practicality, and limited tissue specificity, Professor James A. Wells' team at the University of California, San Francisco (UCSF) developed KineTACs. KineTACs are fully genetically encoded bispecific antibodies composed of a cell factor arm that binds to its homologous cytokine receptor and a binding arm that targets the desired protein. The construction of bispecific antibodies in the KineTAC class is not complicated by light-heavy chain mismatches, a common problem in traditional bispecific antibodies. Furthermore, the KineTAC platform only requires the design of the antigen-binding arm for the target protein, as natural cytokines can recruit the relevant degradation receptors.
To validate the KineTAC platform design concept, the authors developed KineTAC bispecific antibodies targeting different target proteins (PD-L1, HER2, PD-1, EGFR, CDCP1, and TROP2). The N-terminus of the human CXCL12 chemokine was fused to the Fc domain of the bispecific antibody to form the cytokine-binding arm, and the antigen-binding arm contained antigen-binding fragment (Fab) antibody sequences fused to the Fc domain, consistent with a regular antibody. The six KineTACs demonstrated maximum degradation efficiencies (Dmax) of approximately 70%, 51%, 84%, 82%, 93%, and 51%, respectively, demonstrating that the KineTAC platform can degrade a variety of cell surface proteins, offering good technical versatility.
Pretreatment with Bafilomycin (a lysosome inhibitor) reduced PD-L1 degradation, while MG132 (a proteasome inhibitor) treatment did not affect PD-L1 degradation. This suggests that KineTAC mediates degradation by delivering the target protein to lysosomes. Based on the previous research, CXCR7 is the primary receptor responsible for KineTAC-mediated degradation, and alternative cytokines (such as CXCL11 and vMIPII) were also shown to degrade target proteins. Compared to traditional antibody therapies, KineTAC-mediated degradation offers functional advantages as traditional antibodies only bind and inhibit without causing degradation. Pharmacokinetic experiments in vivo showed that KineTAC remained in the plasma for up to 10 days after injection, with a half-life of 8.7 days, similar to the reported half-life of mouse IgG.
While KineTAC demonstrated excellent results in in vitro experiments, its in vivo experiments have not been extensively explored. Only pharmacokinetic studies have been presented, leaving room for further animal experiments to validate whether the in vitro effective dose might lead to potential "cytokine storm" side effects in vivo due to the intensity of cytokine action in vivo.
Proteolysis-Targeting Antibodies (PROTABs)
In 2021, Professor James A. Wells and his team developed AbTACs, and published their findings in JACS. They utilized fully recombinant bispecific antibody AC-1 to recruit the membrane-binding E3 ligase RNF43 for the degradation of the cell surface protein PD-L1, achieving a Dmax of 63%. Although AbTAC represents a novel type in the PROTAC field, using fully recombinant biomolecules for targeting the degradation of cell surface proteins, its industry impact is far less than that of Genentech's PROTAB.
The Genentech-developed PROTAB platform technology constructs bispecific antibodies remarkably similar to those developed by Professor James A. Wells' team. One end targets the E3 ligase N-terminal glycoprotein D site, while the other end binds to the target protein, facilitating degradation. The Genentech team delved into its mechanism of action and found that PROTAB can participate in two degradation pathways: proteasomal and lysosomal. MG132 and Baf can both influence the degradation of the target protein, unlike several other TPD technologies discussed earlier. It also expanded the application scope of the PROTAB technology platform, demonstrating the replicability of PROTAB for different target proteins. Moreover, they optimized the assembly forms of PROTAB to investigate variations in protein degradation efficiency. Although PROTAB exhibits a Hook effect similar to PROTAC, it is believed that combining modular antibody engineering technology can expedite its translation, making more significant contributions to drug development.
Lysosome Targeting Chimeras (LYTACs)
LYTACs use IgG-polysaccharide bioconjugates to effectively clear target proteins. This involves the chemical synthesis and in vitro conjugation of polysaccharides to enable efficient target protein clearance. The mechanism of action is illustrated in Fig. 6, where the target protein ligand portion binds to the extracellular domain of the target protein. Simultaneously, polysaccharides bind to lysosome-targeting receptors (LTRs) on the cell surface, forming a ternary complex. This complex enters cells via clathrin-mediated endocytosis and is transported to endolysosomes. As the endolysosomes become acidified, the ternary complex continues to be transported to lysosomes for degradation. Similar to SignalTAC, the authors targeted CI-M6PR as the receptor and conjugated M6Pn polysaccharide peptides to antibodies that specifically recognize CI-M6PR to create LYTACs with specificity for the degradation of membrane proteins like EGFR and CD71. Constructing LYTACs using this method is intricate, requiring in vitro conjugation reactions. The introduced polysaccharide peptides may have certain immunogenicity, leading to accelerated clearance and reduced degradation efficiency.
GlueBody Targeting Chimeras (GlueTACs)
GlueTAC technology was developed by Professor Peng Chen's team at Peking University and is based on covalent Nb-PROTAC technology. The covalently modified single-domain antibody, GlueBody (modified with proximity-enabled non-natural amino acids PrUAAs), can form a covalent bond with membrane protein antigens like PD-L1 through a proximity-enabled reaction, reducing the dissociation and escape of the target protein during endocytosis and degradation. Additionally, cell-penetrating peptides (CPPs) and lysosome-sorting sequences (LSS) covalently linked to the single-domain antibody facilitate the internalization and lysosomal transport of complexes without the need for specific cell surface proteins, ultimately achieving the targeted degradation of membrane proteins in lysosomes.
The GlueTACs constructed using this method were assessed for their degradation effect on target proteins using a non-small-cell lung cancer cell line, H460. Western blot analysis demonstrated a significantly higher rate of PD-L1 degradation induced by GlueTAC (remaining 11%) compared to NbTAC (remaining 68%), highlighting the crucial role of covalent bonds in target protein degradation. Atezolizumab, Nb-PD-L1, and GlueBody showed lower degradation rates, indicating that individual antibodies or Nb might not be sufficient for target protein degradation, and PD-L1 might be reinternalized and recirculated to the cell membrane after endocytosis.
Studies into the degradation mechanism of GlueTACs revealed that treatment with Chloroquine/Bafilomycin significantly reduced PD-L1 degradation, whereas MG132 treatment did not, suggesting that the degradation process depends on the endolysosome pathway. In vivo experiments showed that both GlueBody and GlueTAC treatment significantly inhibited tumor growth, with tumors treated with GlueTAC having a lower average weight compared to those treated with GlueBody. Based on in vivo results, GlueBody performed slightly better than drug treatment, and the difference between GlueBody and GlueTAC groups was minimal. This might be related to the efficiency of the proximity-enabled covalent binding used in GlueBody. Changing the conjugation method might help enhance the anti-tumor activity of GlueTAC. Replacing GlueBody with antibodies might also improve its anticancer activity, as Fc and FcR interactions contribute to its efficacy. However, this approach might result in some non-specific conjugation, potentially causing the degradation of non-target proteins and side effects. Additionally, the introduction of PrUAA might induce some immunogenicity, leading to faster metabolism in in vivo experiments, masking its therapeutic effect. Overall, GlueTAC technology opens up an important new direction for antibody drug development.
Challenges in the Development of TPD
The development of PROTAC-like technologies still faces numerous challenges. Firstly, there are issues related to their drug-like properties, often including poor cell permeability and low oral bioavailability. Secondly, while the human genome encodes over 600 E3 ubiquitin ligases, only a few (such as VHL, CRBN, IAP, and MDM2) are used for degrading target proteins, and the scope of applications needs further expansion. Additionally, it's worth noting the toxicity concern. PROTACs might potentially exhibit greater toxicity than small molecule inhibitors because they lead to the degradation of the entire target protein rather than merely inhibiting it. Controlling the degree of degradation is an urgent issue in the TPD field.
The development of TPD technologies based on lysosomes has significantly broadened the range of target proteins compared to PROTACs and molecular glues, leading to a surge in research interest in this field. However, it is still in its early stages. Lysosomes, as essential organelles, regulate many important physiological functions aside from protein degradation. It remains unclear whether the "hijacking" of lysosomal degradation pathways may affect the organism's physiological functions. Additionally, the degradation of target proteins may lead to negative feedback regulation in cascading signaling pathways or overactivation of bypass signals, potentially causing more severe side effects, an area where research has yet to be conducted.
Furthermore, it is uncertain whether the excessive activation of specific receptor internalization functions may impair their original biological functions and lead to acquired drug resistance. Despite these challenges, the development of TPD offers a powerful tool for biomedical research and holds great promise for the future of drug development.
References:
Han, Y., et al., Protein labeling approach to improve lysosomal targeting and efficacy of antibody–drug conjugates, Org. Biomol. Chem., 2020, 18, 3229-3233.
Yu, J., et al., Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins, J. Am. Chem. Soc., 2023, 145, 34, 19107–19119.
Pance, K., et al., Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins, Nature Biotechnology, 2023. 41, 273-281.
Cotton, A.D., et al., Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1, J. Am. Chem. Soc., 2021, 143, 593-598.
Banik, S.M., et al., Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature, 2020. 584(7820): 291-297.
Zhang, H., et al., Covalently Engineered Nanobody Chimeras for Targeted Membrane Protein Degradation, J. Am. Chem. Soc., 2021. 143(40),16377-16382.
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Antibody drug conjugate (ADC) research involves more and more targets, such as HER-2, Trop-2, Claudin-18.2, B7-H3 and B7-H4, etc. Trop-2 is a transmembrane glycoprotein. Its high expression is associated with the occurrence of many tumors and poor prognosis. It is a very popular ADC research target, second only to HER2.
Currently, only Gilead's Trop-2 ADC drug Trodelvy has been approved for marketing in the world. It is used to treat adult patients with metastatic triple-negative breast cancer (mTNBC) and locally advanced or metastatic urothelial cancer (UC) who have received at least 2 therapies in the past. Trodelvy's sales have increased steadily since its launch, with sales reaching US$680 million in 2022 and US$482 million in the first half of this year. However, Trodelvy's growth rate did not meet analysts' expectations, mainly due to efficacy and safety issues. Trodelvy's overall efficacy is average, and its safety is a bit worrying. It also has a strong competitor, Enhertu, in the approved breast cancer field. For example, in the ASCENT clinical trial, 9% of patients experienced rash and 5% of patients experienced eye toxicity.
Despite this, enthusiasm for the development of Trop-2 ADCs is high. In addition to Trodelvy, the fastest-growing drugs currently include Daiichi Sankyo/AstraZeneca’s Dato-DXd and Kelun-Biotech’s SKB264 (MK-2870), both of which are in clinical phase 3. Different from the approved indications of Trodelvy, Dato-DXd plans to use advanced non-small cell lung cancer (NSCLC) as the first indication. In addition to single-target Trop-2 ADCs, some companies are also deploying dual-target ADCs. For example, Biocytogen’s Trop-2/PTK7 dual-antibody ADC BCG033, Trop-2/EGFR dual-antibody ADC DM001, and Trop-2/HER2 dual-antibody ADC YH012, etc. However, these are currently in preclinical research, and it will take time to verify how far they will go in the future.
Trop-2-mediated Cellular Pathways and Their Relationship with Tumors
Trophoblast cell surface antigen-2 (Trop-2) is a surface glycoprotein member of the epithelial cell adhesion molecule (EpCAM) family. It is a transmembrane glycoprotein expressed in normal tissues and many epithelial tumors, including breast, cervical, colorectal, esophageal, gastric, and lung cancers. In NSCLC, high Trop-2 expression was observed in 64% of adenocarcinomas and 75% of squamous cell carcinomas (SqCCs). In addition to its function of regulating normal fetal development, Trop-2 is also an intracellular calcium signal transducer, a key component of cell adhesion in human tissues, and plays an important role in stabilizing epithelial tight junctions. Trop-2 mediates several intracellular signaling pathways including PTEN/PIK3CA/Akt, MAPK/ERK, JAK/STAT, ErbB, TGFβ, and WNT/β-catenin (Fig. 1). In addition, Trop-2 is highly expressed in tumors and is associated with poor tumor prognosis. These characteristics all indicate that Trop-2 will become a good target for tumor treatment.
Trop-2 ADC Approved for Marketing - Trodelvy
Trodelvy (sacituzumab govitecan) is a novel Trop-2-targeting ADC originally developed by Immunomedics. In April 2020, it received accelerated approval from the FDA for the treatment of adult patients with metastatic triple-negative breast cancer (mTNBC) who have received at least 2 prior therapies. In September of the same year, Gilead acquired Immunomedics for US$21 billion and also acquired Trodelvy. In April 2021, the FDA granted routine approval for the mTNBC indication. In the same year, Trodelvy received accelerated approval from the FDA for a second indication in patients with locally advanced or metastatic urothelial cancer after treatment with platinum-containing chemotherapy and a PD-1 or PD-L1 inhibitor.
Trodelvy consists of a humanized monoclonal IgG (designated hRS7) that binds Trop-2. IgG is lightly reduced to expose eight sulfhydryl binding sites, which are subsequently coupled to the cytotoxic topoisomerase I inhibitor SN-38 via a CL2A linker. As shown in Fig. 2, the CL2A linker has a short PEG (polyethylene glycol) residue to aid solubility and is coupled to SN-38 at position 20 of the lactone ring, thereby stabilizing the ring from opening to less active carboxylate form. The bond between CL2A and SN-38 is pH-sensitive and is more readily released in low-pH environments (e.g., found in lysosomes and even in the microenvironment of tumors). Binding of SN-38 to IgG protects position 10 of SN-38 from glucuronidation. SN-38, while bound to the antibody, remains in its most potent form before being released. Trodelvy is a very important product of Gilead. It is currently undergoing multiple clinical trials and is used alone or in combination to treat metastatic triple-negative breast cancer (first-line or adjuvant treatment), metastatic breast cancer, non-small cell lung cancer, metastatic Urothelial carcinoma, metastatic castration-resistant prostate cancer, and other solid tumors.
Other Trop-2 ADC Drugs in Development
Datopotamab Deruxtecan
Datopotamab Deruxtecan (Dato-DXd, DS-1062) is a humanized Trop-2 ADC jointly developed by Daiichi Sankyo and AstraZeneca. It is linked to a topoisomerase 1 inhibitor payload (an exatacan derivative) via a tetrapeptide-based cleavable linker. The linker releases DXd upon proteolysis by lysosomal proteases such as cathepsin. Compared to Trodelvy’s 8 DAR, Dato-DXd’s DAR is 4, which is expected to improve security (Fig. 3). In preclinical studies, Dato-DXd significantly reduced the growth of cell lines with high Trop-2 levels but was not effective in inhibiting the growth of cells with low Trop-2 levels. Different from Trodelvy’s approved indications, AstraZeneca/Daiichi Sankyo plan to use advanced non-small cell lung cancer as the first indication for Dato-DXd, turning Dato-DXd into a potential alternative to chemotherapy for the treatment of NSCLC.
SKB-264
SKB264 (MK-2870) is the fastest-growing ADC targeting Trop-2 developed by Kelun-Biotech. Like Trodelvy and Dato-DXd, SKB264’s cytotoxin also uses a topoisomerase I inhibitor. However, the cytotoxin of Trodelvy is SN-38, the cytotoxin of Dato-DXd is an exatacan derivative, and the cytotoxin of SKB264 is Belotecan (KL610023), and the antibodies all use recombinant anti-Trop-2 humanized monoclonal antibodies. The linker of SKB264 is a sulfonylpyrimidine-CL2A-carbonate linker.
DB-1305
DB-1305 is a Trop2 ADC developed by Duality Biologics based on its proprietary Duality Immunotoxin Antibody Conjugate (DITAC) platform. It has demonstrated strong anti-tumor activity in preclinical tumor models, entered clinical phase I/II research (NCT05438329) in June 2022, and demonstrated strong clinical efficacy in NSCLC and other solid tumors. DB-1305 is a targeted ADC formed by coupling a Trop-2 antibody to the novel topoisomerase I inhibitor P1021 via an enzymatically cleaved tetrapeptide linker.
Trop-2 protein is highly expressed in a variety of tumors, such as pancreatic cancer, breast cancer, colon cancer, ovarian cancer, and non-small cell lung cancer. Its high expression is also closely related to shortened survival and poor prognosis of tumor patients, and it is a popular target in the ADC field after HER-2. Currently, there is only one Trop-2 ADC in the world, Trodelvy, approved for marketing to treat TNBC and advanced UC. Sales have risen steadily since its launch, reaching $482 million in the first half of this year. However, as the only Trop-2 ADC on the market, its sales growth rate did not meet expectations, mainly due to efficacy and safety issues. Despite this, many pharmaceutical companies have joined in the layout of Trop-2 ADCs.
References
Claudia, P. et al. TROP-2 directed antibody-drug conjugates (ADCs): The revolution of smart drug delivery in advanced non-small cell lung cancer (NSCLC). Cancer Treatment Reviews. 2023, 118: 102572.
David, M. et al. Antibody-drug conjugates targeting TROP-2 and incorporating SN-38: A case study of anti-TROP-2 sacituzumab govitecan. MAbs. 2019, 11(6): 987-995.
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In recent years, nucleic acid therapy involving various types of ribonucleic acids (RNAs), including messenger RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA), has rapidly emerged as a new cornerstone of modern medicine. Among them, the mRNA-based vaccines developed, particularly the COVID-19 vaccine, have played a significant role in combating the novel coronavirus (SARS-CoV-2) responsible for severe acute respiratory syndrome. The remarkable speed of sequence design and the ease of large-scale production associated with mRNA-based technologies have provided novel possibilities in addressing urgent and ever-evolving medical crises.
In the present context of successful mRNA drugs, the further synthesis of linear mRNA into circular RNA (circRNA) through end-to-end connections to enhance mRNA stability and prolong protein translation time has emerged as a new hot topic in the field of mRNA therapy.
In simple terms, circular RNA is a closed single-stranded RNA molecule formed through covalent linkage at its ends. It can maintain stability without the common 5' cap and 3'-poly(A) tail found in mRNA, owing to its covalently closed structure that impedes degradation by nucleases. Naturally occurring circular RNAs in organisms mostly arise from precursor mRNAs (pre-mRNA) through a process known as back-splicing. These circular RNAs are distributed in the cytoplasm and exhibit half-lives ranging from 18.8-23.7 hours, significantly longer than their linear homologs which have half-lives of only 4.0-7.4 hours.
Although the majority of natural circular RNAs are non-coding RNAs, meaning they cannot be translated into proteins, research has shown that artificially synthesized circular RNAs with internal ribosome entry site (IRES) sequences can be translated both in vivo and in vitro. The investigation of the translation mechanisms of circular RNAs in both cellular and extracellular contexts, along with the advancement of techniques for synthesizing circular RNAs in these environments, has greatly facilitated the progress of the circular RNA therapy field.
Circular RNA Synthesis Method In Vitro
Chemical Synthesis of Circular RNA
Currently, the chemical synthesis of circular RNA primarily relies on phosphoramidite chemistry and solid-phase synthesis, using naturally occurring nucleoside triphosphate derivatives as building blocks. These derivatives replace the reactive amino and hydroxyl groups in natural nucleoside triphosphates with non-reactive protecting groups. This allows for the formation of 3’-5’ phosphodiester bonds and subsequent deprotection, while minimizing the formation of 2’-5’ phosphodiester bonds and other side reactions. Following synthetic processes and purification steps, highly pure short nucleic acid sequences can be obtained. However, the current chemical synthesis technology for circular RNA can only generate circular RNAs with lengths of fewer than 70-80 nucleotides (nt).
Enzymatic Ligation Synthesis of Circular RNA
Enzymatic ligation for circular RNA synthesis is typically achieved through in vitro transcription (IVT) reactions. This reaction requires a DNA template, reaction buffer, and bacteriophage RNA polymerase. Bacteriophage RNA polymerases are commonly derived from T7, SP6, or T3 phages, with T7 RNA polymerase being the more prevalent choice. IVT reactions enable the cost-effective production of longer circular RNA molecules, making enzymatic synthesis the current mainstream method for circular RNA synthesis. Enzymatic ligation can be further categorized into T4 ligase-mediated ligation and ribozyme-mediated ligation.
T4 Ligase for Circular RNA Synthesis
Various T4 ligases derived from bacteriophages can catalyze RNA ligation reactions, including T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2). However, this ligation process requires the acceptor substrate on the linear RNA precursor to possess a 3'-OH group, and the donor substrate to have a 5'-monophosphate, in order to proceed.
T4 Dnl and T4 Rnl 1 are suitable for circularizing RNA without complex secondary structures, while T4 Rnl 2 is more appropriate for linear RNA precursors with double-stranded adapter regions. Therefore, in practical applications, it is necessary to choose different T4 ligases based on the secondary structure of the linear RNA precursor. It should be noted that all of these ligation methods cannot achieve circularization of large RNA fragments and cannot completely avoid side reactions associated with intermolecular end ligation.
Nuclease for Circular RNA Synthesis
1) Group I intron self-splicing system
In 1982, scientists discovered the first type I intron within the rRNA transcript of the ciliate Tetrahymena thermophila. This intron also marked the first identification of a ribozyme, an enzyme capable of self-splicing primary transcript RNA without the assistance of any proteins. Since then, numerous self-splicing type I introns have been found in tRNA, mRNA, and rRNA sequences across various organisms and viral genomes. Despite arising from completely different primary transcripts, these type I introns exhibit strikingly similar catalytic core structures. Based on their secondary structure, type I introns can be divided into ten domains, designated as P1 through P10. The catalytic core is located at the junction of the P4-P6 (P4, P5, P6) and P3-P9 (P3, P7, P8, P9) domains. P1 and P10 represent the 5' and 3' splice sites, respectively, while the surrounding sequences around the conserved catalytic core are responsible for maintaining the stability and correct folding of the ribozyme.
The most common method for in vitro circular RNA synthesis currently is the system engineered by harnessing the self-splicing characteristics of type I introns. This approach, also known as the PIE method (permuted introns and exons), relies on the inherent ability of type I introns to self-splice. It requires only GTP and Mg2+ ions to achieve RNA ligation. The in vitro transcription templates commonly employed for PIE synthesis usually incorporate type I intron sequences derived from the Anabaena tRNALeu gene or the thymidylate synthase (td) gene of T4 bacteriophage. Both of these type I introns have been verified to efficiently facilitate RNA circularization.
Compared to chemical and enzymatic methods, the PIE approach enables circularization of larger linear RNA precursors, and its reaction conditions and purification methods are relatively straightforward. Current research has shown that efficient circularization of almost any sequence of linear RNA precursor can be achieved by designing specific PIE transcription templates. Due to these advantages, the PIE method stands as the most extensively studied and widely employed RNA ligation technique at present.
2) Group II intron self-splicing system
Group II introns are a type of mobile genetic element found in bacterial and organellar genomes. They are considered ancestors of spliceosomal introns and retrotransposons in eukaryotes. Group II introns consist of a catalytically active intron RNA and an intron-encoded protein (IEP), working together to facilitate their proliferation within genomes. The intron RNA of Group II introns catalyzes its own splicing through a transesterification reaction similar to spliceosomal introns, resulting in spliced exons and a released intron in the form of a lariat RNA. The IEP, a multifunctional reverse transcriptase (RT), assists in splicing by stabilizing catalytically active RNA structures. Following splicing, the IEP remains associated with the excised intron RNA, forming a ribonucleoprotein (RNP) complex capable of invading the genomic DNA. Group II introns, discovered in bacterial and organellar genomes, are regarded as precursors of spliceosomal introns and retrotransposons in eukaryotes. They comprise a catalytically active intron RNA ("ribozyme") and an intron-encoded protein (IEP), both cooperating to mediate intron proliferation within genomes. Group II intron RNA catalyzes its own splicing via a transesterification reaction akin to spliceosomal introns, yielding spliced exons and a liberated intron in the form of a lariat RNA. The IEP is a versatile reverse transcriptase (RT) that aids splicing by stabilizing catalytically active RNA structures. Post-splicing, the IEP remains associated with the excised intron RNA, forming an ribonucleoprotein (RNP) capable of genome DNA invasion.
Researchers have developed another in vitro circular RNA synthesis technique based on the self-catalytic properties of Group II introns—utilizing the inverse splicing reaction to connect the 5' splice site of one RNA end with the 3' splice site of the same RNA, thereby forming a circular RNA.
3) Hairpin ribozyme
Hairpin ribozymes (HPRs) can generate circular RNA through rolling circle and self-splicing reactions using single-stranded circular DNA as a template. Linear RNA precursors containing HPR will fold into two conformations with cleavage activity, exposing the 3' and 5' ends. These ends then spontaneously join to form circular RNA. While this method efficiently produces shorter circular RNAs, its drawback lies in the fact that the HPR-catalyzed cleavage and ligation represent a dynamic equilibrium, resulting in the instability of the formed circular RNA. Moreover, the generated circular RNA will contain HPR sequences, which could have detrimental effects on its functionality."
Circular RNA Synthesis Method In Vivo
Unlike the in vitro synthesis of circular RNA, the in vivo synthesis of circular RNA is primarily achieved by overexpressing plasmid vectors containing mini-gene sequences that facilitate circularization within living cells. The sequence intended for circularization undergoes natural circularization through reverse splicing reactions following transcription. Most mini-genes for in vivo RNA circularization encompass at least one exon sequence earmarked for circularization, along with intronic cis-elements at the 5' and 3' ends containing splicing elements. By employing diverse combinations of intron-based cis-elements, it is feasible to circularize RNA sequences of nearly any length, ranging from 100 nucleotides to 5 kilobases. These intron-based cis-elements can be generally categorized as intronic complementary sequences, introns containing specific protein binding sites, introns exhibiting ribozyme activity, and introns originating from metazoan tRNAs.
Intronic Complementary Sequences for Circular RNA Synthesis
Based on the existing high-throughput transcriptome sequencing results, it is evident that the majority of endogenous circular RNAs contain sequences rich in reverse complementary motifs on both sides of their introns. These reverse complementary intronic sequences, located on the flanking introns, bring the upstream receptor and downstream donor splice sites of the circularization sequence closer in spatial distance through complementary base pairing, thereby facilitating the occurrence of reverse splicing reactions. The vast majority of intronic complementary sequences (ICS) are derived from repetitive or non-repetitive segments of endogenous genes in organisms. Additionally, a small portion of ICS has been generated through computer simulation design. Currently, researchers have been able to utilize this endogenous circular RNA formation mechanism to prepare specified RNA sequences into circular RNAs.
Specific Protein Binding Sites for Circular RNA Synthesis
Various RNA binding proteins (RBPs) play a role in promoting reverse splicing within organisms. Therefore, we can incorporate the binding sites of such proteins into overexpressed circularization vectors to mediate the circularization of target RNAs. In addition to RBPs, other proteins such as NF90/110, RBM20, Fus, DHX9, can regulate the intracellular synthesis of circular RNAs.
tRNA Introns for Circular RNA Synthesis
A class of abundant circular RNAs derived from tRNA introns, known as tRNA intron circular RNAs (tricRNAs), can be produced independently of spliceosomes and self-catalytic splicing reactions. They are primarily generated through the cleavage of tRNA precursor molecules containing introns by a set of highly conserved host enzymes. Subsequently, the excised introns are internally ligated by the host ligase RctB to form circular RNA molecules.
Prospects of Circular RNA Synthesis
Circular RNA, owing to its remarkable stability, holds the potential to serve as an effective and safe next-generation vaccine delivery platform. Previously, the artificial synthesis of circular RNA largely relied on in vitro strategies based on biochemistry. However, in recent years, novel molecular biology-based methods have emerged, enabling the direct production of circular RNA within living cells. It is conceivable that in the future, more refined and standardized methods for the fabrication of artificial circular RNA will emerge. These advanced circular RNA preparation techniques are bound to significantly propel the research progress in the field of circular RNA biology, functions, and molecular mechanisms.
References
1. Chen, X.; et al. Circular RNA: Biosynthesis in Vitro. Frontiers in Bioengineering and Biotechnology. 2021, 9.
2. Obi P.; et al. The Design and Synthesis Of Circular RNAs. Methods. 2021(196): 85-103.
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PEG manufacturing (Polyethylene Glycol) involves several steps and processes. Here is a general outline of how to conduct PEG manufacturing in a cGMP (current Good Manufacturing Practices) compliant facility:
Raw material selection: Choose high-quality raw materials, such as ethylene oxide and water, that meet the required specifications for PEG production.
Reactor setup: Set up the reaction vessel or reactor with appropriate equipment to handle the reaction, such as heating and cooling systems, agitation devices, and proper safety measures.
Ethoxylation: The ethylene oxide is introduced into the reactor while controlling the reaction conditions (temperature, pressure, and time) to ensure the desired level of polymerization. This process is known as ethoxylation.
Purification: After the reaction, the PEG mixture undergoes purification to remove any unreacted raw materials, impurities, or by-products. Various techniques like filtration, distillation, or chromatography can be employed for purification.
Concentration and drying: The purified PEG solution is concentrated to achieve the desired concentration level. This can be done through techniques like evaporation or reverse osmosis. The final concentrated PEG solution is then dried, typically through spray drying or freeze drying, to obtain the solid form of PEG.
Quality control: Throughout the manufacturing process, perform rigorous quality control tests to ensure the PEG products, for instance, PEG hydrogels, meet the required specifications and comply with cGMP regulations. These tests may include assessing the molecular weight, purity, moisture content, and other relevant parameters.
Packaging and labeling: Once the PEG product has passed quality control tests, it is packaged and labeled according to cGMP guidelines. This includes using appropriate containers, labeling with necessary product information, and ensuring proper storage conditions for the packaged PEG.
Documentation and record-keeping: Maintain detailed documentation of all manufacturing steps, including raw material usage, processing conditions, quality control results, and any deviations or corrective actions taken. This documentation is critical for GMP compliance and traceability.
Quality assurance and release: Before releasing the manufactured PEG for distribution or use, perform a final batch review and quality assurance check. This ensures that the product meets all required specifications and cGMP standards.
Storage and distribution: Store and distribute the PEG product under appropriate conditions as specified on the label. Maintain proper inventory management and tracking systems to ensure product traceability and prevent product mix-ups or contamination.
It is important to note that the manufacturing process may vary depending on the specific requirements of the PEG product, such as molecular weight, chemical modifications, or intended use. Therefore, it is recommended to consult with experts, such as chemical engineers or process development specialists, to optimize the manufacturing process for your specific application. Additionally, adherence to regulatory requirements and guidelines is crucial throughout the entire manufacturing process to ensure the safety and quality of the PEG product.
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In recent years, bioconjugation has emerged as a powerful strategy for improving drug delivery and enhancing therapeutic efficacy. By linking drugs to various biological entities, such as proteins, antibodies, nanoparticles, or targeting moieties, bioconjugation enables precise and targeted delivery of therapeutic agents to desired sites. This article aims to provide an overview of different bioconjugation strategies employed for various drug classes, highlighting their advantages, challenges, and potential applications.
Bioconjugation involves the covalent linkage of drugs or drug carriers to biological macromolecules, thereby enhancing their stability, solubility, and pharmacokinetics. This conjugation strategy allows for targeted delivery, sustained release, and controlled distribution of drugs while reducing off-target effects, improving efficacy, and minimizing toxicity. We will explore bioconjugation strategies for different drugs, including small molecules, peptides, proteins, and peptide RNA along with their potential applications in disease management.
Bioconjugation of small molecules: Small molecule drugs can be conjugated to biocompatible polymers or carriers to enhance their pharmacokinetics and bioavailability. Different conjugation chemistries such as carbodiimide coupling, click chemistry, and maleimide-thiol reactions have been extensively investigated. These strategies provide controlled release systems, improving drug stability, and reducing rapid clearance. Additionally, targeting moieties like antibodies, aptamers, or peptides can be conjugated to small molecules to achieve selective delivery to specific cellular or tissue targets, enabling personalized medicine.
Protein conjugation: Bioconjugation strategies for proteins include site-directed labeling, protein-polymer conjugation, or antibody-drug conjugation (ADC). These approaches enhance protein stability, prolong circulation time, and target specific cell surface receptors. Antibody-drug conjugates, in particular, have gained significant attention in cancer therapy by combining the specificity of antibodies with the cytotoxicity of small molecules, enabling selective drug delivery to cancer cells while sparing healthy tissues.
Peptide drug conjugate: Peptide-based drugs have gained substantial interest due to their high specificity and low toxicity. Bioconjugation of peptides with nanoparticles, liposomes, or polymers provides protection against enzymatic degradation and improves stability. Moreover, conjugation with cell-penetrating peptides facilitates enhanced cellular uptake and intracellular drug delivery. The precise modification of peptides through bioconjugation provides opportunities for the development of peptide-based therapeutics targeting cancer, neurodegenerative disorders, or metabolic diseases.
Peptide RNA conjugation: It refers to the process of attaching a therapeutic peptide molecule to an RNA molecule to create a novel therapeutic entity. This conjugation approach combines the unique targeting capabilities of peptides with the therapeutic potential of RNA-based molecules, such as siRNA or mRNA. By specifically delivering RNA molecules to target cells or tissues through peptide-mediated interactions, the conjugate enables precise modulation of gene expression, protein synthesis, or other cellular processes. This innovative strategy holds great promise for the development of highly specific, efficient, and customizable therapeutics to treat various diseases, including cancer, genetic disorders, and infectious diseases.
Conclusion: Bioconjugation has emerged as a versatile and effective strategy for enhancing drug delivery and therapeutic efficacy. By conjugating drugs with various biological entities, precise targeting, improved pharmacokinetics, sustained release, and reduced off-target effects can be achieved. By exploring bioconjugation strategies for different drug classes, this article highlights its potential for personalized medicine, thereby opening new avenues for the development of novel therapeutics for a wide range of diseases.
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Antibody-drug conjugates (ADCs), as emerging anticancer drugs, can deliver highly cytotoxic molecules directly to cancer cells to kill them. ADC is a monoclonal antibody covalently bound to cytotoxic chemical substances (payload) through linker. ADC linker plays a key role in the therapeutic effect of ADC, and its characteristics greatly affect the therapeutic index, pharmacodynamics and pharmacokinetics of ADC. For example, the linkage between linker-mAb determines the drug-antibody ratio (DAR), which determines the homogeneity and stability of ADC. In order to ensure the selectivity and efficacy of ADC, linker should strive to achieve three key features :
(1) High cycle stability: Payload will not be released before it reaches the target, thereby minimizing the off-target effects.
(2) High water solubility: it is helpful for coupling and avoids the formation of inactive ADC aggregates.
(3) Efficient release: allowing efficient release of highly cytotoxic linker-payload metabolites.
Cleavable and Non-cleavable Linker
ADC linker can be divided into cleavable linker and non-cleavable linker. Mechanistically, when the non-cleavable linker reaches the lysosome, the mAb is metabolized through the proteolytic mechanism, and the payload, linker and amino acid appendages are released. Substantial modifications to the payload can also yield potent ADCs, such as Kadcyla®, if the key pharmacophore of the payload is not affected. However, non-cleavable linkers are often unable to exert bystander effects due to the lack of cell permeability of charged amino acid appendages. Therefore, the application range of ADCs containing non-cleavable linkers is limited, and they are mainly used for the treatment of hematological cancers or tumors with high antigen expression.
Compared with non-cleavable linkers, cleavable linkers use specific conditions to release drugs at target cells. Cleavable linkers can be further subdivided into chemically cleavable linkers or enzymatically cleavable linkers. Although having a wider range of applications than non-cleavable linkers, cleavable linkers are more unstable in blood circulation. Cleavable linkers' performance thus hinges on their capacity to effectively distinguish between target cell circumstances and blood circulation conditions.
Chemically Cleavable Linker
There are three main types of chemically induced cleavable linkers: acid cleavable, cleavable under reducing conditions (disulfide, etc.), and linkers that can be cleaved by exogenous stimuli.
Acid-cleavable Linker
Acid-cleavable linkers are designed to utilize the acidity of endosomes (pH 5.5-6.2) and lysosomes (pH 4.5-5.0), while maintaining circulation stability under physiological conditions at pH 7.4. This strategy achieved the earliest clinical success with Pfizer's Mylotarg® (AcBut Linker). Although reducible disulfides are also employed, linker contains an acid-sensitive N-acylhydrazone linkage. Therefore, under acid catalysis, Mylotarg® is hydrolyzed into ketone and hydrazide-payload. In addition, during the development of Mylotarg®, the researchers also tested the stability of a series of hydrazone-containing linkers at pH 4.5 and pH 7.4, as well as their in vitro and in vivo stability in mice as part of ADC. Studies have shown that linker, which is stable at pH 7.4 and unstable at pH 4.5, provides the most effective ADC. This kind of linker-payload is also applied to Besponsa®.
In addition to the hydrazone bond mentioned above, the carbonate linker used by Trodelvy® is also a kind of acid cleavage linker. Although ester bonds are theoretically more stable than carbonates in blood circulation, experimental results show that ADCs constructed from the former are less stable in human serum. The serum stability of the ADC was significantly improved (t1/2=36 hrs) by introducing a p-aminobenzyl (PABC) spacer, which showed some selectivity for the acidic lysosomal compartment, with t1/2 at pH 5 2 for 10 hours.
Reductive Cleavage Linker
Despite the clinical success of Mylotarg®, Besponsa®, and Trodelvy®, acid-cleavable linkers are no longer an option for most ADC ligation techniques. Linker's requirement to strictly distinguish between pH 5 and pH 7.4 environments is very difficult. Although in some cases, slow release of payload can produce beneficial results, this method is usually only able to adopt payload of moderate cytotoxicity, and the highly toxic payload preferred by ADC now requires a more stable linker.
The release of payload in Mylotarg ® and Besponsa ® requires not only the acid-sensitive hydrazone bond to play a role, but also the disulfide bond in the linker. The disulfide bond linker is stable at physiological pH, but is susceptible to nucleophilic attack by thiols. In plasma, the main thiol species is the reduced form of human serum albumin (HSA, ~422 μM ), but its reactivity with macromolecules is hindered due to limited exposure of solvents containing free thiol residues. Contrary to the limited reduction capacity of plasma, the cytoplasm contains high levels of glutathione (GSH, 1-10 mM); the reduction conditions of plasma and cytoplasm provide an opportunity for ADC to selectively release the effective load. In addition, compared to normal tissues, tumor-associated oxidative stress typically leads to elevated levels of glutathione, which increases the selectivity of cancer cells. At present, the payload of linkers involving disulfide bond hydrolysis mainly includes the effective load of maytansinoids (DM1, DM3, DM4) and disulfide bond carbamate.
Exogenous Stimulus Cleavable Linker
Although the use of endogenous cleavage ADC linker is the simplest drug release method, the release of payload through external stimuli can have the following advantages: (1) avoid the difference in linker cleavage rate due to biological differences between patients, and (2) ) ADCs can also function when endogenous cleavage is insufficient to effectively release the payload. Akalux®, which has been launched in Japan, uses a near-infrared light-sensitive linker as a cleavage method. After irradiation with 690 nm red light, it releases toxic IRDye700 to play a therapeutic role, which belongs to near-infrared photoimmunotherapy. In addition, there are currently more studies on metal ion (Pt, Pd, Fe, Lu) catalyzed cleavage, UV/Vis and NIR light-sensitive linkers.
Enzyme-mediated Cleavable Linker
According to the traditional mechanism of action of ADC, the enzyme-cleavable linker can be selectively cleaved inside the cell by transporting ADC to the lysosome, which contains a high concentration of certain hydrolytic enzymes. The most successful method at the moment is the use of cathepsin B cleavable linkers. The peptide linker broken by cathepsin B is used by the commercially available medications Adcetris®, Polivy®, Padcev®, Tivdak®, Aidixi®, Lumoxiti®, Zynlonta®, and Enhertu®. Among them, p-aminobenzyl carbamate (PABC) was used as a self-degrading spacer, which spontaneously undergoes 1,6-elimination after proteolysis, releasing payload, CO2 and azaquinone methide. PABC maintains the enzyme activity independent of payload, which increases the scope of application of the peptide linker. All of these linker combinations are stable in isolated human plasma. In addition to cathepsin B cleavable linker, phosphatase cleavable linker, sulfatase cleavable linker, β-galactosidase cleavable linker, β-glucosidase cleavable linker and nitroreductase cleavable linker are also widely used in the construction of ADCs.
Other Cleavable Linker
Based on the characteristics of bioorthogonal chemistry that does not interfere with normal biological processes, high selectivity, fast and simple processing, and non-toxic by-products, bioorthogonal cleavage pairs can also be used as linker cleavage triggers. In addition, IEDDA reactions can also be used for payload release. Introduced by Tagworks Pharmaceuticals, this method uses trans-cyclooctene (TCO) as a cleavable linker to undergo a click reaction with a tetrazine activator to generate a 4,5-dihydropyridazine intermediate, which is then converted to 2,5- and 1 ,4-Tautomers, of which only the latter can undergo subsequent electron cascade reactions, thereby releasing different payloads. The IEDDA and tautomerization steps are affected by substituents on the tetrazine moiety, respectively, and show better drug release properties through the combination of two functional groups with opposite electron-withdrawing and electron-repelling properties.
In Conclusion
A good linker is a crucial assurance of the security and efficiency of ADC. Despite the prevalence of non-cleavable linkers, most ADC medicines prefer cleavable linkers because to the cytotoxicity of free payloads and the significance of bystander effects. Acid-cleavable linkers were initially promising, but the stringent stability requirements for highly toxic payloads reduced their usefulness. In contrast, the vast majority of ADCs currently use peptide technology because they can effectively distinguish blood circulation and target cell conditions, but this aspect still needs further development to better address key issues such as solubility and plasma stability. Furthermore, the applicability of extracellular cleavage to non-internalizing ADCs, eliminating the requirement for antigen internalization, greatly increases the number of possible antigen targets, which may open the door to many new ADC therapies.
References
Albericio, F. et al. Linkers: An Assurance for Controlled Delivery of Antibody-Drug Conjugate. Pharmaceutics. 2022, 14: 396.
Joubert, N. et al. Antibody–Drug Conjugates: The Last Decade. Pharmaceuticals. 2020, 13: 245.
Pillow, T.H. et al. Decoupling Stability and Release in Disulfide Bonds with Antibody-Small Molecule Conjugates. J. Name. 2013, 00: 1-3.
Spring, D.R. et al. Cleavable linkers in antibody–drug conjugates. Chem. Soc. Rev. 2019, 48(16): 4361-4374.
Spring, D.R. et al. Sulfatase-cleavable linkers for antibody-drug conjugates. Chem Sci. 2020, 11(9): 2375-2380.
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Targeted therapies have significantly advanced the field of cancer therapy, and among these, antibody-drug conjugates (ADCs) have emerged as a viable therapeutic strategy. ADCs increase efficacy and decrease systemic toxicity by fusing the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). The manufacture of high-quality and stable payloads by fermentation techniques is an essential component of ADC development.
What are ADC Payloads?
The payloads, also known as cytotoxins, are crucial parts of ADCs because they have a therapeutic effect on cancer cells. Typically, they are small molecules with strong anti-cancer properties. The particular cancer type being targeted and the cytotoxin's mode of action must be taken into consideration when choosing an appropriate payload. A linker molecule allows the payload to be attached to the antibody. The linker must be able to release the drug once the ADC has specifically attached to its target on cancer cells while remaining stable in circulation to prevent premature release of the cytotoxin.
The cytotoxic agents include:
Analogs of DNA bases: 5-fluorouracil and 8-azaguanine
DNA-damaging agents: cisplatin and actinomycin D
Antimetabolites: aminopterin and methotrexate
Tubulin inhibitors: paclitaxel and vincristine derivatives
Mycotoxins, secondary metabolites of molds fungis, have a very toxic effect on human being which can be served as ADC payloads. Fermentation plays a crucial role in the production of ADC payloads, involving strain improvement, metabolic pathway engineering, and fermentation process development.
The Significance of Fermentation in ADC Payload Production
Enabling Large-Scale Production
Large-scale fermentation serves as a key method for production of ADC payloads. By utilizing microbial systems, such as bacteria or yeast, it becomes possible to generate substantial quantities of cytotoxins or drug precursors required for conjugation to the antibody. This scalability is crucial to meet the increasing demand for ADCs in commercial production.
Customization of ADC Payloads
Fermentation allows for the customization and production of diverse payloads. With a wide variety of microorganisms available, each with distinct metabolic pathways, it becomes possible to engineer specific strains for the synthesis of unique cytotoxic agents. This versatility enhances the potential for developing ADCs targeting different types of cancer and specific molecular targets.
Challenges in Fermentation of ADC Payloads
Optimization of Yield and Productivity
Maximizing the yield and productivity of ADC payloads presents a significant challenge in fermentation. The production of cytotoxic compounds often involves complex metabolic pathways, which can be influenced by factors such as media composition, fermentation conditions, and genetic modifications. Fine-tuning these parameters is crucial to achieve high yields while maintaining the desired quality and stability of the payload.
Ensuring Consistent Quality and Purity
Fermentation processes must be carefully controlled to minimize batch-to-batch variability, ensuring that the produced payload meets stringent regulatory requirements. Contamination or impurities in the payload can compromise the safety and efficacy of ADCs, necessitating robust purification steps.
Advances in Fermentation Technologies
Metabolic Engineering
By manipulating the metabolic pathways of microbial hosts, the yield and productivity of ADC payloads could be enhanced. This can be achieved through genetic modifications, such as overexpression of key enzymes, elimination of competing pathways, or introduction of novel biosynthetic pathways.
Process Optimization and Control
Advancements in process optimization and control have contributed to the improved fermentation of ADC payloads. Through advanced analytics and monitoring techniques, such as real-time metabolite analysis, online sensors, and automated feedback systems, it is possible to obtain a better understanding of the fermentation process dynamics.
Outlook of Fermentation of ADC Payloads
Continuous Fermentation
Implementing continuous fermentation strategies can enhance the efficiency and productivity of ADC payload production. Continuous systems offer several advantages over traditional batch processes, including better control of fermentation parameters, reduced downtime between batches, and improved process stability. Exploring continuous fermentation approaches holds the potential to further optimize the scalability and cost-effectiveness of ADC payload production.
Integration of Omics Technologies
The integration of omics technologies, such as genomics, proteomics, and metabolomics, can provide a holistic understanding of microbial host physiology and metabolic pathways. This knowledge can guide the design of more efficient fermentation processes and enable targeted metabolic engineering strategies. Omics technologies offer insights into the complex interactions within the microbial system and can contribute to the development of superior ADC payloads.
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