First time to uses mRNA technology to deliver mucosal dimer IgA to prevent bacterial infection
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Moderna uses mRNA technology to deliver mucosal dimer IgA for the first time to prevent bacterial infection.
The use of mRNA-LNP technology to generate antibodies in vivo has long been attempted, however, IgG antibodies are generally chosen .
In 2018 , Philip J. Santangelo et al. published an article on Nature Communications : Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection .
They delivered mRNA (palivizumab) encoding an RSV neutralizing antibody directly to the lungs through a lung nebulizer (invasive tracheal administration) , thereby inhibiting RSV viral infection.
On October 31, 2022 , Philip J. Santangelo and others published an article in Advanced Science again : Nebulized mRNA-Encoded Antibodies Protect Hamsters from SARS-CoV-2 Infection , in a hamster model, using nebulized inhalation to target lung delivery The mRNA encoding the COVID-19 neutralizing antibody carrying the plasma membrane anchor sequence GPI can efficiently express the neutralizing antibody anchored on the plasma membrane in lung cells, significantly inhibiting weight loss after infection with the new coronavirus, and inhibiting the virus from spreading in the lungs. Replication, lower lung viral load.
mRNA-1944 encodes the heavy and light chains of CHKV-24, a chikungunya virus (CHIKV) -specific monoclonal neutralizing antibody, delivered using LNP.
Moderna has launched a phase I clinical trial (NCT03829384) of mRNA-1944 from 2019 to 2021 , and mRNA-1944 has become the first mRNA-encoded monoclonal antibody to show expression in vivo and detectable neutralizing activity in vitro in clinical trials .
IgG has been the isotype of choice for monoclonal antibodies due to ease of production and favorable pharmacodynamic properties.
In contrast, there are many challenges in the clinical transformation of recombinant protein IgAR .
First, human IgA is highly glycosylated, and N-glycosylation affects protein conformation, thermostability, folding efficiency, solubility, and susceptibility to protease degradation.
In addition, due to incomplete sialylation and inability to be recycled by neonatal Fc receptor ( FcRn) , recombinant protein IgAR will be cleared in the circulation, which makes the serum half-life of recombinant protein IgAR longer than that of endogenous Sexual human IgA short .
Efforts are underway to synthesize IgG/IgA chimeras with ideal Fc-receptor interactions, serum half-life, and mucosal delivery; however, they have not yet reached the clinical stage.
Antibodies in mammals can be divided into 5 isotypes: IgG, IgM, IgA, IgD and IgE. IgA is the predominant antibody isotype in mucosal secretions of the gastrointestinal tract and upper respiratory tract and is the first line of defense against pathogen invasion.
Humans have two IgA subclasses, IgA1 and IgA2, which differ in hinge length, degree of O-linked glycosylation, and mucosal localization.
Serum IgA is usually monomeric m IgA , whereas mucosal IgA is dimeric d IgA or multimer because of the hinge (JC) that covalently links two or more IgA monomers together through their C-termini.
Polymeric immunoglobulin receptor (pIgR) is another important component of the mucosal immune system, which can transport dimeric d IgA instead of monomeric m IgA across the epithelial cell barrier of the intestinal tract and respiratory tract, Causes the secretion of dIgA , which captures pathogens and prevents their attachment to epithelial cell receptors on mucosal surfaces.
Sal4 is an IgA monoclonal antibody that can recognize the O5 antigen from the lipopolysaccharide of Salmonella typhimurium. Passive injection of Sal4 can prevent Salmonella from invading Peyer’s patches in the intestinal tract.
CAM003 is an IgG1 mAb that binds Psl, a Pseudomonas aeruginosa biofilm component, and has shown protective effects in several different PA animal models, including acute pneumonia models.
On January 3, 2023 , the Moderna team uploaded an article on BioRxiv : mRNA delivery of dimeric human IgA protects mucosal tissues from bacterial infection . For the first time, the mRNA technology platform was used to deliver IgA2 mRNA encoding Sal4 or IgA1 mRNA encoding CAM003 to the human body , thereby The body produces powerful protective mucosal antibodies to prevent bacterial infection.
Moderna team leader of IgA antibody delivery project using mRNA technology
Synthesis of Sal4 Ig A2 mRNA at the cellular level or in mice
Encapsulate the mRNA encoding Sal4 heavy chain HC and light chain LC with LNP, transfect HEK293 cells, no matter whether the mRNA encoding hinge JC is co-transfected, the IgA2 mRNA secreted in the cell supernatant can be detected by the Salmonella immobilized on the microtiter plate identified by Bacillus.
However, Sal4 IgA2 mRNA could be recognized by the polymeric immunoglobulin receptor (pIgR) immobilized on the microtiter plate only when JC-mRNA was co-transfected , indicating the formation of dimeric d IgA2 .
It can also be seen from the SEC peak diagram that when JC-mRNA is co-transfected, the Sal4 Ig A2 mRNA synthesized by the cells mainly has the dimer d IgA2 as the main peak; when the JC hinge is lacking, the Sal4 Ig A2 mRNA synthesized by the cells is composed of Monomer m IgA2 and m IgA2 aggregates.
Electron micrographs showed that, similar to the structure of recombinant protein dIgA2 R , dIgA2 mRNA formed a dimer through tail-tail.
The IgA2 mRNA expressed by EXPI293 cells can reduce the infection of Hela cells by Salmonella, and the degree of reduction is similar to that of the recombinant protein d IgA2 R .
The above data confirm for the first time that mRNA technology can be used to encode and produce functional oligomer IgA protein.
Functional Sal4 IgA2 mRNA can be synthesized in cells using mRNA/LNP technology.
Mucosal targeting and oligomeric properties are very attractive features of IgA proteins, however, IgA proteins have poor pharmacodynamics, especially with a very short half-life.
In order to know whether the efficacy of IgA produced by mRNA technology is changed, the researchers injected 5 mg/kg recombinant protein IgA2 R (monomer/dimer mixture ) and 4.5 mg/kg polyclonal antibody human serum IgA (large partly monomeric) and 1 mg/kg mRNA-LNP encoding Ig A2 mRNA .
Similar to previous research results, the half-life of recombinant protein IgA2R in mouse serum is 0.64 days , which can only be detected on the second day after injection, while the half-life of polyclonal antibody human serum IgA is 0.93 days, which can be detected on the 12th day after injection.
What is exciting is that the half-life of IgA2 mRNA in mouse serum can reach 1.63 days , which shows that there is an essential difference in structure between IgA2 mRNA and recombinant protein IgA2 R.
Differences in Half-life of Recombinant Protein IgA2 R , Human Serum IgA and IgA2 mRNA
Glycosylation pattern of Sal4 IgA2 R
According to the substitution of mannose residues, N-glycans (N-glycan) can be roughly divided into three categories: complex , high-mannose structure (high-mannose) and hybrid structure ( hybrid) .
The complex type contains fucose (Fucose) , sialic acid (Sialic-Acid) and α-linked galactose.
A branch of the high mannose core structure contains multiple mannose residues.
In the heterozygous form, mannose is present in the core structure of one branch and the lactosamine sequence is contained in the other branch.
Compared with IgA from human serum, the recombinant protein IgA2 R synthesized by HEK293 cells was undersialylated, resulting in its rapid clearance from the circulation.
To find out whether the better pharmacodynamic profile of IgA2 mRNA was due to differences in glycosylation, the researchers measured the glycosylation pattern and sialylation level of Sal4 IgA2 mRNA purified from mouse serum .
Since serum IgA is in monomeric form, researchers compared serum IgA2 mRNA with recombinant protein monomeric IgA2 R or IgA2 R containing monomeric/dimeric mixed forms , and found that serum IgA2 mRNA exhibits complex glycosyl The degree of hypersynthesis was as high as 90%, while IgA2R / m IgA2R was only 30% and 19%, respectively .
In contrast, the high mannose core structure occupied 66% and 76% in IgA2 R and m IgA2 R , while in IgA2 mRNA , the high mannose core structure was only 5%.IgA2 R and m IgA2 R protein structures exhibit high levels of high mannose core structure and low levels of sialylation (13-14%), in contrast to IgA2 mRNA protein structures that exhibit high levels of sialylation .
For IgA2 mRNA and IgA2 R / m IgA2 R , the detection of N-glycan structures of single amino acids, glycosylation patterns are similar.
Differences in glycosylation patterns and sialylation levels between recombinant protein IgA2R and IgA2 mRNA.
Distribution of Sal4 Ig A2 mRNA in serum and mucosa
Humans were able to detect IgG1 R in the circulating system (serum) by intravenously injecting 2.5 mg/kg IgG1 R into mice , however, after injecting 2.5 mg/kg d IgA2 R into mice, at any time point, No d IgA2 R could be detected in mouse serum .
In contrast, if 1 mg/kg of mRNA/LNP encoding IgG1 mRNA and Ig A2 mRNA was injected into mice, the IgG1 mRNA and Ig A2 mRNA detected in the mouse serum 24h-48h after injection Antibody titers can reach 69ug/ml and 27ug/ml.
Although IgG1 R antibody titers in mouse serum were high, IgG1 R and IgG1 mRNA could not be detected in mouse excreta at 24 h after injection .
After intravenous injection of Sal4 d IgA2 R into mice, Sal4 d IgA2 R could be detected in the excrement of mice at 6 hours after injection , but at all subsequent time points, Sal4 d IgA2 R in the excretion of mice was in the Below the detection line .
On the contrary, the IgA2 mRNA titer in the excrement of mice at 24h after injection can reach 67ng/ml, and the time it can be detected can be suppressed until 96h after injection.
IgA2 mRNA can be expressed in serum and transported to mucosal sites
IgA mRNA prevents bacterial invasion
Salmonella mouse model
Intravenous injection of 1mg/kg mRNA/LNP encoding Sal4 Ig A2 mRNA or IgG1 mRNA and 5mg/kg of the same type of recombinant protein antibody into mice, before the challenge test, the Ig A2 mRNA titer in the mouse serum was 11ug /ml, however, IgA2R was undetectable in serum .
On the contrary, the mouse serum IgG1 mRNA and IgG1 R titers were 291ug/ml and 84ug/ml, which were much higher than the serum IgA antibody titers.
In mouse excreta, Ig A2 mRNAThe antibody titer was the highest at 125.2ng/ul, the IgG1 mRNA antibody titer was 51.66ng/ml, and the IgG1 R antibody titer was 9.71ng/ul.
IgA2 mRNA antibody titers in excretion were much higher than in serum because IgA2 mRNA was secreted into the gut, whereas IgG antibodies detected in excretion were due to leakage.
More importantly, IgA2 mRNA was more effective than IgA2 R or IgG1 in preventing Salmonella intestinal invasion in a mouse model of Salmonella enterica.
Sal4 IgA2mRNA can block Salmonella invasion of intestinal mucosa.
Pseudomonas aeruginosa pneumonia mouse model
After intravenous injection of 1 mg/kg IgG1 or IgA1 antibody encoding CAM003, before the challenge test, IgG1 mRNA in mouse serum can reach 68.73ug/ml, and IgA1 mRNA can reach 7.95ug/ml.
Despite differences in titers of the two types of antibodies in serum, both IgA1 mRNA and IgG1 mRNA were able to protect mice from P. aeruginosa-induced pneumonia death.
Injection of IgG1 or IgA1 antibodies encoding CAM003 protected mice from death in a mouse model of Pseudomonas aeruginosa pneumonia death.
Conclusion
This study is the first to synthesize functional IgA antibodies at the cellular level and in mice using mRNA-LNP technology, which can form dimeric dIgA mRNA when hinge JC- mRNA is present.
Compared with recombinant protein antibodies, mRNA-encoded IgA has better pharmacodynamic characteristics, such as longer half-life, which may be due to differences in glycosylation patterns and sialylation levels.
In Salmonella and Pseudomonas aeruginosa mouse models, intravenous injection of mRNA-LNP encoding IgA was also effective in preventing bacterial infection in the gut and lungs, as was the recombinant protein IgAR .
In conclusion, this article opens a new window for the delivery of IgA mAbs to human mucosal sites using mRNA technology to inhibit bacterial infection.
Moderna uses mRNA technology to deliver mucosal dimer IgA for the first time to prevent bacterial infection.
(source:internet, reference only)
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