The discovery of tight junctions
Thanks to Porter's earlier work (1) on the observation of cells by electron microscopy, Farquhar and his team were able to apply his methods to extend our knowledge of intercellular spaces. Working on the study of tissues of the rat and guinea pig species, and using hemoglobin as a mass tracer, Farquhar et al. (2) were able to demonstrate in 1963 that the intercellular complex was composed of different elements of which:
- the zonula occludens (tight junction),
- the zonula adherens (intermediate junction), whose the major integral membrane proteins are cadherins,
- the macula adherens (desmosome, discovered as early as 1882 by Giulio Bizzozero),
- gap junctions, a dense aggregation of multimeric channels, each of which consists of six identical proteins named connexins.
Comparable to stitches between cells, tight junctions actually involve transmembrane proteins implanted across two adjacent membranes, which they thereby strengthen. Tight junctions form a belt that surrounds the cell at the apical pole and prevents extracellular fluid from passing between the cells, ensuring that the tissue is sealed. By forcing the passage of compounds through the cell membranes, the cells act as a selective sorting of compounds. But that's not all. Tight junctions also control cell proliferation, differentiation and polarity.
Occludin: hydrophobic membrane-spanning domains protein of the tight junction complex
The study of tight junctions then accelerated and led to the work of interest of Furuse et al (3) by the identification of one major component of tight junctions. Starting from chick liver material, and the previous identification of ZO-1, a tight junction-associated protein, the team was able to generate rat monoclonal antibodies targeting this complex. Four rats were immunized with acid-extracted membrane fraction and approximately one hundred clones isolated. Among them, three clones demonstrated specificity for the same antigen but with different epitopes (recognizing four to five bands from 58 to 66 kD in immunobloting profile).
Since the sequence analysis revealed no homology between this membrane protein and other proteins so far identified, Furuse et al decided to give the name “occludin” to this new discovered structure. Like connexins, it appeared that occludin contains four major hydrophobic membrane-spanning domains and two extracellular loops (ECL1 and ECL2).
Claudin: essential transmembrane highly homologous protein of intercellular space
Following the discovery of occludins by Furuse, many international teams were busy investigating the composition of intercellular tight junctions.
And a number of inconsistencies were revealed.
First, Balda et al (4) expressed a mutated chicken occludin that lacked almost the entire COOH-terminal domain in stably transfected MDCK cells. Curiously, they could not see any apparent change in the morphology of the junction when observed by electron microscopy.
Second, using rat monoclonal antibodies specific for occludin, Moroi et al (5) were able to obtain intense signals on Sertoli cells in mice without having any signal for the same cell type in humans and guinea pigs.
The results of these two experiments led Furuse's team to formulate the hypothesis of the intervention of other proteins in addition to occludin in the tight junction zone. This suggestion was confirmed by the work of Saitou et al (6) who developed occludin-deficient embryonic stem cells by knocking out both of the occludin alleles in embryonic stem (ES) cells by homologous recombination. Despite the confirmation of the loss of occludin expression, the tight junctions turned out to be perfectly formed.
By re-examining the isolated junction fraction from chick liver of their previous work (3), Furuse et al were able to isolate two different but very similar proteins of 22kD each, with no homology to occludin and specifically localized in the tight regions. They named these two proteins of four transmembrane domains Claudin 1 and Claudin 2 (7).
We now know that the claudin family has at least 27 members (8).
Potential therapeutic applications of targeting Occludin for infectious diseases, cancer and barrier dysfunction disorders
Hirase et al demonstrated that endothelial cells in neural tissues have a much larger expression of occluding. Investigating this variable expression among tissues, the team showed that multiple factors regulate occludin functions on blood brain barrier permeability, such as matrix metalloproteinases (MMPs), post-translation modifications and pro-inflammatory cytokines Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1 β), Interferon-γ (IFN-γ) (9). Therefore, occludin might be a potential biomarker for early haemorrhagic transformation in ischemic stroke.
Wang et al demonstrated that occludin protein expression was greatly increased in lung cancer patient samples. Furthermore, occludin knockdown inhibited lung cancer cell proliferation in both in vitro and in vivo models. In addition, occludin knockdown promoted apoptosis of lung cancer cell lines and reduced invasive capacity (10).
Ploss et al demonstrated that human occludin (OCLN) is an essential cellular entry factor for hepatitis C virus (11). Therefore, Shimizu and his team decided to develop rat antibodies in the form of monovalent antigen-binding fragments (Fab) and single-chain variable fragments (scFv) specific for occludin, by DNA immunization and cell screening methods (12).
It is estimated that around 71 million individuals globally have chronic hepatitis C virus (HCV) infection, around 3 to 4 million new cases of HCV infection occur globally each year and around 399,000 deaths each year are attributed to hepatitis C virus (HCV) infection. HCV enters hepatocytes through multiple processes involving heparan sulfate, low-density lipoprotein receptor, CD81, the scavenger receptor B1, epidermal growth factor receptor, CLDN-1 and occludin (Colpitts et al, 2015).
Potential therapeutic applications of targeting Claudin for oncology, inflammatory bowel diseases, and barrier dysfunction disorders
Breaking Barriers with Claudin-1 (CLDN-1) Antibody Therapeutic Approaches
Mattern et al (13) are specialists of Metalloproteinase-15 ADAM15, a transmembrane protein involved in protein ectodomain shedding, cell adhesion and signalling. Expressing different isoforms of this metalloproteinase, the mentioned team was able to reveal isoform specific, catalytic function dependent upregulation of Claudin-1. In particular, the PI3K/Akt/mTOR pathway is involved in regulating Claudin-1 expression downstream of ADAM15.
Studies since 2010 have linked abnormal claudin expression to tumor development and prognosis in various cancers, including prostate, bladder, breast, oesophageal and gastric adenocarcinomas, laryngeal carcinoma, lung, and glioblastoma.
Several entities are working on the development of monoclonal antibodies against the Claudin-1 target, such as Alentis Therapeutics, ALE.F02 (generated by the University of Strasbourg), for advanced kidney and lung cancers and liver fibrosis, Cherradi from the University of Montpellier (14) for mCRC, Natascha Roehlen from the University of Strasbourg for fibrosis. We can also note that the latter university has also produced therapeutic antibodies targeting Claudin-1 for the treatment of hepatitis C.
Unlocking the Potential of Claudin-2 (CLDN-2) in Cancer Therapy with Monoclonal Antibodies
In 2011, Tabiès et al demonstrated that Claudin-2 enhances cell/matrix interactions by boosting the presence of α(2)β(1) and α(5)β(1) integrin complexes on breast cancer cell surfaces. The claudin-2-facilitated adhesion to fibronectin and type IV collagen can be inhibited by neutralizing antibodies targeting α(5)β(1) and α(2)β(1) complexes, respectively (16).
To our knowledge, the Hashinomoto team is one of the few to have developed a therapeutic antibody targeting Claudin-2 (17).
The New Frontier in Cancer Treatment: Targeting Claudin-3 (CLDN-3) & Claudin-4 (CLDN-4)
Nearly 80% of ovarian cancer tissues express claudin-3, while 70% express claudin-4, and the correlation between their expression levels and cancer progression has been observed in various cancers (Tracey et al, 2009). Antibodies targeting CLDN-3 or -4, or both, have demonstrated anti-tumor effects in preclinical rodent studies. However, these proteins are also found in normal tissues, including the breast, ovaries, prostate, bladder, and gastrointestinal mucosa, highlighting the importance of thoroughly evaluating the safety profiles of these antibodies to avoid any off-target effects.
The Path to Better Outcomes: Claudin-6 (CLDN-6) as a Target for Cancer Therapy
If there is one subject of primary importance at the moment in the Claudin family, it is Claudin-6 or CLDN-6. CLDN6 is widely expressed in various tumours (like hepatocellular carcinoma, ovarian cancer, endometrial carcinoma) but rarely expressed in healthy adult tissues.
Several companies have announced significant advances on this transmembrane target, including :
|Antibody Drug name||Therapeutic approach|
|Context Therapeutics||CTIM-76||Bispecific antibody to CD3|
IMAB027 (ASP1650 formerly
generated by Ganymed)
|I-MAB Biopharma||GB-7008||Naked antibody|
|BioNTech||CARVac||CAR-T cell amplifying RNA vaccine|
Innovating Cancer Treatment through Claudin-18.2 (CLDN-18) Antibody Therapeutics
And if you think a lot of Biotech and Pharma are interested in Claudin-6 targeting, you haven't seen anything yet because Claudin 18.2 remains the undisputed star of claudins!
Claudin 18.2 (CLDN18.2) is specifically expressed in highly differentiated normal gastric mucosa, but is also highly expressed in tumor cells of 50%–80% of gastric cancer patients and 60% of pancreatic cancer patients. Due to the tight adhesion between cells, it is very difficult for antibody drugs to bind to normal tissues. However, the loose structure of the interstitial space of cancer cells expose CLDN18.2 epitopes on the surface of tumor cells and so make them accessible to macromolecular protein drugs. CLDN18.2 is an ideal target for a new generation of anti-tumor therapy using antibodies and antibody-derived technologies.
We counted no less than 31 different therapeutic antibodies in development.
It is therefore reasonable to ask whether there is still room for new entrants in this market!
|Antibody Drug name||Therapeutic approach|
|Zai Lab||ZL-1211||Naked antibody|
|Turning Point Therapeutics||
(LM-302, generated by
|Antibody Drug Conjugate|
|ABL Bio||ABL111||Bispecific antibody to 41BB|
|SOTIO||SOT102||Antibody Drug Conjugate|
|Elevation Oncology||EO-3021 (SYSA1801)||Antibody Drug Conjugate|
|Triumvira Immuologics||Undisclosed||Autologous and allogeneic T cells|
|KLUS Pharma||SKB315||Antibody Drug Conjugate|
|Luzsana||Undisclosed||Antibody Drug Conjugate|
|Keymed Biosciences||CMG901||Antibody Drug Conjugate|
|Legend Biotech||Undisclosed||Autologous T cell|
|Xencor||ASP2138||Bispecific antibody to CD3|
|Boan Biotech||BA1105||Antibody Drug Conjugate|
|Conjupro Bio||CPO102||Antibody Drug Conjugate|
|Antangene||ATG-022||Antibody Drug Conjugate|
|Phanes Therapeutics||PT886||Bispecific antibody to CD47|
|RemeGen||RC118||Antibody Drug Conjugate|
|Doer Biologics||DR30303||Single domain antibody ScFv|
|Innovent||IBI343||Antibody Drug Conjugate|
|Dragon Boat Pharmaceutical||BC007||Bispecific antibody to CD47|
|Dragon Boat Pharmaceutical||BC008||Antibody Drug Conjugate|
|QureBio||Q-1802||Bispecific antibody to PDL-1|
Overcoming the Obstacles: The Challenge of Generating Antibodies Against Occludin and Claudin Proteins
Generating antibodies against claudin and occludin proteins can be difficult due to several reasons:
- Structural complexity: Claudin and Occludin proteins are highly structured and composed of multiple domains that can change their conformation and interact with other proteins, making it difficult to generate stable antibodies that recognize a specific epitope.
- Low immunogenicity: These tetraspan complex are intracellular proteins that are not naturally exposed on the cell surface, so they are not easily recognized by the immune system.
- Antigenic variability: these transmembrane targets are a family of proteins with high sequence homology, and some members may share similar epitopes, making it difficult to generate antibodies that specifically recognize one claudin over others.
- High sequence homology among humans, mice, and rats: since the sequences of claudins are highly conserved between species, it may be difficult or impossible to generate an anti-human claudin antibody or to create a surrogate antibody in mice and rats without specific immunotolerance-breaking technology.
These factors make it challenging to generate reliable and specific antibodies against claudin proteins, which are important targets in research and medical applications. Nevertheless, several teams have succeeded in raising occludin- and claudin-specific monoclonals, playing with a range of different antigens:
- DNA immunization: anti-CLDN1 mAbs were generated by genetic immunization of rats using a eukaryotic expression vector encoding the full-length human CLDN1 complementary DNA (Fofana et al, 2010), and the same was done for claudin-2 (Hashinomoto et al, 2018).
- Cell immunization: anti-CLDN1 mAbs were raised by injection of 4 million mouse NIH cells transiently transfected with CLDN1 cDNA (NIH-CLDN1 cells) every two weeks (Cherradi et al, 2022).
- Peptide immunization: anti-CLDN6 mAbs were developed by immunizing mice as well as transgenic non-human animals with peptide fragments (Ganymed patent, US9487584B2).
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