Complementary epitopes and favorable developability of monoclonal anti-LAMP1 antibodies generated using two transgenic animal platforms

Cameron, Béatrice; Dabdoubi, Tarik; Berthou-Soulié, Laurence; Gagnaire, Marie; Arnould, Isabelle; Severac, Anne; Soubrier, F; Morales, Jacqueline; Leighton, Philip A.; Harriman, William; Ching, Kathryn H.; Abdiche, Yasmina; Radošević, Katarina; Bouquin, Thomas

doi:10.1371/journal.pone.0235815

Cited by 8 publications

(5 citation statements)

References 27 publications

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“…The copyright holder for this this version posted November 22, 2023. ; https://doi.org/10.1101/2023.11.22.568300 doi: bioRxiv preprint comprises 50% of all lysosomal membrane proteins and is widely used as a cell surface marker of lymphocyte activation and degranulation. Additionally, LAMP-1 is a physiologically essential protein involved in stabilizing lysosomes and regulating autophagy to prevent embryonic lethality [35,36,37]. The expression of CD107 is associated with a decrease in the expression of CD62L.…”

Section: Discussionmentioning

confidence: 99%

Contribution of B-1 cells in the cytotoxicity of CD8 T lymphocytes in encephalitozoonosis

de Oliveira,

Perez,

Alvares-Saraiva

et al. 2023

Preprint

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Encephalitozoon cuniculiis an opportunistic intracellular pathogen that establishes a balanced relationship with immunocompetent individuals depending on the activity of their CD8+T cells lymphocytes. However, lower resistance to experimental infection withE. cuniculiwas found in B-1 deficient mice (Xid), besides increased the number of CD8 T lymphocytes. Here, we evaluated the cytotoxic activity of CD8+T lymphocytes from Balb/c wild-type (WT) or Balb/c Xid mice (with B-1 cell deficiency) on the microbicidal activity of macrophages challenged withE. cuniculi. CD8 T lymphocytes from WT or Xid mice previously infected or not withE cuniculiwere co-cultured with macrophages challenged withE. cuniculi. We evaluated macrophages viability and microbicidal activity, and proliferation, viability, and presence of activating molecules (CD62L, CD69, and CD107a) in CD8 T lymphocytes. Macrophages co-cultured with CD8 T lymphocytes from WT demonstrated high microbicidal activity. CD8 T lymphocytes obtained from uninfected WT mice had a higher proliferative capacity and a higher expression of CD69 and LAMP-1-activating molecules compared to Xid CD8+T lymphocytes. CD8 T lymphocytes from infected Xid mice proliferated more than those from WT mice, however, when the expression of the activating molecule CD69 associated with the expression of CD62L was kept low. In conclusion, the absence of B-1 cells in Xid mice might be associated with the lower expression of activating molecules in CD8+T lymphocytes and their cytotoxic activity. However, after a previous infection withE. cuniculi, CD8 T lymphocytes were more effective in killing macrophages infected withE. cuniculi.

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Section: Discussionmentioning

confidence: 99%

Contribution of B-1 cells in the cytotoxicity of CD8 T lymphocytes in encephalitozoonosis

de Oliveira,

Perez,

Alvares-Saraiva

et al. 2023

Preprint

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Section: Methodsmentioning

confidence: 99%

“…Five thousand events were acquired for each experimental condition and the corresponding images were analyzed using the IDEAS 5 image-analysis software. The internalization score (IS) was then computed as previously described [41] by using the equation below: During the early screening, the normalized IS was plotted. It was the calculated ratio of the IS measured at 37˚C divided by the IS at 4˚C.…”

Section: Antibody Characterization By Flow Cytometry and Internalizationmentioning

confidence: 99%

An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family

et al. 2022

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Background Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier. Method We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry. Results 12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized β1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnβ1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues. Conclusions This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.

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“…While LAMP-1 comprises 50% of all lysosomal membrane proteins and is widely used as a cell surface marker of lymphocyte activation and degranulation, its exact role remains uncertain. 23 , 24 LAMP-1 is a physiologically essential protein involved in stabilizing lysosomes and regulating autophagy to prevent embryonic lethality. Previous studies have demonstrated limited cell surface expression of LAMP-1 in normal tissues and moderate-to-high membrane expression in a number of breast, colorectal, gastric, prostate, lung, and ovarian tumors, 24 making it a target of interest for oncology applications.…”

Section: Introductionmentioning

confidence: 99%

“… 23 , 24 LAMP-1 is a physiologically essential protein involved in stabilizing lysosomes and regulating autophagy to prevent embryonic lethality. Previous studies have demonstrated limited cell surface expression of LAMP-1 in normal tissues and moderate-to-high membrane expression in a number of breast, colorectal, gastric, prostate, lung, and ovarian tumors, 24 making it a target of interest for oncology applications. Some evidence point to a role for LAMP-1 in tumor progression.…”

Section: Introductionmentioning

confidence: 99%

Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold

Pruvost

Mathieu

DuBois

et al. 2023

mAbs

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Delineating the precise regions on an antigen that are targeted by antibodies has become a key step for the development of antibody therapeutics. X-ray crystallography and cryogenic electron microscopy are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, they are labor-intensive and a successful outcome is not guaranteed. We used deep mutational scanning (DMS) of the human LAMP-1 antigen displayed on yeast surface and leveraged next-generation sequencing to observe the effect of individual mutants on the binding of two LAMP-1 antibodies and to determine their functional epitopes on LAMP-1. Fine-tuned epitope mapping by DMS approaches is augmented by knowledge of experimental antigen structure. As human LAMP-1 structure has not yet been solved, we used the AlphaFold predicted structure of the full-length protein to combine with DMS data and ultimately finely map antibody epitopes. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one of the two antibodies with a LAMP-1 luminal domain. Finally, we used AlphaFold models of non-human LAMP-1 to understand the lack of mAb cross-reactivity. While both epitopes in the murine form exhibit multiple mutations in comparison to human LAMP-1, only one and two mutations in the Macaca form suffice to hinder the recognition by mAb B and A, respectively. Altogether, this study promotes a new application of AlphaFold to speed up precision mapping of antibody–antigen interactions and consequently accelerate antibody engineering for optimization.

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Complementary epitopes and favorable developability of monoclonal anti-LAMP1 antibodies generated using two transgenic animal platforms

Cited by 8 publications

References 27 publications

Contribution of B-1 cells in the cytotoxicity of CD8 T lymphocytes in encephalitozoonosis

Contribution of B-1 cells in the cytotoxicity of CD8 T lymphocytes in encephalitozoonosis

An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family

Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold

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