This review focuses on providing insights into the structural basis and clinical relevance of LFA-1 and VLA-4 inhibition by peptides and small molecules as adhesion-based therapeutic strategies for inflammation and autoimmune diseases. Interactions of cell adhesion molecules (CAM) play central roles in mediating immune and inflammatory responses. Leukocyte function-associated antigen (LFA-1, alpha(L)beta(2), and CD11a/CD18) and very late antigen (VLA-4, alpha(4)beta(1), and CD49d/CD29) are members of integrin-type CAM that are predominantly involved in leukocyte trafficking and extravasation. LFA-1 is exclusively expressed on leukocytes and interacts with its ligands ICAM-1, -2, and -3 to promote a variety of homotypic and heterotypic cell adhesion events required for normal and pathologic functions of the immune systems. VLA-4 is expressed mainly on lymphocyte, monocytes, and eosinophils, but is not found on neutrophils. VLA-4 interacts with its ligands VCAM-1 and fibronectin (FN) CS1 during chronic inflammatory diseases, such as rheumatoid arthritis, asthma, psoriasis, transplant-rejection, and allergy. Blockade of LFA-1 and VLA-4 interactions with their ligands is a potential target for immunosuppression. LFA-1 and VLA-4 antagonists (antibodies, peptides, and small molecules) are being developed for controlling inflammation and autoimmune diseases. The therapeutic intervention of mostly mAb-based has been extensively studied. However, due to the challenging relative efficacy/safety ratio of mAb-based therapy application, especially in terms of systemic administration and immunogenic potential, strategic alternatives in the forms of peptide, peptide mimetic inhibitors, and small molecule non-peptide antagonists are being sought. Linear and cyclic peptides derived from the sequences of LFA-1, ICAM-1, ICAM-2, VCAM-1, and FN C1 have been shown to have inhibitory effects in vitro and in vivo. Finally, understanding the mechanism of LFA-1 and VLA-4 binding to their ligands has become a fundamental basis in developing therapeutic agents for inflammation and autoimmune diseases.
A highly productive chemically defined fed-batch process was developed to maximize titer and volumetric productivity for Chinese hamster ovary cell-based recombinant protein manufacturing. Two cell lines producing a recombinant antibody (cell line A) and an Fc-fusion protein (cell line B) were used for development. Both processes achieved product titers of 10 g/L on day 18 under chemically defined conditions. For cell line B, the use of plant derived hydrolysates combined with the optimized chemically defined medium increased the titer to 13 g/L. Volumetric productivities were increased from a base line of about 200 mg/L/d to about 500 mg/L/d under chemically defined conditions and as high as 700 mg/L/d with cell line B using plant derived hydrolysates. Peak cell densities reached greater than 20E6 vc/mL, and cell viabilities were maintained above 80% on day 18 without the use of antiapoptotic genes or temperature shift. A rapid compound screening method was developed to effectively test positive factors within 72 h. Peak volumetric oxygen uptake rates (OUR) more than tripled from the baseline condition. Oxygen demand continued to increase after maximum cell density was reached with a maximal OUR of 3.7 mmol/L/h. The new process format was scaled up and verified at 100 L pilot scale using reactor equipment of similar configuration as used at manufacturing scale.
A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales.
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