Class-Switching of B Lymphocytes by DNA and Cell Immunization for Stereospecific Monoclonal Antibodies against Native GPCR

To develop efficient applications of monoclonal antibodies for therapeutic purposes, stereospecific recognition of the target antigens is needed. DNA immunization is one of the best methods for sensitizing B lymphocytes that can produce conformation-specific antibodies. Here we verified the class-switching of monoclonal antibodies by DNA immunization followed by cell immunization for the generation of stereospecific monoclonal antibodies against native G protein-coupled receptor (GPCR) using the optimized stereospecific targeting (SST) technique. This technology facilitates the efficient selection of sensitized B lymphocytes through specific interaction with the intact antigen via B-cell receptors (BCRs). We demonstrate that multiple DNA immunizations followed by a single cell immunization in combination with a longer sensitization period (three to four months) are an appropriate sensitizing strategy for the generation of IgG-type stereospecific monoclonal antibodies by class-switching, and the characteristics of antibody production could be transferred to hybridoma cells provided by the optimized SST technique.

Self-assembling synthetic nanoadjuvant scaffolds cross-link B cell receptors and represent new platform technology for therapeutic antibody production

Host antibody responses are pivotal for providing protection against infectious agents. We have pioneered a new class of self-assembling micelles based on pentablock copolymers that enhance antibody responses while providing a low inflammatory environment compared to traditional adjuvants. This type of “just-right” immune response is critical in the rational design of vaccines for older adults. Here, we report on the mechanism of enhancement of antibody responses by pentablock copolymer micelles, which act as scaffolds for antigen presentation to B cells and cross-link B cell receptors, unlike other micelle-forming synthetic block copolymers. We exploited this unique mechanism and developed these scaffolds as a platform technology to produce antibodies in vitro. We show that this novel approach can be used to generate laboratory-scale quantities of therapeutic antibodies against multiple antigens, including those associated with SARS-CoV-2 and Yersinia pestis, further expanding the value of these nanomaterials to rapidly develop countermeasures against infectious diseases.

Intratumoral T cell clonality and survival in a randomized phase II study of preoperative autophagy inhibition in combination with gemcitabine and nab-paclitaxel treatment in patients with resectable pancreatic cancer.

e16001 Background: Autophagy is a cell survival mechanism that is upregulated in pancreatic ductal adenocarcinoma (PDAC). PDAC autophagy results in an altered metabolic phenotype that promotes tumor progression, chemotherapeutic resistance, and immune evasion. Methods: We previously completed a randomized phase II clinical trial of preoperative gemcitabine-nab-paclitaxel with (PGH n = 34) and without (PG, n = 30) autophagy inhibition in patients with resectable and borderline resectable PDAC, which demonstrated increased Evans Grade histopathologic and serum CA 19-9 response with autophagy inhibition (IRB 13-074, NCT01128296 ). Utilizing the resected FFPE tumor specimens from evaluable patients, we completed paired multiplex immunohistochemistry (CD4, CD8, FOXP3, CD20, CD68, Pan-CK) and T & B cell receptor RNA sequencing to assess the intratumoral adaptive immune response and correlates of outcome. Results: Autophagy inhibition increased the number of infiltrating CD8 T cells (1133±490 vs 712±460 average cells per high power field, p = 0.01), CD8:CD20 ratio (2.22±3.1 vs 0.96±1.1, p = 0.02) and reduced the CD4:CD8 ratio (2.04±0.87 vs 3.01±2.09, p = 0.03). No effect was observed on the number of immature or mature germinal center-like tertiary lymphoid structures (TLS), though the number of TLS correlated with increased infiltration of CD4 T cells (r = 0.40, p < 0.001), T-regulatory cells (r = 0.26, p = 0.03) and CD20 B cells (r = 0.65, p < 0.001). Although the total number of productive T and B cell receptors increased with autophagy inhibition (167217±105961 vs 97339±5,1628, p = 0.02), no apparent effects were observed on Vαβ TCR or BCR IgH, Igκ, Igλ clonality. Independent of treatment, intratumoral CD8 counts were associated with an improved CA 19-9 response (r = 0.32, p = 0.04) and in a subset of short term ( < 2 years, n = 17) and long term ( > 4 years, n = 10) survivors (LTS), a lowered CD4:CD8 ratio was identified in LTS (1.83±0.63 vs 2.8±0.90, p = 0.01). Dominance of B cell receptors was a prominent feature of the immune repertoire in all patients (average expression: Vα 0.6%, Vβ 0.8%, IgH 18.9%, Igκ 32.3%, Igλ 47.2%) with an IgA skewed immunoglobulin class switching (mean 63% of all BCRs). Increased αβ T cell receptor clonality above the median level was associated with a CA 19-9 response (r = 0.37, p = 0.06) and greater overall survival (median OS 38.3 vs 19.3 months, p = 0.02), indicative of possible tumor specific clonal expansion. Conclusions: Preoperative autophagy inhibition increased the number of tumor infiltrating CD8 T cells in patients with localized pancreatic cancer. Intratumoral αβ T cell receptor clonality was associated with CA 19-9 response and improved overall survival. Combination treatment regimens increasing PDAC specific CD8 responses are warranted. Clinical trial information: NCT01978184.

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A

ABSTRACT Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (VH3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific VH3-IgM via a mechanism requiring CD4+ T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors. IMPORTANCE The LysM domain is found in all kingdoms of life. While their function in mammals is not known, LysM domains of bacteria and their phage parasites are associated with enzymes that cleave or remodel peptidoglycan. Plants recognize microbe-associated molecular patterns such as chitin via receptors endowed with LysM-containing ectodomains. In plants, such receptors play equally important roles in defense and symbiosis signaling. SpA of S. aureus carries a LysM domain that binds glycan strands of peptidoglycan to influence defined B cell responses that divert pathogen-specific adaptive immune responses.