Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

ABSTRACT

Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.     

Generation,characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (K_D) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.     

A monoclonal antibody recognizes a 65 kDa higher plant membrane polypeptide which undergoes cation-dependent association with callose synthase in vitro and co-localizes with sites of high callose deposition in vivo

Summary A monoclonal antibody (MAb) capable of immobilizing detergent-solubilized UDP-glucose: (13)--glucan (callose) synthase activity from higher plants has been selected and characterized. On Western blots this MAb recognizes a polypeptide of about 65 kDa found in membranes isolated from a variety of plant sources. The polypeptide recognized by this MAb does not appear to bind the substrate UDP-glucose, and evidence is presented which indicates that this polypeptide associates with the enzyme complex in a cation-dependent manner under conditions where the callose synthase assumes a larger size. Indirect immunofluorescence localization with this MAb was positive with sieve plates of cucumber (Cucumis sativus) seedlings, and with plasmodesmata of onion (Allium cepa) epidermal cells, both being sites of localized, stress-induced callose deposition.Abbreviations BSA bovine serum albumine - DMSO dimethylsulf-oxide - DTT dithiothreitol - FITC fluorescein isothiocyanate - HB Hepes buffer - HBS Hepes buffer plus 0.15 M NaCl - IgG immuno-globulin G - MAb monoclonal antibody - MSB microtubule stabilizing buffer - NP 40 Nonidet P 40 - PBS phosphate buffered saline - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - UDP uridine diphosphate - UV ultraviolet