The use of an anti-CD40 agonist monoclonal antibody during immunizations enhances hybridoma generation.

ABSTRACT Herein we describe the use of an agonistic anti-murine CD40 MAb as a B cell proliferative agent to enhance the generation of monoclonal antibodies (MAbs) in Balb/c mice. While hybridoma technology has been validated repeatedly over the decades, little work has been described to improve upon the overall numbers of in vivo B cells and specific antibodies obtained from a fusion. To begin to address this situation, strategies to boost B lymphocyte yields for hybridoma production were employed. Anti-CD40 agonist antibodies have been reported to activate and amplify human resting B lymphocytes in vitro, resulting in increased cell numbers available for the generation of human hybridomas or B cell clones. An agonistic anti-murine CD40 MAb was administered to immunized mice 3 days prior to splenic harvest, and B lymphocyte yields were found to be approximately 2-fold higher in treated animals when compared to untreated animals. Moreover, the resulting hybridoma fusions using lymphocytes from treated animals yielded 5- to 10-fold more antigen reactive hybrids when compared to untreated animals. This novel addition to conventional approaches utilizes the proliferative effects of agonistic anti-CD40 MAbs to markedly enhance monoclonal antibody generation.

A rapid and efficient method for generating anti-variable region monoclonal antibodies using type-1 interferons as immune modulators.

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Herein we describe a novel immunization method that utilizes type 1 interferons (IFNs) as immunomodulators coupled with an agonistic anti-CD40 mAb as a B cell proliferative agent to drive immune responses. This novel protocol allows for rapid and robust mAb reagent generation without the use of conventional protein-denaturing adjuvants. The use of IFNs allowed for the generation of comparable and in some cases, increased numbers of anti-variable region mAbs in a dramatically shorter timeframe. This IFN based, immunostimulatory approach utilizing a non-denaturing adjuvant, likely presents conformational epitopes and may optimize the humoral response for the rapid generation of anti-variable region reagents to therapeutic mAb candidates.

A rapid and efficient in vivo method for determining the biologic efficacy of monoclonal antibodies in animal models of cancer.

The selection of efficacious anti-tumor monoclonal antibodies (MAbs) for biological applications is a lengthy and labor-intensive process. In vitro characterization of one hybridoma fusion may reveal large numbers of tumor antigen-specific hybridomas. Very often, many of these tumor-specific antibodies need to be assessed in vivo using several different murine xenograft tumor prevention models to determine biological efficacy. The production and purification of sufficient quantities of many antigen-specific hybridomas is time-consuming, and several months can pass between initial determination of MAb specificity and bioactivity. Moreover, many tumor-specific MAbs selected using in vitro binding studies have no in vivo anti-tumor efficacy. These studies describe an in vivo screening method either to eliminate non-efficacious MAbs or to rank-order several tumor-specific MAbs in an expeditious manner. Proof-of-concept studies were conducted using two hybridomas secreting fully characterized neutralizing human anti-tumor MAbs (CNTO MAbs). Nu-/nu- mice were injected with CNTO MAb-secreting hybridoma cells in Matrigel cell matrix, followed by injection of target human tumor cells 4 days later (when circulating CNTO MAbs were detected in serum). Both the tumor take-rate and the mean tumor volumes were reduced significantly in mice treated with CNTO MAbsecreting hybridomas compared with mice treated with non-antibody-secreting cells. A panel of human antitumor antigen-specific MAbs with unknown biological efficacy was then evaluated by this method. The hybridomas exhibited a varied pattern of anti-tumor protection, indicating that some hybrids were secreting neutralizing anti-tumor MAbs, while others appeared to be less efficacious. These studies demonstrate a rapid, biologically relevant "yes/no" in vivo screening method for the evaluation of anti-tumor antigen MAbs.

A rapid method for generating and characterizing anti-variable region monoclonal antibodies.

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the therapeutic mAb candidate in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Previously, we described a novel immunization method using type 1 interferons (IFNs) coupled with an agonistic anti-CD40 mAb to drive immune responses (Staquet et al., Human Antibodies 15 (2006), 61-69). This protocol allows for rapid and robust generation of large panels of anti-variable region mAbs. In order to quickly characterize and efficiently identify optimal anti-variable region antibody pairs early in the hybridoma process using crude supernatants, an inexpensive, high-throughput ELISA method was developed. The ability to rapidly identify appropriate mAb pairs will save resources by eliminating the time-consuming and laborious process of subcloning irrelevant hybridomas.