Discovery and Control of Succinimide Formation and Accumulation at Aspartic Acid Residues in The Complementarity-Determining Region of a Therapeutic Monoclonal Antibody.

PURPOSE: Succinimide formation and isomerization alter the chemical and physical properties of aspartic acid residues in a protein. Modification of aspartic acid residues within complementarity-determining regions (CDRs) of therapeutic monoclonal antibodies (mAbs) can be particularly detrimental to the efficacy of the molecule. The goal of this study was to characterize the site of succinimide accumulation in the CDR of a therapeutic mAb and understand its effects on potency. Furthermore, we aimed to mitigate succinimide accumulation through changes in formulation. METHODS: Accumulation of succinimide was identified through intact and reduced LC-MS mass measurements. A low pH peptide mapping method was used for relative quantitation and localization of succinimide formation in the CDR. Statistical modeling was used to correlate levels of succinimide with basic variants and potency measurements. RESULTS: Succinimide accumulation in Formulation A was accelerated when stored at elevated temperatures. A strong correlation between succinimide accumulation in the CDR, an increase in basic charge variants, and a decrease in potency was observed. Statistical modeling suggest that a combination of ion exchange chromatography and potency measurements can be used to predict succinimide levels in a given sample. Reformulation of the mAb to Formulation B mitigates succinimide accumulation even after extended storage at elevated temperatures. CONCLUSION: Succinimide formation in the CDR of a therapeutic mAb can have a strong negative impact on potency of the molecule. We demonstrate that thorough characterization of the molecule by LC-MS, ion exchange chromatography, and potency measurements can facilitate changes in formulation that mitigate succinimide formation and the corresponding detrimental changes in potency.

Development of a multiplexing potency assay using upconverting nanoparticles-based luminescence resonance energy transfer.

A homogeneous particle-based immunoassay using upconverting nanoparticles (UCNPs) has been developed for multiplexing potency analysis of two different therapeutic monoclonal antibodies (mAbs) in a fixed-combination formulation.The UCNP, considered as the best donor lumiphore for luminescence resonance energy transfer (LRET), offers long lasting excitation state and increased signal-to-noise (S/N) ratio due to low autofluorescence effect and light scattering from near infrared (NIR) excitation. In this study, the dose-response curves for each therapeutic mAb were generated using two distinct UCNPs. This proof-of-concept LRET-based immunoassay demonstrated a novel approach for increasing testing throughput and analyzing the potency of mixed therapeutic mAbs in co-formulated products.

Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.

Protective efficacy of intranasal cold-adapted influenza A/New Caledonia/20/99 (H1N1) vaccines comprised of egg- or cell culture-derived reassortants.

Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes. However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures. To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt). Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent. When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n. with 7 x 10(10) pfu of NC/wt. In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD50) was less than 1.2 x 10(4) pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells. Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n. vaccines.

Comparative analysis of the effects of packaging signal, transgene orientation, promoters, polyadenylation signals, and E3 region on growth properties of first-generation adenoviruses.

First-generation adenovectors have been developed for gene therapy and vaccine applications. The construction of these adenovectors has entailed the use of numerous types of expression cassettes. It has long been known that first-generation adenovectors can be rescued more easily and to higher titers with some transgenes than with others. This study has systematically shown that there can be marked differences in growth properties of recombinant adenovectors attributable to the use of promoters, the orientation of the transgene within the E1A/E1B-deleted region, and the inclusion of the E3 region. In addition, we had demonstrated the benefit of extending the packaging signal region to include elements V, VI, and VII. The effects of the complete packaging region were studied by plasmid competition studies between original and modified adenovectors. Similar competition studies between E3(+) and E3(-) adenovectors were performed and showed that the E3(+) vector had a growth advantage over its E3(-) counterpart. By making various changes, we have enhanced the growth capacity of our recombinant adenovector by more than 3-fold under serum-free and cell suspension growth conditions. Along with this enhanced growth, our adenovectors have maintained their genetic stability after 21 successive passages in cell culture. This increased robustness will be critical when adapting first-generation recombinant adenovectors to commercial production.

Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene.

Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Rapid method for the characterization of 3' and 5' UTRs of influenza viruses.

A T4 RNA ligase based strategy is demonstrated that allows for the full characterization of 3' and 5' UTR regions of negative strand RNA viruses. Negative strand RNA viruses such as influenza have 3'OH and 5'P terminal ends that are capable of being ligated using T4 RNA ligase. Each segment can form a mixture of linear concatamers between like and different viral segments or can itself form a circular structure upon ligation. RT-PCR can then be performed on these circular RNA segments using gene specific primers subsequently allowing for the characterization of the true terminal sequence for each viral segment. The UTR regions of a number of influenza virus strains were defined accurately using this approach.

Hexon gene switch strategy for the generation of chimeric recombinant adenovirus.

The usefulness of adenovirus as a vehicle for transgene delivery is limited greatly by the induction of neutralizing anti-adenoviral immunity following the initial administration, thereby resulting in shorter-term and reduced levels of transgene expression. In this paper, we outline a strategy for the generation of recombinant Ad5-based adenovectors that have undergone a complete hexon exchange in an effort to circumvent pre-existing anti-vector humoral immunity. Eighteen different chimeric adenoviral vectors (from subgroups A, B, C, D, and E) have been constructed using a combination of direct cloning and bacterial homologous recombination methods. However, only chimeric Ad5-based constructs in which the hexons from Ad1, Ad2, Ad6, and Ad12 are incorporated in place of the Ad5 hexon were successfully rescued into viruses. Despite several attempts, the remaining fourteen chimeric adenovectors were not rescuable. In vivo rodent studies using transgenes for human immunodeficiency virus type 1 (HIV-1) gag and secreted human alkaline phosphatase (SEAP) suggest that the Ad5/Ad6-gag chimera (wherein Ad5 hexon was replaced with that of Ad6) is able to evade neutralizing antibodies generated against Ad5 vector efficiently. However, it appears that cross-reactive cytotoxic T lymphocytes (CTL) may also play a role in controlling in vivo infectivity of Ad5/Ad6-gag chimera. The Ad5/Ad12 chimera was found to be extremely ineffective in the i.m. delivery and expression of HIV-1 gag in mice compared to the Ad5/Ad6 construct. Implications of these results will be discussed.