The CD4-binding domain of human immunodeficiency virus type 1 (HIV-1) gp120 elicits antibodies that are present in infected human sera. Monoclonal antibodies that recognize the HIV-1 gp120 CD4-binding domain have been isolated. Some of these antibodies can neutralize laboratory-adapted strains of HIV-1 and probably mediate neutralization by interfering with virus binding to its cellular CD4 receptor. However, most anti-CD4 binding domain antibodies do not neutralize primary HIV-1 isolates. We used primary HIV-1 isolates in an infectivity reduction assay to test the uniquely derived anti-CD4 binding domain recombinant human monoclonal antibody, IgG1b12. All of the tested HIV-1 isolates were neutralized by this antibody. Additional studies indicated that neutralization of a primary isolate with MAb IgG1b12 did not require continuous exposure of human peripheral blood mononuclear cell cultures to the antibody. Finally, a complete IgG1 molecule of an in vitro-selected b12 FAb mutant with a > 400-fold increase in affinity was assembled, expressed in mammalian cells, and evaluated in the infectivity reduction assay in comparative studies with the parent IgG1b12 antibody. The mutant did not retain the level of primary isolate neutralization potency that was a property of the parent molecule. Thus, we confirm that recombinant IgG1b12 has a unique specificity, and that it can neutralize all primary isolates tested in human PBMC cultures in vitro.

A panel of monoclonal antibodies (MAbs) which neutralize human papillomavirus type 11 (HPV11) in the athymic mouse xenograph neutralization assay and bind HPV11 virus-like particles (VLPs) has been described. We recently presented evidence that the Gly131-Tyr132 residues of the major capsid protein L1 confer type 11-specific binding. However, residues distally located on the primary L1 sequence also were shown to affect binding. This poses the question whether the epitope is principally centered in the region of Gly131-Tyr132 or, alternatively, is comprised of diversely located residues which come into proximity only upon proper assembly. We analyzed the result of numerous substitutions located between Tyr123 and Val142 of the HPV11 L1 sequence. We show that substitutions at five positions result in loss of binding for one or more of these MAbs by an enzyme-linked immunosorbent assay which measures antibody binding to VLPs. We demonstrate that binding of these MAbs is redirected to HPV16 VLPs which harbor eight type 11-like substitutions within the homologous region. Three of these substitutions did not affect binding when individually substituted in HPV11 but yet were still required to transfer binding to substituted HPV16 VLPs. The results demonstrate that the epitope for this class of neutralizing MAbs, although conformational and requiring VLP assembly for presentation, principally lies along a 20-residue stretch of the L1 major capsid protein. This targets the region for evaluation of the possibility of receptor binding and suggests possibilities for the design of peptide inhibitors of virus infectivity.

Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism.

The addition of concentrated nutrient solutions in fed-batch cultures of recombinant GS-NSO cells delays the onset of apoptosis by 24 to 48 hours and decreases the specific rate of apoptotic death approximately two-fold. This decrease in apoptosis appears primarily responsible for the increase in culture longevity that results in five- to ten-fold increases in final product concentrations in fed-batch cultures.

The anti-gp41 virus neutralizing monoclonal antibody 2F5 was infused into chimpanzees, which were then given an intravenous challenge with a primary human immunodeficiency virus type I (HIV-1) isolate. In two control animals, the infection was established immediately, as evidenced by positive cell-associated DNA PCR and serum RNA PCR tests within 1 week, seroconversion by 4 weeks, and development of lymphadenopathy in this acute phase. Serum RNA PCR tests were negative in one of the two antibody-infused animals until week 8 and in the other antibody-infused animal until week 12; both animals seroconverted at week 14. The peak of measurable virus-specific serum RNA was delayed until week 16 in one antibody-infused animal. Virus-specific RNA in the other animal did not reach levels comparable to those in the other animals through 1 year of follow-up studies. Virus was isolated from the week 16 blood sample from one infused animal. Virus was not isolated from peripheral blood of the second animal but was isolated from lymph node cells taken at week 36. The infection of untreated chimpanzees with this primary isolate appears robust. Use of this isolate should widen the scope of possible experiments in the chimpanzee model. This antibody infusion study indicates that neutralizing antibody, when present at the time of challenge, affects the timing and level of infection and remains influential after it can no longer be detected in the peripheral circulation. It is possible that preexisting, neutralizing antibodies (passively administered or actively elicited) affect the course of acute-phase virus replication in humans. It remains to be established whether these immunologically mediated early effects will influence the course of HIV-1 disease.

Empirical scanning of natural or engineered peptide sequences for functional residues is inherently dependent upon efficient expression of large numbers of individual sequence variants to assay their relative functional potency. The insect baculovirus system has been widely used for expression of viral coat proteins, but it generally requires prior isolation and expansion of a plaque-purified recombinant viral stock to generate useful quantities of self-assembled virus-like particles. In search of a more rapid means of expression of analytical levels of the L1 coat protein of cottontail rabbit and human type 11 papilloma-viruses, we found that even brief transient cotransfection of insect cells with baculovirus plasmid transfer vectors and viral DNA yielded assembled particles that were immunologically indistinguishable from particles obtained with plaque-purified viral stocks. Within six days of plasmid/viral DNA cotransfection of Sf9 cells, at least 1-2 micrograms of assembled L1 particles/100-mm plate could be demonstrated, which proved more than sufficient to assay functionality. Transient cotransfection of insect cells should provide general utility for rapid high-level expression of sets of protein sequence variants, as well as other sequence-scanning applications such as sequence optimization in protein engineering.

The oligomerization by chemical cross-linking of a recombinant human antiviral monoclonal antibody (MAb), r447-1, and its characterization are described. This MAb binds to an epitope residing in the hypervariable V3 region of the envelope protein (gp120/160) of HIV-1. A dimeric form of this MAb displays enhanced avidity and was found to be capable of neutralizing a greater variety of lymphoid cell culture-adapted HIV-1 variants and HIV-1 primary isolates than its monomeric form. The superior binding and breadth of reactivity of this antibody suggests it may have utility as a therapeutic and/or prophylactic agent, if it possesses an appropriate safety and immunogenicity profile.

As reviewed in this report and exemplified in a case study, recombinant antibodies (Abs) can be expressed to very high levels in lymphoid cells by optimizing gene expression and process conditions. Myeloma and hybridoma cell lines have been constructed that secrete functionally active, recombinant antibodies at up to 1.5 pg/c/d in non-amplified cell lines and up to 100 pg/c/d in gene-amplified cell lines. Processes have been developed that achieve final titers in fed-batch culture of 1-2 g/L, or reach volumetric productivities in perfusion culture of 0.2-0.6 g/L/d. In the specific example of the production of a humanized anti-CD18 Ab, h1B4, amplified NS0 cell lines were developed that secreted Ab at up to 50 pg/c/d. Periodic addition of concentrated nutrient solutions in fed-batch culture allowed production of the Ab to 1.8 g/L. In perfusion culture, titers of up to 0.5 g/L were obtained with maximum volumetric (i.e. reactor volume) productivities in excess of 0.6 g/L/d.

We have developed a novel strategy to decrease the antibody:antigen off-rate which we call optimized residue substitution. This strategy employs alanine substitution to first identify residues non-optimal for binding, as evidenced by a decrease in off-rate upon alanine replacement. These positions are then individually randomized to all amino acids, and the best replacement for each position determined. Finally, a construct which combines all optimized substitutions is generated and evaluated. We applied this strategy to the heavy chain CDR3 of P5Q, a scFv antibody which recognizes an epitope on the V3 loop of HIV gp120. We identified two amino acid substitutions that together decrease the off-rate by nearly ten-fold. The contributions by the two substitutions were near additive, indicative of independent affects on binding. We suggest that this strategy can be generalized to strengthen protein:ligand and protein:protein interactions in other systems.

We have used a glutamine synthetase expression vector system1 to generate NSO myeloma cell lines expressing high levels of recombinant antibody genes. Medium depletion analysis has been used to design customized fed-batch processes which maintain the cells in viable condition for periods of three to four weeks and yield titers of nearly 1 g/L for unamplified cell lines and 1.8 g/L for amplified cell lines. We have analyzed the quality of the MAb produced in a such a three week fed-batch process and have found consistent protein integrity, molecular weight and antigen binding kinetics. IEF analysis revealed only minor changes with time, indicating that a slight degree of deamidation had occurred. Glycoform analysis, on the other hand, revealed substantial changes during the process. In particular, we observed that MAb with incomplete, mannose-terminated, glycans at the H-chain, N-linked glycosylation site increasingly accumulates in the medium as the fed-batch process proceeds. This accumulation of incompletely glycosylated MAb does not result from either cell lysis or leakage from non-viable cells. Rather, an increasing percentage of the MAb secreted by the viable NSO cells late in fed-batch culture is of the high-mannose type. Finally, we show that the fed batch protocol can be modified to allow for more complete glycosylation of the secreted antibody.

A sensitive, high-throughput assay has been developed which measures hyaluronidase activity in a microplate format. In this assay, hyaluronic acid is suspended in agarose in a microtiter well, the plate is incubated with the hyaluronidase solution, and the undigested hyaluronic acid is precipitated with cetylpyridinium chloride. The precipitate blocks light transmittance and therefore an increase in the visible light transmitted correlates with the amount of digested hyaluronic acid. Using this assay, as little as 0.05 units of hyaluronidase activity can be detected. The assay is highly reproducible and can be run with commercially available reagents. Hyaluronidase activity is easy to quantitate using a microplate reader and this format allows large numbers of samples to be assayed.

The variable heavy and light chain genes of a monoclonal, IgM, anti-MAG antibody from a patient with neuropathy were inserted into expression vectors containing the gamma and kappa constant regions respectively and co-transfected into monkey kidney CV1P cells. The expressed antibody had the same antigenic specificity but significantly lower avidity than the native IgM, anti-MAG, antibody as detected by ELISA. When the variable heavy chain gene of the anti-MAG antibody was co-transfected with the variable light chain gene from another monoclonal, IgM, anti-MAG antibody, a fully assembled antibody was expressed as determined by a trapping ELISA, but it did not bind to (MAG) or to sulfated glucuronic acid paragloboside, indicating that both heavy and light chains contribute to the binding activity.