AIDSWEEKLY Plus, 9 Sep 1996
Daniel J. DeNoon, Senior Editor

The recombinant human monoclonal antibody (mAb) is called IgG1 b12.

"This antibody is being developed for immunoprophylaxis and immunotherapy of HIV-1 infection in humans," said Paul W.H.I. Parren of The Scripps Research Institute, La Jolla, California.

Parren described development and testing of IgG1 b12 at the XI International Conference on AIDS, held July 7-12, 1996, in Vancouver, British Columbia, Canada.

Parren and colleagues began their search for the antibody by generating a large number of antibody fragments (fAbs) from the genes of patients infected with HIV. These fAbs were then displayed on phages (expressed in E. coli) to create a so- called "library" that could be tested for anti-HIV activity.

"The way we prepare our libraries is starting from bone marrow from immunized donors, and in this case these are all long-term, asymptomatic, seropositive individuals," Parren said.

"We take the bone marrow, isolate the RNA and then amplify the genes for the antibodies - the antibody light/heavy chain - and express it in a phage display factor such that a fAb fragment is expressed on the surface of the phage. We then obtain libraries which we pan against an [HIV] antigen such that the fAb on the phage surface will bind to the antigen by virtue of its specific binding."

This process yielded many fAbs that were reactive with the HIV-1 gp120 envelope and gp41 transmembrane glycoproteins.

"Only a very small subset neutralize HIV-1 with high potency," Parren said. "One such antibody we have identified as directed against the CD4 binding site."

This fAb, designated b12, was then reconverted into a whole antibody by cloning it into the context of the IgG1 heavy chain and expressing it into Chinese hamster ovary (CHO cells) to create the mAb IgG1 b12.

IgG1 b12 was able to neutralize 32 of 40 HIV-1 clade B patient isolates, with neutralization defined as a 90 percent reduction in in vitro infectivity at a physiologically relevant antibody dose of <50 (micro)g).

Remarkably, IgG1 b12 was also able to neutralize about 55 percent of HIV-1 isolates from other clades.

Parren and colleagues then tested IgG1 b12 in an in vivo model: SCIDhu mice whose immune systems were repopulated with human peripheral blood lymphocytes (PBL). The mice were treated with IgG1 b12 and then injected with the infectious SF2 strain of HIV-1.

"Increasing doses of IgG1 b12 gives increasing protection with complete protection at a dose of 100 (micro)g per mouse injection," Parren said. "This amounts to about 5 mg/kg."

Because SF2 is a laboratory strain of HIV-1, the researchers went on to test various patient isolates in the SCIDhu mouse model.

"You can also protect these mice completely against infection with primary isolates, although now we need some higher doses: we have to go up to 50 mg/kg to get complete protection," Parren reported.

"Interestingly, we can also now see a post-exposure prophylaxis. You can give the antibody up to eight hours after infection and still find complete protection for the mice."

In the course of their studies Parren and colleagues made an interesting discovery: in vitro assays of antibody potency underestimate the amount of antibody needed for protection in vivo. This was true not only for HIV-1[SF2] but also for primary isolates.

"You need about up to 100 times higher concentration of antibody in the serum to get the same protection as the amount of antibody needed to get 90 percent of the virus in vitro," Parren said.

"We think this pertains to vaccine research because this would indicate that a candidate vaccine should actually be able to induce a high titer of neutralizing antibodies against primary isolates as tested in the currently used in vitro assays to actually be able to protect in vivo."

Parren noted that the ability of IgG1 b12 to neutralize multiple HIV strains has implications for HIV vaccine design.

"Broad neutralization by b12 indicates that there is a conserved feature on the envelope glycoprotein of infectious virions, or at least on the majority of these virions," he said. "We know this feature is associated with the CD4 binding site. We also know that we have many other antibodies reactive with the CD4 binding site that only weakly neutralize. We do also know that the affinity of these weakly neutralizing antibodies to monomeric gp120 is exactly identical, or very similar, to that of b12.

"So what distinguishes b12 from these other antibodies? This becomes apparent when you look at binding to a native oligomer. ... There is about 100-fold higher affinity for b12 compared to the other CD4-binding-site antibodies with weaker neutralizing abilities."

Parren listed several conclusions from the IgG1 b12 studies:

• Recombinant antibody IgG1 b12 defines a neutralizing epitope on the majority of primary isolates of HIV-1.
• Neutralization of HIV-1 does not correlate with binding to recombinant envelope gp120 or gp160 molecules, but does correlate with binding to native envelope on infected cells or virus.
• A vaccine for HIV-1 should present a native multimer to the immune system.
• A substantial proportion of antibody responses to HIV envelope react weakly if at all to native virus.

Parren noted that efforts are underway to map the epitope for b12. He said that preliminary findings suggest that the V2 region of HIV-1 gp120 may be involved.

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