Trispecific Antibody to HIV Developed; May Represent A Real Cure for AIDS.

[NNadir]

HIV, as many people know, is caused by a "retrovirus," that is a virus that does not contain DNA but rather RNA which is "reverse transcribed" into DNA in infected cells, whereupon the DNA activates machinery in order to construct new viral particles which ultimately rupture the cells, releasing more viruses.

The HIV virus thus offers two opportunities for transcription error: During transcription to DNA, and during formation of RNA for new viral particles. Moreover the HIV viral machinery contains no transcription error correcting mechanism. Mutations in HIV thus arise 10X faster than with DNA viruses.

It was my privilege to work, albeit in a peripheral sense, on the first several of the second class of anti-HIV drugs, the protease inhibitors in the mid 1990's; the first class was reverse transcriptase inhibitors like AZT. This class of drugs had a real impact on the disease; the survival of people like, for one case, Magic Johnson, is a testimony to their success.

The HIV protease cleaves viral zymogens, zymogens being proteins that are inactive until a part of each molecule cleaved by the protease, in this case an "aspartyl" protease that cleaves the zymogens at an aspartic acid residue, thus activating the viral proteins reverse transcriptase, integrase, and more of itself, the protease. Without this cleavage the viral proteins are inactive and the virus cannot function as a virus; it is inactivated, but not destroyed.

However, because of the rapidity of mutations in the virus, over ten billion viral particles are produced each day in an infected person with active AIDS, with a new generation of viruses being produced every 2.4 days at a rate of 140 generations per year, resistant strains of the virus can and do arise rapidly.

For the first generation of proteases developed in the 1990's, resistant strains had appeared for all of them by the year 2000.

The amino acid substitutions for the mutant strains to these drugs are listed here, where the letters refer to the codes for specific amino acids, and the numbers refer to the position in the HIV protease:

D30N: Nelfinavir. (Agouron/Pfizer).
M46I/I47V/I50V: Amprenavir (BMS).
L10R/M46I/L63P/V82T/I84V: Indinavir (Merck)
M46I/L63P/A71V/V82F/I84V: Ritinovir (Abbott).
Saquinavir: G48V/L90M (Roche)

The companies in the parentheses are the companies that developed these drugs.

(cf: Protein Science (2000) 9: 1898-1904)

Modern treatment for AIDS is not really curative; it is palliative and relies on a drug cocktail, a reverse transcriptase inhibitor (the class containing AZT), a protease inhibitor, and a third class, a CCR5 inhibitor known as a fusion inhibitor. It is hoped (and happily often observed) that the combination is effective, if expensive, with a failure to observe the regimen actually promoting the generation of resistant strains. (This is also true of other anti-infectives, such as antibiotics; however with antimicrobials such as antibiotics, the rate of evolution of resistant strains is slower.)

These drugs do not kill the virus; they inactivate it or in some (problematic) cases, slow its replication without actually halting it.

It is thus with some excitement that I came across this paper in the most recent issue of Science: Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques

(Xu, Pegu et al, Science 10.1126/science.aan8630: Final Page Numbers Not Yet Assigned) The paper was published by a consortium of scientists from the pharmaceutical company Sanofi, and a team of academic and government institutions, the latter type of institutions being under attack by the orange ignoramus in the White House and his fellow science hating enablers in Congress and his cabinet.

"Trispecific" means that the antibody has multiple "CDR's" or "Complementarity Determining Regions" designed to bind to different areas on cells. Antibodies are, of course, Y shaped proteins that mediate immune responses, and the CDR's are small sequences of amino acids in these proteins that recognize foreign or diseased cells and attach themselves to them result in their destruction or inactivation. Most antibodies are monospecific, designed to attack a single region of display on the foreign body. This protein, by contrast has been designed to simultaneous attack any of three different regions involved in HIV pathology; by doing so it reduces the avenues by which this wily virus can escape destruction and thrive.

From the introductory text of the paper:

A variety of broadly neutralizing antibodies (bnAbs) have been isolated from HIV-1 infected individuals (1–3), but their potential to treat or prevent infection in humans may be limited by the potency or breadth of viruses neutralized (4, 5). The targets of these antibodies have been defined based on an understanding of the HIV-1 envelope structure (6–9). While bnAbs occur in selected HIV-1 infected individ-uals, usually after several years of infection, it remains a challenge to elicit them by vaccination because broad and potent HIV-1 neutralization often requires unusual antibody characteristics, such as long hypervariable loops, interaction with glycans, as well as a substantial level of somatic mutation. Strategies have thus shifted from active to passive im-munization to both protect against infection and to target latent virus (10–14). We and others have begun to explore combinations of bnAbs that optimize potency and breadth of protection, thus reducing the likelihood of resistance and viral escape (15–17). Antibodies directed to the CD4bs, MPER, and variable region glycans are among the combinations that so far provide optimal neutralization (18). In ad-dition, alternative combinations have also been investigated for the immunotherapy of AIDS, by directing T lymphocytes to activate latent viral gene expression and enhance lysis of virally-infected cells (19, 20). Given that multiple antibodies may help to reduce the viral replication that sustains chronic HIV-1 infection, we report here the generation of multi-specific antibodies designed to increasing the efficacy of HIV therapy.

Although individual anti-HIV-1 bnAbs can neutralize naturally occurring viral isolates with high potency, the per-centage of strains inhibited by these mAbs varies (21, 22). In addition, resistant viruses can be found in the same patients from whom bnAbs were isolated, suggesting that immune pressure against a single epitope may not optimally protect or treat HIV-1 infection. We hypothesized that the breadth and potency of HIV-1 neutralization by a single antibody could be increased by combining the specificities against different epitopes into a single molecule.

Glycans are sugar signaling molecules bound to proteins. (They are very challenging molecules with which to work, although spectacular advances in the their characterization are under way.) "Epitope" is the sequence of amino acids that defines the CDR.

Some technical text relating to the design of the antibodies:

To achieve our goal, we used a previously undescribed trispecific Ab format. Three specificities were combined by using knob-in-hole heterodimerization (24) to pair a single arm derived from a normal immunoglobulin (IgG) with a double-arm generated in the CODV-Ig. A panel of bnAbs was evaluated, including those directed against the CD4bs that included VRC01 and N6, as well as PGT121, PGDM1400 and 10E8 (fig. S1). A modified version of the latter, termed 10E8v4, was used because of its greater solubility (25). We first determined which bispecific arms showed the best potency, breadth and yield. This screening analysis revealed that combinations which contained PGDM1400, CD4bs, and 10E8v4 showed the highest level of production and greatest potency of neutralization (fig. S2).

We then evaluated different combinations of single arm and double arm specificities from PGDM1400, CD4bs, and 10E8v4 Abs for their expression levels and activity against a small panel of viruses (fig. S3), leading ultimately to the identification of trispecific antibodies VRC01/PGDM1400-10E8v4 and N6/PGDM1400-10E8v4 as lead candidates. When analyzed against a panel of 208 viruses (18) and com-pared to the parental antibodies alone, the highest neutrali-zation potency and breadth was observed with N6/PGDM1400-10E8v4, with only 1 of the 208 viruses showing neutralization resistance...

The molecule was able to prevent AIDS infections in a model animal, macques. This said, a molecule of this design has not been tested in humans, although human volunteers tolerated a bispecific analogue quite well. It is not known if the antibodies will not generate ADA's or "antidrug antibodies" which are antibodies against antibodies. This risk is always associated with protein drugs, despite their broad success in treating disease and saving lives.

The authors comment thusly:

While further human trials are needed to assess the full potential of the trispecific Ab platform, the data from the NHP challenge study described here, as well as the pre-vious experience in humans with bispecific Abs (44), sug-gests that the approach merits further clinical investigation. Studies in HIV-infected subjects, alone or in combination with other immune interventions, will address the potential of trispecific Abs to provide durable protective immunity against infection or sustained viral control in HIV infected subjects during drug holidays or in the absence of antiretro-viral therapy.

The experimental details of the project are described in the supplementary information, which is apparently open sourced and is here: Supplementary Information

Here one may learn that the technology making this work possible is genetic engineering.

To wit:

Trispecific antibodies were produced by transient transfection of 4 expression plasmids into Expi293 cells using ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Briefly, 25% (w/w) of each plasmid was diluted into Opti-MEM, mixed with pre-diluted ExpiFectamine reagent for 20-30 minutes at room temperature (RT), and added into Expi293 cells (2.5x106 cells/ml). An optimization of transfection to determine the best ratio of plasmids was often used to produce the trispecific antibody with good yield and purity. 4-5 days after transfection, the supernatant from transfected cells was collected and filtered through 0.45 µm filter unit (Nalgene). The trispecific antibody in the supernatant was purified using a 3-step procedure…

All protein drugs are, in fact, GMO, and if you have a politically motivated hatred of genetic engineering and all things GMO because you get your "science" from reading Greenpeace pamphlets, Greenpeace being an organization that hates science with the same intensity as say, Republicans, these kinds of drugs are not for you.

Nevertheless, this is exciting and encouraging work.

Enjoy the weekend.

[Warpy]

1. Please let it be true.

I got out of nursing school just as GRID was starting to hit hard. I lost friends. I have nightmares about the young guys we could do nothing for except keep them clean and try to make them more comfortable as they died within a few hours.

HIV is diabolical.

[Nitram]

2. Wonderful news. If it is affordable.

[NNadir]

3. Regrettably, this kind of drug is likely to be expensive. It may be cheaper, however, than...

...requiring a lifetime of the triad therapy used now.

I know a fair bit about what goes into making a drug like this. The requirements are unbelievable. The work is performed by people who have spent a quarter of century - or more - of education and training - and they'd like to be paid for their effort - and a tremendous amount of equipment and equipment maintenance is required to safely and reliably provide these materials.

I've seen some questionable things in the pharmaceutical industry in decades of experience, to be sure; but I've also seen tremendous courage and tremendous sacrifice.

There were some hard times in my life involved with this work, even some very real danger, but when I look back, I realize that there are people who are alive because of the efforts of millions of people like me, and not just alive, but people who would be blind without that work, people who would have otherwise seriously disabled or crippled.

You cannot imagine a deeper sense of satisfaction.

People somehow think somehow that it's all about profit and greed. It isn't.

The question we need to ask as a society is how much is a human being, particularly a young human being worth. Are a hundred human lives worth the salary of some hoople quarterback on a football field where people are beating each other's brains to a pulp?

I think so, but I'm not necessarily in the majority.

It is interesting to note, in any case, that drugs that cure diseases can be less profitable than drugs that patients need to take their entire lives. This reality has led me to see some of the less satisfying things I've seen; it is why, for example, that the development of new antibiotics is lagging against the evolution of resistant strains.