In Vitro Testing Has Demonstrated the Neutralizing Capability of Our Monoclonal Antibody Clone 3

     The ability of HIV to mutate renders anti-retroviral therapies ineffective against HIV in many cases. Because the Company's monoclonal antibody therapies target a portion of the virus that does not change, such mutations are not expected to affect the monoclonal antibody therapy.
     Significant testing demonstrates the basis for this expectation. Specifically, in in vitro tests conducted in five independent laboratories, CLONE 3 has been tested against 43 clinical HIV isolates (strains) of the virus. CLONE 3 has demonstrated that it successfully neutralizes 41 of them (100% effective against over 95% of the HIV strain against which it was tested).
     Presently, CLONE 3 is the only human monoclonal antibody found to neutralize the Clade C isolate found in Africa, China and India. CLONE 3 also neutralizes the Clade B isolate that is predominate in North America and Europe. In view of these test results, the Company is moving forward with animal and human trials to establish CLONE 3 as a significant means for treatment of those that have HIV/AIDS and to produce a vaccine against HIV.
     These results were the product of testing in 5 independent laboratories. In tests conducted at the University of California at San Francisco (by Jay Levy, M.D.), CLONE 3 Antibody was shown to have in vitro neutralization effect against geographically distinct clinical HIV (primary) isolates. Research conducted at four additional international institutes has confirmed these results of in vitro neutralization. In total, the neutralizing capabilities of CLONE 3 have been verified at the following 5 laboratories:

1.   University of California, San Francisco, CA, USA (Jay Levy, M.D.)

2.   University of South Florida, Tampa, FL, USA (Kenneth Ugen, Ph.D.)

3.   Polymun Scientific, GmbH, Vienna, AUSTRIA, (Hermann Katinger, Ph.D.)

4.   Duke University, Durham, NC, USA (David Montefiori, Ph.D.)

5.   Dana Farber Cancer Institute (DFCI), Harvard

      Medical School, Boston, MA, USA, (Ruth Ruprecht, M.D., Ph.D.)











    In these tests, Clone 3 neutralized [at IC90] 41 of 43 (>95%) of the clinical HIV-1 group M, N, and O isolates. Clone 3 neutralized 3 of 3 group O HIV isolates tested at 10 µg/ml in PBMC-based assay. In another study of Clone 3 tested against one group N primary HIV-1 isolate, results indicated that the IC90 for Clone 3 versus the HIV primary isolate YBF30 was 3.72 µg/ml. Further, Clone 3 has also been demonstrated to effectively neutralize 4 of 4 virulent pediatric South African clade C isolates [ZA349, ZA562, ZA600, ZA737]; and at 10 µg/ml, neutralize [IC99] a pediatric Zambian clade C HIV-1157, as well as the simian immunodeficiency virus construct SHIV-1157ip. Therefore, of the 43 HIV isolates against which Clone 3 has been tested, it neutralized 41 (over 95%).
Clinical Evidence of Effectiveness of Clone 3
    Other laboratory evidence also supports the Company's conclusion that the Clone 3 antibody will neutralize HIV-1 in humans. This evidence includes the following five (5) laboratory findings in the publications listed:
- P.A. Broliden et al. reported that, in a group of HIV-infected infants born to HIV+ mothers, infants who lacked anti-KLIC (Clone 3) antibodies had a rapid progression to symptomatic AIDS.
Vanini et al. reported that deCreasing concentration of anti-KLIC (Clone 3) antibodies directly correlated with HIV disease progression.
- Loomis-Price et al., demonstrated that high antibody reactivity to the peptide containing the (Clone 3) immunogen epitope [KLIC] is associated with slow progression to AIDS.
- Dietrich et al. further validated the significance of the (Clone 3) epitope by reporting the presence of anti-KLlC antibodies identified in HIV+ blood plasma obtained from LTNPs who had normal T?cell counts, no opportunistic infections and were not taking any prescribed adjunct chemotherapeutics or anti-retroviral drugs.
- Cano et al. cited the BioClonetics publications on (Clone 3) mAb, noting that the human monoclonal anti-gp41 antibody was a neutralizing anti-HIV antibody that binds preferentially to a linear peptide in the immunodominant region. The authors found "a correlation between the presence of anti-linear peptide antibodies and endurance of infection".
    The National Institute of Health (NlH) studies, conducted by Anthony Fauci et al., head of the NIH, and published in Nature Medicine November 2001, provide further validation of the efficacy of this linear amino acid epitope, KLIC, as a protective active vaccine immunogen.
     As background for understanding how tests conducted by Dr. Fauci demonstrate that Clone 3 will be effective in humans, one must understand how Clone 3 actually neutralizes the HIV virus. Through collaboration with the Karolinska Institute in Stockholm, Sweden (Britta Wahren, M.D., Professor of Microbiology), the Company identified the linear minimal essential core epitope on the virus to which Clone 3 binds. As published in the Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology in 1996 (reference 3 in Appendix), this minimal core epitope is defined as the amino acid sequence KLIC, located on the portion of the virus designated as glycoprotein (gp) 41.
    In Dr. Fauci's primate phage-display peptide vaccine studies, primates were vaccinated with a peptide immunogen represented as the linear KLVC rather than KLIC. (Fauci et al., Nature Medicine, Volume 7, Number 11, November 2001, pp. 1225-1231). KLVC is a linear peptide having a conservative amino-acid substitution of valine (V) for isoleucine (I). KLIC occurs in the native HIV-1 gp41 amino acid sequence and is essentially seen as the same immunogen.
    The vaccinated primates produced an antibody [anti-KLVC] that is cross-reactive with linear peptide KLIC?an amino acid sequence on HIV gp41 (and SHIV gp41). When the primates were challenged with the SHIV virus [having the gp41 amino acid sequence KLIC], the vaccinated primates'although infected with SHIV were infected at a lower SHIV viral set point than the SHIV challenged non-vaccinated control primate. Moreover, the vaccinated primates retained normal T4 cell counts while the non-vaccinated control primate's T4 cell counts decreased and progressed to AIDS and death within 6 months of SHIV challenge. The KLVC vaccinated primates' health status were that of a Long-Term Non-Progressor (LTNP).
    These tests demonstrate that the Clone 3 active vaccine component prevented progression to AIDS-like illness, and also supports the conclusion that the peptide of the Clone 3 epitope (KLIC) will be effective as an active vaccine against HIV/AIDS.
    Additional evidence allows the company to project that its antibody will neutralize over 98% of the HIV viral strains (clades) worldwide based upon information confirmed by the U.S. Government?s National Laboratory HIV clinical isolate database at Los Alamos, NM. Specifically, to date, Los Alamos has currently reported 4,556 clinical HIV-1 variants or subtypes (February 2015). The company has determined that the site on the virus to which Clone 3 binds in the neutralization step (its epitope) is highly conserved in 98% of the HIV-1 viral strains. Based on this knowledge and other independent in vivo clinical-correlate evidence from five (5) additional independent research institutions, the company's proprietary antibody is expected to provide a safe immunotherapeutic long-term remission for people living with HIV/AIDS and an effective active vaccine that protects uninfected individuals from HIV infection.