Immunotherapy with Leukocyte Interleukin, Injection for Human Papilloma Virus (HPV) Induced Cervical Dysplasia In HIV Patients Taylor G 1,2, Ely L 1, Wolff T 2, Davis C 1,2, Ioffe O 1, Talor E 3, Khanna N 2, Redfield R 1,2, and Tramont E 4. Institute of Human Virology, Baltimore, MD 1 ; University of Maryland, Baltimore, MD 2 CEL-SCI Corporation, Vienna, VA 3 and NIAID, Bethesda, MD 4

ABSTRACT HPV causes greater than 90% of pre-cancerous cervical lesions leading to cervical cancer. Co- infection with HPV in HIV-infected patients is common and cervical cancer is considered an AIDS defining illness. Local immune responses associated with inflammation can clear cervical HPV infection. Therefore, we hypothesized that local immunity with Leukocyte Interleukin, Injection (Multikine TM ) a natural mixture of pro-inflammatory cytokines and chemokines [containing: IL-2, IL- 1ß, IFN-gamma, TNF-alpha, GM-CSF, among others], can improve and/or cure HPV-induced cervical disease. Preliminary results from this on-going Phase I, dose-escalating study, in which 200 IU (as IL- 2) [lowest dose tested] was administered by endocervical/perilesional injections times over a 6 week period to 5 dually infected (HPV/HIV) patient volunteers with moderate to severe cervical dysplasia. All of the patient volunteers tolerated the injections well and without any associated serious adverse reactions. Five out of five patient volunteers had clinical improvement by colposcopic visualization; 3/4 had no evidence of residual dysplasia on punch biopsy 7-8 weeks after the final injection. Biopsies on the remaining patient volunteer had no change in the histopathology. One patient was lost to follow-up. Quantitative HPV PCR data are pending. Leukocyte Interleukin, Injection did not increase peripheral blood HIV levels by PCR. Dose-ranging studies are proceeding. A pivotal trial is in the planning stage..

HPV-ASSOCIATED CONDITIONS Skin warts Anogenital warts Cervical cancer (>90% HPV positive) Vulvar cancer Penile cancer Respiratory papillomas Conjunctival papillomas Oral cavity lesions

HPV and HIV Immunosuppression inhibits clearance of papillomaviruses Immunosuppression promotes HPV reactivation Prevalence of HPV is ~ 5 times higher in the HIV-infected than in the general population Usually more persistent HPV infection Patients have a greater number of precancerous lesions More likely to be infected with multiple types (independent risk factors for progression) Correlation between lower CD4 levels and a higher number of HPV types

HPV/HIV CO-INFECTION HPV detection in HIV infected women may be as high as 83% HIV infected women undergoing colposcopy after an abnormal PAP smear, the detection of HPV is greater than 90% 20% of dually infected women with no evidence of cervical disease will develop cervical disease within 3 years

HPV/HIV CO-INFECTION HIV infected women are less likely to clear HPV and hence are at a greatly increased risk for developing cervical cancer caused by HPV infection. Recurrence rates after traditional therapy is much greater in HIV-infected women than in those who are HIV negative; i.e., >50%, including those whose HIV is under good control.

INCLUSION CRITERIA Signed informed consent years of age HIV/HPV infected women with LSIL, HSIL on histopathology following colpo-directed biopsy for an abnormal PAP smear No prior therapy with any biological response modifiers No immunosuppressive therapy in the previous 3 months Not taking aspirin or NSAID in the previous 7 days CD4 >50 and HIV RNA <55,000, on or off Highly Active Antiretroviral Therapy (HAART)

EXCLUSION CRITERIA Invasive cervical carcinoma Concurrent, untreated vaginal/cervical infection/lesion History of asthma, requiring medical treatment within the previous 24 months Any medical condition that at the discretion of the investigator precludes participation

PROCEDURE Leukocyte Interleukin is injected into the submucosa Daily injections X 5 for 2 weeks; 2 week rest period; then, daily injections X 5 for 2 weeks Follow-up cervical biopsy 7-8 weeks after the last injection

LEUKOCYTE INTERLEUKIN, INJECTION Interleukin-2 GM-CSF TNF-alpha IFN-gamma IL-1-beta Added material: Human Serum Albumin, USP added as a carrier/stabilizer

RESULTS of 200 IU No complaint of pain, pelvic discomfort, or significant bleeding. One volunteer was hospitalized for lactic acidosis, secondary to antiretroviral drugs. Clinical response - 5/5 improved, visually There was complete clearing, visually, in 3/4. One volunteer was lost to follow-up. Biopsy results - In 3/4, there was resolution of dysplasia by histopathology. In 1/4, the histopathological results were unchanged. HPV results are pending

CONCLUSION Leukocyte Interleukin, Injection at the 200 IU dose appears to be safe. In a small number of subjects Leukocyte Interleukin appears to be effective at improving cervical lesions, visually and histopathologically. It is still too early to determine if there is sustainable clearance of the HPV infection. Leukocyte Interleukin, Injection had no detrimental effect on plasma HIV-1 RNA.