Patent Application
20050175634
The present invention relates to immunotherapy of epithelial tumors, particularly tumors that are induced by infectious agents, particularly viruses, and particularly papilloma viruses. The immunotherapy of the present invention relates to the intralesional injection of at least one antigen into a epithelial tumor of a subject in need of treatment, wherein the subject to be injected had previously developed a naturally-occurring delayed type hypersensitivity (DTH) response to the antigen. The immunotherapy of the present invention is particularly useful for treating verrucae, condyloma, cervical carcinoma and bowenoid papulosis.
Verrucae or human warts are benign epidermal tumors caused by human papilloma virus (HPV). HPV is a member of the papovavirus family. HPV is a non-enveloped double-stranded deoxyribonucleic acid (DNA) virus that replicates in epithelial cells. This means that HPV has a predilection for the mucosa and skin. Currently, there are more than 70 distinct HPV types recognized each with at least a 10% genome difference. Because papillomaviruses tend to be host-specific and HPV has not been successfully grown in culture, the majority of the research with papilloma virus has been conducted with animal papillomaviruses. (37) Papillomaviruses are considered responsible for several forms of viral infection ranging from relatively benign warts of the skin or mucous membranes to cancer, the most significant being cervical cancer. Papillomaviruses are known to infect mammals, including humans, rabbits, canines, felines, bovines and equines. Papillomaviruses are highly species and tissue-specific, and are characterized by a specific mode of interaction with the squamous epithelia they infect. These viridae colonize various stratified epithelia like skin and oral and genital mucosae, and induce the formation of self-limited benign tumors, known as warts or condylomas.
Verrucae are transmitted usually by direct human-to-human transmission with a variable incubation period and clinical presentation. Symptomatic disease includes flat warts (verruca plana), common warts (verruca vulgaris), filiform warts, palmar and plantar warts, condyloma acuminata (venereal warts), myrmecia, focal epithelial hyperplasia, epidermodysplasia verruciformis, laryngeal warts, cervical cancer and anogenital cancer. (1) Warts in and of themselves cause significant morbidity and warrant aggressive therapy.
Verrucae have reached epidemic or even pandemic proportions. In 1990, there was an estimated 79% lifetime risk of acquiring HPV with an annual incidence of 8%. (1) Decreasing the burden of visible wart in a community would be expected to decrease infectivity and help stem the epidemic. Aside from the clinical dermatological burden that HPV causes in our society, it is well known that there is an oncogenic burden caused by HPV. HPV is thought to play a causative role in the formation of cervical carcinoma and anogenital carcinoma in immunocompetent hosts. (27, 29-31, 37, 39) HPV is known to be important in the pathogenesis of carcinomas (squamous cell carcinoma mainly) of immunosuppressed individuals, such as those who are iatrogenically immunosuppressed, infected with the human immunodeficiency virus, affected with epidermodysplasia verruciformis, and after organ transplant. (27, 29-31, 39).
In renal transplant patients with actinic keratoses and squamous cell carcinoma, there is evidence that the epithelial tumors are HPV-induced. Also, there is a clear association between cervical carcinoma and HPV infection.
With the exception of flat warts that have a fine almost imperceptible roughness on the surface, warts show fingerlike projections or rough papular projections and scaling which correspond to the papillomatosis noted histopathologically. The verrucous surface is an important diagnostic feature of warts. Since dilated dermal blood vessels are present within the projections, warts commonly bleed when irritated. The diagnosis is usually made clinically but the diagnosis can be confirmed with biopsy, polymerase chain reaction, or in-situ hybridization.
HPV infection clearly is associated with cancer. Squamous cell carcinoma has been shown to contain HPV-16. (1) Dysplastic periungual papillomas have been shown to have HPV-57. Epidermodysplasia verruciformis is a genetic condition of altered cell-mediated immunity in which affected individuals develop chronic HPV infection and squamous cell carcinoma. There are other states of immunosuppression, both congenital and acquired, that lead to heightened HPV infection and HPV-associated malignancies. (3) The risk of malignant transformation may or may not be decreased with treatment. (1) At a minimum, treatment to decrease the spread of HPV may prevent others from developing a cancer-promoting infection. (3)
Finally, small warts are easier to treat than large warts. The best study of the natural history of warts suggests that only 40% of patients with warts would have all of their warts disappear without treatment after two years. (8) Therefore, it is more likely than not that over several years, warts will continue to enlarge, spread, and become more resistant to treatment. Better to destroy clinically visible warts when they are small and immediately treat any recurrent lesions than to wait and see which will disappear and which will pose more serious treatment problems.
There is no perfect treatment for warts. An antiviral wart antibiotic or vaccine is being researched but does not exist for treatment today. Currently, there are destructive, immunomodulative, chemotherapeutic and other modalities used to treat HPV-associated tumors. (5)
Patients often present to the doctor with a wart after they have suffered with it for some time. They frequently have tried over-the-counter and herbal remedies. The mechanism of action of all of the currently available therapies is either destruction (e.g., cryotherapy), chemotherapeutic (e.g., bleomycin) or immunomodulation (e.g., interferon) in nature. There is a multitude of therapies currently available for the treatment of HPV infection. The following is a list of the most widely employed wart therapies: Liquid nitrogen, Cantharidin (a blistering agent derived from Spanish fly extract), surgical excision, Carbon Dioxide (CO2) Laser Ablation, Vascular Lesion Laser, electrosurgery, bleomycin, glutaraldehyde, formaldehyde, podophyllin, topical retinoic acid, Interferon-α (IFN-α), Imiqimod (a non-nucleoside heterocyclic amine that is a potent inducer of IFN a in humans), Dinitrochlobenzene (DNCB), Diphencypropenone (DPCP), radiation therapy, ultrasound, hypnosis, and accupunture. (1-57) There is a 12% to 56% failure rate with podophyllotoxin used for condyloma and a 50% failure rate of external genital warts with IFN-αplus cryotherapy. (1) The rate of recurrence of common warts after surgical excision is 15-30%, after laser ablation is 5-10% and after liquid nitrogen is 39%. (1, 2) The high reported rates of recurrence (the true recurrence may even be higher) may be due to inherent or functional lack of immunity to HPV by the patient.
Recognizing the effectiveness of cryotherapy, there remains a pressing need for additional therapies in the treatment of verrucae. Liquid nitrogen exerts its effects by epidermal and dermal cellular destruction. The effectiveness of cryotherapy is operator dependent. The duration of the freeze-thaw cycle is important since too little liquid nitrogen provides minimal effect whereas too much liquid nitrogen results in adverse effects. The expected adverse effects include scar, pain, burning, edema and possibly ulceration. (23) Many warts are too large for comfortable use of cryotherapy.
Human interferon-α is known to be useful in the treatment of several viral infections, including chronic hepatitis B virus and herpes zoster. U.S. Pat. No. 5,165,921 discloses treating condyloma acuminatum, commonly referred to as genital warts, known as benign, fibro-epithelial tumors associated with various papilloma viruses, with a topical formulation of interferon-α. Additionally, warts can also be treated by direct injection of interferon into the warts. (16,17) Immunotherapy using an unrelated agent to cause an immune response is certainly not a new idea. This technique has proven successful for the treatment of melanoma, multiple myeloma, chronic myeloid leukemia, and bladder carcinoma. (62-66)
Immunotherapy in the treatment of warts has been attempted in the past with trials of sensitization to dinitrochlorbenzene (DNCB) and other chemicals. DNCB is now known to be mutagenic in the Ames assay and therefore rarely used. This immunotherapy approach is also problematic. Sensitization must first be attempted (often unsuccessfully) in order to develop a brisk immune response upon topical application of DNCB to the wart. In contrast to the topical application of DNCB, the immunotherapy of the present invention injects the antigen directly into the wart or tumor, thus evoking a stronger immune response.
Thus, a need exists for an epithelial tumor therapy that provides successful and long lasting results. The present invention is based upon the discovery that successful resolution of epithelial tumors requires a specific immunologic response to the causative agent of the epithelial tumors. The present invention is based upon the discovery that standard antigens currently employed in anergy panels with a high prevalence of reactivity in human and other mammals result in the elicitation of a DTH response. This response which at first glance appears to be non-specific for the causative agent of the epithelial tumor, in fact, results in a very specific response when the standard antigen to which the subject has previously reacted, is directly injected into the epithelial tumor. The results of the studies show that the present immunotherapy offers significant and long lasting cure rates directly related to the induction or stimulation of existing immunity as compared to cryotherapy. The present method takes advantage of this prior sensitization to an unrelated infectious agent through intralesional injection to evoke a strong secondary immune response against the causative agent of the epithelial tumor, such as the papillomavirus. The data obtained from studies support that the present immunotherapy method results in a significant number of patients achieving complete resolution of warts. Additionally, some patients receiving the present immunotherapy to a specific wart or tumor have experienced resolution of untreated warts at sites distant from the site of injection, which suggests that the present immunotherapy induces or stimulates existing papillomavirus specific immunity. This resolution took place slowly and in a timeframe associated with the injection of the primary verruca. One can conclude that specific immunity to the causative agent of the tumor was stimulated or induced by the immunotherapy of the primary wart which resulted in a systemic response targeting the causative agent, such as HPV for example, throughout the skin. This observation heightens the potential therapeutic value of the immunotherapy protocol for treating epithelial tumors, as well as for other causative agents of associated conditions. Papillomavirus-specific immunity is an example of such a causative agent.
The proposed hypothesized mechanism of action of intralesional injection of an antigen is unveiling of the HPV antigen and epitope spreading. (60) This action will lead to a generalized systemic immune response to HPV and can lead to resolution of all present and future clinical tumors caused by HPV.
The present immunotherapy includes diagnosing the subject having epithelial tumors or skin derived tumors, such as melanoma, then testing the subject with antigens from an anergy panel by injecting intradermally small amounts of anergy panel antigens, such as killed mumps virus protein extract, candida extract, trichophyton extract or comparable antigenic extracts, and determining the reaction of the subject to the antigens. The antigen that elicits the strongest cutaneous DTH response in the subject is selected and injected directly into the epithelial tumor over a period of time at designated intervals until the tumor resolves.
In one embodiment, the present invention relates to a method of treating epithelial tumors or skin derived tumors, such as melanoma, comprising injecting an effective amount of a pharmaceutical composition containing at least one antigen into the tumor, wherein the antigen induces or is capable of inducing an cutaneous DTH response in the subject prior to the injection of the antigen into the tumor. This immunotherapy is particularly useful in treating epithelial tumors, such as cutaneous tumors, including warts or verrucae, that are induced by or related to papillomavirus.
In a further embodiment, the invention relates to a method of treating epithelial tumors comprising injecting the tumors with at least one antigen and at least one additional cytokine or colony stimulating factor. The antigen and cytokine or colony stimulating factor may be in the same pharmaceutical composition, thereby injected simultaneously, or may be in two different pharmaceutical compositions and injected sequentially. The cytokine may be interferon-α, interferon-β, interferon-γ, interleukin-2 or interleukin-12. The colony stimulating factor may be granulocyte-macrophage colony stimulating factor. The subject treated by the present immunotherapy is preferably a mammal selected from a human, rabbit, canine, feline, bovine, equine or ovine subject but also could be avian.
In another embodiment, the invention relates to a pharmaceutical composition for treating epithelial tumors comprising at least two antigens, each of which induces or is capable of inducing a cutaneous DTH response in the subject prior to the injection of the antigens into the tumor, and a pharmaceutically acceptable carrier formulated for injection into an epithelial tumor. This pharmaceutical composition may further comprise a cytokine or a colony stimulating factor.
In another embodiment, the invention relates to a syringe and needle suitable for use in injecting the above described pharmaceutical compositions into an epithelial tumor, wherein the pharmaceutical composition is stored within the syringe. In another embodiment, the invention relates to a kit comprising a syringe and needle suitable for injecting the above described pharmaceutical compositions into an epithelial tumor. The kit further comprises one or more containers containing the above described antigens and/or a cytokine or colony stimulating factor.
In one embodiment, the present invention relates to a method of treating epithelial tumors comprising injecting an effective amount of a pharmaceutical composition comprising at least one antigen into the tumor, wherein the antigen induces or is capable of inducing a cutaneous DTH response in the subject prior to the injection of the antigen into the tumors. The epithelial tumor can be induced by a virus, preferably a papillomavirus, and more preferably by a human papillomavirus. Papillomaviruses of other species of mammals can induce epithelial tumors in dogs, cows, horses and other species susceptible to papillomaviruses, such as birds.
The method of the present invention is directed to treating an epithelial tumor in a subject. This tumor can include both benign and malignant tumors. Preferably, the tumor is a verruca (wart), a condyloma (a genital wart), a cervical carcinoma, bowenoid papulosis, a laryngeal papilloma or epidermodysplasia verruciformis but can also include skin derived tumors, such as melanomas. The verruca to be treated can be of a number of subtypes, such as verruca vulgaris, verruca plantaris, verruca palmaris or verruca plana.
The antigen that is injected into the epithelial tumor is selected for its ability to induce a cutaneous DTH response in a subject. There is no currently available compound which is known to unveil HPV and procure an HPV specific systemic immune response. The term “unveiling HPV” as used herein refers to generating an immune response that recognizes and attacks HPV. Injection of cytokines, such as interferon upregulates the immune responses to HPV but does not cause a cutaneous DTH response and interferon is not an antigen. The present invention is not intended to cover the direct injection of interferon into the tumor without either simultaneous or sequential injection of an antigen. Thus, one aspect of the present invention is directed to simultaneous or sequential injection of the antigen and a cytokine or CSF into the tumor. Injection of bleomycin inhibits DNA synthesis. Topical application of DNCB or other contact sensitizers acts by elicitation of contact hypersensitivity at the site of administration. All of these act in a nonspecific manner and do not lead to resolution of distant warts. Intralesional injection of an antigen into one epithelial tumor is shown herein to lead to resolution of distant epithelial tumors.
The induction of the DTH response in the subject is tested by intradermally injecting small amounts of anergy panel antigens and determining the reaction of the subject to the antigens. The antigen that elicits the strongest response in the subject is selected and injected directly into the epithelial tumor over a period of time at designated intervals until the tumor resolves. If more than one antigen gives a strong response measured by an area of induration of at least 5 mm in diameter, then more than one antigen can be selected for injection into the epithelial tumor.
The antigen can be an antigenic determinant of the antigen, a hapten or an epitope that is responsible for inducing the cutaneous DTH response in the subject. The antigen is preferably a biological substance but it can be a chemical substance if the chemical is not carcinogenic or mutagenic as measured by the Ames test or any other art recognized assay that identifies substances as carcinogenic or mutagenic. It is important that no antigens categorized as carcinogenic or mutagenic be injected into the tumors treated by the present method. The antigens useful in the present invention can be of viral, fungal or bacterial origin. It is preferred that the antigens useful in the present invention are derived from naturally occurring infectious agents to which the majority of the subjects of the treated species have naturally acquired immunities or to which the subject to be treated has been immunized against. In other words, the preferred antigens to use are those viral, fungal and bacterial antigens to which most healthy subjects are already currently sensitized. A positive skin test denotes prior antigenic exposure and DTH immunity. Injection of the antigen into the epithelial tumor or skin derived tumor will therefore lead to an immune response that is composed of various known and unknown immune modulators. The immune response may consist of white blood cells including lymphocytes and Langerhans cells as well as the cytokines they secrete. These cytokines are not limited to interferon-α. They include other immune modulators such as other interferons, interleukins, leukoreglins, and growth factors. Therefore, the immune response from injection of an antigen is much greater than that elicited by injection of interferon-α.
Such preferred antigens for treating humans are allergenic extracts for intradermal testing available from a number of different companies, such as Bayer Corporation, Elkhart, Ind. 46515, or as a skin test antigen, such as Mumps Skin Test Antigen USP available from Pasteur Merieux Connaught, Swiftwater, Pa. 18370. Preferred antigens useful to inject into a human epithelial tumor are mumps skin test antigen, candida extract and trichophyton extract, all of which are prepared in combination with a pharmaceutically acceptable carrier, such as isotonic saline and which are known to persons skilled in the art. A preferred candida extract is the Candida albicans Skin Test Antigen available from known commercial sources. The antigens used for injection into the epithelial tumor are preferably not composed of live agents but instead are preferably composed of killed or parts of agents, thus reducing the risk of contracting a disease caused by the live agents.
The present method of treating an epithelial tumor optionally can include injecting at least one additional pharmaceutical composition containing at least one cytokine or colony stimulating factor (CSF) into the tumor. This optional injection can occur simultaneously with or after the injection of the antigen. The CSF preferably is granulocyte macrophage colony stimulating factor, such as LEUKINE® (sargramostim), which is a recombinant human granulocyte macrophage colony stimulating factor (GM-CSF) in a injectable pharmaceutically acceptable carrier obtained from Immunex Corporation, Seattle, Wash. 98101. The GM-CSF boosts the number and function of the Langerhans cells in the epidermis and possibly the dermis. The Langerhans cells present antigen to naïve and memory cells, thus promoting Langerhans cell function, which in turn boosts the DTH response. Any of the cytokines, such as interferon-α, interferon-β, interferon-γ, interleukin-2 or interleukin-12 can be utilized to enhance the treatment of the epithelial tumor. Preferred interferons, such as interferon-α 2a, interferon-α 2b, and interferon-α N3, interferon-β 1a and interferon-β 1b and interferon-γ in pharmaceutically acceptable carriers are useful in the present method. ROFERON®-A is an example of an acceptable recombinant interferon-α 2a that is commercially available and useful in the present invention and which is obtained from Roche Laboratories, Nutley, N.J. 07110.
The method of the invention can utilize any device that injects the antigen into the epithelial tumor so that the injected solution enters at least the epidermis or the dermis of said subject. Particularly useful in the present method is a hypodermic needle or high pressure injection device sufficient for the antigen(s) to enter at least the epidermis or dermis of said subject. These devices and modes of injection can be used to deliver the antigen as well as the cytokine or colony stimulating factor to the epithelial tumor.
Thus, in one embodiment, the invention relates to a pharmaceutical composition comprising at least two of the above described antigens and a pharmaceutically acceptable carrier that has been formulated for injection into an epithelial tumor. Injecting at least two antigens increases the likelihood that the composition will induce a DTH response in the subject. The pharmaceutical composition may contain preservatives and other non-immunogenic additives, according to methods well known in the art. See, e.g. Remington's Pharmaceutical Sciences: Drug Receptors And Receptor Theory, (18th ed.), Mack Publishing Co., Easton, Pa. (1990). In another embodiment, such pharmaceutical composition may also contain one or more cytokines or colony stimulating factors, as described above.
In yet another embodiment, the invention relates to a syringe containing any of the above described pharmaceutical compositions, wherein such pharmaceutical compositions are stored in such syringe and wherein the syringe can be used for injecting the pharmaceutical compositions into an epithelial tumor. In another embodiment, the invention is directed to a kit which comprises one or more containers containing the above described antigens and/or cytokines. Such kit may also contain a syringe and needle suitable for injecting the antigens into an epithelial tumors. The kit may also contain appropriate instructions for use.
Preferably, the present method and pharmaceuticals treat a mammal. More preferably, a human is treated but the present method is useful for treating any mammal that is afflicted by epithelial tumors. Such other non-human mammals are dogs, cats, rabbits, cows or cattle, horses and sheep. Any non-human mammal that is susceptible to and contracts papillomavirus induced epithelial tumors is a subject that can be treated by the present method, such as birds.
In its preferred embodiment of treating humans having HPV-induced epithelial tumors or melanomas, the method of the present invention takes advantage of the prior sensitization to candida and mumps prevalent in the population. Candida and mumps were chosen over other antigens because they are FDA approved traditional DTH controls utilized for anergy testing. Additionally, persons skilled in the art also are familiar with the local induration and erythema that is expected with intradermal injection of these antigens. There are other antigens available for DTH testing, such as trichophyton, but that are not FDA approved for intradermal injection at this time that also would be appropriate for use in the present invention.
The present invention provides for both a prognostic instrument to predict response to standard therapy and to develop a novel treatment option for epithelial tumors, such as verrucae. Verrucae are often recalcitrant to multiple treatment modalities including liquid nitrogen, bleomycin, cantharidin, and podophyllin. Topical immunotherapy with dinitrochlorobenzene is known to be effective by elicitation of contact dermatitis. The present approach to immunotherapy utilizes standard antigens currently employed in anergy panels with a high prevalence of reactivity in humans and elicitations of a DTH response are utilized in our method. Furthermore, since untreated warts resolve with the present approach, elicitation of HPV-specific immunity should provide less relapse and longer remissions.
Any antigen or combination of antigens can be used in the present invention to treat epithelial tumors by intralesional injection of the antigens. Preferably the subject has a preexisting sensitivity to the antigen or antigens used such that the antigens induce a cutaneous delayed type hypersensitivity response in the subject (upon their first intralesional injection). But the method works as long as the antigen or antigens are capable of inducing a delayed type hypersensitivity response in the subject. If the subject does not have a preexisiting sensitivity, the subject will develop a sensitivity over the course of the injections so that the antigen induces a cutaneous delayed type hypersensitivity response on the second, third, or fourth injection.
In particular embodiments, the subject is a human. In particular embodiments, the antigen is an antigen to which the human has a preexisting sensitivity such that the antigen induces a cutaneous delayed type hypersensitivity response in the human subject.
The antigen may be, and typically is, unrelated to an infectious agent that causes the benign epithelial tumor.
In particular embodiments, the antigen is a trichophyton, candida, blastomyces, histoplasma, mumps, measles, rubella, smallpox, polio, diphtheria, tetanus, pertusis, staphylococcus, or streptococcus antigen. These are antigens to which much of the population of the U.S. has been immunized or has otherwise developed an immune response.
In other particular embodiments, the antigen is a trichophyton, candida, blastomyces, histoplasma, mumps, measles, rubella, polio, diphtheria, tetanus, pertusis, staphylococcus, or streptococcus antigen.
In particular embodiments, the antigen is a trichophyton, candida, or mumps antigen.
In particular embodiments the at least one antigen injected intralesionally that induces or is capable of inducing a delayed type hypersensitivity response in the subject includes at least two antigens or at least three antigens from the antigens listed above.
In particular embodiments, the at least one antigen injected intralesionally includes a fungal antigen and a viral antigen, a bacterial antigen and a viral antigen, or a fungal antigen and a bacterial antigen.
Preferably the antigen is not a mycobacterium antigen or a Bacillus Calmette-Guerin (BCG) antigen. These are used to develop immunity to tuberculosis. In the United States we test for exposure to or infection with tuberculosis by testing for a cutaneous delayed type hypersensitivity reaction to tuberculosis antigens. Exposure to a mycobacterium antigen or BCG antigen would cause a human subject to test positive for tuberculosis exposure thereafter and make it impossible or more difficult to determine whether the person really had been exposed to tuberculosis.
In other particular embodiments, the antigen is not a bacillus antigen.
The following description is the protocol for identifying the antigens to be used in the claimed method. After the diagnosis of epithelial tumors, such as verruca, such as is made in the subject, small amounts of antigens, such as mumps and candida, are separately injected intradermally on the surface of the skin, e.g., the volar forearm of a human subject. If the subject reacts to the antigen(s), intralesional injection with the antigen that elicited the greater response is utilized.
While verrucae may respond to destructive mechanisms such as cryotherapy or laser ablation, complete resolution ultimately requires an HPV-directed immunologic response. The present immunotherapy will result in significant and long lasting cure rates directly referable to the induction or stimulation of existing HPV-specific immunity. The support for this hypothesis resides in two preliminary observations. First, a significant number of subjects achieved complete resolution of warts by the present immunotherapy. Second, some subjects receiving immunotherapy to a specific wart in the setting of multiple warts experienced resolution of untreated warts at distant sites.
The following pilot clinical trial was carried out to determine the efficacy of the method and compositions of the present invention. The diagnosis of a wart was made by clinical examination of one or multiple well-circumscribed, hypertrophic papillary tumors. (6) At least one hundred fifty volunteers between the ages of 3 years old and 85 years old are to be evaluated. To be included in the study, the subject must have been willing to comply with all of the requirements of the protocol, had the capacity to understand and provide detailed informed consent prior to enrollment, and been able to return to the evaluation site for all necessary visits. A subject was excluded with a history of Gell-Coombs type I allergy to mumps or candida antigens or with any condition or compliance issue which in the opinion of the investigators might interfere with adequate evaluation or safety, such as pregnancy, infection with the human immunodeficiency virus-1, iatrogenic immunosuppression, primary immunodeficiency, or generalized dermatitis.
Once diagnosed, an anergy panel was placed: 0.1 milliliter of 40 cfu/ml of Connaught manufactured Mumps Skin Test Antigen USP was injected into the intradermal aspect of the left flexor forearm and 0.1 milliliter of Alcon candida antigen was injected intradermally into the right flexor forearm. DTH reactivity was determined 48 hours after intradermal placement by measuring the induration, not the erythema, in millimeters. A reaction was considered positive for either antigen if induration extended at least 5 mm in diameter around the injection site. A reaction was considered negative if the area of induration was less than 5 mm. If there was a lack of DTH response to both antigens, then conventional treatment with cryotherapy was initiated. This treatment consisted of paring every hyperkeratotic wart followed by two freeze-thaw cycles of liquid nitrogen (LN2) for thirty seconds, each every three to six weeks until clinical clearance of the wart was obtained or for a total of ten treatments. If a positive DTH response to one or more antigens was elicited, the subject was randomized either to have conventional cryotherapy as above or therapy with the antigen that elicited the greater response. Randomization occurred according to order of acceptance into the study. That is, the first and subsequent odd numbered subjects received immunotherapy and the second and subsequent even numbered subjects received cryotherapy. Immunotherapy consisted of a titrated amount of the antigen that elicited the greater test response injected intralesionally and into the underlying dermis of only one, preferably the largest, wart. If the DTH response measured by induration was 5 mm to 20 mm then 0.3 ml of the antigen was injected. If the DTH response was 20 mm to 40 mm then 0.2 ml was injected. If the DTH response was greater than 40 mm then 0.1 ml was injected. Evaluation of the clinical response occurred in three-week intervals.
The response was considered complete when there was disappearance of the warts and return of the normal skin markings. The response was considered partial if the warts regressed in size. If there was no decrease in size, then no clinical response was deemed to have occurred. If there was no clinical response or a clinical response of less than 25% by visual examination after 10 treatments or 30 weeks then the study was concluded for that subject. However, if immunotherapy failed to show any sign of resolution of the verruca vulgaris (VV) lesion after three injections, immunotherapy was discontinued and cryotherapy initiated. Telephone follow-up occurred four months after the subject was considered free of warts.
The following Table A provides a flowchart of the protocol.
The data collected in this therapeutic arm of the trial was primarily descriptive in nature. The safety, tolerability, and technical feasibility of this novel technique was confirmed. Each subject was assigned a unique number and a chart. All of the charts were stored in a labeled folder that was stored at the physician workstation to ensure subject confidentiality. Comparative statistical analysis allowed the investigators to determine if there was a significant advantage to intradermal skin testing and treatment with candida and mumps antigens.
The summary of accumulated data is provided in Table B. A chi-square analysis or comparison of two proportions was used. Assuming a 15% difference in cure rates, alpha of 0.05, and power of 80%, this will require 150 subjects to be included in the study.
Lymphocyte Proliferation Assay:
Twenty subjects were included in a concomitant arm of the study to investigate systemic response to relevant antigens in those subjects receiving immunotherapy. This investigation included obtaining 20 milliliters of blood at the initial visit and after two injections with the antigens. Using standard techniques, these samples were examined after Ficoll separation of mononuclear cells to look for a statistically significant increase in T cell proliferation as measured by thymidine incorporation in response to mumps, candida and HPV epitopes. (61)
The data collected in the lymphocyte proliferation assay was analyzed quanitatively for statistically significant differences in T cell proliferative responses referable to immunotherapy.
Histologic Evaluation:
Biopsies were taken of a regressing wart to view the histologic changes. Immunoperoxidase was performed utilizing standard techniques. (58, 59) Markers to Langerhans cells (CD 1a), T cells (CD3), T helper/inducer cells (CD4), T cytotoxic/suppressor cells (CD8), B cells (CD20), interleukin 2 receptors (CD 25), and natural killer cells (CD56) were performed.
The data collected was descriptive in nature. The information demonstrated the nature and relative proportion of inflammatory cell infiltrate subsets.
The general utilization of immunotherapy with antigens is outlined:
Step 3 of the immunotherapy described above may be modified by intradermally injecting more than one antigen. Such a modification would remove the need to inject a number of the antigens separately. The goal of step 3 is to determine one or more antigens that induce a cutaneous DTH response in the subject, and in most cases, it is not necessary to know the specific antigen from a mixture of antigens that causes the cutaneous DTH response as long as the mixture is injected into the tumor.
Step 5 of the immunotherapy described above may be modified by injecting more than one antigen into the tumor. The concentrations and amounts of the antigen may be varied as can be determined by the skilled artisan, a dermatologist. For example, if 0.2 ml of one antigen would be indicated for injection depending upon the cutaneous DTH response, then 0.1 ml of each of two antigens may be used. This dosage would ensure that the absolute concentration of the antigens injected remain approximately the same. The size of the tumor limits the amount (liquid volume) of antigen(s) that can be injected into the tumor. It may be necessary to concentrate the antigen so that a similar dosage is present in a smaller volume. It is well within the skill of the artisan to determine modifications to this immunotherapy using the guidelines of step 5 for injection into the tumor.
Introduction
We and others have shown the effectiveness, in the treatment of common warts, of intralesional injection of antigen preparations of mumps, candida or trichophyton, normally used to assay the status of the cellular immune system via intradermal injection (67,68). In our earlier study, 74% of subjects receiving immunotherapy experienced resolution of the treated wart and 78% of the subjects with multiple warts experienced resolution of untreated, distant warts. 57% of subjects treated in the cryotherapy arm of the same study experienced resolution of the treated wart, while no distant wart responses occurred (67). In a separate report evaluating the effectiveness of this treatment in children, all of whom had failed two treatment modalities, we found that 47% of subjects responded to immunotherapy and 34% cleared untreated distant and anatomically distinct warts (69).
Herein, we extend our observations on intralesional injection of skin test antigens for common warts and report on a single-blinded, randomized and controlled trial comparing immunotherapy, immunotherapy+intralesional interferon alfa 2 b, intralesional interferon alfa 2b alone or saline. Data correlating treatment success or failure in individual subjects to HPV type, major histocompatibility complex (MHC) antigens and degree of pre- and post-treatment peripheral blood mononuclear cell (PBMC) proliferation in response to HPV antigens are presented.
Materials and Methods
The study protocol received approval from the institutional investigational review boards of the University of Arkansas for Medical Sciences, the Central Arkansas Veterans Healthcare System, Arkansas Children's Hospital and the General Clinical Research Center.
Patients and immunotherapy methods
Patients with the clinical diagnosis of 1 or multiple warts (or their guardians) provided informed consent. Patients were randomized after obtaining a positive intradermal pretest (see below) to one of four groups: (1) intralesional injection with mumps (Connaught, Swiftwater, Pa.; mumps antigen is not commercially available), candida (Bayer, Spokane, Wash.) or trichophyton (Alk—Abello, Round Rock, Tex.) skin test antigen preparation, (2) intralesional injection with mumps, candida or trichophyton skin test antigen preparation+interferon alfa 2b (1 million IU), (3) intralesional injection with interferon alfa 2b (1 million IU) or (4) intralesional injection with saline. Mumps antigen is not currently commercially available. Randomization was computer-generated and the sequence provided to the investigators in sealed envelopes. All subjects were tested for existing immunity to each antigen preparation by placing 0.1 ml intradermally into the skin of the forearm (pretest). Determination of a positive reaction necessitated induration of at least 5 mm in diameter. The test antigen that induced the greatest response was then employed for intralesional injection. If equal reactions occurred, then an arbitrary assignment to candida first, then trichophyton and then mumps was employed. If all skin tests were non-reactive, then the patient was excluded from the study.
Intralesional injection was performed using the following volumes per group: (1) 0.3 ml of antigen preparation, (2) 0.3 ml of antigen preparation+0.08 ml of interferon alfa 2b, (3) 0.08 ml of interferon alfa 2b or (4) 0.3 ml of saline. The effectiveness of this amount of antigen preparation was previously established (67). Only the largest wart, based on surface area, was treated in patients with multiple warts. All subjects received injections every three weeks into the same wart until complete clearing of the treated wart was achieved or for a maximum of 5 treatments.
Patients were examined at study initiation and at each episode of treatment with notation as to the number and surface area of warts. At the follow-up visits, presence or absence of response to treatment and approximate decrease in size of warts in responders were recorded. Complete resolution was judged to have occurred when the thickening, hyperkeratosis and dilated vasculature of the treated wart were no longer evident and normal skin markings returned. No response was judged to have occurred if there was less than 25% decrease in surface area of the treated wart. Partial responses were estimated as follows: 25% to 50%, 51% to 75% and greater than 75% but less than 100%. For analytic purposes, only those subjects with >75% improvement in the injected or distantly responding warts were defined as responders.
Exclusion criteria consisted of prior allergic response to any of the antigen preparations, pregnancy, lactation, infection with human immunodeficiency virus type 1, clinical evidence of epidermodysplasia verruciformis, iatrogenic immunosuppression, primary immunosuppression or any generalized dermatitis.
Only subjects with multiple warts were chosen for viral typing and assay of PMBC proliferation since these procedures required biopsy of one wart. Fresh tissue was employed for viral typing as described below. Protein extracted from a portion of the tissue was employed in the PBMC stimulation assay as described below.
Viral Typing
Sample specimens of wart obtained from consenting patients were received in the laboratory and processed for HPV typing. A section of wart tissue, approximately 2 mm3, was placed in a sterile microtube and mixed in 200 μl of HPV digestion solution consisting of a fresh mixture of 1 ml 1M Tris pH 8.0, 1 ml 1M Tris pH 9.0, 80 μl 0.5M EDTA pH 8.0, 200 μl Tween 20 and 37.1 ml sterile dH2O and Proteinase K in a ratio of 197 μl HPV digestion solution to 3 μl Proteinase K. The microtubes were incubated overnight in a 65° C. water bath followed by heating at 95° C. to inactive the Proteinase K. The microtubes were then centrifuged at 6,000 rpm in an Eppendorf microcentrifuge for 5 minutes. The resulting digests were decanted and stored on ice or at −80° C. Viral phenotyping was then performed on the wart digests via PCR utilizing sets of PCR primers specific for the HPV genotypes. Two μl of template (wart digest or negative control) was added to a PCR master mix consisting of the following components: 45 μl of Gibco Supermix (Gibco BRL), 2 μl primer A and 2 μl primer B on ice (primers A and B represent primer pairs). The PCR cycling program was run under the following conditions: Step 1: 2 minutes at 95° C. for initial melt, Step 2: 1 minutes at 95° C., Step 3: 90 sec at 50° C., Step 4: 2 minutes at 72° C., Step 5: repeat steps 2-4 29x, Step 6: 5 minutes at 72° C. and Step 7: Store at 4° C. The PCR products where then run on a 1.2% agarose gel and identified as to HPV genotype by (1) presence or absence of a specific PCR product, and (2) generation of a PCR product of the appropriate band size.
PBMC Proliferation Assay
Venous blood was collected in heparinized test tubes for mononuclear cell isolation prior to treatment and, on average, 4.5 weeks (range=3-6 weeks) after initiation of the protocol. Sample specimens were immediately transferred to the laboratory for processing.
A wart extract from the tissue was prepared by transferring the tissue to a dounce homogenizer containing 1.5 ml sterile saline or PBS. The tissue was homogenized and filtered through a 45 μm filter attached to a 3 ml syringe, into sterile cryovials and stored at −20° C.
Venous blood (15 ml) was transferred to a 50 ml centrifuge tube, diluted to a total volume of 30 ml with saline or PBS, underlayed with Fico/Lite-LymphoH (Atlanta Biologicals) and centrifuged for 20 minutes at 2100 rpm in Eppendorf Model 5804R centrifuge. Interface cells were collected and washed 2× with saling/PBS, centrifuged and resuspended in saline/PBS. Cell counts were performed using a Beckman Coulter Zi particle counter and the cells resuspended in freezing media (RPMI/20% human AB serum) and stored at −70° C. Pre- and post-specimen peripheral blood mononuclear cells from each patient were stored for proliferation assays.
Peripheral blood mononuclear cells were thawed, washed 2× in saline/PBS and resuspended at 5×105 cell/ml in RPMI/10% human AB serum. Cells were plated at 200 μl/well and wart extract (5, 10, and 15 μl in quadruplicate) added to desired wells. Concanavalin A (5 μg/ml) was added as a positive control. Plates were incubated at 37° C., 5% CO2 for 5 days and then pulsed with 1 μCi 3H-thymidine for an additional 6 hours. Cells were harvested and lysed with an automated cell harvester (Packard Filtermate 196), the filter was air-dried and counted in a Matrix 9600 Direct Beta Counter. Results were calculated by averaging the groups and dividing by treated wells with media alone (nonspecific incorporation) to obtain a stimulation index (SI). A positive SI was determined if a subject had a 2-fold increase comparing pre and post blood draws.
MHC Determination
HLA-A, B and DRB typing were performed by a PCR/SSOP (polymerase chain reaction followed by sequence specific oligonucleotide probes) method (70). This method uses locus-specific primers to amplify the polymorphic exons of the gene of interest (exon 2 for HLA-DRB and exons 2 and 3 of HLA-A and HLA-B). Amplified DNA was spotted onto replicate nylon membranes and probed with 30-60 different horseradish peroxidase labled oligonucleotide probes. After a stringency wash, the positive reactions were detected with a chromogenic substrate (tetramethylbenzidine).
Statistical Methods
Power/Sample Size:
The study was originally designed to enroll 100 patients in each of 4 study arms. Assuming 40% clearance of the injected warts in the placebo arm, no effect of GM-CSF (later amended to interferon alfa 2b; see below), and an increase to 60% clearance in each immunotherapy arm, the study had 93% power to detect a difference among treatment arms in the primary comparison of response rates, using a three degree-of-freedom chi-square. If GMCSF/interferon alfa 2b had no effect over placebo but boosted the clearance to 70% when combined with immunotherapy, the power to detect an overall difference among the four study arms was 99%. After enrollment of 233 patients, however, an unplanned interim analysis was conducted, for administrative reasons, with the intention of stopping the trial, if significant treatment effects were evident. Using Lan-DeMets boundaries (71), it was determined that the stopping boundary for a significant result at this point in the trial should be an observed p-value of 0.0052 or less. The observed (uncorrected) p-value for the primary over-all comparison of the four groups was less than 0.0001, and enrollment in the trial was stopped. All patients already enrolled in the trial at that point were followed to planned completion of treatment. Once the decision was made to stop the trial using the Lad-DeMets criteria, further analyses were reported in terms of nominal (uncorrected) p-values.
Randomization:
Randomization was conducted by means of sealed envelopes prepared by the Division of Biostatistics, using randomized blocks of random block sizes. This assured approximately equal numbers of patients in each treatment arm at any point in the study. No stratification factors were used for randomization. After a patient who had agreed to participate and had signed informed consent, was determined to be reactive to at least one of the study antigens, the next sealed envelope was opened in sequence, revealing the assigned study arm. The study was single-blind, in that the patients, but not the study investigators, were blinded to an individual's treatment assignment.
Data Analysis:
Cross-classified categorical data was analyzed using chi-square tests. Characteristics measured on a continuous scale (i.e., age) were compared between responders and non-responders using t-tests or Wilcoxon rank sum tests. The simultaneous effects of treatment and age on the probability of response were modeled using logistic regression.
Results
233 subjects entered the study. The initial design included treatment arms employing granulocyte-macrophage colony stimulating factor (GMCSF) instead of interferon alfa 2b. Because of serious adverse events experienced by subjects receiving GMCSF (see below) these arms were discontinued and the trial proceeded using interferon alfa 2b in place of GMCSF. 23 subjects received GMCSF and are discussed separately, but excluded from analysis except where expressly noted. An additional 10 subjects were randomized, but had no injections or failed to return for even one follow up visit. Thus, 201 subjects formed the main group for analysis. Data on subject age and gender are provided in Table C. There were no significant differences among the four study arms in terms of age or gender. Data regarding positive intradermal skin tests prior to study initiation are given for all 233 enrolled subjects in Table D.
Immunotherapy with Antigen is More Effective Than Interferon Alfa 2b or Saline Alone.
Response data in each arm of the study are shown in Table C. These are from the 201 subjects with at least 1 follow-up. Compared to injection with interferon alfa 2b or saline alone, subjects receiving antigen, with or without concomitant interferon injection were statistically more likely to respond (p<0.0001). The same result obtained when analyzing the four groups separately as well as analyzing two groups—(1) antigen with or without interferon alfa 2b against (2) interferon alfa 2b alone and saline alone. Response to interferon alfa 2b injection alone was similar to saline (p=0.65). Adding interferon alfa 2b did not improve response compared to injection of antigen alone (p=0.20). There were no differences in response among the individual antigens (p=0.48). There was no difference in response based upon gender (p=0.56), but subjects who did not respond were significantly older (39 versus 34 years) than those who did respond (p=0.01, t-test; p=0.042, Wilcoxon test). A multivariate logistic regression analysis indicated that age less than 40 was positively associated with the probability of response, after controlling for antigen injection (p=0.01). Across responders in all groups, the average number of injections to complete response was 4.
Immunotherapy with Antigen Leads to More Distant Responses in Untreated and Anatomically Distinct Warts.
Distant response data in each arm of the study are shown in Table E. 153 subjects had >1 wart. Compared to injection with interferon alfa 2b or saline alone, subjects with >1 wart receiving antigen, with or without concomitant interferon injection were statistically more likely to experience resolution of untreated and anatomically distinct warts (p<0.0001). The same result was obtained when analyzing the four groups separately as well as analyzing two groups, as above. The addition of interferon alfa 2b to antigen did not significantly improve response (p=0.24). There was no significant difference in response between interferon alfa 2b and saline (p=0.23).
Adverse Events were Uncommon.
In 1027 episodes of treatment, 47 subjects reported fever and myalgias (5% of treatment episodes) and 46 subjects reported edema and erythema at the injection site of acral lesions (5% of treatment episodes). Of the 47 subjects experiencing fever and myalgias, 9 received interferon alfa 2b alone, 30 interferon alfa 2b+antigen, 7 mumps antigen alone, and 1 saline. The 46 subjects experiencing edema and erythema were scattered among all treatment groups. In no case were these adverse events a cause of study discontinuation. Oral antipyretic medications were employed to lower temperature. Cool compresses and limb elevation were employed to lessen edema and erythema. Signs and symptoms resolved within 24 hours in all subjects.
GMCSF, While Possibly Effective, Resulted in Serious Side Effects.
Of the 23 subjects receiving GMCSF, 2 developed hypotension, necessitating medical intervention, within 4 hours of intralesional injection. For this, the study arms employing GMCSF were halted with substitution of interferon alfa 2b for GMCSF, utilizing the same randomization protocol. Two of 10 subjects receiving GMCSF alone responded, while 10 or 13 receiving GMCSF+antigen responded. These data are excluded from the remainder of the analysis, except as indicated.
Responding Subjects were More Likely than Nonresponders to Have Positive PBMC Proliferation Assays.
35 subjects consented to the PBMC proliferation assay. Table F summarizes response data. Because only 35 subjects were studied, the data include subjects receiving GMCSF as well as interferon alfa 2b. 19 subjects received antigen or antigen+interferon alfa 2b (or GMCSF), and 16 received interferon alfa 2b (or GMCSF) or saline. Responding subjects were more likely to have a positive PBMC proliferation assay, as defined above, than nonresponders (p=0.002). There was no significant association between use of antigen or antigen+interferon alfa 2b (or GMCSF) versus saline or interferon alfa 2b (or GMCSF) alone and PBMC proliferation assay response (p=1.0). Proliferative responses to candida, mumps or trichophyton antigen were not assayed in order to conserve PBMC for study of HPV response.
There is No Significant Association between Response to Immunotherapy and Viral Type or MHC Antigen.
Viral type was determined in warts from 146 subjects. Results of viral typing are given in Table G. There was no significant association between viral type and response to any injection (p=1.0). This finding was also true among those receiving antigen (p=0.86). 65 subjects underwent MHC determination. There was no association between response and any of the MHC antigens found in this population (data not shown).
There is No Significant Difference in Response Rate between the Different Antigens Used.
Response to therapy did not depend on the type of antigen used—candida, trichophyton, or mumps (Table H). There was no significant association between response rate and the particular antigen used (p=0.48, exact chi-square test). Furthermore, each of the three antigens, administered with or without interferon alfa 2b, (candida 66% response, n=41; trichopyton 63% response, n=16; mumps 53% response, n=38), produced a significantly better response than administration of interferon alfa 2b alone or saline (24% response, n=106) (p<0.05) (Table C).
*IfA = interferon alfa 2b
Discussion
Few prospective, controlled trials of wart therapies exist. We document the clinical efficacy of intralesional immunotherapy employing candida, mumps or trichophyton skin test antigens. The response rate of warts injected with antigen alone was lower in this study than in our earlier publication (60% versus 74%) (combined response data from Table 1, 67). The majority of the subjects reported here were referred for the protocol whereas in the earlier work, subjects were mainly recruited from our general clinic population. We speculate that a selection bias toward more recalcitrant disease exists in the patients constituting the present study group. Still, injection of antigen±interferon alfa 2b resulted in a meaningful clinical response rate when compared with injection of saline or interferon alfa 2b alone (56% versus 23%, p<0.0001). Similarly, resolution of anatomically distinct, untreated warts was more likely when antigen was injected compared to injection of saline or interferon alfa 2b alone (49% versus 16%, p<0.0001). That older subjects (>40 years) were less likely to respond to immunotherapy than younger subjects agrees with other data documenting less robust immunologic responses with increasing age (72, 73). We do not have long term follow-up of these subjects to be able to report on relapse rate.
We sought to enhance local immunity through concomitant injection of cytokines. Injection of interferon alfa 2b together with antigen gave a higher response rate than injection of antigen alone. But the difference did not rise to statistical significance. It is possible that a regimen of more frequent injection and higher dose might enhance outcome, but more frequent visits and larger volumes of injection are inconvenient and uncomfortable for patients. Because we hypothesize that immunologic events in the wart itself contribute to enhanced recognition of epitopes on the human papillomavirus, with subsequent expansion of lymphocyte clones targeting the virus, we initially thought to co-inject GMCSF to affect Langerhans cells and trafficking lymphocytes. While the addition of GMCSF to antigen injection or even injection of GMCSF alone might provide a therapeutic response and while patients in these arms of the study were observed to respond, the occurrence of hypotension in two subjects necessitated discontinuation of GMCSF in the protocol.
Our repeated observation that untreated warts resolve after injection of only one wart indicates that intralesional immunotherapy induces HPV-directed immunity. Indeed, we have observed resolution of hundreds of flat warts in individual patients after injection of only one lesion. We found that increased proliferation of PBMC in response to autologous HPV antigens after initiation of therapy was more likely to be observed among responders than non-responders regardless of the substance injected (p=0.002). It is possible that local and distant responses of warts in subjects who received saline or interferon alfa 2b alone develop by the same mechanism as when antigen is injected and that many triggers of an immune response to HPV exist. While injection of saline is an appropriate control, it is not a true placebo. That noted, local and systemic responses to saline injection in this study were far less likely than when antigen was employed.
While little work has been performed with cutaneous wart types in terms of understanding the interplay between viral infection and host immunity, the interest in developing effective vaccines against HPV types causing cervical dysplasia and carcinoma has led to significant knowledge in this area. T lymphocytes responding to HPV-16 can be identified in vivo in infected humans (74). Lymphocyte proliferative responses are documented in humans in response to vaccination with HPV-18 E6 and E4 fusion proteins (75). HPV-16 LI VLPs vaccination of humans has been shown to result in proliferation of both CD4+ and CD8+ T cells (76). In the same study, high neutralizing antibody titers after initial vaccination correlated with strong cytokine responses after second vaccination 6 months later, suggesting that the cellular and humoral immune responses are linked. Peripheral blood mononuclear cells from the patients with cervical cancer may be stimulated by autologous leukocytes pulsed with HPV-16 E7 to generate cytolytic T cells with appropriate specificity (77). Rhesus macaques vaccinated with VLPs, chimeric VLPs and plasmid DNA against HPV-16 L1 vary in terms of the nature of immune response (humoral versus cellular) and degree of immune response according to type of vaccination (78).
Intralesional immunotherapy is not a direct vaccination strategy but the data of this Example indicate it does induce an immune response to HPV, perhaps based on mechanisms similar to those documented in the literature. We have mostly treated subjects with common warts but have observed complete responses utilizing intralesional immunotherapy for genital warts as well (unpublished observation).
Intralesional immunotherapy employing injection of candida, mumps, or trichophyton skin test antigens is an effective treatment for warts as indicated by significantly higher response rates and distant response rates in subjects receiving antigen. Viral type and MHC antigens did not appear to influence treatment response. Response is accompanied by proliferation of PBMC to HPV antigens, indicating that an HPV-directed cell-mediated immune response plays a role in wart resolution.
All citations to publications, books, patents, patent applications set forth herein are incorporated by reference in pertinent part or in its entirety.
This application is a continuation-in-part of application Ser. No. 09/344,257, filed Jun. 25, 1999, now U.S. Pat. No. 6,350,451, and of application Ser. No. 10/081,185, filed Feb. 25, 2002, which is a divisional application of application Ser. No. 09/344,257. Accordingly, this application claims priority under 35 U.S.C. § 120 to application Ser. No. 09/344,257, filed Jun. 25, 1999, and application Ser. No. 10/081,185, filed Feb. 25, 2002.
Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09344357 | Jun 1999 | US |
| Child | 10081185 | Feb 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10081185 | Feb 2002 | US |
| Child | 11061996 | Feb 2005 | US |
| Parent | 09344257 | Jun 1999 | US |
| Child | 11061996 | Feb 2005 | US |
