Title:  Vaccine adjuvants for immunotherapy of melanoma

United States Patent:  6,716,422

Issued:  April 6, 2004

Inventors:  Gajewski; Thomas F. (Chicago, IL); Fallarino; Francesca (Peruga, IT)

Assignee:  ARCH Development Corporation (Chicago, IL); Genetics Institute Incorporated (Cambridge, MA)

Appl. No.:  168832

Filed:  October 8, 1998

Abstract

The invention provides methods of inducing the production of cytolytic T lymphocytes directed against malignancy or infectious agent by a mammal and treating such disease such that deleterious side effects are minimized and treatment of metastatic melanomas are surprisingly and dramatically improved.

SUMMARY OF THE INVENTION

The present invention provides methods of overcoming shortcomings of the prior art by providing improved methods of treating diseases and infections that are unexpectedly effective in inducing immune responses directed against diseases and infections. In some preferred embodiments the invention relates to treating melanomas, such as metatstatic melanomas, and viral infections. The inventors have discovered that administration of the adjuvants in the described manner are far more effective than would have been predicted based on the prior art or when the adjuvants are administered either alone or in a different combination or order. The invention provides the further advantage of reducing deleterious side effects that have been previously associated with cancer therapies.

As used in this specification and the appended claims and in accordance with long-standing patent law practice, the singular forms "a" "an" and "the" generally mean "at least one", "one or more", and other plural references unless the context clearly dictates otherwise. Thus, for example, references to "a cell", "a peptide" and "an adjuvant" include mixtures of cells, one or more peptides and a plurality of adjuvants of the type described; and reference to "IL-12" includes different species of such IL-12, for example, recombinant human IL-12, and so forth.

As used herein, the term "a recombinant peptide", unless otherwise expressly stated, is used to succinctly refer to a recombinant peptide which is derived from an antigen that can be recognized by T-lymphocytes. "Recombinant peptides" are generally peptide molecules that may be provided to cells (or animals) by the hand of man. The term "recombinant" peptide does not generally extend to amino acid sequence, peptides and proteins that may have been moved by a process of nature such that they have "recombined" in a sequence or order different to the parent cell or organism from which they were derived without intervention by man.

The invention provides a method of inducing a mammalian immune response comprising: providing a composition comprising IL-12 and antigen-presenting cells pulsed with peptide and administering the composition to a mammal in an amount effective to induce an immune response. In one illustrative system the composition, or adjuvant, comprises peptide-pulsed, or loaded, antigen-presenting cells (APCs) and IL-12.

The invention further provides that the APCs comprise autologous cells and in some illustrative embodiments the antigen-presenting cells may comprise B cells activated by lipopolysaccharide, whole spleen cells, dendritic cells, fibroblasts or non-fractionated peripheral blood mononuclear cells (PMBC). Of course, it is understood that one of skill in the art will recognize that other antigen-presenting cells may be useful in the invention and that the invention is not limited to the exemplary cell types which are described herein.

The APCs are pulsed, or loaded, with antigenic peptide or recombinant peptide derived from at least one antigen. In one embodiment the peptide comprises an antigenic fragment capable of inducing an immune response that is characterized by the production of cytolytic T lymphocytes (cytolytic T cells or CTLs) which are directed against a malignancy or infection by a mammal. In a particular exemplary embodiment the peptide comprises one or more fragments of an antigen binding to class I MHC or class II MHC molecules (see Tables 1 and 2 for lists of exemplary tumor antigens). It is understood that the antigens listed in Tables 1 and 2 are provided for illustrative purposes and the skilled artisan will recognize that the described invention is not limited to these illustrative antigens.

In an illustrative system, the peptides comprise one or more fragments of one or more antigens expressed by melanoma tumors or other cancers, or infectious agents such as viruses, bacteria, parasites and the like. In some illustrative embodiments of the invention the peptide comprises MAGE-1, MAGE-3, Melan-A, P198, PIA, gp100 or tyrosinase. Of course, it is understood that one of skill in the art will recognize that peptides comprising one or more fragments of other antigens may be useful in the invention and that the invention is not limited to the exemplary peptides and antigens which are described herein.

APCs may be pulsed with any effective concentration of peptide. In a particular illustrative system, the APCs comprise cells pulsed with about 0.1 .mu.M-1 .mu.mM peptide. In a preferred illustrative system, the APCs comprise cells pulsed with about 1 .mu.M-100 .mu.M peptide, with a further preferred embodiment with about 10 .mu.M-50 .mu.M.

In a further embodiment the malignancy comprises a melanoma or other cancer, such as cancer of the prostate, ovary, kidney, lung, brain, breast, colon, bone, skin, testes or uterus, and the virus comprises a retrovirus, adenovirus, vaccinia virus, herpesvirus, adeno-associated virus, lentivirus, human immunodeficiency virus (HIV), or an arbovirus (arthropod-borne virus) (comprehensive lists and descriptions of arboviruses are provided in Entomology in Human and Animal Health, 7th ed., 1979 and The Biology of Disease Vectors, University Press Colorado, 1996, both of which are incorporated herein by reference). In another embodiment the infection comprises a bacterial or parasitic infection.

Mammals include, but are not limited to, equines, cattle, felines, canines, rats, mice and humans.

In a particular embodiment, the invention provides a method of inducing a mammalian immune response, wherein the peptide-pulsed APCs are administered to a mammal in need thereof, in a single therapeutic dose in combination with a single therapeutic dose of IL-12 followed by multiple therapeutic doses of IL-12.

Dosages may be any that induce an immune response. In certain embodiments, the amount of APCs administered comprises 1x10⁶ -1x10⁹ per dose. In exemplary preferred embodiments the amount of APCs administered comprises about 1x10⁸ per dose. In other embodiments the amount of IL-12 administered comprises 1 ng/kg-1000 ng/kg. In certain preferred exemplary embodiments the amount of IL-12 administered comprises 30-50 ng/kg per dose. Of course, it will be understood by the skilled artisan that the preferred dosage should be individualized to the patient following good laboratory practices and standard medical practices.

In another aspect the invention provides a method of treating a patient with a malignancy or infection comprising administering an adjuvant or composition comprising peptide-pulsed antigen-presenting cells and IL-12.

In a particular embodiment, the invention provides a composition using tumor antigen peptide pulsed autologous PBMC with and without rhIL-12 to produce an immune response in humans. In preferred embodiments, the tumor antigen peptide is Mage3 or MelanA. In further preferred embodiments, rhIL-12 is provided in addition to the Mage3 or MelanA.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention discloses novel methods of using a vaccine adjuvant which specifically induces antigen-specific immune stimulation against an antigen derived from a tumor or infectious agent. Mammalian blood cells that are pulsed by this innovative method have been demonstrated to induce specific cytolytic T lymphocyte (CTL) production and protect from tumorgensis. In general, the method admixes the tumor or disease antigen with autologous peripherial blood cells which are then irradiated and injected back into the animal or patient. The injection is co-administered with IL-12 which helps to stimulate the immune system to promote an anti-neoplastic or anti-disease response in the animal or patient. In exemplary systems, this method has been applied to mice using the mastocytoma tumor antigens P198 and P1A, and to humans using the melanoma antigens MAGE-3 and Melan-A. The use of autologous peripherial blood cells, which can be readily harvested and rapidly prepared in a few hours, provides a significant improvement over other therapies which require lengthy purification and culturing techniques of several weeks thus causing a critical delay in treatment. Further, the combination of autologous peripherial blood cells with antigen and IL-12 yields an unexpectedly high inhibition of tumor growth such that tumor regression or even disappearance occurs, and extraordinarily, living tumor challenges may not result in tumor occurrence.

CTLs are involved directly in the body's defense against any infection and are well-known to kill virus-infected cells. Further, as CTLs recognize foreign antigens in the context of class I MHC molecules, the invention is not restricted to the treatment of cancers but can be useful in the treatment of infectious diseases, especially viral diseases, for which an antigenic peptide that binds to class I MHC molecules can be admixed with autologous peripheral blood cells. It is not necessary for the practice of the invention that the antigenic peptide be provided in a purified or isolated state.

It is envisioned that the methods of the invention will be useful in the treatment of infectious, viral or parasitic diseases that are resistant to other therapies, such as arboviruses or malaria, or for which effective vaccines are not known, such as human immunodeficiency virus (HIV) and herpes viruses and certain arboviruses.

It is further envisioned that this invention can be useful surveillance therapies designed to prevent the recurrence of disease, such as tumor regeneration, and in preventative therapies such as vaccinations against viral or parasitic diseases, such as encephalitis or malaria.

Thus this invention provides novel methodology and immunization protocols which are surprisingly more effective in the generation of CTLs than conventional approaches and have the additional improvement of requiring less time to prepare the vaccine adjuvant or adjuvants compared to other therapies.

A further advantage of this invention is that few, if any, deleteriores side effects occur in the animal or patient through the administration of the vaccine adjuvant.

Peptide-based Immunization Strategies

In order to move towards immunization of melanoma patients, a methodology for peptide-based vaccination was required. Using the well-defined murine P815 system as a preclinical model (Brichard et al., 1995; Van den Eynde et al., 1991; Uyttenhove et al., 1980; Van Pel et al., 1985) and detection of specific CTL in peripheral blood as a surrogate readout, multiple immunization strategies were examined. Three weekly subcutaneous immunizations with peptide alone, peptide in several different adjuvants, or peptide plus IL-12 failed to induce detectable CTL. Next, in order to focus peptide delivery on APC, ex vivo pulsing of purified dendritic cells (DC) followed by their reinjection was attempted. This approach yielded CTL generation in 10-20% of mice. However, injection of peptide-pulsed DC plus IL-12 unexpectedly induced specific CTL in 100% of mice. Although a single injection of peptide-loaded DC on day 1 was sufficient, the IL-12 needed to be given during the several days after the immunization in order to be efficacious.

Generation of purified DC from each melanoma patient would be a cumbersome task, requiring several weeks of cell culture. Therefore, three additional sources of APC were examined: B cells activated by lipopolysaccharide, whole spleen cells, and non-fractionated peripheral blood mononuclear cells (PBMC). Interestingly, each of these cell populations pulsed with tumor antigen peptide also generated CTL in 100% of mice, but only if IL-12 was provided as well. The fact that pulsed PBMC plus IL-12 were sufficient simplifies considerably the procedure required for preparing the tumor antigen peptide-based vaccine. Successful immunization was achieved with two antigenic peptides P198 and P1A.

In order to determine whether peptide-pulsed APC could induce the generation of specific CTL in the human system, activated B cells or dendritic cells were isolated from a normal individual expressing HLA-A2. These cells were incubated with a peptide derived from MAGE-3 predicted to bind HLA-A2, and were used to stimulate CD8⁺ T cells from the same individual. Only if IL-12 was included during the initial stimulation were specific CTL induced after expansion which could lyse melanoma cell lines expressing MAGE-3 (Van der Bruggen et al., 1994). Inasmuch as HLA-A2 is the most frequently expressed HLA allele and MAGE-3 is the most frequently expressed MAGE gene among melanoma samples examined, this peptide/HLA combination is suggested for human immunizations. A peptide derived from another tumor antigen, Melan A, also has been identified that binds to HCA-A2 and can be recognized by CTLs.

Tumor Antigen-Specific Immunization in a Murine Model

It is understood that the skilled artisan will recognize that the described system can be applicable to any number of cancers. Thus an illustrative list of tumors, tumor antibodies, etc. is provided in Tables 1 and 2 for which the described invention may be used. But for the purposes of providing an exemplary illustration, the tumor antigen P815 will be used.

Although P815 is a mastocytoma and not a melanoma cell line, it is likely that the principles of tumor antigen immunity defined with this model system are generally applicable to other tumor types. The advantages of the system are multiple. Five tumor antigens expressed by P815 have been identified according to recognition by CTL clones (Brichard et al., 1995), and the gene P1A encoding two of these antigens has been cloned and characterized (Van den Eynde et al., 1991). The genomic sequence of P1A in P815 tumor cells is identical to that in normal mouse cells, indicating that it is a normal gene that is abnormally expressed. It is expressed by several mastocytoma cell lines but not in normal tissues except for testis and placenta, and in this way mirrors the expression of the human tumor antigen genes of the MAGE family (Van Pel et al., 1995). In addition, immunogenic tum^- variants have been generated by mutagenesis of P815 (Uyttenhove et al., 1980). These variants express at least one neoantigen as a result of point mutations in normally expressed genes (Sibille et al., 1990; Wolfel et al., 1987; De Plaen et al., 1988), resulting in their being rejected by the majority of syngeneic mice. The description of a point mutation generating a human melanoma antigen (Coulie et al., 1995) adds yet another parallel between the P815 system and human tumors. A highly transfectable variant of P815, P1.HTR, also has been generated that facilitates transfection by calcium phosphate precipitation (Van Pel et al., 1995). This variant has been used for all the studies requiring transfection.

Peptides encoded by several of the unique tumor antigens of the tum^- variants have been defined, such as the P198 peptide used in one of the examples herein. The P198 peptide is more hydrophilic than the P1A peptide. Therefore, initial peptide-based immunization studies were performed using the more soluble P198 peptide. Information gained was then examined using P1A peptide as well. Studies with P1A are important in order to measure efficacy of immunization in vivo in terms of protection against living tumor challenge and regression of pre-established tumors. These types of studies would not be possible with P198 because that tumor is rejected spontaneously.

The studies described herein provide convincing evidence that both B7 and IL-12 should be provided during active tumor antigen immunization. Although B7 apparently can be recruited under some circumstances from host immune cells, IL-12 apparently cannot.

Groups of 6-10 female DBA/2 mice were treated for each condition examined. In the first studies, naive (non-tumor-bearing) mice were immunized. The studies were performed by pulsing different APC with P198 peptide at 1 .mu.g/ml. These procedures were performed next using P1A peptide in an identical fashion, with peptide-specific CTL activity from peripheral blood measured as a surrogate readout. Cytokine production, particularly IFN-.gamma. and TNF-.alpha., were assessed in parallel studies following restimulation of effector T cells with peptide-pulsed syngeneic APCs or antigen-expressing tumor cell lines.

The optimal dose of peptide for immunization was determined. Whole syngeneic splenocytes are pulsed with 10 or 1 .mu.g/ml of P1 A peptide, washed, irradiated (2,000 rad), and injected into the mice. The optimal number of injections was assessed. One advantage of using peripheral blood as a source of T lymphocytes to assay is that the mice do not need to be sacrificed in order to measure CTL activity. In this way, levels of CTL activity were examined at weekly intervals prior to each immunization. This approach is analogous to that which is used for patient studies. A general goal of the pre-clinical model was to construct a specific procedure that was then transferred to patient use. The optimal location of immunization is not yet known. Pulsed APC were injected subcutaneously, intradermally, intravenously, and intraperitoneally, and CTL activity were measured as before.

Although non-fractionated lymphoid cell populations can function for immunization, it was not clear whether the few DC present in the mixture were actually responsible for the effect. Both spleen cells and PBMC contain a population of DC precursors. Nonetheless, the inventors reasoned that many cell types can serve as APC for immunization, provided IL-12 is administered as well. The hypothesis was tested rigorously by comparing pulsed purified resting B cells, activated B cells, DC, and fibroblasts. If each of these class I MHC⁺ APC populations induced specific CTL when pulsed with peptide and co-injected with IL-12, then the conclusion that provision of IL-12 makes the nature of the APC irrelevant could be made. Finally, PBMC were isolated from mice and used for immunization in a similar fashion. Isolation of sufficient numbers of mouse PBMC is difficult, but success using this cell population as a source of APC bridges even more closely to the clinical situation, as PBMC constitute the easiest APC population to isolate from humans.

Conditions that generated positive results using CTL induction as a readout were then explored by challenging immunized mice with living P815 or P1.HTR cells to assess for tumor protection. A related tumor, L1210, that has been transfected with the tumor antigen gene P1A was also used. A comparison between the ability to protect against L1210 versus L1210.P1A served as a measure of the antigen specificity of the immune response. The optimal conditions observed in the tumor protection assays were then transferred to the immunization of mice bearing pre-established tumors. Tumors were established subcutaneously or intraperitoneally. Beginning 4, 7, 10, or 14 days later, immunization with P1A-pulsed APC plus EL-12 was initiated. The rate of regression of tumor growth was determined. The inventors deduced that the protocols that are most efficacious at inducing rejection of pre-established tumors in the mouse model may be the most important to apply to human patients, as these individuals will possess pre-established tumors as well.

Peptide-pulsed APC in Humans

Peripheral blood macrophages as a source of APC have been cultured from the blood of melanoma patients, pulsed with a peptide derived from MAGE-1, and injected back into the patients subcutaneously and intravenously (Mukheiji et al., 1995), No major toxicities were observed. Biopsy of the immunization sites revealed the presence of MAGE-1-specific CTL, suggesting that a specific immune response was initiated. Based on the success in the mouse model using non-fractionated PBMC as a source of APC, the inventors reasoned that it may not be necessary to carry out a procedure for in vitro expansion of macrophages or DC to obtain a successful immunization. The use of non-fractionated PBMC would simplify considerably the preparation of the vaccine, and avoid potential sources of toxicity.

Phase I/Phase II Experience with IL-12 in Humans

A Phase I clinical study of recombinant human IL-12 (rhIL-12) in patients with various malignancies was performed. A single test dose of rhIL-12 was administered intravenously, followed in 2 wk by a daily dose for 5 days, every 3 wk. Cohorts of at least 4 patients received rhIL-12 at dose levels of 3, 10, 30, 100, 250, 500, or 1000 ng/kg/day. Toxicities included transient cytopenias (nadirs occurring 2-5 days after treatment), reversible increases of transaminases and bilirubin, transient hyperglycemia, stomatitis, and capillary leak syndrome. The maximally tolerated dose at this schedule was 500 ng/kg/day, and there were several tumor responses observed.

A second Phase I clinical study of rhIL-12 was conducted, employing subcutaneous administration 3 times a wk for 2 wk, followed by one wk off. Patients were treated at dose levels of 3, 10, 30, 100, and 300 ng/kg/day. The maximally tolerated dose was not achieved as the trial was suspended after a clinical hold was placed on the Phase II renal cell carcinoma studies described below.

Two Phase II studies of rhIL-12 administered intravenously to patients with advanced renal cell carcinoma were initiated. The dose of 500 ng/kg/day was administered intravenously 5 times per wk followed by a 16 day rest period. Unexpectedly, 12 of the 17 patients enrolled required hospitalization for adverse events, and there were 4 patient deaths. Two of these were attributed to rhIL-12 and 2 were related to progressive disease. Therefore, the trial was suspended. After lengthy investigation into the potential differences between the Phase I and Phase II trials, it appeared that the toxicity profile was highly dependent on the schedule of administration of rhIL-12. The toxicity in the Phase I study apparently was attenuated by the single test dose given prior to the daily dosing.

Based on these observations, a third Phase I study of rhIL-12 was completed. Cohorts of 6 patients were treated by subcutaneous injection 3 times per wk for 2 wk followed by a 9 day rest period, at doses of 30, 100, and 300 ng/kg/day. There were no major toxicities, and 3 patients were then treated at a 500 ng/kg/day dose. Two renal cell carcinoma patients appeared to have a minor response. This dose range and schedule of rhIL-12 appear to be well tolerated in patients with advanced malignancies.

Overview of the Approach to Tumor Antigen-Specific Immunization

Based on the above preclinical and Phase I results, the inventors conceived of a strategy for tumor antigen-specific immunization of melanoma patients. A Phase I/Phase II study in metastatic melanoma patients was undertaken. Patients were first HLA-typed. HLA-A2-positive patients underwent a tumor biopsy to screen for expression of MAGE-3 and Melan-A using RT-PCR.TM.. Patients with MAGE-3⁺ tumors were eligible for vaccination with MAGE-3 peptide. Patients with tumors that were MAGE-3-negative but Melan-A-positive were eligible for immunization with Melan-A peptide.

Peripheral blood was collected and fractionated by density centrifugation to isolate PBMC as a source of APC. Cells were incubated with the appropriate MAGE-3 or Melan-A peptide, washed, resuspended in PBS, and lethally irradiated. Pulsed cells (50-100x10⁶) were injected subcutaneously at 2 sites, near lymph node locations but not adjacent to a tumor mass. The subcutaneous route was preferred for the reasons of safety, efficacy in the preclinical model, and the goal of targeting the vaccine to a draining lymph node.

Eligible patients were assigned to the respective cohorts as they came, whether being immunized with MAGE-3 or Melan-A peptide. Three to six patients were treated with peptide-pulsed PBMC alone, using either MAGE-3 or Melan-A peptide as indicated. For the remaining cohorts, rhIL-12 was administered subcutaneously near one of the immunization sites on days 1, 3, and 5. The dose of rhIL-12 was escalated in groups of 3-6 patients each, to determine an optimal dose with respect to safety and successful immunization. The dosing schedule was based on the most recent phase I data. Reimmunization was performed at 3 wk intervals, with rhIL-12 administration on days 1, 3, and 5 of each cycle. Prior to each immunization, peripheral blood was collected to assay for peptide-specific CTL activity and production of IFN-.gamma. and TNF-.alpha.. Injection sites also were examined for local inflammation indicative of a delayed-type hypersensitivity reaction. Clinical response was assessed as a secondary outcome.

One major advantage of the tumor antigen-specific immunization approach is the ability to measure a specific immune response independently of an effect on tumor regression which has not been possible with more generic immunotherapies, such as injection of recombinant IL-2, because the antigens expressed by the patient's tumor are not normally analyzed. In addition, any successfully generated response might be directed against antigens that are not yet characterized and therefore would go undetected. A first step to improving upon immunotherapy of cancer is to determine whether or not successful immunization has occurred; only then can vaccination be improved upon in order to determine its true potential in cancer therapy.

The appropriate surrogate readout of immunization is not yet known. It is generally felt that induction of antigen-specific cytolytic activity is the desired endpoint. However, other properties of the effector cells induced might be just as critical. A likely candidate is the ability of the activated CTL to produce the cytokines IFN-.gamma. and TNF-.alpha., a characteristic of a Th1/Tc1 phenotype. Studies in the murine model have suggested that a Th1/Tc1 phenotype might be optimal for mediating rejection of pre-established tumors.

Three measures of successful immunization of patients are examined. First, the serum samples collected from each patient following each immunization are assayed for IFN-.gamma. and TNF-.alpha. content. The inventors reasoned that effectively immunized patients have an increase in these cytokines after each inoculation, and that the magnitude of the increase is greater with each subsequent vaccination. These cytokines are measured by standard ELISA technique well known to those of skill in the art. Serial dilutions of the serum sample are compared to serial dilutions of a standard. The dilutions giving half-maximal absorption are compared and the concentration is determined based on the known concentration of the standard. This surrogate readout can be performed routinely, but the sensitivity of the assay might not be sufficient to detect the expected increases.

The second assay measures MAGE-3- or MelanA-specific cytolytic activity from the cryopreserved PBMC which is assessed by re-stimulating the T lymphocytes with peptide-pulsed APC, expanding the responding cells with IL-2, and measuring lysis of chromium-labeled target cells expressing the correct MHC molecules and pulsed with MAGE-3 or MelanA peptide. Controls include non-pulsed targets and the NK-sensitive target K562. Cold competition is performed with non-radiolabelled K562 cells to eliminate non-specific NK activity.

The third readout is a combination of the first and the second approaches. Because a Th1/Tc2 phenotype might be predictive of anti-tumor efficacy, the effector cells generated upon expansion of specific T cells in the second method are stimulated for 24 hours with peptide-pulsed APC, and the supernatants are assayed for the presence of IFN-.gamma. and TNF-.alpha.. Even if the serum levels are undetectable, cytokine production by the antigen-specific T cells should be easily measurable.

Outline of a Specific Human Vaccination Study

A vaccination study of patients with refractory metastatic disease was conducted using tumor antigen peptide pulsed autologous PBMC with and without rhIL-12. In particular, using Mage3 and MelanA, generation of peptide-specific, IFN-.gamma.-producing CD8⁺ T cells was detected after 1 to 3 immunizations as shown in FIG. 10, FIG. 11. and FIG. 12.

Biological Functional Equivalents

It is understood that the therapeutic regimen described herein can be utilized with any antigenic peptide that binds to class I MHC molecules. For the MAGE-3 and Melan A peptides described, biological functional equivalents are described. As will be understood by those of skill in the art, modification and changes may be made in the structure of the recombinant peptide and still obtain a molecule having like or otherwise desirable characteristics. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of T cell antigen receptors or binding sites on HLA molecules of melanoma cells. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties. It is thus contemplated by the inventor that various changes may be made in the sequence of recombinant proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.

In terms of functional equivalents, it is also well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. In particular, where small peptides are concerned, less amino acids may be changed. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.

It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e.g., residues in the antigenic recognition region, such residues may not generally be exchanged.

Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.3+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.

While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. A table of amino acids and their codons is presented below for use in such embodiments, as well as for other uses, such as in the design of probes and primers and the like.

The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see Codon Table, above).

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

Therapeutic Regimens and Dosage

A therapeutic regimen is described herein; however, the treatment with L-12 may precede or follow administration of peptide-pulsed APC by intervals ranging from seconds to hours to days to even weeks. In embodiments where peptide-pulsed APC and IL-12 are administered separately to the patient, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the combination of the two would still be able to exert an advantageously combined effect on the recipient. In such instances, it is contemplated that one would contact the patients with both agents within about 0.1 to 24 hours of each other and, even, within about 1 to 4 hours of each other, with a delay time of only about 1 hour to about 2 hours being preferred. In some situations, it is desirable to extend the time period for treatment significantly; where several days (1, 2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of peptide-pulsed APC will be desired in certain circumstances in combination with IL-12. Various combinations may be employed, where peptide-pulsed APC is "A" and IL-12 is "B":

A/B/B B/A/A A/A/B

A/B/A B/A/B B/B/A

B/B/B/A B/B/A/B B/A/B/A B/A/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A

A/A/A/B B/A/A/A A/B/A/A

B/A/B/B A/A/B/A A/B/B/B

To achieve tumor cell killing, both agents are delivered to a patient in a combined amount effective to kill the tumor cells. These treatment cycles can be repeated multiple times, or delivered only once.

The skilled artisan will recognize that factors that are well known to influence patient response to drug therapy include, but are not limited to, species, age, weight, gender, health, pregnancy, addictions, allergies, ethnic origin, prior medical conditions, current medical condition and length of treatment. Thus, the skilled artisan will be well acquainted with the need to individualize dosage(s) to each patient.

The skilled artisan will also consider the condition that is to be treated prior to selecting the appropriate dosage. For example, a dosage that is appropriate for the treatment of a cancer, may not be the desired dosage for subsequent surveillance therapy designed to prevent the recurrence of the cancer.

Thus it is recognized that in the practice of the invention a wide variety of dosages may be useful and that the desired dosage is individualized to the patient. In an illustrative case, 10-50 .mu.M peptide is loaded onto APCs, 10x10⁸ APCs are administered per injection and 30-50 ng/kg IL-12 is administered per injection.

Yet the amount of peptide loaded onto APCs may be as little as about 0.1 .mu.M to as much as about 1 mM. It is understood that this range includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, etc.; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.; 20, 21, 22, 23, etc.; 25, 26, 27, 28 etc.; 30, 31, 32, 33, etc.; 35, 36, 37, etc.; 40, 41, 42 etc.; 45, 46, 47, etc.; 50, 51, 52, 53, etc.; 60, 61, 62, etc.; 70, 71, 72, etc.; 80, 81, 82, etc.; 90, 91, 92, etc.; 100, 110, 120, etc.; 150, 160, 170, etc.; 200, 210, 220, etc.; 250, 260, 270 etc.; 300, 310, 320, 330, etc.; 350, 360, 370, etc.; 400, 410, 420, etc.; 450, 460, 470, etc.; 500, 525, 550, 575, etc.; 600, 625, 650, etc.; 700, 725, 750, etc.; 800, 825, 850, etc.; 900, 925, 950, etc.; 1000 .mu.m.

The number of APCs per injection may also be varied from 1x10⁶ -1x10⁹. It is understood that this range is inclusive of all doses between about 1x10⁶ and x10⁹. Thus this range includes 1x10⁶, 2x10⁶, 3x10⁶, 4x10⁶, 5x10⁶, 6x10⁶, 7x10⁶, 8x10⁶, 9x10⁶, 1x10⁷, 2x10⁷, 3x10⁷, 4x10⁷, 5x10⁷, 6x10⁷, 7x10⁷, 8x10⁷, 9x10⁷, 1x10⁸, 2x10⁸, 3x10⁸, 4x10⁸, 5x10⁸, 6x10⁸, 7x10⁸, 8x10⁸ and 9x10⁸ APCs per injection.

The amount of IL12 which can be administered ranges from 1 ng/kg-1000 ng/kg per injection. It is understood that this range is inclusive of all doses between about 1 ng/kg and about 1000 ng/kg. Thus this range includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.; 20, 21, 22, 23, etc.; 25, 26, 27, 28 etc.; 30, 31, 32, 33, etc.; 35, 36, 37, etc.; 40, 41, 42 etc.; 45, 46, 47, etc.; 50, 51, 52, 53, etc.; 60, 61, 62, etc.; 70, 71, 72, etc.; 80, 81, 82, etc.; 90, 91, 92, etc.; 100, 110, 120, etc.; 150, 160, 170, etc.; 200, 210, 220, etc.; 250, 260, 270 etc.; 300, 310, 320, 330, etc.; 350, 360, 370, etc.; 400, 410, 420, etc.; 450, 460, 470, etc.; 500, 525, 550, 575, etc.; 600, 625, 650, etc.; 700, 725, 750, etc.; 800, 825, 850, etc.; 900, 925, 950, etc.; 1000 ng/kg.

Treatment Routes

Peptide-pulsed APC and IL-12 can be administered intravenously, intraarterially, intratumorally, parenterally or intraperitoneally. In the invention, the preferred routes of administration are subcutaneous (SC); however, intravenous (IV), intrarterial, and intraperitoneal (IP) can be used. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be m brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Although it is not envisioned as a preferred route, either or both peptide-pulsed APC and IL-12 may also be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or enclosed in hard or soft shell gelatin capsule, or compressed into tablets, or incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

Screening and Monitoring Effectiveness of Therapy

It is contemplated that in the context of the present invention one may remove cells, either tumor, normal or both tumor and normal cells, from an individual in order to either monitor the progress of treatment or as a part of the treatment. It is expected that one may monitor the effectiveness of treatment by removing such cells and treating such cells with DAPI staining to determine the level of chromatin condensation, measuring the level of apoptosis, measuring the level of neutral sphingomyelinase production or other methods such as the following.

One particular method for determining induction of apoptosis is terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assays, which measure the integrity of DNA (Gorczyca, 1993). This assay measures the fragmentation of DNA by monitoring the incorporation of labeled UTP into broken DNA strands by the enzyme terminal transferase. The incorporation can be monitored by electroscopy or by cell sorting methodologies (e.g., FACS).

Another method with which it is expected that one may monitor the effectiveness of treatment is the use of enzyme linked immunosorbent assays (ELISAs).

ELISAs

Certain preferred immunoassays are the various types of ELISAs and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, ELISPOT, FACS analyses, and the like may also be used.

In one exemplary ELISA, an antibody against a cytokine, such as IFG.gamma., is immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a composition containing the counterpart cytokine is added to the wells. After binding and washing to remove non-specifically bound complexes, the bound cytokine protein complex may be detected. Detection is generally achieved by the addition of an anti-cytokine or anti-tumor protein antibody that is linked to a detectable label. Detection may also be achieved by the addition of a first anti-cytokine or anti-tumor protein antibody, followed by a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described as follows:

In coating a plate with the primary antibody, one will generally incubate the wells of the plate with a solution of the agent, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is neutral with regard to binding to the biological components. These include bovine serum albumin (BSA), casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of proteins onto the surface.

In the ELISAs of the present invention it will probably be more customary to use a secondary or tertiary detection means. Thus, after binding of the first protein to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the second biological protein under conditions effective to allow protein complex formation. Detection of the complex then requires a labeled binding ligand or antibody.

"Under conditions effective to allow protein complex formation" means that the conditions preferably include diluting the tumor antigen and cytolkine proteins, with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The "suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25^o to 27^oC., or may be overnight at about 4^oC. or so.

Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of bound complexes may be determined.

To provide for detection, a first or second antibody will preferably be provided that has an associated label to allow detection. Preferably, the label will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the bound complexes with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid [ABTS] and H₂ O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.

Ex vivo Delivery

In the present invention, it is contemplated that systemic delivery of either or both peptide-pulsed APC and IL-12 may be used. It is further contemplated that in practicing the claimed invention that one will wish to alter the PBMC by ex vivo manipulation. Ex vivo gene therapy refers to the isolation of cells from an animal or patient, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal or individual. This may involve the surgical removal of tissue/organs from an animal or patient or the primary culture of cells and tissues.

APC can be prepared from PBMC isolated by density centrifugation of whole blood. Human mononuclear cells (MNC), prepared from bone marrow also can be used as APC. Bone marrow can be obtained from the tibiae, femora, spine, ribs, hips, sternum, as well as the humeri, radi, ulna, tibiae, and fibulae. Additionally, these cells also can be obtained from cord blood, peripheral blood, or cytokine-mobilized peripheral blood. Other sources of human hematopoietic stem cells include embryonic yolk sac, fetal liver, fetal and adult spleen, and blood. The marrow layer is centrifuged over a density gradient to produce a pellet of red cells at the bottom of the tube, a clear layer of media, an interface layer which contains the MNC and a plasma medium layer on top. The interface layer may then be removed using, for example, suction. Centrifugation of this layer at 1000 g ultimately yields a MNC pellet. This pellet may then be resuspended in a suitable buffer for cell sorting by FACS. The isolated MNC can be cultured in vitro to expand the immunologically active cells. The expanded, therapeutically active cells are then loaded with peptide and provided to the patient to obtain a therapeutic effect.

APC also can be dendritic cells, generated from bone marrow or peripheral blood. Fibroblasts can serve as APC, and then can be cultured from tissues such as the skin.

Claim 1 of 43 Claims

What is claimed is:

1. A method of inducing a therapeutic immune response comprising:

a) providing a composition comprising IL-12;

b) providing a composition comprising antigen-presenting cells pulsed with peptide, wherein said antigen-presenting cells are not purified dendritic cells; and

c) administering said composition comprising IL-12 and said composition comprising antigen-presenting cells pulsed with peptide to a mammal in an amount effective to induce an immune response.

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