CD4+ T-cell activation for immunotherapy of malignancies using Ii-Key/MHC class II epitope hybrid vaccines.

Active immunotherapy is becoming a reality in the treatment of malignancies. Peptide-based vaccines represent a simple, safe, and economic basis for cancer immunotherapeutics development. However, therapeutic efficacy has been disappointing. Some of the reasons for this, such as selection of patients with advanced disease and ignorance of the delayed activity of many immunotherapeutic vaccines, have hampered the entire field of cancer immunotherapy over the last decade. Another reason for this may be that most peptide regimens historically have focused on activation of CD8+ cytotoxic T lymphocytes, having little or only indirect CD4+ T helper (Th) cell activation. We review here evidence for the importance of specific CD4+ Th activation in cancer immunotherapy and the use of Ii-Key technology to accomplish this. Ii-Key (LRMK), a portion of the MHC class II-associated invariant chain (Ii protein), facilitates the direct charging of peptide epitopes onto MHC class II molecules. Directly linking Ii-Key to MHC class II peptide epitopes greatly enhances their potency in activating CD4+ T-cells. The Ii-Key hybrid AE37, generated by linking LRMK to the known HER2 MHC class II epitope HER2 (aa 776-790), has been shown to generate robust, long lasting HER2-specific immune responses both in patients with breast and prostate cancer. Interim data from a phase II study of AE37 in breast cancer patients suggest a possible improvement in clinical outcome. The Ii-Key hybrid technology is compared to other methods for enhancing the potency of peptide immunotherapy for cancer.

Results from a phase I clinical study of the novel Ii-Key/HER-2/neu(776-790) hybrid peptide vaccine in patients with prostate cancer.

PURPOSE: Active immunotherapy is emerging as a potential therapeutic approach for prostate cancer. We conducted the first phase I trial of an Ii-Key/HER-2/neu(776-790) hybrid peptide vaccine (AE37) with recombinant granulocyte macrophage colony-stimulating factor as adjuvant in patients with HER-2/neu(+) prostate cancer. The primary end points of the study were to evaluate toxicity and monitor patients' immune responses to the vaccine. EXPERIMENTAL DESIGN: Thirty-two HER-2/neu(+), castrate-sensitive, and castrate-resistant prostate cancer patients were enrolled. Of these, 29 patients completed all six vaccination cycles with AE37. Immunologic responses in the total patient population were monitored by delayed-type hypersensitivity and IFN-gamma ELISPOT and intracellular staining. Regulatory T-cell (Treg) frequency and plasma HER-2/neu and transforming growth factor-beta levels were also determined. Immunologic responses were also analyzed among groups of patients with different clinical characteristics. Local/systemic toxicities were monitored throughout the study. RESULTS: Toxicities beyond grade 2 were not observed. Seventy-five percent of patients developed augmented immunity to the AE37 vaccine and 65% to the unmodified AE36 peptide as detected in the IFN-gamma-based ELISPOT assay. Intracellular IFN-gamma analyses revealed that AE37 elicited both CD4(+) and CD8(+) T-cell responses. Eighty percent of the patients developed a positive delayed-type hypersensitivity reaction to AE36. Additionally, significant decreases could be detected in circulating Treg frequencies, plasma HER-2/neu, and serum transforming growth factor-beta levels. Patients with less extensive disease developed better immunologic responses on vaccination. CONCLUSION: AE37 vaccine is safe and can induce HER-2/neu-specific cellular immune responses in patients with castrate-sensitive and castrate-resistant prostate cancer, thus emphasizing the potential of AE37 to target HER-2/neu for the immunotherapy of prostate cancer.

A new era in anticancer peptide vaccines.

The use of synthetic peptides as vaccines aimed at the induction of therapeutic CD8-positive T-cell responses against tumor cells initially experienced great enthusiasm, mostly because of advances in vaccine technology, including design, synthesis, and delivery. However, despite impressive results in animal models, the application of such vaccines in humans has met with only limited success. The therapeutic activity of vaccine-stimulated, tumor-specific, CD8-positive T cells can be hampered through the physical burden of the tumor, tolerance mechanisms, and local factors within the tumor microenvironment. Recently, accumulating evidence has suggested that combining a peptide-based therapeutic vaccination with conventional chemotherapy can uncover the full potential of the antitumor immune response, increasing the success of immunotherapy. In addition, therapeutic vaccination in the preventive setting has been extremely effective in eliciting antitumor responses in preclinical tumor models and has demonstrated good promise clinically in patients with minimal residual disease. The rationale behind preventive vaccination is that patients with minimal tumor burden still have a fully competent immune system capable of developing robust antitumor responses. Finally, therapeutic CD8-positive T-cell peptide vaccines have been improved by coimmunization with T-helper epitopes expressed on long peptides.

Induction of potent CD4+ T cell-mediated antitumor responses by a helper HER-2/neu peptide linked to the Ii-Key moiety of the invariant chain.

The Ii-Key fragment from the MHC class II-associated invariant chain (or Ii protein) has been shown to facilitate direct charging of MHC class II epitopes to the peptide binding groove. The purpose of the present study was to test the potential of a series of Ii-Key/HER-2/neu776-790 hybrid peptides to generate increased frequencies of peptide-specific CD4+ T cells over the native peptide in mice transgenic (Tg) for a chimeric human mouse class II molecule (DR4-IE) (H-2b) as well as their antitumor potency. Following in vivo priming, such hybrid peptides induced increased proliferation and frequencies of IFN-gamma producing CD4+ T cells in response to either syngeneic dendritic cells pulsed with native peptide, or HLA-DR4+ human tumor cell lines expressing HER-2/neu. Hybrid peptides were more stable in an off-rate kinetics assay compared to the native peptide. In addition, antigen-specific CD4+ T cells from hybrid peptide immunized DR4-IE Tg mice synergized with HER-2/neu(435-443)-specific CD8+ T cells from HLA-A2.1 Tg HHD (H-2b) mice in producing antitumor immunity into SCID mice xenografted with the HER-2/neu+, HLA-A2.1+ and HLA-DR4+ FM3 human melanoma cell line. High proportions of these adoptively transferred HER-2/neu peptide-specific CD4+ and CD8+ T cells infiltrated FM3-induced tumors (tumor infiltrating lymphocytes; TIL) in SCID mice. CD8+ TIL exhibited long-lasting antitumor activity when cotransferred with CD4+ TIL, inducing regression of FM3 tumors in a group of untreated, tumor-bearing SCID mice, following adoptive transfer. Our data show that Ii-Key modified HER-2/neu776-790 hybrid peptides are sufficiently potent to provide antigen-specific CD4+ TH cells with therapeutic antitumor activity.

Ii-Key/HER-2/neu(776-790) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/neu+ tumors.

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.

Ii-Key/MHC class II epitope peptides as helper T cell vaccines for cancer and infectious disease.

Potent MHC class II antigenic peptide vaccines are created by covalently linking the N-terminus of a MHC class II epitope through a polymethylene bridge to the C-terminus of the Ii-Key segment of the Ii protein. Such hybrids enhance potency of presentation in vitro of the MHC class II epitope about 200 times relative to the epitope-only peptide. In vivo, as measured by IFN-gamma ELISPOT assays, the helper T cell response to vaccination is enhanced up to 8 times. The design of such hybrid vaccine peptides comes from insight into the mechanism of action of the Ii-Key motif within the Ii protein, in regulating antigenic peptide binding into the antigenic peptide binding groove of MHC class II molecules. Here we present the logic and experimental history of the development of these vaccine peptides, with particular attention to the hypothesized mechanism of action. Methods for the design and testing of these peptides are presented. Experience in developing peptide vaccines for immunotherapy of cancer is reviewed, focusing on the clinical potential of Ii-Key/MHC class II epitope hybrids.

Ii-Key/MHC class II epitope hybrids: a strategy that enhances MHC class II epitope loading to create more potent peptide vaccines.

Life-threatening diseases, such as cancer and pandemic influenza, demand new efforts towards effective vaccine design. Peptides represent a simple, safe and adaptable basis for vaccine development; however, the potency of peptide vaccines is insufficient in most cases for significant therapeutic efficacy. Several methods, such as Ligand Epitope Antigen Presentation System and ISCOMATRIX, have been developed to enhance the potency of peptide vaccines. One way of increasing the loading of MHC class II peptides occurs through the use of Ii-Key technology. Ii-Key (LRMK), a portion of the MHC class II-associated invariant chain (Ii), facilitates the direct loading of epitopes to the MHC class II molecule groove. Linking the Ii-Key moiety via a simple polymethylene bridge to an MHC class II epitope, to generate an Ii-Key/MHC class II epitope hybrid, greatly enhances the vaccine potency of the tethered epitope. The combination of such Ii-Key/MHC class II epitope hybrids with MHC class I epitope-containing peptides might generate a potent peptide vaccine for malignancies and infectious diseases. The Ii-Key hybrid technology is compared with other methods that enhance the potency of a peptide vaccine.

Enhanced CD4+ T-cell response in DR4-transgenic mice to a hybrid peptide linking the Ii-Key segment of the invariant chain to the melanoma gp100(48-58) MHC class II epitope.

Linking the Ii-Key functional group LRMK, through a simple polymethylene linker, to the melanoma gp100(48-58) MHC class II epitope significantly enhances the vaccine response to that epitope in DR4-IE transgenic mice. A homologous series of Ii-Key/gp100(46-58) hybrids was synthesized to test the influence of spacer length (between Ii-Key and the gp100(48-58) epitope) on in vivo enhancement of gp100(48-58)-specific CD4+ T-lymphocyte responses. As measured by IFN-gamma and IL-4 ELISPOT cytokine assays, the most effective vaccine hybrid was the one with a shorter linker between Ii-Key and the epitope. Mechanistic reasons for this observation are considered. This structure-activity relationship was seen with bulk and CD4+ purified T cells, and both primary and secondary in vitro restimulation assays. CFA augmented the IFN-gamma response and to a lesser extent the IL-4 response. CpG enhanced a strong IFN-gamma response, with a negligible IL-4 response. The 3- to 5-times enhancement of the total ELISPOT responses (number of spots x mean spot area) observed after vaccination with peptides consisting of an MHC class II epitope engineered into an Ii-Key hybrid indicates a potent vaccine effect. Such constructs can be applied to many diagnostic and therapeutic uses.

Curative antitumor immune response is optimal with tumor irradiation followed by genetic induction of major histocompatibility complex class I and class II molecules and suppression of Ii protein.

Transfecting genes into tumors, to upregulate major histocompatibility complex (MHC) class I and class II molecules and inhibit MHC class II associated invariant chain (Ii), induces a potent anti-tumor immune response when preceded by tumor irradiation, in murine RM-9 prostate carcinoma. The transfected genes are cDNA plasmids for interferon-gamma (pIFN-gamma), MHC class II transactivator (pCIITA), an Ii reverse gene construct (pIi-RGC), and a subtherapeutic dose of adjuvant IL-2 (pIL-2). Responding mice rejected challenge with parental tumor and demonstrated tumor-specific cytotoxic T lymphocytes (CTLs). We have extended our investigation to determine the relative roles of each one of the four plasmids pIFN-gamma, pCIITA, pIi-RGC, and pIL-2 in conjunction with radiation for the induction of a curative immune response. Upregulation of MHC class I with pIFN-gamma or class II with pCIITA, separately, does not lead to a complete response even if supplemented with pIL-2 or pIi-RGC. An optimal and specific antitumor response is achieved in more than 50% of the mice when, after tumor irradiation, tumor cells are converted in situ to a MHC class I+/class II+/Ii- phenotype with pIFN-gamma, pCIITA, pIi-RGC, and pIL-2. We demonstrate further that both CD4+ helper T cells and CD8+ cytotoxic T cells are essential for induction of an antitumor response because in vivo depletion of either subset abrogates the response. The radiation contributes to the gene therapy by causing tumor debulking and increasing the permeability of tumors to infiltration of inflammatory cells.

Linkage of Ii-Key segment to gp100(46-58) epitope enhances the production of epitope-specific antibodies.

Linkage of the Ii-Key segment of the Ii protein to MHC class II epitope gp100(46-58) using a polymethylene linker significantly enhances the production of epitope-specific antibodies in HLA-DR4-IE transgenic mice. This enhancement is not restricted by the spacer length in between the Ii-Key and epitope. The use of either IFA or CFA induced only epitope-specific IgG1. In contrast, CpG adjuvant induced both IgG1 and IgG2a isotypes. These results indicate that the Ii-Key hybrid technology is a novel and potent method to increase the immunogenicity of a MHC class II epitope. It can also be used to more efficiently generate epitope-specific antibodies.

Immunotherapy of cancer by antisense inhibition of Ii protein, an immunoregulator of antigen selection by MHC class II molecules.

Ii protein suppression is a promising antisense drug-based therapy that dramatically enhances the immunogenicity of tumor cell major histocompatibility complex class II-presented antigenic epitopes. The strength of this approach is that the antisense only needs to be transiently effective in a fraction of the tumor cells. The systemic antitumor immune response generated subsequently eradicates both directly treated cells and distant tumor deposits. The drugs and mechanisms of this therapy are considered, in addition to practical developmental questions.

Turning tumor cells in situ into T-helper cell-stimulating, MHC class II tumor epitope-presenters: immuno-curing and immuno-consolidation.

Immunological control or cure of tumors depends on initiating a robust T helper cell response to MHC class II epitopes of tumor-associated antigens. T helper cells regulate the potency of cytotoxic T lymphocyte and antibody responses. We have developed a novel approach to stimulate T helper cells by converting tumor cells into MHC class II molecule-positive, antigen presenting cells. Furthermore, using antisense methods, we suppress expression of the Ii protein, that normally blocks the antigenic peptide binding site of MHC class II molecules during synthesis in the endoplasmic reticulum. In such gene-engineered tumor cells, the MHC class II molecules pick up antigenic peptides, which have been transported into the endoplasmic reticulum for binding to MHC class I molecules. All nucleated cells create such "surveys of self" to detect viral or malignant transformation. Our method extends that survey of self to MHC class II endogenous tumor-associated antigens. Simultaneous presentation of tumor antigens by both MHC class I and II generates a robust and long-lasting antitumor immune response. Injecting murine tumors with genes, which induce MHC class II molecules and suppress Ii protein, cures a significant number of animals with renal and prostate tumors. We have developed analogous human gene vectors that are suitable for most patients and cancers, because they are monomorphic and active in all HLA-DR alleles. We review our findings, and analyze remaining issues for preclinical study and the design of clinical trials.

Ii-Key/HER-2/neu MHC class-II antigenic epitope vaccine peptide for breast cancer.

PURPOSE: Cytotoxic T lymphocytes (CTL)- and T-helper cell-specific, and major histocompatibility complex (MHC) class-I and class-II peptides, respectively, of the HER-2/ neu protein, induce immune responses in patients. A major challenge in developing cancer peptide vaccines is breaking tolerance to tumor-associated antigens which are functionally self-proteins. An adequate CD4+ T-helper response is required for effective and lasting responses. METHODS: Stimulating anti-cancer CD4+ T cell responses by MHC class-II epitope peptides has been limited by their weak potency, at least compared with tight-binding MHC class-I epitope peptides. Previously, a potent T-cell response to a MHC class-II epitope was engineered by coupling the N-terminus of the pigeon cytochrome C [PGCC(95-104)] MHC class-II epitope to the C-terminus of an immunoregulatory segment of the Ii protein (hIi77-81, the Ii-Key peptide) through a polymethylene spacer. RESULTS: In vitro presentation of the MHC class-II epitope to a T hybridoma was enhanced greatly (>250 times). Now, an Ii-Key/HER-2/neu (777-789) MHC class-II epitope hybrid peptide stimulated lymphocytes from both a healthy donor and a patient with metastatic breast carcinoma. The in vitro primary stimulation with the hybrid peptide strongly activated IFN-gamma release, whereas the epitope-only peptide was weakly active. In fact, the hybrid stimulated IFN-gamma release as well as the wild-type peptide when augmented with IL-12; however, the hybrid was comparable to free peptide in stimulating IL-4 release. This pattern is consistent with preferential activation along a non-tolerogenic Th1 pathway. CONCLUSION: Such Ii-Key/MHC class-II epitope hybrid peptides have both diagnostic and therapeutic applications.

Ii-Key/MHC class II epitope hybrid peptide vaccines for HIV.

The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Hybrid peptides linking an immunoregulatory segment of the Ii protein (Ii-Key peptide) through a polymethylene bridge to MHC class II epitopes of HIV gp160 or gag are potent vaccines to elicit CD4(+) T cell responses. More potent responses to two MHC class II epitopes, HIV gp160(843-852) or HIV gag(279-292), occurred in mice immunized with Ii-Key hybrid peptides than with epitope-only peptides, as measured in IL-4 and IFN-gamma ELISPOT assays of splenic lymphocytes stimulated in vitro by epitope-only peptides. Both the number of responding cells and cytokine output per cell were increased. The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Such antigenic peptide constructs create opportunities to enhance greatly Th1 or Th2 responses to MHC class II epitopes for therapeutic purposes.

Tumor immunotherapy by converting tumor cells to MHC class II-positive, Ii protein-negative phenotype.

A potent antitumor CD4(+) T-helper cell immune response is created by inducing tumor cells in vivo to a MHC class II(+)/Ii(- )phenotype. MHC class II and Ii molecules were induced in tumor cells in situ following tumor injection of a plasmid containing the gene for the MHC class II transactivator (CIITA). Ii protein was suppressed by the antisense effect of an Ii-reverse gene construct (Ii-RGC) in the same or another co-injected plasmid. The MHC class II(+)/Ii(- )phenotype of the tumor cells was confirmed by FACS analysis of cells transfected in vitro and by immunostaining of tumor nodules transfected by injections in vivo. Subcutaneous Renca tumors in BALB/c mice were treated by intratumoral injection with CIITA and Ii-RGC, in combination with a subtherapeutic dose of IL-2, to up-regulate the activation of T cells. Significant tumor shrinkage and decrease in rates of progression of established Renca tumors were seen in the groups injected with Ii-RGC, compared with groups in which only IL-2 plus empty plasmid controls were injected. Our method provides an effective immunotherapy warranting further development for human cancers.

Radiation improves intratumoral gene therapy for induction of cancer vaccine in murine prostate carcinoma.

Our goal was to convert murine RM-9 prostate carcinoma cells in vivo into antigen-presenting cells capable of presenting endogenous tumor antigens and triggering a potent T-helper cell-mediated immune response essential for the generation of a specific antitumor response. We showed that generating the major histocompatibility complex (MHC) class I+/class II+/Ii- phenotype, within an established subcutaneous RM-9 tumor nodule, led to an effective immune response limiting tumor growth. This phenotype was created by intratumoral injection of plasmid cDNAs coding for interferon gamma, MHC class II transactivator, and an antisense reverse gene construct (RGC) for a segment of the gene for Ii protein (-92,97). While this protocol led to significant suppression of tumor growth, there were no disease-free survivors. Nevertheless, irradiation of the tumor nodule on the day preceding initiation of gene therapy yielded 7 of 16 mice that were disease-free in a long-term follow up of 57 days compared to 1 of 7 mice receiving radiotherapy alone. Mice receiving radiotherapy and gene therapy rejected challenge with parental RM-9 cells and demonstrated specific cytotoxic T-cell activity in their splenocytes but not the mouse cured by radiation alone. These data were reproduced in additional experiments and confirmed that tumor irradiation prior to gene therapy resulted in complete tumor regression and specific tumor immunity in more than 50% of the mice. Increasing the number of plasmid injections after tumor irradiation induced tumor regression in 70% of the mice. Administering radiation before this novel gene therapy approach, that creates an in situ tumor vaccine, holds promise for the treatment of human prostate carcinoma.