Minzhen Xu

Increasing the potency of MHC class II-presented epitopes by linkage to Ii-Key peptide.

We previously found that peptide Ii77-92 from the immunoregulatory Ii protein significantly enhances the binding of antigenic peptides to MHC class II molecules. Now a series of hybrids have been constructed linking LRMK, the active core region of the Ii77-92 peptide, to an antigenic epitope of cytochrome C. In vitro T cell hybridoma stimulation by some of these hybrids is up to 250 times more potent than by the antigenic peptide. The biological activities of the hybrids were tested in terms of length and composition of the linker. Simple spacers containing a polymethylene bridge (-HN-CH(2)-CH(2)-CH(2)-CH(2)-CO(2)-) were fully active in these hybrids which can enhance vaccination with MHC class II-presented epitopes.

Cathepsin B cleavage and release of invariant chain from MHC class II molecules follow A staged pattern

A staged pattern of cathepsin B cleavage of MHC class II alpha, beta-bound invariant (Ii) chain and release of fragments was defined. Charge-loss mutations in the Ii chain were created in three clusters of cathepsin B putative cleavage sites R78K80K83K86, K137K143, and R151K154. Products of HLA-DR1 alpha, beta and wild type (WT) or mutant Ii genes, co-transfected into COS1 cells, were cleaved by cathepsin B and immunoprecipitated by antibodies either to MHC class II chains or to different Ii epitopes. In WT Ii, cathepsin B digestion generated two forms of p21 Ii fragments: a p21 recognized by anti-C-terminus antibodies and a p21 recognized by an antibody to a determinant near the N-terminus. C-terminal p21 was released from MHC class II alpha, beta chains upon its formation while N-terminal p21 remained associated with MHC class II alpha, beta chains. Mutations at K137K143 inhibited the generation of N-terminal p21 by cathepsin B. Mutation at R78K80K83K86 led to an accumulation of MHC class II-bound N-terminal p21 without the appearance of MHC class II-bound p14, p10, and p6 fragments after cathepsin B digestion. These results indicate that cathepsin B cleaves wild type Ii first about K137K143 to produce a MHC class II-associated N-terminal p21, which is then cleaved about R78K80K83K86 to generate p14, p10 and finally p6 which still associates with MHC class II alpha, beta chains. This pattern of staged cleavage and release of Ii might be related to a concerted mechanism regulating the binding of antigenic peptides to MHC class II molecules.

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

PurposeCytotoxic 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.MethodsStimulating 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.ResultsIn 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-γ release, whereas the epitope-only peptide was weakly active. In fact, the hybrid stimulated IFN-γ 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.ConclusionSuch Ii-Key/MHC class-II epitope hybrid peptides have both diagnostic and therapeutic applications.

Cancer immunotherapy by antisense suppression of Ii protein in MHC-class-II-positive tumor cells.

Abstract This study was aimed at creating a more effective tumor cell vaccine by suppressing Ii protein in the presence of MHC class II molecules within a cancer cell. Absence of the Ii protein, which normally blocks the antigenic-peptide-binding site of MHC class II molecules at synthesis in the endoplasmic reticulum, presumably increases the range of cancer-related epitopes presented to CD4+ helper T cells. Effective suppression of Ii protein was achieved with an antisense, phosphorothioate oligonucleotide, which was selected on the basis of (1) the RNase H activation assay, (2) an assay for Ii protein suppression, and (3) a test for potency with respect to the extent of base sequence (“sequence walking”). The SaI murine sarcoma, which is MHC-class-I+ and MHC-class-II−, Ii-protein−, upon transfection with genes for either interferon γ or the MHC class II transactivator, came to express MHC class II molecules and Ii protein. In each line of transfected tumor cells, the antisense oligonucleotide profoundly suppressed Ii protein in 35%–55% cells, without affecting expression of MHC class II molecules. Inoculation of mice with such Ii-protein-suppressed tumor vaccine cells, after either formaldehyde fixation or X-irradiation, led to much greater protection against challenge with the parental SaI sarcoma than did inoculation with untreated cells. This approach to cancer cell vaccination can be applied in a wide range of human tumors.

MHC Class II Allosteric Site Drugs: New Immunotherapeutics for Malignant, Infectious and Autoimmune Diseases*

The discovery of the interactions of the ‘Ii‐Key’ segment of the Ii protein with the major histocmpatibility complex (MHC) Class II allosteric site, which is adjacent to the antigenic peptide‐binding site, creates therapeutic opportunities by regulating the antigenic peptide binding to MHC class II molecules. The binding of Ii‐Key to the MHC class II allosteric site loosens the hold of the MHC Class II ‘clamshell’ on antigenic peptides and leads to highly efficient antigenic peptide charging to or releasing from the MHC class II antigenic peptide‐binding groove. Ii‐Key peptide‐induced spilling of bound antigenic peptide, or replacement with inert blockers, leads to ‘inert immunosuppression’. Highly efficient replacement of ambient with vaccine peptides by Ii‐Key permits ‘active immunosuppression’ for antigen‐specific control of autoimmune diseases in the absence of cytokines or adjuvants. On the other hand, active immunization against cancer or infectious disease can result from epitope replacement mediated by Ii‐Key and accompanied by cytokines or other adjuvants. Finally, linking the Ii‐Key peptide through a simple polymethylene bridge to an antigenic sequence vastly increases the potency of MHC Class II peptide vaccines. In summary, the discovery of the MHC class II allosteric site allows one to increase the efficiency of MHC class II‐related, antigenic epitope‐specific therapy for malignant, infectious, and autoimmune diseases. The focus of this review is on the mechanism and potential clinical use of such novel allosteric site‐directed, Ii‐key drugs.

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-γ 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-γ response and to a lesser extent the IL-4 response. CpG enhanced a strong IFN-γ response, with a negligible IL-4 response. The 3- to 5-times enhancement of the total ELISPOT responses (number of spots × 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.

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.

More efficient peptide binding to MHC class II molecules during cathepsin B digestion of Ii than after Ii release

The binding of a T cell-presented peptide to MHC class II alpha,beta chains occurs as a concurrent process with the release of the associated invariant chain (Ii) by cathepsin B. Ii was digested by cathepsin B from solubilized, MHC class II alpha,beta,Ii complexes in the presence of N-hydroxysuccinimidyl-4-azidobenzoate-conjugated, 125I-labeled, influenza virus matrix (18-29) peptide. The peptide was crosslinked where it became bound. This HLA-DR1-restricted peptide bound about three times more efficiently to class II alpha,beta chains of DR1-positive B cells when present during cathepsin B digestion of Ii than when added afterward, also at pH 5.0. Binding was competed by similarly DR-restricted peptides. Cathepsin D cleaved Ii but did not enhance peptide binding. However, a trace level of cathepsin D, added to the assay for peptide binding in the presence of cathepsin B, further enhanced peptide binding about three times. These experiments support an hypothesis for the staged release of Ii fragments by cathepsin D and cathepsin B, catalyzing at one point the insertion of a peptide into the antigen binding site formed by class II alpha and beta chains.

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.

Genetic modulation of tumor antigen presentation

An effective cancer-cell vaccine is created by expressing major-histocompatibility-complex (MHC) class II molecules without the invariant chain protein (Ii) that normally blocks the antigenic-peptide-binding site of MHC class II molecules at their synthesis in the endoplasmic reticulum. Such tumor-cell constructs are created either by the transfer of genes for MHC class IIalpha and beta chains, or by the induction of MHC class II molecules and Ii protein with a transacting factor, followed by Ii suppression using antisense methods. Preclinical validation of this approach is reviewed with the goal of using this immunotherapy for metastatic human cancers.