Pease, Larry R.

AbstractOur goal is to develop peptide vaccines that stimulate tumor antigen–specific T-cell responses against frequently found cancers. Previous work has shown that to generate effective T-cell responses, peptides have to be administered in combination with strong adjuvants such as Toll-like receptor agonists. However, most animal tumor model systems used to study peptide vaccines were not truly representative of malignant diseases in humans because they solely used transplantable tumor lines, and instead of true tumor antigens, they used highly immunogenic foreign proteins. Here, we describe a peptide vaccination strategy, which is highly effective in delaying or preventing the occurrence of spontaneous breast tumors. Transgenic female BALB-neuT mice that carry the activated rat HER-2/neu oncogene were vaccinated with a synthetic peptide from the rat HER-2/neu gene product, which represents an epitope for CTLs in combination with a Toll-like receptor agonist adjuvant. Our results show that to obtain tumor antigen-specific CTL responses and antitumor effects, the vaccine had to be administered repetitively, or the function of CD4/CD25 T regulatory cells had to be blocked with anti-CD25 antibody therapy. Mice that were vaccinated with this approach remained tumor-free or were able to control spontaneous tumor growth and exhibited long-lasting CTL responses, not only against the immunizing peptide but also against other peptides derived from rat HER-2/neu product (i.e., epitope spreading). These results suggest that similar strategies should be followed for conducting clinical studies in patients. [Cancer Res 2007;67(3):1326–34]

Summary A patient with Waldenstrom’s macroglobulinaemia expresses a high titre IgM antibody in serum that binds both mouse and human dendritic cells (DC) in a B7-DC (PD-L2)-dependent manner. We have reported previously that purified antibody from patient serum activates immature and mature DC in vitro, enhancing the ability of these professional antigen-presenting cells to activate naive T cells, take up antigen, resist a cytokine-depleted environment and secrete immunomodulatory cytokines, such as interkeukin (IL)-6 and tumour necrosis factor (TNF)-α. Systemic treatment of experimental animals with this antibody induces potent anti-melanoma immunity and modulates protectively the recall response against antigen challenge through the airway in an experimental model of inflammatory airway disease. Here we describe a monoclonal IgM antibody derived from this serum immunoglobulin that recapitulates each of these earlier observations, providing direct evidence that M protein from the Waldenstrom’s patient mediates these potent immunomodulatory effects. Furthermore, cell lines expressing this recombinant form of the human antibody provide the basis for developing this reagent for clinical application.

ABSTRACT Epidemiological studies suggest that an environmental factor (possibly a virus) acquired early in life may trigger multiple sclerosis (MS). The virus may remain dormant in the central nervous system but then becomes activated in adulthood. All existing models of MS are characterized by inflammation or demyelination that follows days after virus infection or antigen inoculation. While investigating the role of CD4 + T cell responses following Theiler's virus infection in mice deficient in STAT4 or STAT6, we discovered a model in which virus infection was followed by demyelination after a very prolonged incubation period. STAT4−/− mice were resistant to demyelination for 180 days after infection, but developed severe demyelination after this time point. Inflammatory cells and up‐regulation of Class I and Class II MHC antigens characterized these lesions. Virus antigen was partially controlled during the early chronic phase of the infection even though viral RNA levels remained high throughout infection. Demyelination correlated with the appearance of virus antigen expression. Bone marrow reconstitution experiments indicated that the mechanism of the late onset demyelination was the result of the STAT4−/− immune system. Thus, virus infection of STAT4−/− mice results in a model that may allow for dissection of the immune events predisposing to late‐onset demyelination in MS.

Abstract A naturally occurring human antibody potentiates dendritic cell function on cross-linking B7-DC (PD-L2), supporting robust T-cell responses in vitro. Moreover, treatment of dendritic cells with B7-DC cross-linking antibody resulted in secretion of interleukin-12, suggesting a TH1 polarization of this response. Here we show an in vivo immunotherapeutic effect of this B7-DC cross-linking antibody using a poorly immunogenic B16 melanoma tumor model. Treatment of mice systemically with antibody at the time of tumor cell engraftment prevented tumor growth in a CD4 and CD8 T-cell-dependent manner. The protective effect of B7-DC cross-linking antibody treatment was independent of endogenous antibody responses. Tumor-specific CTL precursors could be isolated from lymph nodes draining the tumor site in animals treated with B7-DC cross-linking antibody, but not from those treated with isotype control antibodies. The elicited antitumor responses in vivo were specific and long-lasting. More strikingly, treatment of mice with B7-DC cross-linking antibody after the tumors were established in the lungs resulted in protection in a CD8-, perforin-, and granzyme B-dependent fashion. Depletion of natural killer cells did not block the effects of treatment with B7-DC cross-linking antibody. Together, these findings demonstrate that cross-linking B7-DC with the human IgM antibody sHIgM12 can induce a protective immune response against a weakly antigenic experimental tumor and therefore has potential as a novel immunotherapeutic approach for treating cancer.

Dendritic cells (DCs) are classified in two states: immature DCs (iDCs), which perform sentinel functions, sampling for antigen and danger signals, and mature DCs (mDCs), which exhibit enhanced antigen-presenting functions but are no longer capable of acquiring antigen. We now describe DCs with a different activation phenotype: cells having the strong antigen-presenting functions of mDCs and the antigen-acquiring functions of iDCs. We have described an antibody that binds the costimulatory molecule B7-DC and activates DCs. The resulting phenotype is distinct from iDCs or mDCs matured by using Toll-like receptor (TLR) agonists. Ability to take up antigen increases, while expression of B71/2 costimulatory and MHC molecules remains unchanged. DCs matured with TLR agonists and then superactivated through B7-DC exhibit a previously unrecognized phenotype. These DCs recover the ability to take up antigen, which is normally lost after treatment with TLR-3 and TLR-9 agonists, yet retain the high expression of costimulatory and MHC molecules and strong antigen-presenting functions of mDCs. Immunization using TLR agonists and B7-DC XAb (cross-linking antibody) together as adjuvant resulted in substantially increased cytolytic T cell responses, particularly when minimal peptide antigens were used. By stimulating DCs with two distinct activation signals, a previously unrecognized phenotype exhibiting augmented antigen-presenting functions was obtained.

ABSTRACTTheiler's murine encephalomyelitis virus (TMEV) infection of the brain induces a virus-specific CD8+T-cell response in genetically resistant mice. The peak of the immune response to the virus occurs 7 days after infection, with an immunodominant CD8+T-cell response against a VP2-derived capsid peptide in the context of the Dbmolecule. The process of activation of antigen-specific T cells that migrate to the brain in the TMEV model has not been defined. The site of antigenic challenge in the TMEV model is directly into the brain parenchyma, a site that is considered immune privileged. We investigated the hypothesis that antiviral CD8+T-cell responses are initiated in situ upon intracranial inoculation with TMEV. To determine whether a brain parenchymal antigen-presenting cell is responsible for the activation of virus-specific CD8+T cells, we evaluated the CD8+T-cell response to the VP2 peptide in bone marrow chimeras and mutant mice lacking peripheral lymphoid organs. The generation of the anti-TMEV CD8+T-cell response in the brain requires priming by a bone marrow-derived antigen-presenting cell and the presence of peripheral lymphoid organs. Although our results show that activation of TMEV-specific CD8+T cells occurs in the peripheral lymphoid compartment, they do not exclude the possibility that the immune response to TMEV is initiated by a brain-resident, bone marrow-derived, antigen-presenting cell.

ABSTRACT The human monoclonal antibody rHIgM22 enhances remyelination following spinal cord demyelination in a virus‐induced murine model of multiple sclerosis. Using three‐dimensional T2‐weighted in vivo spinal cord magnetic resonance imaging (MRI), we have therefore assessed the extent of spinal cord demyelination, before and after 5 weeks of treatment with rHIgM22, to determine whether antibody enhanced remyelination can be detected by MRI. A significant decrease was seen in T2 high signal lesion volume following antibody treatment. Histologic examination of the spinal cord tissue reveals that this decrease in lesion volume correlates with antibody promoted remyelination. To show that rHIgM22 enters the spinal cord and colocalizes with demyelinating lesions, we used ultrasmall superparamagnetic iron oxide particle (USPIO)‐labeled antibodies. This may be considered as additional evidence to the hypothesis that rHIgM22 promotes remyelination by local effects in the lesions, likely by binding to CNS cells. The reduction in high signal T2‐weighted lesion volume may be an important outcome measure in future clinical trials in humans.

The set of peptides that is bound by a given major histocompatibility complex class I product can be described by one or two properly spaced anchor residues, and two properly spaced peptide termini, approximately 8-10 residues apart. Using radiolabeled peptide libraries, we examined whether mutations in those "pockets" in class I Kb molecules that do not seem critically involved in the interaction with the peptide anchor residues, do exert an effect on the set of preferred peptides. We find that mutations in all the pockets found in the structure of Kb have a significant effect on the peptide preference of the molecule, and their recognition by cytotoxic T cells. Alterations in substrate specificity are also observed for mutations involving residues that interact with main chain atoms in both peptide termini. These findings challenge a static view of the interaction of peptide termini with their respective pockets in the class I molecule, and imply a role for the minor pockets in peptide selectivity.

A chimeric class I glycoprotein was created to investigate the functional contribution of the alpha helices and the beta-pleated sheets in forming the antigen recognition site (ARS) of antigen-presenting molecules. This novel molecule was generated by replacing the DNA sequences encoding the alpha helices of the Ld gene with the corresponding sequences from the Kb gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (Kb alpha Ld beta) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the alpha helices of Kb, as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the alpha helices of Kb can associate with the beta-pleated sheets of Ld to form a stable structure, which is expressed on the cell surface. To address the role of the alpha helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing Kb alpha Ld beta. CTL raised against Kb or Ld as alloantigens showed little, if any, ability to lyse L cells expressing Kb alpha Ld beta. Thus, alloreactive CTL did not recognize structures determined by the alpha helices alone or by the beta sheets of the ARS alone. However, bulk and cloned alloreactive CTL that were generated against the mutant Kb glycoprotein Kbm8 reacted strongly with Kb alpha Ld beta. In addition to the Kb alpha helices, the Kbm8 ARS shares a single polymorphic amino acid at position 24 with Kb alpha Ld beta. Amino acid 24 is located on the beta 2 strand that forms part of the floor of the ARS and has been identified as a component of pocket B in the HLA class I ARS. The substitution of Glu to Ser at this position was shown previously to be the central determinant of the Kbm8 mutant alloantigenicity. The functional significance of this position in determining crossreactivity between bm8 and Kb alpha Ld beta identifies pocket B as a strong anchor for allogenic self-peptides. These findings demonstrate that determinants recognized by CTL on class I alloantigens are formed by interactions involving both the alpha helices and beta sheets of the ARS. These interactions are best explained by the influence of the alpha helices and beta sheets on the peptide-binding properties of these antigen-presenting molecules.

Three cDNA with D region gene features have been identified from the H-2v haplotype. Provisionally, the sequences have been designated as D/Lv1, D/Lv2, and D/Lv3. The coding segments for the antigen binding domain (ABD) of all three D/Lv genes were engineered into a class I genomic expression vector and expressed in L cells. FACS analysis of the three D/Lv-Ld gene transfectants revealed that the D/Lv1 molecules were recognized by both monoclonal antibodies (mAbs) 141 and 142, and the D/Lv2 molecules were recognized by mAb 143. In addition to the D/Lv1 molecules, the mAb 141 also recognized the D/Lv3 molecules. Both the D/Lv1-Ld and D/Lv2-Ld transfectants were killed efficiently by H-2Dv region-specific alloreactive CTL. The D/Lv3 gene is the first identified D region gene other than D and L that is transcribed abundantly in spleen and the D/Lv3 RNA is present as two alternatively spliced forms. Structural analysis of the D/Lv3 hybrid molecules showed that it was susceptible to proteolysis and thermolabile at 37 degrees C, suggesting D/Lv3 is a transcribed pseudogene. A parsimony tree analysis of three D/Lv sequences with a set of class I gene sequences revealed that the H-2v sequences clustered with D region genes. The presence of a third gene with D/L-like features in H-2v, yet structurally different from the known D/L alleles, raises the possibility that the current D/L genes evolved from a family of D/L-like genes, some of which are no longer represented among many of the mouse major histocompatibility complex haplotypes. The observation that D region alleles cluster into subgroups suggests that the alleles are not all related to each other by linear descent through a single locus. We propose that current alleles are derived from more than one ancestral locus in a manner similar to the origin of the gamma 2 a immunoglobulin constant region alleles.