[Targeted molecular therapy and immunotherapy for prostate cancer]. / Molekulare zielgerichtete Therapie und Immuntherapie des Prostatakarzinoms.

For decades, the treatment of advanced prostate cancer was mainly based on the manipulation of the androgen receptor-controlled proliferation pathway. Chemotherapy only played an additional important role with the advent of taxanes. The progress in translational research in recent years has led to innovations in the therapeutic environment. With the decoding of the homologous repair deficiency (HRD) machinery and its ability to be influenced by PARP inhibitors, targeted therapies moved into the therapeutic focus for selected patients. The first positive phase III study for PARP inhibitors is already available. In addition, immunotherapy for the treatment of prostate cancer, which is now widely used in oncology, is also making progress; both checkpoint inhibitors and bispecific antibodies have shown clinically useful activities. Cellular therapies such as CAR T cells, which are directed against prostate-specific membrane antigen (PSMA), are still at an early stage of development. In this review, the authors provide a summary of the basic principles and clinical development of these new therapies.

Tyrosine kinase inhibition increases the cell surface localization of FLT3-ITD and enhances FLT3-directed immunotherapy of acute myeloid leukemia.

The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.

Compartment specific expression of dendritic cell markers in human glomerulonephritis.

Macrophages and dendritic cells are heterogenous and highly plastic bone marrow-derived cells that play major roles in renal diseases. We characterized these cells using immunohistochemistry in 55 renal biopsies from control patients or patients with glomerulonephritis as an initial step towards postulating specific roles for these cells in kidney disease. In proliferative glomerulonephritis numerous CD68 positive (pan monocyte, macrophage and dendritic marker) cells were found in both glomeruli and the tubulointerstitial space, however, a myeloid dendritic cell marker (DC-SIGN) was identified only in the tubulointerstitium. A significant number of plasmacytoid dendritic cells (identified as BDCA-2 positive cells) were seen at sites of interstitial inflammation, including follicular aggregates of inflammatory cells. Langerin positive cells (a marker of Langerhans' cells) were detectable but rare. The area of either CD68 or DC-SIGN positive interstitial cells correlated with serum creatinine. Low levels of DC-SIGN, DC-LAMP and MHC class II mRNA were present in the tubulointerstitial space in controls and increased only in that region in proliferative glomerulonephritis. We demonstrate that the CD68 positive cells infiltrating the glomerulus lack dendritic cell markers (reflecting macrophages), whereas in the tubulointerstitial space the majority of CD68 positive cells are also DC-SIGN positive (reflecting myeloid dendritic cells). Their number correlated with serum creatinine, which further emphasizes the significance of interstitial DCs in progressive glomerular diseases.

The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors.

Members of the heat shock protein (hsp70) family are either constitutively expressed (hsc70) or can be induced by hyperthermic stress (hsp70). Recombinant hsp70 (rhsp70) stimulates cytokine production from monocytes and enhances NK cell proliferation and cytotoxicity. Here we demonstrate that rhsp70 binds to immature dendritic cells (DC) derived from monocyte precursors and induces their maturation as evidenced by an increase in CD40, CD86 and CD83 expression. Immature DC stimulated to mature with rhsp70 show an enhanced ability to present tyrosinase peptide to specific CTL. Mature DC did not bind rhsp70, suggesting a down-regulation in the expression of its receptor. When rhsp70 was added to monocyte precursors at the same time as GM-CSF and IL-4 it reduced the differentiation of monocytes into DC as shown by a decrease in the level of CD40, CD83, CD86 and HLA-DR expression and an increase in CD14 expression. The constitutively expressed hsc70 had neither a stimulatory effect on the maturation of immature DC nor did it reduce the differentiation of monocytes into DC. These findings demonstrate the specific ability of rhsp70 to induce the maturation of immature DC. Therefore rhsp70 may be useful for its adjuvant like properties in DC based immunotherapy of certain tumors.

Gene transfer of human interferon gamma complementary DNA into a renal cell carcinoma line enhances MHC-restricted cytotoxic T lymphocyte recognition but suppresses non-MHC-restricted effector cell activity.

Even though renal cell carcinomas (RCC) are thought to be immunogenic, many tumors express variations in surface molecules and intracellular proteins that hinder induction of optimal antitumor responses. Interferon gamma (IFNgamma) stimulation can correct some of these deficiencies. Therefore, we introduced the complementary DNA (cDNA) encoding human IFNgamma into a well-characterized RCC line that has been selected for development of an allogeneic tumor cell vaccine for treatment of patients with metastatic disease. Studies were performed to determine how endogenous IFNgamma expression influences tumor cell immunogenicity. IFNgamma transductants showed minimal increases in surface expression of MHC class I and adhesion molecules but expression of class II molecules was induced. Proteins of the transporter associated with antigen processing (TAP) and low molecular weight polypeptide (LMP) were constitutively expressed at high levels. The transductants stimulated allospecific cytotoxic T lymphocytes (CTL); however, they were not better than unmodified tumor cells in this capacity. Endogenous IFNgamma expression enhanced tumor cell recognition by MHC-restricted, tumor antigen-specific CTL but suppressed recognition by non-MHC-restricted cytotoxic cells. Thus, the functional consequences of IFNgamma expression varied with respect to the type of effector cell and were not always beneficial for tumor cell recognition.

Non-MHC-restricted CD4+ T lymphocytes are regulated by HLA-Cw7-mediated inhibition.

Natural killer cells (NK cells) represent an important component of innate immunity with the capacity to kill many tumor and virus-infected cells. The discovery of several classes of killer cell inhibitory receptors expressed by NK cells that bind specific MHC class I ligands on target cells provides detailed insight into the regulation of NK cells. Inhibitory receptors deliver negative signals following MHC ligand binding that abrogate cytotoxicity and, thus, determine the specificity of NK effector cell function. Here, we describe a novel subset of human memory CD4(+) T lymphocytes that display an NK-like pattern of regulation. These CD4(+) T cells display non-MHC-restricted cytotoxicity that is governed by HLA-Cw7 mediated inhibition. In NK cells, such specificity is associated with expression of the inhibitory receptor p58.2. In contrast, neither p58.2 nor other known inhibitory receptors were detected on these non-MHC-restricted CD4(+) T cells. This suggests that these cells are regulated by a hitherto unknown inhibitory receptor. The finding that interactions with MHC molecules downregulate the function of these CD4(+) T cells suggests that these non-MHC-restricted T cells may function to detect and eliminate cells with aberrant MHC expression.

Cellular and molecular analyses of major histocompatibility complex (MHC) restricted and non-MHC-restricted effector cells recognizing renal cell carcinomas: problems and perspectives for immunotherapy.

Renal cell carcinomas belong to the small group of tumors that are able to induce antitumor responses. Here we describe two general types of cytotoxic effector lymphocytes that can eliminate autologous tumor cells and discuss the role that major histocompatibility complex encoded molecules play in governing their specificities. Improved understanding of the cellular and molecular basis of renal cell carcinoma recognition opens new avenues of research with the potential to develop better immunotherapies for patients with metastatic disease.

HLA-derived peptides which inhibit T cell function bind to members of the heat-shock protein 70 family.

Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.

Dissociation of beta 2-microglobulin from HLA class I heavy chains correlates with acquisition of epitopes in the cytoplasmic tail.

A rabbit antiserum "ABR2" was raised against a peptide with sequence identity to 10 amino acids of the cytoplasmic tail of HLA class I heavy chains. Western blotting and immunoprecipitation analyses demonstrate that ABR2 reacts with HLA class I heavy chains. The antiserum reacts poorly with beta 2-microglobulin (beta 2-m)-associated heavy chains and reacts strongly with free heavy chains. ABR2 reacts with immature heavy chains from the endoplasmic reticulum that have yet to bind beta 2-m and mature heavy chains that have dissociated from beta 2-m at the plasma membrane. Comparison with HC10, a mAb that recognizes an epitope defined by polymorphism at residue 62 of the alpha 1 helix of free HLA class I heavy chains, shows that ABR2 reacts with overlapping populations of free heavy chains (for those allotypes that react with both Abs), but it also identifies populations that bind to one Ab and not the other. ABR2 induces dissociation of beta 2-m from HLA-B38 molecules expressed by the human B cell line "TEM," a phenomenon not detected with other allotypes or with the same allotype in a different cell line. This study shows that association of beta 2-m with the extracellular domains of HLA class I heavy chains can cause a change in the cytoplasmic tail that prevents binding of Abs present in the ABR2 antiserum. Similar findings have been made for mouse H-2 class I molecules, which suggests that this is a general property of class I MHC molecules.

Species-specific differences in chaperone interaction of human and mouse major histocompatibility complex class I molecules.

Previous studies have shown that immature mouse class I molecules transiently associate with a resident endoplasmic reticulum protein of 88 kD that has been proposed to act as a chaperone for class I assembly. Subsequently, this protein was demonstrated to be identical to calnexin and to associate with immature forms of the T cell receptor complex, immunoglobulin, and human class I HLA heavy chains. In this paper we define further the interaction of human class I HLA heavy chains with chaperone proteins and find key differences with the complexes observed in the mouse system. First, calnexin and immunoglobulin binding protein (BiP) both associate with immature HLA class I heavy chains. The two chaperones are not found within the same molecular complex, suggesting that calnexin and BiP do not interact simultaneously with the same HLA class I heavy chain. Second, only free HLA class I heavy chains, and not beta 2-microglobulin (beta 2m)-associated heavy chains are found associated with the chaperones. Indeed, addition of free beta 2m in vitro induces dissociation of chaperone-class I HLA heavy chain complexes. The kinetics for dissociation of the class I HLA heavy chain-chaperone complexes and for formation of the class I HLA heavy chain-beta 2m complex display a reciprocity that suggests the interactions with chaperone and beta 2m are mutually exclusive. Mouse class I heavy chains expressed in human cells exhibit the mouse pattern of interaction with human chaperones and human beta 2m and not the human pattern, showing the difference in behavior is purely a function of the class I heavy chain sequence.

Microheterogeneity in HLA-B35 alleles influences peptide-dependent allorecognition by cytotoxic T cells but not binding of a peptide-restricted monoclonal antibody.

Strong peptide dependency of HLA-B*3501-specific alloreactive T-cell clones was observed in the recognition of cells bearing closely related B35 variants. The single amino acid exchange in the beta-pleated sheet of B*3503 completely abolished the responses of all clones, whereas an amino acid exchange in the alpha 2 helix of the newest B35 member (B*3508) only altered allorecognition of one T-cell clone, demonstrating the differential impact of these positions on peptide binding to B35 molecules. In contrast to T cells, a mAb (TU165) recognizing the B35 specificity in a peptide-dependent manner bound to the B35 variants irrespective of their sequence heterogeneity. However, quantitative binding differences were detected with cells bearing the same B35 alleles. This is most likely due to variations in the amount of peptide(s) that associates with B35 and forms the ligand seen by this mAb. These results reveal how naturally occurring single amino acid substitutions have led to generation of functionally distinct molecules of another multimember HLA class I cluster.

Soluble T cell receptor-like properties of an HLA-B35-specific monoclonal antibody (TU165).

A mouse monoclonal antibody of IgM class (TU165) was produced using Epstein-Barr virus (EBV)-infected mutant cells derived from the human BJAB-B95.8.6 cell line as immunogen. Binding studies with several HLA deletion mutant cell lines indicated that TU165 recognized the HLA-B35 molecule. In a panel of 89 EBV-transformed lymphoblastoid cell lines, all HLA-B35+ cells (n = 24) reacted with TU165 while all but two HLA-B35- lines (n = 65) were unreactive (r = 0.95). Surprisingly, peripheral blood lymphocytes of HLA-B35+ donors were unreactive; however, strong enhancement of TU165 recognition was observed with B cells of one of these individuals after transformation with EBV (B95.8 strain). Transfection of both HLA-B35 and human beta 2-microglobulin genomic DNA into mouse P815 cells led to high expression of HLA-B molecules; yet, expression of the TU165 epitope was not observed. Furthermore, the EBV-negative cell line BJAB as well as the EBV-infected (P3HR1 strain) line BJAB-HR1K were only weakly reactive, whereas the BJAB-B95.8 cell line was strongly positive. These results indicate that EBV-encoded or -controlled peptide(s) must be bound by HLA-B35 antigens to create the epitope which allows efficient binding of TU165.