Presence of breast cancer antigens in uninvolved axillary lymph nodes

Monoclonal antibodies which bind to breast cancer have been used to evaluate the detection of metastatic disease in axillary lymph nodes. Three monoclonal antibodies (H59, H71, and H72) were reacted with tissue sections of primary tumors and axillary nodes from 24 mastectomy specimens and four specimens from glandular mastectomies for benign disease. All three antibodies had been shown to react with subsets of normal and malignant breast tissue; did not bind erythroid, myeloid, or lymphoid tissue; and recognized antigens in paraffin-embedded tissue. The antibodies recognized cell surface antigens, and H59 and H72 bound to glycoproteins which are either sloughed or secreted. Primary tumors and tumors in lymph nodes from the same specimen were always bound by the same antibodies. Antibodies detected unrecognized microscopic tumor in nodes from one previously node-negative specimen and two specimens with positive nodes. This suggests that monoclonal antibodies may be useful for detecting metastatic breast cancer in nodes which by light microscopy are negative. Moderate binding of H59 and H72 antibodies to sinus histiocytes and perivascular cells was observed in all uninvolved nodes with sinus hyperplasia obtained from benign and malignant specimens. Thus, breast antigens can be identified in hyperplastic nodes in patients with no evidence of breast cancer. The antigens are detected predominately in the lymphoid sinuses and are bound to nonneoplastic cells. Therefore, breast antigens are regularly being processed and presented by normal lymphoid cells within the sinus. The binding of these monoclonal antibodies to axillary lymph nodes does not necessarily indicate the presence of metastatic disease. Dense binding to paracortical single cells was observed in tumor-containing lymph nodes and in uninvolved nodes obtained from mastectomy specimens with breast cancer. These cells are infrequent, and their number in an uninvolved node correlates with the pathological stage. They represent either binding to isolated lymphoid cells or metastatic tumor. Studies are under way to determine the origin of these cells.     

Prostate-specific Membrane Antigen (PSMA)-specific Monoclonal Antibodies in the Treatment of Prostate and Other Cancers

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein that is expressed by prostate epithelial cells. PSMA-specific monoclonal antibodies have been utilized to characterize the biologic function and in vivo biodistribution of PSMA. PSMA is an attractive target protein for monoclonal antibody directed imaging or therapeutics for prostate cancer since its expression is relatively restricted to prostate epithelial cells and is over-expressed in prostate cancer, including in advanced stages. Currently, clinical usage of PSMA specific monoclonal antibodies has been limited to diagnostic immunohistochemistry and imaging of patients with prostate cancer. Novel applications for these antibodies will be discussed.     

Isolation of native human monoclonal autoantibodies to breast cancer

Using a unique fusion partner cell line, MFP-2, and B-lymphocytes from breast cancer patients, we developed a set of fully human monoclonal antibodies (MAbs) that bind with high specificity and sensitivity to breast cancer cells. Immunofluorescent staining of normal tissues, primary tumors, and metastatic lymph nodes demonstrates that these antibodies are specific for breast cancer of autologous and allogeneic origin. We have also determined that many of the antibodies selected based on specific binding to breast cancer cells and tissue also bind prostate cancer cells and tissue with high specificity and sensitivity. The targets of these antibodies have been localized to the cytoplasm and membrane. Biological assays for internalization and cytotoxicity demonstrated the ability of three antibodies to rapidly internalize. Our study demonstrates that isolation of native human MAbs from the natural antibody repertoire, targeted to cancer cells, is feasible and may provide a source of tools for immunotherapy.     

Preparation and characterization of new anti-PSMA monoclonal antibodies with potential clinical use

Monoclonal antibodies with high specificity for prostate cancer tissue are of interest for diagnostic and therapeutic applications employing targeted therapy. The prostate-specific membrane antigen (PSMA) is a protein predominantly found in epithelial cells of prostate tissue origin and its expression correlates with tumor aggressiveness. Here, we report the development and characterization of new antibodies against PSMA. Murine monoclonal antibodies (MAb) were obtained by immunizing mice with a peptide corresponding to PSMA extracellular residues 490-500 -- GKSLYESWTKK (PSMA(490-500)). The MAbs react specifically to PSMA and to the prostate cancer cell line LNCaP with an affinity for PSMA in the low nanomolar range. This study also demonstrates the potential use of these antibodies for targeted drug delivery to prostate cancer cells. Nanomolar concentrations of PSMA-specific MAb in association with a molecule with cytotoxic potential were sufficient to allow for binding and uptake by LNCaP cells within minutes, leading to complete cell death within 3 days. These MAbs have potential clinical value in the development of diagnostic and therapeutic applications for prostate cancer.     

Targeting immunosupportive cancer therapies: accentuate the positive, eliminate the negative

In this Commentary we aim to provide an overview of some specific examples of cancer therapeutics, including targeted approaches using monoclonal antibodies and kinase inhibitors, as well as highlight novel approaches for enhancing immunological responses against tumors. We point out that a fundamental property of the cancer cell, genomic instability, confounds the targeted therapies that aim to induce cell death directly while simultaneously enhancing the potential for immunological attack by creating a large number of neoantigens. We argue for combinatorial strategies with agents that target tumor cells to release these antigens together with innovative therapies that enhance immunological responses by interfering with inhibitory checkpoints.     

Spatial distribution of tumor-specific monoclonal antibodies in human melanoma xenografts.

The time-dependent (1-72-h) spatial distribution of three biotinylated anti-melanoma monoclonal antibodies (MAbs), a control MAb, and several macromolecular tracers was studied in two small (4-12-mg), well-characterized human melanoma xenografts (SK-MEL-2, M21) growing in the s.c. space of athymic nude mice. The specific MAbs (436, IND1, and 9.2.27) recognize two different melanoma cell surface antigens (Mr 125,000 glycoprotein melanoma-associated antigen and high molecular weight melanoma-associated antigen) and have equilibrium association constants differing by two orders of magnitude (10(8)-10(10) M-1). SK-MEL-2 tumors were poorly vascularized and were composed of one or several collections of tumor cells with few intratumor blood vessels. In contrast, M21 tumors induced a strong angiogenic response and were organized into multiple small tumor cell nests separated from each other by fine blood vessels. Neither tumor developed extensive connective tissue stroma. In both tumors, hyperpermeable blood vessels were concentrated at the tumor-host interface but some intratumor vessels in M21 tumors were also leaky. Macromolecular tracers extravasated extensively from leaky vessels into tumor stroma but penetrated poorly into tumor parenchyma. All three tumor-specific MAbs stained tumor cell surfaces in a time-dependent fashion such that one-half or more of all tumor cells were stained by 24-48 h. Tumor cell staining was favored by increased density of tumor cell antigens but, at the doses studied, was little affected by differences in affinity among tumor-specific antibodies. The distribution of MAb staining was nonuniform in two respects: (a) peripherally situated tumor cells were more likely to be stained than centrally placed cells, and only in the smallest tumors did MAb reach centrally placed tumor cells; and (b) staining was nonuniform in different parts of the same tumor. The inhomogeneity of tumor cell staining by tumor-specific MAb was attributable to several factors, including: tumor blood vessel number, distribution, perfusion and permeability; distribution of tumor connective tissue stroma; small volume of the parenchymal interstitial space and relatively impaired diffusion of macromolecules in that space (low effective diffusivity of MAb); and interactions between specific MAbs and tumor cells. Of these factors, those associated with the parenchymal compartment apparently were rate limiting, and strategies that enhance parenchymal penetration are likely to improve solid tumor therapy with MAbs.     

Profiling of internalizing tumor-associated antigens on breast and pancreatic cancer cells by reversed genomics

Human antibodies directed towards functionally associated tumor antigens have great potentials as adjuvant treatment in cancer therapy. Here we describe an efficient subtractive approach to select single chain Fv (scFv) antibodies, specifically binding to unknown rapidly internalizing tumor-associated antigens (TAA) on human breast and pancreatic carcinoma cell lines. After re-engineering the scFv into intact IgG molecules, these fully human antibodies displayed individual binding profiles to TAAs on breast, pancreatic, colorectal and prostate carcinomas, while showing no reactivity to lymphomas. The ability of the selected antibodies to undergo receptor-mediated internalization was verified by confocal microscopy, thus proving our strategy to provide a unique set of human antibodies, potentially useful in immunotherapy.     

Comparative study of monoclonal antibodies TURP-27 and HNK-1: their relationship to neural cell adhesion molecules and prostate tumor-associated antigens.

The murine monoclonal antibodies TURP-27 and HNK-1 have been shown to detect antigens which are heavily expressed by benign prostatic hyperplasia and carcinoma of the prostate. Western blot analysis of prostate extracts showed that monoclonal antibodies TURP-27 and HNK-1 bound glycoproteins with molecular weights of 180,000, 140,000, 120,000, 100,000, 90,000, and 69,000. Studies have shown that the HNK-1 carbohydrate epitope may be involved in cell adhesion and that it is a component of several characterized adhesion proteins. TURP-27 was found to bind at least three of these adhesion proteins: neural cell adhesion molecules; myelin-associated glycoprotein; and a second myelin glycoprotein, P0. Western blot analysis of prostate extracts showed that an antineural cell adhesion molecule serum bound the Mr 180,000 and 140,000 proteins. Based on reciprocal blocking and chemical tests, it was determined that the TURP-27 and HNK-1 epitopes are not identical. These data imply that the TURP-27 epitope may be a variant of the HNK-1 epitope or that the two epitopes are closely linked and that the TURP-27 and HNK-1 epitopes on prostate cells are positioned on neural cell adhesion molecule-like proteins.     

Human bladder cancer cell-surface antigens recognized by murine monoclonal antibodies raised against T24 bladder cancer cells

Seven hybridoma clones producing monoclonal antibodies (HBJ8, HBJ27, HBJ67, HBJ71, HBJ98, HBJ104 and HBJ127) were selected from hybridomas prepared by fusion between P3 X Ag8.653 mouse myeloma cells and spleen cells of a BALB/c mouse immunized with T24 human urinary bladder cancer cells, and the binding specificity of, and the molecular characters of the antigens defined by, these monoclonal antibodies were examined. The cell-surface antigens detected with these monoclonal antibodies from T24 bladder cancer cells were as follows: 1) HBJ27-defined gp85 antigen common in all human cells or tissues tested, 2) HBJ98- or HBJ127-defined gp125 antigen distributed in all epithelial and non-epithelial human tumor cell lines tested and in basal layers of the skin or esophagus, proximal tubules of the kidney, and crypts of the gastric and intestinal mucosa, 3) HBJ8-defined gp(40/90) and HBJ67-defined gp83 antigens distributed in a characteristic portion of epithelial tumor cell lines, 4) HBJ71-defined antigen of protein nature and HBJ104-defined antigen of unknown character, both being detected from immunizing T24 cells and a few epithelial tumor cell lines. All these monoclonal antibodies could bind with certain portions of fresh bladder cancer tissues from patients.     

A novel tumor-specific broad-spectral monoclonal antibody to PL2L60 is highly effective for the treatment of various types of cancers from human and mouse

There are numerous antibodies used for cancer therapy in clinic, but they are essentially less efficacy than expected. None of them has tumor-specific and broad-spectral properties. PIWIL2-like (PL2L) protein 60 (PL2L60) is a product of alienated activation of PIWIL2 gene, and has been found to be specifically and widely expressed in various types of cancers, including hematopoietic and solid ones. Current study aims to investigate whether a monoclonal antibody (mAb) to PL2L60 has both tumor-specific and broad-spectral properties, which can be used universally to treat various types of cancers. The expression of PL2L60 protein in the cell surface and cytoplasm were determined in a panel of human and mouse tumor cell lines by flow cytometry, immunofluorescent microscopy and Western Blotting. The apoptosis and the cell cycle arrest of the tumor cells treated with mAb KAO3 were evaluated by flow cytometry. The tumorigenesis of the mAb KAO3-pretreated tumor cells was determined by tumor incidence and tumor size, and the efficacy of mAb KAO3 treatment on tumor growth in tumors-bearing mice were kinetically evaluated. Complement-dependent cytotoxicity (CDC) assay was used to determine the capacity of mAb KAO3 to kill tumor cells. Treatment of human or mouse tumor cells from hematopoietic or solid tumors with mAb KAO3 at the time of inoculation efficiently inhibited tumorigenesis in the severe combined immunodeficient (SCID) mice. Moreover, injection of mAb KAO3 into established tumors significantly inhibited their growth, and prolonged survival of the tumor-bearing mice, including lymphoma, breast cancer, lung cancer and cervical cancer. The efficacy of mAb KAO3 treatment is likely associated with its binding to PL2L60 expressed on tumor cell surface, which may lead to cancer cell death through blocking cell cycling and/or activation of complement. In conclusion, we have identified a tumor-specific mAb to PL2L60 (KAO3), which may be used potentially to treat all the types of human cancers including from both hematopoietic and solid ones.     

Prostate cancer vaccines: current status

Recent insights into cell-mediated immunotherapy have led to a wave of new trials involving immunotherapy for prostate cancer. Vaccines have evolved from nonspecific immune stimulants like Bacillus Calmette-Guerin (BCG) to much more specific and potent strategies. Techniques currently being investigated include passive immunotherapy with monoclonal antibodies, adoptive transfer of activated effector T cells, and active immunotherapy involving immunization with whole-cell or antigen-specific vaccines. These therapies are being modified with cytokines and other immune modulating agents. Understanding the mechanisms of antitumor immunity and identifying relevant tumor-specific antigens will likely improve these vaccine strategies and provide them with a niche in the future of prostate cancer therapy.     

MUC1/X protein immunization enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target malignant cells

MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most antibodies against MUC1 recognize epitopes within the highly immunogenic alpha chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 alpha chain is shed into the peripheral circulation, sequesters circulating antitandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recognizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 alpha subunit binding the membrane-tethered beta subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding full-length MUC1 (MUC1/TM) followed by boosting with the alternatively spliced MUC1/X isoform from which the tandem repeat array has been deleted, we generated monoclonal antibodies, designated DMC209, which specifically bind the MUC1 alpha/beta junction. DMC209 is exquisitely unique for this site; amino acid mutations, which abrogate MUC1 cleavage, also abrogate DMC209 binding. Additionally, DMC209 specifically binds the MUC1 alpha/beta junction on full-length MUC1/TM expressed by breast and ovarian cancer cell lines and on freshly obtained, unmanipulated MUC1-positive malignant plasma cells of multiple myeloma. DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly and as an immunotoxin conjugate. Moreover, the novel immunization procedure used in generating DMC209 can be used to generate additional anti-MUC1 alpha/beta junction antibodies, which may, analogously to Herceptin, have cytotoxic activity. Lastly, sequential immunization with MUC1/TM cDNA acting as a nonspecific adjuvant followed by protein of interest may prove to be a generalizable method to yield high-titer specific antibodies.     

Screening of HLA-A24-restricted epitope peptides from prostate-specific membrane antigen that induce specific antitumor cytotoxic T lymphocytes.

PURPOSE: Prostate-specific membrane antigen (PSMA), which is a transmembrane glycoprotein predominantly expressed in prostate cancer, is an attractive target for tumor-specific immunotherapy. To identify human leukocyte antigen (HLA)-A24-restricted epitope peptides from PSMA for further application of the dendritic cell (DC)-based immunotherapy targeting prostate cancer, we have screened several PSMA-encoded HLA-A24-binding peptides for their capabilities to elicit specific antitumor CTL response in vitro. EXPERIMENTAL DESIGN: The amino acid sequence of PSMA was screened for peptides consisting of 9 or 10 amino acids, which possess the known HLA-A24-binding motif. Nine candidate peptides were screened for binding to HLA-A24 molecules. Then, each of these nine peptides was studied to determine whether CTL responses could be induced by primary in vitro immunization of CD8(+) T cells using peptide-pulsed autologous DCs derived from peripheral blood mononuclear cells of HLA-A24(+) healthy donor as antigen-presenting cells. The antigen specificity of the CTL lines was confirmed using several tumor cell lines as target cells, which were genetically modified to express both HLA-A24 and PSMA. RESULTS: Two peptides, LYSDPADYF and NYARTEDFF, were demonstrated to elicit CTL lines that lyse peptide-pulsed, HLA-A24(+) B-lymphoblastoid cells. Each of the CTL lines recognized their specific PSMA-expressing target cells in a HLA-A24-restricted manner. The capability to release IFN-gamma by the CTL lines was specifically inhibited by anti-MHC class I and anti-CD8 monoclonal antibodies but not by anti-MHC class II and anti-CD4 monoclonal antibodies. CONCLUSION: Two novel HLA-A24-restricted PSMA-derived epitopes were identified in this study. These epitopes can be used to further evaluate the clinical utility of DC-based immunotherapeutic strategies for treatment of hormone-refractory prostate cancers.     

Targeted toxin therapy for the treatment of cancer

Protein toxins such as Pseudomonas exotoxin, diphtheria toxin, and ricin may be useful in cancer therapy because they are among the most potent cell-killing agents. One molecule of a toxin delivered to the cytoplasm of a cancer cell will be lethal for that cell. However, to be therapeutically useful, these toxins need to be targeted to specific sites on the surface of cancer cells, then be internalized and ultimately reach the cell cytoplasm. This process is accomplished by eliminating binding to toxin receptors and redirecting the cell-killing activity of the toxin to receptors or antigens present on cancer cells. Typically, toxins are conjugated to cell-binding proteins such as monoclonal antibodies or growth factors. These conjugates bind and kill cancer cells selectively while normal cells, which don't bind the conjugates, are spared. Because the genes for many protein toxins have been cloned, it is possible to make genetic modifications to their structure. By deleting the DNA that codes for the toxin binding region and replacing it with various complementary DNA encoding other cell-binding proteins, it has been possible to make chimeric toxins that kill cells on the basis of the newly acquired binding activity. The ability to make these chimeras may be useful in designing future toxin-based anticancer therapies.     

Targeting CUB domain-containing protein 1 with a monoclonal antibody inhibits metastasis in a prostate cancer model

Through a whole-cell panning approach, we previously identified a panel of antibodies that bound to prostate cancer cell surface antigens. One such antigen, CUB domain-containing protein 1 (CDCP1), was recognized by monoclonal antibody 25A11 and is a single transmembrane molecule highly expressed in several metastatic cancers as well as on CD34(+)CD133(+) myeloid leukemic blast cells. We show CDCP1 expression on prostate cancer cell lines by real-time quantitative PCR (RT-qPCR), flow cytometry, and immunohistochemistry and on prostate cancer patient samples by RT-qPCR and immunohistochemical staining. In cell-based assays, antibody 25A11 inhibited prostate cancer cell migration and invasion in vitro. Further characterization showed that CDCP1 is internalized on antibody binding. When 25A11 was coupled to the cytotoxin saporin either directly or via a secondary antibody, both resulted in prostate cancer cell killing in vitro. In vivo targeting studies with an anti-CDCP1 immunotoxin showed significant inhibition of primary tumor growth as well as metastasis in a mouse xenograft model. These data provide support for continued evaluation of anti-CDCP1 therapy for potential use in cancer in primary and metastatic disease.     

Distribution,cellular localization,and therapeutic potential of the tumor-associated antigen Ku70/80 in glioblastoma multiforme

Antibodies specifically targeting tumor-associated antigens have proved to be important tools in the treatment of human cancer. A desirable target antigen should be unique to tumor cells, abundantly expressed, and readily available for antibody binding. The Ku70/80 DNA-repair protein is expressed in the nucleus of most cells; it is, however, also present on the cell surface of tumor cell lines, and antibodies binding Ku70/80 at the cell surface were recently shown to internalize into tumor cells. To evaluate the potential of Ku70/80-antigen as a therapeutic target for immunotoxins in glioblastoma multiforme, we investigated binding and localization of Ku70/80-specific antibodies in tissue samples from glioblastomas and normal human brains, and in glioma cell cultures. Furthermore, the internalization and drug-delivery capacity were evaluated by use of immunotoxicity studies. We demonstrate that Ku70/80 is localized on the cell plasma membrane of glioma cell lines, and is specifically present in human glioblastoma tissue. Antibodies bound to the Ku70/80 antigen on the cell surface of glioma cells were found to internalize via endocytosis, and shown to efficiently deliver toxins into glioblastoma cells. The data further imply that different antibodies directed against Ku70/80 possess different abilities to target the antigen, in relation to its presentation on the cell surface or intracellular localization. We conclude that Ku70/80 antigen is uniquely presented on the plasma membrane in glioblastomas, and that antibodies specific against the antigen have the capacity to selectively bind, internalize, and deliver toxins into tumor cells. These results imply that Ku70/80 is a potential target for immunotherapy of glioblastoma multiforme.     

Diagnostic and therapeutic utility of monoclonal antibodies in urologic oncology

Remarkable advances in the treatment of urologic malignancies have recently been made. Monoclonal antibodies selective for a variety of normal and malignant urologic tissues have been useful in defining normal antigens and tumor-associated antigens and have potential as diagnostic and immunotherapeutic agents. In renal cancer, monoclonal antibodies can define serum markers, radiolabel tumor xenografts, and assist in specific tissue diagnosis. Additionally, there is potential for these antibodies either alone or as conjugates to localize and kill tumors. Monoclonal antibodies to bladder cancer associated antigens are able to demonstrate differential antigen expression on superficial versus invasive tumors, to refine urinary cytologic diagnosis of bladder cancer, and to predict invasive recurrence of superficial cancer. Monoclonal antibodies have localized bladder tumor xenografts and can inhibit tumor growth when conjugated to radioisotopes or toxins. In prostate cancer monoclonal antibodies to prostate antigens are not usually tumor specific. Monoclonal antibodies to prostate antigen (PA) and prostatic acid phosphatase (PAP) are able to localize prostate cancer metastases. Chemotherapy-conjugated anti-PAP monoclonal antibodies have demonstrable inhibition on human prostate cancer xenografted tumor growth. Monoclonal antibodies have defined normal and tumor-associated antigens in urologic cancers and are expected to be useful in immunodiagnosis and cancer therapy in the near future.     

Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer

Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA is expressed in the basal cells of normal prostate and in more than 80% of prostate cancers. The purpose of the present study was to examine PSCA protein expression in clinical specimens of human prostate cancer. Five monoclonal antibodies were raised against a PSCA-GST fusion protein and screened for their ability to recognize PSCA on the cell surface of human prostate cancer cells. Immunohistochemical analysis of PSCA expression was performed on paraffin-embedded sections from 25 normal tissues, 112 primary prostate cancers and nine prostate cancers metastatic to bone. The level of PSCA expression in prostate tumors was quantified and compared with expression in adjacent normal glands. The antibodies detect PSCA expression on the cell surface of normal and malignant prostate cells and distinguish three extracellular epitopes on PSCA. Prostate and transitional epithelium reacted strongly with PSCA. PSCA staining was also seen in placental trophoblasts, renal collecting ducts and neuroendocrine cells in the stomach and colon. All other normal tissues tested were negative. PSCA protein expression was identified in 105/112 (94%) primary prostate tumors and 9/9 (100%) bone metastases. The level of PSCA expression increased with higher Gleason score (P=0.016), higher tumor stage (P=0.010) and progression to androgen-independence (P=0. 021). Intense, homogeneous staining was seen in all nine bone metastases. PSCA is a cell surface protein with limited expression in extraprostatic normal tissues. PSCA expression correlates with tumor stage, grade and androgen independence and may have prognostic utility. Because expression on the surface of prostate cancer cells increases with tumor progression, PSCA may be a useful molecular target in advanced prostate cancer.     

Human tumor cells killed by anthracyclines induce a tumor-specific immune response

Immunogenic cell death is characterized by the early surface exposure of chaperones including calreticulin and HSPs, which affect dendritic cell (DC) maturation and the uptake and presentation of tumor antigens. It has also been shown that it is characterized by the late release of high mobility group box 1 (HMGB1), which acts through Toll-like receptor 4 (TLR4) and augments the presentation of antigens from dying tumor cells to DCs. Most of the data on immunogenic tumor cell death were obtained using mouse models. In this study, we investigated the capacity of clinically used chemotherapeutics to induce immunogenic cell death in human tumor cell lines and primary tumor cells. We found that only anthracyclines induced a rapid translocation of calreticulin, HSP70, and HSP90 to the cell surface and the release of HMGB1 12 hours after the treatment. The interaction of immature DCs with immunogenic tumor cells led to an increased tumor cell uptake and induces moderate phenotypic maturation of DCs. Killed tumor cell-loaded DCs efficiently stimulated tumor-specific IFN-γ-producing T cells. DCs pulsed with killed immunogenic tumor cells also induced significantly lower numbers of regulatory T cells than those pulsed with nonimmunogenic tumor cells. These data indicate that human prostate cancer, ovarian cancer, and acute lymphoblastic leukemia cells share the key features of immunogenic cell death with mice tumor cells. These data also identify anthracyclines as anticancer drugs capable of inducing immunogenic cell death in sensitive human tumor cells.