Induction of in vitro and in vivo NK cell cytotoxicity using high-avidity immunoligands targeting prostate-specific membrane antigen in prostate carcinoma

Cancer that might develop as host natural killer (NK) cells fail to detect ligands for their activating NK receptors. Immunoligands represent promising immunotherapeutic tools to overcome this deficit. These are fusion proteins containing a single-chain antibody fragment (scFv) to target an available tumor antigen and ULBP2 to activate host NK cells by targeting the activatory receptor NKG2D. Prostate-specific membrane antigen (PSMA) is an integral non-shed type 2 membrane protein that is highly and specifically expressed on prostate epithelial cells and strongly upregulated in prostate cancer. Here, we compare the impact of various anti-PSMA immunoligand formats on the therapeutic efficacy against prostate carcinoma cells by activating NK cells via NKG2D. Shortening of the linker separating the heavy and light chain antibody domain leads to the formation of dimers, trimers, and higher molecular mass oligomers. NK cells are most efficiently activated by multimeric immunoligands, thus showing an altered cytokine release pattern. The high avidity format is also superior in in vitro NK-mediated tumor cell targeting as shown in cytotoxicity assays. Finally, the efficacy of a multimeric immunoligand is shown in a prostate carcinoma mouse xenograft model showing a strong activity against advanced established tumors.     

Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors

Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell-mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA-transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer.     

A phase 1 study of a vaccine targeting preferentially expressed antigen in melanoma and prostate-specific membrane antigen in patients with advanced solid tumors

Preferentially expressed antigen in melanoma (PRAME) and prostate-specific membrane antigen (PSMA) are tumor-associated antigens implicated in cellular differentiation, genetic stability, and angiogenesis. MKC1106-PP is an immunotherapeutic regimen cotargeting PRAME and PSMA, comprised of a recombinant plasmid (pPRA-PSM encoding fragments derived from both antigens) and 2 peptides (E-PRA and E-PSM derived from PRAME and PSMA, respectively). This multicenter study evaluated MKC1106-PP with a fixed plasmid dose and 2 different peptide doses, administered by intralymph node injection in a prime-boost sequence in human leukocyte antigen-A*0201 and tumor-antigen-positive patients with progressing metastatic solid tumors who had failed standard therapy. Immune monitoring was done by tetramer and enzymatic-linked immune spot analysis. The treatment was well tolerated, with no significant differences in safety, immune response, and clinical outcome relative to peptide doses. Fifteen of 24 evaluable patients showed an immune response, as defined by the expansion of PRAME-specific or PSMA-specific T cells in the blood. There were no partial or complete responses by the Response Evaluation Criteria in Solid Tumors. Seven patients showed stable disease (SD) for 6 months or longer, or prostate specific antigen decline: 4 of 10 with prostate carcinoma, 2 of 2 with renal clear cell carcinoma, and 1 of 10 with metastatic melanoma. In addition, there was an association between the induction and persistence of antigen-specific T cells in blood above baseline levels and disease control, defined as SD for 6 months or longer. These results support further development of MKC1106-PP in specific clinical indications.     

复合前列腺特异性抗原、总前列腺特异性抗原及其比值在前列腺癌诊断中的应用

目的:研究复合前列腺特异性抗原、总前列腺特异性抗原及其比值在鉴别前列腺癌和良性前列腺增生中的应用价值。方法:采用化学发光法检测30例前列腺癌患者、40例良性前列腺增生患者和30例对照组患者复合前列腺特异性抗原、总前列腺特异性抗原,计算并比较其比值。结果:前列腺癌患者的复合前列腺特异性抗原、总前列腺特异性抗原均高于对照组和良性前列腺增生患者,差异均具有统计学意义(P<0.05);其比值前列腺癌患者高于良性前列腺增生患者,差异有统计学意义(P<0.05);其相关性和共线性均不如前列腺癌组病例。结论:前列腺癌患者总前列腺特异性抗原的升高以复合前列腺特异性抗原升高为主,测定复合前列腺特异性抗原更有意义,结合其比值进行综合分析可以有效区分良性前列腺增生和前列腺癌。     

Prostate specific membrane antigen (PSMA) ligands for diagnosis and therapy of prostate cancer

Introduction: Prostate specific membrane antigen (PSMA) has become an attractive diagnostic and therapeutic target for small molecule ligands. Radionuclide-chelating ligands can be labeled with either ⁶⁸Ga for positron-emission-tomography (PET) or ¹⁷⁷Lu for radionuclide therapy.

Areas covered: In this literature review we evaluate the diagnostic value of ⁶⁸Ga PSMA PET/CT and the therapeutic potential of ¹⁷⁷Lu PSMA radioligand therapy (RLT) in patients with prostate cancer. ⁶⁸Ga PSMA PET/CT is more accurate than CT for nodal staging and superior to conventional imaging in patients with biochemical recurrence, translating into major changes in clinical management. The preliminary data for ¹⁷⁷Lu PSMA indicates >50% reduction of PSA levels in up to 59% of patients. Severe adverse events occurred <10% of patients after RLT.

Expert commentary: PSMA ligands for diagnostic and therapeutic purpose will significantly impact the management of patients with prostate cancer.     

In vitro antigen-induced antibody responses to hepatitis B surface antigen in man. Kinetic and cellular requirements.

In this report we define the parameters of the human immune response after immunization with hepatitis B vaccine. 2 wk after booster immunization, there is significant spontaneous secretion of antibody to hepatitis B surface antigen (anti-HBs IgG), which is not further augmented by stimulation with antigen or pokeweed mitogen (PWM). By 4 wk there is little spontaneous secretion of specific antibody, and low doses of antigen or PWM produce significant increases in the amount of anti-HBs IgG produced. By 8 wk the peripheral blood mononuclear cells are refractory to stimulation by antigen, but anti-HBs IgG is produced in response to PWM. 0.5 yr or more after the last immunization, some individuals will manifest an antigen-induced specific antibody response. This induction of anti-HBs IgG by hepatitis B surface antigen (HBsAg) is monocyte- and T cell-dependent. Note that there is a dichotomy in the T cell response to HBsAg. The specific antibody response is clearly T cell dependent, but no in vitro T cell proliferative response to HBsAG could be demonstrated in the immunized individuals. Although the precise reason for the absent proliferative response to HBsAg despite well-established humoral immunity has not been determined, there was no evidence to suggest nonspecific suppression by HBsAg or the presence of an adherent suppressor cell population. The ability to evaluate antigen-induced, antigen-specific responses to HBsAg will be useful in defining the unique interaction between the human immune response and this clinically important viral agent.     

Ultrastructural localizations of beta-microseminoprotein, a prostate-specific antigen, in human prostate and sperm: comparison with gamma-seminoprotein, another prostate-specific antigen

Immunohistochemical localizations of beta-microseminoprotein and gamma-seminoprotein, which are prostate-specific antigens, were examined by light and electron microscopy with peroxidase-labeled antibody. In normal, hypertrophic, and neoplastic prostate glands, beta-microseminoprotein was found in glandular epithelium but not in stroma cells. beta-Microseminoprotein may be as useful as gamma-seminoprotein in the pathologic examination of prostatic diseases, especially in histogenic classification of tumors or metastatic tumors. In an immunoelectron microscopic study, the primary localizations of beta-microseminoprotein and gamma-seminoprotein in the cell were demonstrated to be secretory granule and lysosome, respectively, suggesting that beta-microseminoprotein is a secretory protein. Weak distributions of beta-microseminoprotein and gamma-seminoprotein were observed in rough endoplasmic reticulum and Golgi apparatus. In spermatozoa, beta-microseminprotein was found attached to the cell membrane of the head but not in the tail, and gamma-seminoprotein was not found at all.     

Enhancement of in vitro and in vivo antigen-specific antibody responses by interleukin 11

The availability of large quantities of highly purified recombinant interleukin 11 (rhuIL-11) has allowed us to investigate the effects of rhuIL-11 on sheep red blood cell (SRBC)-specific antibody responses in the murine system. The results showed that rhuIL-11 was effective in enhancing the generation of mouse spleen SRBC-specific plaque-forming cells (PFC) in the in vitro cell culture system in a dose-dependent manner. These effects of rhuIL-11 were abrogated completely by the addition of anti-rhuIL-11 antibody, but not by the addition of preimmunized rabbit serum. Cell-depletion studies revealed that L3T4 (CD4)+ T cells, but not Lyt-2 (CD8)+ T cells, are required in the rhuIL-11-stimulated augmentation of SRBC-specific antibody responses. The effects of rhuIL-11 on the SRBC-specific antibody responses in vivo were also examined. RhuIL-11 administration to normal C3H/HeJ mice resulted in a dose-dependent increase in the number of spleen SRBC-specific PFC as well as serum SRBC-specific antibody titer in both the primary and secondary immune responses. In mice immunosuppressed by cyclophosphamide treatment, rhuIL-11 administration significantly augmented the number of spleen SRBC-specific PFC as well as serum SRBC-specific antibody titer when compared with the cyclophosphamide-treated mice without IL-11 treatment. These results demonstrated that IL-11 is a novel cytokine involved in modulating antigen-specific antibody responses in vitro as well as in vivo.     

Expression and purification of prostate-specific membrane antigen in the baculovirus expression system and recognition by prostate-specific membrane antigen-specific T cells.

Antigen-specific immunotherapy of cancer depends on a consistent source of well-defined protein antigen. Production of recombinant protein offers the obvious solution to this problem but few comparisons of recombinant and native proteins in cellular immune assays have been reported. We report expression of a putative immunotherapy antigen, prostate-specific membrane antigen (PSMA), in insect cells using a baculovirus vector. T cells stimulated with recombinant PSMA or native PSMA derived from the LNCaP cell line recognized both native PSMA and recombinant, baculoviral PSMA. These data indicate that PSMA produced in Sf9 cells is immunologically cross-reactive with native PSMA and therefore suitable for immunotherapy as it is recognized by both cellular and humoral immune responses.     

In vitro and in vivo removal of anti-A erythrocyte antibody by adsorption to a synthetic immunoadsorbent

A solid-phase immunoadsorbent column using blood group A trisaccharide was tested in vitro and in vivo in dogs. This column was demonstrated to specifically remove anti-A antibody from human plasma in vitro and in vivo dogs specifically immunized with synthetic human blood group A trisaccharide. These studies indicate that this technique will be useful in removing anti-A and B antibody in marrow transplant recipients of incompatible marrow.     

Uses and limitations of prostate-specific antigen in the laboratory diagnosis of prostate cancer

We have studied the clinical utility of a recently developed assay for a prostate specific protein (prostate-specific antigen). Histochemical demonstration of prostate-specific antigen has a higher sensitivity and specificity for the diagnosis of prostate carcinoma than does prostatic acid phosphatase. Similarly, measurement of prostate-specific antigen by immunoassay in serum is a more sensitive indicator of tumor stage and recurrence after therapy than prostatic acid phosphatase. More information is needed, however, regarding the variation in this protein with different therapeutic methods.     

Novel splice variants of prostate-specific antigen and applications in diagnosis of prostate cancer

ObjectivesWe aimed to identify novel splice variants of prostate-specific antigen/or human kallikrein 3 (PSA/KLK3), the most widely used serum biomarker for case-finding, screening and monitoring of prostate cancer.Design and methodsThe full-length sequences of splice variants were assembled as contigs from human ESTs that displayed homology to the cDNA sequence encoding PSA. Expression of variants in clinical samples was analyzed by semi-quantitative RT-PCR.ResultsEST database mining led to the identification of seven previously unidentified splice variants encoding PSA-like proteins that are predicted to contain epitope sequences recognized by PSA-specific antibodies, therefore, expression of these isoforms may affect the amount of total PSA measured by established immunoassays. Analysis of the differential expression profile of isoform PSA-SV5 in patients with benign prostate hyperplasia and prostate cancer showed that it is specifically expressed in prostate cancers.ConclusionsA novel splice variant of PSA was identified, PSA-SV5, that may be exploited in clinical diagnosis to distinguish prostate cancer from benign prostate hyperplasia.     

Disulfide-constrained peptides that bind to the extracellular portion of the prostate-specific membrane antigen

The prostate-specific membrane antigen (PSMA) is a well-characterized surface antigen, overexpressed in the most advanced, androgen-resistant human prostate cancer cells. We sought to exploit PSMA cell surface properties as a target for short peptides that will potentially guide protein-based therapeutics, such as viral vectors, to prostate cancer cells. Two separate phage display peptide strategies were applied, in parallel, to purified PSMA protein bound to two separate substrates. We reasoned that peptide sequences common to both substrate selections would be specific binders of PSMA. Additionally, the design allowed for stringent cross-selections, where phage populations from one selection condition could be applied to the alternative substrate. These strategies resulted in a series of phage displayed peptides able to bind to PSMA by ELISA and direct binding assays, both with purified protein and in prostate cancer cells. Cell binding is competitively inhibited by purified PSMA. The synthesized peptides are capable of enhancing PSMA carboxypeptidase enzymatic activity, suggesting protein folding stabilization. The discovery of these peptides provides the foundation for subsequent development of peptide targeted therapeutics against prostate cancer.     

In vitro cell-mediated immune responses to the male specific(H-Y) antigen in mice

C57BL/10 female mice were primed to the male specific antigen H-Y, either by grafting with syngeneic male tail skin or by i.p. injection of syngeneic male spleen cells. Primed female spleen cells, either unseparated or filtered through nylon wool to remove most of the B lymphocytes, were then cultured for 5 days in vitro with irradiated syngeneic male spleen cells and assayed against 51Cr-labeled target cells. Both unseparated and nylon wool filtered female cells displayed significant cytotoxic activity restricted to male target cells. Pretreatment of sensitized female cells with antitheta serum and complement just before assay abolished cytotoxic responses. We were unable to demonstrate cell-mediated cytotoxic responses into two nonresponding strains, CBA and B10.A, which fail to reject male isografts. The cytotoxic activity of C57BL/10 female cells was restricted to male target cells histocompatible with C57BL/10 over at least a portion of the major (H-2) histocompatibility complex. We conclude that secondary in vitro cytotoxic responses against the H-Y antigen are mediated by cytotoxic T lymphocytes, and that the H-Y target cell antigen may be specified by the H-2 complex.     

Vaccination of cytotoxic T lymphocyte-directed peptides elicited and spread humoral and Th1-type immune responses to prostate-specific antigen protein in a prostate cancer patient

The authors studied humoral and CD4+ T-cell responses in an HLA-A24+ prostate cancer patient vaccinated with cytotoxic T lymphocyte (CTL)-directed peptides, including a prostate-specific antigen (PSA)248-257 peptide, to understand what kinds of immune responses are elicited in peptide-vaccinated patients. The levels of immunoglobulin G (IgG) reactive to the administered PSA248-257 peptide or the PSA protein were kinetically examined. The level of IgG reactive to the PSA248-257 peptide drastically increased after the peptide vaccination, with a peak after the seventh vaccination, whereas that of IgG reactive to the PSA protein continued to increase throughout the vaccination period. IgG reactive to the PSA protein after the 13th vaccination showed no reactivity to the administered PSA peptides. However, HLA-DRB1*1302-restricted and PSA protein-recognizing TH1-type CD4+ T-cell clone and line, with different specificity, were successfully established from the post-7th and post-13th peripheral blood mononuclear cells, respectively. Both CD4+ T cells produced interferon-gamma in response to naturally processed PSA secreted from prostate cancer cells, whereas their reactivity to the administered PSA248-257 peptide was undetectable or negligible. These findings indicate that vaccination with CTL-directed peptides, including a PSA-derived peptide, was able to elicit and spread humoral and TH1-type immune responses to the PSA protein.     

Augmentation of in vitro humoral immune responses in the mouse by an antibody to IgD

Heterologous anti-delta-chain antibodies have an adjuvant effect on specific in vivo humoral immune responses to simultaneously, or subsequently, injected antigens in the rat and rhesus monkey. We have used a hybridoma-secreted antibody that binds murine delta-chain of the allotype (4.22aM delta a) to study this phenomenon in the mouse and to investigate the mechanism of this effect. Injection of 4.22aM delta a into BALB/c mice removes almost all surface IgD (sIgD) from splenic B lymphocites. sIgD does not reappear until the serum level of 4.22aM delta a decreased 5-7 d after injection. 4.22aM delta a fails to induce detectable proliferation or to raise total serum Ig levels substantially above control values. However, 4.22aM dalta a injected 24 h before antigen elicits an approximately twofold enhancement of serum IgM and a 3- to 10-fold enhancement of serum IgG anti-trintriphenyl (TNP) antibodies in response to immunization with optimal doses of TNP- Ficoll or TNP-sheep red blood cells (TNP-SRBC). 4.22aM delta a injected 1 wk before or 3 d after TNP-SRBC, however, has no effect on IgG anti- TNP levels. The adjuvant effect of anti-delta-chain antibody was markedly decreased when suboptimal antigen doses were used. Furthermore, even in the case of TNP-Ficoll, a relatively T-independent antigen, the ability of 4.22aM dalta a to enhance the anti-TNP antibody response was T cell dependent. Our data suggest that the binding of anti-delta-chain antibody to cell sIgD may partially activate B lymphocytes and make them more capable of differentiating into antibody- secreting cells when stimulated by antigen-specific T cell help.