Pharmacokinetics and PET imaging properties of two recombinant anti-PSMA antibody fragments in comparison to their parental antibody.

BACKGROUND: Radioimmunoimaging with disease-specific tracers can be advantageous compared to that with nonspecific tracers for the imaging of glucose metabolism and cell proliferation. Monoclonal antibodies (mAbs) or their fragments are excellent tools for immuno-positron emission tomography (PET). In this study, PSMA-specific mAb 3/F11 and its recombinant fragments were compared for the imaging of prostate cancer in xenografts. METHODS: Recombinant anti-PSMA antibody fragments D7-Fc and D7-CH3 were constructed by genetically fusing the binding domains of mAb 3/F11 (D7) to the human IgG3 CH3 or CH2-CH3 (Fc) domain. The fragments and the mAb 3/F11 were DOTA conjugated, tested in vitro, and radiolabeled with (64) Cu. PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used for PET-MR imaging and for ex vivo biodistribution. RESULTS: The constructs showed strong and specific binding to PSMA-positive C4-2 cells in vitro which did not decrease after DOTA conjugation. Both tested fragments showed stable accumulation in PSMA-positive C4-2 tumors at all measured time points but reduced uptake compared to the full-length antibody. Other organs and PSMA-negative tumors showed a very low tracer uptake only 3 hr after injection, with the exception of the kidneys, which demonstrated high radioactivity uptake due to rapid renal clearance of the mAb fragments. CONCLUSION: Stable tumor uptake and fast serum clearance of the tested radiolabeled fragments was observed in this preclinical study compared to the full length mAb. Since the fragments show rapid and specific tumor uptake, the tested fragments might serve as tools for theranostic imaging with suitable isotopes for radioimmunotherapy.

Antitumor activities of PSMA×CD3 diabodies by redirected T-cell lysis of prostate cancer cells.

AIM: Although prostate cancer is one of the most commonly diagnosed malignancies in men, there is no effective curative therapy for the advanced disease. Therefore, the aim of the present study was to generate prostate-specific membrane antigen (PSMA)×CD3 diabodies as a novel treatment option for this tumor. METHODS: A PSMA×CD3 diabody and a covalently linked single-chain diabody were constructed from the anti-PSMA single-chain Fv fragment D7 and an anti-CD3 single-chain Fv fragment. The fusion proteins were periplasmatically expressed in Escherichia coli. The binding properties were tested on PSMA-expressing C4-2 prostate cancer cells and CD3(+) Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability assay was used. T-cell activation was determined by flow cytometry. In vivo activity of the diabody was tested in SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts. RESULTS: Bacterial expression levels were significantly higher for the diabody (1-1.5 mg/l culture) compared with the single-chain diabody (0.2-0.4 mg/l culture). Specific binding on CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown with both diabody formats. In vitro, both diabodies proved to be potent agents for retargeting human CD4(+) and CD8(+) lymphocytes to lyse C4-2 prostate cancer cells. The formation of conjugates between T cells and target cells with clustering of the diabody at sites of interaction could be shown. SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts with the diabody showed an efficient inhibition of tumor growth. CONCLUSION: Both diabody formats showed a highly efficient and specific T cell-mediated killing of prostate cancer cells and are encouraging for further development in preclinical and clinical studies.

Effective targeting of prostate cancer by lymphocytes redirected by a PSMA × CD3 bispecific single-chain diabody.

BACKGROUND: For redirecting T-lymphocytes to induce prostate cancer cell lysis, we constructed a novel bispecific single-chain (bsc) diabody directed to the prostate specific membrane antigen (PSMA) and the T-cell receptor (TCR)-associated CD3 molecule on T-cells. METHODS: The PSMA × CD3 bsc diabody was generated from an anti-CD3 single chain Fv fragment (scFv) and the anti-PSMA scFv D7. It was expressed in E. coli and purified from the periplasmic extract and culture supernatant by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST-1) was used and activation of T-cells was determined by measuring the surface marker expression of CD25 and CD69. For in vivo evaluation, the diabody was administered in combination with human peripheral blood lymphocytes (Ly) in a C4-2 xenograft-SCID mouse model. RESULTS: Specific binding of the PSMA × CD3 bsc diabody both to CD3-positive Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the PSMA × CD3 bsc diabody proved to be a potent agent for retargeting CD4+ and CD8+ human lymphocytes to lyse C4-2 prostate cancer cells. Treatment of SCID mice bearing C4-2 tumor xenografts with the diabody and human lymphocytes efficiently inhibited tumor growth. CONCLUSIONS: The PSMA × CD3 bsc diabody bears a high potential for the immunotherapy of prostate cancer.

Influence of structural variations on biological activity of anti-PSMA scFv and immunotoxins targeting prostate cancer.

BACKGROUND: In this study, different variants of the anti-PSMA single-chain antibody fragment (scFv) D7 were cloned, varying in linker type, position of hexahistidine tags and VH-VL orientation. From these scFv, Pseudomonas exotoxin A-based immunotoxins were constructed and their biological activities against prostate cancer cells were compared. MATERIALS AND METHODS: Binding of the constructs to PSMA-expressing prostate cancer cells was determined by flow cytometry. ADP-ribosyltransferase activity was analysed and cytotoxicity was measured with WST-1 assays. RESULTS: Different linker types did not influence the characteristics of the scFv or immunotoxins. The addition of an N-terminal hexahistidine-tag, however, resulted in decreased expression, binding and cytotoxicity. scFv in VH-VL orientation showed the highest expression and binding, whereas immunotoxins in VL-VH orientation exhibited the best binding and cytotoxicity. CONCLUSION: The present study showed how structure influences the characteristics of scFv and immunotoxins. It is therefore suggested that for each individual construct the optimal structure has to be determined separately.

Preclinical evaluation of a recombinant anti-prostate specific membrane antigen single-chain immunotoxin against prostate cancer.

The prostate-specific membrane antigen (PSMA) is abundantly expressed on prostate cancer epithelial cells and its expression correlates with tumor progression. Therefore, a specific immunotherapy against this antigen may be a novel therapeutic option for the management of prostate cancer. We generated an anti-PSMA single-chain antibody fragment (scFv), called D7, by phage display from the monoclonal antibody 3/F11. By C-terminal ligation of the toxic domain of Pseudomonas Exotoxin A (PE40) to the genes of D7, the immunotoxin D7-PE40 was generated. D7 and D7-PE40 specifically bound to PSMA transfectants and to the PSMA expressing prostate cancer cell line C4-2. In addition, D7-PE40 showed a high serum stability and induced a 50% reduction of viability (IC50) in C4-2 cells at a concentration of 140 pM. In vivo, D7-PE40 was well tolerated in SCID mice up to a single dose of 20 microg, whereas higher doses induced severe hepatotoxicity with deaths of the animals. Immunotoxin treatment of mice bearing C4-2 tumor xenografts caused a significant inhibition of tumor growth, whereas mice with PSMA-negative DU 145 tumors remained unaffected. Owing to its high and specific cytotoxicity and its capability to inhibit prostate tumor growth in vivo the immunotoxin D7-PE40 represents a promising candidate for the immunotherapy of prostate cancer.

Comparison of gene expression in LNCaP prostate cancer cells after treatment with bicalutamide or 5-alpha-reductase inhibitors.

INTRODUCTION: Androgen deprivation is the preferred treatment for disseminated prostate cancer. However, it mostly leads to the development of incurable androgen-independent disease. The aim of the present study was to compare gene expression changes that occur after treatment with either the antiandrogen bicalutamide or the 5-alpha-reductase inhibitors finasteride (MK906) and MK386. MATERIALS AND METHODS: LNCaP cells of low passages were treated with MK906 and MK386 at 5 microM each or with bicalutamide at 10 microM for 48 h. In these cultures we analyzed the expression of 22,500 transcripts on the Affymetrix Human U133+ 2.0 GeneChip platform. Gene expression was verified by real-time quantitative Taqman PCR. RESULTS: Our studies revealed 312 differentially regulated genes upon bicalutamide treatment and 68 differentially regulated genes upon treatment with the 5-alpha-reductase inhibitors. There were 35 genes equally regulated by both drugs. This subset of genes included those with the highest fold change in both treatment groups. In the subset KlK2, TMPRSS2, TRGC2, PMEPA1 and TM4SF1 were downregulated, whereas EGR1, DDC and OPRK1 were upregulated. CONCLUSIONS: A cohort of interesting genes that are differentially expressed after androgen withdrawal could be found in this study. Investigation into these genes could contribute to a better understanding of antiandrogen treatment and development of androgen-independent prostate cancer.

Target-dependent T-cell activation by coligation with a PSMA x CD3 diabody induces lysis of prostate cancer cells.

Recently, we have described a bispecific PSMA x CD3 diabody with one binding site for the T-cell antigen receptor (TCR-CD3) and another for the Prostate Specific Membrane Antigen (PSMA). It effectively eliminates human prostate cancer cells by redirecting T-lymphocytes in vitro and in vivo. Here, we show that activation of the T-cells and killing of the tumor cells, only occurred when the T-cells were coincubated with PSMA-positive tumor cells and the PSMA x CD3 diabody. Both CD4+ and CD8+ human T-lymphocytes were activated. Surprisingly, they were equally potent in their cytotoxic activity, proliferation, and up-regulation of activation markers. Both, CD4+, and CD8+ T-cells mainly used the perforin-granzyme- based pathway and to a somewhat lesser extent the FasL pathway to lyse tumor cells. When Jurkat T-cells were stimulated with the diabody alone, the TCR-CD3 was not triggered. In contrast, when the diabody was clustered with a secondary antibody the TCR-CD3 was stimulated as detected by Ca(2+)-influx and Erk, IkappaB, and linker of activated T-cell phosphorylation. Clustering of the diabody could also be achieved by the dimeric PSMA antigen expressed on tumor cells. Thus, although the diabody binds to all T-cells, only those in contact with PSMA-expressing cancer cells are activated. In conclusion, the PSMA x CD3 diabody is suitable for a controlled polyclonal T-cell therapy of prostate cancer.

A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is an excellent candidate for targeting prostate cancer by virtue of its restricted expression on prostatic epithelial cells and its upregulation on prostatic carcinoma cells. PSMA is expressed on the cell surface displaying a specific three-dimensional structure. Therefore, only antibodies with a high cell binding activity will have an important impact on antibody-based imaging and therapy. METHODS: Monoclonal antibodies (mAbs) and single chain antibody fragments (scFvs) were prepared from spleen cells of mice that had been immunized either with purified PSMA or a cell lysate of prostate cancer LNCaP cells containing native PSMA. mAbs and scFvs were screened for reactivity with purified PSMA and binding to PSMA-expressing LNCaP cells. RESULTS: From mice immunized with purified PSMA, we obtained three mAbs (K7, K12, D20) and four scFvs (G0, G1, G2, G4), which were highly reactive with the isolated antigen, but showed weak or no reaction with viable LNCaP cells. From mice immunized with unpurified LNCaP lysate, we obtained three mAbs (3/E7, 3/F11, 3/A12), and one scFv (A5), which were reactive with purified PSMA, also showing a strong and specific binding to viable LNCaP cells and PSMA-transfected cells. CONCLUSIONS: Our results suggest that only the mAbs and scFvs, that were elicited with unpurified LNCaP lysate and not with purified PSMA will be useful agents for diagnostic imaging and therapeutic applications of prostate cancer.

Different basal expression of type T1 and T2 cytokines in peripheral lymphocytes of patients with adenocarcinomas and benign hyperplasia of the prostate.

BACKGROUND: It has been proposed that a reduced immunological competence and a dysregulation in the T1/T2 balance may be implicated in the development of cancer. In order to study the immunological situation in vivo, we compared the mRNA expression of the T1 cytokine IFN-gamma and the T2 cytokine IL-10 in freshly isolated, not in vitro stimulated, peripheral blood lymphocytes from patients with localized prostate carcinomas (PCa) and benign prostate hyperplasia (BPH). MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation. CD4(+)- and CD8(+)-lymphocytes were selected with magnetic beads against these determinants. In these lymphocyte preparations, mRNA expression of IL-10 and IFN-gamma was determined by using a semiquantitative RT-PCR, standardized to the beta-actin expression. RESULTS: In the PBMC from 35 patients with localized PCa a significantly lower expression of IFN-gamma and IL-10 was found, compared to 28 patients with BPH. Cytokine expression also was lower in the isolated CD4(+)- and CD8(+)-lymphocytes of the carcinoma patients. However, the difference was statistically significant only for IL-10. CONCLUSION: Our data reflect a reduced basal cytokine expression in peripheral lymphocytes of patients with localized PCa compared to BPH. Since the T2 response was more strongly reduced than T1, there seems to be a decreased immune activation, but a relative T1 dominant cytokine pattern in the cancer patients.

Different expression of Fas and Fas ligand in tumor infiltrating and peripheral lymphocytes of patients with renal cell carcinomas.

BACKGROUND: The presence of tumor infiltrating lymphocytes (TIL) in renal cell carcinomas (RCC) is thought to reflect a host-mediated immune response against the tumor and the Fas/Fas ligand--based lytic pathway is a major mechanism of T lymphocyte--mediated cytotoxicity. The aim of the present study was to investigate the expression of Fas and Fas ligand (FasL) in freshly-isolated pure TIL as compared to autologous peripheral blood lymphocytes (PBL), in order to determine their activation status. MATERIALS AND METHODS: The mRNA expression of Fas and FasL was compared in freshly-isolated CD3(+)-, CD4(+)- and CD8(+)-TIL and matched autologous CD3(+)-, CD4(+)- and CD8(+)-PBL. The TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with magnetic beads. In these pure lymphocyte preparations the constitutive expression of Fas and FasL was determined by using a semiquantitative PCR-assisted mRNA amplification assay. RESULTS: In the CD3(+)-TIL of 20 patients with RCC (group 1), mRNA levels of FasL were significantly higher than in the matched autologous CD3(+)-PBL (p < or = 0.01) whereas Fas expression was not different in both populations. In a second group of 20 patients (group 2), we found a significantly higher expression of FasL in the CD8(+)-TIL compared to the CD8(+)-PBL (p < or = 0.001) and also in the CD4(+)-TIL compared to the CD4(+)-PBL (p < or = 0.001). CONCLUSION: The higher expression of FasL in the TIL compared to autologous PBL reflects an in vivo activation and lytic potency of the lymphocytes in the tumor surrounding.