Dual energy can accurately differentiate uric acid-containing urinary calculi from calcium stones.

PURPOSE: To retrospectively evaluate the accuracy of dual-energy CT (DECT) in the detection of the chemical composition of urinary calculi in correlation with infrared spectroscopic stone analysis. METHODS: We reviewed the CT scans of 255 patients who underwent DECT due to a clinical suspicion of urolithiasis. Out of this group, we included 64 patients with clinically symptomatic urolithiasis requiring stone removal. After surgical removal of the stone by ureterorenoscopy, chemical composition was analyzed with infrared spectroscopy. We correlated DECT stone characterization results with chemical stone composition based on dual-energy indices (DEI). A total of 213 renal and ureteral stones could be removed and chemically analyzed. RESULTS: A total of 213 calculi were evaluated. Thirty eight out of sixty four (59 %) patients had >1 stone. DECT was used to differentiate stones by using DEI. Stones harboring calcium (CA) were color-coded in blue, while stones containing uric acid (UA) were colored red. Median DEI in UA-containing stones were 0.001. Non-UA-containing stones had a DEI between 0.073 for pure CA stones and 0.077 containing CA and other substances (p = 0.001; p = 0.03, respectively). Sensitivity of DECT was 98.4 % for differentiation of UA from non-UA-containing calculi. Specificity was 98.1 %. Mean effective radiation dose of DECT was 4.18 mSv (0.44-14.27 mSv), thus comparable to conventional CT scans of the abdomen. Conventional measurement of Hounsfield units did not correlate with stone composition. CONCLUSION: DECT with image post-processing reliably discriminates UA-containing calculi from all other stones, but the study offered limitations. Discrimination within the non-UA stones cannot be reliably achieved but is clinically insignificant.

Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism.

Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.

T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE antibody construct.

Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML.

A novel recombinant bispecific single-chain antibody, bscWue-1 x CD3, induces T-cell-mediated cytotoxicity towards human multiple myeloma cells.

The development of antibody-based strategies for the treatment of multiple myeloma (MM) has been hampered so far by the fact that suitable plasma cell-specific surface antigens have been missing. However, recently a novel monoclonal antibody, designated Wue-1, has been generated that specifically recognizes normal and malignant human plasma cells. Therefore, Wue-1 is an interesting and promising candidate to develop novel immunotherapeutic strategies for the treatment of MM. One variant for an antibody-based strategy is the bispecific antibody approach. Recombinant bispecific single-chain (bsc) antibodies are especially interesting candidates because they show exceptional biological properties. We have generated a novel MM-directed recombinant bsc antibody, bscWue-1 x CD3, and analyzed the biological properties of this antibody using the MM cell line NCI-H929 and primary cells from the bone marrow of patients with MM. We were able to show that bscWue-1 x CD3 induces efficient and selective T-cell-mediated cell death of NCI-H929 cells and primary myeloma cells in nine out of 11 cases. The bscWue-1 x CD3 Ab is efficacious even at low E:T ratios, and with or without additional T-cell pre- or costimulation. Target cell lyses were specific for Wue-1 antigen-positive cells and could be blocked by the Wue-1 monoclonal antibody.

Efficient elimination of chronic lymphocytic leukaemia B cells by autologous T cells with a bispecific anti-CD19/anti-CD3 single-chain antibody construct.

Recently, we have shown that a novel recombinant bispecific single-chain antibody construct (bscCD19 x CD3), induces highly efficacious lymphoma-directed cytotoxicity mediated by unstimulated peripheral T lymphocytes. Functional analysis of bscCD19 x CD3 has so far been exclusively performed with human B lymphoma cell lines and T cells from healthy donors. Here we analysed the properties of bscCD19 x CD3 using primary B cells and autologous T cells from healthy volunteers or patients with B-cell chronic lymphocytic leukaemia (B-CLL). We show that bscCD19 x CD3 induces T-cell-mediated depletion of nonmalignant B cells in all four cases and depletion of primary lymphoma cells in 22 out of 25 cases. This effect could be observed at low effector-to-target (E:T) ratios and in the majority of cases without additional activation of autologous T cells by IL-2. Even in samples derived from patients heavily pretreated with different chemotherapy regimens, strong cytotoxic effects of bscCD19 x CD3 could be observed. The addition of bscCD19 x CD3 to patients' cells resulted in an upregulation of activation-specific cell surface antigens on autologous T cells and elevated levels of CD95 on lymphoma B cells. Although anti-CD95 antibody CH-11 failed to induce apoptosis in lymphoma cells, we provide evidence that B-CLL cell depletion by bscCD3 x CD3 is mediated at least in part by apoptosis via the caspase pathway.

Induction of a T helper cell response against the tumor-associated antigen HER2 using monocyte-derived dendritic cells.

Tumor-reactive CD4(+) T helper (Th) cells play a criticalrole in antitumor immunity, due to their ability to induceCD8(+) T cell-mediated cytotoxic activity and humoralresponse. This study focuses on the in vitro generationand expansion of Th cells specific for the tumor-associatedantigen ;human epidermal growth factor receptor-2' (HER2). Aprotocol for efficient HER2 presentation was developed usingautologous monocyte-derived dendritic cells (DC) as antigenpresenting cells (APC) and purified HER2 protein as antigensource. Our data suggest that DC pulsed with recombinantprotein of the extracellular domain (ECD) of HER2 (ECD/HER2)induce an ECD/HER2-specific Th cell response. This finding mayfacilitate the development of immunotherapy regimens withoutrequiring defined immunogenic epitopes of the antigen.

Bispecific single-chain antibodies as effective tools for eliminating epithelial cancer cells from human stem cell preparations by redirected cell cytotoxicity.

High-dose chemotherapy (HDC) with autologous bone marrow or peripheral stem cell transplantation is discussed as one option to treat the extensive stage of a variety of tumors. Effective methods to eliminate contaminating tumor cells from human bone marrow or stem cell grafts may improve the outcome of the patients. We investigated 3 recombinant bispecific single-chain antibodies (bscAbs) directed against 17-1A (EpCAM), c-erbB-2 (HER-2/neu) and LeY on the one and CD3 on the other binding site for their ability to induce lysis of epithelial tumor cells by retargeting autochthonous T lymphocytes present in bone marrow mononuclear cells (BMMC) and in peripheral stem cell mononuclear cells (PSMC). The bscAbs showed remarkable specific lysis of different epithelial tumor cell lines with BMMCs as well as with PSMCs as effector cells. Investigation of the alpha 17-1A-alpha CD3 bscAb revealed a significant correlation between the percentage of CD3(+) cells present in the BMMCs and the rate of lysis as well as the absence of detrimental effects on the viability of hematopoietic progenitor cells as determined by colony-forming unit assays (CFUs). Our results indicate that recombinant bispecific single-chain antibodies could be new tools for purging of human bone marrow and peripheral stem cell grafts from contaminating epithelial cancer cells for patients receiving autologous stem cell transplantation after HDC.

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens.

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients.

Minimal costimulatory requirements for T cell priming and TH1 differentiation: activation of naive human T lymphocytes by tumor cells armed with bifunctional antibody constructs.

Direct priming of naive human CD8+ and CD4+ T cells by tumor cells devoid of any intrinsic antigen presentation properties, but passively armed with recombinant proteins mediating primary and costimulatory T cell signals, was investigated. Bifunctional antibody constructs were used to specifically target costimulatory molecules such as B7-1, B7-2 and LFA-3 to the epithelial cell adhesion molecule (EpCAM), a surface antigen successfully used as target for antibody therapy of minimal residual colorectal cancer. T cell priming was monitored by flow cytometric analysis of CD45 isoform expression and confirmed by measuring typical effector functions of primed T cells known to be absent from naive T lymphocytes. Accordingly, CD8+ T cells were tested for cytotoxic activity and secretion of TNF-alpha, while secretion of IFN-gamma, IL-5 and IL-4 was determined for CD4+ T cells. B7, known to be required for the initial activation of naive T cells, also proved to be sufficient for T cell priming when present as the only costimulatory molecule together with an appropriate primary signal. The requirement of dendritic and other antigen presenting cells (APCs) for T cell priming through non-APCs such as tumor cells could be ruled out. Under minimal priming conditions, naive CD4+ T cells were found to exclusively enter the TH1 developmental pathway, while several factors thought to favor TH2 polarization, like weak primary signals and B7-2 versus B7-1 costimulation, could be excluded as dominant TH2 promoters.

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon alpha and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells.

Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon gamma (IFNgamma), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3+CD56+ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNalpha but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis(Y) antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors.

A recombinant bispecific single-chain antibody, CD19 x CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes.

Although bispecific antibodies directed against malignant lymphoma have been shown to be effective in vitro and in vivo, extended clinical trials so far have been hampered by the fact that conventional approaches to produce these antibodies suffer from low yields, ill-defined byproducts, or laborious purification procedures. To overcome this problem, we have generated a small, recombinant, lymphoma-directed, bispecific single-chain (bsc) antibody according to a novel technique recently described. The antibody consists of 2 different single-chain Fv fragments joined by a glycine-serine linker. One specificity is directed against the CD3 antigen of human T cells, and the other antigen-binding site engages the pan-B-cell marker CD19, uniformly expressed on the vast majority of B-cell malignancies. The construct was expressed in Chinese hamster ovary cells and purified by its C-terminal histioline tag. Specific binding to CD19 and CD3 was demonstrated by fluorescence-activated cell sorter analysis. By redirecting unstimulated primary human T cells derived from the peripheral blood against CD19-positive lymphoma cells, the bscCD19 x CD3 antibody showed significant cytotoxic activity at very low concentrations of 10 to 100 pg/mL and at effector to target cell ratios as low as 2:1. Moreover, strong lymphoma-directed cytotoxicity at low antibody concentrations was rapidly induced during 4 hours even in experiments without any T-cell prestimulation. Thus, this particular antibody proves to be much more efficacious than the bispecific antibodies described until now. Therefore, the described bscCD19 x CD3 molecule should be a suitable candidate to prove the therapeutic benefit of bispecific antibodies in the treatment of non-Hodgkin lymphoma. (Blood. 2000;95:2098-2103)

Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow.

PURPOSE: This study was designed to evaluate the potential of reverse-transcriptase polymerase chain reaction (RT-PCR) analyses for the detection of micrometastatic carcinoma cells in bone marrow (BM). PATIENTS AND METHODS: The specificity of RT-PCR assays with primers specific for various tumor-associated and organ-specific mRNA species was examined by analysis of 53 BM aspirates from control patients with no epithelial malignancy. In addition, BM samples from 63 patients with prostate cancer (n = 53) or breast cancer (n = 10) were analyzed by RT-PCR with primers specific for prostate-specific antigen (PSA) mRNA. As a reference method, all samples were analyzed simultaneously by an established immunocytochemical assay, using monoclonal antibodies (mAbs) against cytokeratins (CK) for tumor-cell detection. RESULTS: Seven of eight marker species could be detected in a considerable number of BM samples from control patients: epithelial glycoprotein-40 (EGP-40; 53 of 53 samples), desmoplakin I (DPI I; five of five), carcinoembryonic antigen (CEA; five of 19), erb-B2 (five of seven), erb-B3 (six of seven), prostate-specific membrane antigen (PSM; four of nine), and CK18 (five of seven). Only PSA mRNA was not detected in any of the 53 control BM samples. In serial dilution experiments, the PSA RT-PCR assay was able to detect five LNCaP prostate carcinoma cells in 4 x 10(6) BM cells. CK-positive cells were found in 20 patients (37.7%) with prostate cancer, while PSA mRNA was found in only 15 (28.3%; P = .04). Moreover, despite the recent observation that PSA is also expressed in mammary carcinomas, none of the 10 CK-positive BM samples were PSA mRNA-positive. CONCLUSION: Limiting factors in the detection of micrometastatic tumor cells by RT-PCR are (1) the illegitimate transcription of tumor-associated or epithelial-specific genes in hematopoietic cells, and (2) the deficient expression of the marker gene in micrometastatic tumor cells.

Biologic properties of a bispecific single-chain antibody directed against 17-1A (EpCAM) and CD3: tumor cell-dependent T cell stimulation and cytotoxic activity.

Anti-CD3 x anti-tumor-bispecific Abs have been used to redirect cytotoxic T cells to tumor cells in an MHC-unrestricted fashion and to induce their rejection in vivo. We have recently described a recombinant bispecific single-chain Ab that combines four different V regions of two Abs, anti-17-1A and anti-CD3, on one polypeptide chain. It folds correctly to a 60-kDa globular protein and is secreted in fully functional form by a high producer Chinese hamster ovary cell line. In this work, we report that its remarkable cytotoxicity against 17-1A+ tumor cells is exerted via T cells without an apparent engagement of a detectable costimulatory pathway. T cells are activated only by the bispecific Ab when coincubated with 17-1A+ target cells. In a chromium release assay, CD8+ T cells reach maximal tumor cell cytotoxicity within 4 h, while CD4+ T cells need about 20 h to reach similar levels of cytotoxicity. Addition of costimulatory CD28 Abs did not lead to a further increase in cytotoxicity. Its remarkable stability at 37 degrees in serum, the ease of production, and purification by affinity chromatography via polyhistidine tail make this smaller version of a bispecific Ab a promising candidate for a therapeutic trial in patients with solid tumor. Because adjuvant therapy with an intact, much less cytotoxic IgG2a Ab against the 17-1A target had already increased the 7-yr survival of colorectal cancer patients by 30%, the presented small bispecific construct lacking the immunogenic murine Fc region as well as autochthonous T lymphocyte stimulatory activity warrants a therapeutic trial in patients with minimal residual 17-1A+ cancer.

Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer.

Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL17-1A-VH17-1A-VHCD3-VLCD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkable, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor alpha, interleukin-6 and interferon gamma. Maximal cytotoxicity (51Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8+ as well as CD4+ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8+ cells induced high 51Cr release within 4 h while CD4+ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in vitro.

Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

AIDS patient monocytes target CD4 T cells for cellular conjugate formation and deletion through the membrane expression of HIV-1 envelope molecules.

The human immunodeficiency virus (HIV) causes in humans the acquired immunodeficiency syndrome (AIDS). It replicates at a high rate in lymphoid organs even before it causes clinical symptoms. It binds to CD4 cell surface markers and destroys T lymphocytes that express the receptor. The immune system replenishes CD4 T cells at a formidable rate but, unable to keep up with the losses, allows the CD4 T cell compartment to disintegrate gradually. The net loss of CD4 T cells is an indicator for disease progression. How the virus destroys CD4 T cells and whether their loss accounts for the ensuing immunodeficiency have not been fully explained. We have reported evidence, and confirm here, that HIV-infected subjects deposit on monocytes immune complexes containing the virus or its envelope molecule gp120. Armed with these immune complexes monocytes form specific cellular conjugates with CD4 T cells and kill them. The destruction of normal CD4 T cells by monocytes from AIDS patients can be blocked by soluble CD4 and by free gp120. Normal monocytes and macrophages can be armed with CD4-binding gp120, and so induced to destroy CD4 T cells, by incubating them with gp120 and gp120-specific antibody. CD4-reactive HIV-1 components have a short half-life on the phagocyte surface. Removed from the HIV-infected environment, monocytes clear their surfaces of antibody-complexed viral components within hours, which abrogates their ability to destroy CD4 T cells. Rearming the monocytes with gp120-anti-gp120 complexes restores their capacity to destroy CD4 T cells. The data imply that for uninterrupted deletion of CD4 T cells, monocytes require a continued productive HIV-1 infection of their host.

A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity.

Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.

Recombinant CD4-IgE, a novel hybrid molecule, inducing basophils to respond to human immunodeficiency virus (HIV) and HIV-infected target cells.

Basophils and mast cells, as the main effector cells in IgE-mediated type I hypersensitivity, are involved in the elimination of parasites and, according to recent findings, may also play an important role in the defense against bacterial and viral infections. Using a genetic engineering approach we wanted to redirect this potent IgE-mediated defense system against intruding human immune deficiency virus. We constructed a recombinant CD4-IgE molecule, consisting of the two N-terminal domains of CD4 and the CH2-4 domains of the IgE heavy chain, thus providing the IgE with specificity for the gp120 of human immunodeficiency virus (HIV). The binding properties of hybrid CD4-IgE to the high-affinity receptor for IgE (Fc epsilon RI) on basophils as well as to the low-affinity receptor (Fc epsilon RII or CD23) for IgE on lymphoid cells were found to be similar to those of native IgE. At the same time, the CD4 domains of the recombinant molecule retained the gp120 binding specificity with an affinity similar to that of the native CD4. By functional tests, we demonstrated that CD4-IgE armed basophils can be triggered by free HIV and by HIV-infected cells to release their mediators. We further show that HIV-triggered basophils lead to a decreased replication of HIV in susceptible T cells. We, therefore, conclude that the type I hypersensitivity effector cells can be engaged in the elimination of HIV-infected cells, at least in vitro. Because of the strong binding of the CD4-IgE construct to the Fc epsilon RI, we assume that CD4-IgE has a short t1/2 in serum, but may similarly to IgE exhibit prolonged resident time on basophils and mast cells, which are located close to mucosal surfaces or in the connective tissue. Thus CD4-IgE could play an important role in the elimination of HIV also in vivo.

Prion protein mutation at codon 102 in an Italian family with Gerstmann-Sträussler-Scheinker syndrome.

We present the first family from Italy with the Gerstmann-Sträussler-Scheinker syndrome (GSS) and a substitution of leucine for proline at codon 102 of the prion protein gene. This mutation is associated with the ataxic form of GSS in a number of reported families. The clinical presentation of our family includes amyotrophic changes in some affected family members in addition to ataxia.