In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33 Acute Myeloid Leukemia.

Genetic engineering of T cells with chimeric T-cell receptors (CARs) is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV-) specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34(+) hematopoietic progenitors. Moreover, after intravenous administration into CD33(+) human acute myeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Artificial T-cell receptors.

Artificial T-cell receptors are generated by joining an Ag-recognizing domain (ectodomain) to the transmembrane and intracellular portion of a signaling molecule (endodomain). The ectodomain is most often derived from Ab variable chains, but may also be generated from T-cell receptor variable chains, as well as from other molecules. Various alternative ectodomain designs exist, with some comparative studies suggesting optimal forms. The endodomain most often used is the intracellular portion of CD-zeta. Although signaling by CD-zeta leads to IFN-n release and cell killing, it fails to transmit a full activation signal. Recently, unions of different signaling molecule segments have facilitated transmission of more potent signals, stimulating T-cell proliferation and overcoming this major limitation. Artificial T-cell receptors allow grafting of nearly any specificity to T cells. This allows generation of large numbers of specific T cells, without laborious selection and expansion procedures. Efficacy against tumors has been demonstrated in animal models. Phase I and II studies of T-cells transduced with artificial T-cell receptors as therapy for HIV infection have been performed. This rapidly advancing technology will make new strategies of adoptive immunotherapy possible.

Serum cystatin C in patients with myeloma.

BACKGROUND: Cystatin C is a low molecular weight protein thought to be synthesised by all nucleated cells and freely filtered by the kidney. It has been proposed as a marker for GFR; however, it has been suggested that there may be limitations to its use, because it may be over-expressed in some tumour cells and the abnormal tissue growth may also lead to an increased circulating level. METHODS: We investigated the serum cystatin C levels in 60 patients with myeloma, comparing results with those for serum creatinine, beta(2)-microglobulin and the paraprotein concentration. RESULTS: We found no correlation between cystatin C and the paraprotein concentration in these patients. CONCLUSION: These results suggest that disease burden does not correlate to the circulating level of cystatin C in patients with myeloma.

Developments in the assessment of glomerular filtration rate.

The assessment of the glomerular filtration rate (GFR) is the most commonly used test of renal function. The accepted reference procedure employs an exogenous clearance marker whilst the most popular test is that of serum or plasma creatinine. All of these tests have limitations, although the surrogate endogenous markers are the most practical. Cystatin C, a low molecular weight protein which can be measured by light scattering immunoassay, possesses many of the attributes required of the ideal GFR marker. Data on reference ranges indicate that circulating cystatin C levels reflect the variation in GFR throughout life and the marker demonstrates a better correlation with the reference procedure than serum creatinine.

Adult reference ranges for serum cystatin C, creatinine and predicted creatinine clearance.

Serum cystatin C measurement has been previously shown by ourselves and others to be a better indicator of changes in glomerular filtration rate (GFR) than serum creatinine. However, the available literature on reference values for cystatin C concentration remains surprisingly sparse; we thus set out to determine an adult reference range. Blood was taken from 309 healthy blood donors and creatinine and cystatin C concentrations were measured using commercially available automated methodologies. In addition, predicted creatinine clearances were calculated using the Cockcroft and Gault formula. The 95% reference intervals for creatinine, predicted creatinine clearance and cystatin C for all blood donors, regardless of gender, were 68-118 mumol/L, 58-120 ml/min/1.73 m2 and 0.51-0.98 mg/L, respectively. For women, the intervals were 68-98 mumol/L, 60-119 ml/min/1.73 m2 and 0.49-0.94 mg/L; for men, they were 78-123 mumol/L, 57-122 ml/min/1.73 m2 and 0.56-0.98 mg/L. This mean 95% reference interval for cystatin C in all donors under 50 years of age was 0.53-0.92 mg/L; for those over 50 years of age it was 0.58-1.02 mg/L. The small difference between make and female ranges meant that a single reference range for cystatin C could be established for all adults under 50 years of age without adjustment for body surface area. Serum cystatin C measurement offers a simpler and more sensitive screening test than serum creatinine for early changes in GFR.

Reference ranges for plasma cystatin C and creatinine measurements in premature infants, neonates, and older children.

AIM: To establish a reference range in the paediatric population for the new glomerular filtration rate (GFR) marker, cystatin C, and to compare it with that of creatinine. METHODS: Cystatin C and creatinine were measured by particle enhanced nephelometric immunoassay (PENIA) and fixed interval Jaff¿e methods, respectively, in 291 children aged 1 day to 17 years, including 30 premature infants with gestational ages ranging from 24 to 36 weeks. RESULTS: In the premature infants, concentrations of both cystatin C and creatinine were significantly raised compared with term infants, with cystatin C concentrations being between 1.10 and 2.06 mg/litre and creatinine between 32 and 135 micromol/litre. In premature infants, there was no significant relation between gestational age and cystatin C or creatinine concentration. Creatinine concentrations fell to a nadir at 4 months of age, rising gradually to adult values by about 15-17 years of age, in contrast to cystatin C, which fell to a mean concentration of 0.80 mg/litre by the 1st year of life, and remained constant throughout adulthood up to the age of 50 years. Neither analyte showed any influence of sex. CONCLUSION: The measurement of cystatin C, rather than creatinine, is more practical for monitoring GFR changes in the paediatric population.

PDGFbeta receptor blockade inhibits intimal hyperplasia in the baboon.

BACKGROUND: We have evaluated the use of a mouse/human chimeric anti-platelet-derived growth factor-beta receptor antibody in combination with heparin to inhibit intimal hyperplasia in the saphenous artery of the baboon after balloon angioplasty. METHODS AND RESULTS: The study evaluated lesion development in sequential injuries made 28 days apart. Each animal received control treatment after the first injury and antibody/heparin therapy after the second injury to the contralateral artery. The antibody was administered by bolus intravenous injections (10 mg/kg) on study days 1, 4, 8, 15, and 22 and heparin coadministered by continuous intravenous infusion at a dose of 0.13 mg/kg per hour. Morphometric analysis of tissue sections showed a 53% decrease in intimal area after antibody/heparin treatment (P=0.005), corresponding to a 40% decrease in the intima-to-media ratio (P=0.005). Smooth muscle cell proliferation in the injured wall, measured at both 4 and 29 days after balloon injury, were similar in the control and antibody/heparin-treated animals. CONCLUSIONS: These data suggest that platelet-derived growth factor plays a key role in the development of intimal lesions at sites of acute vascular injury in the nonhuman primate.

Changes in microvessel endothelial cell gene expression in an in vitro human breast tumour endothelial cell model.

Selective targeting of tumour-endothelium has been proposed as a means of therapy. The successful exploitation of this approach will rely on the identification of suitable targets expressed specifically on the tumour-associated endothelium. In an attempt to identify novel tumour-endothelium associated targets we have used differential mRNA display to identify genes up-regulated in an in vitro breast tumour-endothelial cell culture model. Confluent monolayers of human mammary microvessel endothelial cells (HuMMEC) were incubated for 5 days with MDA-MB-231 breast adenocarcinoma cell-conditioned medium (TCM). mRNAs isolated from TCM-treated and control cells were amplified using 104 combinations of four 3(') anchored T(12)VN primers and 26 'random' 10mers by RT-PCR and the products examined on DNA sequencing gels. Seventy-four sequences were cloned and the differential expression of five genes was confirmed using dot-blots. These were identified as procollagen type-IV, Tie-2/Tek receptor tyrosine kinase, NADH dehydrogenase subunit-6, and ferritin heavy-chain, which were up-regulated, and insulin-like growth factor binding protein-5, which was down-regulated. Increased endothelial expression of basement membrane proteins and tyrosine kinase receptors is known to occur during angiogenesis. Our data support the use of this model for further in vitro investigation of tumour angiogenesis.

Plasma cystatin C determinations in a healthy elderly population.

Plasma cystatin C measurement has been previously shown to be a better indicator of changes in glomerular filtration rate (GFR) than plasma creatinine. The available literature on reference intervals for cystatin C concentration encompasses only paediatric and adult populations up to 60 years of age, therefore we set out to determine an elderly reference range. Blood was taken from 401 subjects (65-101 years) and cystatin C and creatinine concentrations measured using commercially available methodologies. The availability of height and weight measurements allowed the additional calculation of predicted creatinine clearances using the Cockcroft and Gault formulae. Whilst no notable gender difference in cystatin C values was observed (female, 1.48 mg/l; male, 1.53 mg/l), concentrations rose with increasing age (60-79 years, 1.39 mg/l; >80 years, 1.70 mg/l). Conversely, there was a significant (P<0.0001) gender difference in creatinine values (female, 99 micromol/l; male, 120 micromol/l) but none between age groups (60-79 years, 105 micromol/l; >80 years, 113 micromol/l). Calculated GFR determinations resulted in a predicted creatinine clearance range of 21-81 ml/min per 1.73 m2 (n=361). There was no significant difference between gender (male, 18-88 ml/min per 1.73 m2; female, 24-69 ml/min per 1.73 m2), but a very significant 20% decrease in predicted GFR per decade. Sex-related reference intervals for creatinine were established (female, 66-149 micromol/l; male, 71-204 micromol/l); whilst age-related reference intervals were established for both cystatin C (60-79 years, 0.93-2.68 mg/l; >80 years, 1.07-3.35 mg/l) and predicted creatinine clearance (60-79 years, 27-89 ml/min per 1.73 m2; >80 years=18-55 ml/min per 1.73 m2). Plasma cystatin C measurement offers a simple, more sensitive screening assay for early changes in GFR and reflects the decreasing GFR that occurs with increasing age.

Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product.

Single chain Fv chimeric receptors, or T-bodies, are described with intracellular sequences comprising the costimulatory signaling domain of CD28 in series with the zeta-chain from the TCR complex. Using an engineered human single chain Fv derived from P67, an mAb with specificity for human CD33, and a spacer comprising an Ab hinge region with either Fcgamma or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to mediate both primary signaling and costimulation from one extracellular binding event. Constructs with the CD28 signaling domain proximal and the zeta-chain distal to the membrane were found to express more efficiently in Jurkat than constructs with the opposite orientation and were capable of mediating up to 20 times more IL-2 production on stimulation with solid phase Ag when compared with transfectants expressing chimeric receptors with zeta-chain intracellular signaling domains only. IL-2 production was specific to Ag challenge and was completely inhibited by incubation with free Ab of the same specificity as the extracellular binding site of the construct, but not by an isotype-matched control Ab. The CD28 intracellular domain of these fusion proteins was shown to be capable of binding the p85 subunit of phosphatidylinositol 3'-kinase. These constructs represent the first of a new generation of single gene multidomain chimeric receptors capable of mediating both primary and costimulatory signaling specifically from a single extracellular recognition event.

Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems (BNA, BN II).

Serum cystatin C has been suggested as a new marker of glomerular filtration rate (GFR). We describe a fully automated and rapid particle-enhanced nephelometric immunoassay (PENIA) for measuring serum cystatin C on the Behring nephelometer systems (BNA, BN II). Each sample is analyzed in 6 min with as many as 75 samples per batch. The assay covers the range 0.23-7.25 mg/L, up to seven times the upper limit of normal. The intra- and interassay imprecision are < 3.3% and < 4.5%, respectively. There is absolute linearity across the assay range (r2 = 0.997), with analytical recovery by cystatin C addition between 95% and 109% (mean 102%). Hemoglobin (< or = 8.0 g/L), bilirubin (< or = 488 microL), triglycerides (< or = 23 mmol/L), rheumatoid factor (< or = 2000 kIU/L), and myeloma paraprotein (< or = 41 g/L) do not interfere with the assay. This assay agreed well with an in-house particle-enhanced turbidimetric immunoassay (PETIA) (mean difference = 1.73 +/- 2.10) and a commercial PETIA (mean difference = 1.13 +/- 0.86). This is a new assay by which cystatin C may be effectively used as a marker of GFR estimation.

Preparation and preclinical evaluation of humanised A33 immunoconjugates for radioimmunotherapy.

A humanised IgG1/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg l-1 in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD = 1.3 nM) was shown to be equivalent to that of the murine antibody in a cell-binding assay. hA33 labelled with yttrium-90 using the macrocyclic chelator 12N4 (DOTA) was shown to localise very effectively to human colon tumour xenografts in nude mice, with tumour levels increasing as blood concentration fell up to 144 h. A Fab' variant of hA33 with a single hinge thiol group to facilitate chemical cross-linking has also been constructed and expressed with yields of 500 mg l-1. Trimaleimide cross-linkers have been used to produce a trivalent Fab fragment (hA33 TFM) that binds antigen on tumour cells with greater avidity than hA33 IgG. Cross-linkers incorporating 12N4 or 9N3 macrocycles have been used to produce hA33 TFM labelled stably and site specifically with yttrium-90 or indium-111 respectively. These molecules have been used to demonstrate that hA33 TFM is cleared more rapidly than hA33 IgG from the circulation of animals but does not lead to accumulation of these metallic radionuclides in the kidney. 90Y-labelled hA33 TFM therefore appears to be the optimal form of the antibody for radioimmunotherapy of colorectal carcinoma.

Humanization of an anti-mucin antibody for breast and ovarian cancer therapy.

Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.

Nephritis secondary to chronic cerebrospinal fluid - vascular shunt infection: "shunt nephritis".

Chronic indolent shunt infection can lead to intermittent transient bacteremia and secondary deposition of immune complexes in the kidney. The infective organism is usually one of low virulence, resulting in vague symptoms and a delay in diagnosis. Resolution of the infection usually results in return of renal function to normal.

Atlantooccipital instability. Case report.

A patient is presented who had neck pain and transient episodes of visual-field defect followed by unconsciousness when he hyperextended his neck. Radiographic examination revealed atlantooccipital instability on flexion and extension views. Treatment by fusion of the occiput to C-2 resulted in relief of symptoms.

Focal seizure disorder secondary to multiple shunt revisions. A case report.

A case of focal seizure disorder secondary to multiple ventricular shunt revision is presented. The literature reviewed indicates this complication of shunting is probably due to low grade infections or bleeding at the puncture site at the time of shunt revisions.

Fibrous dysplasia of the skull with progressive cranial nerve involvement.

The purpose of this report is twofold. First, to discuss fibrous dysplasia of bone as it relates to the neurosurgeon, and second to present a case of fibrous dysplasia of the skull with progressive involvement of the optic and trigeminal nerves. Of special interest in this case is the progression of the disease after puberty and the technical problems of decompression of the involved nerves. A combined approach to the lesion with otolaryngological assistance was undertaken.