Binders from the deepest vaults.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
P Carter
Publications and source records attributed to P Carter.
'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization.
'Knobs-into-holes' was originally proposed by Crick in 1952 as a model for the packing of amino acid side chains between adjacent alpha-helices. 'Knobs-into-holes' is demonstrated here as a novel and effective design strategy for engineering antibody heavy chain homodimers for heterodimerization. In this approach a 'knob' variant was first obtained by replacement of a small amino acid with a larger one in the CH3 domain of a CD4-IgG immunoadhesin: T366Y. The knob was designed to insert into a 'hole' in the CH3 domain of a humanized anti-CD3 antibody created by judicious replacement of a large residue with a smaller one: Y407T. The anti-CD3/CD4-IgG hybrid represents up to 92% of the protein A purified protein pool following co-expression of these two different heavy chains together with the anti-CD3 light chain. In contrast, only up to 57% of the anti-CD3/CD4-IgG hybrid is recovered following co-expression in which heavy chains contained wild-type CH3 domains. Thus knobs-into-holes engineering facilitates the construction of an antibody/immunoadehsin hybrid and likely other Fc-containing bifunctional therapeutics including bispecific immunoadhesins and bispecific antibodies.
Antibodies↗
Nursing staff back injuries: prevalence and cost in long term care facilities.
This report describes the period prevalence and cost of back injuries to nursing staff of long term care facilities in comparison to nurses employed industry wide and to other occupations industry wide. The period prevalence of back injuries to nursing staff in long term care facilities was highest for nurse aides, followed by LPNs and then RNs. Nurses (combined) had a period prevalence of back injuries nearly 1.5 times higher than all employees of long term care facilities and 6 times higher than all occupations combined industry wide. Within long term care facilities, nurses sustaining back injuries were younger and had been employed for a shorter period of time than the average for all nurses employed in long term care facilities. Back injuries accounted for more than half of the indemnity and medical costs for all injuries incurred in nursing homes and industry wide. The findings highlight the need for better prevention and rehabilitation
Adult↗
Translational enhancement of FGF-2 by eIF-4 factors, and alternate utilization of CUG and AUG codons for translation initiation.
The FGF-2 mRNA of most mammals contains a long and inhibitory 5' UTR in addition to at least two CUG codons upstream and in frame with the AUG start codon. These CUGs are used for translation initiation to generate several polypeptides. Cells overexpressing the translation initiation factor 4E produce and secrete large amounts of FGF-2, and particularly the largest, CUG1-initiated form. Overexpression of FGF-2 is due to a translational enhancement of its mRNA, as indicated by its association with large polyribosomes in contrast to control cells, where it partitions mostly in fractions lighter than 80 S or small polyribosomes. Breast carcinomas expressing elevated eIF-4E also exhibited the large FGF-2 isoforms, which could play an important role in tumor angiogenesis. Translation of in vitro transcribed rat FGF-2 in reticulocyte lysates resulted in synthesis of four polypeptides, of similar size to those observed in vivo. Addition of purified eIF-4F preferentially increased translation of CUG1- and AUG-initiated isoforms. Since the different isoforms of FGF-2 may have different roles and intracellular distribution, the effects of the eIF-4 factors on FGF-2 expression could be important for the control of cell proliferation and differentiation.
Animals↗
Identification of heavy chain residues in a humanized anti-CD3 antibody important for efficient antigen binding and T cell activation.
A previously identified humanized anti-CD3 Ab variant, v9, binds T cells with > 100-fold higher efficiency than the original variant, v1, and almost as efficiently as a chimeric molecule containing corresponding murine variable domains. Variants v1 and v9 differ at six positions in the H chain second CDR. Here a mutational analysis was used to identify which of these six replacements are primarily responsible for the difference in binding efficiency. These anti-CD3 variants were used to probe the relationship between Ag binding efficiency and potency in stimulating T cell proliferation. The human to mouse mutations T57S and V63F increase the binding efficiency of variant v1 for T cells by 8- and 12-fold, respectively, and together in variant M18 enhance binding by 26-fold to within 4-fold of variant v9. A framework mutation, 169L, was identified that enhances the binding of variants v1 and M18 by 14- and 3-fold, respectively. The Ag binding efficiencies of anti-CD3 variants correlate directly with their potencies in stimulating the proliferative activity of both resting human PBMC and IL-2-activated human T lymphocytes. Humanized variant v9 is equipotent to the murine parent Ab in stimulating ATL activity. PBMC activated by variants v1 and v9 IgG in a short term culture are equally cytotoxic against human breast carcinoma cells. Thus, high efficiency Ag binding by anti-CD3 variants is important for stimulating efficient T cell proliferation, but not cytotoxicity, in vitro.
Amino Acid Sequence↗
Engineering high affinity humanized anti-p185HER2/anti-CD3 bispecific F(ab')2 for efficient lysis of p185HER2 overexpressing tumor cells.
We previously constructed a humanized anti-p185HER2/anti-CD3 bispecific antibody variant, BsF(ab')2 v1 which retargets the cytotoxic activity of human T cells in vitro against human breast tumor cells which overexpress the p185HER2 product of the HER2/neu (c-erbB-2) protooncogene. Subsequently we identified an improved anti-CD3 variant, v9, which binds to T cells with approx. 100-fold higher affinity than the original variant, v1. Here we demonstrate that BsF(ab')2 v9 is more potent than BsF(ab')2 v1 in stimulating the proliferation of both resting peripheral blood lymphocytes (PBL) and IL-2-activated, long-term cultured T lymphocytes (ATL). In addition, at low concentrations (0.01-1 ng/ml) BsF(ab')2 v9 is much more efficient than BsF(ab')2 v1 in directing lysis of p185HER2-overexpressing tumor cells by IL-2 activated PBL. In contrast, at higher concentration BsF(ab')2 v9 and BsF(ab')2 v1 have similar potency in retargeted cytotoxicity. At BsF(ab')2 v9 concentrations of > or = 1 ng/ml the susceptibility of p185HER2-expressing tumor cells to lysis is apparently independent of the level of p185HER2 expression. At lower concentrations of BsF(ab')2 v9 and/or lower ratios of effector to target cells the extent of lysis is reduced, in some cases improving the selectivity of lysis of high p185HER2 expressors over low expressors. Thus selection of a high affinity anti-CD3 arm is likely important in the design of BsF(ab')2 for retargeting the cytotoxicity of T cells to tumors. The dose of BsF(ab')2 v9 in any future clinical evaluation will require optimization to maximize anti-tumor efficacy whilst minimizing potential toxicity against normal tissue expressing p185HER2.
Antibodies, Bispecific↗
Development of anti-p185HER2 immunoliposomes for cancer therapy.
The product of the HER2 protooncogene, p185HER2, represents an attractive target for cancer immunotherapies. We have prepared anti-p185HER2 immunoliposomes in which Fab' fragments of a humanized anti-p185HER2 monoclonal antibody with antiproliferative properties (rhuMAb-HER2) were conjugated to either conventional or sterically stabilized liposomes. These immunoliposomes bind specifically to p185HER2-overexpressing breast cancer cells (SK-BR-3 and BT-474). High-affinity binding of anti-p185HER2 immunoliposomes is comparable to that of free rhuMAbHER2-Fab' or the intact antibody. Empty immunoliposomes inhibit the culture growth of p185HER2-overexpressing breast cancer cells, and this antiproliferative effect is superior to that of free rhuMAbHER2-Fab', indicating that liposomal anchoring of these anti-p185HER2 Fab' fragments enhances their biological activity. Efficient internalization of anti-p185HER2 immunoliposomes, demonstrated by light and electron microscopy, occurs by receptor-mediated endocytosis via the coated pit pathway and also possibly by membrane fusion. Doxorubicin-loaded anti-p185HER2 immunoliposomes are markedly and specifically cytotoxic against p185HER2-overexpressing tumor cells in vitro. Anti-p185HER2 immunoliposomes administered in vivo in Scid mice bearing human breast tumor (BT-474) xenografts can deliver doxorubicin to tumors. These results indicate that anti-p185HER2 immunoliposomes are a promising therapeutic vehicle for the treatment of p185HER2-overexpressing human cancers.
Animals↗
Development of a humanized disulfide-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug.
The humanized anti-p185HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185HER2-overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) beta-lactamase fusion protein for use in antibody-dependent enzyme-mediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the VL/VH interface and secreted from Escherichia coli. One variant, dsFv3 (VL L46C VH D101C0, has similar affinity for antigen (Kd = 0.7 nM) as the wild-type Fv and was used to construct a fusion protein in which beta-lactamase, RTEM-1, is joined to the carboxy terminus of VH. The dsFv3-beta-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, kcat/km = 1.5 x 10(5) s-1 M-1). PRODOX is approximately 20-fold less toxic than free doxorubicin against breast tumor cell lines SK-BR-3 and MCF7, which express p185HER2 at elevated and normal levels, respectively. Prebinding the dsFv3-beta-lactamase fusion protein specifically enhances the toxicity level of PRODOX to that of doxorubicin against SK-BR-3 but not MCF7 cells. The fusion protein retains both antigen-binding plus kinetic activity in murine serum and is cleared rapidly as judged by pharmacokinetic analysis in nude mice (initial and terminal half-lives of 0.23 and 1.27 h, respectively). Development and characterization of the dsFv3-beta-lactamase fusion protein is an important step toward targeted prodrug therapy of p185HER2-overexpressing tumors.
Animals↗
Bispecific HER2 x CD3 antibodies enhance T-cell cytotoxicity in vitro and localize to HER2-overexpressing xenografts in nude mice.
Recently, we reported the development of fully humanized bispecific F(ab')2 antibodies with dual binding specificities to human T-lymphocytes and to tumor cells overexpressing HER2. These antibodies were shown to effectively mediate targeted HER2-overexpressing tumor cell killing by freshly isolated human T-cells. In this report we extend our studies to describe the interaction of the bispecific antibody with activated T-lymphocytes (ATL) maintained in culture for an extended period of time. A microtiter plate radioreceptor assay was used to elucidate the affinity of bispecific antibody binding to ATL. The data show that ATL maintained in vitro for up to 5 weeks continued to express high-affinity CD3 surface markers that bound to bispecific antibody with a Kd of 2.49 nM and exerted cytolytic activities against targets overexpressing HER2. In addition, we demonstrated the specific localization of HER2 x CD3 bispecific antibody to HER2-overexpressing tumor xenografts in nude mice. Furthermore, HER2 x CD3 bispecific antibody has the ability to inhibit the proliferative activities of breast tumor (SKBR-3) cells in vitro. The clinical implications of these data are discussed.
Animals↗
Synthesis and beta-lactamase-mediated activation of a cephalosporin-taxol prodrug.
BACKGROUND: Enzyme-activatable prodrugs in conjunction with antibody-enzyme fusion proteins may enhance the anti-tumor efficacy of antibodies and reduce the toxic side effects of conventional chemotherapeutics. Cephalosporins have proven to be highly versatile triggers for the enzymatic activation of such prodrugs. RESULTS: A cephem prodrug of taxol (PROTAX) was synthesized by substituting the C-3' position of cephalothin with 2'-(gamma-aminobutyryl) taxol. Hydrolysis of PROTAX by beta-lactamase rapidly released 2'-(gamma-aminobutyryl) taxol (kcat/K(M) = (1.4 +/- 0.1) x 10(5) s-1 M-1), which yielded taxol following intramolecular displacement. PROTAX is inactive in a microtubule assembly assay in vitro but has similar activity to taxol following prolonged activation with beta-lactamase. PROTAX is approximately 10-fold less toxic than taxol against SK-BR-3 breast tumor cells in vitro but has activity approaching that of taxol following prolonged activation with a fusion protein comprising beta-lactamase fused to a tumor-targeting antibody fragment. CONCLUSIONS: Tubulin polymerization activity is abolished and cytotoxicity is reduced in the PROTAX prodrug compared to taxol. Activation of PROTAX by beta-lactamase followed by self-immolation restores the activity of PROTAX to that of free taxol.
Animals↗
Malignant islet cell tumor associated with hypercalcemia.
BACKGROUND: Three cases of islet cell tumors of the pancreas with hypercalcemia were studied, and 16 similar cases have been found in a 25-year review of the English-language literature. The purpose of the study was to review the cause of the hypercalcemia and the clinical characteristics of the tumors. METHODS: Tumor tissue retrieved from paraffin-embedded blocks was studied immunohistochemically for both parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH was measured in the serum in each patient and the serum PTHrP was measured by immunoassay in one patient. RESULTS: One of our patients had a fatal serum calcium level of 26.4 mg/dl. PTHrP stains were positive in two of our tumors, and one patient had an elevated PTHrP serum level. Serum PTH levels were normal or low in each patient. All three tumors were malignant and extremely vascular. The total group of 19 patients have in common hypercalcemia associated with a normal or low serum PTH level. Although the cause of hypercalcemia has not been proved, the tumors apparently produce PTHrP, because seven of eight tumors stained positive for PTHrP and each of the four patients tested had an elevated PTHrP serum titer. The tumors are extremely vascular, are usually malignant (17 of 18), and become large, but they are compatible with a relatively long patient survival time. CONCLUSIONS: These neuroendocrine tumors associated with hypercalcemia share several characteristics, but a claim that they represent another type of "functioning islet cell tumor" should await a clearer delineation of the cause of the hypercalcemia.
Adenoma, Islet Cell↗
Toward the production of bispecific antibody fragments for clinical applications.
The clinical potential of bispecific antibodies (BsAb) has been hindered by the difficulty of obtaining clinical grade material, together with the immunogenicity of rodent-derived BsAb in patients. The supply issue is being directly addressed by recombinant methods for BsAb fragment production reviewed here. The immunogenicity issue will likely be overcome by the use of humanized or human antibodies. Currently, three technologies appear suitable for the production of BsAb fragments for clinical applications: BsF(ab')2 assembled from Fab' fragments expressed in Escherichia coli, BsF(ab')2 assembled using leucine zippers, and diabodies.
Animals↗
Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity.
We developed a novel bivalent antibody fragment, the linear (L-) F(ab')2, comprising tandem repeats of a heavy chain fragment VH-CH1-VH-CH1 cosecreted with a light chain. Functional humanized L-F(ab')2 directed against p185HER2 was secreted from Escherichia coli at high titer (> or = 100 mg/l) and purified to homogeneity. The L-F(ab')2 binds two equivalents of antigen with an apparent affinity (Kd = 0.46 nM) that is within 3-fold of the corresponding thioether-linked F(ab')2 fragment. The N-terminal site binds antigen with an affinity (Kd = 1.2 nM) that is approximately 4-fold greater than that for the C-terminal site, as shown by the comparison of L-F(ab')2 variants containing a single functional binding site. L-F(ab')2 has greater antiproliferative activity than the thioether-linked F(ab')2 against the p185HER2-overexpressing tumor cell line BT474. Linear and thioether-linked F(ab')2 have very similar pharmacokinetic properties in normal mice, and their serum permanence times are respectively 7- and 8-fold longer than the corresponding Fab fragment. L-F(ab')2 offers a facile route to bivalent antibody fragments that are potentially suitable for clinical applications, and that may have improved biological activity compared with thioether-linked F(ab')2 fragments.
Animals↗
Impedance measurement of the Nucleus 22-electrode array in patients.
Explore the source record for details and available documents.
Cochlear Implants↗
Electrospray ionization mass spectrometry of recombinantly engineered antibody fragments.
Mass spectrometry has been used to confirm the correct protein expression of Fab and F(ab')2 fragments of the humanized anti-p185HER2 antibody huMAb4D5. These data demonstrate that electrospray ionization mass spectrometry can be used routinely to measure the masses of purified proteins as large as 100 kDa, with an accuracy of < or = 0.02%. This level of accuracy is helpful in demonstrating the faithful translation and proper posttranslational modification of these proteins. Accurate and reliable mass assignments by electrospray ionization mass spectrometry are achieved by calibrating with protein mass standards, optimizing resolution, and using methods to improve sample purity. On-line reversed-phase high-performance liquid chromatography/mass spectrometry has been employed to improve sample purity and thereby increase sensitivity. Disulfide reduction to yield the component subunits provides additional confirmation of the correct amino acid sequence with greater absolute mass accuracy. A volatile reducing agent such as tributylphosphine provides a convenient method to generate subunits for mass measurement while requiring little or no further sample purification.
Humans↗
Interaction of cationic porphyrins with DNA.
Utilizing linear dichroism (LD), circular dichroism (CD), and fluorescence energy transfer, the binding geometries of a series of Co(3+)-porphyrins and their free ligands were examined. The compounds studied were Co-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) and its free ligand (H2-TMPyP), Co-meso-tetrakis(N-n-butylpyridinium-4-yl)porphyrin (CoTBPyP) and its free ligand (H2TBPyP), and Co-meso-tetrakis(N-n-octylpyridinium-4-yl)porphyrin (CoTOPyP). The two non-metalloporphyrins exhibit negative LD, having angles of roughly 75 degrees relative to the DNA helix axis. They also display negative CD and a significant contact energy transfer from the DNA bases. On the other hand, the three metalloporphyrins display orientation angles of roughly 45 degrees between the porphyrin plane and the helix axis of DNA. Furthermore, they exhibit positive CD and no contact energy transfer from DNA bases. These observations show that the metalloporphyrins are not intercalated whereas non-metalloporphyrins having four freely rotating meso-aryl groups intercalate between the base pairs of DNA. In the presence of KHSO5, the cobalt porphyrins cleave closed circular PM2 DNA in a single strand manner, i.e., a single activation event on the porphyrin leads to a break in one of the DNA strands. A kinetic analysis of the cleavage data revealed that cleavage rates are in the order CoTMPyP > CoTBPyP > CoTOPyP with the difference being due to different DNA affinities rather than differences in cleavage rate-constants. Based on these and earlier observations, the metalloporphyrins appear bound to a partially melted region of DNA.
Animals↗
X-ray structures of fragments from binding and nonbinding versions of a humanized anti-CD18 antibody: structural indications of the key role of VH residues 59 to 65.
X-ray crystal structures of fragments from two different humanized anti-CD18 antibodies are reported. The Fv fragment of the nonbinding version has been refined in space group C2 with a = 64.2 A, b = 61.3 A, c = 51.8 A, and beta = 99 degrees to an R-value of 18.0% at 1.9 A, and the Fab fragment of the tight-binding version has been refined in space group P3 with a = 101. A and c = 45.5 A to an R-value of 17.8% at 3.0 A resolution. The very large difference in their binding affinity (> 1000-fold) is attributed to large and local structural differences in the C-terminal part of CDR-H2, and from this we conclude there is direct contact between this region and antigen when they combine. X-ray structures of antibody-antigen complexes available in the literature have yet to show this part of CDR-H2 in contact with antigen, despite its hypervariable sequence. Implications of this result for antibody humanization are discussed.
Amino Acid Sequence↗
Towards an immunotherapy for p185HER2 overexpressing tumors.
Explore the source record for details and available documents.
Animals↗