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Biomedical subjects

P Carter

Publications and source records attributed to P Carter.

At least 19 recordsLinked to original sources
PubMed1992

Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments.

The murine monoclonal antibody 4D5 (anti-p185HER2) inhibits the proliferation of human tumor cells overexpressing p185HER2 in vitro and has been "humanized" [Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W.-L. T., Rowland, A. M., Kotts, C., Carver, M. E., & Shepard, H. M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)] for use in human cancer therapy. We have determined the antigen binding thermodynamics and the antiproliferative activities of chimeric 4D5 Fab (ch4D5 Fab) fragment and a series of eight humanized Fab (hu4D5 Fab) fragments differing by amino acid substitutions in the framework regions of the variable domains. Fab fragments were expressed by secretion from Escherichia coli and purified from fermentation supernatants by using affinity chromatography on immobilized streptococcal protein G or staphylococcal protein A for ch4D5 and hu4D5, respectively. Circular dichroism spectroscopy indicates correct folding of the E. coli produced Fab, and scanning calorimetry shows a greater stability for hu4D5 (Tm = 82 degrees C) as compared with ch4D5 Fab (Tm = 72 degrees C). KD values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the delta H and delta Cp for binding were determined by using isothermal titration calorimetry. ch4D5 Fab and one of the humanized variants (hu4D5-8 Fab) bind p185HER2-ECD with comparable affinity (delta G degrees = -13.6 kcal mol-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Humanization of an anti-p185HER2 antibody for human cancer therapy.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

Adenocarcinoma↗
PubMed1992

CD20 and CD5 expression in B-chronic lymphocytic leukemia.

In order to quantitate a previously noted decrease in CD20 fluorescence intensity (FI) on B-CLL lymphocytes, binding capacities [BC x 10(3) +/- 1SD = number of antibodies bound per cell] were calculated. The mean (N = 5) BC x 10(3) +/- 1SD of CD20 reagents for normal B-PBL and B-CLL lymphocytes confirmed this observation. B-PBL and B-CLL were 56 +/- 11 and 61 +/- 14, and 19 +/- 15 and 18 +/- 16, respectively, for Leu 16 and B1. Although adequate compensation standards for the determination of CD5 and CD20 coexpression are not available, qualitatively, the density of CD5 on both normal B-PBL and B-CLL is less compared to the expression of CD5 by normal T cells. CD5 expression on B-CLL seems to be linked to the lower levels of CD20, whereas CD5 expression may appear to be absent on CLL lymphocytes expressing normal levels of CD20. Levels of CD20 in B-CLL suggest involvement of one or two genes (alleles) whose decreased expression may be linked to CD5 expression.

Antigens, CD↗
PubMed1992

Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene.

The HER2 protooncogene encodes a 185-kD transmembrane phosphoglycoproteins, human epidermal growth factor receptor 2 (p185HER2), whose amplified expression on the cell surface can lead to malignant transformation. Overexpression of HER2/p185HER2 is strongly correlated with progression of human ovarian and breast carcinomas. Recent studies have shown that human T cells can be targeted with bispecific antibody to react against human tumor cells in vitro. We have developed a bispecific F(ab')2 antibody molecule consisting of a humanized arm with a specificity to p185HER2 linked to another arm derived from a murine anti-CD3 monoclonal antibody that we have cloned from UCHT1 hybridoma. The antigen-binding loops for the anti-CD3 were installed in the context of human variable region framework residues, thus forming a fully humanized BsF(ab')2 fragment. Additional variants were produced by replacement of amino acid residues located in light chain complementarity determining region 2 and heavy chain framework region 3 of the humanized anti-CD3 arm. Flow cytometry analysis showed that the bispecific F(ab')2 molecules can bind specifically to cells overexpressing p185HER2 and to normal human peripheral blood mononuclear cells bearing the CD3 surface marker. In additional experiments, the presence of bispecific F(ab')2 caused up to fourfold enhancement in the cytotoxic activities of human T cells against tumor cells overexpressing p185HER2 as determined by a 51Cr release assay. These bispecific molecules have a potential use as therapeutic agents for the treatment of cancer.

Amino Acid Sequence↗
PubMed1992

High level Escherichia coli expression and production of a bivalent humanized antibody fragment.

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

Amino Acid Sequence↗
PubMed1992

Weyers' ulnar ray/oligodactyly syndrome and the association of midline malformations with ulnar ray defects.

We describe a two generation family with variable ulnar and radial ray reduction and midline craniofacial abnormalities. The features suggest a diagnosis of Weyers' ulnar ray/oligodactyly syndrome originally described in two isolated cases. Syndromes of ulnar ray reduction are briefly reviewed and the relationship between limb bud and midline development discussed.

Abnormalities, Multiple↗
PubMed1992

Engineering a humanized bispecific F(ab')2 fragment for improved binding to T cells.

We recently constructed a humanized bispecific antibody (BsF(ab')2v1) by separate E. coli expression of each Fab' arm followed by directed chemical coupling in vitro. BsF(ab')2 v1 (anti-CD3/anti-p185HER2) was demonstrated to retarget the cytotoxic activity of human CD3+ CTL in vitro against the human breast-tumor cell line, SK-BR-3, which over-expresses the p185HER2 product of the proto-oncogene HER2. Our minimalistic humanization strategy is to install as few murine residues as possible into a human antibody in order to recruit antigen-binding affinity and biological properties comparable to that of the murine parent antibody. This strategy proved very successful for the anti-p185HER2 arm of BsF(ab')2 v1. In contrast BsF(ab')2 v1 binds to T cells via its anti-CD3 arm much less efficiently than does the chimeric BsF(ab')2 which contains the variable domains of the murine parent anti-CD3 antibody. Here we have constructed additional BsF(ab')2 fragments containing variant anti-CD3 arms with selected amino acid replacements in an attempt to improve antibody binding to T cells. One such variant, BsF(ab')2 v9, was created by replacing 6 residues in the second hypervariable loop of the anti-CD3 heavy chain variable domain of BsF(ab')2 v1 with their counterparts from the murine parent anti-CD3 antibody. BsF(ab')2 v9 binds to T cells (Jurkat) much more efficiently than does BsF(ab')2 v1 and almost as efficiently as the chimeric BsF(ab')2. This improvement in the efficiency of T-cell binding of the humanized BsF(ab')2 is an important step in its development as a potential therapeutic agent for the treatment of p185HER2 over-expressing cancers.

Amino Acid Sequence↗
PubMed1991

Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN'.

A mutant of the serine protease, subtilisin BPN', in which the catalytic His64 is replaced by Ala (H64A), is very specific for substrates containing a histidine, presumably by the substrate-bound histidine assisting in catalysis [Carter, P., & Wells, J.A. (1987) Science (Washington, D.C.) 237, 394-399]. Here we probe the catalytic mechanism of H64A subtilisin for cleaving His and non-His substrates. We show that the ratio of aminolysis to hydrolysis is the same for ester and amide substrates as catalyzed by the H64A subtilisin. This is consistent with formation of a common acyl-enzyme intermediate for H64A subtilisin, analogous to the mechanism of the wild-type enzyme. However, the catalytic efficiencies (kcat/KM) for amidase and esterase activities with His-containing substrates are reduced by 5000-fold and 14-fold, respectively, relative to wild-type subtilisin BPN, suggesting that acylation is more compromised than deacylation in the H64A mutant. High concentrations of imidazole are much less effective than His substrates in promoting hydrolysis by the H64A variant, suggesting that the His residue on the bound (not free) substrate is involved in catalysis. The reduction in catalytic efficiency kcat/KM for hydrolysis of the amide substrate upon replacement of the oxyanion stabilizing asparagine (N155G) is only 7-fold greater for wild-type than H64A subtilisin. In contrast, the reductions in kcat/KM upon replacement of the catalytic serine (S221A) or aspartate (D32A) are about 3000-fold greater for wild-type than H64A subtilisin, suggesting that the functional interactions between the Asp32 and Ser221 with the substrate histidine are more compromised in substrate-assisted catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1990

Functional interaction among catalytic residues in subtilisin BPN'.

Variants of the serine protease, subtilisin BPN', in which the catalytic triad residues (Ser-221, His-64, and Asp-32) are replaced singly or in combination by alanine retain activities with the substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPF-pna) that are at least 10(3) to 10(4) above the non-enzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564-568, 1988]. A possible source of the residual activity was the hydrogen bond with the N delta 2 of Asn-155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild-type enzyme. Replacing Asn-155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF-pna by 150-fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcat is actually 5-fold greater than for the S221A enzyme. Thus, the catalytic role of Asn-155 is dependent upon the presence of Ser-221. The residual activity of the N155G:S221A enzyme (approximately 10(4)-fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. The mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1990

RO 23-6457 prolongs survival of vascularized allografts in rodents and primates.

The ability of RO 23-6457, a retinoid compound with marked in vitro immunosuppressive properties, to prolong vascularized allografts was examined in several in vivo transplantation models. In the murine heterotopic heart model, efficacy was shown in two H-2 incompatible strain combinations, with indefinite graft survival at some doses. In the rat heterotopic heart model, oral administration prolonged Wistar-Furth grafts in Lewis hosts an average of 1 week, with no long-term survivors at a variety of doses. Given subcutaneously, grafts were further prolonged, but the compound proved toxic. In a bilaterally nephrectomized renal transplant model in cynomolgus monkeys treated intravenously at a dose of 2 mg/kg/day, host survival was prolonged to 18, 32, 33, and 74 days, compared with 11, 11, 12, 13, and 26 days in untreated controls (P less than 0.05 by rank-sum testing). The three shorter surviving recipients died from anorexia and weight loss with normal renal function, while the longest survivor rejected its kidney when exhaustion of iv sites precluded further treatment. The toxic effects of the compound resemble the syndrome of hypervitaminosis A. RO 23-6457 will prolong graft survival as a single agent, justifying further preclinical testing and efforts to reduce toxicity.

Animals↗
PubMed1989

Engineering subtilisin BPN' for site-specific proteolysis.

A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.

Amino Acid Sequence↗
PubMed1989

Plunging ranula: literature review and report of three cases.

Three cases of plunging ranula are described and the literature is reviewed. In many cases, a plunging ranula is iatrogenic and follows surgery to an oral ranula. In the cases presented, the cervical swelling was associated with prolongations of sublingual gland into or through the mylohyoid muscle. All patients were cured by partial or total excision of the sublingual gland.

Adult↗
PubMed1989

The structures of human C1r and C1s and their relationship to other serine proteases.

The recent sequencing of the C1 subcomponents has allowed comparison with other molecules of homologous primary structure. Where tertiary structures are available for at least one member of the family it is possible to make further progress by modelling the amino acid sequence of the complement protein into the three-dimensional coordinates of the directly determined structure, thereby obtaining an approximation of the structure of the complement protein. Molecular modelling allows structure-function relationships to be explored and suggests further experiments that may be amenable to techniques such as site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1989

Molecular defects of the C1-inhibitor gene in hereditary angioedema.

Family studies of hereditary angioedema are discussed with particular emphasis on the molecular basis of the more common form of the disease (type I), characterized by quantitative C1-Inhibitor deficiencies. Results obtained in several laboratories can be interpreted in the light of the restriction maps and of the exon-intron structure of the normal C1-Inhibitor gene that have been published recently. Several partial deletions of either exon 4 or exon 7 or of larger portions of the 5' end of the gene and a duplication of exon 4 have been described. These gene alterations are essentially family-specific and they account for at least fifteen-percent of the defects in type I hereditary angioedema. Detailed sequences analyses of cloned defective genes indicate that clusters of repetitive sequences of the Alu family, located within several introns, are responsible for the relatively high frequency of gross gene alterations observed at the C1-Inhibitor locus. In contrast, most of the defects that lead to production of dysfunctional proteins appear to be point mutations within the P1 residue of the reactive site, whose codon features a hypermutable CpG dinucleotide.

Angioedema↗