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PubMed1991

Application of engineered chimeric antibodies to the calibration of human antibody standards.

Immunoassay quantitation of human antibody depends on the availability of reference sera that have been characterized in terms of their antibody content. This report expands upon the concept of "cross-calibration", using reference (dose-response) curves that have been generated with heterologous antibody as a means of calibrating prospective human antibody standards. Engineered human-mouse chimeric antibody, with a single human isotype and specificity, is used to construct the heterologous dose-response curve from which test antibody response data are interpolated. Transfer experiments are used to define and correct for the percentage of the total chimeric antibody that is immunoreactive and bound to solid phase antigen. The chimeric antibody-based calibration procedure is illustrated using H. influenzae Type B as the model antigen and an engineered chimeric IgG1 antibody that has a known mass per volume content and specificity for the hapten, 4-hydroxy-3-nitro-phenylacetyl (NP).

Animals↗
PubMed1990

Natural history of mixed chimerism after bone marrow transplantation with CD6-depleted allogeneic marrow: a stable equilibrium.

Mixed hematopoietic chimerism (MC) is a common finding after allogeneic bone marrow transplantation (BMT), but the natural history of this phenomenon remains unclear. To understand the evolution and the implications of this finding, we performed a prospective analysis of the development of mixed chimerism in 43 patients with hematologic malignancies who received bone marrow (BM) from human leukocyte antigen (HLA)-identical sibling donors. T-cell depletion in vitro with anti-T12 (CD6) monoclonal antibody and rabbit complement was used as the only method of graft-versus-host disease (GVHD) prophylaxis. Overall, MC was identified in peripheral blood (PB) and BM in 22 of 43 (51%) patients evaluated. MC was found by restriction fragment length polymorphism (RFLP) analysis in 21 of 40 (53%) patients, by cytogenetic analysis in 6 of 29 (21%) patients, and by red blood cell phenotyping in 4 of 9 (44%) patients. RFLP studies were performed at 0.5, 1, 3, 6, 9, and 12 months post-BMT and then every 6 months, and showed a high probability of developing MC in the first 6 months after BMT followed by stabilization after 12 months. Cytogenetic analysis was less sensitive in detecting MC. Once MC was detected after BMT, the percentage of recipient cells increased very slowly over more than 3 years of follow-up, and no patient reverted to complete donor hematopoiesis (CDH). Thus, recipient and donor cells remained in a relative state of equilibrium for prolonged periods that seemed to favor recipient cells over donor cells. Patient's disease, remission status, or intensity of the transplant preparative regimen did not influence the subsequent development of mixed chimerism. Early immunologic reconstitution was the only factor that correlated with the subsequent chimeric status of the patients. The percentage and absolute number of T3 (CD3) and T4 (CD4) positive cells at day 14 after BMT were significantly higher in the patients who maintained CDH but NK cell reconstitution was similar in both groups, suggesting that early reconstitution with T cells may play a role in preventing recovery of recipient cells after BMT. GVHD was also associated with maintenance of CDH, but the probability of relapse, survival, and disease-free survival was identical in patients with MC and CDH.

Antigens, CD↗
PubMed1990

IL2-PE664Glu, a new chimeric protein cytotoxic to human-activated T lymphocytes.

To produce a molecule that will kill activated T cells as well as lymphomas and leukemias expressing interleukin 2 (IL2) receptors, we have created a recombinant chimeric protein in which IL2 is attached in peptide linkage to a truncated mutant form of Pseudomonas exotoxin (PE) (Lorberboum-Galski, H., FitzGerald, D.J.P., Chandhary, V.K., Adhya, S., and Pastan, I. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1922-1926). Although this molecule was very active on rodent cells, it had lower activity on some human cell types. A new chimeric protein termed IL2-PE664Glu has been constructed that is extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes. The chimeric gene encoding this protein was constructed by fusing a cDNA clone for human interleukin 2 to the 5' end of a mutated cDNA encoding a full-length PE molecule. Four amino acids in domain I of PE were changed thus decreasing its nonspecific toxicity. IL2-PE664Glu is a much more active cytotoxic molecule for primate and human-activated T cells than IL2-PE40 which is a chimeric protein that was found to be an effective immunosuppressive agent in rodent models. Our results indicate that IL2-PE664Glu should be evaluated as an immunosuppressive agent for the treatment of human immune disorders in which activated T cells expressing the IL2 receptor are prominent.

ADP Ribose Transferases↗
PubMed1991

Pharmacokinetics, immune response, and biodistribution of iodine-131-labeled chimeric mouse/human IgG1,k 17-1A monoclonal antibody.

Pharmacokinetics, immunogenicity, and biodistribution of a 131I-labeled mouse/human chimeric monoclonal antibody (C-17-1A) was studied in six metastatic colon cancer patients. Pharmacokinetics obtained from serum radioactivity or chimera concentration were identical after 5 mCi of 131I-C-17-1A with mean alpha half-lives of 17.6 +/- 2.3 and 19.7 +/- 2.9 and mean beta half-lives of 100.9 +/- 16.1 and 106.4 +/- 14.1 hr, respectively. HPLC analysis documented the monomeric chimeric 17-1A without evidence of immune complexes or free 131I. None of the patients developed antibody after 131I-chimeric 17-1A exposure. Radiolocalization occurred in known areas of disease greater than 4 cm in all patients. The half-life of total-body radioactivity was 58 +/- 7 hr by whole-body counts and 64 +/- 13 hr by urine measurements. Whole-body and bone marrow dose estimates ranged from 0.75-1.03 and 0.76-1.05 rad/mCi, respectively. These studies confirm the prolonged circulation and reduced immunogenicity of chimeric 17-1A versus murine 17-1A. Marrow radiation exposure using antibodies with prolonged circulation is a critical factor in planning for radioimmunotherapeutic applications.

Adenocarcinoma↗
PubMed1990

Expression of chimeric cDNAs in cell culture defines a region of UDP glucuronosyltransferase involved in substrate selection.

The cDNAs encoding two forms of UDP glucuronosyltransferase have been expressed in cultured cells to demonstrate that one form, UDPGTr-3, glucuronidates testosterone, whereas the second form, UDPGTr-4, is mainly active toward etiocholanolone (Mackenzie, P. I. (1986) J. Biol. Chem. 261, 14112-14117; Mackenzie, P. I. (1987) J. Biol. Chem. 262, 9744-9749). In order to localize areas of the polypeptide chain involved in substrate selection, the 5' regions of UDPGTr-3 and -4 cDNAs were exchanged to form two chimeric cDNAs. A 53-kDa protein was synthesized in COS cells transfected with the chimeric UDPGTr-3.4 cDNA, which encodes the amino-terminal 298 residues of UDPGTr-3 and the carboxyl-terminal 232 residues of UDPGTr-4. This protein glucuronidated testosterone rather than etiocholanolone and had a faster electrophoretic mobility when transfected COS cells were cultured in the presence of tunicamycin, an inhibitor of N-linked glycosylation. The unglycosylated variant produced by this treatment also glucuronidated testosterone. In contrast, a 50-kDa protein that was more active toward etiocholanolone as substrate was synthesized in COS cells transfected with UDPGTr-4.3, a chimeric cDNA that encodes the amino-terminal region of UDPGTr-4 joined to the carboxyl-terminal region of UDPGTr-3. The electrophoretic mobility of this chimeric protein was unaffected by tunicamycin treatment. These results demonstrate that amino acid sequences that specify substrate specificity are localized in the amino-terminal half of the UDP glucuronosyltransferase polypeptide chain and that the presence of N-linked oligosaccharide chains on the protein does not affect the choice of substrate.

Amino Acid Sequence↗
PubMed1990

Genetically distinct cell populations in naturally occurring bone marrow-chimeric primates express similar MHC class I gene products.

The cotton-top tamarin (Saguinus oedipus) is a naturally occurring "A" + "B"----"A" bone marrow-chimeric species. These primates usually are born as dizygotic twins and, due to placental vascular anastomoses, develop sharing each others' bone marrow elements. Strikingly, almost 50% of the PBL of a member of a twin pair are derived from the hematopoietic stem cells of its cotwin. To clarify the mechanisms underlying the maintenance of tolerance in these stable chimeras, MHC gene products have been biochemically characterized in cloned, genetically distinct, male and female lymphocytes from two male/female cotton-top tamarin twin pairs. Extensive MHC class II sharing between the genetically distinct cell populations was not seen in the two twin pairs. This was consistent with the MHC class II polymorphism seen in the species. However, the MHC class I gene products expressed by one member of a twin pair were almost identical to those expressed by its cotwin. A human minisatellite probe demonstrated restriction fragment length polymorphism in DNA from these animals, indicating extensive polymorphism. Thus, MHC class I sharing did not occur due to inbreeding in these animals. Additionally, another bone marrow-chimeric primate species, the common marmoset (Callithrix jacchus), expresses MHC class I molecules with low levels of variation. These studies suggest that the stable chimerism of bone marrow-chimeric primates may be facilitated by MHC class I similarity between the genetically distinct bone marrow derived-cell populations in their circulation.

Animals↗
PubMed1990

Importance of suppressor T cells in cyclophosphamide-induced tolerance to the non-H-2-encoded alloantigens. Is mixed chimerism really required in maintaining a skin allograft tolerance?

Mechanisms of cyclophosphamide (CP)-induced tolerance were studied. When AKR/J Sea (AKR: H-2k) mice were primed i.v. with 5 x 10(7) spleen cells plus 1 x 10(7) bone marrow cells from [C57BL/6 Slc (B6; H-2b) x C3H/He Slc (C3H; H-2k)]F1 (B6C3F1) mice and treated i.p. with 200 mg/kg CP 2 days later, the survival of C3H skin was moderately prolonged, but the survival of either B6 or B6C3F1 skin was not prolonged. By this treatment, however, mixed chimerism of B6C3F1 cells in the AKR mice was not established. When C3H cells were used as the tolerogen, a minimal degree of mixed chimerism associated with profound tolerance to C3H skin was established. Similar results were observed in various donor-recipient combinations. When C3H skin was grafted in the AKR mice 12 wk after the treatment with C3H cells and CP, or B6C3F1 cells and CP, survival of the grafted C3H skin was prolonged remarkably or moderately, respectively, although mixed chimerism was not detectable at the timing of grafting in either of the groups. In this late stage of tolerance, a strong level of tolerogen-specific suppressor cell activity was observed in those tolerant AKR mice. The suppressor activity was mainly attributable to T cells. These results suggest that the role of Ts cells in order to maintain skin tolerance is important in our CP-induced tolerance system, especially in the late stage of tolerance. Moreover, the generation of the Ts cells does not necessarily require the establishment of a long term mixed chimeric state.

Animals↗
PubMed1990

Recombinant human IgG1-murine IgE chimeric Ig. Construction, expression, and binding to human Fc gamma receptors.

We have constructed a set of chimeric Ig by exchanging corresponding H chain C domains between human (hu) IgG1 and murine (m) IgE. We used this set of Ig to dissect the interaction of individual Ig domains with human Fc gamma receptors. Only one of the chimeras, epsilon/C gamma 2,3 (an mIgE with C epsilon 3 and C epsilon 4 replaced by C gamma 2 and C gamma 3 from huIgG1), binds tightly to the human Fc gamma RI on U937 cells. We found that epsilon/C gamma 2,3 has only twofold lower affinity for Fc gamma RI as compared to huIgG1. The gamma/C epsilon 4 (huIgG1 with C epsilon 4 replacing C gamma 3) binds weakly to Fc gamma RI. The other chimeric Ig, epsilon/C gamma 3, epsilon/C gamma 2, and gamma/C epsilon 3, as well as mIgE do not bind detectably to Fc gamma RI. From these data we conclude that the C gamma 2 domain is crucial for binding and contains the majority of the binding site for Fc gamma RI on IgG1. The C gamma 3 domain makes a smaller contribution to the binding, and the C gamma 1 domain and the hinge region have very little effect on the Fc gamma RI-IgG1 interaction. The chimeric epsilon/C gamma 2,3 and huIgG1 both mediate the formation of rosettes between K562 cells and antigen-sensitized E with similar concentration dependences. These results suggest similar ability to bind to Fc gamma RII. The other chimeric Ig do not cause rosettes in this assay system. Hence, both C gamma 2 and C gamma 3 seem to be required for binding to Fc gamma RII, but the C gamma 1-hinge region has no detectable effect.

Animals↗
PubMed1990

Single crystals of a chimeric T7/T3 RNA polymerase with T3 promoter specificity and a nonprocessive T7 RNAP mutant.

Two RNA polymerases homologous to bacteriophage T7 RNA polymerase, bacteriophage Sp6 and T3 RNA polymerases, were screened for crystallization under conditions identical or similar to those reported for the growth of large single crystals of T7 RNA polymerase (Sousa, R., Rose, J. P., Chung, Y. J., Lafer, E. M., and Wang, B.-C. (1989) Proteins 5, 266; Sousa, R., Lafer, E. M., and Wang, B.-C. (1990) J. Crystal Growth, in press; Sousa, R., and Lafer, E. M. (1990) Methods 1, in press). A number of mutant T7 RNAPs were also screened under these conditions as were three chimeric RNA polymerases consisting of T7 RNAP N-terminal and T3 RNAP C-terminal sequences. One chimeric polymerase and two mutant polymerases crystallized readily under T7 RNAP crystallization conditions. The chimeric polymerase crystallized in a space group different from T7 RNA polymerase: orthorombic with unit cell parameters a = 75 A, b = 98 A, c = 159 A; space group P2(1)2(1)2(1) and 4 molecules/unit cell. This chimeric enzyme exhibits T3 promoter specificity and will make it possible to investigate how structural differences between the T3 and T7 RNA polymerase promoter recognition domains determine their different promoter specificities. One of the mutant polymerases successfully crystallized was an enzyme which can carry out promoter recognition and abortive transcription but cannot carry out processive transcription. Its structure may provide information on the nature of the conformational changes undergone by T7 RNAP in the abortive-processive switch. Crystals of the second mutant T7 RNA polymerase were unsuitable for x-ray analysis.

Chimera↗
PubMed1989

Characterization of a human-mouse chimeric antibody reactive with a human melanoma associated antigen.

The data presented demonstrate that a human-murine chimeric antibody has been generated that retains the immunoreactivity and has similar pharmacokinetic properties of its parent murine monoclonal antibody NRML-05. A competitive ELISA assay demonstrated that antigen reactivity of both parent and chimera were nearly identical. In a direct cell binding assay, NRML-05 and chimeric antibodies were immunoreactive, 81% and 83%, respectively. Scatchard Analysis of the antibodies indicate very similar affinities for NRML-05 (4.4 x 10(9) M-1) and chimera (2.7 x 10(9) M-1). Localization of the two antibodies to tumor xenografts in nude mice were very similar in biodistribution studies. The chimeric antibody is not as well recognized by antiglobulin from patients who have responded to non-idiotypic murine antibody determinants. Although this data does not predict how immunogenic a chimera would be in the clinical setting, it does suggest that patients without significant anti-idiotypic antiglobulin response could benefit from second and subsequent administrations with chimeric antibody. Even though anti-idiotypic responses may occur with the chimera, these may be reduced as a result of the presentation of these epitopes on the less immunogenic human constant domains.

Animals↗
PubMed1989

Secretion of a homodimeric V alpha C kappa T-cell receptor-immunoglobulin chimeric protein.

Transfection into lymphoid cells of a chimeric T-cell receptor-immunoglobulin gene has been used to generate a secreted water-soluble form of the variable (V) domain of a human T-cell receptor alpha chain for use in structural (i.e. x-ray crystallographic) studies. The chimeric protein consists of the V alpha region of the T-cell receptor of a diphtheria toxoid-specific human T-cell clone fused to a human immunoglobulin kappa light chain constant (C) region. It is efficiently secreted by myeloma cells as a noncovalent homodimer of 65-kDa molecular mass in the absence of either immunoglobulin heavy or light chain. The V alpha C kappa protein is extensively glycosylated, and its secretion is glycosylation-dependent. Chimeric genes containing the V beta region of this particular T-cell receptor linked to immunoglobulin C kappa or C gamma 2 regions are expressed intracellularly, but the products, although glycosylated, are not secreted, nor do they assemble with the V alpha C kappa protein. This suggests that the chimeric beta chain-immunoglobulin proteins are incorrectly folded and/or processed due either to the design of the gene fusions themselves or to the absence of vital T-cell-specific accessory molecules in the myeloma host.

Amino Acid Sequence↗
PubMed1989

Induction of immunological tolerance by chimeric thymus transplantation.

The role of bone marrow derived cells (BMD) within the thymus during the induction of immunological tolerance was investigated using transplantation of chimeric thymuses. Chimeric thymuses were constructed by reconstituting lethally irradiated BALB/C recipients with T-cell depleted C3H bone marrow (BM) cells. Two to three months post bone marrow transplantation the thymuses of these animals, in which the epithelium was of BALB/C origin and BMD cells of C3H origin (verified by immunoperoxidase staining), were transplanted into thymectomized, lethally irradiated BALB/C recipients which were reconstituted with T-cell depleted syngeneic (BALB/C) bone marrow cells. Induction of specific tolerance to the BMD cells (C3H origin) present in the chimeric thymus could be demonstrated in MLR at 3 to 6 months post chimeric thymus transplantation. It is concluded that bone marrow derived cells are able to induce immunological tolerance within the thymus.

Animals↗
PubMed1989

Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor.

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.

Amino Acid Sequence↗
PubMed1989

Chimeric protein: abrin B chain-trypsin inhibitor conjugate as a new antitumor agent.

Abrin B chain and trypsin inhibitor isolated from Acacia confusa (ACTI) were covalently linked to form a chimeric protein (ANB-ACTI) with N-succinimidyl-3-(-2-pyridyldithio)propionate. The chimeric protein had 31% of trypsin inhibitory activity of ACTI and 7% of hemagglutinating activity of abrin B chain, but no inhibition on protein biosynthesis. ANB-ACTI had strong inhibitory effects on the growth of sarcoma 180 cells and Hela cell culture while the mixture of an equivalent amount of free abrin B chain and ACTI did not. The results suggests that abrin B chain of chimeric protein may act as a vector to carry ACTI into the tumor cells. ACTI into the tumor cells. ACTI in the chimeric protein potentiates its antitumor activity as well as its resistance to tryptic digestion.

Abrin↗
PubMed1988

Incomplete chimerism in erythroid, myeloid and B lymphocyte lineage after T cell-depleted allogeneic bone marrow transplantation.

In 22 transplant patients who received T cell-depleted allogeneic bone marrow (alloBMT) we investigated the chimeric state using a mixed agglutination technique and isoenzyme determinations for the red cell lineage, an isoenzyme assay for the myeloid cells, immunoglobulin allotyping for B lymphocytes and cytogenetics for karyotyping. The median duration of follow-up for these patients was 25 months after BMT. Chimerism was evaluated after 6 months. In 14 (64%) of the 22 patients incomplete chimerism was found when the results of the different techniques were combined. Recipient type cells were detected in the red cell lineage in 36% of cases, recipient type myeloid cells in 7% of cases and recipient type mitotic cells in 25% of cases where the marker could provide discrimination. In 100% of the evaluable patients recipient type immunoglobulins persisted after BMT. The data indicate a high frequency of incomplete chimerism following T cell-depleted BMT which involved the broad series of hematological lineages. This frequency appears higher than that following non-T cell-depleted BMT.

Anemia, Aplastic↗
PubMed1988

Cloning and high level expression of a chimeric antibody with specificity for human carcinoembryonic antigen.

A mouse/human chimeric antibody has been constructed by using variable light and variable heavy regions from a murine hybridoma specific for human carcinoembryonic antigen (CEA) (CEM231.6.7). These V regions were combined with kappa and gamma-1 constant region genes cloned from human lymphocytes. The chimeric constructs were sequentially electroporated into murine non-Ig-producing myeloma (P3.653) and hybridoma (SP2/0) cell. Significant differences were seen in expression levels between the two cell types. High levels of expression (24 to 32 micrograms/ml/10(6) cells) were seen with several of the anti-CEA SP2/0 transfectomas but not with the P3.653 cells. The SP2/0 transfectoma lines were adapted to serum-free, chemically defined media and grown in large scale fermentation cultures where they continued to secrete high levels of antibody. The chimeric antibodies remain reactive against human CEA with affinity constants comparable to that of the parental hybridoma antibody. High level expression will make practical the production of chimeric antibodies for in vivo therapeutic and diagnostic purposes.

Amino Acid Sequence↗
PubMed1988

[Genotypic status of different tissues and organs in aggregated chimeras of BALB/c----C57BL/10 and BALB/c----B10.D2 with discovered chimeric drift].

Genotypic composition of different tissues and organs in BALB/s----C57BL/10 and BALB/c----B10.D2 mice in which chimeric drift was discovered in peripheral blood, erythrocyte population was studied. The proportion of cells of parental components was defined visually in coat, by electrophoresis of allozyme variants at the Gpi-1 locus in blood, spleen, brain, by differential C-staining of X and Y chromosome in cells of bone marrow and by mating of chimeras with BALB/c mice in gametes population. Chimeric drift in the erythrocyte population is not caused by advantage of BALB/c parental component over C57BL parental component in embryonal or postnatal development of whole chimeric organism, but result from selective advantage of hemopoietic BALB/c cells over C57BL cells in chimeric hemopoietic organs.

Animals↗
PubMed1987

Biological properties of chimeric antibodies. Interaction with complement.

Genetic engineering allows the production of chimeric immunoglobulins where the same variable region is expressed in conjunction with constant regions of different species. We have compared the capacity of mouse and chimeric (mouse variable region/human constant region) immunoglobulin M (IgM) to trigger complement-dependent lysis and to bind complement component C1. Guinea pig and human C1 were bound more efficiently by mouse IgM than by chimeric IgM, whereas rat and rabbit C1 were bound more efficiently by chimeric IgM. Comparable results were obtained for complement as measured by the lysis of sensitized erythrocytes. These results indicated that differences between the human and mouse mu heavy chain constant regions define structures that are important in the C1-IgM interaction. Furthermore, species-specific differences in C1 also influence this interaction.

Animals↗