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A human monoclonal anti-hepatitis B antibody preparation (TUVIRUMAB) was administered 6 times over a 2-week period in a dose-escalating scheme to chronic hepatitis B patients pre-treated with lamivudine. The capacity of the TUVIRUMAB antibody to "neutralize" hepatitis B surface antigen in the circulation was investigated by means of experimental enzyme-immunoassays. Monoclonal antibody conjugates enabled the detection of HBsAg, TUVIRUMAB, and HBsAg/TUVIRUMAB complexes. The results showed that (1) TUVIRUMAB was able partially to "neutralize" in vitro and in vivo, (2) HBsAg/TUVIRUMAB complexes can be traced by assays that capture the complex at either its HBsAg or its TUVIRUMAB component, (3) the final concentration of TUVIRUMAB at the end of therapy varied greatly but seemed to be related to HBsAg production at the start of therapy, (4) for at least 14 days after discontinuation of therapy, a minimal HBsAg level could be maintained in the presence of a declining TUVIRUMAB titer in patients with less than 3 microg/ml HBsAg before the start of therapy, (5) three months after therapy, all HBsAg levels had returned to pre-treatment levels and TUVIRUMAB had disappeared.

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The feasibility to raise nonhuman primate antibodies against selected components of the human immune system was tested. The immunogens were whole cells (human T lymphocytes) or purified, recombinant human proteins (cytokines: TNF alpha or GM-CSF; soluble forms of cell surface antigens: sCD4 or sCD25). Significant immunizations, yielding functionally relevant antibodies, were readily achieved in rhesus monkeys, but, not surprisingly, may be less frequent in chimpanzees. The results suggest a general strategy for production of therapeutically useful MAB.

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The protective efficacy of a human monoclonal antibody directed against the a determinant of hepatitis B virus (HBV) surface antigen was studied in a chimpanzee. A single high dose of 5 mg/kg (body weight) of monoclonal antibody SDZ OST 577 was intravenously administered to a chimpanzee, followed by intravenous challenge with 10(3.5) chimpanzee infectious doses of a wild-type HBV, the MS-2 strain (ayw subtype). The passively acquired antibody to HBV surface antigen could be detected for 40 weeks. Serum HBV DNA tested by a "nested" polymerase chain reaction assay was negative through the 36th week after virus challenge but became positive by the 38th week. The chimpanzee subsequently developed acute hepatitis B approximately 1 year after challenge. The nucleotide sequence of the a determinant of the surface gene of the replicated virus was identical with that of the inoculated wild-type virus. Thus, a human monoclonal antibody directed against the a determinant of HBV surface antigen delayed but did not prevent experimental infection of HBV and hepatitis in the chimpanzee. Our results indicate an incomplete ability of this antibody to protect against HBV infection in vivo after a single infusion.

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Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.

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Antigenic domain 1 (AD-1) on glycoprotein gp58 of human cytomegalovirus was characterized in detail, using mouse and human monoclonal antibodies as well as human convalescent sera. Series of procaryotically expressed fusion proteins and synthetic peptides of various lengths were used as sources of antigen. Binding of antibodies was found to depend on a continuous sequence of more than 70 amino acids between residues 552 and 635 of gp58. The fine specificities for sequences involved in antibody binding were (i) amino acids 557 to 635 for neutralizing as well as nonneutralizing mouse monoclonal antibodies, (ii) amino acids 552 to 630 for a neutralizing human monoclonal antibody, and (iii) amino acids 557 to 630 for antibodies present in human sera. Experiments involving fragments of AD-1, presented either as procaryotically expressed fusion protein or as synthetic peptides, indicated that the intact structure was required for recognition of AD-1 by antibodies.

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A gene region encoding a segment of the major surface protein, HBsAg, of hepatitis B virus was analyzed from serum samples after orthotopic liver transplantation of three hepatitis B virus chronic carrier patients treated with a human anti-hepatitis B virus monoclonal antibody (SDZ OST 577). Each of these three patients became HBsAg negative after transplantation and therapy with the human anti-hepatitis B virus monoclonal antibody but returned to HBsAg positivity (first detected 143,251 and 252 days after the transplantation). Polymerase chain reaction DNA amplification was performed on DNA from serum samples showing low levels of recurrent HBsAg and reduced antigen reactivity with SDZ OST 577 antibody. Polymerase chain reaction DNA included a 230-bp highly conserved, major S gene region that was cloned into M13 bacteriophage; analysis of this DNA segment provided a consensus of DNA sequences for the serum samples exhibiting altered reactivity with the therapeutic monoclonal. Analysis of independent DNA clones from serum samples of patients exhibiting low but detectable recurrent serum levels of posttherapy HBsAg revealed the presence of S protein variant sequences when compared with polymerase chain reaction DNA derived from the original infected liver or pretherapy serum HBsAg. Genetic variation was predominant in a highly conserved peptide domain that has previously been implicated in antibody binding and neutralizing antibody epitopes. In independent patients infected with either adw or ayw hepatitis B virus subtypes, single nucleotide changes resulted in one to two amino acid differences for each variant allele (residues 124, 129, 131, 137, 140 and/or 145) when compared with pretherapy viral DNA. Administration of serum containing one of these variant viruses to a single hepatitis B-naive chimpanzee resulted in subclinical hepatitis and detectable levels of circulating anti-HBs and anti-HBc antibodies 49 and 70 days after virus administration, respectively. Hepatitis B virus DNA was recovered on liver biopsy between 6 and 8 wk after inoculation, although the animal remained persistently seronegative for HBsAg. DNA sequence analysis of both primate and patient liver hepatitis B virus confirmed the presence of the DNA encoding the S protein variant and associates this DNA with the predominant hepatotropic virus in liver infection.

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Four mouse monoclonal anti-idiotypic antibodies (anti-Id) were generated against human monoclonal and polyclonal antibodies to hepatitis B surface antigen (HBsAg). These monoclonal anti-Id, along with a polyclonal anti-Id raised in rabbits, were used to characterize the idiotype (Id) specificity of the human antibody response to HBsAg (anti-HBs). The anti-Id reagents identified distinct private and cross-reactive Id expressed on monoclonal and polyclonal human anti-HBs preparations respectively. The anti-Id recognized both HBsAg combining site and non-combining site related private Id, and HBsAg combining site related cross-reactive Id. The Id specificities recognized by two of the monoclonal anti-Id were associated with the H chain alone, whereas two of the monoclonal anti-Id, along with the polyclonal anti-Id appeared to recognize Id determinants associated with both isolated H and L chains. These data suggest that Id heterogeneity exists within the human antibody response to HBsAg. The knowledge that Id heterogeneity exists is of importance in understanding the observed variability in the immune response during hepatitis B virus infection.

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Four hybridoma cell lines stably secreting human monoclonal antibodies directed against hepatitis B surface antigen have been isolated. The monoclonal antibodies have been characterized by determining several allotypes, measuring affinity for HBsAg, binding to two HBsAg subtypes, and kinetics of antibody binding to solid adsorbed antigen. In addition, the relative position of the epitopes have been determined by competition in binding to HBsAg. The results indicate that human anti-HBsAg monoclonal antibodies of quite different properties can be isolated. Pharmacokinetics of one antibody have been measured in two rhesus monkeys. This monoclonal antibody, OST 577, has been compared to hyperimmune gamma globulins in a Bureau of Biologics approved radioimmunoassay. It is expected that these antibodies will be useful in preventing and treating hepatitis B.

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The safety and pharmacokinetics of the two neutralizing human IgG1 monoclonal antibodies to cytomegalovirus (CMV) SDZ 89-104 and 89-109 in bone marrow transplant (BMT) recipients was assessed in an open phase I trial. Thirteen patients, 8 seropositive and 5 seronegative for CMV, were treated with allogeneic or autologous bone marrow transplantation. SDZ 89-104 was given to 5 and SDZ 89-109 to 8 patients. Patients were divided into high- and low-dose groups. A fixed prestudy dose of 0.1 mg/kg was given 4 days before BMT. On days 3, 17, 31, 45, 59, and 73, patients were treated with either 0.5 or 2 mg/kg of the respective antibody. Results indicate that doses of 2 mg/kg of SDZ 89-104 or SDZ 89-109 in alternating weeks can be safely administered to BMT patients. Serum trough levels measured by antiidiotype ELISA were approximately 10 micrograms/ml after administration of 0.5 mg/kg and approximately 50 micrograms/ml after treatment with 2 mg/kg of SDZ 89-104 or SDZ 89-109. High serum levels defined by antiidiotype ELISA techniques closely paralleled increased neutralizing activity. Serum half-lives calculated from these data were approximately 6 days.

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This study examined the safety and pharmacokinetic profile of a potentially therapeutic and fully human anti-CMV monoclonal antibody (SDZ MSL-109) in a phase I dose escalation trial in patients receiving allogeneic bone marrow transplants. Fifteen adult marrow transplant patients, twelve with chronic myelogenous leukemia and three with acute nonlymphocytic leukemia, in cohorts of five patients each, were administered monoclonal antibody intravenously at doses of 50, 250, and 500 micrograms/kg at approximately three-week intervals for six months. Administration of the monoclonal antibody was associated with minimal side effects and no dose-related toxicity. Antibody elimination curves in all dose groups were consistent with a two-compartment model with an alpha half-life at the low, middle, and high dose groups of 1.03, 0.82, and 0.79 days, and a beta half life of 13.9, 14.0, and 16.5 days, respectively. The volume of distribution decreased with repetitive dosing to approximate the plasma volume in each patient and the pharmacokinetic profile was comparable to that of human IgG. There was no host antiidiotypic or antiallotypic antibody formation, indicating that MSL-109 was not immunogenic. Further studies are warranted to assess the potential efficacy of human monoclonal anti-CMV disease in marrow transplant recipients and other patients with immunodeficiency disorders.

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The nucleotide sequence of the Fc and hinge regions of a chimpanzee monoclonal antibody has been determined. Most of the sequence is similar to the human IgG1 sequence. However, the chimpanzee hinge regions differs from the human hinge region in six of 48 nucleotides, which leads to three amino acid substitutions. Two of the amino acid changes are not conservative and may lead to differences in flexibility of the hinge. The chimp hinge sequence seems to be a combination of the human IgG1 hinge and the hinge sequence of a human IgG pseudogene. The implications of this difference for the evolution of human IgG subgroup is discussed. Despite differences in the hinge regions, the chimpanzee monoclonal antibody differs from the most closely related human IgG1 allotype only slightly more than the two most distantly related human allotypes differ from each other.

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The nucleotide sequence of the mRNA that codes for Fab fragments from two chimpanzee monoclonal antibodies has been determined. Both antibodies have high affinity and good specificity for digoxin. Four domains from the two antibodies have been sequenced: the constant domains of the kappa and lambda light chains, the variable domain of the lambda light chain, and the CH1 domain of the IgG1 heavy chain. There are very few differences between the chimpanzee and human sequences; the nucleotide sequences differ by less than 2%.

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Human monoclonal antibodies, owing to their decreased immunogenicity, are expected to be an improvement over mouse monoclonal antibodies for in vivo therapy. Human and primate monoclonal antibodies are best produced with a human x mouse heteromyeloma. Several human chromosomes are stable in the human x (human x mouse) hybrids. Chimpanzee anti-digoxin monoclonal antibodies were prepared and characterized. Because they are structurally very similar to human antibodies, they should be well tolerated in humans. The anti-digoxin antibodies can be used for therapy of extreme overdoses or as an in vivo diagnostic tool for slight overdoses. Because the advantage of using human monoclonal antibodies is their lack of immunogenicity, preparation of the antibody must be scrupulous so as not to introduce extraneous immunogens. Analysis to ensure the purity of the preparation can be complicated by the presence of high concentrations of the antibody and the low levels of contamination that must be detected. We describe a Western blot assay for Protein A that is sensitive even in the presence of human IgG.

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We have shown previously that human monoclonal antibodies are not very immunogenic in rhesus monkeys, with only one monkey out of five mounting an anti-monoclonal antibody response. Two additional monkeys have been injected multiple times with much larger amounts of one human monoclonal antibody. No anti-antibody response has been detected in these monkeys. Structural changes that occur in the monoclonal antibodies over time in vivo have been investigated by Western Blots using anti-idiotypic antisera. Sodium dodecyl sulfate gel electrophoresis reveals that very little antibody has altered molecular weight. Isoelectric focusing patterns change more dramatically. Forms of the antibodies with lower isoelectric points appear in the serum. These forms have a similar in vivo half-life as the freshly prepared antibody. Very low pI forms of the monoclonal antibodies are not detected in the serum. Isoelectric focusing can be used to determine the in vivo or in vitro condition of a monoclonal antibody preparation. Finally, the monkey anti-human IgG that arose in the single monkey studied previously has been further characterized.

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The complete amino acid and nucleotide sequences of the variable regions of the heavy and light polypeptide chains of a human neutralizing IgGl anti-cytomegalovirus (CMV) antibody reveal a striking homology to IgM rheumatoid factors (RFs) of the Wa idiotypic family. The anti-CMV antibody and Wa RFs have in common VKIIIb, JKl, and VHIa gene segments but use different DH and JH gene segments. The anti-CMV antibody does not have RF activity and does not express the Wa idiotype. The Wa RFs do not have anti-CMV activity. A subset of Wa RFs, however, and the anti-CMV antibody do share several idiotypes on the VHIa and VKIIIb polypeptides. Since there are major differences in the antigen binding characteristics and some of the other expressed idiotypes, these data suggest that the D and J region amino acids are crucial to such specificities. Although the use of such highly homologous gene segments in different immune responses is well-documented in murine systems, these data represent the first such example in the human.

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Five rhesus monkeys were injected multiple times over several months with two different human IgG1 monoclonal antibodies, both directed against human cytomegalovirus. Three monkeys each injected four times with monoclonal antibody EV2-7 for over 200 days showed no response other than a normal decay in antibody level. The in vivo half life of this antibody was substantially longer when measured with an idiotype-specific two site immunoassay than with radiolabeled antibody, indicating that the iodination procedure greatly affected the stability of the antibody. Although there was considerable individual variation in the half-life of EV2-7, from 8.9 to 30.5 days, the half-life was fairly long, especially considering the size of the monkeys. Two monkeys were injected with monoclonal antibody EV1-15. One monkey has responded in a similar manner to the EV2-7-injected monkeys. However, the other monkey has produced an anti-idiotypic antibody (or antibodies) of high affinity. It is possible that this response was triggered by the unusual physical nature of antibody EV1-15 or the effect of the species difference between human and rhesus monkey. In any case, the results from these five monkeys indicate that human monoclonal antibodies should have a significant advantage over mouse monoclonal antibodies for in vivo therapeutic applications.

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Animal cell technology is attracting considerable interest because of the capacity of animal cell cultures to synthesize or transform complex compounds such as virus vaccines, immunochemicals, hormones or enzymes. For the growth of surface-dependent cells, microcarrier technology is gaining importance. Here, we have attempted to immobilize surface-independent cells, normally grown in suspension, by entrapping them in polymer microbeads. Such entrapment should give increased stability to the normally fragile animal cells, allow for high cell densities to be achieved within the beads and make such preparations suitable for continuous operation. At the same time, the need for separation of the desired product from the cells is obviated. With the model systems studied, we showed that hybridoma, as well as other cell lines entrapped in agarose microbeads, remained viable. Both immunoglobulins and lymphokines were exported through the microbeads into the medium for 1-3 weeks, at levels corresponding well to those produced with free cells.

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