Evaluation of the in vivo biodistribution of yttrium-labeled isomers of CHX-DTPA-conjugated monoclonal antibodies

We evaluated the in vivo stability and biodistribution of four isomers (CHX-A', CHA-A", CHX-B' and CHX-B") of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriaminepentaaceti c acid (CHX-DTPA), a recently developed backbone-substituted derivative of DTPA. METHODS: The ligands were conjugated to monoclonal antibody B3, a murine IgG1 kappa, and labeled with 88Y at 55.5-66.6 MBq/mg (1.5-1.8 mCi/mg). Nontumor-bearing nude mice were injected intravenously with 55.5-66.6 kBq (1.5-1.8 microCi) of 88Y-labeled B3 conjugates and with 125I-labeled B3 as an internal control. The mice were then killed at 6, 24, 48, 96 and 168 hr postinjection. RESULTS: At 168 hr, the concentration of 88Y in processed bone of either CHX-A' [4.6% injected dose (ID)/g] or CHX-A" (4.0%ID/g) was less than that of either the CHX-B' (21.9%ID/g) or B" (12.1%ID/g) ligands. The two ligands CHX-B" and CHX-B' were not acceptable for yttrium labeling of antibody because of their high and progressive bone accumulation. The accumulation of 88Y in bone of CHX-B' was five times greater than that of CHX-A' at 168 hr. The CHX-A" cleared from the circulation slightly faster than CHX-A' without releasing the yttrium and showed the lowest uptake by bone of any of the four isomers. The accumulation in the other normal organs was similar for all four isomers of 88Y-CHX-B3 conjugates. CONCLUSION: Although the CHX-B" and CHX-B' were not acceptable for labeling with yttrium, the CHX-A' and CHX-A" were suitable, indicating that differences in stereochemistry can greatly influence stability of radionuclide in the chelate.     

Radiolabelled monoclonal antibodies (McAb): an alternate approach to the conventional methods for the assessment of cardiomyocyte damage in an experimental brain-death pig model

BACKGROUND: According to the Ministry of Health and Welfare AIDS Surveillance Committee's report on vertically transmitted human immunodeficiency virus (HIV) infection, there have been eight children with acquired immune deficiency syndrome (AIDS) and 18 children with HIV infection in Japan, totalling 26 in all as of February 1997. A search of the literature fails to reveal any report that deals with many cases of vertically transmitted HIV infection in Japan. METHODS: A primary questionnaire survey was taken of the main medical institutions across the country, followed by a secondary questionnaire survey of physicians and pediatricians who treated the disease. A clinical review was made of 19 children with vertically transmitted HIV infection (including eight AIDS children) according to the 1994 Revised Classification System for HIV Infection in Children. RESULTS: The mean age at diagnosis was 14.5 months and the diagnosis was made at less than 18 months of life in approximately 70% of infected children. In the mean observation period of 16 months, six of eight AIDS children (75%), and one child of group B died. The mean period of observation for the seven dead children was 7 months, and six of seven children died by 36 months of life. The survival period after the diagnosis of AIDS was 15 months. The diagnosis of HIV infection was made based on the clinical symptoms of all children with AIDS. Of 11 children, six (45%) presented with symptoms of HIV infection by 6 months of life, and 10 of 11 children (91%) presented with symptoms by 26 months of life. The noteworthy clinical findings included hepatomegaly, splenomegaly, recurrent respiratory tract infection, lymph node swelling, oral candidiasis, hepatitis, wasting syndrome, HIV encephalopathy and severe pneumonia. The favored age for the start of complications and the magnitude of decrease in the HIV helper cell count varied with each case of complications of HIV infection (wasting syndrome, HIV encephalopathy) or opportunistic infections (cytomegalovirus infection, Mycobacterium avium complex infection). Anti-HIV drugs (mainly zidovudine) had been used in five of eight children with AIDS and were effective in two long survivors alone. CONCLUSIONS: Children who are diagnosed with HIV infection, based on their clinical symptoms, carry a poor prognosis. In this respect, early diagnosis and progress in anti-HIV therapy are necessary.     

Characterization of murine monoclonal anti-endothelial cell antibodies (AECA) produced by idiotypic manipulation with human AECA

The IgG fraction of human anti-endothelial cell antibodies (AECA) obtained from a patient with Wegener's granulomatosis was used as immunogen to raise AECA mAb in mice selected among those which developed vasculitis-like lesions after immunization. Three mAb (BGM, 3C8 and 7G2), selected by cyto-ELISA and flow cytometry analyses, featured a specific reactivity with human umbilical vein endothelial cells (HUVEC) and the mouse endothelial cell line H5V; on the contrary, HEp2 cells, the murine melanoma B16 cell line, the extracellular matrix as well as several other antigens tested were not recognized. BGM mAb, an IgG3 precipitating a 70 kDa structure from HUVEC, was able to induce endothelial cells to secrete amounts of IL-6 significantly higher than irrelevant controls or mAb binding different endothelial antigens (i.e. CD31, CD29, ICAM-1 and HLA class I). BGM mAb induced significant levels of antibody-dependent cell cytotoxicity (13 +/- 2.5 versus 0.6 +/- 0.03%). To the best of our knowledge, BGM is the first murine mAb specific for human endothelial cells generated by idiotypic manipulation; secondly, its biological properties further support the notion of a pathogenic role for AECA in autoimmune-mediated diseases.     

Analysis of the roles of antilipopolysaccharide and anti-cholera toxin immunoglobulin A (IgA) antibodies in protection against Vibrio cholerae and cholera toxin by use of monoclonal IgA antibodies in vivo

Secretory immunoglobulin A (IgA) antibodies (sIgA) directed against cholera toxin (CT) and surface components of Vibrio cholerae are associated with protection against cholera, but the relative importance of specific sIgAs in protection is unknown. A monoclonal IgA directed against the V. cholerae lipopolysaccharide (LPS), secreted into the intestines of neonatal mice bearing hybridoma tumors, was previously shown to provide protection against a lethal oral dose of 10(7) V. cholerae cells. We show here that a single oral dose of 5 to 50 micrograms of the monoclonal anti-LPS IgA, given within 2 h before V. cholerae challenge, protected neonatal mice against challenge. In contrast, an oral dose of 80 micrograms of monoclonal IgA directed against CT B subunit (CTB) failed to protect against V. cholerae challenge. A total of 80 micrograms of monoclonal anti-CTB IgA given orally protected neonatal mice from a lethal (5-micrograms) oral dose of CT. Secretion of the same anti-CTB IgA antibodies into the intestines of mice bearing IgA hybridoma backpack tumors, however, failed to protect against lethal oral doses of either CT (5 micrograms) or V. cholerae (10(7) cells). Furthermore, monoclonal anti-CTB IgA, either delivered orally or secreted onto mucosal surfaces in mice bearing hybridoma tumors, did not significantly enhance protection over that provided by oral anti-LPS IgA alone. These results demonstrate that anti-LPS sIgA is much more effective than anti-CT IgA in prevention of V. cholerae-induced diarrheal disease.     

Poly(anhydride-co-imides): in vivo biocompatibility in a rat model

The degradation and tissue compatibility characteristics of a novel class of biodegradable poly(anhydride-co-imide) polymers: poly[trimellitylimidoglycine-co-1,6-bis(carboxyphenoxy)hexan e] (TMA-gly: CPH) (in 10:90; 30:70 and 50: 50 molar ratios) and poly[pyromellitylimidoalanine-co-1,6-bis(carboxyphenoxy)hexa ne] (PMA-ala:CPH) (in 10:90 and 30:70 molar ratios) were investigated and compared with control poly(lactic acid/glycolic acid) (PLAGA in 50:50 molar ratio) matrices, a well-characterized biocompatible polymer, in rat subcutaneous tissues for 60 days. Polymers were compression-molded into circular discs of 14 mm x 1 mm in diameter. On post-operative days 7, 14, 28 and 60, histological tissue samples were removed, prepared by fixation and staining, and analyzed by light microscopy. PLAGA matrices produced mild inflammatory reactions and were completely degraded at the end of 60 days, leaving implant tissues that were similar to surgical wounds without implants. TMA-gly:CPH (10:90 and 30:70) matrices produced mild inflammatory reactions by the end of 60 days, similar to those seen with PLAGA. TMA-gly: CPH (50: 50) produced moderate inflammatory reactions characterized by macrophages and edema. PMA-ala:CPH matrices elicited minimal inflammatory reactions that were characterized by fibrous encapsulation by the end of 60 days. In vivo degradation rates of poly(anhydride-co-imides) were similar to PLAGA. Both PMA-ala:CPH and TMA-gly: CPH matrices maintained their shapes and degraded at a constant rate over the period of two months. These polymers, possessing good mechanical properties and tissue compatibility, may be useful in weight-bearing applications in bone.     

Human recombinant anti-Rh(D) monoclonal antibodies: improvement of biological properties by in vitro class-switch

The aim of this study was to investigate how intrauterine growth retardation affects body proportions in VLBW infants. The cohort consisted of 135 surviving and 80 deceased preterm infants weighing less than 1250 grams at birth. Gestational age varied between 24 and 36 weeks (mean age 29.7 and 27.5 weeks, respectively). Birth weight was more than 2 SD below the mean birth standard values in 32% of the surviving, and in 27% of the deceased infants. Reduction of weight, length and head circumference at birth was analysed using Z scores based on Swedish birth standards. Z scores of weight, length and head circumference were highly correlated in the surviving and the deceased infants (r = 0.78 to 0.94 and 0.65 to 0.97, respectively). Length was significantly more affected by growth retardation than weight. Weight and head circumference were proportionately reduced. Intrauterine growth retardation influences body proportions in VLBW infants differently than in larger preterm and term infants.     

In vivo and in vitro production of monoclonal antibodies: current possibilities and future perspectives. Discussion

It is well known that various perceptual abnormalities exist in autism. However, because perceptual phenomena are intersubjective, a phenomenological approach is required for getting hold of the reality of the modes of perception involved in autism. From this standpoint, the author has proposed the concept of 'perception metamorphosis phenomenon' (PMP) as the mode of perception peculiar to autistics. This mode of perception is notable to some degree in infancy and adolescence, and points to the appearance of behavior that is indicative of the environmental world being perceived in a manner different from before by the autistic child. The phenomenon has been classified into three basic categories according to the aspect of perception: (i) visual PMP; (ii) auditory PMP; and (iii) situational PMP. The proposal of this concept was made with the objective of capturing the onset of autism or the mechanism of appearance of the various symptoms from a more phenomenological viewpoint, to serve as a possible starting point for understanding the inner world of autistics. The proposal was made emphasizing the validity of this approach in mapping out new therapeutic approaches and for re-investigating the relationship between autism and schizophrenia.     

Two chicken B cell lines resistant to ouabain for the production of chicken monoclonal antibodies

Two chicken B cell lines, termed MuH1 and MuH4, which are resistant to ouabain, were established as fusion partners for production of chicken monoclonal antibody from the parental cell lines R27H1 and R27H4 which were deficient in thymidine kinase activity. The MuH1 synthesized, but did not secrete mu chain, whereas the MuH4 secreted non-specific IgM. Fusions of the MuH1 and MuH4 with spleen cells from chickens immunized with human IgG resulted in successful preparations of hybridomas secreting antibodies specific to human IgG. Transformed cells insensitive to HAT appeared in many culture wells after cell hybridizations with R27H4, but on hybridization with MuH1 or MuH4 the appearance of such unfavorable cells could be avoided by the inclusion of ouabain in HAT medium. Thus, in comparison with the R27H4 line, the MuH1 and MuH4 lines did not provide higher fusion efficiencies, but gave increased opportunities to obtain hybridomas producing specific antibodies. Among a total of eight hybridomas, four derived from HuM1 and four derived from MuH4, seven secreted both IgG and IgM, and the remaining one derived from MuH1 secreted only IgG. In all eight hybridoma lines IgG was specifically reactive to human IgG.     

In vivo observations and electron microscopy in the treatment of experimental HSV keratitis with monoclonal antibodies

In vivo observations and electron microscopy showed that topical use of anti-HSV monoclonal glycoprotein antibodies produced marked antiviral effects in inhibiting the development of experimental herpetic keratitis in rabbits and in protecting the susceptible corneal cells. As a new biological product, the anti-HSV monoclonal antibodies may provide a new approach to the treatment of HSV keratitis.     

Analysis of the bovine respiratory syncytial virus fusion protein (F) using monoclonal antibodies

Seven monoclonal antibodies (MAbs) directed against bovine respiratory syncytial virus (BRSV) fusion (F) protein were produced and characterized by radioimmunoprecipitation and immunofluorescence assays. These seven MAbs together with the previously described MAbs (Beeler and Van Wyke Coelingh, 1989) to the F protein of human respiratory syncytial virus (HRSV) were used to study the antigenic variation of 12 strains of ungulate RSV. All except one MAbs specific for the HRSV-F protein reacted with ungulate RSV strains less efficiently, indicating that some epitopes are conserved, and others are not conserved on the F proteins of HRSV and BRSV strains. Three MAbs specific to the BRSV-F protein neutralized virus infectivity and reacted with all the ungulate RSV strains, suggesting that these epitopes are well conserved. Based on the reactivity of three other MAbs specific to the BRSV-F protein, ungulate RSVs could be grouped into two subgroups. The results indicated that there are antigenic variations in the F protein among ungulate RSV strains.     

Further analyses of epitopes for human monoclonal anti-basement membrane zone antibodies produced by stable human hybridoma cell lines constructed with Epstein-Barr virus transformants

We previously established Epstein-Barr virus (EBV)-transformed bullous pemphigoid (BP) patient lymphoblastoid cell lines, which produced human monoclonal anti-basement membrane zone antibodies. In the present study, we established two independent human-human hybridomas by fusion of these EBV transformants with a human B-cell line. These hybridomas, designated 5E-HY-4B and 10D-HY-8B, were very stable and showed a high yield of monoclonal antibody (MoAb) secretion. Each cell line was tetraploid and showed combined rearranged segments of immunoglobulin heavy-chain gene derived from both an EBV transformant and a parent cell. Immunoblot analysis showed that the 5E-HY-4B MoAb recognized the 230-kDa BP antigen but that the 10D-HY-8B MoAb did not show any reactivity. In contrast, both MoAbs precipitated the 230-kDa BP antigen with immunoprecipitation. These results indicate that the two MoAbs reacted with different epitopes on the 230-kDa BP antigen: a continuous epitope for the 5E-HY-4B MoAb and a conformation-dependent epitope for the 10D-HY-8B MoAb. This speculation was confirmed at the molecular level by the result that the fusion protein produced by a partial cDNA for the 230-kDa mouse BP antigen reacted with the 5E-HY-4B MoAb but not with the 10D-HY-8B MoAb. Furthermore, the study of the reactivity with fusion proteins of a series of deleted clones restricted the epitope for the 5E-HY-4B MoAb within the region with 114 amino acid residues in the C-terminal domain of the 230-kDa BP antigen.     

Characterization in vitro and in vivo of the pig analogue of human CD59 using new monoclonal antibodies

CD59 is the sole characterized regulator of the complement membrane attack complex in humans. It is very widely and abundantly distributed, being present on all circulating cells, endothelia and epithelia, and in most tissues. CD59 analogues in rodents are distributed similarly. Interest in complement regulation in the pig has developed out of the current enthusiasm to exploit this species as a donor in xenotransplantation of organs to humans. We have recently isolated and cloned the pig analogue of human CD59. We here report the development and characterization of monoclonal antibodies against pig CD59. We have used these antibodies to develop efficient methods for the purification of pig CD59 to homogeneity from erythrocyte membranes and have obtained new information on the structure and function of the purified protein. The antibodies were found to function well in immunohistochemistry and have been used to perform a comprehensive survey of the expression and distribution of pig CD59 on cells and in organs of normal pigs. Pig CD59, like human CD59, is broadly expressed but there are some striking differences in tissue distribution, notably the apparent lack of pig CD59 on circulating platelets and on a subset of leucocytes in blood and lymphoid organs. The reported findings have important implications for the current approaches to avoiding complement-mediated hyperacute rejection in pig-to-human xenografts.     

PTHrP and cell division: expression and localization of PTHrP in a keratinocyte cell line (HaCaT) during the cell cycle

Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were actively dividing. Taken together, these results support a role for PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.     

Clinical comparison of radiolocalization of two monoclonal antibodies (mAbs) against the TAG-72 antigen

Ten patients with colorectal cancer metastases received 125I-B72.3 and 131I-CC49 prior to laparotomy (five patients received 1 mg, and five 20 mg of each mAb). Tumor:serum ratios of 131I-CC49 were better than those of 125I-B72.3 (P < 0.01 at 1 mg; P = 0.05 at 20 mg; P < 0.01 at both doses). All known lesions > or = 1 cm in diameter were visualized at the 20 mg dose. There was no difference in absolute tumor uptake of 125I-B72.3 or 131I-CC49. We conclude that mAb CC49 has better relative uptake in colorectal cancers than mAb B72.3.