Co-secretion of two distinct kappa light chains by the mu-9 hybridoma.

Mu-9 is a monoclonal antibody (MAb) specific for the CSAp antigen (Ag) expressed by colorectal cancers. By using variable (V)-region-specific primers, the respective VH and VL sequences of Mu-9 were polymerase chain reaction (PCR)-amplified. However, chimeric Ab (cMu-9-1) constructed from these PCR-amplified V sequences failed to bind the CSAp Ag. Although the light chain of murine Mu-9 was not glycosylated, that of cMu-9-1 was found to be O-glycosylated, as confirmed by reducing SDS-PAGE analyses, glycoprotein blotting and O-linked specific deglycosylation studies. Removal of O-linked oligosaccharides either by enzymatic digestion or by blocking O-glycosylation with a specific inhibitor did not restore the immunoreactivity of cMu-9-1, indicating that light chain O-glycosylation was not the cause for lack of immunoreactivity. We reported earlier that screening of a Mu-9 cDNA library uncovered the presence of an additional light chain sequence that was later proven to be the authentic light chain of Mu-9. Analyses of the cDNA sequence encoding the nonimmunoreactive light chain, however, revealed no defects that would preclude the sequence from being translated and secreted by the murine hybridoma. By adapting the Mu-9 hybridoma culture to serum-free conditions, we confirmed the secretion of low levels of O-glycosylated light chain. The biological significance of the O-glycosylation as well as the cosecretion of both light chains with respect to allelic exclusion are discussed.

Humanization of Immu31, an alpha-fetoprotein-specific antibody.

Immu31 is a murine monoclonal antibody (Ab) specific for alpha-fetoprotein (AFP), a tumor-associated marker. The excellent tumor targeting ability of Immu31 has led to the development of a Immu31-based radioimmunodiagnostic agent, AFP-Scan, for hepatocellular carcinoma and other AFP-producing tumors. To enhance the capability of Immu31-based immunoconjugates being used in diagnostic and therapeutic procedures in humans, a humanized version of Immu31 (hImmu31) was constructed by grafting the complementarity determining regions (CDRs) of murine variable domains for the heavy (VH) and kappa (Vkappa) chain to the respective human VH and Vkappa framework regions (FRs). The cDNA encoding the VH and Vkappa of Immu31 was cloned by reverse transcription-PCR from hybridoma cells, and a chimeric Immu31 (cImmu31) composed of murine V and human C domains was constructed. Competitive ELISA assays showed identical AFP binding activity between the chimeric and murine Abs, confirming the authenticity of the cloned V genes. Based on sequence homology, the EU FR1, FR2, and FR3 and the NEWM FR4 were selected as the scaffold for grafting VH CDRs and REI FRs for Vkappa CDRs of Immu31. The amino acid residues in murine FRs that are considered to be in contact with the CDRs of the Ab were maintained in the humanized version. hImmu31, thus constructed and expressed, showed comparable immunoreactivity in a competitive binding ELISA assay to that of murine Immu31 and cImmu31. High-level production was achieved by expressing hImmu31 in a dhfr-based amplifiable system, and the productivity has exceeded 100 mg/liter in terminal cultures.

Generation and monitoring of cell lines producing humanized antibodies.

Antibody humanization has eliminated or reduced the human antimouse antibody response associated with the administration of murine antibodies. We have successfully humanized three different antibodies: (a) hMN-3 (granulocyte targeting); (b) hMu-9 (colorectal cancer targeting); and (c) hWI2 (anti-idiotype to the anti-carcinoembryonic antigen antibody MN-14). All humanized antibodies demonstrated immunoreactivities comparable to their parent counterparts. Previously, we reported the generation of high productivity cell lines for hMN-14 and hLL2 using the amplifiable vector pdHL2. Through amplification, selection, and cloning procedures, cell lines capable of large scale production were established, and further enhancement of production was achieved by a fed-perfusion bioreactor process. Using a similar and improved approach, we have enhanced the production of the above-mentioned humanized antibodies by gene amplification induced by a stepwise increase in the concentration of methotrexate in the culture media. A reliable IgG determination method is essential to monitor amplification, especially at the final cloning stage, for the selection of the subclones with the highest productivity. We found that measurement of humanized IgG concentration in culture media supplemented with more than 1 microM methotrexate by a standard ELISA assay could be unreliable and misleading. Whereas the determination of antibody by adsorption/elution on protein A from a 100-ml culture is accurate and reproducible, the method is time-consuming, tedious, and labor intensive. We have recently developed a Western blot assay that enables us to monitor the productivity of the cultures. The assay is simple and sensitive, and it makes simultaneous determinations of relative antibody production from individual clones at the 96-well stage feasible. With this method, amplification, cloning, and adaptation to serum-free conditions of multiple cell lines can be monitored in an efficient manner.

The effects of domain deletion, glycosylation, and long IgG3 hinge on the biodistribution and serum stability properties of a humanized IgG1 immunoglobulin, hLL2, and its fragments.

Antibody (Ab) fragments are preferred agents for imaging applications because of their rapid clearance from the blood, thereby providing high tumor:blood ratios within a few hours. Several preclinical studies have also suggested that Ab fragments might be preferred for therapeutic applications over an intact IgG. The purpose of this project was to develop engineered Ab fragments using a humanized anti-carcinoembryonic antigen and anti-CD22 Ab as the parent. Three types of variants were prepared: a deltaCH2 (deletion mutant missing the CH2), a gamma3 F(ab')2 containing the human IgG3 hinge, and three glycosylated variants. The gamma3 F(ab')2 and glycosylated variants were developed because of the potential for site-specific linkage to the Ab in its divalent or monovalent fragment. The gamma3 F(ab')2 variant contains 10 cysteine residues that could be used for direct coupling using thiol chemistry, whereas the glycosylated variants have N-linked glycosylation sites engineered in the CH1 domain (two variants) as well as the VK domain (one variant). All of these variants were successfully prepared and shown to react with the target antigen. All Abs could be purified to a single peak by size-exclusion HPLC, but the deltaCH2 variant showed two distinct peaks, which were believed to be both the divalent and monovalent forms of this fragment. The two CH1 glycosylated variants showed differences in the extent of glycosylation. Modeling studies suggest that one variant would be better suited for site-specific coupling than the other because the carbohydrate chain is extended further away from the antigen-binding site. The Abs were radioiodinated to determine their pharmacokinetic behavior in mice. All of the humanized Ab divalent fragments cleared nearly 20 times faster from the blood than the murine parent F(ab')2 over a 24-h period. The glycosylated fragments showed some added stability compared to the other fragments over 4 h, but by 24 h, they had cleared to the same extent. Size-exclusion high-performance liquid chromatography of blood samples indicated that the humanized Ab fragments were quickly degraded in the blood. Thus, there is an inherent instability of the divalent fragments from these humanized IgG1 constructs that may affect their utility in imaging or therapy applications.

Bypassing immunization: optimized design of "designer T cells" against carcinoembryonic antigen (CEA)-expressing tumors, and lack of suppression by soluble CEA.

Tumor-associated antigens are typically nonimmunogenic in cancer patients, "immune surveillance" having manifestly failed. The fact that most tumor antigens are normal human proteins presents significant obstacles to current cancer immunization approaches that researchers are presently striving to overcome. An alternative strategy bypasses immunization altogether by direct genetic alteration of autologous patient T cells, to create "designer T cells" specific to a particular antigen. Chimeric immunoglobulin-T cell receptors (IgTCR) with a specificity for carcinoembryonic antigen (CEA) were created to evaluate the optimal IgTCR structure for cancer therapy. Antigen-binding domains of a humanized antibody were combined with TCR signaling chains to yield four different chimeric IgTCR: single chain Fv fragment (sFv)-zeta, fragment antigen-binding (Fab)-zeta, sFv-epsilon, and Fab-epsilon. All of the IgTCR were well expressed on T cells, and all showed specific binding and activation, as demonstrated by IL-2 production on contact with immobilized or cellular CEA, excepting sFv-epsilon alone which was inert solely against cellular targets for steric reasons unique to this construct. In contrast to prior studies of isolated TCR chains that related increased tyrosine-based activation motifs in zeta as a reason for superior signaling potency, these tests are the first to show that epsilon and zeta are indistinguishable for T cell signaling when assayed in the context of the intact TCR complex. Further, Fab was equivalent to sFv as an IgTCR component for expression and antigen binding, establishing an important alternative for IgTCR antigen recognition because sFvs may often lose antigen affinity. When IgTCR was expressed on normal human T cells, cytotoxic potency was demonstrated at low E:T ratios, with T cell recycling and progressive tumor cell destruction. Contrary to recent speculations, these observations prove that high affinity TCR interactions are not an impediment to serial target engagement and disengagement by cytotoxic T cells. The multivalent intercellular interactions of target cell binding, activation, and cytotoxicity were resistant to inhibition by soluble CEA. These studies establish a potentially important new immunotherapeutic modality for the treatment of CEA-expressing tumors.

A single-chain immunotoxin against carcinoembryonic antigen that suppresses growth of colorectal carcinoma cells.

We have engineered an anti-carcinoembryonic antigen (CEA) single-chain immunotoxin derived from humanized anti-CEA antibody (hMN14) and a truncated Pseudomonas exotoxin (PE), PE40. The purified anti-CEA immunotoxin (hMN14(Fv)-PE40) was first measured for binding affinity against a CEA-positive colorectal carcinoma cell line and compared with its parental IgG and the monovalent Fab fragment. The Ka of sFv-PE40, Fab, and IgG were 5 x 10(7), 6 x 10(7), and 3 x 10(8) M(-1), respectively. There was no significant affinity loss by conversion of Fab to the single-chain Fv, but these monovalent forms were 5-6-fold reduced in affinity compared with the parental IgG. In cytotoxicity assays, the hMN14(Fv)-PE40 showed specific growth suppression of CEA-expressing colon cancer cell lines MIP-CEA (high CEA) and LS174T (moderate CEA) with IC50s of 12 ng/ml (0.2 nM) and 69 ng/ml (1.1 nM). These IC50s correlated inversely with the surface expression of CEA, such that 50% killing was equivalent for each cell type when expressed in toxin molecules bound/cell (3000-5000). The presence of soluble CEA up to 1000 ng/ml did not affect the cytotoxicity against CEA-expressing cells, with 50% suppression only at 4000 ng/ml that correlated with the binding Kd of the single-chain Fv. The stability of the hMN14(Fv)-PE40 molecule at 37 degrees C was confirmed by bioassay and by lack of aggregation. Our hMN14(Fv)-PE40 may be clinically useful for tumors with high CEA expression without affecting normal tissues with low or absent CEA, even in patients with high soluble antigen levels.

Carbohydrates engineered at antibody constant domains can be used for site-specific conjugation of drugs and chelates.

To improve the efficiency of site-specific conjugation of chelates and drugs to antibodies, and to minimize the incidence of immunoreactivity perturbation to the resultant immunoconjugates, Asn-linked oligosaccharide moieties were designed and engineered into the constant domains of a humanized anti-CD22 monoclonal antibody, hLL2. From 10 potential glycosylation mutants, two CH1 domain glycosylation sites, HCN1 and HCN5, were identified that were positioned favorably for glycosylation. The carbohydrate (CHO) chains attached at these sites were differentially processed so that HCN5-CHOs were physically larger than HCN1-CHOs. Although both the CH1-appended CHOs, and the LL2 Vkappa-appended CHOs conjugated efficiently with small chelates, the HCN5-CHOs, due to the structural and positional superiority, appear to be a better conjugation site for large drug complexes, such as 18 kDa doxorubicin (DOX)-dextran.

Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2).

BACKGROUND: LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2. METHODS: The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed. RESULTS: By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen. CONCLUSIONS: By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

Chimerization of Mu-9: a colon-specific antigen-p antibody reactive with gastrointestinal carcinomas.

BACKGROUND: Mu-9 is a murine monoclonal antibody that is directed against affinity-purified colon-specific antigen-p (CSAp). CSAp is a tumor-associated antigen that is present in 60% of colorectal carcinomas. Preclinical and clinical studies have shown Mu-9 to have excellent targeting abilities. However, as administration of the murine immunoglobulin G (IgG) provoked a human anti-mouse antibody response, chimerization of Mu-9 is warranted for decreasing immunogenicity. METHODS: Polymerase chain reaction and cDNA library screening methods were used for the cloning of Mu-9 heavy and light chain variable regions for the construction of chimeric Mu-9. RESULTS: The functional chimeric Mu-9 antibody binds to the CSAp antigen in the GW-39 extracts. It has immunochemical properties similar to that of murine Mu-9. CONCLUSIONS: The V-region sequence information will be used for design of humanized Mu-9, which will be evaluated for targeting gastrointestinal carcinomas.

Site-specific modifications of light chain glycosylated antilymphoma (LL2) and anti-carcinoembryonic antigen (hImmu-14-N) antibody divalent f1agments.

Site-specific introduction of metal-chelating groups into F(ab')2 fragments of an antilymphoma antibody (LL2) possessing a natural Asn-linked light chain carbohydrate and an anti-carcinoembryonic antigen antibody (hImmu-14-N) grafted with a light chain carbohydrate site is described. For this purpose, four yttrium- (and indium)-chelating agents were used, containing a primary amino group for antibody binding and 1-(4-substituted benzyl)diethylenetriaminepentaacetic acid as the metal-chelator, separated by structurally different additional linkers. Conjugates were prepared by reacting excess chelator with oxidized carbohydrate of F(ab')2 fragments, with or without a subsequent reduction step. The conjugates, with up to an average of 5.5 chelating groups attached to a F(ab')2 fragment, were readily labeled with 90Y and 111In and were found to retain antigen-binding ability in in vitro assays. Tumor targeting was demonstrated using a 88Y-labeled hImmu-14-N F(ab')2 carbohydrate-modified conjugate. 2-Pyridyldithiopropionic hydrazide was conjugated to the carbohydrate region, and the disulfide was selectively deprotected to the thiol group, which is reactive with reduced 99mTc. These initial experiments establish that light chain carbohydrate modification of F(ab')2 is as facile as with the Fc-region carbohydrate of intact IgG, and thereby offer the possibility of designing site-specifically substituted F(ab')2 fragments with favorable pharmacokinetic properties.

Treatment of non-Hodgkin's lymphoma with radiolabeled murine, chimeric, or humanized LL2, an anti-CD22 monoclonal antibody.

LL2 is a murine IgG2a anti-CD22 monoclonal antibody found to react with virtually all non-Hodgkin's lymphomas (NHLs). Twenty-one patients with chemotherapy-resistant NHL received nonmyeloablative doses of 131I-labeled LL2 IgG and F(ab')2 ranging from 15 to 343 mCi given in cycles of 15-50 mCi, for up to seven treatment cycles. The cumulative protein dose ranged from 1.1 mg IgG to 157 mg F(ab')2. Seventeen patients were assessable for treatment response, and antitumor effects were seen in five (one complete remission, two partial remissions, and two minor or mixed responses). In addition, one complete response was seen in a patient who received only "diagnostic" doses of 131I-LL2 IgG. Thus, a total of six patients had responses according to the defined response criteria. Three additional patients have been treated with potentially myeloablative doses of 131I-LL2 IgG at a starting dose level of 90 mCi/m2 (100 mg). Two patients were evaluable, and both had partial remissions lasting 8 and 3 months, respectively. Chimeric and complementarity-determining region-grafted LL2 have been developed. Initial clinical studies have shown that these agents have targeting properties similar to the murine LL2 and, therefore, may be suitable alternatives to murine LL2 in the treatment of NHL. LL2 is a promising agent for the treatment of lymphoma, particularly when the maximum tolerated dose is given either with or without autologous bone marrow transplantation.

Bacterial expression of a kemptide fusion protein facilitates 32P labeling of a humanized, anti-carcinoembryonic antigen (hMN-14) antibody fragment.

Despite the potential advantages of 32P over other isotopes for radioimmunotherapy, its development as a therapeutic has been hindered by the difficulty of the labeling chemistry. Recently, a heptapeptide [Kemptide (KPT)] has been chemically conjugated to antibodies, and the conjugates have successfully been labeled with 32P enzymatically by using bovine protein kinase. By using genetic engineering, we have produced a chimera (Fab.KPT) consisting of the Fab' moiety of the complementarity-determining region-grafted anti-carcinoembryonic antigen-monoclonal antibody, MN14, and a heptapeptide derivative of KPT (Trp-Arg-Arg-Ala-Ser-Leu-Gly). The recombinant protein was expressed in Escherichia coli as a soluble secretory product. The presence of the KPT derivative downstream of the COOH terminus of the hinge region did not impair the binding affinity of the antibody fragment. The Fab.KPT was enzymatically phosphorylated with 32P by bovine protein kinase, without significant effect on the resultant immunoreactivity; 100% of the 32P-labeled Fab.KPT was complexed with liquid carcinoembryonic antigen. The 32P-labeled humanized MN-14 Fab.KPT is expected to have longer blood circulation half-life, allowing for an improved therapeutic efficacy in radioimmunotherapy.

Development and evaluation of the specificity of a rat monoclonal anti-idiotype antibody, WN, to an anti-B-cell lymphoma monoclonal antibody, LL2.

Anti-idiotype monoclonal antibodies (Mabs) to mLL2, an anti-B-cell lymphoma and CD22-specific murine IgG2a-kappa Mab, were generated by hybridoma technology from splenocytes of Copenhagen rats immunized with mLL2 F(ab')2. Mab WN, an IgG2a-kappa, was selected based on its specific binding to mLL2 and not other IgG isotypes or anti-B-cell Mabs. In a radioimmunoassay, WN was found to inhibit the binding of 125I-labeled mLL2 to Raji cells and to have no effect on the binding of other B-cell-reactive antibodies. Using high performance liquid chromatography analysis, WN was shown to complex specifically with both mLL2 and mLL2 Fab'. Meanwhile, we have constructed chimeric (cLL2) and humanized (hLL2) versions of LL2. Both cLL2 and hLL2 were demonstrated to retain the original antigen specificity and affinity of mLL2 [S.O. Leung et al., Proc. Am. Assoc. Cancer Res., 2872 (abstract), 34: 481, 1993]. The specific binding of WN to either radioiodinated or peroxidase-conjugated mLL2 was inhibited in a dose-response manner, and to a similar extent by mLL2, cLL2, and hLL2. Since the mLL2 complementarity-determining regions are the only sequences common to mLL2, cLL2, and hLL2, the result confirms that WN is specific to the antigen-binding complementarity-determining regions. A WN binding assay is currently being evaluated as a substitute for the tedious, and sometimes inconsistent, Raji cell-binding assay for the determination of LL2 immunoreactivity. In conclusion, we have developed an anti-idiotype Mab, WN, to mLL2. Its potential use as a surrogate antigen for B-cell lymphoma is under investigation.

Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL2.

The murine monoclonal antibody, LL2, is a B-cell (CD22)-specific IgG2a which has been demonstrated to be clinically significant in the radioimmunodetection of non-Hodgkin's B-cell lymphoma. The antibody carries a variable region-appended glycosylation site in the light chain and is rapidly internalized upon binding to Raji target cells. Humanization of LL2 was carried out in order to develop LL2 as a diagnostic and immunotherapeutic suitable for repeated administration. Based on the extent of sequence homology, and with the aid of computer modeling, we selected the EU framework regions (FR) 1, 2 and 3, and the NEWM FR4 as the scaffold for grafting the heavy chain complementarity determining regions (CDRs), and REI FRs for that of light chains. The light chain glycosylation site, however, was not included. Construction of the CDR-grafted variable regions was accomplished by a rapid and simplified method that involved long DNA oligonucleotide synthesis and the polymerase chain reaction (PCR). The humanized LL2 (hLL2), lacking light chain variable region glycosylation, exhibited immunoreactivities that were comparable to that of chimeric LL2 (cLL2), which was shown previously to have antigen-binding properties similar to its murine counterpart, suggesting that the VK-appended oligosaccharides found in mLL2 are not necessary for antigen binding. Moreover, the hLL2 retained its ability to be internalized into Raji cells at a rate similar to its murine and chimeric counterparts.

Effect of VK framework-1 glycosylation on the binding affinity of lymphoma-specific murine and chimeric LL2 antibodies and its potential use as a novel conjugation site.

A potential asparagine (Asn)-linked glycosylation site was identified in the VK FRI sequence of an anti-B lymphoma monoclonal antibody (MAb), LL2.SDS-PAGE analysis and endo-F treatment of both murine and chimeric LL2 antibodies indicated that this site was glycosylated; however, no differences in the binding affinity to Raji cells were observed between the native murine LL2 and the endo-F-deglycosylated murine LL2 antibodies. Elimination of the glycosylation site from the chimeric LL2 antibody was accomplished by an Asn to Gln mutation in the tri-acceptor site found in the light chain. The resultant aglycosylated chimeric LL2 exhibited a similar Raji cell binding affinity to that of the glycosylated form. The results are in agreement with computer modeling studies which suggested the lack of interactions between the oligosaccharide moiety and the CDRs. The finding is interesting because it enables a wider choice of human framework sequences, which in most cases do not have a corresponding glycosylation site, for the humanization of the LL2 VK domain, as well as a greater latitude of host expression systems. Most importantly, the LL2 VK carbohydrate moiety might be used as a novel conjugation site for drugs and radionuclides without compromising the immunoreactivity of the antibody.

Chimerization of LL2, a rapidly internalizing antibody specific for B cell lymphoma.

LL2 is a murine monoclonal antibody (MAb) that has been shown to be effective for the diagnosis and treatment of patients with non-Hodgkin's B cell lymphoma. Studies have also shown that radiolabeled murine LL2 (mLL2) or mLL2 and fragments thereof coupled to Pseudomonas exotoxin (PE) can effectively target human B cell lymphoma in mice. We have obtained the DNA sequences encoding the VK and VH domains of mLL2, an IgG2a MAb, which were combined with their respective human kappa and IgG1 constant region domains and expressed in SP2/0 cells. Like its murine counterpart, the chimeric LL2 (cLL2) antibody is glycosylated in the light chain variable region. Chimerization did not interfere with the immunoreactivity of the antibody, as determined by a competitive binding assay, where either antibody shows equivalent inhibition of the binding of its counterpart to the Raji cell membrane surface antigen, CD22. Both antibodies bind and are rapidly internalized by Raji cells, whereas an irrelevant humanized antibody did not bind and was not internalized under similar conditions. The internalization rates of the bound murine or chimeric antibodies were nearly identical, with Ke values of 0.106 and 0.118 min-1 for mLL2 and cLL2, respectively. The observed close equivalence between the murine and chimeric antibodies suggests potential advantages of the latter as a less immunogenic agent. Studies are currently underway to evaluate the chimeric antibody as a potential therapeutic immunoconjugate.

An extended primer set for PCR amplification of murine kappa variable regions.

The design and successful usage of an extended primer set for the PCR amplification of murine variable kappa light chain sequences from mouse hybridomas are described. Since some of these primer pairs also amplify the endogenous SP2/0 aberrant light chain sequence, strategies to distinguish the irrelevant SP2/0 from the sequence of interest are also provided.

Effect of membrane phase transition on long-time calcium-induced fusion of phosphatidylserine vesicles.

Dynamic light scattering has been used to study the temperature dependence of the extent of long-time calcium-induced fusion of sonicated vesicles composed of various natural and synthetic phosphatidylserine with different acyl chains. The vesicles of each composition are found to exhibit a peak temperature in the vicinity of which the extent of fusion shows a distinct maximum. The fusion peak temperature increases as the bilayer gel-to-liquid-crystal phase transition temperature increases. The results suggest a role played by membrane fluidity in determining fusion efficiency.

Transcriptional and translational analysis of the human theta globin gene.

The human theta-globin gene in man appears to be functional, based on its sequence and evolutionary conservation. However its physiological role is unknown and furthermore its deletion in some individuals appears to have no effect on erythroid development. We have therefore analysed the transcriptional and translational competence of the theta globin gene to assess whether or not it is a silent or active globin gene. First, we demonstrate that theta globin mRNA is correctly spliced, by sequencing its cDNA. Second, using this theta cDNA, we generated synthetic theta globin mRNA and were able to demonstrate that this mRNA is translated into theta globin protein in wheat germ in vitro translation extracts. Similarly, the theta globin gene transfected into an erythroid cell line produces a protein product that comigrates with theta globin. Finally, we analysed the unusual promoter of the theta globin gene. The GC rich sequence directly adjacent to the multiple cap sites of theta globin mRNA functions as a promoter element in both erythroid and non-erythroid cell lines, while the more usual CCAAT and ATA box regions (found in all other globin genes) which are displaced by the GC rich promoter sequence, do not possess detectible promoter activity. Taken together, these results suggest that theta globin may have some as yet undetermined role in human erythropoiesis.

The immunosuppressive activities of two abortifacient proteins isolated from the seeds of bitter melon (Momordica charantia).

Two abortifacient proteins, alpha- and beta-momorcharin, have been purified from the seeds of the bitter melon (Momordica charantia). It was found that non-cytotoxic concentrations of these plant proteins can significantly inhibit the mitogenic responses of mouse splenocytes to concanavalin A, phytohaemagglutinin and lipopolysaccharide in a dose-dependent manner. In addition, the alloantigen-induced lymphoproliferation and the in vitro generation of a primary cytotoxic lymphocyte response were severely suppressed in the presence of these proteins. In contrast, the cytolytic activity of cytotoxic lymphocytes and natural killer cells was unimpaired by in vitro exposure to momorcharin. On the other hand, a clear decrease in the functional capacity of macrophages, such as the cytostatic and phagocytic activities, was observed under similar conditions. In vivo studies have shown that single injections of nontoxic microgram amounts of momorcharin into mice resulted in a significant depression of the delayed-type hypersensitivity response as well as the humoral antibody formation to sheep red blood cells. Similarly, the thioglycollate-induced in vivo migration of macrophages was also suppressed. Interestingly, the in vivo activation of natural killer cells was not appreciably affected. Our data suggests that the observed potent immunosuppressive effect of alpha- and beta-momorcharin is unlikely to be due to direct lymphocytotoxicity or due to a shift in the kinetic parameter of the immune response.