Molecular analysis of the t(2;14) translocation of childhood chronic lymphocytic leukemia.

Two rare cases of chronic lymphocytic leukemia (CLL) in children have been studied; both are associated with a previously undescribed chromosomal translocation [t(2;14) (p13;q32)]. In one patient the translocation was reciprocal and the breakpoint on chromosome 14 occurred just 5' of the C gamma 2 region on the productive immunoglobulin heavy-chain allele. The breakpoint on chromosome 2 does not involve the K locus but lies within an uncharacterized region that coincides with the position of a constitutive fragile site that occurs within normal lymphocytes. Data on the second patient are consistent with these findings and suggest that these cases represent a rare but distinct subgroup of CLL's with a specific cytogenetic change.

Chimerization of antitumor antibodies via homologous recombination conversion vectors.

Homologous recombination vectors were designed to convert murine hybridoma cell lines expressing IgG3, IgG1, or IgG2a heavy chains into chimeric human IgG1 producers. These conversion vectors included homology both upstream and downstream of the target sequences and consistently resulted in a higher frequency of successful gene targeting than an insertion vector bearing a single region of homology. A human kappa light chain conversion vector was also constructed and used to complete chimerization of the anticarcinoma hybridoma cell line BR96. The resulting cell line expressed antigen-specific chimeric antibody at comparable levels to those found in the murine parental cell line. Southern blots confirm that recombination occurred within the upstream and downstream regions of homology for both vectors, resulting in the loss of murine constant region sequences.

Genetic construction, expression, and characterization of a single chain anti-carcinoma antibody fused to beta-lactamase.

We report the genetic construction and expression of a fusion protein between an antibody single chain-linked variable domain fragment specific for human carcinomas and beta-lactamase II from Bacillus cereus. Sequences encoding the variable regions of the L6 monoclonal antibody were assembled so as to be separated from each other by an 18-amino acid linker and from the mature form of beta-lactamase by a 6-amino acid linker. The construct was placed under the transcriptional regulation of the lac promoter, and the PelB signal sequence was used to direct export of the fusion protein to the periplasmic space of Escherichia coli. After induction, biologically active material was recovered from both culture supernatants and cell lysates. Affinity chromatography yielded about 2.5 micrograms of protein/ml of initial culture volume. The fusion protein was shown to bind to tumor cells at least as well as chemically prepared F(ab') and to maintain beta-lactamase activity at a level similar to that of the native enzyme. Tumor cells coated with the fusion protein were sensitive to a cephalosporin mustard prodrug in a dose-dependent fashion comparable to that of enzyme chemically conjugated to F(ab'). This article demonstrates the feasibility of using single chain-linked variable domain-enzyme fusion proteins for the activation of anticancer prodrugs.

Epitope mapping and use of anti-idiotypic antibodies to the L6 monoclonal anticarcinoma antibody.

Mouse monoclonal anti-idiotypic antibodies (anti-ids) were raised against L6, a murine IgG2a monoclonal antibody specific for a cell surface antigen expressed by many human carcinomas. Ten distinct anti-ids were generated. Eight anti-ids were shown to inhibit the binding of L6 to its target antigen and were characterized in detail. The heavy and light chain variable region gene segments of the monoclonal antibody L6 linked to human constant regions (chimeric L6) were expressed separately or together, to map the epitopes recognized by the anti-ids. Individual anti-ids were shown to recognize heavy chain, light chain, or combinatorial variable region determinants. Defining these specificities enabled us to select particular anti-ids for assays to monitor the pharmacokinetics of either murine or chimeric L6 antibodies in the circulation of human patients. A quantitative enzyme-linked immunosorbent assay developed with two anti-ids accurately detects less than 5 ng/ml. Anti-ids specific for light chain variable region-encoded determinants were capable of recognizing L6 antigen-binding fragments bound to the surface of human carcinoma cells. These anti-ids can be used to study the binding of chimeric L6 antibody at the surface of tumor cells in histological sections of tumor biopsies.

Betacellulin-Pseudomonas toxin fusion proteins bind but are not cytotoxic to cells expressing HER4; correlation of EGFR for cytotoxic activity.

Betacellulin (BTC) is a member of the EGF ligand family that directly binds to both EGFR and HER4 and induces the growth of certain epithelial cell types. Fusion proteins composed of the terminal 48 or 50 amino acids of mature betacellulin and a binding defective form of Pseudomonas exotoxin (BTC-TX48 and BTC-TX50, respectively), have been produced. BTC-TX50 induced tyrosine phosphorylation of both EGFR and HER4, whereas BTC-TX48 induced phosphorylation of HER4 but to a much lesser extent EGFR, indicating that the presence of two additional amino acid residues, Arg62 and Lys63, contribute to full kinase activity. BTC-TX50 was up to 300-fold more active at inhibiting protein synthesis than BTC-TX48 on cell lines expressing EGFR, most likely due to the >tenfold higher affinity of BTC-TX50. MDA-MB-453 breast carcinoma cells which express HER4 but not EGFR, were not sensitive to either BTC-TX form. These data indicate that despite the ability of BTC-TX to bind and phosphorylate HER4, it was only cytotoxic to cells expressing EGFR. The inability of BTC-TX to kill cells was likely due to its failure to internalize through HER4.

Regional localization of the human c-rel locus using translocation chromosome analysis.

The human cellular homolog of v-rel, the transforming gene of reticuloendotheliosis virus, strain T, was previously localized to 2 cent-2p13 by a combination of somatic cell hybrid and in situ hybridization analyses. In this study, we use translocation chromosome analysis to refine c-rel's genetic assignment to 2p12-2p13.

An immunoglobulin light chain dimer with CD4 antigen specificity.

A subclone that had lost its IgG1 heavy chain was derived from a hybridoma cell line G17-2 that produces an anti-CD4 monoclonal antibody. This subclone was found to secrete a kappa light chain dimer (LCD) that could bind to the CD4 antigen expressed on a subset of human T lymphocytes. The light chain dimer bound to the same or similar epitope as the parental antibody since it blocked the binding of the parenteral anti-CD4 MAb but not the binding of another anti-CD4 MAb G19-2 that recognizes a different epitope. A rabbit anti-idiotype prepared against G17-2 crossreacted with the LCD and could block the antigen binding of both G17-2 and the LCD. Scatchard analysis performed with 35S-methionine or 3H-leucine labelled LCD showed an association constant Ka of 2.2 x 10(7) M-1, whereas the G17-2 parental antibody showed an association constant Ka of 2.5 x 10(9) M-1. These data indicate that the antigen specificity of the G17-2 parental MAb is conferred to a large extent by its light chain. The LCD was expressed on the surface of the LCD-producing hybridoma cells. Southern blot analysis with C kappa and J kappa probes demonstrated a single kappa transcription units which does not have any unusual DNA rearrangements and is distinct from the NS-1 kappa genes. To our knowledge, this LCD is unique in its ability to bind to a large (55,000 mol. wt) glycoprotein antigen.

Role of epidermal growth factor receptor family members in growth and differentiation of breast carcinoma.

Members of the epidermal growth factor (EGF) family of tyrosine kinase receptors are involved in the regulation of cell growth and differentiation, and are found to be expressed in many types of cancers. Activation of these receptors can be elicited by multiple ligands, resulting in the formation of a spectrum of heterodimer complexes and a number of biological outcomes. A clear demonstration of biological activation by a single complex has been difficult to address because of the endogenous expression of HERs (human EGF-like receptors) in many cell lines. We have generated a collection of cell lines expressing all HERs alone or in all pairwise combinations in a clone of NIH 3T3 cells (3T3-7d) devoid of detectable EGF receptor family members. Transformation, as measured by growth in soft agar, only occurred in cells expressing two different HER family members. Transformation with activated Neu and the rate of in vivo tumour formation were also correlated with the expression of multiple HERs in the same cell. To further our understanding of the role of heterodimer signalling, we demonstrated that, within a breast carcinoma cell line, activation of HER-3 results in cellular differentiation, prolonged activation of extracellular-signal-related kinase 1 (ERK1) activity and an increase in p21CIP1/WAF1 nuclear staining. In contrast, activation of HER-4 is mitogenic, induces transient activation of ERK1 activity and decreases the nuclear staining of p21CIP1/WAF1. These differences in biochemical and biological responses are correlated with the contrasting abilities of HER-3 and HER-4 to be down-regulated from the cell surface. The cell-surface localization of HER-3 does not change in response to ligand, whereas activation of HER-4 results in a loss of cell-surface staining followed by accumulation into a perinuclear compartment.

Identification of serum components that inhibit the tumoricidal activity of amphiphilic alpha helical peptides.

Antimicrobial peptides that can form amphiphilic alpha helices were tested for their ability to lyse various human tumor cell lines in vitro. These peptides include C18G, whose sequence is a derivative of the carboxyl terminus of human platelet factor IV, and 399, an idealized amphiphilic alpha helix. Both peptides exhibited potent antitumor activity against all cell lines tested, unlike magainin 2, a naturally occurring antimicrobial peptide of similar structure, which was relatively inactive under the same conditions. Also, the lytic activity of C18G is specific for tumor cells versus human red blood cells. The effects of serum can be important when evaluating the potency of lytic peptides, since other tumoricidal peptides have been shown to be completely inactivated by low serum levels. Experiments with C18G and 399 revealed that their activity was indeed reduced in the presence of human serum, but that significant lytic activity remained even at relatively high serum concentrations. Various serum components were tested for their inhibitory activity. Whereas albumin and high-density lipoprotein had only slight inhibitory properties, low-density lipoprotein was found to be a potent inhibitor of peptide-mediated cell lysis. The peptide 399, which is more sensitive to serum inhibition than C18G, also binds more extensively to all serum components tested.

Cognate interaction between T helper cells and B cells. VII. Role of contact and lymphokines in the expression of germ-line and mature gamma 1 transcripts.

Complete reconstitution of Th cell function for B cell growth and differentiation was provided by plasma membranes (PM) derived from activated Th (PMAct) in combination with IL-4 and IL-5 (IL-4/IL-5). IL-5 has been shown to be essential for the secretion of all Ig isotypes by resting, conventional B cells activated by PMAct and IL-4. It was shown that in the presence of PMAct/IL-4/IL-5, a high frequency of resting B cells differentiated to express IgG1. IL-4 alone transiently induced the expression of germ-line gamma 1 transcripts. PMAct alone were ineffective at inducing germ-line gamma 1 transcript expression by resting B cells suggesting that Th-B cell contact was an insufficient signal to cause a detectable increase in the steady-state levels of gamma 1 germ-line transcripts. PMAct alone or PMAct/IL-4 did not induce the appearance of transcripts for secreted mu or mature gamma 1. IL-5, in combination with PMAct/IL-4, provided the necessary signal(s) required for the expression of secreted mu and mature gamma 1 transcripts. Therefore, IL-5 appeared to be an important if not essential differentiation factor for conventional B cells that have been activated by cognate help. It appeared that IL-5 promoted the secretion of Ig by inducing the synthesis of mature Ig transcripts.

Cognate interactions between helper T cells and B cells. VI. TGF-beta inhibits B cell activation and antigen-specific, physical interactions between Th and B cells.

TGF-beta is a 25-kDa homodimeric protein that has been shown to have multiple roles in the regulation of lymphocyte activation. Previous studies have shown that TGF-beta is an inhibitor of numerous T and B lymphocyte activities. This study shows that TGF-beta is able to inhibit Th- and mitogen-induced murine B cell proliferation, as well as mitogen-induced B cell cycle entry and immunoglobulin secretion. Acridine orange analysis established that TGF-beta inhibits the LPS-induced B cell transition from G0 to G1A phase of the cell cycle. Evaluation of the effects of TGF-beta on the interactions between Th and B cells showed that TGF-beta inhibited antigen-specific Th-B cell physical interactions. Flow cytometric data showed that the ability of TGF-beta to interfere with the formation of Th-B cell conjugates was not due to decreased expression of IgD, IgM, class I, class II, LFA-1, or ICAM1 by the B cell. Taken together, these data establish that TGF-beta is able to act at multiple sites within the immune system.

Cross-linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells.

OX40 is a member of the TNF/NGF-receptor family expressed on activated T cells, whose ligand is found on activated T and B cells. In the present study, we show that cross-linking of OX40L on CD40L-stimulated B cells, alpha IgD dextran-stimulated B cells, or both results in a significantly enhanced proliferative response with no change in the cell survival rate. Furthermore, OX40 stimulation increases immunoglobulin heavy chain mRNA levels and immunoglobulin secretion, which could not be blocked by anti-cytokine antibodies. In additional molecular studies, we show that OX40L cross-linking results in the down-regulation of the transcription factor BSAP. This, in turn, leads to a change in the in vivo binding pattern of the immunoglobulin heavy chain gene 3' alpha enhancer, suggesting its activation. This effect may thus be one mechanism for OX40-induced increase in immunoglobulin secretion. In conclusion, our data suggest that the OX40-OX40L interaction is a novel pathway in T cell-dependent B cell proliferation and differentiation.

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli.

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30 degrees C or 37 degrees C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic beta-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30 degrees C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A660 at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.

Prevention of immunotoxin-mediated vascular leak syndrome in rats with retention of antitumor activity.

Immunotoxins are hybrid molecules composed of a cell-surface binding domain and a protein toxin moiety that together target specific cell populations for elimination. These agents represent a promising approach for the treatment of many human diseases, most notably cancer. However, it has recently become clear that many immunotoxins when used in human clinical trials induce vascular leak syndrome (VLS), restricting the administration of doses necessary to achieve good therapeutic responses. The lack of an appropriate animal model has hindered efforts to understand and prevent immunotoxin-induced VLS. We have found that in rats, intravenous administration of the single-chain immunotoxin BR96 sFv-PE40 results in symptoms that closely resemble VLS seen in human immunotoxin trials. A large fluid accumulation in the thoracic cavity was observed, along with an increase in hematocrit and body weight and a decrease in serum albumin. The VLS was apparent within 24 hr after administration of immunotoxin and was seen in both immunocompetent and athymic rats. Similar symptoms were not found in mice even at lethal doses. Prophylactic administration of the corticosteroid dexamethasone resulted in prevention of VLS and survival of rats injected with what would otherwise be lethal doses of BR96 sFv-PE40. Prophylactic treatment with dexamethasone in rats xenografted with human tumors either did not inhibit or minimally inhibited the antitumor activity of BR96 sFv-PE40. The use of prophylactic corticosteroids should be considered for immunotoxin clinical trials, since it may improve therapeutic efficacy by decreasing the dose-limiting toxicity of VLS.

HER4-mediated biological and biochemical properties in NIH 3T3 cells. Evidence for HER1-HER4 heterodimers.

The EGF receptor family of tyrosine kinase growth factor receptors is expressed in a variety of cell types and has been implicated in the progression of certain human adenocarcinomas. The most recent addition to this family of receptors, HER4, was expressed in NIH 3T3 cells to determine its biological and biochemical characteristics. Cells expressing HER4 were responsive to heregulin beta2 as demonstrated by an increase in HER4 tyrosine phosphorylation and ability to form foci on a cell monolayer. HER4 exhibited in vitro kinase activity and was able to phosphorylate the regulatory subunit of phosphatidylinositol 3-kinase and SHC. Peptide competition studies identified tyrosine 1056 of HER4 as the phosphatidylinositol 3-kinase binding site and tyrosines 1188 and 1242 as two potential SHC binding sites. Interestingly, transfection of HER4 into NIH 3T3 cells conferred responsiveness to EGF with respect to colony formation in soft agar. It was also found that in response to heregulin beta2, endogenous murine HER1 or transfected human HER1 became phosphorylated when HER4 was present. This demonstrates that HER1 and HER4 can exist in a heterodimer complex and likely activate each other by transphosphorylation.

Construction, expression, and characterization of anticarcinoma sFv fused to IL-2 or GM-CSF.

Local production of cytokines by genetically engineered tumor cells decreases their tumorigenicity and elicits protective immune responses against the parental tumor cells. An alternative approach to elicit a therapeutic immune response is to use fusion proteins that can target tumor cells and simultaneously activate effector cells. Fusion proteins between human IL-2, murine or human GM-CSF, and sFv of antihuman carcinoma antibody L6 have been constructed, expressed in both COS and Chinese hamster ovary (CHO) cells, and purified by affinity chromatography. The biologic activity of L6 sFV-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF was tested on human T cell blasts, factor-dependent FDCP-1, and TF-1 cells, respectively. The ability of soluble L6 sFv-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF to stimulate the proliferation of the indicator cells was found to be comparable to that of recombinant hIL-2, mGM-CSF, or hGM-CSF. Tumor cells coated with L6 sFV-mGM-CSF or L6 sFv-hGM-CSF were also tested in this way and were found to be potent stimulators, indicating that the cytokines were functionally active when bound to the tumor cell surface. This work demonstrates the feasibility of targeting sFv-cytokine fusion proteins for the activation of effector cells as an alternative to cytokine gene therapy.

Genetic construction and characterization of a fusion protein consisting of a chimeric F(ab') with specificity for carcinomas and human IL-2.

A genetic construct was created incorporating gene fragments encoding the H chain V region of the human carcinoma specific antibody L6, the CH1 domain of human IgG1, a linker region, and human IL-2. This construct was cotransfected with a chimeric L6 L chain construct into the murine myeloma cell line Ag8.653 for expression. First round clones produced the fusion protein at an estimated 5 to 10 micrograms/ml based on idiotypic reactivity. Dual binding activity was demonstrated through specific interaction with the L6 Ag on human tumor cells and the IL-2R on activated human T cells. The IL-2 portion of the molecule was shown to support the growth of the IL-2-dependent T cell line CTLL2, and the qualitative nature of the IL-2 signal was found to be the same as rIL-2 with respect to induction of tyrosine-phosphorylation of intracellular protein substrates. Tumor cells coated with the fusion protein were shown to cause T cell proliferation and the presence of the fusion protein was found to enhance cell-mediated destruction of human tumor cells.

Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting.

We demonstrate that murine myeloma cells can efficiently mediate homologous recombination. The murine myeloma cell line J558L was shown to appropriately recombine two transfected DNA molecules in approximately 30% of cells that received and integrated intact copies of both molecules. This activity was then exploited to direct major reconstructions of an endogenous locus within a hybridoma cell line. Production of antigen-specific chimeric heavy chain was achieved by targeting the human IgG1 heavy chain constant region (C gamma 1) exons to the genomic heavy chain locus of a hybridoma cell line secreting antibody specific for a human tumor-associated antigen. The frequency of productive genomic recombinations was approximately 1 in 200 transfectants, with accumulation of the chimeric protein reaching greater than 20 micrograms/ml in culture supernatants.

Structure-activity analysis of the antitumor and hemolytic properties of the amphiphilic alpha-helical peptide, C18G.

The in vitro antitumor and hemolytic activities of analogs of peptide C18G were compared in order to elucidate important structural features which affect cytotoxicity. The sequence of C18G, a basic peptide which can form an amphiphilic alpha-helix, is a derivative of the carboxyl terminus of human platelet factor IV. The results demonstrate that both amphiphilicity and helicity are essential for peptide activity, and that addition of a negatively charged amino acid results in decreased cell lysis. Whereas peptides exhibiting various degrees of potency did not differ with respect to helical content, an increase in peptide hydrophobicity did correlate with an increase in antitumor and hemolytic activity, as well as susceptibility to inhibition by serum. Higher hydrophobicity could be associated with improved ability to insert into the cell membrane. The position or context of specific residues within an amphiphilic peptide can also be important for activity. Furthermore, an increase in tumoricidal activity is not always accompanied by an increase in hemolytic activity or susceptibility to inhibition by serum. Possible reasons for the lower sensitivity of RBCs versus tumor cells to peptide cytotoxicity are discussed. Finally, compared with structurally idealized amphiphilic alpha-helical peptides, non-idealized peptides can possess higher tumoricidal activity, but are less hemolytic and less susceptible to serum inhibition.