Mouse models of human chromosomal translocations and approaches to cancer therapy.

Cancer arises because of genetic changes in somatic cells, eventually giving rise to overt malignancy. Principle among genetic changes found in tumor cells are chromosomal translocations which give rise to fusion genes or enforced oncogene expression. These mutations are tumor-specific and result in production of tumor-specific mRNAs and proteins and are attractive targets for therapy. Also, in acute leukemias, many of these molecules are transcription regulators which involve cell-type-specific complexes, offering an alternative therapy via interfering with protein-protein interaction. We are studying these various features of tumor cells to evaluate new therapeutic methods. We describe a mouse model of de novo chromosomal translocations using the Cre-loxP system in which interchromosomal recombination occurs between the Mll and Af9 genes. We are also developing other in vivo methods designed, like the Cre-loxP system, to emulate the effects of these chromosomal abnormalities in human tumors. In addition, we describe new technologies to facilitate the intracellular targeting of fusion mRNAs and proteins resulting from such chromosomal translocations. These include a masked antisense RNA method with the ability to discriminate between closely related RNA targets and the selection and use of intracellular antibodies to bind to target proteins in vivo and cause cell death. These approaches should also be adaptable to targeting point mutations or to differentially expressed tumor-associated proteins. We hope to develop therapeutic approaches for use in cancer therapy after testing their efficacy in our mouse models of human cancer.

Masked antisense: a molecular configuration for discriminating similar RNA targets.

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These 'masked' antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR-ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.

Analysis of a positive feedback mechanism in the anti-Sm autoantibody response of MRL/MPJ-lpr/lpr mice.

The mechanism of induction of anti-Sm antibodies by passive transfer of anti-Sm mAb in MRL/lpr mice was investigated. No idiotypic relationship was detected between the inducing monoclonal antibody KSm2 and either the induced circulating anti-Sm antibodies or the products of anti-Sm-producing hybridomas derived from spleen cell fusion of treated mice. Treatment of mice with ribonucleoprotein Sm antigen, alone or as an immune complex, induced anti-Sm and anti-ribonucleoprotein antibodies similarly to treatment with KSm2. This suggests that autoantigen contributes to the development of the anti-Sm response in MRL mice.

Differential induction of lupus associated antinuclear antibodies in MRL mice by monoclonal anti-Sm antibodies.

The effect of monoclonal autoantibodies on immunoregulation was investigated in MRL/MpJ-lpr/lpr mice. Passive transfer of KSm2 (a monoclonal IgG2a antibody directed against the 16 kD polypeptide of Sm) induced IgG antibodies to the other major immunoreactive polypeptides of Sm (28 and 29 kD) in all mice studied, and to polypeptides of the closely related antigen nRNP/Sm in 63% of the mice. In addition an increment in IgG anti-dsDNA antibodies, and in IgA and IgM anti-Sm antibodies, over control levels was observed. These effects were not due to polyclonal activation since anti-histone antibody levels were unaffected. Two other IgG2a monoclonal antibodies: KSm5 (directed against the 28 and 29 kD Sm polypeptides) and OX 12 (directed against an irrelevant antigen) failed to modulate the autoimmune responses of the mice in any way. These results demonstrate specific antibody-mediated connectivity between B cell clones producing autoantibodies against three distinct antigens.

Individual variation of anti-Sm autoantibodies in the MRL/MP-lpr/lpr mouse: age-related increase in diversity.

The prevalence and diversity of anti-Sm antibodies (which are a marker of human lupus) were investigated in MRL/MP-lpr/lpr mice. The low prevalence of anti-Sm reported by other workers was confirmed. Anti-Sm antibodies bound to either the 28 kD or both 28 and 16 kD polypeptides on immunoblots. Antibodies to the 16 kD polypeptide, detected on spectrotyping blots, were not detected on immunoblots probed with sera from three mice, suggesting that these antibodies were binding only to an additional conformational epitope. Isoelectric focusing of the sera showed that the anti-Sm antibodies were restricted in clonal diversity and in some cases resembled the pattern of a monoclonal antibody. A longitudinal increase in antibody titre was correlated in some mice with an increase in the diversity of clones expressing anti-Sm antibody. Anti-nRNP antibodies were also detected in the mouse serum but had a lower prevalence than anti-Sm antibodies.

Antibodies to La, Jo-1, nRNP and Sm detected by multi-track immunoblotting using a novel filter holder: a comparative study with counterimmunoelectrophoresis and immunodiffusion using sera from patients with systemic lupus erythematosus and Sjögren's syndrome.

The use of Western blotting or immunoblotting to detect autoantibodies in the serum of patients with autoimmune connective tissue diseases was investigated. An apparatus suitable for simultaneously screening 16 sera on immunoblots was used to show that a complex pattern of antibody binding polypeptides was present in whole HeLa cells. A simpler and readily interpreted pattern of binding was achieved using affinity-purified rabbit thymus antigens. Seventy-seven patients with systemic lupus erythematosus, 44 with primary Sjögren's syndrome and 50 normals were screened for anti-Sm, anti-La, anti-nRNP and anti-Jo-1 by immunoblotting and the results compared with those obtained by counterimmunoelectrophoresis and immunodiffusion. It was shown that both IgG and IgM antibodies must be analysed on immunoblots to detect the maximum number of positive sera, and that the immunoblot detects many anti-La sera which do not form precipitins.

Administration of monoclonal anti-Sm antibody prolongs the survival and renal function of MRL-lpr/lpr mice.

The repeated administration of a monoclonal anti-Sm antibody (KSml) resulted in a significant prolongation of life in MRL-lpr/lpr lupus mice with a 50% mortality of 36 weeks compared with 18-24 weeks in the control groups. Control animals injected with APC11 (a myeloma protein of the same isotype) lived no longer than an untreated group. In addition the renal function as assessed by blood urea levels was less impaired in the KSml-injected mice than in the controls. All KSml-injected mice showed the presence of circulating anti-Sm antibodies which had a different Sm polypeptide binding specificity from that of the injected monoclonal antibody; the increased prevalence of these antibodies compared to the control mice (10-30%) suggested that the anti-Sm antibody response had been induced. The increased longevity in the KSml-treated animals was not associated with alterations in the anti-dsDNA antibody response. The data suggest that administration of anti-Sm antibodies modifies the course of murine lupus.

Murine lupus monoclonal antibodies define five epitopes on two different Sm polypeptides.

Monoclonal anti-Sm (Smith) antibodies derived from the mouse strain MRL/lpr were isolated and characterized by binding to purified antigen, immunoprecipitating characteristic uridine-rich RNAs from Hela cell extracts, and by Western blot analysis using rabbit thymus extract. Five different Sm epitopes were demonstrated by epitope blockade and probing Western blots with the monoclonal antibodies. Human anti-Sm serum inhibited each monoclonal antibody from binding to antigen, indicating that both human and mouse antibodies bind to the same Sm epitopes. Human anti-Sm antibodies bound to 28,000 and 16,000 MW polypeptides, a small number also binding to a 14,000 MW polypeptide. The monoclonal antibodies also bound to the 28,000 and/or the 16,000 polypeptide, and provided evidence to suggest that these two Sm polypeptides bear some structural similarities, but are distinct molecules.

Antibody class differences are detected in anti-nRNP and anti-Sm antibodies directed against distinct antigen subunits.

Antibodies to nRNP, Sm and La were detected and characterised by immunoblot analysis. A comparison was made between IgG and IgM autoantibodies in 77 patients with systemic lupus erythematosus (SLE) and 50 normal subjects. No antibodies were detected in the normal subjects. In all 3 antigen specificity groups, a heterogeneity of antibody class was observed between patients. Antibodies to the 2 nRNP-specific polypeptides (33 and 67 kD) were approximately equally frequent. Although IgG antibodies to the 67 kD polypeptide were detected in 88% of patients with antibodies to this polypeptide, IgG antibodies to the 33 kD polypeptide were only detected in 43% of patients with antibodies to this polypeptide. This suggested either that anti-33 kD antibody is produced by a B cell which cannot mature to an IgG-secretor, or that anti-33 kD antibody production succeeds an initial immune response producing anti-67 kD. Reactivity with the 29 kD, Sm-specific polypeptide appeared to be the most frequent in anti-Sm sera compared with the 16 kD polypeptide suggesting that this polypeptide may be the primary immunogenic component of Sm.