Summary FMC"63 is an lgCi2a mouse monoclonal antibody belonging to the CDI9 cluster, CDI9 aniibodics react witlui 95kDa protein expressed by cells of ihe B lymphoc\tc hneage. from pre-B cells to mature B l\niphocytcs. CDl^ antibodies have heen suggested as candidates for immunological attack on leukaemic and lyniphoma cells of the B lineage because ihe antigen is restricted lo ihc B lineage, Wiih the polcntial use of FMC63 in immunolberapy in mind, we ha\e produced a minisc-human chimera in which the genes coding for the \'DJ region of Ihe hea\y chain and the VJ region of the liglu chain derive from the FMC63 n-iousc hybridoma. while the C region genes code for human lg(il. The genes have been transfectcd liack into a mouse myeloma line, which secretes low levels ol' immunoglobulin, {Ig). This Ig was puriticd and biolinylaied in order to determine the speci licily of the an! i body. The chimeric antibody has a read ion proltle concordant with llie original FM(63 antibodv. bul has the properties of a human IgCil. including iho ability to fi,\ human complement. However, the antJbod) is not cytoloxic in vilro in the presence of complement or cells capable of mediating antibody-dependeni cellular cytoloxicity. Possible reasons for this and ways of using the antibody are discussed.
Class switch of immunoglobulin from mu to gamma occurs by recombination between two repetitive switch sequences: S mu and S gamma. However, there are no such sequences in the mu-delta introns of human and mouse genomes. Although the frequency of IgD-secreting cells is extremely low in mouse about 1% of patients with myeloma produce IgD in human. In a previous report (Nucleic Acids Res. 1988. 16: 9497) we reported that a 442-bp DNA sequence located in the JH-mu intron (defined as sigma mu) was inserted into the mu-delta intron (defined as sigma mu) in human genome. There is no such insertion in mouse. We analyzed Ig H chain gene loci of two human IgD myelomas: one was analyzed by cloning and sequencing and the other by Southern hybridization. We found that recombination had occurred between these two homologous DNA sequences, resulting in loss of the DNA segment from sigma mu to sigma mu. On the other hand, in a Burkitt lymphoma, Daudi, the DNA fragment from sigma mu to sigma mu was duplicated. These results suggest that homologous recombination between sigma mu and sigma mu sequences mediates class switch from mu to delta in human and that it occurs via unequal crossing-over between sister chromatids or daughter chromosomes.
The thermal stability of Fv fragments was examined by circular dichroism (CD) spectrometry and high-performance liquid chromatography. We analyzed three Fv fragments: that of a monoclonal antibody D1.3 and two derivatives of it. After separation of wild-type VH and V L fragments, thermal denaturation of each fragment was monitored by CD spectrometry. The results indicated that the dissociation of Fv into VH and VL fragments seemed to be coupled with the denaturation of each fragment and that the thermal denaturation of VH and VL fragments was prevented when they were associated with one another. The analysis of the three Fv fragments also indicated that, in some cases, differences in amino acids even within the CDRs could have significant effects on the thermal stability of the complex between VH and VL fragments.
The variable region of immunoglobulin (Ig) heavy chain is encoded by three separate genes: variable (VH), diversity (DH) and joining (JH) genes on the germ-line genome. In mice, most complementarity determining region (CDR) III of the heavy chains of myelomas and hybridomas sequenced so far can be assigned to one of the 12 already identified germ-line DH genes by the homology of nucleotide sequences of DH gene-coding regions although extranucleotides, the so-called N segments, are found at the boundaries between DH and JH as well as VH and DH. On the other hand, Siebenlist et al. (Nature 1981. 294:631) identified two DH gene families in human genome: DHQ52, located at 45 bp upstream of the JH gene cluster, and another family encoded at 9-kb regular intervals possibly between VH and JH gene clusters. However, the somatic DH sequences found in VH-DH-JH structure (the somatic DH segment being defined as the region which is not encoded either by germ-line VH or JH gene) are relatively long and apparently random, and do not seem to have the homology to any of the germ-line DH sequences. To explain the origin of high diversity in the CDR III of human Ig heavy chains, Siebenlist et al. predicted the presence of another mechanism, namely DH-DH joinings. In the present study, we identified five DH genes in one of the above 9-kb repeats. This suggests that the total number of germ-line DH genes is much higher in man than in mouse. The comparison between somatic DH sequences and germ-line DH sequences indicates that most somatic DH sequences in human Ig heavy chains are also produced by VH-DH and DH-JH joinings without the joining of multiple DH gene segments.
Noncoding regions within the cluster of immunoglobulin heavy chain constant genes in the human genome contained a number of repeats. In the mu-delta intron, two repeating units were contained. One 442-base-long fragment located JH-mu intron (defined as "sigma mu(sigma mu)") occupied the position in the mu-delta intron. The other 1166-base-long fragment located somewhere in front of S (class switch) region of C gamma gene was also found in the mu-delta intron. We defined the repeats in the mu-delta intron as "SIGMA (sigma)". The polarities of the longer repeats in the genome were opposite between the mu-delta intron and the upstreams of C gamma genes. These inverted copies (defined as sigma gamma 3 and sigma gamma 4), located 6 kb upstream of their respective C gamma's, were apparently transcribed in vitro, via RNA polymerase III and transcripts should have contained tRNA-like structures. Small DNA fragments capable of encoding tRNA-like structures were also found in corresponding regions of mouse Ig C gamma cluster.
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