Gene targeting in mouse embryonic stem (ES) cells was used to replace (i) the mouse immunoglobulin heavy chain (IgH) Cγ2a gene segment (mCγ2a) with the human Cγ1 gene segment (hCγ1), and (ii) the mouse immunoglobulin light chain (IgL) Cκ gene segment (mCκ) with its human counterpart (hCκ). ES cells carrying these gene conversions were used to generate chimeric mice that transmitted the human alleles through the germ line. Mice homozygous for both gene alterations were generated by breeding. Serum from homozygous mutant mice contained comparable amounts of antibodies with chimeric κ or mouse λ light chains but only small fractions of basal serum IgG or antibodies elicited against immunizing agents contained chimeric heavy chains. A relative increase in immunogen-specific hCγ1 antibodies was seen following immunization in combination with the saponin adjuvant QS-21. The effect of this was to shift the IgG1-dominated response to an IgG subclass profile that included significant amounts of IgG2a, IgG2b and IgG3 and chimeric IgG. The amounts of antibody secreted by hybridomas derived from mutant and wild-type mice were similar. Sequencing confirmed correct splicing of hCγ1 and hCκ gene segments to mouse J gene segments in hybridoma Ig gene transcripts. In conclusion, IgHhCγ1/IgLhCκ double mutant mice provide a useful animal model for deriving humanized antibodies with potential applications in immunotherapy and diagnostics in vivo as well as for investigating hCγ1 associated functions.

Carcinoembryonic antigen (CEA) has been shown to be one of the best markers for in vivo tumor targeting of radiolabeled antibodies, despite the fact that it is localized predominantly at the apical side of human colon carcinoma cells within the fairly closed pseudolumen structures formed by these tumors. Due to this particular histological localization, a large proportion of the CEA molecules may remain inaccessible to the intravenously injected radiolabeled anti-CEA antibodies of IgG isotype, which are widely used in the clinic. In order to improve targeting, we made a recombinant dimeric IgA, which should have the capacity to translocate from the basolateral to the apical side of the pseudolumen formed by colon carcinoma cells after binding to the poly Ig receptor (plgR). A genomic chimeric mouse-human IgA2 construct was made using one of our most specific anti-CEA hybridomas, CE-25. The chimeric IgA (chlgA) was expressed in the Sp2/0 myeloma cell line. The secreted recombinant antibody was found to consist mostly of a dimeric form of IgA with a molecular weight of about 350 kDa. The dimeric chlgA was shown to translocate efficiently in vitro across a monolayer of epithelial cells expressing the plgR and to retain full CEA binding activity.

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and, therefore, has potential as a therapeutic agent in patients with IgE-mediated allergies such as hay fever, food and drug allergies and extrinsic asthma. The clinical usefulness of mouse antibodies is limited, however, due to their immunogenicity in humans. Mouse C21 antibody was humanized by complementarity determining region (CDR) grafting with the aim of developing an effective and safe therapeutic for the treatment of IgE-mediated allergies. The CDR-grafted, or reshaped human, C21 variable regions were carefully designed using a specially constructed molecular model of the mouse C21 variable regions. A key step in the design of reshaped human variable regions is the selection of the human framework regions (FRs) to serve as the backbones of the reshaped human variable regions. Two approaches to the selection of human FRs were tested: (i) selection from human consensus sequences and (ii) selection from individual human antibodies. The reshaped human and mouse C21 antibodies were tested and compared using a biosensor to measure the kinetics of binding to human IgE. Surprisingly, a few of the reshaped human C21 antibodies exhibited patterns of binding and affinities that were essentially identical to those of mouse C21 antibody.

Tumour localisation and tumour to normal tissue ratios of a chimeric anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), in intact form and as an F(ab')2 fragment labelled with 125I and 131I, were compared in groups of nude mice bearing four different colon cancer xenografts, T380, Co112 or LoVo, of human origin, or a rat colon cancer transfected with human CEA cDNA, called '3G7'. For each tumour, three to four mice per time point were analysed 6, 12, 24, 48 and 96 h after MAb injection. In the different tumours, maximal localisation of intact MAb was obtained at 24 to 48 h, and of F(ab')2 fragment 12 to 24 h after injection. Among the different tumours, localisation was highest with colon cancer T380, with 64% of the injected dose per gram (% ID/g) for the intact MAb and 57% for its F(ab')2 fragment, while in the three other tumours, maximal localisation ranged from 14 to 22% ID g-1 for the intact MAb and was about 11% for the F(ab')2. Tumour to normal tissue ratios of intact MAb increased rapidly until 24 h after injection and remained stable or showed only a minor increase thereafter. In contrast, for the F(ab')2 fragment, the tumour to normal tissue ratios increased steadily up to 4 days after injection reaching markedly higher values than those obtained with intact MAb. For the four different xenografts, tumour to blood ratios of F(ab')2 were about 2, 3 and 5 to 16 times higher than those of intact antibodies at 12, 24 and 96 h after injection, respectively.

We report the isolation, sequence, and identification of a cDNA encoding the human kinesin light-chain (KLC) protein. The cDNA molecule consisted of 276 nucleotides of 5' untranslated region, the complete coding sequence of 1,710 nucleotides, and 322 nucleotides of 3' untranslated region. It encoded a polypeptide of 569 amino acids and a deduced molecular mass of 64,789 daltons. The predicted secondary internal structure of the KLC molecule consisted of about 27 contiguous repeats, each of approximately 21 amino acid residues, and could be divided into three domains. The amino-terminal domain consisted of heptad repeats typical of the rod domain of several cytoskeletal proteins. The central and carboxy-terminal domains consist of 21-mer repeats. KLC mRNA was expressed in most tissues analyzed. The gene, which was expressed in bacteria and Chinese hamster ovary cells, was provisionally assigned to the long arm of human chromosome 14.

Based on previous experiments in nude mice, showing that fluoresceinated monoclonal antibodies against carcinoembryonic antigen localized specifically in human carcinoma xenografts and could be detected by laser-induced fluorescence, we performed a feasibility study to determine whether this immunophotodiagnosis method could be applied in the clinic. Six patients, with known primary colorectal carcinoma, received an i.v. injection of 4.5 or 9 mg of mouse-human chimeric anti-carcinoembryonic antigen monoclonal antibody coupled with 0.10-0.28 mg of fluorescein (molar ratio 1/10 to 1/14). The monoclonal antibody was also labeled with 0.2-0.4 mCi of 125I (1 Ci = 37 GBq). Photodetection of the tumor was done ex vivo on surgically resected tissues for the six patients and in vivo by fluorescence rectosigmoidoscopy for the sixth patient. Upon laser irradiation, clearly detectable heterogeneous green fluorescence from the dye-antibody conjugate was visually observed on all six tumors; almost no such fluorescence was detectable on normal mucosa. The yellowish tissue autofluorescence, which was emitted from both tumor and normal mucosa, could be subtracted by real-time image processing. Radioactivity measurements confirmed the specificity of tumor localization by the conjugate; tissue concentrations of up to 0.059% injected dose per g of tumor and 10 times less (0.006%) per g of normal mucosa were found. The overall results demonstrate the feasibility of tumor immunophotodiagnosis at the clinical level.

Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors, and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for "humanization" of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or used for therapeutic strategies which similarly take advantage of the ability of the antibodies to target cytotoxic agents selectively to tumor cells. The application of these novel procedures for manipulating MAb structure presents entirely new opportunities for diagnosis and treatment of human colorectal cancer.

Mouse-human chimeric monoclonal antibodies (MAbs) of 3 different human IgG sub-classes directed against carcinoembryonic antigen (CEA) have been produced in SP-0 cells transfected with genomic chimeric DNA. F(ab')2 fragments were obtained by pepsin digestion of the purified chimeric MAbs of human IgG1, IgG2 and IgG4 sub-class and of parental mouse MAb IgG1. The 4 F(ab')2 fragments exhibit similar molecular weight by SDS-PAGE. They were labelled with 125I or 131I and high binding (80 to 87%) to purified unsolubilized CEA was observed. In vivo, double labelling experiments indicate that the longest biological half-life and the highest tumour-localization capacity is obtained with F(ab')2 from chimeric MAb of human IgG2 sub-class, whereas F(ab')2 from chimeric MAb IgG4 give very low values for these 2 parameters. F(ab')2 from chimeric MAb IgG1 and from parental mouse MAb yield intermediate results in vivo. Our findings should help to select the appropriate human IgG sub-class to produce chimeric or reshaped MAb F(ab')2 to be used for tumour detection by immunoscintigraphy and for radioimmunotherapy.

Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by the Tp1 family of highly repetitive retrotransposon-like sequences. Tp1 elements consist of two terminal direct repeats of 277bp which flank an internal domain of 8.3kb. They are the major sequence component in the hypermethylated (M +) fraction of the genome where they have been found exclusively in scrambled clusters of up to 50kb long. Scrambling is thought to have arisen by insertion of Tp1 into further copies of the same sequence. In the present study, sequence analysis of cloned Tp1 elements has revealed striking homologies of the predicted amino acid sequence to several highly conserved domains characteristic of retrotransposons. The relative order of the predicted coding regions indicates that Tp1 elements are more closely related to copia and Ty than to retroviruses. Self-integration and methylation of Tp1 elements may function to limit transposition frequency. Such mechanisms provide a possible explanation for the origin and organisation of M + DNA in the Physarum genome.

Radioimmunodetection of tumours with monoclonal antibodies is becoming an established procedure. Positron emission tomography (PET) shows better resolution than normal gamma camera single photon emission tomography and can provide more precise quantitative data. Thus, in the present study, these powerful methods have been combined to perform radioimmuno PET (RI-PET). Monoclonal antibodies directed against carcinoembryonic antigen (CEA) an IgG, its F(ab')2 and a mouse-human chimeric IgG derived from it were labelled with 124I, a positron-emitting radionuclide with a convenient physical half-life of four days. Mice, xenografted with a CEA-producing human colon carcinoma, were injected with the 124I-MAb and the tumours were visualized using PET. The concentrations of 124I in tumour and normal tissue were determined by both PET and direct radioactivity counting of the dissected animals, with very good agreement. To allow PET quantification, a procedure was established to account for the presence of radioactivity during the absorption correction measurement (transmission scan). Comparison of PET and tissue counting indicates that this novel combination of radioimmunolocalization and PET (RI-PET) will provide, in addition to more precise diagnosis, more accurate radiation dosimetry for radioimmunotherapy.

The pregnancy-specific glycoprotein (PSG) genes encode a group of proteins which are found in large amounts in placenta and maternal serum. In situ hybridization analyses of metaphase chromosomes reveal that all the human pregnancy-specific glycoprotein (PSG) genes are located on the long arm of chromosome 19 (19q13.2–13.3), overlapping the region containing the closely-related carcinoembryonic antigen (CEA) gene subgroup. Higher resolution analyses indicate that the PSG genes are closely linked within an 800kb SacII restriction endonuclease fragment. This has been confirmed through restriction endonuclease mapping and DNA sequence analyses of isolated genomic clones, which show that at least some of these genes are located in very close proximity. Further, these studies have helped to identify a new member of the PSG gene sub-family (PSG7). DNARNA hybridization analyses, using gene-specific oligonucleotide probes based on published sequences, showed that five from six PSG genes tested are coordinately transcribed in the placenta. Due to the close proximity of these genes and their coordinated expression pattern, common transcriptional regulatory elements may exist.

The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by Tp1, a family of retrotransposon-like sequences. Tp1 elements are arranged in scrambled clusters probably arising from integration of the element into copies of its own sequence. The present report describes a second sequence family, Tp2, which has been identified within cloned DNA segments of scrambled Tp1 sequences. Like Tp1, the Tp2 element is structurally related to retrotransposons, having long terminal direct repeats and being flanked by an apparent target site duplication, but its relatively short length (1.68 kb) indicates that it is probably incapable of encoding all the functions necessary for its own mobilisation. Analysis of the coding potential of the Tp2 element supports this view, although a striking homology to a nucleic acid binding domain common to many retrotransposons was identified. As with Tp1, putative regulatory signals can be identified in the LTRs of Tp2. Identical arrangements of Tp2 with respect to Tp1 in more than one independently derived clone indicate that non-functional copies of Tp2 may be mobilised as part of a Tp1 transcriptional unit.

The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma DNA are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis.

A procedure was devised for the identification and specific cloning of functionally rearranged variable region immunoglobulin (Ig) gene segments from genomic DNA of a murine hybridoma cell line which produces a high‐affinity monoclonal antibody (MAb) directed against human carcinoembryonic antigen (CEA). The cloned, functionally‐rearranged murine Ig H‐chain and L‐chain variable region gene segments were incorporated into plasmid vectors capable of directing the expression of a chimaeric mouse‐human antibody molecule with human (Y4,K) constant region sequences. Expression plasmids were transfected into a mouse myeloma cell line by electro‐poration and transfectomas secreting functional chimaeric antibody selected. Chimaeric antibody generated by transfectomas was analysed and shown to compete effectively with its murine counterpart for binding to the CEA epitope, and to have an equivalent antigen‐binding affinity. This anti‐CEA recombinant antibody should find application in in vivo diagnosis by immunoscintigraphy of human colonic carcinoma, and possibly also in therapy of the disease, overcoming some of the difficulties associated with the repeated use of non‐human immunoglobulins in human patients.

Human pregnancy-specific beta 1-glycoprotein (SP1) plays an essential role in normal pregnancy. It is also a well-characterized oncodevelopmental antigen, expressed aberrantly by all trophoblastic tumors and some other malignant cell types. Here we report the identification of a human placental cDNA encoding the SP1 polypeptide sequence. The coding sequence shows 95% identity at the nucleotide level with a distinct, recently published SP1 cDNA sequence (PSG16). Unexpectedly, the sequence is also highly homologous to the published sequence of human carcinoembryonic antigen (CEA). SP1, CEA and CEA-related nonspecific cross-reacting species thus belong to a group of closely related though antigenically diverse tumor-associated glycoproteins. Comparison of the deduced amino acid sequence of the SP1 cDNA with that of CEA provides insight into the modular nature of these related proteins. This may have implications for the genomic organization and evolution of the CEA gene family.