Sequential treatment with vindesine-ifosfamide-platinum (VIP) chemotherapy followed by platinum sensitized radiotherapy in stage IIIB non-small cell lung cancer: a phase II trial

Monoclonal antibody (mAb) A33 detects a glycoprotein homogeneously expressed by > 95% of human colon cancers and by normal colon cells. The A33 antigen is not secreted or shed and after mAb A33 binds to antigen on the cell membrane, a fraction of membrane-bound mAb A33 is internalized into endosomes. Phase I 131I-mAb A33 biodistribution studies have shown consistent, specific tumor-targeting, and phase I radioimmunotherapy trials with 131I- or 125I-mAb A33 have demonstrated antitumor effects. Here we describe a nude mouse model that was established using a human colon cancer cell line, SW1222, which grows as a relatively hypovascular, invasive heterotransplant when injected i.m. Peak uptake of 131I-labeled or 111In-chelated mAb A33 was observed at 48-96 h, with a mean of 34% (SE +/- 5.0) and 46.7% (SE +/- 1.7) injected dose per gram of tumor tissue, respectively. 111In-mAb A33 was retained in tumor tissue longer than halide radioimmunoconjugates. The specificity of antibody localization was assessed using a control antibody (tumor uptake and pharmacokinetics), a control tumor, corrections for vascular antibody blood-pooling in tumor tissue, and blocking of radiolabeled mAb A33 localization by pretreating mice with excess unlabeled mAb A33. These experiments demonstrate that mAb A33 localization in tumor was specific, and they emphasize the unexpected rapidity with which the antibody localizes. Our conclusions were confirmed by immunohistochemical techniques which allowed direct visualization of localization and distribution of the humanized version of mAb A33 in tumor tissue. Furthermore, antibody doses approximating tumor-saturating doses demonstrated that a homogeneous distribution of antibody in tumor is possible. This model will be valuable for studies focusing on general physiologic aspects of antibody-to-tumor cell localization and critical as a guide to the evaluation of various A33 antibody constructs and combinations with other therapies for the treatment of colon cancer.     

CTX, a Xenopus thymocyte receptor, defines a molecular family conserved throughout vertebrates

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) member comprising one variable and one constant C2-type Igsf domain, a transmembrane segment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, the variable domain is not encoded by somatic rearrangement of the gene but by splicing of two half-domain exons. The C2 domain, also encoded by two exons, has an extra pair of cysteines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70 amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chicken homologue of CTX, has the same structural and genetic features, and both molecules are expressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNA and genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similarity with recently described human cell surface molecules, A33 antigen and CAR (coxsackie and adenovirus 5 receptor), and a number of expressed sequence tags leads us to propose that CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extending beyond the immune system. Strong homologies within vertebrate sequences suggest that the V and C2 CTX domains are scions of a very ancient lineage.     

Specificity of LIM domain interactions with receptor tyrosine kinases

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.     

Surgical treatment of obstructive jaundice

Genetically engineered structural variants of human beta2-microglobulin (beta2m) were produced by sequence exchange with mouse beta2m for the purpose of examining species-specific antigenic determinant expression. For aggregate mapping, mouse and human beta2m, which differ by 30% in their primary sequence of 99 amino acids, were prepared as chimeric (human X mouse) molecules and expressed in the FO-1 beta2m-null human melanoma cell line. A chimera containing residues 1-69 from human beta2m (and residues 70-99 from mouse beta2m) induced expression of the epitopes defined by the anti-beta2m monoclonal antibodies (mAb) BBM.1, NAMB-1, and L368; the reverse chimera did not, although HLA class I heavy chain was evident on the cell surface as determined with the TP25.99 mAb. For fine dissection of the epitopes defined by these mAbs, site-directed mutants of beta2m were prepared by replacement of individual amino acids in human beta2m with the dimorphic residue from mouse beta2m. Substitutions were made at each divergent residue between positions 1 and 66 and, as controls for COOH-terminal modification, a series of residues between positions 75 and 94. Replacement of amino acids 38, 44, and 45, but not 16 other dimorphic residues in the linear stretch from residue 1 to residue 66, resulted in the loss of, or gross reduction in, binding by mAbs BBM.1 and NAMB-1. A reduction in binding was also observed for mAb L368. These data provide strong evidence that the antigenic epitopes defined by these mAb map to a region including S3 and its adjacent intra-beta-strand turn of the three-stranded beta-pleated sheet of beta2m. The mapping of these epitopes is consistent with their accessibility in the assembled major histocompatibility complex class I molecule and indicates that the region from amino acid 38 to 45 is an important structural feature in the "foreignness" of human and mouse beta2m.     

Identification of novel transmembrane gene sequence and its use for cell-surface targeting of beta subunit of human chorionic gonadotropin

We identified a 685-nucleotide gene fragment that codes for the transmembrane and cytoplasmic domains of glycoprotein of the LEP strain rabies virus and carried out experiments designed to express a novel fusion protein on the cell surface. The cDNA encoding the membrane anchor sequence was fused in the correct reading frame to the 3' end of the cDNA encoding the beta subunit of human chorionic gonadotropin (beta(h)CG), a secretory glycoprotein that is used as an antigen for a contraceptive vaccine being developed in our laboratory. The fusion gene cassette was placed under the control of a vaccinia virus early promoter and cloned in a host-restricted fowlpox viral vector. The recombinants, when used to infect mammalian cells that do not allow the replication of fowlpox virus, expressed the N-terminal 135 amino acid residues of beta(h)CG anchored in the cell membrane by the 75-amino acid C-terminal sequence derived from rabies virus glycoprotein. This hybrid protein is correctly processed post-translationally and transported efficiently to the plasma membrane of non-permissive cells such that the anchored beta(h)CG molecule retains the correctly folded native antigenic epitope(s).     

Characterization of a Helicobacter pylori neutrophil-activating protein

Antibody-blocking studies have demonstrated the role of CD6 in thymocyte-thymic epithelial (TE) cell adhesion. Here we report that CD6 expressed by COS cells mediates adhesion to TE cells and that this interaction is specifically blocked with an anti-CD6 monoclonal antibody (mAb) or with a mAb (J4-81) that recognized a TE cell antigen. We isolated and expressed a cDNA clone encoding this antigen and show that COS cells transfected with this cDNA bind a CD6 immunoglobulin fusion protein (CD6-Rg). This antigen, which we named ALCAM (activated leukocyte-cell adhesion molecule) because of its expression on activated leukocytes, appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. We prepared an ALCAM-Rg fusion protein and showed that it binds to COS cell transfectants expressing CD6, demonstrating that ALCAM is a CD6 ligand. The observations that ALCAM is also expressed by activated leukocytes and that both ALCAM and CD6 are expressed in the brain suggest that ALCAM-CD6 interactions may play a role in the binding of T and B cells to activated leukocytes, as well as in interactions between cells of the nervous system.     

VEGI, a novel cytokine of the tumor necrosis factor family, is an angiogenesis inhibitor that suppresses the growth of colon carcinomas in vivo

A novel member of the tumor necrosis factor (TNF) family has been identified from the human umbilical vein endothelial cell cDNA library, named vascular endothelial growth inhibitor (VEGI). The VEGI gene was mapped to human chromosome 9q32. The cDNA for VEGI encodes a protein of 174 amino acid residues with the characteristics of a type II transmembrane protein. Its amino acid sequence is 20-30% identical to other members of the TNF family. Unlike other members of the TNF family, VEGI is expressed predominantly in endothelial cells. Local production of a secreted form of VEGI via gene transfer caused complete suppression of the growth of MC-38 murine colon cancers in syngeneic C57BL/6 mice. Histological examination showed marked reduction of vascularization in MC-38 tumors that expressed soluble but not membrane-bound VEGI or were transfected with control vector. The conditioned media from soluble VEGI-expressing cells showed marked inhibitory effect on in vitro proliferation of adult bovine aortic endothelial cells. Our data suggest that VEGI is a novel angiogenesis inhibitor of the TNF family and functions in part by directly inhibiting endothelial cell proliferation. The results further suggest that VEGI maybe highly valuable toward angiogenesis-based cancer therapy.     

Tension relaxation induced by pulse photolysis of caged ATP in partially crosslinked fibers from rabbit psoas muscle

We previously isolated gp17, a human seminal plasma glycoprotein, which specifically interacts with the D1-D2 region of CD4, a T cell surface molecule involved in antigen recognition mediated by helper T cells also acting as a receptor for the human immunodeficiency virus. In this study we report that monoclonal antibodies (mAb) reacting with gp17 are able to inhibit the binding of gp17 to immobilized soluble CD4. An immunohistochemical analysis shows that gp17 is also expressed in mammary tumor cells upon hormone treatment and in biopsies from breast cancer patients. A structural characterization of gp17, including amino acid sequencing, indicates that the protein has an extensive structural similarity with a glycoprotein designated as seminal actin-binding protein (SABP), also secreted by male sexual glands. SABP is in turn identical to gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP), a factor known as a highly specific and sensitive marker of primary and metastatic apocrine breast cancer. To establish further the correspondence of gp17 and GCDFP-15/PIP/SABP, the latter was expressed in bacteria from a cloned cDNA and purified by affinity chromatography to either anti-gp17 mAb-Sepharose or CD4-Sepharose. The purified recombinant protein is shown to inhibit the binding of labeled, pure g17 to immobilized soluble CD4. The finding that breast cancer cells express a protein able to interact with the CD4 domains involved in the recognition of class II major histocompatibility antigens suggests a possible mechanism by which a tumor may affect the activity of tumor-infiltrated CD4+ T cells.     

Stch encodes the 'ATPase core' of a microsomal stress 70 protein

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome-associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP-binding domains and truncates the carboxyl terminal peptide-binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane-bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium-inducible, microsome-associated ATPase activity with properties similar to a proteolytically cleaved N-terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.     

Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family

F4/80 is a monoclonal antibody that recognizes a murine macrophage-restricted cell surface glycoprotein and has been extensively used to characterize macrophage populations in a wide range of immunological studies. Apart from the tightly regulated pattern of expression of the F4/80 antigen, little is known about its possible role in macrophage differentiation and function. We have sought to characterize the molecule at the molecular level, through the isolation of cDNA clones, and now describe the sequence of the F4/80 protein. The primary amino acid sequence demonstrates homology to two protein superfamilies. The NH2-terminal region consists of seven epidermal growth factor-like domains, separated by approximately 300 amino acids from a COOH-terminal region that shows homology to members of the seven transmembrane-spanning family of hormone receptors. The potential role of these distinct domains is discussed with respect to the possible function of the F4/80 molecule.     

Isolation and characterization of human fast skeletal beta troponin T cDNA: comparative sequence analysis of isoforms and insight into the evolution of members of a multigene family

A cDNA encoding human fast skeletal beta troponin T (beta TnTf) has been isolated and characterized from a fetal skeletal muscle library. The cDNA insert is 1,000 bp in length and contains the entire coding region of 777 bp and 5' and 3' untranslated (UT) segments of 12 and 211 bp, respectively. The 3' UT segment shows the predicted stem-loop structure typical of eukaryotic mRNAs. The cDNA-derived amino acid sequence is the first available sequence for human beta TnTf protein. It is encoded by a single-copy gene that is expressed in a tissue-specific manner in fetal and adult fast skeletal muscles. Although the human beta TnTf represents the major fetal isoform, the sequence information indicates that this cDNA and the coded protein are quite distinct from the fetal and neonatal TnTf isoforms reported in other mammalian fetal muscles. The hydropathy plot indicates that human beta TnTf is highly hydrophilic along its entire length. The protein has an extremely high degree of predicted alpha-helical content involving the entire molecule except the carboxy-terminal 30 residues. Comparative sequence analysis reveals that the human beta TnTf shares a high level of sequence similarity in the coding region with other vertebrate TnTf and considerably reduced similarity with slow skeletal and cardiac TnT cDNAs. The TnT isoforms have a large central region consisting of amino acid residues 46-204 which shows a high sequence conservation both at the nucleotide and amino acid levels. This conserved region is flanked by the variable carboxy-terminal and an extremely variable amino-terminal segment. The tropomyosin-binding peptide of TnT, which is represented by amino acid residues 47-151 and also includes a part of troponin I binding region, is an important domain of this central segment. It is suggested that this conserved segment is encoded by an ancestral gene. The variable regions of vertebrate striated TnT isoforms reflect the subsequent addition and modification of genomic sequences to give rise to members of the TnT multigene family.     

Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor

A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (Ki = 1.1 and 0.31 nM, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.     

Cloning and sequencing of a cDNA encoding bovine intercellular adhesion molecule 3 (ICAM-3)

A cDNA encoding a putative bovine intercellular adhesion molecule (ICAM)-3, a ligand of the leukocyte integrin LFA-1 (CD11a/CD18), was sequenced and compared with human ICAM sequences. The 1635-bp bovine sequence codes for a protein of 544 amino acids (aa). This putative bovine ICAM-3 has five immunoglobulin (Ig)-like domains similar to human ICAM-1 and ICAM-3, and belongs to the Ig gene superfamily. The overall identities of the deduced aa sequence with those of human ICAM-3 and ICAM-1 are 61% and 58%, respectively. The predicted number and positions of Cys residues are all conserved between the bovine and human ICAM 3 aa sequences.