Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells

Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells

Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the a-chain of the human interleukin-2 receptor

The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the a-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2Rcc chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy

β1 Integrin Is Essential for Teratoma Growth and Angiogenesis

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of β1 integrin during teratoma formation, we compared teratomas induced by normal and β1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, β1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of β1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in β1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface

LST1 promotes the assembly of a molecular machinery responsible for tunneling nanotube formation

Carefully orchestrated intercellular communication is an essential prerequisite for the development of multicellular organisms. In recent years, tunneling nanotubes (TNT) have emerged as a novel and widespread mechanism of cell-cell communication. However, the molecular basis of their formation is still poorly understood. In the present study we report that the transmembrane MHC class III protein LST1 induces the formation of functional nanotubes and is required for endogenous nanotube generation. Mechanistically, we found LST1 to induce nanotube formation by recruiting the small GTPase RalA to the plasma membrane and promoting its interaction with the exocyst complex. Furthermore, we determined LST1 to recruit the actin-crosslinking protein filamin to the plasma membrane and to interact with M-Sec, myosin and myoferlin. These results allow us to suggest a molecular model for nanotube generation. In this proposal LST1 functions as a membrane scaffold mediating the assembly of a multimolecular complex, which controls the formation of functional nanotubes

LST1 promotes the assembly of a molecular machinery responsible for tunneling nanotube formation

Carefully orchestrated intercellular communication is an essential prerequisite for the development of multicellular organisms. In recent years, tunneling nanotubes (TNT) have emerged as a novel and widespread mechanism of cell-cell communication. However, the molecular basis of their formation is still poorly understood. In the present study we report that the transmembrane MHC class III protein LST1 induces the formation of functional nanotubes and is required for endogenous nanotube generation. Mechanistically, we found LST1 to induce nanotube formation by recruiting the small GTPase RalA to the plasma membrane and promoting its interaction with the exocyst complex. Furthermore, we determined LST1 to recruit the actin-crosslinking protein filamin to the plasma membrane and to interact with M-Sec, myosin and myoferlin. These results allow us to suggest a molecular model for nanotube generation. In this proposal LST1 functions as a membrane scaffold mediating the assembly of a multimolecular complex, which controls the formation of functional nanotubes

Full-Length L1CAM and Not Its Δ2Δ27 Splice Variant Promotes Metastasis through Induction of Gelatinase Expression

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression

Identification de protéines altérées au cours du processus métastasique

Le cancer constitue une des causes majeures de mortalité dans les pays économiquement développés où la durée moyenne de vie ne cesse de s'allonger. Afin d'identifier de nouveaux gènes susceptibles de contribuer à la progression métastasique du cancer, nous avons utilisé l'approche des micro-puces à ADN pour établir le profil d'expression génétique de lignées cellulaires tumorales isogéniques à caractère métastasique défini. L'analyse d'un premier modèle cellulaire a mis en évidence 59 gènes associés au potentiel métastasique, mais ne nous a pas permis d'identifier d'ESTs dérégulées. Nous avons donc étudié un deuxième modèle cellulaire, BSp73. De nombreux gènes (457) et ESTs (1555) sont clairement dérégulés au sein de la lignée métastasique par rapport à la lignée non métastasique. Le nouveau gène smagp (small cell adhesion glycoprotein) a été identifié à partir d'une sélection d'ESTs surexprimées. Il s'agit d'une glycoprotéine de faible poids moléculaire contenant des domaines de liaisons aux protéines 4.1 et MAGUK. SMAGP est exprimée dans la membrane latérale des structure épithéliales de tissus normaux de côlon et du sein. Dans les tumeurs invasives et peu différenciées de ces organes respectifs, l'expression de SMAGP est diminuée et délocalisée dans le cytoplasme, illustrant le potentiel de SMAGP en tant que marqueur de dé-différenciation et de progression tumorale. L'étude fonctionnelle de SMAGP par expression ectopique dans des transfectants stables a montré que SMAGP inhibe l'adhésion cellulaire à la vitronectine. Nous avons proposé que SMAGP soit impliquée dans la polarisation et l'adhésion cellulaire, et soit associée à la perte adhésion et de différenciation relatives à la progression tumorale.Cancer is one of the leading cause of death in industrialised countries, since life expectancy is constantly increasing. In order to identify new genes implicated in the metastatic progression of cancer, we have been using the DNA chip technology to establish the transcriptionnel profil of isogenic tumor cell lines with defined metastatic potential. The analysis of the first cellular model showed 59 genes associated with metastatic potential, but no deregulated ESTs were detected. We then worked on a second cellular model, BSp73. Numerous genes (457) and ESTs (1555) were clearly deregulated in the metastatic cell line in comparaison to the non metastatic cell line. The new gene smagp (small cell adhesion glycoprotein) was identified from a selected and upregulated EST. This gene code for a small glycoprotein with binding domains for protein 4.1 and MAGUKs. SMAGP is expressed in the lateral cellular membrane in epithelial structure of colon and breast normal tissue. In corresponding invasive tumours, SMAGP is downregulated and delocalised to the cytoplasma, illustrating the potential of SMAGP as de-differentiation and tumor progression marker. The function of the SMAGP protein was analysed with SMAGP stable transfectants, and it was demonstrated that SMAGP expression inhibits cellular adhesion to vitronectin. We have suggested that SMAGP is implicated in cellular polarisation and adhesion, and is associated to loss of adhesion and loss of differenciation relative to tumour progression.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF