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PubMed1988

Effect of human blood mononuclear cell populations in antibody dependent cellular cytotoxicity (ADCC) using two murine (CO17-1A and Br55-2) and one chimeric (17-1A) monoclonal antibodies against a human colorectal carcinoma cell line (SW948).

Peripheral blood mononuclear cells (PBMC) from healthy individuals were studied for their lytic capability in ADCC using SW948 (a human colorectal carcinoma cell line) as target cells. Three monoclonal antibodies (MAbs) were used: two mouse MAbs (IgG2A) against the antigenic structures CO17-1A and BR55-2 respectively and one chimeric MAb 17-1A (IgG1) (mouse-human). Three kinds of effector cells were prepared. PBMC were purified on a Ficoll-Isopaque gradient (FIP cells) (a mixture of lymphocytes and monocytes). To obtain pure monocytes (greater than 90%), PBMC were centrifuged on a Nycodenz gradient (Nycodenz cells). Highly purified lymphocytes (greater than 98%) were obtained by treatment of FIP cells with iron powder and removal of phagocytic cells (PBL cells). Monocytes had the highest lytic capability. FIP cells were less effective than monocytes. PBL cells had the poorest killing activity. In reconstitution experiments addition of increasing amount of monocytes to PBL resulted in an augmented cytotoxicity. The numbers of Leu-M3+ cells, Leu-M5+ cells (monocytes) and CD16+ cells correlated positively to cytotoxicity. Higher concentration of MAb 17-1A was required to reach the same level of cytotoxicity using FIP cells as effector cells as compared to monocytes. MAb BR55-2 induced the same cytotoxic activity as MAb 17-1A. Combination of these two MAbs did not increase the lytic capability. Chimeric MAb 17-1A mediated ADCC in a dose-dependent fashion. The chimeric MAb was consistently more effective than the mouse MAb.

Animals↗
PubMed1993

Cloning of cDNAs encoding the variable domains of antibody BrE-3 and construction of a chimeric antibody.

BrE-3 is an IgG1,kappa murine monoclonal antibody that binds to human breast epithelial mucin and that has been shown to be promising for imaging and treatment of breast cancer. We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of BrE-3. VL belongs to group II and resulted from a V kappa-J kappa 1 fusion. VH belongs to group IIIc and arose from a V-D-JH3 non-conservative fusion which left little or nothing of the original D minigene. Thus, the third VH CDR contains only 4 amino-acids. We constructed an IgG1,kappa human/mouse chimeric antibody (by joining the murine variable domains to human constant domains) and expressed it in SP2/0 myeloma cells. This chimeric monoclonal antibody stains breast carcinoma tissue sections by the ABC immunoperoxidase method. Its affinity for the BrE-3 antigen is 2.68 x 10(8) M-1, which, considering the experimental error, is indistinguishable from the affinity of the original murine antibody (3.75 x 10(8) M-1). The VL and VH domains alone are respectively 73%, and 63% identical to the human V kappa II and VHIII consensus sequences. If the CDRs are excluded, these numbers become respectively 82% and 80%. Therefore, we expect the reported chimeric BrE-3 to be considerably less immunogenic to humans than the original murine antibody, while retaining the original binding properties.

Amino Acid Sequence↗
PubMed1999

cDNA sequence analysis of monoclonal antibody FU-MK-1 specific for a transmembrane carcinoma-associated antigen, and construction of a mouse/human chimeric antibody.

Mouse monoclonal antibody (MAb) FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a transmembrane antigen, GA733-2, present on most adenocarcinomas and seems to be of potential utility for immunodiagnosis and immunotherapy of those cancers. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (V(H) and Vkappa) of FU-MK-1 using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch FU-MK-1, by fusing the FU-MK-1 V(H) and Vkappa genes to the human Cgamma1 and Ckappa genes, respectively, and by ligating the chimeric H and L chain genes to each other in a mammalian cell expression vector. The final gene construct was transfected into mouse non-Ig-producing hybridoma cells by electroporation. The Ch FU-MK-1 antibody thus prepared bound to human adenocarcinoma cells and competitively inhibited the binding of the parental FU-MK-1 to the adenocarcinoma cells. Ch FU-MK-1 also showed a potent antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral blood mononuclear cells as effectors against the adenocarcinoma cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.

Adenocarcinoma↗
PubMed2000

Accelerated assessing of antisense RNA efficacy using a chimeric enhanced green fluorescent protein-antisense RNA-producing vector.

The selection of suitable parts of a gene as antisense RNA sequences is largely a matter of trial and error and, as a consequence, a rather time-consuming process. In this study, we present a rapid and reproducible method to bypass this protracted procedure by using a chimeric enhanced green fluorescent protein (EGFP)-antisense RNA-producing vector. The combination of a reporter gene and antisense RNA allows easy measurement by flow cytometry of antisense RNA efficacy in successfully transfected cells shortly after transfection. Four chimeric EGFP-p185c-erbB-2-antisense RNA vectors were constructed and transfected into the p185-c-erbB-2-overexpressing cell line SKBR3. Within 1 week, we were able to estimate the inhibitory capacities of the different antisense RNA sequences used in this study. Our results strongly suggest that a chimeric EGFP-antisense RNA vector is an appropriate tool to expedite the laboratory work and time in screening the efficacy of antisense RNA strategies.

Cell Division↗
PubMed2005

Potent inhibitory activity of chimeric oligonucleotides targeting two different sites of human telomerase.

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.

Base Sequence↗
PubMed1998

Clonal analysis of urothelial carcinomas in C3H/HeN<-->BALB/c chimeric mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine.

The histological background for multifocal and metachronous development of urothelial carcinomas remains equivocal, although accumulated genetic evidence suggests monoclonal origin of multiple urothelial carcinomas. Clonal development of various preneoplastic and neoplastic urothelial lesions of C3H<-->BALB/c chimeric mice induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was immunohistochemically investigated using a C3H strain-specific antibody. All tumor masses induced in the mice treated with 0.05% BBN for 20 weeks were composed of neoplastic cells of a single parental type, which is indicative of monoclonal lesions. Three of 10 animals harbored two or more separate carcinomas of different clonal type, which is indicative of multicentric development applicable in this model. Using DNAs derived from urothelial carcinomas and tumor-adjacent urothelium of chimeric mice, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing were performed for p53 gene exons 5-7. p53 mutations were identified in four of 11 (36%) dysplasias and non-invasive carcinomas (carcinoma in situ and pTa tumor) and 13 of 22 (59%) invasive carcinomas. Only in a single case were identical p53 mutations found in separate urinary bladder carcinomas. In contrast to the random distribution of urothelial proliferating units in chimeric mice without chemical supplement, invasive carcinomas in BBN-treated mice were accompanied by widely-distributed preneoplastic and neoplastic lesions of the same clonality, which occasionally had frequent foci of microinvasion. This is indicative of lateral clonal expansion of the clones, which precedes the bulk of invasive carcinomas. Thus, two aspects of 'field change' of the urothelium became evident in this model: either independent transformation events or lateral clonal expansion might, respectively, result in multicentric and monoclonal carcinoma development in the urinary tract.

Animals↗
PubMed2003

Human galectin-1 recognition of poly-N-acetyllactosamine and chimeric polysaccharides.

Human galectin-1 is a dimeric carbohydrate binding protein (Gal-1) (subunit 14.6 kDa) widely expressed by many cells but whose carbohydrate binding specificity is not well understood. Because of conflicting evidence regarding the ability of human Gal-1 to recognize N-acetyllactosamine (LN, Galbeta4GlcNAc) and poly-N-acetyllactosamine sequences (PL, [-3Galbeta4GlcNAcbeta1-]n), we synthesized a number of neoglycoproteins containing galactose, N-acetylgalactosamine, fucose, LN, PL, and chimeric polysaccharides conjugated to bovine serum albumin (BSA). All neoglycoproteins were characterized by MALDI-TOF. Binding was determined in ELISA-type assays with immobilized neoglycoproteins and apparent binding affinities were estimated. For comparison, we also tested the binding of these neoglycoconjugates to Ricinus communis agglutinin I, (RCA-I, a galactose-binding lectin) and Lycopersicon esculentum agglutinin (LEA, or tomato lectin), a PL-binding lectin. Gal-1 bound to immobilized Galbeta4GlcNAcbeta3Galbeta4Glc-BSA with an apparent K(d) of approximately 23 micro M but bound better to BSA conjugates with long PL and chimeric polysaccharide sequences (K(d)'s ranging from 11.9 +/- 2.9 microM to 20.9 +/- 5.1 micro M). By contrast, Gal-1 did not bind glycans lacking a terminal, nonreducing unmodified LN disaccharide and also bound very poorly to lactosyl-BSA (Galbeta4Glc-BSA). By contrast, RCA bound well to all glycans containing terminal, nonreducing Galbeta1-R, including lactosyl-BSA, and bound independently of the modification of the terminal, nonreducing LN or the presence of PL. LEA bound with increasing affinity to unmodified PL in proportion to chain length. Thus Gal-1 binds terminal beta4Gal residues, and its binding affinity is enhanced significantly by the presence of this determinant on long-chain PL or chimeric polysaccharides.

Carbohydrate Conformation↗
PubMed1993

Effects of isotype and Fc region on in vitro function of a mouse/human chimeric CD4 antibody.

Murine CD4 mAbs have shown potential for the treatment of allograft rejection and autoimmune disorders including rheumatoid arthritis. Clinical usefulness of the murine mAbs has been limited by immunogenicity and a short circulating half-life. Mouse/human chimeric antibodies have been constructed, composed of the variable region of M-T412 (a murine G2a mAb specific for the human CD4 molecule) and human G1 (cM-T412 G1) or G4 (cM-T412 G4) Fc regions. F(ab')2 and F(ab) fragments of the murine G2a and chimeric G1 mAbs were generated by enzymatic digestion. The chimeric mAbs and all fragments retained the avidity and specificity of the murine M-T412 and were evaluated in in vitro assays measuring Ig production by pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC), sIL-2R produced by phytohemagglutinin-stimulated PBMC, and proliferation in response to tetanus toxoid, CD3 mAb plus IL-2, and mixed lymphocyte response (MLR). When PBMC were stimulated with tetanus toxoid, 10 ng/ml of cM-T412 G1 inhibited proliferation by 90%, while neither the cM-T412 G4, M-T412 G2a, nor any mAb fragment produced > 65% inhibition, even at 1000-fold higher concentrations. A similar pattern of inhibition was observed in MLR assays. In contrast, the F(ab')2 fragment of the cM-T412 G1 was as effective as the whole antibody in inhibiting PWM-stimulated IgM synthesis and PBMC proliferation in response to stimulation by a CD3 mAb plus IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Induction of cell death by chimeric L-selectin-Fas receptors.

In the present study, we have established a system where engagement of an adhesion molecule triggers a death signal into cells. L-selectin, which is a well characterized adhesion receptor involved in the initial adhesion between lymphocyte and endothelium, was fused to the intracellular domain of an apoptosis-inducing molecule, Fas. Ligation of the chimeric receptors with a carbohydrate ligand for L-selectin, fucoidin or a mAb that recognizes the lectin domain of L-selectin, induced apoptosis in receptor-expressing cells. However, ligation with an anti-L-selectin mAb reactive with a non-ligand binding site did not induce apoptosis, indicating that stimulation through the lectin domain of L-selectin in the chimeric receptor leads to signal delivery. Upon activation L-selectin shows a unique proteolytic cleavage at the membrane proximal site on the extracellular (EC) domain, of which the significance is also unclear. We found that truncations in the EC domain which abrogate the proteolytic cleavage of L-selectin did not influence induction of apoptosis, suggesting that the cleavage on the EC domain itself is not important for the signaling function of the chimeric receptor. This is the first demonstration that an adhesion signal can be converted to a signal that leads to apoptotic cell death.

Animals↗
PubMed2005

Peptide-loaded chimeric influenza virosomes for efficient in vivo induction of cytotoxic T cells.

Virus-specific CD8(+) T cells are thought to play an important role in resolving acute hepatitis C virus (HCV) infection as viral clearance has been associated with a strong and sustained CD8(+) T cell response. During the chronic state of HCV infection virus-specific T cells have a low frequency and a reduced responsiveness. Based on this, a therapeutic vaccine increasing the frequency of specific T cells is a promising alternative for the treatment of chronic HCV infection. We improved an existing vaccine platform based on immunopotentiating reconstituted influenza virosomes (IRIVs) for efficient delivery of peptide epitopes to the MHC class I antigen presentation pathway. IRIVs are proteoliposomes composed of phospholipids and influenza surface glycoproteins. Due to their fusogenic activity, IRIVs are able to deliver encapsulated macromolecules, e.g. peptides to immunocompetent cells. We developed a novel method based on chimeric virosomes [chimeric immunopotentiating reconstituted influenza virosomes (CIRIVs)] combining the high peptide-encapsulation capacity of liposomes and the fusion activity of virosomes. This new approach resulted in a 30-fold increase of the amount of incorporated soluble peptide compared with current preparation methods. To study the immunogenicity of chimeric virosomes HLA-A2.1 transgenic mice were immunized with CIRIVs containing the HCV Core132 peptide. Core132-CIRIVs efficiently induced specific cytotoxic and IFNgamma-producing T cells already with low peptide doses. Vaccine formulations, which include combinations of different HCV-derived CTL epitopes could be used to induce not only a strong but also a multi-specific CTL response, making them potential candidates for therapeutic and maybe prophylactic T cell vaccines in humans.

Adjuvants, Immunologic↗
PubMed1988

Mouse/human chimeric antibodies to a tumor-associated antigen: biologic activity of the four human IgG subclasses.

Variable region genes from mouse monoclonal antibody 17-1A (gamma 2a kappa) with specificity for human gastrointestinal malignancies have been paired with human immunoglobulin constant region genes (for heavy and light chains) to produce mouse/human chimeric immunoglobulin molecules (chIgG) for each of the four human IgG subclasses. Mouse 17-1A and the four chIgG bound similarly to two human colon cancer cell lines and had comparable binding affinities. The chIgG1 and chIgG3 molecules mediated lymphocyte and monocyte antibody-dependent cell-mediated cytotoxicity (ADCC) to colon cancer tumor cell lines comparable to that of the parent murine 17-1A. The chIgG2 and chIgG4 molecules were able to mediate ADCC to colon cancer cell lines but were clearly inferior to the chIgG1 and chIgG3 reagents. None of the chIgG antibodies or the murine 17-1A was able to mediate complement lysis of colon cancer cell lines. These studies demonstrate the ability to produce all four human IgG subclass chimeric molecules which retain biologic activity. We have confirmed the subclass preferences of human lymphocyte and monocyte Fc receptors for human IgG subclasses previously determined by studies with monomeric or aggregated IgG. These data may aid in the selection of chimeric antibodies for in vivo trials.

Animals↗
PubMed1993

Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.

Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure--function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides at noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (kcat = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (kcat) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.

Animals↗
PubMed1995

Correct splicing of a group II intron from a chimeric reporter gene transcript in tobacco plastids.

An in vivo test system was developed to study group II intron splicing in higher plant chloroplasts. The chimeric reporter gene uidA was constructed by translational fusion of an intron-containing segment of the plastid atpF gene with the coding region of a plastid uidA reporter gene. The chimeric uidA gene was inserted into the tobacco plastid genome by the biolistic transformation procedure using a plastid targeting vector. Correct intron excision was confirmed by Northern blot analysis, by sequencing amplified cDNAs and by accumulation of the encoded beta-glucuronidase (GUS), the expression of which was dependent on intron removal. Removal of the intron from the uidA mRNA is less efficient (< 50%) than from the atpF mRNA (> 90%). The efficiency of atpF mRNA splicing is not affected in the plasmid transformants indicating that inefficient splicing of the highly-expressed uidA mRNA is not due to depletion of factor(s) required for the atpF intron removal. A derivative of uidA, with a stop codon introduced into the loop of domain VI, was also tested. The mutations did not affect the splicing efficiency. The chimeric uidA splicing system will facilitate the study of structural and sequence requirements for group II intron splicing in plastids of higher plants.

Base Composition↗
PubMed2000

Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains.

This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme FOK:I. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by approximately 4 bp with a 5' overhang, as for native FOK:I. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of the VH and V kappa genes of an anti-CD3 monoclonal antibody, and construction of a mouse/human chimeric antibody.

Mouse monoclonal antibodies against CD3 on human T lymphocytes have been used for therapy in organ-transplant patients as a potent immunosuppressive agent or for treatment of cancer as a potent T cell activating agent. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (VH and V kappa) of a mouse anti-human CD3 monoclonal antibody (OKT3) using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch OKT3, by fusing the OKT3 VH and V kappa genes to the human heavy and light chain constant region genes (C gamma 1 and C kappa) derived from a human plasma cell leukemia line (ARH77), respectively. The chimeric gene constructs were sequentially co-transfected into mouse non-Ig-producing hybridoma cells (Sp2/0) by electroporation. The Ch OKT3 antibody thus prepared bound to human peripheral blood mononuclear cells and competitively inhibited the binding of the parental MAb OKT3 to the blood mononuclear cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.

Amino Acid Sequence↗
PubMed1993

Preparation of mouse-human chimeric antibody to an embryonic carbohydrate antigen, Lewis Y.

Stage-specific embryonic antigen-1 (SSEA-1) is a well-known carbohydrate antigen that is specifically expressed on the surface of cancer cells as well as embryonic cells. In this study, starting with a previously established hybridoma producing a monoclonal antibody (named H18A) to Lewis Y antigen, which is closely related to SSEA-1, we cloned genomic DNA encoding active variable regions both of heavy and light chains of the antibody. Sequence analysis showed that VH and V kappa genes of H18A were in the VH 7183 family and V kappa C1 family, respectively. A transfected cell line named HC-H18A-7 expressing a recombinant chimeric H18A composed of mouse-derived antigen-binding variable regions and human-derived constant regions was established. The chimeric H18A was purified to homogeneity and shown to bind purified Lewis Y antigen with the same dose-response curve as the original H18A. The chimeric H18A looks more promising for clinical application than the original mouse-derived H18A because its antigenicity is expected to be reduced.

Amino Acid Sequence↗
PubMed2005

A chimeric AtMYB23 repressor induces hairy roots, elongation of leaves and stems, and inhibition of the deposition of mucilage on seed coats in Arabidopsis.

We reported previously that a chimeric repressor, in which a transcription factor was fused to the EAR motif repression domain, acted as a dominant repressor and suppressed the expression of target genes, such that resultant phenotypes were similar to those associated with loss-of-function alleles. We report here that expression of the chimeric AtMYB23 repressor induced a variety of morphological changes, namely the ectopic formation of root hairs, a short primary root, elongation of leaves and of inflorescence stems, and absence of the accumulation of mucilage on seed coats, in addition to disruption of the development of trichomes. The short primary root and the elongation of leaves and stems appeared to be due to the reduced and enhanced lengthwise expansion, respectively, of epidermal cells. Expression of the GL2 gene, which is involved in the formation of root hairs and the accumulation of mucilage, was suppressed in both the roots and siliques of the transgenic plants. In contrast, the expression of genes related to cell elongation, such as DWF1, SAUR, AQP, AGP15, DET3 and XET-1, was enhanced in leaves of the transgenic plants. Results suggest that the AtMYB23 transcription factor has the molecular function of regulating the development of epidermal cells not only in leaves but also in stems, roots and seeds. We describe that this type of chimeric repressor can be exploited as a useful tool for the functional analysis of redundant transcription factors.

Amino Acid Sequence↗
PubMed1999

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding.

The crystallographic study of chimeric B72.3 antibody illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.

Amino Acid Sequence↗