Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family‐specific oligonucleotide probes

Marks, James D.; Tristem, Michael; Karpas, Abraham; Winter, Greg

doi:10.1002/eji.1830210419

Cited by 209 publications

(96 citation statements)

References 36 publications

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“…We constructed six phage-display libraries of Fvs isolated from patients who had developed neutralizing antibodies to RITs (10,11), and selected for those phage that bound specifically to PE38. Fig.…”

Section: Resultsmentioning

confidence: 99%

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Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

Liu

Onda

Lee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

143

140

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Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydrophilic amino acids to alanine, yet retained full antitumor activity. Here we present results in which we identify and silence human B-cell epitopes in the RIT HA22. We obtained B cells from patients with antibodies to RITs, isolated the corresponding variable fragments (Fvs), and constructed a phage-display library containing Fvs that bind to the RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently overlap but are not identical. Most mutations that remove mouse epitopes did not remove human epitopes. Using the epitope information, we constructed a variant immunotoxin, HA22-LR-LO10, which has low reactivity with human antisera, yet has high cytotoxic and antitumor activity and can be given to mice at high doses without excess toxicity. The toxin portion of this RIT (LR-LO10) can be used with Fvs targeting other cancer antigens and is suitable for clinical development.cancer treatment | immunotherapy | leukemia | mesothelioma | protein engineering

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Section: Resultsmentioning

confidence: 99%

“…PAXgene tubes were used to extract RNA from blood samples of six patients who had made neutralizing antibodies to immunotoxins (PreAnalytiX/Qiagen) (Dataset S1). The scFv phage library was constructed as described previously; scFv fragments were digested with NcoI and NotI and subcloned into pCANTAB 5E (10,11).…”

Section: Methodsmentioning

confidence: 99%

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

Liu

Onda

Lee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

143

140

View full text Add to dashboard Cite

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“…For analysis of V H genes a set of 5 0 primers specific for human V H 1/V H 5/V H 7, V H 2, V H 3, V H 4 and V H 6 gene families containing a PstI restriction site [12] in combination with a 3 0 primer specific for the first exon of the constant region (position 134-139) [1] containing a SalI restriction site was employed (Fig. 1).…”

Section: Amplification Of V H Genesmentioning

confidence: 99%

VH genes expressed in peripheral blood IgE-producing B cells from patients with atopic dermatitis

Tilgner

Golembowski

Kersten

et al. 1997

Clinical and Experimental Immunology

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SUMMARYIn this study we analysed the V H genes expressed in IgE-producing peripheral B cells in two patients with severe atopic dermatitis. We found a diverse antibody repertoire containing V H genes of five different V H families, no V H 2 and 7 genes, with no biased usage of one particular V H gene family. Some V H germ-line segments were found to be used several times within the e transcripts (V3-23, V3-30).Comparison of the IgE-encoding V H genes with the respective germ-line elements revealed the expression of nearly germ-line identical as well as somatically mutated V H genes. In these latter V H genes base substitutions were distributed over both complementarity determining and framework regions. Because of the similarities between the IgE committed B cell repertoire in atopic patients and the peripheral B cell repertoire in non-atopic individuals we conclude that the atopic V H gene repertoire probably reflects the result of an increased class switch of polyclonal B cells, including virgin and mature B cells, in response to a Th2 cytokine profile.

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“…Both the V H and V L gene repertoires were PCR amplified by using the cDNA and a previously constructed scFv-phage library plasmid (28) as templates. To amplify the V H and V L genes from the cDNA and plasmid template, the primers were designed based on those published previously and the most recent gene segments entered in the V-Base sequence directory (29,(35)(36)(37). All primary PCR reactions were carried out with separate backward primers and combined forward primers.…”

mentioning

confidence: 99%

A method for the generation of combinatorial antibody libraries using pIX phage display

Gao

Mao

Kaufmann

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

117

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For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 ؋ 10 9 members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis (''ricin'') agglutinins (RCA60 and RCA120), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens.

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Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family‐specific oligonucleotide probes

Cited by 209 publications

References 36 publications

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

VH genes expressed in peripheral blood IgE-producing B cells from patients with atopic dermatitis

A method for the generation of combinatorial antibody libraries using pIX phage display

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