Identification of a panel of tumor-associated antigens from breast carcinoma cell lines, solid tumors and testis cDNA libraries displayed on lambda phage

Kenton Labs, c/o Sigma Tau, Pomezia, Rome, Italy.
BMC Cancer (Impact Factor: 3.36). 11/2004; 4(1):78. DOI: 10.1186/1471-2407-4-78
Source: PubMed


Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX).
Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer.
A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27).
Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.

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    • "This study also utilized the λ KM8 and λ KM10 phage constructs, which were originally constructed based on KM4 phage that can accept inserts of up to 3 kb in length without disturbing λ packaging (Minenkova et al. 2003). Both the use of an N-terminal fusion and engineered phage allowed for simpler assembly of chimeric phage, but by using enhanced green fluorescent protein, or eGFP, as a fusion partner, an N-terminal fusion would not allow eGFP to fold properly, and hence fluorescence would not be possible (Pavoni et al. 2004). Zucconi et al. (2001) determined that the number of tolerable fusion proteins displayed on the capsid surface was based on the length and composition of the foreign amino acid sequence. "
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    ABSTRACT: Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique formation of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future applications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control.
    Applied Microbiology and Biotechnology 01/2014; 98(7). DOI:10.1007/s00253-014-5521-1
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    • "patients with colo - rectal carcinomas is biased toward intracellular antigens ( Roovers et al . , 2001 ) . It is quite common that naturally occurring anti - bodies recognize preferably intracellular tumor antigens . Also in SEREX - based screening ( Sahin et al . , 1995 ) and related methods ( Sioud and Hansen , 2001 ; Minenkova et al . , 2003 ; Pavoni et al . , 2004 ) , where tumor cDNA libraries are expressed in bacteria and specific tumor antigens inducing immune response in autolo - gous patients are identified by antibodies from patient sera , the same phenomenon was noted . The antigens identified by SEREX are prevalently cytoplasmic proteins . Cytoplasmic proteins are probably more immunogeni"
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    ABSTRACT: Generation of human recombinant antibody libraries displayed on the surface of the filamentous phage and selection of specific antibodies against desirable targets allows production of fully human antibodies usable for repeated administration in humans. Various lymphoid tissues from immunized donors, such as lymph nodes or peripheral blood lymphocytes from individuals with tumor or lymphocytes infiltrating tumor masses may serve as a source of specific anti-tumor antibody repertoire for generation of tumor-focused phage display libraries. In the case of lack of tumor-associated antigens in the purified form, high affinity anti-tumor antibodies can be isolated through library panning on whole cells expressing these antigens. However, affinity selection against cell surface specific antigens within highly heterogeneous population of molecules is not a very efficient process that often results in the selection of unspecific antibodies or antibodies against intracellular antigens that are generally useless for targeted immunotherapy. In this work, we developed a new cell-based antibody selection protocol that, by eliminating the contamination of dead cells from the cell suspension, dramatically improves the selection frequency of anti-tumor antibodies recognizing cell surface antigens.
    Molecular Immunology 11/2013; 57(2):317-322. DOI:10.1016/j.molimm.2013.10.009
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    • "At least five copies of α-peptide per phage tail were incorporated in the phage bearing a normal copy of the V gene and grown on host strain expressing α-peptide as a C-terminal fusion to full-length gpV [37]. In our two-gene based vector system, λKM10 [17], the recombinant bacteriophages AP/α-CEA-λ or GFP/α-CEA-λ have mosaic structure of their tails, composed of native gpV and truncated gpV fused to scFv gene. In this case about half of total gpV incorporated in the phage tail are fused to anti-CEA scFv antibody. "
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    ABSTRACT: Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA. In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles.
    BMC Biotechnology 09/2013; 13(1):79. DOI:10.1186/1472-6750-13-79
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