Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies

Abstract

The human hybridoma technique offers an important approach for isolation of human monoclonal antibodies. A diversity of approaches can be used with varying success. Recent technical advances in expanding the starting number of human antigen-specific B cells, improving fusion efficiency, and isolating new myeloma partners and new cell cloning methods have enabled the development of protocols that make the isolation of human monoclonal antibodies from blood samples feasible. Undoubtedly, additional innovations that could improve efficiency are possible. HISTORY OF HYBRIDOMASMonoclonal antibodies (mAbs) have revolutionized the conduct of science since their first description in 1975 (1). The use of these specific reagents also has made possible improved clinical diagnostics in the medical arena, and many antibodies have found their way to clinical use as prophylactic or therapeutic agents. Nevertheless, the potential of mAbs derived specifically from technology based on human hybridomas remains largely unfulfilled. The principal reason for the lack of a large number of hybridoma-derived mAb therapeutics has simply been the technical difficulty in generating stable hybridomas that secrete human mAbs of high affinity and functional activity. This chapter reviews recent efforts to develop and employ novel methods for the efficient generation of human hybridomas secreting human mAbs for clinical use.The principal advantage of the use of human hybridoma technology for mAb generation is that this approach preserves the authentic sequence and pairing of antibody DNA from a natural B cell for the expression of a naturally occurring full-length human mAb. There are significant theoretical advantages for expressing cDNAs encoding authentic heavy and light chains with chains that are paired using the coding sequence as it was generated naturally through B cell selection, class switch, and affinity maturation. No genetic modification of these sequences is required. Since antibody expression is typically very stable in hybridomas, sequence amplification of antibody variable genes is achieved easily if recombinant production or manipulation is desired. The resulting recombinant mAb retains most features of naturally occurring human antibodies, as the clone retains the native amino acid sequence and heavy/light chain pairing. Since the native constant region of the antibody in the original human B cell is retained in the mAb expressed by the resulting hybridoma, the functional properties of the particular Fc region can be studied for Fc-mediated activities, such as antibodydependent cellular cytotoxicity.

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

“…We isolated nine human mAbs from four human donors targeting the postfusion RSV F protein using human hybridoma technology (16). Transformed B cells generated from the B cells of adult human donors were screened by ELISA for reactivity to the RSV A2 F protein.…”

Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies

“…The data presented here suggest that surrogate systems such as chimeric VSV expressing filovirus GP may produce misleading results and therefore appear to be suboptimal for the reliable characterization of filovirus antibodies. The simultaneous circulation of multiple lineages of filoviruses in the same outbreak (46,47), along with the emergence of "new" filoviruses (5, 6), and improved methods for isolation of MAbs from survivors (34,38,48), along with the utility of their use in therapeutics, strongly support the need for improved efficient means of rapid screening and characterization of large panels of MAbs. Although a previous study demonstrated the possibility of the generation of viable ebolaviruses with some genes replaced with counterparts from a heterologous ebolavirus (49), the present study demonstrates that EBOV can easily tolerate exchange of GP not only from ebolaviruses but also from more distantly related marburgvirus and cuevavirus.…”

Monoklonal Antikor Teknolojisinin Dünü, Bugünü Ve Geleceği

“…Söz konusu hücre hattının EBV ile transforme edilmiş hücrelerden, bademcik ve kan lenfosit hücrelerine kadar farklı hücreler ile birleştirilerek stabil biçimde antikor salgılama yetisine sahip hibritler oluşturabildikleri ve bunların uzun süreli ve yeterli seviyelerde antikor salgılayabildikleri rapor edilmiştir 11 . Ancak farklı kaynaklar tarafından yapılan bağımsız çalışmalarda başarılı sonuçların elde edilebildiği durumların oldukça nadir olduğu ifade edilmektedir 19 . …”

“…Bu kapsamda kullanılan heteromiyeloma hücreleri, belirli bir insan hücre hattı (sıklıkla miyeloma hücreleri) ile fare miyeloma hücrelerinin füzyonu sonucunda elde edilmektedir 19 . Bu kapsamda yapılan çalışmalarda geliştirilen heteromiyeloma hücre hatlarında insan kökenli partner olarak insan miyeloma hücre hatlarının yanı sıra [22][23][24][25] , IgA miyeloma hastası bireyden izole edilen kemik iliği mononükleer © Kocaeli Üniversitesi Sağlık Bilimleri Dergisi, 2016 SBD16-1.2 SBD 2016 S.1, C.2, s.6-14…”

“…Bu kapsamda, SARS enfeksiyonuna maruz kalarak, kurtulan bir hastadan 35 ayrı antikor izole edilebilmiştir 11 . Bu teknik sayesinde ayrıca aşağıda daha detaylı olarak bahsi geçen RT-PCR (ters transkriptaz ile polimeraz zincir reaksiyonu) ile ilgilenilen antikora ait Fv protein zincirine (Çizim-3) ulaşılması işlemi daha kolay uygulanabilir bir hal almıştır 19 . Bununla beraber, söz konusu ölümsüzleştirme, fiilen hücrelerin ancak füzyon sürecine kadar geçen zaman içerisindeki karakterizasyon vb.…”