High efficiency creation of human monoclonal antibody-producing hybridomas

Dessain, Scott; Adekar, Sharad P.; Stevens, Jennifer B.; Carpenter, Katherine A.; Skorski, Maria L.; Barnoski, Barry L.; Goldsby, Richard A.; Weinberg, Robert A.

doi:10.1016/j.jim.2004.05.005

Cited by 44 publications

(22 citation statements)

References 51 publications

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“…Reverse Transcription-PCR Amplification of Immunoglobulin Variable Domain Sequences-Antibody variable domain cDNA sequences were amplified with consensus primer sets specific for human HC ␥, , and LCs (36). RNA was isolated from hybridomas using RNA Stat 60 (Tel-Test, Friendswood, TX).…”

Section: Methodsmentioning

confidence: 99%

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

Adekar

Klyubin

Macy

et al. 2010

Journal of Biological Chemistry

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We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of A␤, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig ␥ heavy chains. A ␥ 1 heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloidladen tissue sections. F1 did not bind to native A␤ monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig ␥ heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble A␤ oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig ␥ heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules. Alzheimer disease (AD)3 is approaching global epidemic proportions and is the most common of over 25 incurable protein misfolding diseases that are termed the amyloidoses (1, 2). A hallmark of the disease is deposition of amyloid fibril-containing neuritic plaques that are formed by abnormal processing of ␤-amyloid protein (A␤), a proteolyzed transmembrane 39 -43 fragment of amyloid precursor protein (3). Diverse experimental studies support a central pathogenic role for aggregated A␤, which in the brain initiates a cascade of events that ultimately lead to neuronal dysfunction and death (4 -6). The most neurotoxic A␤ species are low molecular weight oligomers (5), which include noncovalently linked species (7-9) and dityrosine cross-linked ␤-amyloid protein species (CAPS) (10).Active vaccination with A␤ or passive administration of anti-A␤ antibodies has shown promise in transgenic AD animal models and in some AD patients by inducing neuritic plaque clearance, neutralizing neurotoxic soluble A␤ oligomers, and/or improving cognitive functioning (6,8,(11)(12)(13). Immunotherapy has generally targeted linear sequence epitopes on A␤ (13). However, antibodies that do not distinguish between pathologic A␤ conformers and the monomeric peptide may have adverse effects because the native peptide has been implicated in normal lipid and cholesterol homeostasis (14). Of potentially greater use are antibodies that cross-react with conformational epitopes on pathogenic aggregated A...

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Section: Methodsmentioning

confidence: 99%

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

Adekar

Klyubin

Macy

et al. 2010

Journal of Biological Chemistry

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“…Recently, a heterohybridoma fusion partner cell line was derived from the murine myeloma cell line Sp2 and modified to coexpress genes encoding murine interleukin-6 and human telomerase catalytic subunit (TERT) (81). The expression of murine IL-6 directly stimulates proliferation as well as immunoglobulin production from the resulting hybridoma.…”

Section: Fusion Partnersmentioning

confidence: 99%

“…The Sp2/mIL-6/hTERT heterohybridoma line then was demonstrated to produce stable hybridomas able to secrete fully human mAbs by fusing splenic B cells from a patient immunized with a Streptococcus pneumoniae vaccine. The authors succeeded in creating hybridomas that secrete human mAbs specific for S. pneumoniae antigens (81).…”

Section: Fusion Partnersmentioning

confidence: 99%

Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies

Smith

Crowe

2015

Microbiol Spectr

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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.

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“…[29][30][31][32] In addition, a promising human myeloma cell line allowing efficient generation of stable and highly productive human hybridomas was described, even though a more widespread use of this cell line awaits the production of a subline lacking expression of an endogenous l chain. 33 Finally, the use of genetically engineered mouse myeloma cells allows for highly efficient formation of stable heterohybridomas, 34 with a propensity to produce affinity-matured IgG antibodies when appropriately pre-treated CD27-positive B cells are used for fusion. 35 As mentioned above, human hybridoma technology not only suffered from the lack of suitable fusion partners, but also from a shortage of antigen-stimulated B cells.…”

Section: Non-combinatorial Miningmentioning

confidence: 99%