Cloning and sequence analysis of κ and γ cynomolgus monkey immunoglobulin cDNAs

Lewis, Alan P.; Barber, Karen A.; Cooper, Helen J.; Sims, Martin; Worden, Jenny; Crowe, J.S.

doi:10.1016/s0145-305x(05)80010-2

Cited by 22 publications

(16 citation statements)

References 35 publications

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“…Furthermore, although many synthetic molecules have been designed to inhibit the enzymatic activity of LF (15,24,26,32,37,42,52,55), neither their bioavailability nor their tolerance is guaranteed. We report here the first recombinant neutralizing antibody directed against LF, obtained after immunization of a nonhuman primate (NHP) and showing human-like framework regions (FRs), as expected (10,31). This antibody may be suitable for medical use (7,16,40).…”

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confidence: 89%

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

Pelat

Hust

Laffly

et al. 2007

Antimicrob Agents Chemother

102

112

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The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 ؋ 10 8 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ؎ 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

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confidence: 89%

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

Pelat

Hust

Laffly

et al. 2007

Antimicrob Agents Chemother

102

112

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“…Four IGG genes from the closely related rhesus have been cloned: RH1, RH2, RH3, and RH4 (http://www.ncbi.nlm.nih. gov/nuccore accession numbers AF045537, AF045539, AF045538, and AY292520.1, respectively) (9)(10)(11)(12). Three rhesus IgG constant domain subclasses have also been characterized by using subclassspecific Abs, further demonstrating that there are at least three H chain (HC) subclasses expressed in rhesus (12).…”

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confidence: 99%

“…Three rhesus IgG constant domain subclasses have also been characterized by using subclassspecific Abs, further demonstrating that there are at least three H chain (HC) subclasses expressed in rhesus (12). Additional studies have revealed at least one IGG C region sequence for the cynos as well as a k L chain (LC) C region gene (GenBank number CS101580.1) (11).…”

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confidence: 99%

Molecular and Functional Characterization of Cynomolgus Monkey IgG Subclasses

Jacobsen

Padaki

Morris³

et al. 2011

The Journal of Immunology

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Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc–FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.

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“…Primate antibodies are more similar in sequence to human, versus murine, antibodies and they are less likely to be immu-nogenic in humans (25,36). The V-domains of antibodies of nonhuman primate origin can be fused to the constant regions of human immunoglobulin Gs (IgGs) to produce primatized Ab suitable for clinical use.…”

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confidence: 99%

Selection of a Macaque Fab with Framework Regions Like Those in Humans, High Affinity, and Ability To Neutralize the Protective Antigen (PA) of Bacillus anthracis by Binding to the Segment of PA between Residues 686 and 694

Laffly

Danjou

Condemine

et al. 2005

Antimicrob Agents Chemother

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Human anthrax infection cannot always be treated successfully by antibiotics, as highlighted by recent bioterrorist attacks; thus, adjunct therapies are clearly needed for the future. There is a particular need to further develop adjunct therapies that can neutralize secreted toxins, such as antibodies directed towards the 83-kDa protective antigen (PA 83 ). In the absence of human donors, we immunized a macaque (Macaca fascicularis) with PA 83 to obtain such antibodies suitable as an adjunct therapy for human anthrax infection. By using bone marrow as a template, we PCR amplified specific Fab-encoding genes and cloned them as an immune library (10 7 clones). We isolated a high-affinity ( Bacillus anthracis, the causative agent of anthrax, produces a toxin related to the classic A-B family, which plays a major role in disease pathogenesis. The 83-kDa protective antigen (PA 83 ) is the common cell-binding domain (38) that, after proteolytic activation, can interact with two enzymatically active domains that elicit cell damage, the edema factor (EF; 89 kDa) and lethal factor (LF; 90 kDa). LF is a metalloproteinase specific for mitogen-activated protein kinase kinases. EF is a calmodulin-dependent adenylate cyclase that causes dramatic increases in the intracellular concentration of cAMP. These proteins are secreted by the bacterium as nontoxic monomers and assemble on the surface of receptor-bearing cells to form toxic complexes. After binding to its cellular receptor (4,30,44), PA 83 is cleaved by a furin-like cellular protease(s) (34), causing the release of an amino-terminal 20-kDa fragment and leaving the carboxy-terminal 63-kDa moiety (PA 63 ) bound to its receptor. PA 63 then spontaneously oligomerizes to form a ringshaped homoheptamer that binds LF and/or EF (38) to form the lethal toxin and/or the edema toxin, respectively. Heptamers are internalized by acidic endosomes, and then EF and LF are translocated into the cytosol (32).This pathogenesis and the prophylactic/therapeutic approaches used to treat anthrax are subjects of great interest due to concerns over intentional or inadvertent exposure to aerosols of Bacillus anthracis spores (47

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Cloning and sequence analysis of κ and γ cynomolgus monkey immunoglobulin cDNAs

Cited by 22 publications

References 35 publications

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

Molecular and Functional Characterization of Cynomolgus Monkey IgG Subclasses

Selection of a Macaque Fab with Framework Regions Like Those in Humans, High Affinity, and Ability To Neutralize the Protective Antigen (PA) of Bacillus anthracis by Binding to the Segment of PA between Residues 686 and 694

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