Cross-reactive staphylococcus aureus antibody sequences

A staphylococcus, golden yellow technology, applied in the direction of antibodies, antibody medical components, antibacterial drugs, etc., can solve problems such as unreported toxin cross-reactive mAbs

Inactive Publication Date: 2016-08-17
ARSANIS BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In conclusion, to date, no cross-reactive mAbs against different two-component S. aureus toxins or against α-hemolysin and any of the two-component toxins have been reported

Method used

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  • Cross-reactive staphylococcus aureus antibody sequences
  • Cross-reactive staphylococcus aureus antibody sequences
  • Cross-reactive staphylococcus aureus antibody sequences

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0606] Example 1 Generation of Hla–two-component toxin cross-reactive mAbs that bind to a single toxin component with high affinity

[0607] method:

[0608] Production of recombinant toxins. The genes of S-component and F-component are derived from TCH1516USA300 bacterial strain, codon optimization is used for Escherichia coli expression, produces (Genescript, USA) by gene synthesis, (see Figure 7 ), cloned as pET44a, produced protein in BL21, Rosetta or Tuner DE3 strains without signal peptide sequence (determined by PrediSi program; Hiller, Nucleic Acids Res., 2004, 32: W375-W379).

[0609] LukS, LukF, LukE, LukD, HlgA, HlgC and HlgB expressed in soluble form with N-terminal NusA / His 6 marker, which is proteolytically removed after the first purification step. Purification usually involves three chromatographic steps 1) IMAC (immobilized metal ion affinity column) 2) cation exchange or IMAC, and 3) size exclusion chromatography. Clarified cell extracts were loaded ...

example 2

[0615] Example 2H1a - Improved Binding Affinity of Two-Component Toxin Cross-Reactive mAbs to LukF and LukD in vitro toxin neutralizing potency

[0616] method:

[0617] In vitro assay of toxin-mediated cell lysis. Toxin potency in target cells was assessed by measuring ATP levels in intoxicated cells (polymorphonuclear cells (PMN), differentiated HL60 or A549 cells). Briefly, HIa or equimolar mixtures of F- and S-components were serially diluted in assay medium for intoxication of cells. Then, using a commercial kit (Cell Luminescent Cell Viability Assay; Promega, USA), the cell viability of PMN, differentiated HL60 and A549 cells was determined according to the manufacturer's instructions. % survival was calculated relative to the mock-treated control.

[0618] For Hla, two different in vitro assays were performed using the human lung epithelial cell line A549 or rabbit erythrocytes. The day before, A549 cells (HPACC#86012804) were trypsinized and cultured at a den...

example 3

[0628] Example 3H1a - Improvement of the binding affinity of two-component toxin cross-reactive mAbs to LukF and LukD In vivo protection

[0629] method:

[0630] Passive protection of mice by monoclonal antibodies. The protective effect of anti-S. aureus antibodies was evaluated in several murine models. Passive immunization with mAbs was performed intraperitoneally 24 hours before lethal challenge with recombinant toxin. A group of 5 mice (BALB / c) each received a single mAbs dilution in PBS at a dose of 5 or 10 mg / kg (100 or 200 ug / mouse, respectively). Control groups received only PBS or the same dose of isotype-matched non-specific mAb. Toxin challenges were performed using HlgA-HlgB or HlgA-LukD toxin pairs at doses of 0.2 and 1 ug per mouse (each component), respectively.

[0631] result:

[0632] Such as Figure 4 mAbs AB-28-3 (K D =18pM) and AB-28-9 (K D = 5 pM) showed that cross-reactive Hla mAbs that bind to HlgB with an affinity <20 pM were very effe...

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Abstract

The invention refers to a cross-neutralizing antibody comprising at least one polyspecific binding site that binds to alpha-toxin (Hla) and at least one of the bi- component toxins of Staphylococcus aureus, which antibody comprises at least three complementary determining regions (CDR1 to CDR3) of the antibody heavy chain variable region (VH), wherein A) the antibody comprises a) a CDR1 comprising or consisting of the amino acid sequence YSISSGMGWG (SEQ ID 1); and b) a CDR2 comprising or consisting of the amino acid sequence SIDQRGSTYYNPSLKS (SEQ ID 2); and c) a CDR3 comprising or consisting of the amino acid sequence ARDAGHGVDMDV (SEQ ID 3); or B) the antibody comprises at least one functionally active CDR variant of a) the parent CDR1 consisting of the amino acid sequence of SEQ ID 1; or b) the parent CDR2 consisting of the amino acid sequence of SEQ ID 2; or c) the parent CDR3 consisting of the amino acid sequence of SEQ ID 3; wherein the functionally active CDR variant comprises at least one point mutation in the parent CDR sequence, and comprises or consists of the amino acid sequence that has at least 60% sequence identity with the parent CDR sequence. It further refers to such cross-neutralizing antibody which is a functionally active variant antibody of a parent antibody that comprises a polyspecific binding site of the VH amino acid sequence of SEQ ID 20, and the VL amino acid sequence of SEQ ID 39, which functionally active variant antibody comprises at least one point mutation in any of the framework regions (FR) or constant domains, or complementarity determining regions (CDR1 to CDR6) in any of SEQ ID 20 or SEQ 39, and has an affinity to bind each of the toxins with a Kd of less than 10-8M, preferably less than 10-9M.

Description

[0001] The present invention relates to cross-neutralizing antibodies comprising at least one multispecific binding site for binding to at least one of the alpha toxin (Hla) and the two-component toxin of Staphylococcus aureus, characterized by a specific amino acid sequence . Background technique [0002] Staphylococcus aureus infection is a major unsolved medical problem. Staphylococcus aureus is one of the most common causes of associated infections, and mortality is particularly high in patients who develop pneumonia, bacteremia, and / or sepsis. Another point of concern is the development of antibiotic-resistant clones (methicillin-resistant Staphylococcus aureus associated with hospitals and communities: HA- and CA-MRSA), highlighting the need for novel treatments. [0003] The inability of new antibiotics to address this medical problem is largely due to the rapid development of resistance and the inability of antibiotics to eliminate mechanisms of toxicity, such as cyto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12
CPCC07K2317/33C07K2317/34C07K16/1271A61P27/02A61P31/04A61K39/085A61K39/395A61K2039/505C07K2317/565C07K2317/515G01N33/56938G01N2800/26
Inventor E·纳吉A·巴达罗H·劳哈G·纳吉I·米尔基娜Z·毛焦里奇Z·维斯拉姆M·B·巴托斯B·D·普林茨T·S·贾殷
Owner ARSANIS BIOSCI
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