Bispecific tetravalent antibodies and methods of making and using thereof

A bispecific, binding specificity technology, applied in chemical instruments and methods, antibodies, antibody medical components, etc., can solve the problems of suboptimal and high cost of monoclonal antibodies

Pending Publication Date: 2017-09-26
SYSTIMMUNE INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the efficacy of using simple combinations of two or more mAbs is suboptimal...

Method used

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  • Bispecific tetravalent antibodies and methods of making and using thereof
  • Bispecific tetravalent antibodies and methods of making and using thereof
  • Bispecific tetravalent antibodies and methods of making and using thereof

Examples

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example 1

[0062] To assess the growth inhibitory potential of anti-HER2 antibodies, the effect on the proliferation of BT-474 cells (ATCC HTB-20, Manassas, Va.), a breast ductal carcinoma tumor cell line, was tested. Cells were seeded into 96-well tissue culture plates at a density of 6000 cells / well with 100 μl RPMI-1640 medium containing 1% fetal calf serum. After 4 hours, the test antibodies were added at different concentrations ranging from 0.0061 nM to 400 nM. Cells were cultured for 7 days in the presence of test antibodies. 20 μl of MTS reagent (Promega, Madison, Wl) was added to each well and the cells were incubated at 37°C for 2 hours. MTS is readily taken up by actively proliferating cells, reduced to formazan (which readily absorbs light at 490 nm), and then secreted into the culture medium. After incubation, OD490 values ​​were measured using a BioTek (Winooski, VT) ELx800 absorbance reader. OD490 values ​​for control cells (treated with medium only) were also obtained ...

example 2

[0065] The ability of anti-Her2 antibodies to be internalized by BT-474 cells was tested. A 1 mg aliquot of antibody standard PBS solution was reacted with Alexa Fluor 488 carboxylic acid, TFP ester (Thermo Fisher #A-10235, Waltham, MA) for 1 hour at room temperature. Unbound dye was removed by gel filtration using a Bio-Gel P-30 column. After conjugation, incubate an aliquot of 3x105 BT-474 cells with 50 nM of each Alexa488-labeled antibody in complete medium (RPMI-1640 + 10% FBS) at 37°C or 4°C (on ice) 1 hour. After incubation, cells were washed twice with ice-cold PBS in a cold centrifuge. Cells were then resuspended in 500 nM quenched rabbit-anti-Alexa488 antibody (Thermo Fisher #A-11094, Waltham, MA) or 500 nM rabbit IgG isotype control antibody (Jackson ImmunoResearch Laboratories #011-000-003, WestGrove, PA ) in either and incubated on ice for 30 minutes. Add two volumes of 2% paraformaldehyde to each sample and incubate for 10 min at room temperature. Cells were ...

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Abstract

A bispecific tetravalent antibody comprising an IgG having a pair of heavy chains and a pair of light chains, and two scFv components being connected to either C or N terminals of the heavy or light chains. The bispecific tetravalent antibody may have a binding specificity for two different epitopes on HER2 receptor.

Description

[0001] Cross references to related patent applications [0002] This application claims priority to US Provisional Application No. 62095348, filed December 22, 2014, entitled "BISPECIFIC ANTIBODIES," which is incorporated herein by reference in its entirety. [0003] sequence listing [0004] The Sequence Listing related to this application is provided in text format in lieu of a paper copy and is incorporated into the specification by reference. The text file containing the sequence listing is named Sequence Listing_ST25_0003PCT2.txt. The text file is approximately 164KB, created on December 18, 2015, and submitted electronically via EFS-Web. technical field [0005] The present disclosure relates generally to the technical field of antibodies, and more specifically to bispecific antibodies. Background technique [0006] HER2, a member of the ErbB / HER receptor family, is overexpressed and / or deregulated in several cancers of the breast and ovary (King, Kraus and Aaronson...

Claims

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Application Information

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IPC IPC(8): A61K39/395
CPCC07K16/32A61K2039/505C07K2317/35C07K2317/64C07K2317/73C07K2317/732C07K2317/92C07K2317/94A61K47/6803A61K47/6849A61K47/6851C07K16/2863C07K2317/31C07K16/3015A61P35/00A61P43/00A61K39/3955A61K45/06C07K2317/21C07K2317/24C07K2317/622C07K2317/76A61K39/39558A61K2039/5152C07K16/468A61K47/6879
Inventor 高泽人朱义菲尔·谭布莱尔·伦肖布莱恩·科瓦切维奇
Owner SYSTIMMUNE INC
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