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Anti-CD5 antibodies

An antibody, antibody molecule technology, applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc.

Inactive Publication Date: 2011-07-27
SYMPHOGEN AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, another question regarding the role of antibody therapeutics is the interaction of the antibody Fc region with the recruited effector molecules

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0493] Example 1: Cloning of anti-CD5 antibodies

[0494] immunity

[0495] Female BALB / c strain A (8-10 weeks old) was used for immunization by injection of CD5-human growth hormone (hGH) fusion protein.

[0496] Recombinant CD5 extracellular domain (ECD) prepared in-house was used for all immunizations. CD5-ECD was produced as a fusion protein consisting of the ECD of CD5 and human growth hormone (hGH), isolated by the tobacco etch virus (TEV) cleavage site.

[0497] CD5-hGH was diluted in PBS and then mixed 1:1 with Freund's Adjuvant. Adjuvants are used to enhance and modulate the immune response. For the first immunization, complete FA's adjuvant (CFA) was used, while incomplete FA's adjuvant (IFA) was used for subsequent immunizations. IFA is an oil-in-water emulsion containing mineral oil and CFA is IFA with added heat to kill dry mycobacterial species. Both adjuvants have a reservoir effect. CFA produces a long-lasting immune response and is used in the first ...

Embodiment 2

[0531] Example 2: Mammals prepare anti-CD5 antibodies

[0532] Anti-EGFR antibodies were transiently expressed using the Freestyle MAX CHO Expression System (Invitrogen). Antibodies were expressed in volumes of 200-2000ml.

[0533] About 24 hours before transfection, subculture CHO-S cells to reach 0.5×10 6 cell concentration per cell / ml. Plasmid was diluted in OptiPro serum-free medium (1.25 μg per ml of cell culture medium) and mixed with a solution of FreeStyle MAX transfection reagent as recommended by the supplier. The transfection reagent was transferred to the cell culture and the supernatant was collected 8 days later.

[0534] The expressed antibodies were purified from the culture supernatants using an affinity chromatography step using a protein A sepharose column (MabSelect Sure, GE Health Care) to purify IgGl molecules. Antibody was eluted from the column using 0.1 M Glycine 2.7. Antibody-containing fractions, as determined by absorbance measurements at 280...

Embodiment 3

[0535] Example 3: Determining Epitope Specificity

[0536] Competition ELISA using reference antibody

[0537] A competition ELISA that distinguishes the binding epitopes of anti-CD5 antibodies was developed by using a reference antibody that binds CD5 by incubation with a secondary reagent that is specific for the human Fc region of an anti-CD5 antibody and does not display Cross activity with murine IgG Fc. The ELISA was adapted from the description published in Ditzel et al., 1995, The Journal of Immunology, Vol. 154, No. 2, 893-906.

[0538] Epitope blocking ELISA was performed by diluting CD5ECD antigen to 1 μg / ml in PBS and coating 50 μl / ELISA well overnight at 4°C. The next morning, wells were washed twice with PBS-T and blocked with PBS-T-1% BSA for 1 hour at room temperature, followed by 4 washes in PBS-T. Next, 25 μl of murine reference mAbs were added to individual ELISA wells at dilutions known from previous experiments to saturate all epitopes on CD5 at this ...

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Abstract

The present invention relates to the field of compositions comprising anti-CD5 antibodies. In particular, the present invention concerns an antibody composition comprising at least two anti-CD5 antibodies capable of binding distinct CD5 epitopes. The invention further concerns bi-specific molecules having the binding specificities of said antibody compositions. The invention also relates topharmaceutical compositions, use of antibody compositions and methods for manufacturing antibody compositions. The invention further relates to cell banks and a method for killing cells.

Description

[0001] All patent and non-patent references cited in the above-cited applications or in this application are hereby incorporated by reference in their entirety. technical field [0002] The present invention is in the field of compositions comprising anti-CD5 antibodies. Background technique [0003] Antibodies are molecules produced by the immune system when challenged by invading pathogens such as bacteria and viruses. Antibody molecules consist of two heavy chains (HC) and two light chains (LC) connected by disulfide bridges to form a V-shaped molecule with variable binding domains present on the tips of each arm. These molecules are characterized by a high variability and an extremely strong binding to foreign substances, typically proteins, so-called antigens. Antibodies function by binding to specific epitopes on antigens. After binding, different effector functions can be mediated through the constant part of the Fc region of the antibody. Depending on the antibody...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/395A61P19/02C07K16/28A61P35/00A61P35/02
CPCC07K16/2896C07K2317/92A61K2039/507A61P19/02A61P35/00A61P35/02A61K39/395C07K16/28C07K16/46
Inventor 约瑟芬·L·K·克利特加德查尔斯·派克米克尔·W·佩德森克劳斯·凯福德
Owner SYMPHOGEN AS
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