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Antibodies

A technology of antibody and human antibody, which is applied in the field of antibody binding to CXCR4, to achieve a favorable and safe effect

Inactive Publication Date: 2013-03-27
AFFITECH RESEARCH AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While human antibodies are generally considered superior, it is well known that the development of human antibodies with high enough affinity and appropriate functionality to be candidates for successful human therapy is far from straightforward

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0622] Example 1: Novel Antibody

[0623] Five human antibodies that could specifically bind to CXCR4 were identified. The single-chain form of the antibody was cloned into the pHOG21 plasmid containing c-myc and the 6xHis tag epitope (Kipryanov et al., 1997). TG1 bacteria were transformed and expressed scFv after IPTG induction. Binding of purified antibodies was confirmed by EasyCyte.

[0624] The nucleotide sequences of the heavy and light chains of the antibodies from which the clones were generated were sequenced. Antibodies were designated C-9P21, B-1M22, C-1I24, D-1K21 and 9N10. The nucleotide sequence and amino acid sequences of the light and heavy chains of C-9P21 are shown in figure 1 . The CDR regions of the light and heavy chains of C-9P21 are shown in Table 1. The nucleotide sequence and amino acid sequences of the light and heavy chains of B-1M22 are shown in figure 2 . The CDR regions of the light and heavy chains of B-1M22 are shown in Table 2. The n...

Embodiment 2

[0632] Example 2: Binding specificity of antibody clones to CXCR4 expressing cells

[0633] Four antibodies, B-1M22, C-9P21, C-1124 and D-1K21, were tested for their CXCR4 specificity at the scFv level by their ability to bind CXCR4 expressing cells. Transformed cells differed from their untransformed counterparts only in the expression of CXCR4.

[0634] Materials and methods

[0635] DT40 and HEK293T / 17 cells were maintained as described above.

[0636]DT40+CXCR4, HEK293T / 17+CXCR4, DT40 and HEK293T / 17 cells were collected from culture flasks, washed twice with PBS (400xg, 5min, 4°C) and resuspended in a medium containing 0.2% BSA and 0.09% NaN 3 in the PBS. 1x10 5 Cells / well were aliquoted into V-shaped 96-well plates (Greiner Bio-One, Frikenhausen, Germany). Cells were centrifuged (400x g, 5 min, 4°C) and resuspended in 50 μl ScFvs (10 μg / ml) in PBS (containing 0.2% BSA and 0.09% NaN 3 ). After incubation for 1 hour (4°C), the cells were washed with 100 μl PBS (c...

Embodiment 3

[0645] Example 3: Anti-CXCR4 antibodies interfere with ligand binding

[0646] Four antibodies B-1M22, C-9P21, C-1I24 and D-1K21 bind to CXCR4-transfected cells and constitutively through them at the scFv level in the presence and absence of two molecules that specifically bind to CXCR4 The ability of CXCR4-expressing lymphoblastoid cells to test their CXCR4 specificity. One molecule is SDF-1, the natural ligand of CXCR4, and the other is AMD3100, a highly specific competitive inhibitor of SDF-1α binding to CXCR4.

[0647] Materials and methods:

[0648] Jurkat and Ramos cells were maintained as described above.

[0649] The scFv clones were expressed on a large scale and purified as monomeric fractions by SEC fractionation. Harvest native CXCR4+ expressing cell lines Jurkat and Ramos from culture flasks, wash with PBS (with 0.2% BSA and 0.09% NaN 3 Buffer replenishment) washed 2 times and with 1x10 5 Cells / well were aliquoted into V-shaped 96-well plates (Greiner Bio...

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Abstract

The present invention provides antibodies which bind to CXC chemokine receptor (4) (CXCR4) and which do not induce significant apoptosis of CXCR4 expressing cells. Also provided are inter alia immunoconjugates and compositions comprising such antibodies and methods and uses involving such antibodies, particularly in the medical and diagnostic fields.

Description

technical field [0001] The present invention generally relates to the fields of antibodies, CXCR4 biology and related treatments. More particularly, it provides antibodies that bind to CXCR4. Such anti-CXCR4 antibodies are useful in the diagnosis and treatment of diseases and conditions associated with CXCR4, for example, the treatment of cancer and viral infections (especially HIV), the treatment of inflammatory and immune diseases, such as monitoring by imaging tumor blood vessels or Predict tumor growth and progression, inhibit or attenuate metastasis formation and inhibit or reduce angiogenesis. The antibody-based compositions and methods of the invention also extend to the use of immunoconjugates and other therapeutic articles, kits and methods. Background technique [0002] With >800 members, G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signaling, accounting for more than 2% of the total genes encoded by hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395C12N15/13G01N33/53A61P35/00A61P31/12A61P29/00A61P37/00
CPCC07K16/2866C07K2317/33C07K2317/622C07K2317/21C07K2317/734C07K2317/732C07K2317/76C07K2317/73A61P9/00A61P19/02A61P29/00A61P31/12A61P31/18A61P35/00A61P35/04A61P37/00A61P37/02A61P37/04A61P43/00A61K39/3955A61K2039/505C07K2317/14C07K2317/565C07K2317/567C07K2317/92
Inventor 阿妮塔·卡夫利谢尔盖·米哈伊洛维奇·库普里亚诺夫
Owner AFFITECH RESEARCH AS
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