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Blocking leukocyte emigration and inflammation by interfering with CD99l2

一种CD99L2、-CD99L2的技术，应用在抗炎症领域，能够解决不是等问题

Inactive Publication Date: 2013-10-02
CORNELL RES FOUNDATION INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, while PECAM is the only molecule that has been identified to play a unique role in TEM, it is clearly not the only molecule involved in TEM

Method used

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  • Blocking leukocyte emigration and inflammation by interfering with CD99l2
  • Blocking leukocyte emigration and inflammation by interfering with CD99l2
  • Blocking leukocyte emigration and inflammation by interfering with CD99l2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0212] Example 1: murine CD99L2

[0213] Several murine cDNA libraries were probed with human CD99 cDNA clones to discover murine CD99.

[0214] A breakthrough came when mouse genome sequences began to accumulate in public databases. A putative open reading frame predicted to encode a putative protein called XAP89 (X chromosome-associated protein 89) based on three ESTs and one genome sequence cosmid (Genbank accession numbers AI466980, AW227546, AW320831, and AF125314, respectively) Has several regions of high homology to human CD99 (Butcher, E.C. 1991 Cell 67:1033-1036). However, its nucleotide sequence differs significantly from human CD99 due to the use of alternative codons. This sequence was used to design primers to complete the cDNA sequence by 5'-RACE using the mouse thymus cDNA library. A cDNA clone of 843bp ( figure 1 ) encodes a 237 amino acid protein that has significant homology to human CD99 along several highly conserved segments, especially in the trans...

Embodiment 2

[0217] Example 2: The role of muCD99L2 in the transformation in vivo

[0218] Preliminary data show that rat anti-muCD99L2 serum can block inflammation in a thioglycolate peritonitis model. Anti-muCD99L2 serum, but not normal rat serum, blocked 60% of PMN accumulation and reduced monocyte influx to near background levels when measured 18 hours after thioglycolate administration ( Figure 7 ). Similar data were obtained using polyclonal rabbit IgG.

[0219] We also developed a muCD99L2-Fc chimera in which the extracellular domain of CD99 was fused to the Fc portion of human IgG1. We have successfully used a similar PECAM-Fc chimera to block shifts in PECAM-dependent steps in vitro (34) and in vivo (35, 36). The muCD99L2-Fc chimera was purified from the supernatant of transfected cells and showed the expected Mr (for dimer) of 100 kD on SDS-PAGE ( Figure 8 ). Due to the high level expression of muCD99L2 on leukocytes and the low level expression of muCD99L2 on RBC (see ...

Embodiment 3

[0220] Example 3: Generation of anti-muCD99L2 monoclonal antibody

[0221] Female Fisher rats were immunized intraperitoneally with 100 μg of muCD99L2-Flag protein produced in COS cells, and boosted twice with the same material. Four days after the last booster immunization, spleens were harvested from individual rats, and spleen cells were aseptically fused with Y3 rat myeloma cells using polyethylene glycol. Surviving hybrid cells were fed at 1×10 per well 5 Densities of cells were seeded in HAT medium in 11 Costar 96-well plates (only the middle 60 wells were used per plate).

[0222] Hybridoma supernatants from wells with abundant cell growth were screened by immunoperoxidase cytochemistry as previously described (Muller et al., 1989, J. Exp. Med. 170, 399-414). Briefly, cultured monolayers of muCD99L2-expressing COS cells or parental cells were fixed in paraformaldehyde and air-dried. A 2 [mu]l droplet of hybridoma culture supernatant was placed on top of the cells a...

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Abstract

The present invention provides methods and compositions for modulating transendothelial migration (TEM) of leukocytes. In particular, inhibition of TEM can provide a potent therapeutic approach to treating inflammatory conditions. The invention specifically relates to the discovery that the adhesion molecule CD99L2 mediates TEM of leukocytes. CD99L2 is present on endothelial cells and leukocytes and mediates leukocyte-endothelial cell adhesion. Blockade of CD99L2 by use of a specific antibody blocks migration of leukocytes into a site of inflammation. CD99L2 shows functional analogy to the structurally-related molecule, CD99, inhibition of which, in conjunction with inhibition of PECAM, causes near total blockade of TEM. Thus, blocking CD99L2 on either endothelial cells or monocytes can block migration 80-90%. In conjunction with PECAM inhibitors, TEM blockade can approach 100%. Therapeutic treatments involving inhibition of CD99L2 show significant promise in remediation of inflammatory conditions.

Description

[0001] Research leading to the present invention was supported in part by Grant No. HL64774 from the National Institutes of Health. Accordingly, the US Government may have certain rights in this invention. technical field [0002] The present invention relates to anti-inflammatory methods, in particular to modulating transendothelial migration of leukocytes, and compositions for blocking transendothelial migration of leukocytes. Background technique [0003] References cited by numbers throughout this specification are listed in the "References" section following the examples. [0004] Previous studies (1-12) have demonstrated the transendothelial migration of platelet / endothelial cell adhesion molecule-1 (PECAM) in neutrophils (PMN), monocytes (Mo) and natural killer cells (NK) (TEM) in the key role. However, even under optimal circumstances, anti-PECAM agents block only 80-90% of leukocyte influx. While this is as good or better than the block of inflammation obtained b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/385
CPCC07K16/2896A61K2039/505
Inventor W·A·马勒A·R·申凯尔
Owner CORNELL RES FOUNDATION INC
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