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Antibodies specific to human nectin4

A connexin and antibody technology, applied in the direction of animal/human protein, anti-animal/human immunoglobulin, antibody, etc., can solve problems such as unmet needs

Pending Publication Date: 2020-12-15
YISSUM RES DEV CO OF THE HEBREWUNIVERSITY OF JERUSALEM LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Despite the success in cancer immunotherapy, there remains an unmet need for other methods of boosting the cells of the immune system to attack tumor cells and for more effective and specific agents and drug combinations

Method used

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  • Antibodies specific to human nectin4
  • Antibodies specific to human nectin4
  • Antibodies specific to human nectin4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0376] Example 1. Generation and selection of anti-connexin-4 mAbs

[0377] The immunogen (Nec4-Fc) expression technology is based on mammalian HEK 293T cells and is a technology of choice giving the best quality, stability, solubility and yield especially in the case of glycoproteins. Nec4-Fc protein This fusion protein of the ectodomain of connexin-4 and the Fc domain of human IgG1 was recombinantly produced and purified as follows:

[0378] The coding sequence of human connexin 4 was cloned as a fusion with the Fc fragment of human IgG1 to generate a recombinant Fc-fusion protein. The ectodomain (extracellular) portion of the human connexin 4 molecule, which spans residues 32 to 349, was used. The C-terminal serine residue at position 349 of the connexin 4 amino acid sequence was fused to the heavy chain hinge of deglycosylated Fc (N297A) of human IgGl, followed by the CH2 and CH3 constant regions. The open reading frame (ORF) of the recombinant protein is codon-optimized...

Embodiment 2

[0385] Example 2. Affinity of anti-connexin-4 mAbs for human connexin 4

[0386] The affinity of antibodies hNec4.01 and hNec4.05 to fluorophore-labeled human connexin 4-Ig molecules was further determined using a microthermophoresis assay (Wienken et al., 2010, Nat. Commun. 1:100). Measurements were repeated using at least three independent protein preparations. Such as image 3 As shown in , very high binding affinities were observed for both antibodies. The calculated Kd was 272±154 pM for clone hNec4.05 and 107±115 pM for the hNec4.01 clone.

Embodiment 3

[0387] Example 3. Sequencing of anti-connexin-4 mAbs

[0388] The two hybridoma clones No. 0.1 and No. 0.5 that demonstrated the best inhibition of Connexin 4-TIGIT binding were sent for nucleotide and amino acid sequencing.

[0389] method

[0390] comply with Reagents (Ambion, cat. no. 15596-026), total RNA was isolated from the hybridoma cells. Then follow PrimeScript TM Total RNA was reverse transcribed into cDNA using isotype-specific antisense primers or universal primers from the technical manual of the First Strand cDNA Synthesis Kit (Takara, cat#6110A). Amplify V according to GenScript's Rapid Amplification of cDNA Ends (RACE) standard operating procedure (SOP) H and V Lantibody fragments. The amplified antibody fragments are cloned individually into standard cloning vectors. Colony PCR was performed to screen for clones with inserts of the correct size. For each fragment, no fewer than 5 clones with inserts of the correct size were sequenced. The sequences ...

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Abstract

The present invention provides monoclonal antibodies that recognize human Nectin4 with high affinity and specificity and inhibit its binding to T cell immunoreceptor with Ig and ITIM domains (TIGIT).The present invention further provides pharmaceutical compositions comprising the antibodies and methods for their use in cancer immunotherapy and in diagnosis.

Description

technical field [0001] The present invention belongs to the field of immunotherapy, and relates to antibodies and fragments thereof bound to human protein nectin 4 (Nectin4), polynucleotide sequences encoding these antibodies and cells producing these antibodies. The invention also relates to therapeutic and diagnostic compositions comprising these antibodies, and methods of using these antibodies for the treatment and diagnosis of diseases, especially cancer. Background technique [0002] Immunotherapy is one of the most promising advances in cancer treatment made in the past decade. Cancer immunotherapy is used to generate and enhance an anti-tumor immune response, for example, by treatment with antibodies specific to antigens on tumor cells, by treatment using fusions of antigen-presenting cells and tumor cells, or by activation of anti-tumor T cells. The ability to recruit immune cells, such as T cells, against tumor cells in patients offers a treatment modality to comb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K38/17A61K39/395A61K39/00
CPCC07K16/2803C07K2317/565C07K2317/732A61K39/395A61K2039/505A61P35/00A61K45/06C07K2319/03C07K14/7051Y02A50/30A61K39/4613A61K39/464466A61K39/4631A61K2239/38C12N5/0636C12N5/0646A61K39/4611A61P31/12G01N33/574G01N33/563C07K14/705C07K2317/622C07K2319/02
Inventor 奥夫尔·曼德尔波姆阿迪·雷切斯斯提潘·约尼奇平沙斯·楚克尔曼
Owner YISSUM RES DEV CO OF THE HEBREWUNIVERSITY OF JERUSALEM LTD
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