Human antibodies and specific binding sequences thereof for use in stroke and ischemia or ischemic conditions
A specific, antibody technology, applied in the field of constructs and compositions, human natural antibodies, stroke or ischemia-related agents, especially cerebral ischemia, recombinant antibodies and their fragments, alleviating or treating stroke or ischemia
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Embodiment 1
[0260]
[0261] Autoantibodies are often considered pathogenic. In contrast, as demonstrated herein, anti-neuron autoantibodies (sHIgM12 and sHIgM42 and recombinant antibodies based thereon) did not kill neurons. Instead these IgMs protect neurons from death, promote neurite extension, increase NAA in vivo, protect axons in vivo in a TMEV model, and improve nocturnal spontaneous function in TMEV-affected mice.
[0262] The neuron-binding antibodies IgM12 and IgM42 target neurons in CNS lesions and may be useful for reversing neuronal loss and / or ameliorating the effects of neuronal injury or disease. Neuron-binding human IgM represents a new class of therapeutic agents uniquely useful for various diseases and conditions involving neurodegeneration, neuronal injury or neuronal death. Such diseases include, without limitation, MS, spinal cord injury, ALS, Alzheimer's disease, traumatic brain injury, cerebrovascular event or stroke. These human IgMs are minimally antigenic w...
Embodiment 2
[0272] Example 3: Recombinant antibody derived from sHIgM12
[0273] Two recombinant forms of sHIgM12 were constructed. Each used the same expression vector as previously used for the heavy and light chain recombinant IgM22 antibody (rHIgM22) (22, 28, WO0185797). The vector includes an selectable dHfR gene expressed under the control of the SV40 promoter. A partially human recombinant IgM12 antibody format with a mouse J chain was initially constructed as follows, after which the antibody was produced in the human / mouse hybridoma line F3B6 cells.
[0274] Using the primers described below, in a manner similar to that described previously (6), by inserting the heavy chain variable region cDNA with a leader sequence from the nucleotide database and the complete light chain with a linked leader sequence from the nucleotide database Strand cDNA for recombinant IgM12 antibody (PAD12) vector construction. Figure 4 The vector is depicted in . Figure 5 The heavy and light chain ...
Embodiment 3
[0276] (1) 5' primer with BspEI site:
[0277]
[0278] (4) 3' primer with PAC I site:
[0279] .
[0280] The primers used to prepare rHIgM12Vk and splice it with the leader sequence of the database (lower case letters, accession number X59312) by overlap extension (soe) are as follows:
[0281] (1) 5'-Ck primer of soe lym 12 Vk-Ck:
[0282] (2) 3'Ck primer with Xho 1:
[0283] (3) 5' Lym 12 Vk with NheI:
[0284] .
[0285] As previously described (6), vectors with synthetic antibody genes were introduced into F3B6 hybridoma cells by electroporation and subjected to methotrexate (MTX) amplification.
[0286] Briefly, 8 million F3B6 mouse / human heterohybridoma cells (American Type Culture Collection: ATCC) were linearized with BglII in 800 μl serum-free medium. Incubate with 10μg PAD12 carrier for 10 minutes, then place it in Biorad Gene Pulser TM (Biorad, Hercules, CA, USA) at 0.2 V for electroporation. After 10 minutes of incubation on ice, cells wer...
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