Recombining single chained three specific antibodies of anti CCA, anti CD 3, anti CD 28 through genetic engineering

A specific antibody and single-chain antibody technology, applied in genetic engineering, recombinant DNA technology, antibodies, etc., can solve the problems of increasing expression and purification procedures and production costs, and achieve the effect of avoiding non-specific killing reactions and broad application prospects

Inactive Publication Date: 2005-01-12
弘业新创抗体技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the combined use of two bispecific antibodies will bring a series of inconveniences in specific applications, such as increasing the operating procedures and production costs of expression and purification, and the relative ratio of the two antibodies must be considered in clinical applications, etc.

Method used

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  • Recombining single chained three specific antibodies of anti CCA, anti CD 3, anti CD 28 through genetic engineering
  • Recombining single chained three specific antibodies of anti CCA, anti CD 3, anti CD 28 through genetic engineering
  • Recombining single chained three specific antibodies of anti CCA, anti CD 3, anti CD 28 through genetic engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1. Overlapping PCR splicing is used to construct the multiple cloning site DNA fragment of the trispecific antibody expression vector pTRI

[0114] The flowchart of overlapping PCR splicing is shown in Figure 1. The oligonucleotide fragments used in the splicing reaction are as follows:

[0115] 1.5'-TAT ACC ATG GGT CTC GAG-3' (SEQ ID NO: 5)

[0116] 2.5’-TAT ACC ATG GGT CTC GAG ATG TAC CCG CGC GGT AAC ACT AGT GAA

[0117] TTC AAC AGC ACG TA-3' (SEQ ID NO: 6)

[0118] 3.5’-AGC CAG TCC TGG TGC AGT ACG GTG AGG ACG CTT ACA ACC CGG TAC

[0119] GTG CTG TTG AAT TC-3' (SEQ ID NO: 7)

[0120] 4.5’-CTG ​​CAC CAG GAC TGG CTG AAT GGC AAG GAA TAC AAA TGC AAG AGT

[0121] ACT TCT AGA ATG TA-3' (SEQ ID NO: 8)

[0122] 5.5’-CGA ACC AGC AGC GCA TTC TGG AAG TCG ACG TTA CCG CGC GGG TAC

[0123] ATT CTA GAA GTA CT-3' (SEQ ID NO: 9)

[0124] 6.5’-AAT GCG CTG CTG GTT CGT TAC ACC AAG AAA GTA CCC CAA GTG TCA

[0125] ACT CCA ACT CCT GT-3' (SEQ ID NO: 10)

[0126] ...

Embodiment 2

[0152] Embodiment 2: Construction of CEA-TsAb

[0153] Refer to Figure 4 for the specific construction process. A simplified diagram of the various vectors used during construction is shown in Figure 5. The specific steps of the construction process are as follows:

[0154] (1) Construction of vector pTRI:

[0155] The multiple cloning site DNA fragment and pTMF empty vector (Zhang et al., 2002) were subjected to NcoI / BamHI double enzyme digestion reaction at the same time, and the digested product of the multiple cloning site DNA fragment and the large fragment product of pTMF digestion were recovered, ligated The TOP10 strain was transformed, and the plasmid of the positive transformed clone was extracted and identified by PCR to obtain the correct connection product. The plasmid of the correctly ligated product was named pTRI and used in the next step.

[0156] The specific operations of the enzyme cleavage reaction, ligation reaction, bacterial competent preparation an...

Embodiment 3

[0232] Example 3: Low temperature-induced intracellular soluble expression of CEA-TsAb

[0233] (1) Transform CEA-TsAb / pTRI into Escherichia coli BL21(DE3) (Novagen)

[0234]According to the method for preparing competent cells described in Example 2, Escherichia coli BL21(DE3) competent cells were prepared. The plasmid CEA-TsAb / pTRI was extracted according to the instructions of the plasmid extraction kit (Shanghai Huashun Co., Ltd.), the transformation experiment was carried out according to the step (1) of Example 2, and the identification was carried out as described in Example 2.

[0235] (2) Low temperature induced expression

[0236] Coat BL21(DE3) containing CEA-TsAb / pTRI on LB-K plate, culture overnight at 37°C, then pick a single clone, inoculate in 5ml LB-K liquid medium, culture in a large test tube on a shaker at 37°C Overnight (200 rpm). The next day, take the overnight culture and transfer it to 250ml LB-K liquid medium at a ratio of 1 / 100, and continue to cu...

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PUM

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Abstract

The invention relates to a recombinant single chain tri-specific antibody, and it is characterized by that it is made by successively connecting antibody of antineoplastic related antigen, FC onnecting peptide, antihuman CD3 antibody, HSA connecting peptide and antihuman CD28 antibody. More specifically, said invention relates to a recombinant single chain tri-specific antibody; CEA-TsAb of anti-CEA, anti-CD3 and anti-CD28. Said antibody is made up by using three antibody fragments (unti-CEA single chain antibody, anti-CD3 single chain antibody and anti-CD28 single chain antibody), utilizing two connecting peptides (FC connecting peptide and HSA connecting peptide) and series-connecting them together, and on its C end C-myc tag and (His)6-tag are added. Said invention also relates to a method for constructing expressing and purifying said antibody, including exonic DNA sequence of said antibody, expression vector and host cell containing said vector.

Description

technical field [0001] The present invention relates to the field of genetically engineered antibodies, more specifically, to a genetically engineered recombinant anti-CEA, anti-CD3, and anti-CD28 single-chain trispecific antibody; methods for constructing, expressing and purifying the antibody; a vector containing the antibody and an Escherichia coli host bacteria. Background technique [0002] In the human body, the activation of T cells requires the action of dual signaling pathways: the MHC / antigen peptide complex on the surface of antigen presenting cells (APC: Antigen Presenting Cells) interacts with the TCR / CD3 complex expressed by T cells to generate the first Signal transmission pathway; co-stimulatory signal molecule receptors (such as B7) on the surface of APC cells interact with co-stimulatory signal molecules (such as CD28) on the surface of T cells to generate a second signal, namely the co-stimulatory signal transmission pathway. It is generally believed that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P35/00C07K16/18C07K16/28C07K16/30C07K19/00C12N15/12C12N15/13C12N15/63C12N15/70
CPCC07K2317/34C07K16/2818C07K2317/626C07K16/3007C07K16/00A61K2039/505C07K16/2809C07K2317/73A61P35/00A61P43/00
Inventor 王祥斌黄华樑赵宝峰赵琦朴锦华林晴
Owner 弘业新创抗体技术股份有限公司
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