CN113773393A

CN113773393A - Multi-specific single-chain antibody and application thereof

Info

Publication number: CN113773393A
Application number: CN202010522517.2A
Authority: CN
Inventors: 黄朝峰; 陈焕鹏; 韦凤娇
Original assignee: Guangzhou Changfeng Bio Tech Co ltd
Current assignee: Guangzhou Changfeng Bio Tech Co ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2021-12-10
Anticipated expiration: 2040-06-10
Also published as: CN113773393B

Abstract

The invention relates to a multi-specificity single-chain antibody and application thereof, wherein the multi-specificity single-chain antibody sequentially comprises a signal peptide, a variable region sequence of a first antibody, a variable region sequence of a first connecting peptide and a variable region sequence of a second antibody from an N end to a C end, and does not comprise constant region sequences of the first antibody and the second antibody; the first antibody and the second antibody are capable of specifically binding to two surface marker antigens of a T cell, respectively, and the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence, which are linked by a second linker peptide. The multi-specificity single-chain antibody can effectively adsorb and separate T cells from whole blood, simplifies the separation steps of the T cells, lightens the damage to the T cells, reduces the misoperation risk and equipment requirements brought by multi-step operation, and can reduce the probability of cross contamination and influence among different samples in T cell immunotherapy.

Description

Multi-specific single-chain antibody and application thereof

Technical Field

The invention relates to the technical field of biochemical molecules, in particular to a multi-specificity single-chain antibody and application thereof.

Background

T cell isolation is the first step in CAR-T cell preparation and is also an important technology in T cell preparation. At present, when T cells are separated from whole blood, it is usually necessary to separate monocytes from whole blood by using lymphocyte separating fluid or other density gradient centrifugation reagents, and then to sort the desired T cells by using antibody separation technology through antibodies combined with T cell characteristic molecules CD3, TCR molecules and other costimulatory molecules such as CD 28. The common separation techniques include magnetic bead separation, flow separation and the like.

However, the process of separating monocytes from whole blood requires multiple centrifugation, which is cumbersome, and the lysis of cells with erythrocyte lysate, which also has a certain damage to T cells and affects the activity of T cells.

Disclosure of Invention

Based on this, there is a need to provide a multispecific single-chain antibody that can be used to isolate T cells directly from whole blood.

A multi-specific single-chain antibody for separating T cells from whole blood, having a signal peptide, a variable region sequence of a first antibody, a variable region sequence of a first linker peptide and a variable region sequence of a second antibody in this order from N-terminus to C-terminus, and not having constant region sequences of the first antibody and the second antibody; the first antibody and the second antibody are capable of specifically binding to two surface marker antigens of a T cell, respectively, and the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence, which are linked by a second linker peptide.

In one embodiment, the first antibody is a CD3 antibody and the second antibody is a CD28 antibody.

In one embodiment, the C-terminus of the multispecific single-chain antibody further has a tag sequence.

In one embodiment, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1, the amino acid sequence of the first connecting peptide is shown as SEQ ID NO. 2, the amino acid sequence of the second connecting peptide is shown as SEQ ID NO. 3, and the amino acid sequence of the tag sequence is shown as SEQ ID NO. 4.

In one embodiment, the amino acid sequence of the multispecific single-chain antibody is shown in SEQ ID NO. 5.

An expression gene capable of expressing the multispecific single-chain antibody.

In one embodiment, the nucleic acid sequence is set forth in SEQ ID NO 6.

A recombinant cell comprising the above-mentioned gene or having the gene integrated into the genome of the recombinant cell.

A T cell isolation method comprising the steps of: the multispecific single-chain antibody is labeled with a first label, and then mixed with a whole blood sample to obtain a mixed solution, and T cells bound with the multispecific single-chain antibody are captured from the mixed solution by using a second label capable of specifically binding with the first label.

In one embodiment, the method of capturing T cells bound to the multispecific single-chain antibody comprises the steps of: and adding a second marker labeled magnetic bead into the mixed solution, incubating and then carrying out magnetic sorting to obtain the T cells combined with the multispecific single-chain antibody.

In one embodiment, the method of capturing T cells bound to the multispecific single-chain antibody comprises the steps of: and (3) allowing the mixed solution to flow through a cell culture bag filled with the cell culture carrier coated with the second marker in a gravity drip mode, washing the cell culture bag with physiological saline, and adding a culture medium for culture.

A T cell separation kit comprises the multi-specific single-chain antibody, a first marker and a second marker, wherein the first marker and the second marker can be specifically combined.

The invention takes a first antibody and a second antibody which can respectively and specifically bind two surface marker antigens of a T cell as a basis, removes a constant section in the first antibody and the second antibody, and combines a signal peptide and a connecting peptide to construct a multi-specificity single-chain antibody molecule which can be expressed by a single chain. The variable region sequences of the first antibody and the second antibody are connected together, so that two antigen molecules can be simultaneously combined, the binding force of a single-chain antibody molecule and an antigen is enhanced, and the variable region sequences do not contain constant region sequences, so that the variable region sequences can be directly used for separating corresponding T cells from whole blood, the processes of removing red blood cells and separating mononuclear cells from the whole blood are omitted, and the damage to the T cells in the separation process is reduced. Therefore, the multispecific single-chain antibody can be directly added into whole blood to separate T cells, red blood cells can not adsorb the antibody, the antibody can be ensured to be combined on corresponding T cells, and meanwhile, the multispecific single-chain antibody can better make up for the problem of weak binding force of the single-chain antibody after a constant region is removed. Experimental results show that the multi-specificity single-chain antibody can effectively adsorb and separate T cells from whole blood, simplifies the separation steps of the T cells, lightens the damage to the T cells, reduces the misoperation risk and equipment requirements caused by multi-step operation, and can reduce the probability of cross contamination and influence among different samples in T cell immunotherapy.

Drawings

FIG. 1 shows the results of the expression purification of the anti-CD 3CD28 bispecific single chain antibody molecule of example 1;

FIG. 2 is a graph showing the results of flow analysis of TCRb-positive cells among the positive cells after magnetic sorting in example 1;

FIG. 3 is a graph showing the results of flow analysis of CD4+ T cells and CD8+ T cells in positive cells after magnetic sorting in example 1;

FIG. 4 is a graph showing the results of flow analysis of TCRb-positive cells among the positive cells after magnetic sorting in example 1;

FIG. 5 is a graph showing the results of flow analysis of CD4+ T cells and CD8+ T cells in unsorted whole blood in example 1;

FIG. 6 is a graph showing the results of flow analysis of CD3+ T cells in the cells cultured for seven days in example 2;

FIG. 7 is a graph showing the results of flow analysis of CD4+ T cells and CD8+ T cells in the cells after seven days of culture in example 2;

FIG. 8 is a graph showing the results of flow analysis of comparative example 1;

fig. 9 is a graph showing the results of the flow analysis of comparative example 2.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein.

The term "antibody" is a class of immunoglobulins that specifically bind to an antigen. Antibodies exist as one or more wye-shaped monomers, each of which consists of 4 polypeptide chains, comprising two identical heavy chains and two identical light chains, the light and heavy chains being named according to their molecular weight size. The variable region is located at the top of the Y-shaped structure and is an antigen binding site. Each heavy chain has two regions, a constant region and a variable region, and all antibodies of the same type have the same constant region and differ from one type to another. Each light chain also has two domains, a constant region and a variable region, connected in tandem.

The term "signal peptide" is a short (5-30 amino acids in length) peptide chain that directs the transfer of a newly synthesized protein to the secretory pathway. In the host cell, the foreign protein is usually not present in the cell. After the signal peptide is connected with the exogenous gene, secretory expression can be obtained in an expression system. The signal peptide can guide the secretion of the foreign protein to the extracellular culture medium, thereby improving the yield of the protein, simplifying the process of protein recovery and avoiding the difficulty of protein purification caused by cell disruption and the like.

The multi-specific single-chain antibody for separating T cells from whole blood according to an embodiment of the present invention has a signal peptide, a variable region sequence of a first antibody, a variable region sequence of a first linker peptide and a variable region sequence of a second antibody in this order from N-terminus to C-terminus, and does not have a constant region sequence of the first antibody and the second antibody. The first antibody and the second antibody can respectively and specifically bind to two surface marker antigens of the T cell, and the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence which are connected through a second connecting peptide.

Since antibodies can bind non-specifically to the surface of erythrocytes, in a typical T cell separation procedure, mononuclear cells are first separated from whole blood using a lymphocyte separation medium or other density gradient centrifugation reagent before the antibody separation step can begin. It has been found that the main reason for the non-specific binding of the antibody by erythrocytes is that the constant segments of the antibody can bind to the antibody-binding receptor on the surface of the erythrocyte membrane. Thus, theoretically removing the constant region of the antibody would prevent binding by red blood cells. However, many antibody separation reagents rely on the recognition of constant segments by the adsorption reagents (e.g., magnetic beads). Meanwhile, the stability of the antigen recognition segment remaining after the removal of the constant segment is reduced, and originally one antibody can recognize two antigen molecules, while the segment remaining after the removal of the constant segment can only recognize one antigen molecule, and the binding force is reduced, thereby affecting the separation efficiency.

In one particular example, the first antibody is a CD3 antibody and the second antibody is a CD28 antibody. It will be appreciated that other antibodies may be selected for the primary and secondary antibodies for use in isolating T cell subsets, such as CD4, CD8, CD25, CD30 and the like.

In one particular example, the C-terminus of the multispecific single-chain antibody also has a tag sequence. Protein detection and protein purification can be more conveniently performed by using the tag sequence. Specifically, the tag sequence is selected from FLAG, glutathione S-transferase, polyhistidine, maltose binding protein, thioredoxin, and dihydrofolate reductase, and the like, preferably polyhistidine.

In a specific example, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1, the amino acid sequence of the first connecting peptide is shown as SEQ ID NO. 2, the amino acid sequence of the second connecting peptide is shown as SEQ ID NO. 3, and the amino acid sequence of the tag sequence is shown as SEQ ID NO. 4. It is to be understood that the specific amino acid sequences of the signal peptide, the first linker peptide and the second linker peptide are not limited thereto and may be selected as desired.

In one specific example, the amino acid sequence of the multispecific single-chain antibody is shown in SEQ ID NO. 5, i.e., the structure from N-terminus to C-terminus is: signal peptide-CD 3 antibody light chain variable region- (GGGGS)₃Linker peptide-CD 3 antibody heavy chain variable region-inter linker-CD28 antibody heavy chain variable region- (GGGGS)₃The peptide-CD 28 antibody light chain variable region-His tag was linked.

The expression gene of one embodiment of the present invention is capable of expressing the multispecific single-chain antibody.

In one specific example, the nucleic acid sequence is shown as SEQ ID NO 6. It will be appreciated that due to the degeneracy of the codons, the nucleic acid sequences capable of expressing the same protein have a variety of forms, the above being codon optimized nucleic acid sequences, but are not limited thereto.

The recombinant cell according to an embodiment of the present invention contains the above-described expression gene, or has the above-described expression gene integrated into its genome.

The T cell separation method of one embodiment of the invention comprises the following steps: a first label is labeled on the multispecific single-chain antibody, and then mixed with a whole blood sample to obtain a mixed solution, and T cells bound with the multispecific single-chain antibody are captured from the mixed solution by using a second label capable of being specifically bound with the first label.

In one specific example, the first label is biotin and the second label is avidin. It is to be understood that the specific types of the first label and the second label are not limited thereto, and other combinations of substances capable of specifically binding may be selected as desired.

In one particular example, a method of capturing T cells bound to a multispecific single chain antibody comprises the steps of: and adding magnetic beads marked by a second marker into the mixed solution, incubating and then carrying out magnetic sorting to obtain the T cells combined with the multi-specificity single-chain antibody.

In one particular example, a method of capturing T cells bound to a multispecific single chain antibody comprises the steps of: and (3) allowing the mixed solution to flow through a cell culture bag filled with the cell culture carrier coated with the second marker in a gravity drip mode, washing the cell culture bag with physiological saline, and adding a culture medium for culture. Optionally, the cell culture carrier is a cellulose membrane, but is not limited thereto. So, directly fix the cell of separation in the culture bag through the cell culture carrier, reduced the link of the application of magnetic bead and middle centrifugation collection transfer cell, can reduce the operating procedure effectively, alleviate the injury to T cell, promoted cell culture's efficiency and maneuverability, establish the basis for realizing full-automatic isolated culture simultaneously.

The T cell isolation kit according to an embodiment of the present invention includes the aforementioned multi-specific single-chain antibody, a first label, and a second label, and the first label and the second label are capable of specifically binding to each other.

The following are specific examples.

Example 1

Expression and purification of anti-CD 3CD28 bispecific single chain antibody

The sequences of the anti-CD 3CD28 bispecific single chain antibody molecules are shown in the following table, the related sequences were synthesized by nucleotides, then cloned into pcDNA3.1 vector, expressed by transfection into 293T cell line, the supernatant was collected, the expressed antibody was purified by nickel chromatography column, and the results after purification are shown in FIG. 1.

From the expression purification results of FIG. 1, it was revealed that the anti-CD 3CD28 bispecific single chain antibody has been successfully obtained and the molecular weight is also correct.

Second, Whole blood separation experiment

The purified anti-CD 3CD28 bispecific single chain antibody molecule was biotinylated using the biotin labeling kit and purified according to the manufacturer's instructions.

And (2) taking 5mL of whole blood, adding 10mL of physiological saline for dilution, then adding 5mg of the biotin-labeled anti-CD 3CD28 bispecific single-chain antibody molecule, then adding streptavidin-labeled nano magnetic beads (nutria biology) for uniformly mixing, incubating for 5 minutes at room temperature, and sorting positive cells in a magnetic frame to obtain the T cells through separation.

Third, purity and efficiency of flow analysis separation

The cells sorted by the magnetic frame were labeled with anti-human TCRb-PE, CD4-FITC, CD8a-PE-Cy7 antibody (available from ebioscience), wherein the TCRb-positive cells represent T cells, and the CD 4-positive and CD 8-positive cells represent two T cell subsets, CD4+ T cells and CD8+ T cells, respectively, and the results are shown in FIG. 2 and FIG. 3. Meanwhile, we retained a portion of magnetic sorting negative cells and unsorted whole blood, stained with the above-mentioned antibody after lysing erythrocytes, and examined the sorting efficiency and the effect on the CD4+ T cells and CD8+ T cell subsets after sorting in the same flow assay, and the results are shown in fig. 4 and 5.

Fourth, result analysis

As shown in fig. 2, the proportion of TCRb-PE cells was 69.8%, indicating that the sorted T cells reached about 70% purity; the results shown in figure 3 indicate that CD4+ T cells and CD8+ T cells were 48.2% and 37.2%, respectively; the results shown in fig. 4 indicate that the proportion of T cells in the sorted negative cells was 0.04%, indicating that the proportion of sorted positive cells was very high, which could reach 95% or more; the results shown in fig. 5 indicate that the proportion of CD4+ T cells and CD8+ T cells before sorting was 50.7% and 36.4%, respectively, and as can be seen from a comparison of the results in fig. 3, there was no large change in the proportion of cells of the two subpopulations before and after sorting.

Example 2

The bispecific single chain antibody against CD3CD28 was obtained by expression and purification as described in example 1.

The bispecific single chain antibody molecule expressing and purifying the CD3CD28 is labeled by biotin with a biotin labeling kit according to the instructions of manufacturers, so that the feasibility of the bispecific single chain antibody molecule is verified by a whole blood separation experiment. Meanwhile, a cellulose membrane coated by streptavidin protein is prepared, and the coated cellulose membrane is placed into a cell culture bag with communicated infusion tubes in the upper and lower directions.

50mL of whole blood was diluted with 100mL of physiological saline, 50mg of biotin-labeled anti-CD 3CD28 bispecific single chain antibody molecule was added, the mixture was mixed and incubated at room temperature for 5 minutes, unbound cells were discharged through a liquid transfer tube on the lower side of the culture bag by gravity drip-flow through a cellulose membrane coated with streptavidin, the contents of the culture bag were washed once with 200mL of physiological saline, and then 10mL of serum-free medium (Behcet Co.) was added for culture for seven days, and cell counting and flow analysis were collected.

The obtained cells were labeled with anti-human CD3-PE, CD19-PB, CD4-FITC, CD8a-PE-Cy7 antibodies (ebioscience), wherein the CD3 positive cells represent T cells, and the CD4 positive and CD8 positive cells represent two T cell subsets, CD4+ T cells and CD8+ T cells, respectively, as shown in FIG. 6 and FIG. 7.

The results shown in FIG. 6 indicate that the proportion of CD3-PE cells was 89.9%, indicating that the purity of T cells reached around 90%; the results shown in figure 7 indicate that CD4+ T cells and CD8+ T cells are 46.1% and 43.5%, respectively, within the range of normal CD4+/CD8+ T cell ratios.

Comparative example 1

Compared with the antibody of the example 1, the antibody of the comparative example adds a constant region sequence, namely, the constant region sequence is added at the N terminal of the HIS tag of the CD3CD28 bispecific single chain antibody of the example 1, and the structure is as follows: signal peptide-CD 3 antibody light chain variable region- (GGGGS)₃Linker peptide-CD 3 antibody heavy chain variable region-inter linker-CD28 antibody heavy chain variable region- (GGGGS)₃The peptide-CD 28 antibody light chain variable region-Fc segment-HIS tag was linked, and the constant region (Fc) sequence was referenced to GENE BANK: AF 237583.1.

The whole blood separation experiment steps are the same, and the flow analysis result is shown in FIG. 8, which shows that the T cell separation effect is obviously inferior to that of example 1.

Comparative example 2

In this comparative example, the flow analysis results obtained after 7 days of culture after removing erythrocytes and then sorting T cells by the conventional separation method are shown in fig. 9, which is similar to the results shown in fig. 6 of example 2, indicating that the multispecific single-chain antibody and T cell separation method of the present invention can achieve the same effects as the conventional protocol.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims

1. A multispecific single-chain antibody for separating T cells from whole blood, which has, in order from the N-terminus to the C-terminus, a signal peptide, a variable region sequence of a first antibody, a variable region sequence of a first linker peptide and a variable region sequence of a second antibody, and does not have constant region sequences of the first antibody and the second antibody; the first antibody and the second antibody are capable of specifically binding to two surface marker antigens of a T cell, respectively, and the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence, which are linked by a second linker peptide.

2. The multispecific single-chain antibody of claim 1, wherein the first antibody is a CD3 antibody and the second antibody is a CD28 antibody.

3. The multispecific single-chain antibody according to claim 2, wherein the C-terminus of the multispecific single-chain antibody further comprises a tag sequence.

4. The multi-specific single-chain antibody of claim 3, wherein the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1, the amino acid sequence of the first connecting peptide is shown as SEQ ID NO. 2, the amino acid sequence of the second connecting peptide is shown as SEQ ID NO. 3, and the amino acid sequence of the tag sequence is shown as SEQ ID NO. 4.

5. The multispecific single-chain antibody of claim 4, wherein the amino acid sequence of the multispecific single-chain antibody is set forth in SEQ ID NO 5.

6. An expressed gene capable of expressing the multispecific single-chain antibody according to any one of claims 1 to 5.

7. The expressed gene of claim 6, wherein the nucleic acid sequence is set forth in SEQ ID NO 6.

8. A recombinant cell comprising the expressible gene of claim 6 or 7 or having the expressible gene integrated into the genome of the recombinant cell.

9. A method of T cell isolation comprising the steps of: a mixture obtained by labeling the multispecific single-chain antibody according to any one of claims 1 to 5 with a first label, mixing the mixture with a whole blood sample, and capturing T cells bound to the multispecific single-chain antibody from the mixture with a second label capable of specifically binding to the first label.

10. The method of T cell isolation of claim 9, wherein the method of capturing T cells bound to the multispecific single-chain antibody comprises the steps of: and adding a second marker labeled magnetic bead into the mixed solution, incubating and then carrying out magnetic sorting to obtain the T cells combined with the multispecific single-chain antibody.

11. The method of T cell isolation of claim 9, wherein the method of capturing T cells bound to the multispecific single-chain antibody comprises the steps of: and (3) allowing the mixed solution to flow through a cell culture bag filled with the cell culture carrier coated with the second marker in a gravity drip mode, washing the cell culture bag with physiological saline, and adding a culture medium for culture.

12. A T cell isolation kit comprising the multi-specific single-chain antibody according to any one of claims 1 to 5, a first label and a second label, wherein the first label and the second label are capable of specifically binding to each other.