CN113773393B

CN113773393B - Multispecific single-chain antibody and application thereof

Info

Publication number: CN113773393B
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: 2023-11-28
Anticipated expiration: 2040-06-10
Also published as: CN113773393A

Abstract

The application relates to a multi-specific single-chain antibody and application thereof, wherein the multi-specific 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 N end to 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 T cells, respectively, and the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence, and the light chain variable region sequence and the heavy chain variable region sequence are connected through a second connecting peptide. The multi-specific single-chain antibody can effectively adsorb and separate T cells from whole blood, simplify the separation step of the T cells, reduce the damage to the T cells, reduce the risk of misoperation and equipment requirements caused by multi-step operation, and reduce the probability of cross contamination and influence among different samples in T cell immunotherapy.

Description

Multispecific single-chain antibody and application thereof

Technical Field

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

Background

T cell isolation is the first link in CAR-T cell preparation and is also an important technology in T cell preparation. Currently, it is generally necessary to separate T cells from whole blood by separating monocytes from the whole blood using lymphocyte separation fluid or other density gradient centrifugation reagent, and then separating the desired T cells by using antibody separation technology through binding to antibodies of T cell characteristic molecules CD3, TCR molecules and other co-stimulatory molecules such as CD28, etc. The common separation technology is a method of magnetic bead separation, flow separation and the like.

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

Disclosure of Invention

Based on this, it is necessary to provide a multi-specific single chain antibody that can be used to isolate T cells directly from whole blood.

A multi-specific single chain antibody for isolating T cells from whole blood, having a signal peptide, a variable region sequence of a first antibody, a first connecting peptide and a variable region sequence of a second antibody in this order from the N-terminus to the C-terminus, and having no 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 T cells, respectively, and the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence, and the light chain variable region sequence and the heavy chain variable region sequence are connected through a second connecting peptide.

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

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

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 multi-specific single chain antibody described above.

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

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

A method of T cell isolation comprising the steps of: labeling the multi-specific single-chain antibody with a first label, mixing the labeled multi-specific single-chain antibody with a whole blood sample to obtain a mixed solution, and capturing T cells bound with the multi-specific single-chain antibody 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 multi-specific single chain antibody comprises the steps of: and adding magnetic beads marked by a second marker into the mixed solution, incubating, and then magnetically separating to obtain the T cells combined with the multi-specific single-chain antibody.

In one embodiment, the method of capturing T cells bound to the multi-specific single chain antibody comprises the steps of: the mixed solution was flowed through a cell culture bag filled with the second label-coated cell culture carrier by gravity instillation, the cell culture bag was washed with physiological saline, and then culture medium was added for culture.

A T cell isolation kit comprising the multi-specific single chain antibody described above, a first label and a second label, said first label and said second label being capable of specific binding.

The application is based on a first antibody and a second antibody which can respectively specifically bind to two surface marker antigens of T cells, wherein a constant section is removed, and a binding signal peptide and a connecting peptide are combined to construct a multi-specific single-chain antibody molecule capable of single-chain expression. By connecting the variable region sequences of the first antibody and the second antibody together, two antigen molecules can be combined simultaneously, the binding force of the single-chain antibody molecules and the antigens is enhanced, and the variable region sequences are not contained, so that the variable region sequences can be directly used for separating corresponding T cells from whole blood, the processes of removing erythrocytes from the whole blood and separating monocytes are omitted, and the damage to the T cells in the separation process is reduced. Therefore, the multi-specific single-chain antibody can be directly added into whole blood for T cell separation, red blood cells can not adsorb the antibody, the antibody can be ensured to be combined with corresponding T cells, and meanwhile, the problem of weak single-chain antibody binding force after the constant region is removed can be well overcome by utilizing multi-specificity. Experimental results show that the multi-specific single-chain antibody can effectively adsorb and separate T cells from whole blood, simplify the separation step of the T cells, reduce the damage to the T cells, reduce the risk of misoperation and equipment requirements caused by multi-step operation, and 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 magnetically sorted positive cells 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 magnetically sorted positive cells 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 of example 1;

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

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

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

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

Detailed Description

The present application will be described more fully hereinafter in order to facilitate an understanding of the present application, and preferred embodiments of the present application are set forth. This application 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 application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings 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 Y-shaped monomers, each consisting 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. The top of the Y-shaped structure is a variable region which is an antigen binding site. Each heavy chain has two regions, namely a constant region and a variable region, and antibodies of all the same type have the same constant region, and antibodies of different types have differences. Each light chain also has two domains, one constant and one variable, connected in tandem.

The term "signal peptide" is a short (5 to 30 amino acids in length) peptide chain that directs the transfer of a newly synthesized protein to the secretory pathway. Foreign proteins are mostly intracellular non-proteins in host cells. After the exogenous gene is connected with the signal peptide, secretory expression can be obtained in an expression system. The signal peptide can guide the secretion of the exogenous protein to the extracellular culture medium, so that the yield of the protein can be improved, the protein recovery process is simplified, and the difficulty in protein purification caused by cell disruption and the like is avoided.

The multi-specific single-chain antibody for separating T cells from whole blood according to an embodiment of the present application has a signal peptide, a variable region sequence of a first antibody, a first connecting peptide and a variable region sequence of a second antibody in order from the N-terminus to the C-terminus, 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 T cells, respectively, and the variable region sequences include a light chain variable region sequence and a heavy chain variable region sequence, which are linked by a second linking peptide.

Since antibodies can bind non-specifically to the surface of erythrocytes, in a typical T cell separation method, it is necessary to separate monocytes from whole blood using lymphocyte separation fluid or other density gradient centrifugation reagents before the antibody separation step can begin. It was found that the main reason for the non-specific binding of erythrocytes to antibodies is that the constant region of the antibody can bind to the antibody binding receptor on the surface of erythrocyte membranes. Thus, the removal of the constant segment of the antibody theoretically prevents binding by erythrocytes. However, the adsorption of reagents (e.g., magnetic beads) in many antibody separation reagents is dependent on the recognition of constant regions. Meanwhile, the stability of the remaining antigen recognition section after the constant section is removed is reduced, and the original one antibody can recognize two antigen molecules, while the remaining section after the constant section is removed can only recognize one antigen molecule, the binding force is reduced, and the separation efficiency is further affected.

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

In a specific example, the C-terminal end of the multispecific single-chain antibody further 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, etc., 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 connecting peptide and the second connecting peptide are not limited thereto, and may be selected as desired.

In a specific example, the amino acid sequence of the multispecific single-chain antibody is shown in SEQ ID NO. 5, namely, the structure from the N end to the C end is as follows: signal peptide-CD 3 antibody light chain variable region- (GGGGS) ₃ Connecting peptide-CD 3 antibody heavy chain variable region-inter linker-CD28 antibody heavy chain variable region- (GGGGS) ₃ The linker peptide-CD 28 antibody light chain variable region-His tag.

The expressed gene of an embodiment of the present application is capable of expressing the multi-specific single chain antibody described above.

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

The recombinant cell of an embodiment of the application comprises the above expressed gene or has the above expressed gene integrated into its genome.

The T cell separation method of one embodiment of the application comprises the following steps: labeling a first marker on the multi-specific single-chain antibody, mixing the labeled first marker with the whole blood sample to obtain a mixed solution, and capturing T cells bound with the multi-specific single-chain antibody from the mixed solution by using a second marker capable of specifically binding with the first marker.

In one specific example, the first label is biotin and the second label is avidin. It will be appreciated that the particular type of first and second labels is not limited thereto and that other combinations of substances capable of specifically binding may be selected as desired.

In a specific 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 performing magnetic separation to obtain the T cells combined with the multi-specific single-chain antibody.

In a specific example, a method of capturing T cells bound to a multispecific single-chain antibody comprises the steps of: the mixed solution was flowed through a cell culture bag filled with a cell culture carrier coated with a second marker by gravity instillation, the cell culture bag was washed with physiological saline, and then the culture medium was added for culture. Alternatively, the cell culture carrier is a cellulose membrane, but is not limited thereto. Therefore, the separated cells are directly fixed into the culture bag through the cell culture carrier, the application of magnetic beads and the link of collecting and transferring the cells through intermediate centrifugation are reduced, the operation steps can be effectively reduced, the damage to T cells is lightened, the efficiency and operability of cell culture are improved, and meanwhile, the foundation is laid for realizing full-automatic separation culture.

The 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 following are specific examples.

Example 1

1. Expression and purification of anti-CD 3CD28 bispecific single chain antibodies

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

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

2. Whole blood separation experiment

The bispecific single chain antibody molecule expressing purified anti-CD 3CD28 was biotin-labeled with a biotin labeling kit and purified by labeling according to the manufacturer's instructions.

Taking 5mL of whole blood, adding 10mL of normal 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 (beaver organisms), uniformly mixing, incubating for 5 minutes at room temperature, and separating positive cells in a magnetic rack to obtain the T cells.

3. Purity and efficiency of flow analysis separation

Cells obtained by the above-mentioned magnetometric frame sorting were labeled with anti-human TCRb-PE, CD4-FITC, CD8a-PE-Cy7 antibody (product of ebioscience Co.), wherein TCRb positive cells represent T cells, CD4 positive and CD8 positive represent CD4+ T cells and CD8+ T cells of two T cell subsets, respectively, and the results are shown in FIGS. 2 and 3. Meanwhile, we left part of cells which were negative in magnetic sorting and whole blood which were not sorted, stained with the above antibodies after lysing erythrocytes, and examined in the same flow, and observed the sorting efficiency and the effect on CD4+ T cells and CD8+ T cell subsets after sorting, and the results are shown in FIGS. 4 and 5.

4. Analysis of results

As shown in FIG. 2, the proportion of TCRb-PE cells was 69.8%, indicating that the purity of the sorted T cells reached about 70%; the results shown in fig. 3 demonstrate that cd4+ T cells and cd8+ T cells are 48.2% and 37.2%, respectively; the results shown in FIG. 4 indicate that the proportion of T cells in the sorted negative cells is 0.04%, indicating that the proportion of sorted positive cells is very high and can reach more than 95%; the results shown in FIG. 5 demonstrate that the ratios of CD4+ T cells and CD8+ T cells before sorting are 50.7% and 36.4%, respectively, and that there is no significant change in the ratio of cells between the two subsets before and after sorting as can be seen by comparing the results of FIG. 3.

Example 2

The anti-CD 3CD28 bispecific single chain antibody was obtained by expression purification as described in example 1.

The expressed and purified anti-CD 3CD28 bispecific single chain antibody molecules are subjected to biotin labeling by a biotin labeling kit, and are subjected to labeling and purification according to the specification of the manufacturer, so that the feasibility of the whole blood separation experiment is verified later. And preparing a cellulose membrane coated by streptavidin proteins, and placing the coated cellulose membrane into a cell culture bag with a communicating infusion tube in the upper and lower directions.

50mL of whole blood was taken, diluted with 100mL of physiological saline, then 50mg of biotin-labeled anti-CD 3CD28 bispecific single chain antibody molecule was added, mixed well, incubated at room temperature for 5 minutes, and the unbound cells flowed out through a transfusion tube filled with a streptavidin-coated cellulose membrane by gravity instillation, the contents of the culture bag were washed once with 200mL of physiological saline, then 10mL of serum-free medium (Pinctada martensii) was added for seven days, and cell counts and flow analyses were collected.

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

The results shown in FIG. 6 indicate that the proportion of CD3-PE cells is 89.9%, indicating that the purity of T cells reaches about 90%; the results shown in FIG. 7 demonstrate that CD4+ T cells and CD8+ T cells are 46.1% and 43.5%, respectively, within the normal CD4+/CD8+ T cell ratio range.

Comparative example 1

The antibody of this comparative example has a constant region sequence added to the N-terminus of the CD3CD28 bispecific single chain antibody HIS tag of example 1, compared with example 1, and has the structure: signal peptide-CD 3 antibody light chain variable region- (GGGGS) ₃ Connecting peptide-CD 3 antibody heavy chain variable region-inter linker-CD28 antibody heavy chain variable region- (GGGGS) ₃ The linker peptide-CD 28 antibody light chain variable region-Fc segment-HIS tag, constant region (Fc) sequence was referenced to GENE BANK: AF237583.1.

The whole blood separation experiment was identical, and the results of the flow analysis are shown in FIG. 8, and it can be seen that the T cell separation effect was significantly inferior to that of example 1.

Comparative example 2

The comparative example adopts the conventional separation method to remove erythrocytes first, then carries out T cell separation, and cultures for 7 days, and the obtained flow analysis result is shown in FIG. 9, which is similar to the result of FIG. 6 of example 2, which shows that the multi-specific single chain antibody and the T cell separation method of the application can achieve the same effect as the conventional scheme.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Sequence listing

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Claims

1. A multi-specific single chain antibody for isolating T cells from whole blood, characterized by having a signal peptide, a variable region sequence of a first antibody, a first connecting peptide and a variable region sequence of a second antibody in this order from the N-terminus to the C-terminus, and having no constant region sequences of the first antibody and the second antibody; the first antibody and the second antibody can respectively specifically bind to two surface marker antigens of T cells, the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence, and the light chain variable region sequence and the heavy chain variable region sequence are connected through a second connecting peptide; the amino acid sequence of the multispecific single-chain antibody is shown as SEQ ID NO. 5.

2. An expressed gene, characterized in that it is capable of expressing the multi-specific single chain antibody of claim 1.

3. The expressed gene according to claim 2, wherein the nucleic acid sequence is shown in SEQ ID NO. 6.

4. A recombinant cell comprising the expressed gene of claim 2 or 3, or having the expressed gene integrated into the genome of the recombinant cell.

5. A method of T cell isolation comprising the steps of: labeling a first label on the multi-specific single-chain antibody according to claim 1, then mixing with a whole blood sample to obtain a mixed solution, and capturing the T cells bound to the multi-specific single-chain antibody from the mixed solution by using a second label capable of specifically binding to the first label.

6. The method of claim 5, wherein the method of capturing T cells bound to the multi-specific single chain antibody comprises the steps of: and adding magnetic beads marked by a second marker into the mixed solution, incubating, and then magnetically separating to obtain the T cells combined with the multi-specific single-chain antibody.

7. The method of claim 5, wherein the method of capturing T cells bound to the multi-specific single chain antibody comprises the steps of: the mixed solution was flowed through a cell culture bag filled with the second label-coated cell culture carrier by gravity instillation, the cell culture bag was washed with physiological saline, and then culture medium was added for culture.

8. A T cell isolation kit comprising the multi-specific single chain antibody of claim 1, a first label and a second label, said first label and said second label being capable of specifically binding.