CN116023499A - Bispecific antibody for CD3 and CD20 - Google Patents
Bispecific antibody for CD3 and CD20 Download PDFInfo
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Abstract
The invention provides a bispecific antibody aiming at CD3 and CD20, a nucleic acid molecule containing an antibody coding sequence and application thereof in preparing medicines for diagnosing and treating malignant tumors.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a bispecific antibody, in particular to a bispecific antibody aiming at CD3 and CD 20.
Background
Bispecific antibodies are an emerging tumor immunotherapy technology that can recognize and bind two different epitopes simultaneously, and can be generally divided into two according to the routes of action, one that acts by blocking two different signal pathways simultaneously through synergism, and the other that acts by activating effector cells (T cells, NK cells, etc.).
The bispecific antibody technology platform is mainly divided into two major classes according to structure: bispecific antibodies containing an Fc region (IgG-like bispecific antibodies) and bispecific antibodies not containing an Fc region (non-IgG-like bispecific antibodies); most conventional antibodies are Fc region-containing. While BiTE (Bispecific T cell engager, T cell engager antibody) is a T cell dependent bispecific antibody. The antibody mainly consists of two single-chain antibodies (scFv), has the molecular weight of about 50kDa, has the advantage of being easier to penetrate through tumor tissues compared with the traditional IgG type bispecific antibody, and in addition, due to lack of an Fc segment, not only is toxic and side effects caused by interaction between the Fc segment and in vivo FcRn-expressing cells avoided, but also systemic immune side effects caused by the Fc segment can be avoided, and the specificity of the antibody is further ensured.
Compared with traditional chemotherapy, the monoclonal antibody aiming at the CD20 target has better clinical effect in clinic, and compared with high expression of CD20 by tumor cells, the monoclonal antibody aiming at the CD20 on the surface of the tumor cells has limited tumor treatment effect with low expression level of CD 20; monoclonal antibodies to CD3 targets can recruit T cells to the vicinity of target cells, directly killing the target cells by activating the T cells; the CD3/CD20 double antibody can combine with T cells and tumor cells simultaneously, recruit the T cells to the tumor cells expressing CD20, and activate the T cells to secrete granzyme, perforin and the like to kill CD20 positive tumor cells.
Most of the double-antibody platform technologies of CD3xCD20 currently under clinical or preclinical research retain the Fc structure of antibodies, and no report on the miniaturization of double antibodies of CD3xCD20 is available; because the technical platform containing the Fc region has the advantages of convenient purification, good stability and the like, the technical platform has the disadvantages that some structures (such as Crossmab/KIH) CMC are complex, and the problems of higher polymers, mismatching, low purification yield and the like are likely to occur. In addition, the antibody has too large molecular weight, has poor permeability to tumor tissues, is difficult to penetrate through the cortex and the cell gap in blood vessels so as to reach tumor cells in the deep part of the solid tumor, and therefore has an inferior therapeutic effect on the solid tumor compared with that of blood tumor.
There is a need in the art for a CD3xcd20 bispecific antibody that has high specificity, low side effects, and good pharmacokinetic properties.
Disclosure of Invention
In order to solve the above problems, in a first aspect, the present invention aims to provide a bispecific antibody comprising a first protein sequence and a second protein sequence, wherein the first protein sequence comprises a CD20 binding fragment and the second protein sequence comprises a CD3 binding fragment, the first protein sequence and the second protein sequence being linked by a flexible linker peptide sequence 2.
In a preferred embodiment, the amino acid sequence of flexible linker peptide sequence 2 is shown in SEQ ID No. 5.
In a specific embodiment, the bispecific antibody of the present invention comprises the first protein sequence, the flexible linker peptide sequence 2 and the second protein sequence in that order.
In a specific embodiment, said first protein sequence in said bispecific antibody of the present invention comprises a heavy chain variable region CD20VH and a light chain variable region CD20VL that bind to CD20, and the heavy chain variable region CD20VH and said light chain variable region CD20VL are linked by a flexible linker peptide sequence 1. In a preferred embodiment, the amino acid sequence of the heavy chain variable region CD20VH is shown in SEQ ID No.1 and the amino acid sequence of the light chain variable region CD20VL is shown in SEQ ID No. 2. In a more preferred embodiment, the amino acid sequence of the flexible linker peptide sequence 1 may be as shown in SEQ ID No.6
In a specific embodiment, said second protein sequence in said bispecific antibody of the present invention comprises a heavy chain variable region CD3VH and a light chain variable region CD3VL that bind to CD3, and the heavy chain variable region CD3VH and said light chain variable region CD3VL are linked by a linking sequence CD 3L. In a preferred embodiment, the amino acid sequence of the heavy chain variable region CD3VH is shown in SEQ ID No.3 and the amino acid sequence of the light chain variable region CD3VL is shown in SEQ ID No. 4. In a more preferred embodiment, the amino acid sequence of the linker sequence Linke 3 may be as shown in SEQ ID No.7
In a preferred embodiment, the second protein sequence comprises in sequence the heavy chain variable region CD3VH, the flexible connecting peptide sequence 3 and the light chain variable region CD3VL.
In a specific embodiment, the C-terminal end of the bispecific antibody of the invention may be linked to a tag sequence for protein purification, such as a histidine tag sequence.
In a second aspect, the present invention aims to provide a nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antibody as in the first aspect.
In a third aspect, the present invention aims to provide the use of a bispecific antibody as in the first aspect, a nucleic acid molecule as in the second aspect, for the manufacture of a medicament for the diagnosis and treatment of malignant tumors. In a preferred embodiment, the malignancy may be a malignancy having CD20 protein expression.
Compared with the prior art, the bispecific antibody has the following advantages:
(1) The preparation method of the bispecific antibody has the advantages of high antibody assembly efficiency and no mismatch of light chains and heavy chains of the antibody.
(2) The bispecific antibodies of the invention can target malignant tumors to which CD20 protein expression is bound with high affinity;
(3) The bispecific antibody has a binding effect with CD3, has lower affinity, can well activate T cells and can not continuously activate the T cells to cause cytokine storm; the safety of the CD3 end bispecific molecule can be improved;
(4) The bispecific antibody lacks an Fc segment, can avoid systemic immune side effects caused by the Fc segment, further ensures the specificity of the antibody, simultaneously retains the advantage that a BiTE form is easy to penetrate through tumors, and has good pharmacokinetic properties;
(5) The bispecific antibody can effectively mediate the killing effect of T cells on tumor cells, and the killing effect is remarkable.
Drawings
FIG. 1 is a schematic structural diagram of exemplary bispecific antibodies A and B of the invention and control antibody C.
FIG. 2 is a graph showing a fit of binding/dissociation of an exemplary bispecific antibody of the invention and a control antibody to CD20 protein; wherein fig. 2a is a binding/dissociation fit of antibody a to CD20 protein, fig. 2B is a binding/dissociation fit of antibody B to CD20 protein, fig. 2C is a binding/dissociation fit of control antibody C to CD20 protein, and fig. 2d is a binding/dissociation fit of rituximab to CD20 protein.
FIG. 3 is a graph showing a fit of binding/dissociation of an exemplary bispecific antibody of the present invention to a CD3 protein; wherein fig. 3a is a fitted curve of binding/dissociation of antibody a with CD3 protein and fig. 3B is a fitted curve of binding/dissociation of antibody B with CD3 protein.
FIG. 4 is a graph showing exemplary bispecific antibody A and B mediated killing of target cells by T cells of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings.
Example 1: construction of expression vectors
(1) Molecular design of bispecific antibody CD3xCD20 and control antibody
The amino acid sequences of the bispecific antibodies A and B are shown in SEQ ID No.8 and SEQ ID No.9, wherein the sequence SEQ ID No.8 comprises the following amino acid sequences in sequence: SEQ ID No.2 (light chain variable region CD20 VL), SEQ ID No.6 (flexible connecting peptide sequence 1), SEQ ID No.1 (heavy chain variable region CD20 VH), SEQ ID No.5 (flexible connecting peptide sequence 2), SEQ ID No.3 (heavy chain variable region CD3 VH), SEQ ID No.7 (flexible connecting peptide sequence 3), SEQ ID No.4 (light chain variable region CD3 VL) and histidine tag (6 His); the sequence SEQ ID No.9 comprises the following amino acid sequences in sequence: SEQ ID No.1 (heavy chain variable region CD20 VH), SEQ ID No.6 (flexible linker sequence 1), SEQ ID No.2 (light chain variable region CD20 VL), SEQ ID No.5 (flexible linker sequence 2), SEQ ID No.3 (heavy chain variable region CD3 VH), SEQ ID No.7 (flexible linker sequence 3), SEQ ID No.4 (light chain variable region CD3 VL) and a histidine tag (6 His).
The coding nucleotide sequences of the bispecific antibody are shown as SEQ ID No.10 and SEQ ID No. 11.
In addition, a control antibody C was designed, the amino acid sequence of which is shown in SEQ ID No.12, comprising the following amino acid sequences in order: SEQ ID No.3 (heavy chain variable region CD3 VH), SEQ ID No.7 (flexible linker sequence 3 sequence), SEQ ID No.4 (light chain variable region CD3 VL), SEQ ID No.5 (flexible linker sequence 2) SEQ ID No.2 (light chain variable region CD20 VL), SEQ ID No.6 (flexible linker sequence 1), SEQ ID No.1 (heavy chain variable region CD20 VH), and histidine tag (6 His). The coding nucleotide sequence of the control antibody is shown as SEQ ID No. 13.
(2) Construction of antibody expression plasmids
The invention obtains the coding nucleotide sequences shown as SEQ ID No.10, SEQ ID No.11 and SEQ ID No.13 by a gene synthesis technology, and all three coding sequences use human IL-2 secretion signal peptide so as to be capable of secretory expression in CHO cells; this fusion frame obtained by cloning was inserted into p2U-dhfr vector (purchased from Vega Biolab LLC Co., U.S.A.) to obtain plasmid A (bispecific antibody), plasmid B (bispecific antibody) and plasmid C (control antibody), and gene sequencing was performed, and the sequencing result was aligned with the designed sequence to confirm that the constructed plasmids A, B and C were correct.
Example 2: transient transfection and expression of antibodies
The results obtained in example 1 were constructed by the method of liposome (kit purchased from gibco) transfectionPlasmid A, plasmid B and plasmid C were transfected into the ExpiCHO-S host, respectively; adjusting CHO-S cell density to 7-10e with ExpiCHO Expression Medium medium 6 Per ml, transfection was performed as per the liposome kit instructions, and the transfected cell suspension was placed at 36.5℃in 6% CO 2 Shaking seed culture at 100RPM, cooling to 32deg.C on day 1, and adding feed on day 1 and day 5 respectively, when cell viability is reached<At 70%, the culture was terminated and the cell supernatant was collected.
Example 3: purification of antibodies
Cell supernatants were collected by centrifugation, the cell supernatants were filtered with 0.22 μm filters, then bispecific antibodies were captured with protein L affinity chromatography packing (purchased from GE), target proteins were eluted by 100mM acetic acid-sodium acetate pH3.5 buffer, and the pH of the samples was adjusted to around 6.5 with 2M Tris-HCl buffer. Further purified to a purity of more than 90% by analysis by means of a purity (SEC-HPLC), antibody sample a (bispecific antibody), sample B (bispecific antibody) and sample C (control antibody) were obtained.
Example 4: affinity detection with antigen CD20 protein and antigen CD3 protein, respectively
(1) Affinity detection with the antigen CD20 protein
Sample a, sample B, rituximab (purchased from roche) and sample C were first labeled with biotin, respectively, and then captured with an SA probe (strepitavidin from gator); the capturing height is 1-1.3nm; CD20 protein (ex ACRO) as analyte at 200nM concentration as initial concentration and total dilution of 7 concentration points at 2-fold dilution; running an experimental program, fitting a binding and dissociation curve (see fig. 2), and calculating affinity KD values; the results are shown in Table 1:
table 1: binding of sample to CD20 target protein
Kon reflects the binding rate of the sample to the CD20 target protein, the greater the number, the stronger the binding rate, the no rising signal value (ordinate) is observed in the binding step except for the lack of binding signal between sample C and the target protein (fig. 2C), the binding rate between sample a, sample B and rituximab and the CD20 target protein are not significantly different, and the Kon values are on the same order of magnitude. Koff reflects the rate of dissociation of the sample from the target protein, the larger the value, the faster the dissociation; the KD value (Koff/Kon ratio) reflects the strength of the sample in terms of its binding capacity to the target protein.
Table 1 data shows: sample A and rituximab have similar binding strength to the target protein, and KD values are in the same order of magnitude; sample B had a slightly lower binding strength than rituximab and sample C did not bind to CD20 protein.
(2) Affinity detection with antigen CD3 protein
First, CD3 protein (ex ACRO) was captured with a Human Fc probe (ex Gator), sample a and sample B were each used as analyte, starting at 1500nM concentration, and diluted 2-fold, each at 5 concentration points; the experimental procedure was run, the binding and dissociation curves were fitted (see fig. 3), and affinity KD values were calculated, the results are shown in table 2.
Table 2: binding of sample to CD3 protein
| Sample name | KD(M) | Feature map |
| Sample A | 1.89E-07 | See FIG. 3a |
| Sample B | 8.62E-08 | See FIG. 3b |
The results in table 2 show that: both sample A and sample B have binding effect with CD3 protein, can activate T cells better and activate T cells continuously, so that the risk of cytokine storm is low; improving the safety of the CD3 end bispecific molecule.
Example 5: detection of killing Activity of bispecific antibodies against tumor cells
In order to further verify the killing effect of samples A and B on tumor cells at the cellular level, we selected fluorescein-labeled Raji cells (Raji-luc) as target cells and HUT78-CRM cells (T cells) as effector cells, and verified that samples A and B can play a bridging role between the two cells to mediate the killing effect of the T cells on the target cells; wherein the decrease in fluorescent signal is measured to reflect cell death of the target cell.
The specific experimental method is as follows: the cell density was adjusted using log-phase HUT78-CRM cells (effector cells) and Raji-Luc cells (target cells) to control the effective target ratio to 10:1, the initial concentration of the sample is 6000ng/ml, the sample is diluted to 11 concentration points according to a quadruple dilution method, HUT78-CRM cells, raji-Luc cells and samples with different concentrations are incubated for 24 hours in an incubator, a 96-well plate is taken out and returned to room temperature, and 200 mu l of Steady-
Luciferase Assay System, placing the reagent on a 96-well plate mixing instrument, incubating for 15min at room temperature in a dark place, detecting fluorescent values by an enzyme-labeled instrument, performing four-parameter fitting by using Magellan software, and analyzing the result.
As shown in the experimental results in FIG. 4, the samples A and B can effectively mediate HUT-78 cells (T cells) to kill Raji cells (tumor cells), the killing effect is remarkable, and the maximum killing rate can reach about 75%; the EC50 of sample A was 15.330ng/ml and the EC50 of sample B was 24.357ng/ml.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, and that the foregoing embodiments and description are merely illustrative of the principles of this invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, and these changes and modifications fall within the scope of the invention as hereinafter claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A bispecific antibody, wherein the antibody comprises a first protein sequence and a second protein sequence, wherein the first protein sequence comprises a CD20 binding fragment and the second protein sequence comprises a CD3 binding fragment, wherein the first protein sequence and the second protein sequence are linked by a flexible linker peptide sequence 2; preferably, the amino acid sequence of the flexible connecting peptide sequence 2 is shown as SEQ ID No. 5.
2. The bispecific antibody of claim 1, wherein the bispecific antibody comprises the first protein sequence, the flexible linker peptide sequence 2, and the second protein sequence in that order.
3. The bispecific antibody of claim 1 or 2, wherein the first protein sequence comprises a heavy chain variable region CD20VH and a light chain variable region CD20VL that bind to CD20, and wherein the heavy chain variable region CD20VH and the light chain variable region CD20VL are linked by a flexible linker peptide sequence 1; preferably, the amino acid sequence of the heavy chain variable region CD20VH is shown as SEQ ID No.1 and the amino acid sequence of the light chain variable region CD20VL is shown as SEQ ID No. 2; more preferably the amino acid sequence of the flexible linker peptide sequence 1 is shown in SEQ ID No. 6.
4. The bispecific antibody of claim 1 or 2, wherein the second protein sequence comprises a heavy chain variable region CD3VH and a light chain variable region CD3VL that bind to CD3, and wherein the heavy chain variable region CD3VH and the light chain variable region CD3VL are linked by a flexible linker peptide sequence 3; preferably, the amino acid sequence of the heavy chain variable region CD3VH is shown as SEQ ID No.3 and the amino acid sequence of the light chain variable region CD3VL is shown as SEQ ID No. 4; more preferably the amino acid sequence of the flexible linker peptide sequence 3 is shown in SEQ ID No. 7.
5. The bispecific antibody of claim 4, wherein the second protein sequence comprises in order the heavy chain variable region CD3VH, the flexible linker peptide sequence 3, and the light chain variable region CD3VL.
6. A nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1-5.
7. Use of the bispecific antibody of any one of claims 1-5, the nucleic acid molecule of claim 6 for the preparation of a medicament for the diagnosis and treatment of malignancy; preferably, the malignancy is a malignancy with CD20 expression.
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