Bispecific antibodies targeting CD24 and CD3 and their applications

Abstract

The present invention belongs to the technical field of genetic engineering antibodies, and specifically relates to bispecific antibodies targeting CD24 and CD3 and their applications. The present invention discloses a bispecific antibody and configuration that simultaneously targets human leukocyte differentiation antigen CD24 and human CD3. The construction method is simple, avoids the possibility of mismatching of two groups of light chains and heavy chains of heterologous bispecific antibodies, reduces the difficulty of antibody purification, and obtains a bispecific antibody with high purity and strong killing effect on CD24-positive liver cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to a bispecific antibody that can simultaneously target human leukocyte differentiation antigen (CD24) and CD3, can effectively activate T cell activation-related signals, and can kill CD24-positive liver cancer cells. The present invention is a genetically engineered bispecific antibody that has the function of bridging T cells and liver cancer cells. Background Art

[0002] Liver cancer is a malignant tumor with high morbidity and mortality. Due to its insidious onset, most patients are diagnosed in the late stage of the tumor and cannot be treated by surgical resection. The first-line drug for liver cancer is the kinase inhibitor sorafenib, but sorafenib still does not meet clinical needs in terms of prolonging patient survival and improving tolerance. More effective therapies for liver cancer need to be developed. Immunotherapy has attracted more attention because of its advantages of relying on the body's own immune system and being able to target and kill tumor cells without killing normal cells. It is an effective tumor treatment method.

[0003] Tumor immunotherapy includes monoclonal antibodies, bispecific antibodies, adoptive cell therapy and CAR-T, among which bispecific antibodies have the characteristics of dual targeting compared with monoclonal antibodies and can exert better therapeutic effects, so they have become a hot direction for antibody research and development. From the perspective of mechanism of action, bispecific antibodies are divided into multiple types such as cell bridging, dual target blocking, immune cell activation and promotion of protein complex formation. Cell bridging bispecific antibodies refer to bispecific antibodies in which one arm acts on tumor cell surface antigens to play a targeting role, and the other arm acts on immune cells, mostly effector T cells and NK cells, to play a recruiting role. Cell bridging bispecific antibodies can shorten the distance between effector cells and tumor cells to achieve the purpose of killing tumors.

[0004] Due to the continuous antigen stimulation and the presence of immunosuppressive factors in the tumor microenvironment, tumor-infiltrating T cells show an exhausted state, and their proliferation ability and effector function are weakened or lost. The T cell surface receptor TCR recognizes the antigen stimulation presented by MHC, and the recognition signal is transmitted to the cell by the CD3 protein complex. CD3 is a co-stimulatory molecule of the T cell surface activation receptor TCR. The intracellular domain includes the ITAM signal motif, which can transmit the T cell activation signal to the cell, thereby activating T cells. Monoclonal antibodies targeting CD3 can bind to and activate T cells. Activated T cells can effectively fight tumor cells, and this process does not depend on MHC molecules.

[0005] CD24 is a protein expressed on early-stage B cells, neural cells, and a variety of tumor cells. It is GPI-anchored to the cell membrane. Its protein core skeleton consists of 33 amino acid residues, contains abundant N-sugar and O-glycosylation sites, and has a high glycosylation characteristic. Its expression is closely related to tumor growth, metastasis, and tumor cell drug resistance. CD24 is highly expressed in a variety of tumor tissues, including liver cancer, and high expression of CD24 has also been detected in the plasma of patients with advanced liver cancer. Siglec-10 molecules are members of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family. They interact with CD24 through the sialic acid epitope of CD24 molecules. Siglec-10 proteins contain ITIM signal motifs in cells and can transmit inhibitory signals. Siglec-10 is expressed on the surface of a variety of immune cells. In the tumor microenvironment, Siglec-10 protein on the surface of macrophages interacts with CD24 protein on the surface of tumor cells, activates downstream ITIM signaling motifs, and inhibits the phagocytic function of macrophages. Studies have found that antibodies targeting CD24 can effectively restore the phagocytic function of macrophages. This shows that CD24 is expected to become an effective target for tumor immunotherapy of liver cancer.

[0006] Therefore, the present invention aims to develop a cell-bridging bispecific antibody that simultaneously targets CD24 and CD3, so that it can target liver cancer cells while recruiting and activating T cells, thereby exerting a killing effect on liver cancer cells. Summary of the invention

[0007] Purpose of the Invention

[0008] The present invention provides two bispecific antibodies (GT03 and GT04), which simultaneously target CD24 and CD3, target liver cancer cells, and simultaneously recruit and activate T cells to kill liver cancer cells.

[0009] Technical Solution

[0010] A bispecific antibody, characterized in that it is formed by connecting an anti-CD24 full-length antibody and an anti-CD3 antibody scFv through a linker, wherein the heavy chain of the bispecific antibody is composed of an anti-CD24 antibody heavy chain, and its variable region amino acid sequence is SEQNO.1; the light chain is composed of an anti-CD24 antibody light chain and an anti-CD3 antibody scFv, the CD24 antibody variable region amino acid sequence is SEQNO.2, and the CD3 antibody scFv amino acid sequence is SEQNO.3 or SEQNO.4; wherein the linker amino acid sequence is (GGGGS)n, and the value of n is 3.

[0011] The bispecific antibody molecule is characterized in that the bispecific antibody has a symmetrical structure, the heavy chain is composed of the CD24 antibody heavy chain, and its constant region is of human IgG1 type; the light chain comprises the CD24 antibody VL, κ chain CL, linker and CD3 antibody scFv from the N-terminus to the C-terminus, wherein the connected linker amino acid sequence is (GGGGS)n, and the value of n is 3.

[0012] The bispecific antibody is characterized in that the heavy chain constant region includes an amino acid sequence as shown in SEQ NO.5, and the amino acid at position 234 and the amino acid at position 235 of the heavy chain according to EU numbering are alanine, and the amino acid at position 329 is glycine. It is emphasized here that this constant region has mutations, and these two mutations will reduce the ADCC and CDC effects of the antibody, that is, prevent the bispecific antibody and T cells from being cleared due to antibody-dependent cell-mediated cytotoxicity (ADCC effect) and complement-dependent cytotoxicity (CDC effect).

[0013] The bispecific antibody is characterized in that the bispecific antibody is GT03 or GT04, wherein the amino acid sequence of GT03 is SEQNO.8, and the amino acid sequence of GT04 is SEQNO.9.

[0014] A nucleic acid molecule, characterized in that it encodes the bispecific antibody.

[0015] The nucleic acid molecule encoding the above-mentioned has a nucleotide sequence of SEQ NO.6 or SEQ NO.7

[0016] An expression vector contains the nucleic acid molecule.

[0017] The use of the bispecific antibody in the preparation of drugs for treating cancer.

[0018] The application is characterized in that the cancer is liver cancer.

[0019] Beneficial Effects

[0020] The variable region sequences of the CD24 antibody of the present invention are shown in SEQNO.1 and SEQNO.2, and have high affinity and specificity with CD24. The scFv sequences of the CD3 antibody are shown in SEQNO.3 and SEQNO.4. The two antibodies encoded by the two sequences are huSP34 and huOKT3, respectively, which are low-affinity and high-affinity antibodies for CD3, respectively. The prepared bispecific antibody has a large difference in affinity with CD3 positive cells; for CD3, low affinity is not a bad thing, because CD3 antibody activates T cells with specific recognition epitopes and spatial orientation. Although GT03 has a low affinity, the recognition epitope can be beneficial to its activation of T cells. And high-affinity CD3 antibodies may cause higher cytokine release, leading to more serious side effects. At the same time, the bispecific antibody of the present invention has the bispecific antibody constant region and mutations. These two mutations will reduce the ADCC and CDC effects of the antibody, that is, prevent the bispecific antibody and T cells from being cleared due to antibody-dependent cell-mediated cytotoxicity (ADCC effect) and complement-dependent cytotoxicity (CDC effect).

[0021] Therefore, as shown in the results of Example 6, GT03 is better than GT04, but the bispecific antibodies described in both can bind to CD24-positive liver cancer cells and CD3-positive cells, and have a strong killing effect on liver cancer cells, and the killing effect is better than the combination of two monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The diagram shows the molecular configuration of a bispecific antibody. The diagram shows that αCD3 scFv can be huSP34 or huOKT3, and the corresponding bispecific antibodies are GT03 and GT04, respectively.

[0023] Figure 2 It is the Western Blot result of bispecific antibody molecule; Lane M is marker; Lane 1 is the non-reducing band of bispecific antibody GT03, Lane 2 is the reducing band of bispecific antibody GT03, the molecular weight of antibody light chain and heavy chain are close, about 50kDa, and they are displayed as a single band; Lane 3 is the non-reducing band of bispecific antibody GT04; Lane 4 is the reducing band of bispecific antibody GT04, the molecular weight of antibody light chain and heavy chain are close, about 50kDa, and they are displayed as a single band.

[0024] Figure 3 Figure 1 shows the flow cytometry detection of the binding of bispecific antibodies to liver cancer cells and Jurkat cells. A: The binding of bispecific antibodies to BEL-7402 cells; B: The binding of bispecific antibodies to Jurkat cells.

[0025] Figure 4This is a comparison chart of the binding strength of two bispecific antibodies to Jurkat cells detected by flow cytometry.

[0026] Figure 5 Biomembrane interferometry was used to detect the binding of bispecific antibodies to recombinant human CD24 antigen and recombinant human CD3e antigen. A. GT03 binds to recombinant human CD24 protein; B. GT03 binds to recombinant human CD3e protein; C. GT04 binds to recombinant human CD24 protein.

[0027] Figure 6 The results of the LDH release method to detect the dual antibody-induced killing of liver cancer cells by PBMC. A. The results of the dual antibody GT03 inducing the killing of BEL-7402 by PBMC and the comparison with the combination of two monoclonal antibodies; B. The results of the dual antibody GT04 inducing the killing of BEL-7402 by PBMC and the comparison with the combination of two monoclonal antibodies. DETAILED DESCRIPTION

[0028] Example 1 Preparation of bispecific antibodies:

[0029] Construction of bispecific antibody light and heavy chain expression vector and eukaryotic expression of bispecific antibodies

[0030] 1) Designing upstream and downstream primers, using PCR to obtain the variable region of CD24 antibody, the constant region of IgG1 heavy chain and the scFv of CD3 antibody, respectively; splicing the variable region of CD24 antibody heavy chain and the constant region of IgG1 heavy chain by overlap-PCR to obtain the full-length fragment of the heavy chain of the bispecific antibody; splicing the light chain of CD24 antibody and the scFv of CD3 antibody to obtain the full-length fragment of the light chain of the bispecific antibody; using BamHI and KpnI to double-digest the expression vector; connecting the full-length fragments of the light and heavy chains of the bispecific antibody and the digested vector by a one-step cloning method to obtain the heavy and light chain expression vectors of the bispecific antibody.

[0031] 2) The recombinant plasmids encoding the light and heavy chains of the bispecific antibody were transfected into HEK293 cells at a ratio of 2.5:1. After culturing for 5 days, the supernatant was collected by centrifugation at 4°C and purified by protein A column to obtain the bispecific antibody. The bispecific antibody prepared has the following configuration: Figure 1 As shown, huSP34 is a low affinity CD3 antibody, and huOKT3 is a high affinity CD3 antibody. When the CD3 antibody scFv comes from huSP34, the bispecific antibody is numbered as GT03; when the CD3 antibody scFv comes from huOKT3, the bispecific antibody is numbered as GT04.

[0032] Example 2 Bispecific Antibody Western Blot Detection:

[0033] 1) Reduced and non-reduced samples of the bispecific antibody were prepared respectively and separated by 10% SDS-PAGE electrophoresis.

[0034] 2) The separated samples were transferred to a PVDF membrane and blocked by incubation with 5% skimmed milk powder at 37°C for 2 h.

[0035] 3) After washing with TBST for 10 min, HRP-labeled goat anti-human IgG antibody (Sangon Biotechnology, 1:50,000 dilution) was added as a secondary antibody and incubated at 37°C for 1 h.

[0036] 4) Rinse the PVDF membrane with PBST for 10 min, three times.

[0037] 5) Add ECL chemiluminescent colorimetric solution (Yisheng Bio, A and B solution mixed in a ratio of 1:1) evenly onto the surface of the PVDF membrane and place it in an exposure instrument for exposure. Figure 2 As shown, lane M is a marker; lane 1 is a non-reducing band of the bispecific antibody GT03, lane 2 is a reducing band of the bispecific antibody GT03, the molecular weight of the antibody light chain and heavy chain are close, about 50 kDa, and are displayed as a single band; lane 3 is a non-reducing band of the bispecific antibody GT04; lane 4 is a reducing band of the bispecific antibody GT04, the molecular weight of the antibody light chain and heavy chain are close, about 50 kDa, and are displayed as a single band.

[0038] Example 3 Flow cytometry detection of the binding of bispecific antibodies to liver cancer cells (highly expressing CD24) and Jurkat cells (highly expressing CD3):

[0039] 1) The binding of the bispecific antibody to CD24+ hepatoma cells and CD3+ Jurkat cells can be detected by flow cytometry. BEL-7402 cells have a high CD24 expression level, and Jurkat cells have a high CD3 expression level. The cells were cultured in 1640 medium containing 10% FBS at 37°C and 5% CO2.

[0040] 2) For liver cancer cells, trypsinize the cells in good growth state, add fresh medium to resuspend and count, and take 2*10 5 For Jurkat cells, gently blow the cells in good growth condition, centrifuge at 1000 rpm for 4 min, discard the supernatant, add fresh culture medium to resuspend the cells, and take 2*10 cells per tube. 5 Cells. Centrifuge the treated cell suspension at 4°C, 1500 rpm for 3 min, discard the supernatant. Add 100 ul of pre-cooled PBS containing 2% FBS and incubate for 20 min.

[0041] 3) Centrifuge at 4℃, 1500rpm for 3min, discard the supernatant. Add 100μL of 100nM bispecific antibody or monoclonal antibody as primary antibody, resuspend the cells, add 100nM irrelevant antibody as isotype control, incubate at 4℃ for 1h to allow antibody and cells to bind; centrifuge at 4℃, 1500rpm for 3min, discard the supernatant. Add 500μl pre-cooled PBS to wash once.

[0042] 4) Add 100 μL of AlexaFluor488-labeled goat anti-human IgG antibody (Yisheng Bio, 1:200 dilution) as the secondary antibody, resuspend the cells, and incubate at 4°C for 30 min.

[0043] 5) Centrifuge at 4°C, 1500 rpm for 3 min, discard the supernatant. Add 500 μL pre-cooled PBS to wash twice. Add 250 μL PBS to resuspend, and use flow cytometry for detection. The results are as follows Figure 3 As shown, the bispecific antibody of the present invention can effectively bind to BEL-7402 cells with high expression of CD24 and Jurkat cells with high expression of CD3, and has good tumor cell targeting ability and T cell binding ability.

[0044] Example 4 Flow cytometry detection of the binding strength of two bispecific antibodies to Jurkat cells (highly expressing CD3):

[0045] 1) The present invention uses two CD3 antibodies huSP34 and huOKT3 with greatly different affinities, and the constructed bispecific antibodies are GT03 and GT04, respectively. The binding strength of the two bispecific antibodies to CD3+Jurkat cells can be determined by setting a concentration gradient and detecting the mean fluorescence intensity by flow cytometry. Jurkat cells with good growth conditions were gently blown evenly, centrifuged at 1000 rpm for 4 min, the supernatant was discarded, and fresh culture medium was added to resuspend the cells. 2*10 5 The treated cell suspension was centrifuged at 4°C, 1500 rpm for 3 min, and the supernatant was discarded. 500 μl of pre-cooled PBS containing 2% FBS was added and washed twice.

[0046] 2) Add 100 μL of a bispecific antibody with a concentration of 0.07-500 nM as the primary antibody, resuspend the cells, add 100 nM of an irrelevant antibody as an isotype control, and incubate at 4°C for 1 hour to allow the antibody to bind to the cells; the subsequent steps are the same as in Example 2. Figure 4 As shown, because part of the antibodies of GT03 come from huSP34 with low CD3 affinity, and GT04 comes from huOKT3 with high CD3 affinity, the binding of GT03 to Jurkat cells (high CD3 expression) is significantly lower than that of GT04 (the significance analysis method is Wilcoxon signed rank test, P<0.05), which is consistent with the expected results.

[0047] Example 5 Biomembrane interferometry to detect the binding of bispecific antibodies to recombinant human CD24 antigen and recombinant human CD3e antigen: 1) Kinetic detection of antigen-antibody binding can be performed by biomembrane interferometry, and the K of antigen-antibody binding can be calculated. D In this embodiment, the sensor used is an AHC sensor, the bispecific antibody is immobilized on the sensor, 6.25-100 nM of recombinant human CD24 antigen or recombinant human CD3e antigen is added for binding, and dissociation is carried out in a PBS system.

[0048] The results are as follows Figure 5 As shown. A: GT03 binds to recombinant human CD24 protein; B. GT03 binds to recombinant human CD3e protein; C. GT04 binds to recombinant human CD24 protein.

[0049] result:

[0050] Table 1 Equilibrium dissociation constants of bispecific antibody binding to antigen

[0051]

[0052]

[0053] Example 6 LDH release method to detect the promotion of dual antibodies to PBMC killing of liver cancer cells:

[0054] 1. PBMC Extraction

[0055] 1) Take 10 mL of blood from a healthy person and dilute it with an equal volume of PBS. Take 3 mL of lymphocyte separation solution (Ficoll) and add it to the bottom of a 10 mL EP tube. Slowly add 6 mL of diluted blood to form layers.

[0056] 2) Adjust the centrifuge speed to 1, adjust the speed to 0,800g and centrifuge for 20 minutes. After the centrifugation, aspirate the white band in the EP tube. 3) Add two volumes of PBS buffer to the aspirated liquid, mix well, centrifuge at 2000rpm for 8 minutes, and discard the supernatant. Add an appropriate amount of red blood cell lysis buffer to the precipitate for 3 minutes, add 3 volumes of PBS buffer to terminate the reaction, and centrifuge at 2000rpm for 8 minutes.

[0057] 4) Discard the supernatant and add 5 mL of PBS buffer to wash the precipitate twice.

[0058] 5) Discard the supernatant, resuspend the cells at the bottom with 2 mL of fresh 1640 medium and count them.

[0059] 2. Dual antibody promotes PBMC to kill tumor cells detection

[0060] 1) BEL-7402 cells with good growth condition were digested with trypsin, resuspended in fresh 1640 medium and counted, and 10,000 BEL-7402 cells, 50,000 extracted PBMC cells and bispecific antibodies with a concentration range of 7-500 nM were co-incubated. At the same time, a BEL-7402 and PBMC co-incubation group, a BEL-7402 maximum killing or minimum killing group, and a 1640 medium group were set.

[0061] 2) After incubation for 36 hours, centrifuge and add 10% volume of LDH release reagent (Biyuntian, lactate dehydrogenase cytotoxicity detection kit) to the BEL-7402 maximum killing group 1 hour before the end of incubation. Take the supernatant as the test sample to detect LDH release in the sample.

[0062] 3) Prepare LDH detection working solution, add 120 μL of the sample to be tested and 60 μL of LDH detection working solution to a new 96-well plate, react at room temperature for 30 minutes in the dark, and detect the absorbance at a wavelength of 490 nm.

[0063] 4) The killing rate was calculated as (OD490 of the double-antibody group - OD490 of the minimum killing group) / (OD490 of the maximum killing group - OD490 of the minimum killing group)*100%;

[0064] The results are as follows Figure 6 As shown. A: The killing effect of the GT03 group and the αCD24 and huSP34 combination group was compared. The bispecific antibody can play a stronger tumor killing effect, and the effect is better than the combination of two monoclonal antibodies; B: The killing effect of the GT04 group and the αCD24 and huOKT3 combination group was compared. The bispecific antibody can play a stronger tumor killing effect, and the effect is also better than the combination of two monoclonal antibodies (the significance analysis method is Wilcoxon signed rank test, P<0.05). GT03 can play a better tumor cell killing effect than GT04. The reason may be that CD3 antibodies have specific recognition epitopes and spatial orientations for activating T cells. Although GT03 has a lower affinity, the recognition epitope can help it activate T cells. In addition, high-affinity CD3 antibodies may cause higher cytokine release, leading to more serious side effects.

Claims

1. A bispecific antibody, characterized in that: The bispecific antibody is formed by connecting a full-length anti-CD24 antibody and an anti-CD3 antibody scFv through a linker. The heavy chain of the bispecific antibody is composed of the anti-CD24 antibody heavy chain, and its variable region amino acid sequence is SEQ NO.1; the light chain is composed of the anti-CD24 antibody light chain and the anti-CD3 antibody scFv, the CD24 antibody variable region amino acid sequence is SEQ NO.2, and the CD3 antibody scFv amino acid sequence is SEQ NO.3 or SEQ NO.4; the linker amino acid sequence is (GGGGS)n, and the value of n is 3.

2. The bispecific antibody according to claim 1, characterized in that The bispecific antibody has a symmetrical structure, wherein the heavy chain is composed of the heavy chain of the CD24 antibody, and its constant region is of human IgG1 type; the light chain comprises the VL of the CD24 antibody, the κ chain CL, the linker and the CD3 antibody scFv from the N-terminus to the C-terminus, wherein the amino acid sequence of the connected linker is (GGGGS)n, and the value of n is 3.

3. The bispecific antibody according to claim 2, characterized in that The bispecific antibody is GT03 or GT04, wherein the amino acid sequence of GT03 is SEQ NO.8, and the amino acid sequence of GT04 is SEQ NO.

9.

4. A nucleic acid molecule, characterized in that It encodes the bispecific antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, wherein the nucleotide sequence is SEQ NO.6 or SEQ NO.

7.

6. An expression vector comprising the nucleic acid molecule according to claim 4 or 5.

7. Use of the bispecific antibody according to any one of claims 1 to 3 in the preparation of a drug for treating cancer, wherein the cancer is liver cancer.

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