WO2016180034A1 - Anti-ctla-4 and anti-pd-1 dual variable domain immunoglobulin - Google Patents

Abstract

Disclosed is a dual variable domain immunoglobulin (DVD-Ig) specifically binding to PD-1 and/or CTLA-4, and use thereof in the preparation of drugs for treating tumours.

Description

Dual variable domain immunoglobulin against CTLA-4 and PD-1 Technical field

The invention relates to the field of biotechnology, in particular to a method for constructing, screening and preparing anti-CTLA-4 and PD-1 antibody dual variable domain immunoglobulin (DVD-Ig) and its potential in tumor Application in therapy.

Background technique

The immune checkpoint is an inhibitory signaling pathway present in the immune system that protects against tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and is involved in maintaining tolerance to autoantigens (Pardoll DM. The blockade) Of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264.). The study found that tumor cells use the inhibitory signaling pathway of the immune checkpoint to inhibit the activity of T lymphocytes, thereby evading the killing effect of the immune system, especially T lymphocytes, on tumors (Yao S, Zhu Y, Chen L. Advances in targeting Cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146.). Important immune checkpoint molecules are mainly CTLA-4, PD-1, VISTA (Wang, L et al.. VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses. J. Exp. Med. 2011208,577-592), LAG-3 (Triebel, F et al. (LAG-3, a novel lymphocyte activation gene closely related to CD4.J.Exp.Med. 1990,.171, 1393-1405.) and TIM -3 (Sakuishi et al. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J. Exp. Med. 2010207, 2187-2194.) and the like.

Cytotoxic T cell-associated antigen 4 (CTLA-4/CD152) and CD28 are both members of the immunoglobulin supergene family, which have 70% homology and are transmembrane receptors expressed on the surface of T cells. CD28 and CTLA-4 are a pair of important costimulatory molecules with positive and negative regulatory functions, which compete for binding to B7 molecules, but CTLA4 and its ligands are 20 times more potent than CD28. Binding of CD28 to B7-1 and B7-2 on APC is capable of transmitting costimulatory signals to T lymphocytes, enhancing the action of CD3/TCR complexes or CD2 molecules, inducing T lymphocyte activation and secretion of various cytokines. CTLA-4 inhibits the proliferation and activation of T lymphocytes and induces apoptosis of activated T lymphocytes, which are the key links in the maintenance of the body's immune system.

Programmed death-1 (PD-1) is another important inhibitory receptor on the surface of T cells. It shares homology with CD28 and CTLA-4 and is inducibly expressed in activated T. Cells, B cells, macrophages, dendritic cells, and monocytes, on the surface of resting lymphocytes expression. Its ligand is B7-homolog 1 (B7-homolog 1, B7-H1) and B7DC. B7-H1 is also called programmed death ligand-1 (PD-L1). Up-regulation of PD-1 expression on activated lymphocyte surface can result in inhibition of acquired or innate immune responses. The binding of PD-1/PD-L1 plays an important role in regulating T lymphocyte activation and maintaining peripheral immune tolerance. Many tumor cells have high expression of PD-1 and PD-L1 on the surface, and tumor cells use this important mechanism to inhibit T lymphocyte activity and escape the immune system.

In 2013, Science magazine ranked tumor immunotherapy as the top ten scientific breakthroughs in 2013. CTLA-4 is one of the immunological checkpoint molecules. It has been marketed for its two immunological fusion proteins, Abatacept (listed in 2005) and Belatacept (listed in 2011). The former is a common CTLA-4 fusion protein. CTLA-4 was mutated to increase its affinity for ligands for the treatment of autoimmune diseases and renal transplant rejection. CTLA-4 antibody drug Ipilimumab (trade name)

Is a fully human IgG1 antibody molecule, Ipilimumab does not directly kill tumor cells, and its binding to CTLA-4 inhibits the function of CTLA-4, relieves the immune tolerance of antigen-specific T lymphocytes and directly inhibits TCR signaling, and improves The expression of IL-2, thereby enhancing the function of T effector cells, and reducing the inhibition of Treg cells on the patient's immune system. In 2011, it was approved by the FDA for the treatment of melanoma. Clinical studies have shown that Ipilimumab is used in patients with advanced melanoma and can trigger a sustained immune response with an objective response rate of 10% to 15% and can effectively prolong the patient's life. The PD-1/PD-L1 pathway antibodies mainly include anti-PD-1 and PD-L1 monoclonal antibodies, as well as products targeting PD-L2. Anti-PD-1 antibody studies were more mature with Nivolumab from BMS and Lambrolizumab (Pembrolizumab) from Merck. Nivolumab (trade name)

) is a fully human IgG4 antibody molecule, Lambrolizumab (trade name)

) is a humanized IgG4 antibody molecule. When they bind to PD-1, they inhibit binding to ligands PD-L1 and PD-L2, promote T lymphocyte proliferation and cytokine production, and abolish the inhibition of tumor-active T lymphocyte immune surveillance by PD-1 system. From 2008 to 2012, Nivolumab treated 107 patients with advanced melanoma. The results showed that patients with advanced refractory melanoma had a total survival of nearly 17 months after receiving Nivolumab. Tumor remission after drug withdrawal can be maintained for a long time and is highly safe. Of particular concern is that 62% of these patients have previously received 2 to 5 courses of systemic therapy and are in advanced refractory patients. 33 patients (31%) received objective response to the tumor. In a phase 3 clinical-to-head comparison of the efficacy and safety of squamous advanced non-small cell lung cancer treated before Opdivo and docetaxel treatment, patients were randomized to receive 3 mg intravenously every 2 weeks/ Kg of Nivolumab treatment (n = 135), or intravenous injection of 75 mg/m2 of docetaxel every 3 weeks (n = 137). Compared with the docetaxel group, the OS improvement in the Nivolumab group was more significant and statistically significant. The median OS of the Nivolumab group was 9.2 months (95% CI: 7.3, 13.3) and the docetaxel group was 6 months (95% CI: 5.1, 7.3). The effectiveness of Nivolumab on squamous NSCLC was further confirmed in a one-arm trial involving 117 squamous non-small cell lung cancers. Participants in the study developed disease progression after undergoing platinum-based therapy and at least another systemic treatment regimen. Total remission occurred in 15% of patients, and 59% of patients had a remission time of up to 6 months or longer. Opdivo treatment for melanoma and non-small cell lung cancer indications was approved for sale in September 2014 and March 2015, respectively. Lambrolizumab (MK-3475) received FDA breakthrough drug qualifications and was approved for the treatment of melanoma indications in September 2014. The current MK-3475 clinical program includes more than 24 clinical trials across 30 different tumor types, including 4 new Phase III studies.

Both CTLA-4 and PD-1 are T lymphocyte activation inhibitors, which block the activation of T lymphocytes, but the mechanisms of their blocking are completely different. CTLA-4 mediated inhibition is shown to inhibit Akt phosphorylation, whereas PD-1-mediated phosphorylation of Akt inhibits CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K), PD -1 The ability to inhibit PI3K/Akt activation is dependent on the cytoplasmic immunoreceptor tyrosine kinase switching motif, and PD-1 is more effective in inhibiting CD3/CD28 (Parry RV et al. CTLA-4 and PD-1receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol 2005; 25:9543-53.). CTLA-4 has an effect in the early stages of the immune response, which is affected by regional lymph node antigen-presenting cells and untreated T-cell levels. PD-1 is expressed on the surface of activated T cells, and PD-1/PDL-1 is in peripheral tissues when effector T cells play a role. Both CTLA-4 and PD-1 can enhance the activation of T cells and the immunological activity of tumors by inhibitory antibodies, thereby becoming effective tumor therapeutic drugs. Since CTLA-4 and PD-1 act on different stages of immune cell activation, these two drugs can be used in different populations and can even be used in combination. Anti-CTLA-4 and anti-PD-1 antibodies play complementary and synergistic roles in the immune effects that inhibit tumor growth. In the preclinical B16 melanoma tumor model study, the combination of anti-CTLA-4 and anti-PD-1 antibodies resulted in the infiltration of T effector cells in tumor tissues. T effector cells (Teff) in T cells compared to T regulatory cells (Treg) in tumor tissues. The higher the ratio, the higher the effect of inhibiting tumors than the anti-CTLA-4 or anti-PD-1 antibodies alone. The synergistic effect of anti-CTLA-4 and anti-PD-1 antibodies is also manifested in an increase in the ratio of T effector cells (Teff) to myeloid-derived suppressor cells. IFN-γ expression also increased. Bristol-Myers Squibb (BMS) announced in April 2015 the positive data of the immunological combination therapy Opdivo+Yervoy for the first-stage clinical phase of melanoma treatment. Compared with Yervoy monotherapy, Opdivo+Yervoy combination therapy achieved a very high objective response rate (61% vs 11%), a complete response rate of 22%, and a 60% reduction in the risk of disease progression or death. Similar results were achieved with the Opdivo+Yervoy regimen for BRAF mutant melanoma, with a median progression-free survival (PFS: 8.5 months vs 2.7 months) and a 60% reduction in the risk of disease progression or death. In addition, the objective response rate (ORR) was independent of the PD-L1 status: ORR was 58% in PD-L1 positive tumors and 55% in PD-L1 negative tumors.

A bispecific antibody is an artificial antibody containing two specific antigen binding sites and is a genetically engineered antibody. One kind of body has become a hot spot in the field of antibody engineering, and has broad application prospects in tumor immunotherapy. Preparation methods include: chemical coupling method, hybridization-hybridization method, genetic engineering antibody preparation method and the like. The genetically engineered antibody preparation method has good homogeneity due to the bispecific antibody prepared by the same, and combines the current expression technology to have high-efficiency expression characteristics. One of them is called a dual variable domain immunoglobulin (DVD-Ig), (US Pat. No.: 7612181). This patent provides a binding protein that binds two or more antigens with high affinity, which is called Dual variable domain immunoglobulin (DVD-Ig), and 748802, 9005612, 8258268, 8735546, 8716450, 8716450, 8188237, 8722855, and 8566714 derived therefrom.

Summary of the invention:

Both preclinical and clinical studies of anti-PD-1 and CTLA-4 antibody combinations have demonstrated their definitive efficacy in the treatment of tumors, especially when they are used together to produce a synergistic anti-tumor effect. On the one hand, it is convenient for clinical patients and doctors to use, on the other hand, it makes the antibody drugs easier to produce and quality control, and reduces costs. The object of the present invention is to provide a human bispecific novel antibody which can specifically bind PD-1 and/or CTLA-4, which is an anti-PD-1 and CTLA-4 antibody, which is a dual variable domain immunoglobulin. (dual variable domain immunoglobulin, DVD-Ig), as shown in Figure 1.

The flexible fragment between the two heavy chain variable domains may be "AKTTPKLEEGEFSEAR", "AKTTPKLEEGEFSEARV", "SAKTTPKLEEGEFSEARV", "AKTTPPSVTPLAP", "ASTKGPSVFPLAP", "GGGGSGGGGSGGGGS", "TVAAPSVFIFPPTVAAPSVFIFPP", "ASTKGPSVFPLAPSS", preferably "ASTKGPSVFPLAPSS" and "ASTKGPSVFPLAP";

The flexible fragment between the two light chain variable domains may be "AKTTPKLEEGEFSEAR", "AKTTPKLEEGEFSEARV", "SAKTTPKLEEGEFSEARV", "AKTTPPSVTPLAP", "ASTKGPSVFPLAP", "GGGGSGGGGSGGGGS", "TVAAPSVFIFPPTVAAPSVFIFPP", "ASTKGPSVFPLAPSS", preferably "TVAAPSVFIFPPS";

More specifically, based on the analysis of the anti-PD-1 and CTLA-4 antibodies and their antigen-binding variable domain structures, the present invention discloses two novel bispecific antibodies that bind both PD-1 and CTLA-4. The structure, wherein when the anti-PD-1 antibody variable domain is located inside the molecule, the anti-CTLA-4 antibody is located outside the molecule; the other anti-PD-1 antibody variable domain is located outside the molecule, then the anti-CTLA- 4 The antibody is located inside the molecule. The two antibodies were expressed in CHO cells, respectively. It was confirmed after purification that both structures specifically bind to PD-1 and/or CTLA-4 molecules. Both have the potential to treat tumors.

The technical solution of the present invention is as follows:

The present invention provides a bispecific antibody capable of binding to PD-1 and/or CTLA-4 antigen, wherein the heavy chain comprises an HVD1-L1-HVD2-CH structure, wherein

HVD1 is the first heavy chain variable domain,

L1 is a heavy chain flexible joint,

HVD2 is the second heavy chain variable domain.

The CH region is a heavy chain constant domain;

Wherein the light chain comprises an LVD1-L2-LVD2-CL structure, wherein

LVD1 is the first light chain variable domain.

L2 is a light chain flexible joint,

LVD2 is the second light chain variable domain.

CL is a light chain constant domain.

The bispecific antibody provided by the present invention, wherein HVD1 is selected from SEQ ID No 2, HVD2 is selected from SEQ ID No 4, 6, 8, LVD1 is selected from SEQ ID No 1, and LVD2 is selected from SEQ ID No 3, 5. 7;

Or wherein HVD1 is selected from SEQ ID No 4, 6, 8, then HVD2 is selected from SEQ ID No 2, LVD1 is selected from SEQ ID No 3, 5, 7, and LVD2 is selected from SEQ ID No 1.

The bispecific antibody provided by the present invention, wherein the flexible linker between the L1 two heavy chain variable domains is selected from the group consisting of "AKTTPKLEEGEFSEAR", "AKTTPKLEEGEFSEARV", "SAKTTPKLEEGEFSEARV", "AKTTPPSVTPLAP", "ASTKGPSVFPLAP", "GGGGSGGGGSGGGGS", " TVAAPSVFIFPPTVAAPSVFIFPP", "ASTKGPSVFPLAPSS". Preferred are "ASTKGPSVFPLAPSS" and "ASTKGPSVFPLAP".

The bispecific antibody provided by the present invention, wherein the flexible linker between the L2 two light chain variable domains is selected from the group consisting of "AKTTPKLEEGEFSEAR", "AKTTPKLEEGEFSEARV", "SAKTTPKLEEGEFSEARV", "AKTTPPSVTPLAP", "ASTKGPSVFPLAP", "GGGGSGGGGSGGGGS", " TVAAPSVFIFPPTVAAPSVFIFPP", "ASTKGPSVFPLAPSS". It is preferably "TVAAPSVFIFPPS".

The bispecific antibody provided by the present invention may have any of the antibody heavy chain types of IgG1, IgG2, IgG3, and IgG4, and is preferably IgG4.

The bispecific antibody provided by the present invention may have a total antibody light chain type of κ and λ, preferably a κ type.

The present invention provides a bispecific antibody having a variable domain of anti-PD-1 and/or CTLA-4 located inside or outside the antibody and capable of retaining binding ability to PD-1 and/or CTLA-4 .

The present invention also provides a cell for expressing a bispecific antibody, which may be any one of CHO, HEK293, NS0 and Per 6, preferably CHO cells.

The invention also provides the use of the bispecific antibody described in the manufacture of a medicament for treating a tumor. Drawing Description

Figure 1: Molecular structure of the DVD-Ig bispecific antibody.

Figure 2: BY18.1 and BY18.2 bispecific antibody SDS-PAGE electrophoresis 1, protein molecular weight standard; 2, BY18.1; 3, BY18.2.

Figure 3: ELISA assay for binding of BY18.1 and BY18.2 antibodies to recombinant human CTLA4 protein.

Figure 4: ELISA assay for binding of BY18.1 and BY18.2 antibodies to recombinant human PD-1 protein.

detailed description

The invention is described in more detail with reference to the following examples. However, the invention is not limited to these embodiments.

Example 1. Anti-CTLA-4 antibody and anti-PD-1 antibody sequence

Anti-CTLA-4 antibody sequence:

Anti-PD-1 antibody sequence:

Example 2. Synthesis of Ipi(out)-L-Niv(in) DVD-Ig bispecific antibody BY18.1 gene and construction of expression vector

1) Anti-PD-1 antibody Nivolumab sequence according to CTLA-4 antibody Ipilimumab sequence. Entrusted Shanghai Jierui Bioengineering Co., Ltd. to synthesize Ipi-L(out)-Niv-L(in)DVD-Ig bispecific antibody light chain (L chain) encoding gene, and optimize the coding gene to be suitable for CHO The gene encoding the gene. The vector was ligated to the p327.7 expression vector by XhoI-EcoRI (patent application number: 201410441296.0). Name it BY18.1L.

among them

with

The start and stop codons are respectively, "GCCACC" is the Kozak sequence, and "CTCGA" and "GAATTC" are the XhoI-EcoRI restriction sites, respectively. The corresponding amino acids are encoded as:

Wherein "METDTLLLWVLLLWVPGSTG" is a signal peptide sequence,

For the two variable interval Linker, the underlined sequence is the Ipilimumab light chain variable region sequence, the italic sequence is the Nivolumab light chain sequence, and the other is the light chain constant region sequence. The DVD-Ig bispecific antibody light chain is of the kappa type.

2) According to the CTLA-4 antibody Ipilimumab sequence, the anti-PD-1 antibody Nivolumab sequence. Entrusted Shanghai Jierui Bioengineering Co., Ltd. to synthesize the Ipi-H(out)-Niv-H(in)DVD-Ig bispecific antibody heavy chain (H chain) encoding gene, and optimized the coding gene to be suitable for CHO Cell The expressed gene was ligated to the p327.7 expression vector of cloned BY18.1L by double digestion with XbaI-SaII. Named BY18.1.

among them

with

The start and stop codons, respectively, "GCCACC" is the Kozak sequence, and "TCTAGA" and "GTCGAC" are the XbaI-SalI restriction sites, respectively. The corresponding amino acids are encoded as:

Wherein "METDTLLLWVLLLWVPGSTG" is a signal peptide sequence,

As the Linker of the two-chain variable interval, the underline sequence is the Ipilimumab heavy chain variable region sequence, the italic sequence is the Nivolumab heavy chain sequence, and the other is the heavy chain constant region sequence. The DVD-Ig bispecific antibody light chain is of the IgG4 class.

Example 3: Synthesis of Niv(out)-L-Ipi(in) DVD-Ig Bispecific Antibody BY18.2 Gene and Construction of Expression Vector

1) According to the CTLA-4 antibody Ipilimumab sequence, the anti-PD-1 antibody Nivolumab sequence. Entrusted Shanghai Jierui Bioengineering Co., Ltd. to synthesize the Niv-L(out)-L-Ipi-L(in) DVD-Ig bispecific antibody light chain (L chain) encoding gene, and optimize the coding gene to optimize The gene encoding the expression in CHO cells. The vector was ligated to the p327.7 expression vector by XhoI-EcoRI (patent application number: 201410441296.0). Name it BY18.2L.

among them

with

The start and stop codons are respectively, "GCCACC" is the Kozak sequence, and "CTCGAG" and "GAATTC" are the XhoI-EcoRI restriction sites, respectively. The corresponding amino acids are encoded as:

Wherein "METDTLLLWVLLLWVPGSTG" is a signal peptide sequence,

For the two variable interval Linker, the underlined sequence is the Ipilimumab light chain variable region sequence, the italic sequence is the Nivolumab light chain sequence, and the other is the light chain constant region sequence. The DVD-Ig bispecific antibody light chain is of the kappa type.

2) According to the CTLA-4 antibody Ipilimumab sequence, the anti-PD-1 antibody Nivolumab sequence. Entrusted Shanghai Jierui Bioengineering Co., Ltd. to synthesize the Niv-H(out)-L-Ipi-H(in)DVD-Ig bispecific antibody heavy chain (H chain) encoding gene, and optimize the coding gene to optimize The gene expressed in CHO cells was ligated to the p327.7 expression vector of cloned BY18.2L by XbaI-SaII double digestion. Named BY18.2.

among them

with

The start and stop codons, respectively, "GCCACC" is the Kozak sequence, and "TCTAGA" and "GTCGAC" are the XbaI-SalI restriction sites, respectively. The corresponding amino acids are encoded as:

Wherein "METDTLLLWVLLLWVPGSTG" is a signal peptide sequence,

For the Linker of the two-chain variable interval, the underlined sequence is the Ipilimumab heavy chain variable region sequence, the italic sequence is the Nivolumab heavy chain sequence, and the other is the heavy chain constant region sequence. The DVD-Ig bispecific antibody heavy chain is of the IgG4 class.

Example 4, screening of whole antibody stable cells

CHO cells were acclimated and adherently grown in 10% FBS, CD OptiCHO (product purchased from Invitrogen) containing 4 mM glutamine; cell concentration was adjusted after trypsinization, and inoculated into a 75 cm ² flask, the number of cells was approximately 5× 10 ⁶ /bottle; after 24 hours, transfect BY18.1 and BY18.2 according to Lipofectin 2000 (Invitrogen product) instructions; after transfection for 24 hours, trypsinize and collect transfected cells; adjust cell concentration, inoculate to 96-well culture plate, 0.8×10 ⁴ /well, screening medium composition 10% D-FBS, CD OptiCHO, containing 25 μM L-Methionine sulfoximine (MSX, product purchased from Sigma); 5- After 7 days, 50 μl/well of fresh medium was added; clones were grown in about 15 days, and 50 μl of sample double antibody sandwich ELISA method was used to semi-quantitatively detect the protein content in the culture medium, and the clones with relatively high protein expression were screened; The cells were cultured in suspension in 24-well plates, and the medium was changed to CD OptiCHO medium without D-FBS. After several days, the relative protein expression in 24-well plates was also detected by ELISA; further cultured in 6-well plates and 75 cm ² Bottle and 250ml shake flask suspension culture; combined Semi-quantitative results of ELISA and cell growth status were used to screen cells with higher expression levels.

Example 5, Expression and Purification of BY18.1 and BY18.2 Bispecific Antibodies

The highly expressed antibody cells were cultured in serum-free CD OptiCHO, and the culture supernatant was collected after a certain period of time. Balance HiTrap MabSelect SuRe 1ml column with PBS in pH 7.4 (GE Healthcare Life Sciences product, Cat. No: 11-0034-93) 10 bed volumes, flow rate 0.5 ml/min; culture supernatant was filtered through a 0.45 μm filter at a flow rate of 0.5 ml/min. A further 5-10 bed volumes were washed with a pH 7.4 PBS solution at a flow rate of 0.5 ml/min; eluted with 100 mM citrate buffer (pH 3.6) at a flow rate of 0.5 ml/min, and the elution peak was collected. The purified product was subjected to non-reduction electrophoresis and reduced SDS-PAGE electrophoresis to determine the molecular weight.

Purified BY18.1 and BY18.2 bispecific antibody reduction SDS-PAGE electrophoresis is shown in the figure. The SDS-PAGE electrophoresis BY18.1 and BY18.2 molecular size are basically the same, the heavy chain is about 66kDa, the light chain is about 36kDa, which is larger than the common antibody. 50kDa and 25kDa. The theoretical molecular weight of the BY18.1 and BY18.2 antibodies is about 199.0 kDa, the weight chain theory expects the molecular weight to be about 62.8 kDa, and the light chain size is about 36.5 kDa. The measured values are basically consistent with the theoretical expectations. The results are shown in Fig. 2.

Example 6. ELISA method for detection of binding of BY18.1 and BY18.2 bispecific antibodies to human CTLA-4 protein and recombinant human PD-L1 protein

Recombinant human CTLA-4/CD152 protein and recombinant human PD-L1 protein (both purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product numbers: 11159-H03H and 10084-H02H) were coated at 1 μg/ml and 0.8 μg/ml, respectively. EIA 96-well plate. Different purified BY18.1 and BY18.2 antibodies were diluted to 5 μg/ml, then diluted 5-fold, and 4 gradients were diluted. 50 μl was added to the coated 96-well plate and incubated at 37 ° C for 2 hours. After washing 3 times, horseradish peroxidase-labeled goat anti-human secondary antibody (purchased from Beijing Zhongzhe Jinqiao Company, product number: ZDR-5301) was added and incubated at 37 ° C for 1 h. After washing 3 times, TMB substrate color developing solution (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number: CW0050) was added at 50 μl/well. After 10 min, 2N H ₂ SO ₄ to terminate the color development.

RESULTS: BY18.1 and BY18.2 bispecific antibodies have good binding activity to human CTLA-4 protein and recombinant human PD-L1 protein, as shown in Figure 3 and Figure 4. The difference in binding affinity between BY18.1 and BY18.2 and antigen was not judged from the results, and further testing was further carried out by molecular interaction instrument Biacore.

Example 7. Biacore method for detecting the affinity of BY18.1 and BY18.2 bispecific antibodies to PD1 and CTLA-4

The anti-human IgG antibody was first immobilized on the CM5 chip by amino coupling method, and then the bispecific antibody was sequentially captured, and then the bispecific antibody and the antigen CTLA-4 were separately detected by using the Biacore T100 instrument (purchased to Yiqiao Shenzhou Biotechnology Co., Ltd., Product No.: 11159-H08H) and PD1 (purchased to Yiqiao Shenzhou Biotechnology Co., Ltd., product number: 10377-H08H) interaction and affinity. Finally, the data was fitted to the curve to calculate the antibody affinity. The experimental results are shown in the following table:

Affinity results of BY18.1 and BY18.2 bispecific antibodies with PD1 and CTLA-4

antibody

antigen

Ka(1/Ms)

Kd(1/s)

KD(M)

BY18.1	PD1	1.89E+04	7.79E-04	4.12E-08
BY18.1	CTLA4	2.05E+05	7.76E-04	3.78E-09
BY18.2	PD1	9.29E+04	7.15E-04	7.70E-09
BY18.2	CTLA4	9.33E+04	4.07E-04	4.36E-09

The results showed that all four pairs of combinations had stronger affinity. For PD1 antigen, BY18.1 affinity is relatively low, BY18.2 affinity is relatively high; for CTLA4, BY18.1 affinity is relatively high, BY18.2 affinity is relatively low. It can be seen that the ability of the bispecific antibody internal antibody of the DVD structure to bind to the antigen is weaker than the ability to bind the corresponding antigen when it is external.

Claims (11)

1. A bispecific antibody capable of binding to a PD-1 and/or CTLA-4 antigen, wherein the heavy chain comprises an HVD1-L1-HVD2-CH structure, wherein

   HVD1 is the first heavy chain variable domain,

   L1 is a heavy chain flexible joint,

   HVD2 is the second heavy chain variable domain.

   The CH region is a heavy chain constant domain;

   Wherein the light chain comprises an LVD1-L2-LVD2-CL structure, wherein

   LVD1 is the first light chain variable domain.

   L2 is a light chain flexible joint,

   LVD2 is the second light chain variable domain.

   CL is a light chain constant domain.
2. The bispecific antibody according to claim 1, wherein HVD1 is selected from SEQ ID No 2, HVD2 is selected from SEQ ID No 4, 6, 8 and LVD1 is selected from SEQ ID No 1, LVD2 is selected From SEQ ID No 3, 5, 7;

   Or wherein HVD1 is selected from SEQ ID No 4, 6, 8, then HVD2 is selected from SEQ ID No 2, LVD1 is selected from SEQ ID No 3, 5, 7, and LVD2 is selected from SEQ ID No 1.
3. The bispecific antibody according to claim 2, wherein the flexible linker between the L1 two heavy chain variable domains is selected from the group consisting of "AKTTPKLEEGEFSEAR", "AKTTPKLEEGEFSEARV", "SAKTTPKLEEGEFSEARV", "AKTTPPSVTPLAP", "ASTKGPSVFPLAP" ", "GGGGSGGGGSGGGGS", "TVAAPSVFIFPPTVAAPSVFIFPP" or "ASTKGPSVFPLAPSS".
4. The bispecific antibody according to claim 3, wherein the flexible link between the L1 two heavy chain variable domains

   For "ASTKGPSVFPLAPSS" and "ASTKGPSVFPLAP".
5. The bispecific antibody according to claim 2, wherein the flexible linker between the L2 two light chain variable domains is selected from the group consisting of "AKTTPKLEEGEFSEAR", "AKTTPKLEEGEFSEARV", "SAKTTPKLEEGEFSEARV", "AKTTPPSVTPLAP", "ASTKGPSVFPLAP" ", "GGGGSGGGGSGGGGS", "TVAAPSVFIFPPTVAAPSVFIFPP" or "ASTKGPSVFPLAPSS".
6. The bispecific antibody according to claim 5, wherein the flexible linker between the L2 two light chain variable domains is "TVAAPSVFIFPPS".
7. The bispecific antibody according to claim 1, wherein the antibody has a total antibody heavy chain type of any one of IgG1, IgG2, IgG3, and IgG4, and is preferably IgG4.
8. The bispecific antibody according to claim 1, wherein the full antibody light chain type of the antibody is kappa and lambda, preferably kappa.
9. The bispecific antibody according to any one of claims 1 to 8, wherein the anti-PD-1 and/or CTLA-4 variable domain of the antibody is located inside or outside the antibody and can be maintained with PD -1 and / or CTLA-4 binding ability.
10. The bispecific antibody according to any one of claims 1 to 8, wherein the expression cell of the antibody is any one of CHO, HEK293, NS0 and Per 6, preferably CHO cells.
11. Use of the bispecific antibody according to any one of claims 1-8 for the preparation of a medicament for treating a tumor.

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