WO2020177627A1 - Bispecific antibody - Google Patents

Abstract

Provided is a bispecific antibody, in particular a bispecific antibody simultaneously targeting tumour cell surface antigens and immune checkpoint proteins. The antibody comprises a first binding domain targeting the immune checkpoint proteins of a first target cell and a second binding domain binding with tumour cell surface antigens of a second target cell surface. The antibody binds with high affinity to tumour cell surface antigens by means of polypeptides, aggregating fragments (e.g. PD-L1 antibodies) targeting the immune checkpoints fused thereto onto the tumour cells, or near to same and in the tumour microenvironment, exerting the specific killing effect of the effector cells on tumour cells.

Description

A bispecific antibody Technical field

The present invention relates to a bispecific antibody, especially a bifunctional antibody directed against tumor cell surface antigen and immune checkpoint protein at the same time, and a pharmaceutical composition and application thereof.

Background technique

The launch of immune checkpoint inhibitors represented by programmed death receptor 1 (PD-1) and its ligands (PD-L1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a milestone in tumor treatment In the event, there are currently 7 immune checkpoint inhibitors approved by the U.S. Food and Drug Administration (FDA) for use in advanced tumor immunotherapy. They are not only safe, but also effective in advanced melanoma, non-small cell lung cancer (NSCLC), Renal cell carcinoma (RCC), urothelial carcinoma, and non-Hodgkin’s lymphoma have demonstrated exciting therapeutic effects, effectively prolonging the survival period of patients (Li Hanzhong et al. Peking Union Medical College 2018; 9(4): 289- 294).

Although the successful clinical application of immune checkpoint inhibitors has changed the treatment mode of many cancers, due to the high heterogeneity of human tumors, in addition to classic Hodgkin’s lymphoma, fibroproliferative melanoma, and Merkel Cell carcinoma and cancers with highly unstable microsatellites show a higher response rate (40%-80%). The objective response rate of a single immune checkpoint inhibitor is in NSCLC, RCC, head and neck cancer, liver cancer, and urinary tract. Skin cancer and other solid tumors usually do not exceed 20-30% (Borghaei H, et al. NEJM 2015, 373(17): 1627-1639; Motzer RJ, et al. NEJM 2015 373(19): 1803-1813), At the same time, there will be an inevitable problem of drug resistance, and there are still few patients who can obtain a lasting effect.

Based on these limitations, in recent years, immune checkpoint inhibitors have been combined with other drugs to play a complementary role in tumor killing and overcome the insufficient response rate of immune checkpoint inhibitors, which has become one of the research hotspots in the field of tumor immunity. Although the combination medication greatly improves the objective response rate of immune checkpoint inhibitors, its side effects also increase. For example, Larkini J et al. combined the immune checkpoint inhibitors Nivolumab and Ipilimumb, and the objective remission in the treatment of melanoma was 58%, which was much higher than the objective remission rates of Nivolumab and Ipilimumab alone (43.7% and 19%, respectively). The incidence of grade 3-4 adverse reactions related to combination medication was 55.0%, and the rate of drug withdrawal due to medication toxicity was as high as 36.4%, which was much higher than the ratio when Nivolumab or Ipilimumab were used alone: with Nivolumab and Ipilimumab alone The incidence of treatment-related grade 3-4 adverse reactions was 16.3% and 27.3%, respectively, while the rates of treatment termination due to the toxicity of Nivolumab and Ipilimumab were 7.7% and 14.8%, respectively (Larkini J et al. NJEM 2015; 373: 23-34). Hammers HJ et al. combined the PD-1 inhibitor nivolumab and CTAL-4 inhibitor ipilimumab in the treatment of advanced renal cell carcinoma with an objective response rate of 42.1% to 36.8%, and a 2-year survival rate of 69.6% to 67.3%, suggesting that the combined treatment effect may be superior In monotherapy, but the side effects of combined therapy are more serious than monotherapy (Hammers HJ et al. J Clin Oncol 2017; 35(34):3851-3858). The study of PD-1 inhibitor combined with vascular endothelial growth factor inhibitor targeted drugs in the treatment of renal cancer has also shown the superiority of combined therapy. The objective response rate of nivolumab combined with sunitinib in the treatment of metastatic clear cell renal cell carcinoma is 52%. 30% of patients have stable disease, and even a few patients can get complete remission, suggesting that the therapeutic effect may be better than single-agent therapy. However, the incidence of adverse reactions of combination therapy is also high. The incidence of grade 3 and 4 adverse reactions of nivolumab (2mg/kg) and nivolumab (5mg/kg) combined with sunitinib are 71.4% and 84.6%, respectively. The patient developed renal failure and pneumonia. The side effects of the nivolumab combined with pazopanib regimen were more serious, which led to the suspension of the study, although it showed an objective remission rate better than monotherapy (Hammers HJ et al. J Immunother Cancer. 2018; 6:109).

Therefore, there is still a problem in the field of what strategy can be adopted to greatly improve the objective remission rate of immune checkpoint inhibitors without increasing the incidence of toxicity or treatment-related adverse reactions.

The present invention uses the first binding domain (e.g., polypeptide) that specifically targets the tumor surface antigen as a delivery vehicle, and aggregates the immune checkpoint inhibitor (e.g., anti-PD-L1 antibody) connected to it on tumor cells, or The attachment and the tumor microenvironment greatly reduce the extensive immune activation caused by the use of conventional immune checkpoint inhibitors in the body.

Brief description of the invention

The present invention relates to a bispecific antibody, which comprises: a first binding domain targeting an immune checkpoint protein and a second binding domain targeting a tumor cell surface antigen.

In some specific embodiments, the first binding domain of the bispecific antibody of the present invention targets the immune checkpoint protein PD-L1, and the second binding fragment targets the tumor cell surface antigen MC1R.

In some specific embodiments, the first binding domain of the bispecific antibody of the present invention is an antibody targeting PD-L1, and the second binding domain is a polypeptide α-MSH, ΔMSH or (NDP)MSH targeting MC1R.

In some embodiments, the C-terminus of the second binding domain of the bispecific antibody of the present invention is connected to the N-terminus of the heavy chain or light chain variable region of the first binding domain through a connecting peptide.

In some embodiments, the N-terminus of the second binding domain of the bispecific antibody of the present invention is connected to the C-terminus of the heavy chain or light chain variable region of the first binding domain through a connecting peptide.

In some specific embodiments, the N-terminus and or C-terminus of the second binding domain of the bispecific antibody of the present invention is inserted into the CDR1-H, CDR2-H, CDR3-H, Within CDR1-L, CDR2-L or CDR3-L.

In some embodiments, the second binding domain of the bispecific antibody of the present invention is coupled to the first binding domain through a chemical linker.

On the other hand, the present invention also relates to a nucleic acid, which encodes the bispecific antibody of the present invention.

In another aspect, the present invention also relates to an expression vector comprising the nucleic acid of the present invention.

In another aspect, the present invention also relates to a host cell comprising the expression vector of the present invention.

In another aspect, the present invention also relates to a pharmaceutical composition comprising the bispecific antibody of the present invention.

On the other hand, the present invention also relates to the use of bispecific antibodies in the preparation of medicines for the treatment of autoimmune diseases or cancer.

The present invention fuses tumor surface antigen-specific polypeptides such as α-MSH or ΔMSH with antibodies against immune checkpoint proteins, such as heavy or light chains of anti-PD-L1, or combines (NDP) MSH with antibodies against immune checkpoint proteins For example, by coupling the heavy or light chain of anti-PD-L1, the resulting bifunctional antibody can simultaneously target the MC1R antigen and PD-L1 antigen on tumor cells, antagonize the function of MC1R, and block the relationship between PD-L1 and PD-1 The inter-signal pathway specifically promotes the immune cells surrounding tumor cells, such as T cells from an incompetent state to an activated state, and exerts the specific killing effect of immune cells on tumor cells.

In the present invention, the anti-tumor cell surface antigen polypeptide α-MSH, ΔMSH or (NDP) MSH is used as a delivery carrier, and it is connected to the binding domain of the targeted immune checkpoint protein (such as anti-PD-L1) through a connecting peptide or chemical linker. Antibody) connection, by targeting specific targets on the surface of tumor cells with high affinity, and gathering functional molecules for immune checkpoints on tumor cells, or its vicinity and within the tumor microenvironment, the immune checkpoints can be regulated (inhibited Or enhance) the specific killing effect of effector cells on tumor cells is limited to the tumor or tumor microenvironment, which greatly reduces the extensive immune activation caused by the use of conventional immune checkpoint modulators in the body; at the same time, with the help of delivery vectors The high affinity of tumor-specific antigens can adjust the affinity or functional activity of effector molecules to immune checkpoints within a certain range, and has a wide range of clinical applications.

Description of the drawings

Figure 1 is a bispecific antibody SDS-PAGE, where M is the protein marker, Ave is the Avelumab antibody, A is the MSH-SynL fusion antibody (MSH-SynL fusion), and B is the MSH-Ate antibody light chain fusion. Antibody (MSH-AteL fusion), C is the fusion of MSH and Ate antibody heavy chain (MSH-AteH fusion),-means no reducing agent DTT is added when loading, + represents adding reducing agent DTT when loading

Figure 2 shows the results of antibody gel exclusion chromatography

Figure 3 shows the comparison of the binding ability of different antibody conjugates or different fusion forms to the antigen PD-L1

Figure 4 shows the binding of different antibody conjugates or different fusion forms to MC1R on the surface of HER293-MC1R cells (Figures 4A and 4C), and free MSH inhibitory antibody conjugates or fusion double antibodies to HER293-MC1R cells Surface bonding (Figure 4B and 4D)

Figure 5 shows the binding of different antibody conjugates or different fusion forms of double antibodies to B16.SIY cells

Figure 6 shows that different antibody conjugates or different fusion forms of double antibodies stimulate signal transduction in HER293-MC1R cells

Figure 7 shows the serum stability of different antibody conjugates

Figure 8 shows the effect of 5mg/kg (NDP) MSH-Ave conjugate on the size of tumor mass in B16-SIY tumor-bearing mice, where the arrow indicates the time point of administration

Figure 9 shows the proportion of tumor-infiltrating lymphocytes (TILs) in B16-SIY tumor-bearing mice after receiving different doses of double antibodies. Group a: normal saline group; group b: 5 mg/kg Ave injection group; group c: 1 mg/kg Ave injection group; group d: 5 mg/kg NR-Ave conjugate injection group; group e: 1 mg/kg NR-Ave conjugate Injection group; group f: 5mg/kg (NDP) MSH-Ave conjugate injection group; group g: 1mg/kg (NDP) MSH-Ave conjugate injection group.

Detailed description of the invention

The present invention is described in detail here by using the following definitions and references to examples. The contents of all patents and publications mentioned herein, including all sequences disclosed in these patents and publications, are expressly incorporated herein by reference.

Bispecific antibody

The "bispecific antibody" of the present invention is an antibody with two different antigen binding specificities. Where the antibody has more than one specificity, the recognized epitope can bind a single antigen or bind more than one antigen. Antibody specificity refers to the selective recognition of specific epitopes of an antigen by antibodies. Natural antibodies are, for example, monospecific.

The antibody of the present invention is directed against two different antigens, one of which is tumor cell surface antigen and the other is immune checkpoint protein.

In some embodiments, in the bispecific antibody of the present invention, the first antigen-binding fragment and the second antigen-binding fragment are chemically coupled.

In some embodiments, the first antigen-binding fragment and the second antigen-binding fragment of the bispecific antibody of the present invention are connected by a connecting peptide.

Tumor surface antigen

As used herein, the term "tumor surface antigen" includes proteins or polypeptides that are preferentially expressed on the surface of tumor cells. As used in this context, the expression "preferentially expressed" means that the antigen is expressed on tumor cells at least 10% higher than the expression level of the antigen on non-tumor cells (e.g., 10%, 20%, 30%, 40%, 50%). %, 60%, 70%, 80%, 90%, 100%, 110%, 150%, 200%, 400% or higher). In certain embodiments, the target molecule is an antigen that is preferentially expressed on the surface of selected tumor cells (such as solid tumors or hematological tumor cells): non-limiting examples of specific tumor-associated antigens include, for example, EGFR, HER2, HER3, HER4, MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20, VEGFR-1 (FLT1), VEGFR-2 (KDR/FIK-1 ), VEGFR-3, PDGF-RA, PDGF-RB, IGF-1R, IGF2B3, K-RAS, N-RAS, Bly-S (BAFF), BAFF-R, EpCAM, SAGE, XAGE-1b, BAGE, MAGE Protein (such as MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-9, MAGE-10, MAGE-12), GAGE-1, GAGE-2, GAGE-8, GAGE- 3. GAGE-4, GAGE-5, GAGE-6, GAGE-7, XAGE-1b/GAGED2a, RAGE-1, RBAF600, CD2, CD3, CD19, CD-11α, CD16A, CD19, CD20, CD21, CD22, dipeptidyl-peptidase 4(CD26), CD30, CD32B, CD33, CD38, CD40, CD45, CD52, CD70, CD80, CD60, CD62, CD72, CD79a, CD79B, SLAMF7 (CD139), CD123, Ly6D, Ly6E, Ly6K, gp100 /Pmel17, EDAR, GFRA1 (GDNF-Ra1), MRP4, RET, STEAP1, STEAP2, TENB2, E16 (LAT1, SLC7A5), SLC35D3, MPF, SCL34A2, Sema 5b, PSCAhIg, ETBR, MSG783, FcRH1, FcRH2, NCA, MDP, IL20Ra, EphA2, EphA3, EphB2R, ASLG659, GEDA, CXCR5, P2X5, LY64, IRTA2, TMEF1, TMEM46, TMEM118, LGR5, GPR19, GPR172A, GPC3, CLL1, RNF43, KISS1R, ASPHD1, CXORF61, epidermal adjustment , Amphiregulin, lipophilin, AIM-2, ALDH1A1, a-actinin-4, ARTC1, BING-4, CALCA, CASP- 5. CASP-8, cdc27, CDK4, CDKN2A, CLPP, COA-1, CPSF, Cw6, RANKL, DEK-CAN, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), ETV6-AML1, EZH2, FLT3- ITD, FN1, G250, MN, CAIX, GnTVf, GPNMB, HERV-K-MEL, hsp70-2, IDO1, IL13Ra2, intestinal carboxyl esterase, kallikrein 4, KIF20A, KK-LC-1, KM- HN-1, LAGE-1, LDLR-Salcosyltransferase AS fusion protein, Lengsin, M-CSF, lactoglobulin-A, MART-1, Melan-A/MART-1, MART2, MCSP, mdm-2 , ME-1, Meloe, MMP-2, MMP-7, Mucin, MUM-1, MUM-2, MUM-3, Myosin Class I, NA88-A, PAP, neo-PAP, NFYC, NY- BR1, NY-BR62, NY-BR85, NY-ESO1, NY-ESO-1/LAGE-2, RAB38/NY-MEL-1, OA1, OGT, OS-9, p53, PAX3, PAX5, PBF, PML- RARa, PRAME, PRDX5, PSMA(FOLH1), PTPRK, RGS5, Rho, RhoC, RNF43, RU2AS, Protein Isolate 1, SIRT2, SNRPD1, SOX10, Sp17, SSX-2, SSX-4, Survivin, SYT-SSX1 or -SSX2, TAG-1, TAG-2, telomerase, TGF-β, TGF-beta RII, TRAG-3, triose phosphate isomerase, TRP-2, TRP2-INT2, VEGF, WT1, TRPM4, CRIPTO , Glycoprotein IIb/IIIa receptor, glycolipid GD2, GD3, folate receptor 1 (FOLR1), IFNγ, IFNα, β, ω receptor 1, TROP-2, Glyco-protein NMB, MMP9, GM3, mesothelin, fibronectin extra-domain B, endoglin , Rhesus D, plasma kallikrein, CS, thymic stromal lymphopoietin, mucosal address in cell adhesion molecule, nectin 4, NGcGM3, DLL3, DLL4, CLEC12A, KLB , FGFR1C, CEA, BCMA, p-cadherin, FAP, DR1, DR5, DR13, PLK, B7-H3, c-Met, gpA33, gp100/Pmel17, gp100, TRP-1/gp75, BCR-ABL, AFP, ALK , Β-chain protein, BRCA1, BORIS, CA9, Caspase-8, CDK4, CTLA4, Cyclin-B1, Cyclin D1, Cyclin-A1, CYP1B1, Fra-1, GloboH, Phosphatidyl muscle Glycan-3, GM3, HLA/B-RAF, hTERT, LMP2, Mesothelin, ML-IAP, NA17, OX40, p15, PPLR, PCTA-1, PLAC1, PRLR, PRAME, SART-1, SART-3, TAG-72, TMPRSS2, Tn, Tyrosinase and Urinary Plaque-3. CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCL27, CCL28, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, androgen receptor (AR), calcitriol Receptor (CR), Estrogen Receptor (ER), Adrenergic Releasing Hormone Receptor (CRHR), Glucagon Receptor (GCGR), Gonadotropin Receptor (FSHR, LHR), or Melanocortin 1 Receptor (MC1R, MSHR)

Immune checkpoint protein

Immune examination is a type of signal that regulates T cell receptor (TCR) antigen recognition during the immune response. Including costimulatory immune signals that stimulate immunity and co-suppressive immune signals that suppress immunity. Immune examination can prevent autoimmune damage caused by excessive activation of immune cells (for example, T cells). Tumor cells use the protective mechanism of the human immune system to overexpress immune checkpoint proteins, thereby inhibiting the anti-tumor response of the human immune system and forming an immune escape. Immune checkpoint therapy uses costimulatory signal agonists or co-inhibitory signal antagonists to allow the immune system to function normally. Common immune checkpoint proteins include CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1 , PD-L1, PD-L2, TIM-3, VISTA, GARP, PS, CSF1R, CD94/NKG2A, TDO, GITR, TNFR and FasR/DcR.

Immune checkpoint proteins are mainly expressed on the surface of immune cells. Immune checkpoint proteins are also expressed on the surface of tumor cells. For example, PD-L1 is highly expressed on the surface of many tumor cells, such as lung cancer, breast cancer, malignant melanoma, esophageal cancer, gastric cancer, and pancreatic cancer.

Pharmaceutical composition

The pharmaceutical composition as described herein is prepared by mixing the bifunctional antibody of the present invention with the desired purity and one or more optional pharmaceutically acceptable carriers, which is in the form of a lyophilized preparation or an aqueous solution. The pharmaceutically acceptable carrier is generally non-toxic to the recipient at the dose and concentration used.

The bifunctional antibody of the present invention can be used as a single active ingredient or administered in combination with, for example, adjuvants or with other drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory agents, for example for the treatment or prevention of acute lymphoblastic leukemia ( ALL), acute medullary leukemia (AML), adrenal cortical cancer, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, primordial cancer Special lymphoma, cancer of unknown primary origin, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, Craniopharyngioma, skin T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal glioma, fibrous histiocytoma, Ewing sarcoma, eye cancer, reproduction Cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma , Hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, Lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous neck cancer, midline cancer involving NUT gene , Oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, granuloma fungoides, myelodysplastic syndrome, myelodysplastic/myelodysplastic neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, Neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer , Pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyomas, salivary gland cancer, Sezari syndrome, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratomoid tumor, testicular cancer, throat cancer, thymoma and thymic cancer, thyroid cancer, urethra Cancer, uterine cancer, vaginal cancer, vulvar cancer and Wilms tumor.

Example

The following examples further illustrate the present invention. These examples are only for illustration and do not constitute any limitation on the scope of the present invention.

The meanings of the abbreviations are as follows: "h" means hours, "min" means minutes, "s" means seconds, "ms" means milliseconds, "d" means days, "μl" means microliters, "ml" means milliliters, "L" "Refers to liters, "bp" refers to base pairs, "mM" refers to millimoles, and "μM" refers to micromoles.

Example 1 Construction of eukaryotic expression vector

1.1 Construction of Atezolizumab, Avelumab or Synagis antibody eukaryotic expression vector

PfuUltra II DNA polymerase (Agilent Technologies, Inc., CA) was used to amplify the heavy chain of atezolizumab (hereinafter referred to as "Ate") antibody Fab (Ate FabH), the light chain of Ate antibody Fab (Ate FabL), and avelumab antibody ( Hereinafter referred to as "Ave") Fab heavy chain (Ave FabH), Ave antibody Fab light chain (Ave FabL), palivizumab antibody (hereinafter referred to as "Syn") Fab heavy chain (Syn FabH), Syn antibody Fab light chain (Syn FabL) (synthesized by IDT). Clone the amplified Ate FabH, Ave FabH or SYN FabH fragments into the pFuse-hIgG1-Fc2 vector (InvivoGen, CA) through the Gibson assembly kit (NEB, MA) to obtain pFuse-AteHC, pFuse-Ave HC, pFuse-Syn HC, clone the amplified Ate FabL, Ave FabL or Syn FabL into the pFuse2-CLIg-hK vector (InvivoGen, CA) to obtain pFuse-Ate LC, pFuse-Ave LC, pFuse-Syn LC. All constructed vectors are verified by sequencing.

1.2 Construction of a bispecific antibody expression vector fused with MSH and Ate, SYN or Ave

Synthesize α-MSH polypeptide (hereinafter referred to as "MSH") (SEQ ID NO. 2) or ΔMSH polypeptide (SEQ ID NO. 4), and clone them into the above-constructed Ate HC, Ate LC, Ave HC through the connecting peptide, respectively , Ave LC, HC, Syn LC N-terminal or C-terminal. All constructed vectors are verified by sequencing.

Table 1 Sequence name

Example 2 Antibody expression and purification

The heavy chain and light chain of the antibody expression vector constructed in Example 1 were transiently transfected into FreeStyle HEK293 cells (ThermoFisher)) and co-transfected separately. The amount of plasmid for the heavy chain and the plasmid for the light chain during transfection was 1:1 by molar ratio) ：Inoculate 28ml FreeStyle HEK 293 (3×10 ⁷ cells/ml) into a 125ml cell culture flask, and the plasmid is diluted with 1ml Opti-MEM (Invitrogen) and added to 1ml Opti-MEM containing 60μl 293Fectin (Invitrogen), statically at room temperature Leave it for 30 minutes, add the plasmid-293Fectin mixture to the cell culture medium at 125 rpm, 37°C, and 5% CO2. The cell culture supernatant was collected at 48h and 96h after transfection, purified by Protein A Resin (Thermo Fisher Scientific, IL), and detected by SDS-PAGE.

From the SDS-PAGE results in Figure 1, it can be seen that the monoclonal antibody and the fusion double antibody have been successfully expressed.

Example 3 Preparation of chemically coupled bispecific antibodies

The Ave expressed in Example 2 was incubated with BCN-NHS (sigma, Cat#744867) at room temperature for 1 h, and NDP-MSH with PEG linker (Azido-PEG24-SYS-Nle-EHfRWGKPV-NH2, Nle=Norleucine, f ＝D-form Phe) or NR-MSH with PEG linker (Azido-PEG24-SEGYHKSfRP-Nle-WV-NH2) (synthesized by Innopep Inc.) was added to the reaction solution and placed at room temperature for 24h.

The reaction product obtained above and the fusion double antibodies MSH-AteL fusion and MSH-AteH fusion of Example 2 were subjected to gel exclusion chromatography using GE's AKTA chromatography. The chromatography column used is Superdex 200 Increase10/300 GL gel exclusion chromatography column, and the solution used for gel exclusion chromatography is PBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH 7.4 ), the flow rate used in gel exclusion chromatography is 0.4 ml/min.

It can be seen from the gel exclusion chromatography of Figure 2A that the chemically coupled bispecific antibody has relatively high purity. The results of Figure 2B and Figure 2C showed that the purity of the fusion antibody was as expected.

Example 4 Mass Spectrometry Analysis

The fusion bi-antibody obtained in Example 2 and the chemically coupled bi-specific antibody obtained in Example 3 were incubated with PNGase F (NEB) at 37°C for 8 hours, and then treated with 10 mM dithiothreitol and passed through ESI-qTOF -MS (Agilent, USA) analysis. The drug/antibody ratio (DAR) of chemically coupled bispecific antibodies is calculated by molecular weight.

The results are shown in Table 2 and Table 3. The number of (NDP) MSH conjugated in 90% (NDP) MSH-Ave conjugate is between 1-6, and the average number of (NDP) MSH conjugated per Ave antibody is DAR The number of (NDP) MSH conjugated in 90%-98% (NDP) MSH-Ate conjugates is between 1-7, and the average number of (NDP) MSH conjugated per Ate antibody DAR is 2.2.

Table 2 Bispecific antibody ESI-Q-TOF-MS

Table 3 DAR of different antibody conjugates and their corresponding abundance

Example 5 In vitro activity identification of bispecific antibodies

5.1 Bispecific antibody binding to human PD-L1 ELISA detection

Coated with hPD-L1-hIGg1Fc (SinoBiological) (100ng/well) (DPBS buffer, pH 7.4) in a 96-well plate, incubated overnight at 4°C; blocked in DPBST containing 2% skimmed milk powder for 1 hour at room temperature, containing 0.05% Tween- After washing 3 times with 20 DPBS, add gradient dilution MSH-αPD-L1 or NR-αPD-L1/αPD-L1 and incubate for 2h at room temperature. After washing 4-5 times with 0.05% Tween-20 in DPBS, add anti-human kappa light chain (Cat.A18853, Thermo Fisher Scientific, 1:2000) secondary antibody incubated for 2 hours at room temperature, washed 4-5 times in DPBS containing 0.05% Tween-20 Figure 6, QuantaBlu fluorescent peroxidase substrate (Life technologies , Cat.15169) after color development, read at 325nm and 420nm. Prizm Graphpad software uses log(agonist) vs.response model to perform nonlinear regression on the data.

The result is shown in Figure 3. After coupling (NDP) MSH to Ave or Ate antibody, its ability to bind PD-L1 is almost unaffected (Figure 3A and Figure 3B); in addition, (NDP) MSH is fused to the N-terminal of the heavy or light chain of the Ate antibody Later, the affinity of Ave and PD-L1 was almost unaffected (Figure 3C).

5.2 Bispecific antibodies bind to cell surface MC1R ELISA and competitive ELISA

Cultivate HEK293 cells (HEK293-MC1R) overexpressing MC1R (DMEM medium containing 10% FBS, 1% double antibody). After trypsinization, 2x104/well HEK293-MC1R cells were placed on a 96-well flat-bottomed blackboard at 37°C, 5% CO2 culture overnight to make it adherent. After washing 3 times with PBS, centrifuge to discard the supernatant, add 8% formalin solution and incubate at room temperature for 15 min. After aspirating the formalin solution, directly add different concentrations of MSH-αPD-L1 or αPD-L1 for cell binding analysis, or incubate 30nM MSH-αPD-L1 or αPD-L1 with different concentrations of MSH and cells. Competitive combination analysis. The rest of the ELISA test procedure is carried out according to the In-Cell ELISA (ICE) Support Pack (Cat.ab111542, Abcam) manual. In the final detection step, add HRP-labeled anti-human IgG (Fc) (ELITechGroup, Netherlands) secondary antibody (1:1000, solution: PBS/5％BSA/0.1％Tween) to the test well and incubate for 1h at room temperature, wash with PBS After 3 times, QuantaBlu fluorescent peroxidase substrate (Life technologies, Cat. 15169) was added to develop color and read at 325nm and 420nm. Prizm Graphpad software uses log(agonist) vs.response model to perform nonlinear regression on the data.

The results are shown in Figure 4. Both (NDP) MSH-Ave conjugate and (NDP) MSH-Ate conjugate bind to HEK293-MC1R cells in a dose-dependent manner (Figures 4A and 4C), and the binding can be inhibited by free MSH competition (Figures 4B and 4D) . Similarly, MSH-AteL fusion and MSH-AteH fusion also bind to HEK293-MC1R cells in a dose-dependent manner (Figure 4C), and the binding can be inhibited by free MSH competition (Figure 4D). Interestingly, the binding ability of the fused double antibody to HEK293-MC1R is stronger than that of the chemically conjugated double antibody.

5.3 Flow cytometric detection of bispecific antibody binding to B16-SIY (MC1R ⁺ /PD-L1 ⁺ ) cells

Cultivate B16-SIY cells (DMEM medium containing 10% FBS, 1% double antibody), take 2x105 cells and wash 3 times with pre-chilled PBS, block with 2% FBS (dissolved in PBS) and incubate with samples of different concentrations at 4℃ 2h, 2% FBS (dissolved in PBS) to wash away unbound antibodies, incubate with APCanti-human IgG Fc (KPL, Inc., MD) at 4°C for 1 h, 2% FBS (dissolved in PBS) and then use LSR II Flow cytometry (Becton Dickinson, NJ) detection, and FlowJo software (TreeStar, OR) for analysis. Prizm Graphpad uses log(agonist) vs.response model to perform nonlinear regression on data. The result is shown in Figure 5.

As shown in Figure 5, compared with Ate, conjugated double antibody ((NDP) MSH-Ate conjugate, (NDP) MSH-Ave conjugate) or gene fusion double antibody (MSH-AteH fusion and MSH-AteL fusion) The ability to bind to B16-SIY cells is stronger than that of Ate, suggesting that chemical coupling or gene fusion does not affect the binding of Ate to B16-SIY cells.

5.4 MC1R activation experiment

Cultivate HER293 cells overexpressing MC1R and CRE-Luc (DMEM medium, 10% FBS), inoculate the cells into 384-well plates at 5000 cells/well, use different concentrations of bifunctional antibodies or controls at 37°C, 5% CO2 After 24 hours, the fluorescence intensity was detected by One-Glo (Promega, WI) according to the instructions provided by the manufacturer, and the Prizm Graphpad software used log (agonist) vs. response model to perform nonlinear regression on the data.

The results are shown in Figure 6. Both (NDP) MSH-Ave conjugate and (NDP) MSH-Ate conjugate can activate and induce the signal transduction of target cell HEK293-MC1R, but its activation effect is weaker than (NDP) MSH or free MSH Activation of target cells (Figures 6A and 6B). The activation effect of MSH-AteL fusion and MSH-AteH fusion on target cells is similar to that of the conjugate (Figure 6C)

Example 6. Thermal stability analysis of bispecific antibodies

Mix the sample with freshly prepared thermal shift dye and shift buffer buffer (Protein Thermal Shift ^TM Dye Kit (Cat.4461146, ThermoFisher Scientific) at the ratio recommended by the manufacturer, using ViiA ^TM 7 Real-Time PCR System at 0.05C/s The heating rate is 25-99°C for thermal scanning. The Tm value is calculated by the "Area under curve (AUC)" analysis model of GraphPad Prism7 software. Each set of data is repeated twice to ensure the repeatability of the results. The results are shown in Table 4. As shown, the Tm value of (NDP) MSH-Ave conjugate is 64°C, similar to Ave; the melting curve of (NDP) MSH-Ate conjugate is similar to Ate, but its Tm value is slightly lower than that of Ate; MSH-AteL fusion and MSH- The thermal stability of AteH fusion is also similar to that of Ate.

Table 4 Tm values of bispecific antibodies of different antibody conjugates or different fusion forms

sample T _m 1(℃) T _m 2(℃) Ate 67.15 85.68 Ave 67 / MSH-AteL fusion 67.42 85.95 MSH-AteH fusion 66.89 85.95 (NDP)MSH-Ave conjugate 64 / (NDP)MSH-Ate conjugate 65 83

NR-Ave conjugate 64 /

Example 7 Bispecific antibody serum stability test

The bispecific antibody or control was added to a tube containing 100ul of freshly isolated mouse serum (final concentration 1uM), and incubated at 37°C for 0h, 6h, 24h, 48h and 72h. The incubated samples were quickly frozen with liquid nitrogen and placed at -80°C for later use. The content of antibody in each tube was detected by PD-L1 combined with sandwich ELISA, and the detection and result analysis process was as described in Example 5.1.

As shown in Figure 7A and Figure 7B, the conjugate of (NDP) MSH and antibody (Ave or Ate) is very stable in mouse serum. After incubating at 37°C for 72 hours, no (NDP) MSH-Ate conjugate or (NDP) MSH-Ate conjugate is degraded.

Example 8 Study on pharmacokinetics of bispecific antibody in mice

The samples were intraperitoneally injected (I.P.) C57BL/6 mice (3 mice per group, 4 mg/kg). Heparin anticoagulant blood was collected from the tail vein or saphenous vein, and the blood collection time was as follows: 30min, 1h, 2h, 4h, 6h, 4h, 48h, 3d, 4d, 6d, 8d, 10d, 12d and 14d. After centrifugation, plasma was collected and stored at -80°C for later use. Sandwich ELISA detects the content of the sample in the plasma bound to PD-L1. The ELISA detection process is as described in Example 5.1. According to the standard curve (the abscissa is the sample concentration, the ordinate is the fluorescence signal value), the content of the sample in the plasma is calculated. The pharmacokinetic parameters are estimated using modeling program WinNonlin (Pharsight).

As shown in Table 5. (NDP) The coupling of MSH and Ave does not change the pharmacokinetics of Ave in mice, and its metabolic parameters are similar to Ave.

Table 5 PK parameters of mice at a dose of 4 mg/kg

sample R ² T _max (h) C _max (nM) T _1/2 (h) AUC _0-t (h.nM) Ave 0.88 4 523.13 17.83 16782 (NDP)MSH-Ave conjugate 0.94 6 317.64 16.27 13432 NR-Ave conjugate 0.90 6 405.6 17.89 16,923

Example 9 Effectiveness Study on Animal Model

(NDP) The effectiveness of MSH-Ave conjugate was studied in 6-week-old female C57BL/6 mice (Jackson Laboratory). 1.5x106 B16-SIY melanoma cells were injected subcutaneously (SC) into the right flank of mice (Day 0). On the 5th day after injection, the sample or PBS was injected intraperitoneally at a dose of 5mg/kg or 1mg/kg, once every 3 days , A total of 4 injections (Day-5, Day-8, Day-11, Day-14). Measure the size of the tumor with calipers, three times a week. The tumor volume is calculated by the following formula: tumor volume=width*length*height. The mice were sacrificed on Day-23, and tumors were collected for subsequent analysis.

As shown in Figure 8 and Table 6, compared with the control Ave, 5mg/kg (NDP) MSH-Ave conjugate significantly reduced the volume of tumors in tumor-bearing mice, showing stronger tumor suppressor activity.

Table 6 Effect of 5mg/kg (NDP) MSH-Ave conjugate and Ave on tumor volume

Example 10 Analysis of tumor infiltrating lymphocytes (TIL)

The tumor cell suspension is obtained by enzymatically hydrolyzing the tumor block. The specific steps: place the tumor block in HBSS (Life) containing 1mg/ml collagenase, 0.1mg/ml DNAse I, 2.5U/ml hyaluronidase (Sigma-Aldrich) Technologies), stirring at room temperature for 2h. Filter the cell suspension with a 70-um cell sieve, wash once with HBSS, resuspend it to 1x 106 cells/ml in PBS containing 3% BSA, and add FITC-labeled anti-mouse CD3 antibody (eBioscience, San Diego, CA) After incubation, the LSR II flow cytometer (Becton Dickinson, NJ) was used to detect and analyze with FlowJo software (TreeStar, OR). GraphPad prism software uses unpaired t-test (tew-tailed) for data processing, and P<0.05 is considered to be statistically significant.

As shown in Figure 9, the control group Ave and NR-Ave conjugate at concentrations of 1 mg/kg and 5 mg/kg, both showed no significant changes in CD3-positive infiltrating T lymphocytes in the tumor, which was consistent with the performance of the saline injection group. Compared with the control group 5mg/kg Ave and 5mg/kg NR-Ave conjugate, 5mg/kg (NDP) MSH-Ave conjugate treatment has significantly increased CD3-positive infiltrating T lymphocytes in mouse tumors. The tumor suppressor activity shown is consistent. Compared with the control group 1mg/kg Ave and 1mg/kg NR-Ave conjugate, the 1mg/kg (NDP) MSH-Ave conjugate treatment did not show a significant increase in CD3-positive infiltrating T lymphocytes in mouse tumors. The weaker tumor suppressor activity was consistent.

Claims (29)

A bispecific antibody comprising: a first binding domain of an immune checkpoint protein targeting a first target cell, and a second binding domain that binds to a surface antigen of a second target cell, wherein the first binding domain is An antibody structure comprising a constant region, a heavy chain variable region and a light chain variable region, the second binding domain is connected to the heavy chain or light chain of the first binding domain, wherein the second target cell is a tumor cell, and the first target The cells are tumor cells or immune cells. The bispecific antibody of claim 1, wherein the antibody targets two different antigens on the same tumor cell. The bispecific antibody of claim 1, wherein the antibody targets two different antigens on tumor cells and immune cells. The bispecific antibody of any one of the preceding claims, wherein the immune cells are selected from NK cells, T lymphocytes and B cells. The bispecific antibody of any one of the preceding claims, wherein the immune checkpoint protein is selected from PD-L1, PD-1, CTLA-4, LAG-3, OX40, CD28, CD40, CD47, CD70, CD80, CD122, GTIR, A2AR, B7-H3 (CD276), B7-H4, IDO, KIR, Tim-3 or 4-1BB (CD137). The bispecific antibody of any one of the preceding claims, wherein the tumor cell surface antigen is selected from the family of growth factor receptors, chemotactic factor receptors, hormone receptors or mucin family. The bispecific antibody of claim 6, wherein the growth factor receptor is selected from the group consisting of Wnt receptor (WntR) family, epidermal growth factor receptor family (EGFR, HER2, HER3, HER4), neurotrophic factor receptor (NTR), Fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (FLT1, KDR/Flk-1, VEGFR-3), hepatocyte growth factor receptor (HGFR), nerve growth factor receptor (NGFR), Insulin-like growth factor receptor (IGFR) or platelet-derived growth factor receptor (PDGFR). The bispecific antibody of claim 6, wherein the chemotactic factor receptor is selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCL27, CCL28, CX3CR1, CXCR1, CXCR2, CXCR3 , CXCR4, CXCR5 or CXCR6. The bispecific antibody of claim 6, wherein the mucin family is selected from mucin1 (MUC1), MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19 Or MUC20. The bispecific antibody of claim 6, wherein the hormone receptor is selected from the group consisting of melanocortin 1 receptor (MC1R, MSHR), androgen receptor (AR), calcitriol receptor (CR), estrogen receptor (ER), Adrenergic Releasing Hormone Receptor (CRHR), Glucagon Receptor (GCGR) or Gonadotropin Receptor (FSHR, LHR). The bispecific antibody of any one of the preceding claims, wherein the antibody targets the MC1R and PD-L1 antigens. The bispecific antibody according to any one of the preceding claims, wherein the second binding fragment is melanocyte stimulating hormone (MSH) or an analog thereof. The bispecific antibody of claim 12, wherein the melanocyte-stimulating hormone is selected from the group consisting of α-MSH, α-MSH variant, ΔMSH, β-MSH, β-MSH variant, γ-MSH or γ-MSH variant. The bispecific antibody of claim 12, wherein the MSH analogue includes afamelanotide (melanotan I, (NDP) MSH), melanotan II (melanotan II) or bremelanotide (bremelanotide). The bispecific antibody according to claim 13, wherein the melanocyte-stimulating hormone α-MSH has the amino acid sequence shown in SEQ ID No.2, and ΔMSH has the amino acid sequence shown in SEQ ID No.4. The bispecific antibody of claim 14, wherein the MSH analogue (NDP) MSH has the following amino acid sequence: SYS-Nle-EHfRWGKPV-NH2, wherein Nle is norleucine and f is D-Phe. The bispecific antibody according to any one of the preceding claims, wherein the first binding fragment is an anti-PD-L1 antibody. The bispecific antibody of claim 17, wherein the anti-PD-L1 antibody has two chains with the following heavy chain and light chain amino acid sequences: heavy chain SEQ ID No. 10 and light chain SEQ ID No. 8; heavy chain SEQ ID No. 6 and light chain SEQ ID No. 12; heavy chain SEQ ID No. 14 and light chain SEQ ID No. 10; heavy chain SEQ ID No. 6 and light chain SEQ ID No. 16; heavy chain SEQ ID No. .18 and light chain SEQ ID No. 22; heavy chain SEQ ID No. 18 and light chain SEQ ID No. 24. The bispecific antibody according to any one of the preceding claims, wherein the second binding domain is coupled to the first binding domain via a chemical linker. The bispecific antibody of claim 19, wherein the chemical linker is a PEG linker or a polymer of a PEG linker and an NHS ester. The bispecific antibody of any one of claims 1 to 18, wherein the second binding domain is connected to the first binding domain through a connecting peptide. The bispecific antibody of claim 21, wherein the C-terminus of the second binding domain is connected to the N-terminus of the heavy chain or light chain variable region of the first binding domain through a connecting peptide. The bispecific antibody of claim 21, wherein the N-terminus of the second binding domain is connected to the C-terminus of the heavy chain or light chain variable region of the first binding domain through a connecting peptide. The bispecific antibody of claim 22 or 23, wherein the N-terminus and C-terminus of the second binding domain are respectively inserted into the CDR1-H, CDR2-H, CDR3-H, CDR1 of the first binding domain through a connecting peptide. -L, CDR2-L or CDR3-L. A nucleic acid encoding the bispecific antibody according to any one of claims 1-24. An expression vector comprising the nucleic acid of claim 25. A host cell characterized by comprising the expression vector according to claim 26. A pharmaceutical composition characterized by comprising the bispecific antibody according to any one of claims 1-24. The use of the antibody according to any one of claims 1-24 in the preparation of a medicament for the treatment of autoimmune diseases or cancer.

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