Pharmaceutical preparation containing polynucleotide

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a pharmaceutical preparation containing polynucleotide, the pharmaceutical preparation comprises polynucleotide of a bifunctional antibody and a delivery system, the bifunctional antibody comprises a PD-1-targeted first functional area and a CTLA-4-targeted second functional area, the first functional area is used for coding a CTLA4-and PD-1-combined bifunctional antibody, and the second functional area is used for coding a CTLA4-and PD-1-combined bifunctional antibody and is used for coding a CTLA4-and PD-1-combined bifunctional antibody. The first functional area is an anti-PD-1 heavy-chain antibody or an antigen binding fragment thereof, and the second functional area is an anti-CTLA-4 heavy-chain antibody or an antigen binding fragment thereof. The polynucleotide is high in expression quantity and long in expression time, and can be used for preparing medicines for treating cancers.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a pharmaceutical preparation containing a polynucleotide, wherein the pharmaceutical preparation contains a polynucleotide encoding a bifunctional antibody that binds CTLA4 and PD-1 and a delivery system, wherein the polynucleotide is mRNA or saRNA. Background Art

[0002] Over the past decade, immunotherapy has become an important component of the treatment of various cancers [1]. Programmed cell death-1 (PD-1) receptors are mainly expressed on the surface of activated T lymphocytes. Binding of PD-1 to its ligands PD-L1 and PD-L2 can inhibit T cell proliferation and cytokine production. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is expressed on the surface of activated CD4+ and CD8+ T cells. Binding to its ligands B7.1 and B7.2 can inhibit T cell activity. Therefore, CTLA-4 is a negative regulator of T cell activity [2]. PD-L1 and PD-L2 are upregulated in some tumor cells. Signaling through this pathway can inhibit the immune surveillance of tumors by activated T cells. PD-1 and CTLA-4 are co-expressed in tumor-infiltrating lymphocytes, but not in normal peripheral tissue lymphocytes [3].

[0003] Monoclonal antibodies targeting the immune checkpoint PD-1 have been accepted as the standard of care for many tumor types. Many combination therapies with anti-PD-1 antibodies to improve the efficacy of PD-1 monotherapy have been extensively studied. Current clinical studies have shown that combination therapy with anti-CTLA-4 and anti-PD-1 antibodies can produce significant immune responses and improve the efficacy of some difficult-to-treat cancer types, such as renal cell carcinoma, gastric cancer, and small cell lung cancer, but its application is limited due to severe toxicity [4]. Bispecific antibodies are a new generation of therapeutic antibodies that improve the efficacy and safety of cancer patients. Currently, Kangfang Bio's PD-1 & CTLA4 dual antibody has been approved in China for the treatment of cervical cancer [2].

[0004] However, bispecific antibodies are expensive to use in therapy, have a short half-life in serum, and most bispecific antibodies face manufacturing challenges, including poor stability during long-term storage, easy aggregation over time, and the presence of various impurities[5].

[0005] mRNA has been shown to be a promising way to continuously express antibodies due to its simple production, rapid preparation process, and the fact that it does not require complex and expensive experimental facilities for protein purification[6], which can accelerate the clinical development of new bispecific antibodies. In addition, compared with mRNA technology, self-replicating mRNA (saRNA) technology is considered to have more advantages, such as low dosage, fewer side effects, long-term expression[7], and the production of more protein in the body.

[0006] In order to promote the expression of bispecific antibodies in vivo, the inventors now provide a series of polynucleotides with high expression levels to accelerate the application of nucleic acid-based immunotherapy in the field of cancer treatment.

[0007] References:

[0008] [1] Wojtukiewicz MZ, Rek MM, Karpowicz K, et al. Inhibitors of immune checkpoints–PD-1, PD-L1, CTLA-4–new opportunities for cancer patients and a new challenge for internists and general practitioners. Cancer Metastasis Rev. 2021; 40(3):949–82.

[0009] [2]Akeso Inc. Cadonilimab: Chinese prescribing information. Zhongshan: Akeso Inc; 2022

[0010] [3]Wu XH,Ji JF,Lou HM,et al.Efficacy and safety of cadonilimab,ananti-PD-1 / CTLA4 bi-specific antibody,in previously treated recurrent ormetastatic(R / M)cervical cancer:A multicenter,open-label,single-arm,phase IItrial[abstract].Society of Gynecologic Oncology(SGO)Meeting.2022

[0011] [4] Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, Schadendorf D, Dummer R, Smylie M, Rutkowski P, et al. Combined nivolumab andipilimumab or monotherapy in MABS 9untreated melanoma. N Engl J Med. 2015; 373:23–34.

[0012] [5]Vasilenko EA,Mokhonov VV,Gorshkova EN,et al.BispecificAntibodies:Formats and Areas of Application[J].Molekuliarnaia biologia,2018,52(3):380-393.

[0013] [6] Stadler CR, Bahr-Mahmud H, Celik L, et al. Elimination of largetumors in mice by mRNA-encoded bispecific antibodies (vol 23, pg 815, 2017) [J]. Nature medicine, 2017(10):23.

[0014] [7]Huang C, Duan Summary of the invention

[0015] The object of the present invention is to provide a polynucleotide encoding a bifunctional antibody that binds to CTLA4 and PD-1.

[0016] The specific technical solutions are as follows:

[0017] A pharmaceutical preparation containing a polynucleotide, the pharmaceutical preparation containing a polynucleotide encoding a bifunctional antibody that binds to CTLA4 and PD-1 and a delivery system, the bifunctional antibody containing: a first functional region targeting PD-1, and a second functional region targeting CTLA-4, wherein the first functional region is an anti-PD-1 heavy chain antibody or an antigen-binding fragment thereof, the second functional region is an anti-CTLA-4 heavy chain antibody or an antigen-binding fragment thereof, the complementary determining region of the anti-PD-1 heavy chain antibody comprises P-CDR1, P-CDR2 and P-CDR3, wherein P-CDR1 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO: 1 or 4, P-CDR2 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO: 1 or 4, NO:2 or 5, and P-CDR3 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO:3 or 6, and the complementary determining regions of the anti-CTLA-4 heavy chain antibody include C-CDR1, C-CDR2 and C-CDR3, wherein C-CDR1 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO:7, C-CDR2 comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO:8, NO:8 has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical, and C-CDR3 includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO:9.

[0018] In one or more embodiments, the anti-CTLA-4 heavy chain antibody has the sequence shown in SEQ ID NO:12, or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, and wherein each CDR sequence maintains the same sequence as SEQ ID NO:12.

[0019] In one or more embodiments, the first functional area and the second functional area are independently 1, 2 or more than 2.

[0020] In one or more embodiments, the first functional region and the second functional region are fused via a linker. Preferably, the linker is (GGSGG)p or (G4S)mGn, where m, n and P are each independently a positive integer of 1-10. Preferably, m, n and P are each independently a positive integer of 1-6 or 1-4.

[0021] In one or more embodiments, the linker has a sequence as shown in SEQ ID NO: 13-17.

[0022] In one or more embodiments, the anti-PD-1 heavy chain antibody is a chimeric antibody, preferably a humanized antibody.

[0023] In one or more embodiments, the anti-CTLA-4 heavy chain antibody is a chimeric antibody, preferably a humanized antibody.

[0024] In one or more embodiments, the bispecific antibody further contains: an Fc region; preferably, the Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4.

[0025] In one or more embodiments, the Fc region further comprises a mutation, and after the mutation, the affinity constant of the bispecific antibody to FcγRIIIa and / or C1q is reduced compared to before the mutation.

[0026] In one or more embodiments, the Fc region is an Fc region of IgG1, and according to the EU numbering system, the Fc region has one or more mutations selected from the following: L234A, L235A, G237A; or the Fc region is an Fc region of IgG4, and according to the EU numbering system, the Fc region has one or more mutations selected from the following:

[0027] S228P, E233P, F234V, L235A, D254A, L309V, R409K.

[0028] A polynucleotide encoding a bifunctional antibody that binds to CTLA4 and PD-1, wherein the bifunctional antibody contains the amino acid sequence shown at positions 21-284 of SEQ ID NO:46 or the amino acid sequence shown at positions 19-282 of SEQ ID NO:47.

[0029] In a specific embodiment, the polynucleotide can be any one of mRNA and saRNA.

[0030] The mRNA is messenger RNA, which is translated into Chinese as "messenger ribonucleic acid". It is a type of single-stranded ribonucleic acid that is transcribed using a strand of DNA as a template, carries genetic information and can guide protein synthesis.

[0031] The saRNA is a self-replicating mRNA, which is based on the traditional mRNA protein-encoding gene and adds a replicase-encoding gene from an alphavirus. After the saRNA is introduced into the cell, it can initiate RNA replication.

[0032] It should be understood that the types of nucleosides in the polynucleotides of the present invention may be conventional nucleosides, such as adenine, uridine, cytidine and guanosine; or they may be corresponding modified nucleotides or oxidized nucleotides and other common unconventional nucleosides. The modified nucleotides may be selected from one or more of 7-methyl-guanosine, 2'-oxymethyl-guanosine, pseudouridine, 1-methyl-pseudouridine, 5-methyl-cytidine, 4-acetyl-cytidine and 6-methyl-adenine.

[0033] In some embodiments, the polynucleotide can be prepared by in vitro transcription (IVT).

[0034] In a specific embodiment, the delivery system of the drug preparation comprises any one of lipids, liposomes, cationic liposomes, lipid nanoparticles (LNP), polymeric compounds, peptides, proteins, cells, nanoparticle mimics, nanotubes or conjugates, preferably liposomes, cationic liposomes, lipid nanoparticles.

[0035] Lipid nanoparticles (LNPs) are nanocarriers for delivering nucleic acid substances to cells, containing ionizable lipids, auxiliary lipid molecules, cholesterol or cholesterol derivatives, polymer-conjugated lipid molecules. For example, as described in PCT / CN2022 / 118198. The entire text of PCT / CN2022 / 118198 is incorporated herein by reference.

[0036] Cationic liposomes generally comprise (1) a cationic lipid, and (2) one or both of a helper lipid and cholesterol. Optionally, the cationic liposomes may also include polymer-conjugated lipid molecules.

[0037] In some embodiments, cationic liposomes contain DOTMA, DOPE and cholesterol, such as described in CN 109331176 B. Cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids form "lipoplexes (LPX)", which can deliver nucleic acids into cells.

[0038] In a specific embodiment, the ionizable lipid compound is selected from any one of the following,

[0039]

[0040]

[0041] In each formula, a, b, c, d, e and f are each independently an integer from 0 to 20, preferably an integer from 8 to 16, preferably, a, b, c, d, e and f are the same integer from 0 to 20, preferably the same integer from 8 to 16, more preferably the same integer from 9 to 15; preferably, the ionizable lipid compound is selected from any one of the following,

[0042]

[0043] In addition, the ionizable lipid compound can also be selected from 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl) [6-oxo-6-(undecyloxy) hexyl] amino] octanoic acid 1-octylnonyl ester (SM-102), 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino) butyryl) oxy) heptadecanedioate (Lipid L319), heptadecan-9-yl-8-((2-hydroxyethyl)(8-nonyloxy)-8-oxooctylamino) caprylate (Lipid 5), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleyl-3-dimethylamino-propane (DODMA), Lipid A9, Lipid C24, 93-O17S, DBAMEA-O61B, DLin-MC2-MPZ.

[0044] In some embodiments, the auxiliary lipid molecule is selected from any one of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidonylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DBPC), di(tetracosyl)phosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin, ceramide, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), preferably DOPE or DSPC.

[0045] In some embodiments, the polymer-conjugated lipid molecule is selected from PEG2000-DMG (PEG2000-dimyristyl glycerol, PEG2000-DSPE (PEG2000-phosphoethanolamine phospholipids), PEG2000-DPG (PEG2000-dipalmitoyl glycerol), PEG2000-DSG (PEG2000-distearoyl glycerol), PEG5000-DMG (PEG5000-dimyristyl glycerol), PEG5000-DPG (PEG5000-dipalmitoyl glycerol), PEG5000-DSG (PEG5000-distearoyl glycerol). -distearoylglycerol), PEG-cDMA (N-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine), PEG-C-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine), polyethylene glycol (PEG)-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), preferably PEG2000-DSPE or PEG2000-DMG.

[0046] In the above liposome nanoparticles, the molar ratio of the ionizable lipid, the auxiliary lipid molecule, cholesterol or cholesterol derivative, and the polymer-conjugated lipid molecule is 60-5:60-5:50-5:10-1, preferably 40-10:30-10:50-20:8-1.

[0047] The invention also provides the use of the above-mentioned pharmaceutical preparation containing polynucleotides in the preparation of drugs for preventing and / or treating cancer.

[0048] Preferably, the cancer is a PD-1 and / or CTLA-4 related cancer,

[0049] More preferably, the cancer is selected from the group consisting of melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, etc., kidney tumors, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer and ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 :Comparison of effectiveness and safety of different treatments for NSCLC.

[0051] Figure 2 : The ability of antibodies to bind to PD1 antigen.

[0052] Figure 3 :The ability of antibodies to bind to CTLA4 antigen.

[0053] Figure 4 :The ability of the antibody to bind to CTLA4-positive cells.

[0054] Figure 5 : The ability of the antibody to bind to PDL1-positive cells.

[0055] Figure 6 :1194nla bispecific antibody RGA results.

[0056] Figure 7 : RGA results of 1194nla and C43z11 bispecific antibodies.

[0057] Figure 8 :Results of bispecific antibody CTLA4 ligand binding inhibition experiment.

[0058] Fig. 9 :The results of the bispecific antibody CTLA4 ligand binding inhibition experiment. Bi-KLH: isotype control, a non-functional bispecific antibody of the same type.

[0059] Fig.10 : The secretion amount of mRNA sequence 1-21 encoding dual antibody 1194-NLA+12Z1-Fc in CHO cells.

[0060] Fig.11 : The secretion amount of mRNA sequence 22-43 encoding dual antibody Z15+12Z1-Fc in CHO cells.

[0061] Fig.12 : The secretion of IL2 after co-culture of mRNA sequences 22 and 26 encoding the dual antibody Z15+12Z1-Fc with freshly sorted CD4+T cells.

[0062] Fig.13: Expression levels of mRNA sequence 22 and sequence 43 encoding dual antibody Z15+12Z1-Fc in C57 BL / 6 mice.

[0063] Fig.14 : The secretion amount of saRNA sequences 44 and 45 encoding dual antibody Z15+12Z1-Fc in CHO cells. DETAILED DESCRIPTION

[0064] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0065] The inventors creatively fused two types of humanized nanoantibodies into new antibodies through protein recombination, and obtained a humanized bifunctional antibody that can bind to CTLA4 and PD-1. The antibody can effectively bind to cells expressing CTLA4 and / or PD-1, has good functional activity, and has no tissue cross-reaction. It has the potential to be used in the preparation of drugs for the prevention and treatment of cancers such as lung cancer, melanoma, kidney tumors, ovarian cancer, leukemia, etc.

[0066] The present invention provides a bispecific antibody, comprising: a first functional region targeting PD-1, and a second functional region targeting CTLA4. The first functional region is an anti-PD-1 heavy chain antibody or an antigen-binding fragment thereof, and the second functional region is an anti-CTLA-4 heavy chain antibody or an antigen-binding fragment thereof. The first functional region and the second functional region are each independently 1, 2 or more than 2, preferably 1 each. The connection order of the first functional region and the second functional region is not particularly limited. In some embodiments, the bifunctional antibody has the following structure from N-terminus to C-terminus: a first functional region targeting CTLA4, an optional linker, and a second functional region targeting PD-1. In some embodiments, the bifunctional antibody has the following structure from N-terminus to C-terminus: a first functional region targeting PD-1, an optional linker, and a second functional region targeting CTLA4.

[0067] The first functional region and the second functional region can be directly connected or connected through a linker, and the linker sequence is not limited and can be any amino acid linker sequence known in the art that is suitable for heavy chain antibodies or antigen-binding fragments thereof. An exemplary linker can be (GGSGG)p or (G4S)mGn, wherein m, n and P are each independently a positive integer of 1-10 (preferably 1-6, more preferably 1-4). Exemplarily, the linker is (G4S)mG4, and m is a positive integer of 1-4. Preferably, the linker has a sequence shown in SEQ ID NO: 13-17.

[0068] As used herein, when referring to the amino acid sequence of CTLA4 protein (Cytotoxic T-Lymphocyte Antigen 4), it includes the full length of CTLA4 protein, or the extracellular fragment of CTLA4 or its fusion protein. However, those skilled in the art understand that in the amino acid sequence of CTLA4 protein, mutations or variations (including but not limited to substitutions, deletions and / or additions) may be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "CTLA4 protein" shall include all such sequences, including its natural or artificial variants. And, when describing the sequence fragment of CTLA4 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.

[0069] As used herein, when referring to the amino acid sequence of PD-1 protein (NCBI GenBank: NM_005018), it includes the full length of PD-1 protein, or the extracellular fragment of PD-1 or its fusion protein. However, those skilled in the art understand that in the amino acid sequence of PD-1 protein, mutations or variations (including but not limited to substitutions, deletions and / or additions) may be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" shall include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of PD-1 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.

[0070] As used herein, the term EC50 refers to half maximal effect concentration, which refers to the concentration that can induce 50% of the maximal effect.

[0071] As used herein, the term "antibody" includes monoclonal antibodies, antibody compositions and single-chain molecules having multiple epitope specificities, and antibody fragments, especially antigen-binding fragments, e.g., Fab, F(ab')2 and Fv. As used herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0072] The "heavy chain antibody" described herein is an antibody derived from a camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), and only contains 2 heavy chains consisting of a variable region (VHH) and other constant regions, and the variable region is connected to the constant region by a hinge region structure. Each heavy chain of the camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the cartilaginous fish heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragment of the heavy chain antibody includes VHH or a single-chain heavy chain antibody. By fusion with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0073] As used herein, the terms "nanoantibody", "single domain antibody", "heavy chain variable region domain of heavy chain antibody", and "VHH" are used interchangeably to refer to VHH that specifically recognizes and binds to an antigen. VHH is the variable region of a heavy chain antibody. Typically, VHH contains three CDRs and four FRs. Preferably, in the present invention, a heavy chain antibody or VHH against PD-1 has P-CDR1 shown in SEQ ID NO: 1 or 4, P-CDR2 shown in SEQ ID NO: 2 or 5, and P-CDR3 shown in SEQ ID NO: 3 or 6; a heavy chain antibody or VHH against CTLA-4 has P-CDR1 shown in SEQ ID NO: 1 or 4, P-CDR2 shown in SEQ ID NO: 2 or 5, and P-CDR3 shown in SEQ ID NO: 3 or 6.

[0074] VHH has C-CDR1 shown in SEQ ID NO: 7, C-CDR2 shown in SEQ ID NO: 8, and C-CDR3 shown in SEQ ID NO: 9. Single-domain antibodies are the smallest functional antigen-binding fragments. Usually, antibodies that naturally lack the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0075] The term "variable" refers to the situation that some segments in the variable domain differ widely in the antibody sequence. The variable domain (or variable region) contains an antigen binding site, mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed over all amino acids spanned by the variable domain. For heavy chain antibodies or VHH, the variable region includes CDR1, CDR2, and CDR3. The more highly conserved parts in the variable domain are called framework regions (FRs). The variable domains of natural heavy chains (and light chains) each include four FR regions (FR1, FR2, FR3, and FR4), most of which adopt a β-folded conformation, connected by three HVRs that form a loop connection and in some cases form a part of the β-folded structure. The CDRs in each chain are kept together very closely by the FR region, and together with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibody.

[0076] In one or more embodiments, the heavy chain antibody of the present invention or its VHH has a FR1 as shown in SEQ ID NO: 26, 30, 34, 40 or 44, a FR2 as shown in SEQ ID NO: 27, 31, 35, 41 or 45, a FR3 as shown in SEQ ID NO: 28, 32, 36, 42 or 46, and a FR4 as shown in SEQ ID NO: 29, 33, 37, 43 or 47. The sequences of the above FRs can be used in any combination for the PD-1 heavy chain antibody or CTLA-4 heavy chain antibody of the present invention. Exemplarily, the PD-1 heavy chain antibody or VHH of the present invention has a FR1 as shown in SEQ ID NO: 26, 30, 40 or 44, a FR2 as shown in SEQ ID NO: 27, 31, 41 or 45, a FR3 as shown in SEQ ID NO: 28, 32, 42 or 46, and a FR4 as shown in SEQ ID NO: 29, 33, 43 or 47. The CTLA-4 heavy chain antibody or VHH of the present invention has FR1 as shown in SEQ ID NO: 34, as

[0077] FR2 as shown in SEQ ID NO:35, FR3 as shown in SEQ ID NO:36, and FR4 as shown in SEQ ID NO:37.

[0078] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the antibody heavy chain, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells for recognizing the immune response mediated by the Fc region of antibodies. After the Fab region of the antibody recognizes the antigen, its antibody Fc region binds to the Fc receptor on immune cells (such as killer cells) to initiate the response functions of the immune cells, such as phagocytosis and ADCC. The human IgG family includes four members, IgG1, IgG2, IgG3 and IgG4, and there are amino acid differences in the crystallizable fragment (Fc) region of their heavy chain constant regions, resulting in different affinities between them and FcγRs. In order to improve the cell killing effect, herein, the bispecific antibody or its functional regions may have a native sequence Fc or a variant Fc.

[0079] "Antibody fragments" include a portion of an intact antibody, preferably the antigen binding region and / or variable region of an intact antibody. Antibody fragments are preferably antigen binding fragments of antibodies. Examples of heavy chain antibody fragments include Fv fragments; diabodies; linear antibodies; single domain antibodies (VHH); single chain antibody molecules; scFv-Fc fragments; and any fragments that should be able to increase half-life by chemical modification or by incorporation into liposomes.

[0080] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. For heavy chain antibodies, this fragment consists of the heavy chain variable domain. "Single chain Fv" can also be abbreviated as "sFv" or "scFv", which is an antibody fragment containing the heavy chain antibody VHH domain connected into a single polypeptide chain.

[0081] In some embodiments, the antigen-binding fragment of the anti-PD-1 heavy chain antibody has a sequence as shown in SEQ ID NO: 10, 11, 38 or 39, or a sequence having at least 80% sequence identity thereto, wherein each CDR sequence remains the same as SEQ ID NO: 10, 11, 38 or 39. The antigen-binding fragment of the anti-CTLA-4 heavy chain antibody has a sequence as shown in SEQ ID NO: 12, or a sequence having at least 80% sequence identity thereto, wherein each CDR sequence remains the same as SEQ ID NO: 12.

[0082] The first functional region targeting PD-1 described herein may be a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen-binding fragment thereof comprising one, two or more anti-PD-1 single domain antibodies described herein. The first functional region targeting CTLA-4 described herein may be a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen-binding fragment thereof comprising one, two or more anti-CTLA-4 single domain antibodies described herein. The anti-PD-1 heavy chain antibody and / or the anti-CTLA-4 heavy chain antibody further comprises a heavy chain constant region, such as a constant region of a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.

[0083] The present invention also includes the antibody derivatives and analogs. "Derivatives" and "analogs" refer to polypeptides that substantially maintain the same biological function or activity as the antibodies of the present invention. The derivatives or analogs of the present invention may be (i) polypeptides having a substitution group in one or more amino acid residues, or (ii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a cmyc and / or 6His tag). According to the teachings herein, these derivatives and analogs are within the scope known to those skilled in the art. For example, a polypeptide may be connected in series with cmyc and 6His to form a polypeptide-cmyc-his structure. A linker sequence may be present between the polypeptide and cmyc and between cmyc and his, and an exemplary cmyc-his containing two linker sequences is shown in residues 260-285 of SEQ ID NO:18.

[0084] Without substantially affecting the activity of the antibody, a person skilled in the art may change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For another example, adding one or more amino acids to the C-terminus and / or N-terminus will generally not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.

[0085] Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention.

[0086] In some embodiments, the sequence of the variant of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with its source sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters is used. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0087] In one or more embodiments, the bispecific antibody has an anti-PD-1 single domain antibody, a linker, and an anti-CTLA-4 single domain antibody from the N-terminus to the C-terminus, wherein the complementary determining regions CDR1-CDR3 of the anti-PD-1 single domain antibody are shown as SEQ ID NOs: 4, 5, and 6, respectively, the complementary determining regions CDR1-CDR3 of the anti-CTLA-4 single domain antibody are shown as SEQ ID NOs: 7, 8, and 9, respectively, and the linker sequence is shown in any one of SEQ ID NOs: 13-17. In one or more embodiments, the bispecific antibody has an anti-PD-1 single domain antibody, a linker, and an anti-CTLA-4 single domain antibody from the N-terminus to the C-terminus, wherein the complementary determining regions CDR1-CDR3 of the anti-PD-1 single domain antibody are shown as SEQ ID NOs: 1, 2, and 3, respectively, the complementary determining regions CDR1-CDR3 of the anti-CTLA-4 single domain antibody are shown as SEQ ID NOs: 7, 8, and 9, respectively, and the linker sequence is shown in SEQ ID NOs: 14 or 15. In one or more embodiments, the bispecific antibody has an anti-CTLA-4 single domain antibody, a linker and an anti-PD-1 single domain antibody from N-terminus to C-terminus, wherein the complementary determining regions CDR1-CDR3 of the anti-CTLA-4 single domain antibody are shown as SEQ ID NOs: 7, 8, and 9, respectively, and the complementary determining regions CDR1-CDR3 of the anti-PD-1 single domain antibody are shown as SEQ ID NOs: 4, 5, and 6, respectively, and the linker sequence is shown as SEQ ID NO: 15.

[0088] In one or more embodiments, the bispecific antibody has an anti-PD-1 single domain antibody, a linker, and an anti-CTLA-4 single domain antibody from the N-terminus to the C-terminus, wherein the sequence of the anti-PD-1 single domain antibody is selected from one of SEQ ID NOs: 10, 11, 38, or 39, the sequence of the anti-CTLA-4 single domain antibody is as shown in SEQ ID NO: 12, and the linker sequence is as shown in any one of SEQ ID NOs: 13-17, preferably as shown in SEQ ID NO: 14. In one or more embodiments, the bispecific antibody has an anti-CTLA-4 single domain antibody, a linker, and an anti-PD-1 single domain antibody from the N-terminus to the C-terminus, wherein the sequence of the anti-CTLA-4 single domain antibody is as shown in SEQ ID NO: 12, the sequence of the anti-PD-1 single domain antibody is selected from one of SEQ ID NOs: 10, 11, 38, or 39, and the linker sequence is as shown in

[0089] Any one of SEQ ID NOs: 13-17, preferably SEQ ID NO: 15.

[0090] In one or more embodiments, the bispecific antibody has, from N-terminus to C-terminus: an anti-PD-1 single domain antibody as shown in SEQ ID NO: 11, a linker as shown in SEQ ID NO: 13 or 14 or 15, and an anti-CTLA-4 single domain antibody as shown in SEQ ID NO: 12, or an anti-PD-1 single domain antibody as shown in SEQ ID NO: 38, a linker as shown in SEQ ID NO: 14, and an anti-CTLA-4 single domain antibody as shown in SEQ ID NO: 12, or an anti-PD-1 single domain antibody as shown in SEQ ID NO: 39, a linker as shown in SEQ ID NO: 14, and an anti-CTLA-4 single domain antibody as shown in SEQ ID NO: 12.

[0091] CTLA-4 single domain antibody, or the anti-PD-1 single domain antibody shown in SEQ ID NO:10, the linker shown in SEQ ID NO:15, and the anti-CTLA-4 single domain antibody shown in SEQ ID NO:12, or the anti-PD-1 single domain antibody shown in SEQ ID NO:10, the linker shown in SEQ ID NO:14, and the anti-CTLA-4 single domain antibody shown in SEQ ID NO:12, or the anti-CTLA-4 single domain antibody shown in SEQ ID NO:12, the linker shown in SEQ ID NO:15, and the anti-PD-1 single domain antibody shown in SEQ ID NO:11.

[0092] In one or more embodiments, the bispecific antibody has the sequence shown in SEQ ID NO: 48-55. The bispecific antibody with cmyc-his tag is shown in SEQ ID NO: 18-25, respectively.

[0093] In one or more embodiments, the bispecific antibody further contains an Fc region; preferably, the Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4; and the IgG1, IgG2, IgG3 or IgG4 is of human origin.

[0094] Since the Fc region can cause significant ADCC and CDC effects, it may cause immune cell damage and have negative pharmacological effects. Therefore, Fc is usually modified (also referred to as variant Fc herein), and the affinity constant of the bispecific antibody to FcγRIIIa and / or C1q is reduced compared to before the mutation through site mutation, thereby improving the efficacy of the antibody drug. The currently disclosed mutation sites, according to the EU numbering system, include: Fc region mutation sites in IgG1 include L234A, L235A, L235E, L235G, G236A, G237A, N297A, G318A, L320A, L322A; Fc region mutation sites in IgG3 include V234A, G237A, P238S, H28A, V309L , A330S, P331S; Fc region mutation sites in IgG3 include Leu281, Leu282, Gly283, Gly284, Asn344, Pro378; Fc region mutation sites in IgG4 include S228P, E233P, F234V, L235A, F243L, D254A, R292P, Y300L, L309V, R409K. The variant Fc herein includes but is not limited to the Fc of each IgG having the above mutation sites.

[0095] In one or more embodiments, the Fc region is an Fc region of IgG1, and according to the EU numbering system, the Fc region has one or more of the following mutations: L234A, L235A, G237A. In one or more embodiments, the Fc region is an Fc region of IgG4, and according to the EU numbering system, the Fc region has one or more of the following mutations: S228P, E233P, F234V, L235A, D254A, L309V, R409K.

[0096] As used herein, the term "lipid" refers to an amphiphilic molecule comprising a hydrophilic portion (e.g., a polar head group) and a lipophilic or hydrophobic portion. The lipophilic or hydrophobic portion may comprise at least one branched or straight chain saturated or unsaturated fatty acid moiety or a derivative or analog thereof (e.g., a fluorocarbon). The fatty acid moiety is substantially composed of a hydrocarbon portion / chain (particularly an acyl chain). Preferably, the fatty acid moiety or a derivative or analog thereof has a length of 10 to 30, more preferably 12 to 25, even more preferably 14 to 22 carbon atoms. In the case where a lipid comprises more than one (e.g., two or three) fatty acid moieties or derivatives or analogs thereof, these fatty acid moieties or derivatives or analogs thereof may be identical or different.

[0097] The term "lipid" includes cationic lipids and non-cationic lipids (i.e., neutral lipids or anionic lipids). Lipids can include phospholipids or derivatives thereof, glycerolipids or derivatives thereof, sphingolipids (e.g., sphingomyelin) or derivatives thereof, or sterol lipids (e.g., cholesterol) or derivatives thereof. Glycerolipids are composed of glycerol that is monosubstituted, disubstituted or trisubstituted by fatty acid moieties. The phospholipids whose hydrophilic part contains phosphate groups can be glycerophospholipids. Lipids can also be functionalized / modified, for example, functionalized / modified by (oligo) peptides, polymers (e.g., PEG) or other functional groups. In aqueous media, lipids can be further organized supramolecularly, for example, in the form of lipid-based particles or lyotropic phases (e.g., liposomes, lamellar phases, hexagonal phases and reverse hexagonal phases, cubic phases, micelles and reverse micelles composed of monolayers). The stabilizing effect according to the present invention is applicable to all types of supramolecular lipid organizations. Preferably, the lipid used according to the present invention is pharmaceutically acceptable, for example, suitable as an excipient, as a component of a drug delivery formulation and / or for transfecting nucleic acids into cells.

[0098] If the disclosure refers to an electric charge (e.g., positive, negative, or neutral charge) or a cationic, negative, or neutral compound, this generally means that the charge exists at a selected pH (e.g., physiological pH). For example, the term "cationic lipid" refers to a lipid having a positive net charge at a selected pH (e.g., physiological pH). The term "neutral lipid" refers to a lipid that does not have a positive or negative net charge at a selected pH (e.g., physiological pH), which may exist as a non-charged molecule or a neutral zwitterionic (or zwitterionic) molecule.

[0099] Cationic lipids preferably comprise cationic head groups. The polar head groups of cationic lipids preferably comprise amine derivatives, such as primary amines, secondary amines and / or tertiary amines, quaternary ammonium, multiple amine combinations, amidine (amidinium) salts or guanidine and / or imidazole groups and pyridine (pyridinium), piperazine and amino acid head groups such as lysine, arginine, ornithine and / or tryptophan. More preferably, the polar head groups of cationic lipids comprise amine derivatives. Most preferably, the polar head groups of cationic lipids comprise quaternary ammonium. The head groups of cationic lipids can comprise single cationic charge or multiple cationic charges.

[0100] The anionic lipid preferably comprises an anionic head group, such as a phosphate group. The head group of the anionic lipid may comprise a single anionic charge or multiple anionic charges.

[0101] As used herein, the term "nucleic acid lipid complex" or "lipoplex" refers to a complex of a lipid and a nucleic acid (e.g., mRNA or saRNA). Lipoplexes spontaneously form when cationic lipids (e.g., in the form of cationic liposomes, which typically also contain a neutral helper lipid), cationic polymers, and other substances with a positive charge are mixed with nucleic acids and have been shown to deliver nucleic acids into cells.

[0102] The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art (for example, reference to "Molecular Cloning Experiment Guide" by J. Sambrook, Huang Peitang, etc., the third edition, Science Press) or according to the product instructions. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased and obtained on the market.

[0103] In the bispecific Nanobodies provided in the Examples herein,

[0104] The amino acid sequence of the epitope that binds to PD-1 is:

[0105] C43-z11 sequence:

[0106] ELQLVESGGGLVQPGGSLRLSCAASGHSFSIYDMGWFRQAADKERESVAVINF GRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGIDRRQYGLGIPP LADHWGQGSQVTVSS(SEQ ID NO:10)

[0107] C43-z14 sequence:

[0108] QLQLVESGGGLVQPGGSLRLSCSASGHSFSIYDMGWFRQAPGKERESVAVINF GRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGIDRRQYGLGIPP LADHWGQGSQVTVSS(SEQ ID NO:38)

[0109] C43-z15 sequence:

[0110] QLQLVESGGGLVQPGGSLRLSCSASGHSFSIYDMGWFRQAPDKERESVAVINF GRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGIDRRQYGLGIPP LADHWGQGTQVTVSS(SEQ ID NO:39)

[0111] 1194nla sequence:

[0112] EVQLVESGGGLVQPGGSLRLSCAASGRPFSIYDMGWFRQAPDKERESVAVINL ARGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYSCGVDRRQYGLGIP PLADHWGQGTQVTVSS(SEQ ID NO:11)

[0113] The amino acid sequence of the epitope that binds to CTLA-4 is:

[0114] 12-z1 sequence:

[0115] EVQLVESGGGLVQPGGSLRLSCAASGFSSDYYDIGWFRQAPGKEREMVSCIRSSGGSTKYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCGLAPISPVHAVCNQHYFGYWGQGTRVTVSS(SEQ ID NO:12)

[0116] The CDR and FR regions of the above three VHHs are shown in the table below.

[0117] Table 1. FR and CDR regions of three VHHs

[0118]

[0119]

[0120] The amino acid sequences of the bispecific nanobodies 1194-NLA-L1-12-Z1, 1194-NLA-L2-12-Z1, 1194-NLA-L3-12-Z1, C43-Z14-L2-12-Z1 (abbreviated as C43-Z14-12-Z1), C43-Z15-L2-12-Z1 (abbreviated as C43-Z15-12-Z1), 12-Z1-L3-1194, C43-z11-L3-12-Z1, and C43-z11-L2-12-Z1 used in the examples are shown in SEQ ID NOs: 48-55, respectively, and the corresponding amino acid sequences of the bispecific nanobodies with cmyc-his tags are shown in Table 2.

[0121] Table 2. Bispecific antibody names and sequences (letters in the names are not case sensitive)

[0122]

[0123]

[0124] Example 1. Preparation

[0125] The bispecific antibody was commissioned to Bio-Innovative Biotechnology for preparation, expressed in HEK293, purified by Ni column, and had a his tag. The storage solution was PBS. The preparation data is shown in Table 3.

[0126] Table 3. Antibody preparation data (provided by Bio-Bio)

[0127]

[0128]

[0129] Example 2, affinity test

[0130] (1) SPR detection of binding kinetics

[0131] Protein-level affinity detection: Surface plasmon resonance technology (SPR) was used to measure the binding kinetics and affinity of bispecific nanobodies or PD1 nanobodies (1194nla, C43z11) or CTLA4 nanobodies (12-z1) to human PD-1.IgG and CTLA4.IgG antigens.

[0132] Purified PD-1.IgG or CTLA4.IgG antigens flowed through a CM5 chip pre-coated with IgG (GE, cat#BR-1008-39), and the antigens were captured by the IgG on the chip. Then, different concentrations of bispecific nanoantibodies or PD1 nanoantibodies and CTLA4 nanoantibodies were used as mobile phases to detect binding kinetics and affinity. Biacore EvaluationSoftware 2.0 (GE) was used to analyze the association rate (kon), dissociation rate (koff) and equilibrium constant (KD). At the same time, the marketed PD-1 antibody drugs Keytruda and Opdivo were selected as controls.

[0133] As shown in the table below, the ability of the bispecific antibody to bind to PD1 is comparable to that of the PD1 nanobody, and the ability of the bispecific antibody to bind to CTLA4 is comparable to that of the CTLA4 nanobody.

[0134] Table 4. Antibody binding kinetics and affinity

[0135]

[0136]

[0137] (2) ELISA detection and antigen binding ability

[0138] ELISA plates were coated with 1 μg / mL PD.fc or CTLA4.fc protein, and 50 μL of the antibody to be tested in different gradient dilutions was added to the ELISA plates for incubation. After washing, HRP Streptavidin (Biolegend) was added and incubated at room temperature for 1 hour. After washing, TMB colorimetric solution was added, and then the reaction was terminated with 2M HCl, and the OD450 value was read using an enzyme reader.

[0139] ELISA test and antigen binding ability show that the ability of different bispecific antibodies to bind to PD1 antigen or CTLA4 antigen is relatively small ( Figure 2 , Figure 3 ).

[0140] (3) FACS detection of cell binding ability

[0141] HEK293T cells expressing CTLA4 or CHO / K1 cells expressing PD1 were plated in 96-well plates, with 3×10 5 Cells were incubated with gradiently diluted nanoantibodies, incubated on ice for half an hour, and then incubated with secondary antibodies for detection, and then detected using a CytoFLEX flow cytometer. The EC50 of the antibody was calculated by fitting the curve. Isotype is the isotype control (negative control).

[0142] The binding ability of different nanobodies to PD1-positive cells or CTLA4-positive cells was detected by flow cytometry. Figure 4 As shown: The difference in EC50 between the dual antibodies on 293T-CTLA4 cells: C43-Z11-(G4S)-3G4-12-Z1 EC50 is slightly better than other dual antibodies.

[0143] On CHO / K1-PD1 cells, the difference in EC50 between the two antibodies: C43-Z11-(G4S)-3G4-12-Z1 EC50 is slightly better than other two antibodies. Figure 5 shown.

[0144] Based on the above data, at the ELISA level, the EC50 values ​​of bispecific antibodies connected by different linkers for PD1 and CTLA4 antigens were not much different; on cell lines overexpressing CTLA4 or PD1, different bispecific antibodies showed certain differences in EC50 values, among which C43z11-L3-12-Z1 had better binding ability for stable cell lines than other bispecific antibodies (Table 5).

[0145] Table 5. Summary of antibody ELISA and FACS binding data

[0146]

[0147]

[0148] Example 3: Ligand Binding Inhibition

[0149] (1) PD1 / PDL1 reporter gene assay

[0150] First, 10,000 GS / C2-PDL1 (GenScript) target cells were resuspended in 20ul antibiotic-free medium and plated in a 384-well plate for 16-20h. Nanoantibodies were gradiently diluted with RMPI1640+10% FBS buffer, and 40,000 GS-J2 / PD1 (GenScript) effector cells were dissolved in 20ul antibiotic-free medium; the old medium in the 384-well plate was removed, and 20μL of antibody and 20μL of GS-J2 / PD1 effector cells were added to each well and co-cultured for 6h. Finally, 40μL One-Glo substrate was added to each well and reacted for 10 minutes before detection by microplate reader.

[0151] Reporter Gene Assay results Figure 6 or Figure 7As shown, the IC50 order of the inhibitory effect of 1194nla bispecific antibodies with different linkers on PD1 / PDL1 binding is: 1194NLA-L2>1194NLA-L1>1194NLA-L3, all of which have little difference from the IC50 values ​​of 1194nla antibody; under the same linker condition, the inhibitory effect on PD1 / PDL1 binding is greater for the bispecific antibody with 1194nla in front (1194nla-L3) than for the bispecific antibody with 12-z1 in front (12-z1-L3).

[0152] (2) CTLA4 ligand binding inhibition assay

[0153] The GS-J1 / CD28 cell density was adjusted to 2×10 in Assay buffer (RMPI1640+10% FBS buffer). 6 / mL; GS-C1 / CD80 cells (GenScript) target cells were adjusted to a density of 1×10 in antibiotic-free medium. 6 / mL. The antibody to be tested was gradiently diluted with Assay buffer, and the CTLA-4 protein was diluted to 100μg / mL with Assay buffer. 20μL GS-J1 / CD28, 20μL GS-C1 / CD80 cells, 20μL CTLA-4 protein, 20μL antibody to be tested or Assay buffer were added to each well of the 384-well plate in sequence, and the 384-well plate was placed in a cell culture incubator for 24 hours. After 24 hours, the cell supernatant was centrifuged and the IL-2 concentration was quantified using the IL-2 quantitative detection kit (Cisbio). The test results were curve fitted using Graphprism software to calculate the EC50 of the antibody.

[0154] CTLA4 ligand binding inhibition experiment reporter gene assay results are as follows Figure 8 and Fig. 9 As shown ( Figure 8 and Fig. 9 are experimental results of different batches), the IC50 order of the inhibitory effect of 1194nla bispecific antibodies with different linkers on CTLA4 / CD80 binding is: 1194NLA-L2>1194NLA-L3>1194NLA-L1; under the same linker condition, for the inhibitory effect on CTLA4 / CD80 binding, the effect of the bispecific antibody with 1194nla in front (1194nla-L3) is less than that of the bispecific antibody with 12-z1 in front (12-z1-L3).

[0155] Based on the results of the PD1 / PDL1 reporter gene experiment and the CTLA4 ligand competition experiment, 1194nla-L2-12-z1 and C43z11-L2-12-z1 had better effects.

[0156] Example 4. Tissue Cross-Reactivity (TCR)

[0157] 34 kinds of tissues were selected for frozen sections, dried at room temperature, and then soaked in PBST for 5-10 minutes. Use reagent A and reagent B of the endogenous biotin blocking kit (Sangong, E674001) for blocking, add imported sheep serum working solution for incubation for 10-15 minutes after washing; add primary antibody for incubation at room temperature for 10-15 minutes, and then add horseradish peroxidase-labeled streptavidin (Abcam, ab7403) for incubation for 15 minutes after washing. Use DAB for color development and hematoxylin for counterstaining, dehydrate with alcohol, seal with neutral plastic, and air dry naturally for microscopic examination. The positive control is Anti-PD-1 antibody (abcam), and the negative control is IgG4 isotype control. All antibody concentrations in this experiment are 10ug / ml.

[0158] The experimental results are shown in Table 6. The TCR results of the four bispecific antibodies 1194nla-L2-12-z1, C43-z14-12-z1, C43-z15-12-z1 and C43z11-L3-12-z1 are relatively clean and have good specificity.

[0159] Table 6. Bispecific antibody tissue cross-reactivity results

[0160]

[0161]

[0162] Example 5. Synthesis of polynucleotides encoding bifunctional antibodies

[0163] The bispecific nanoantibodies 1194-NLA-L2-12-Z1 and C43-Z15-L2-12-Z1 were selected, and after adding the Fc region, they were labeled as 1194-NLA+12Z1-Fc, with the amino acid sequence shown in SEQ ID NO: 101; Z15+12Z1-Fc, with the amino acid sequence shown in SEQ ID NO: 102.

[0164] Baidu RNA sequence optimization online tool PaddieHelix (https: / / rna.baidu.com / ) was used to select online parameters Beam Size: 100-400; Double-stranded (stacking) Length Limit: S1≤33; S2≤15; CAIλ: 1-4; Less Structures at 5'-end to generate a polynucleotide sequence encoding the PD1&CTLA4 bifunctional antibody, and then the endonuclease recognition sites NcoI, AflII, and BspQI corresponding to cloning and linearization were removed by synonymous codon replacement to obtain the sequences of mRNA and saRNA as shown in Table 7.

[0165] Among them, mRNA sequence numbers 1-21 encode 1194-NLA+12Z1-Fc, mRNA sequence numbers 22-43 encode Z15+12Z1-Fc, and saRNA sequence numbers 44-45 encode Z15+12Z1-Fc.

[0166] The above polynucleotides are prepared using an in vitro transcription (IVT) process, and the IVT system comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. NTPs may be selected from, but are not limited to, natural and non-natural (modified) NTPs described herein. Polymerases may be selected from, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, and mutant polymerases, such as, but not limited to, polymerases capable of incorporating modified nucleic acids. Any number of RNA polymerases or variants may be used to synthesize the polynucleotides of the present invention.

[0167] Table 7. Polynucleotide sequences encoding bifunctional antibodies

[0168]

[0169]

[0170] Example 6. Verification of expression of mRNA encoding bifunctional antibody

[0171] 6.1 mRNA electroporation into CHO cells

[0172] (1) Preparation before the experiment: CHO culture medium was prepared by mixing DMEM culture medium and RPMI 1640 culture medium in a ratio of 1:1, and adding 10% fetal bovine serum, 1% glutamine, 1% HT, and 1% P / S.

[0173] (2) Cell harvesting: CHO-K1 cells in the logarithmic growth phase were digested and terminated with trypsin. The digested cells were added to a 50 mL centrifuge tube and centrifuged at 400 g for 5 min. The supernatant was discarded and an appropriate amount of CHO culture medium was added to resuspend the cell pellet. The cells were mixed and counted.

[0174] (3) Washing: According to the counting results, the cells were divided into 1.5 mL EP tubes at 1.5E6 cells / tube, centrifuged at 400 g for 5 min, and the supernatant was discarded. 1 mL PBS was added to each tube for washing again, and centrifuged at 400 g for 5 min. The supernatant was discarded until only the cell pellet remained.

[0175] (4) Preparation of electroporation mixture: Add 100 μL of Lonza 2B electroporation solution (solution I:solution II=82:18) and 20 μg of the corresponding mRNA to each EP tube, mix well on a vortex shaker and set aside.

[0176] (5) Electroporation: Add the electroporation mixture in step (4) to the cell pellet collected in step (3) above, mix well and add to a Lonza 2B electroporation cup. Place the cup in a Lonza 2B electroporator and select the CHO-K1 (ATCC) high viability (H-014) program for electroporation.

[0177] (6) Cultivation: After electroporation, use the micropipette in the kit to aspirate the preheated culture medium and the electroporation suspension and mix them evenly. Quickly transfer the mixture to a 6-well plate containing 3 mL of preheated culture medium. Mix well and place the plate in a 37°C, 5% CO2 incubator for culture.

[0178] (7) Supernatant collection: After electroporation, culture for 24 h. Collect 500 uL of supernatant from each group and store at -20°C for later use.

[0179] 6.2 ELISA detection of mRNA

[0180] (1) Antigen coating: 10 mL coating solution + 100 uL human PD-1, His Tag (100 ug / mL), add 100 μL / well to the ELISA plate using a dispenser and incubate at 4°C overnight.

[0181] (2) Take out the supernatant prepared in 2.1 from -20°C in advance, thaw it, and centrifuge it at 400g for 5 min.

[0182] (3) Wash the plate three times with PBST, 300 μL / well, shaking for 30 seconds each time. After washing, pat dry with absorbent paper.

[0183] (4) Blocking: Add 300 μL of blocking solution (PBST + 3% BSA) to each well and incubate in a 37°C biochemical incubator for 2 hours.

[0184] (5) Wash the plate three times with PBST, 300 μL / well, shaking for 30 seconds each time. After washing, pat dry with absorbent paper.

[0185] (6) Dilution of standards and samples: The standards and samples were diluted with diluent (PBST + 3% BSA). The Z15-12Z1-Fc standard was diluted 2-fold from below 12.5 ng / mL into 7 gradients and 0 ng / mL. The 1194NLA-Z12-Fc standard was diluted 2-fold from below 3.125 ng / mL into 7 gradients and 0 ng / mL. The samples were diluted 50X and 500X.

[0186] (7) Add the diluted standards and samples to the corresponding wells, 100 μL / well, set up 2 replicate wells, and incubate at 37°C for 1.5 h.

[0187] (8) Wash the plate three times with PBST, 300 μL / well, shaking for 30 seconds each time. After washing, pat dry with absorbent paper.

[0188] (9) Add detection antibody: Use diluent to dilute the detection antibody (MonoRab TM Rabbit Anti-Camelid VHHAntibody (83E7 Clone) [HRP], mAb) was diluted 1:10000, mixed and added to the corresponding well plate, 100 μL / well, and incubated in a 37°C biochemical incubator for 1 h.

[0189] (10) Wash the plate three times with PBST (300 μL / well), shaking for 30 seconds each time. After washing, pat dry with absorbent paper.

[0190] (11) Add TMB colorimetric solution (100 μL / well) and develop the color for 5-10 min at room temperature in the dark.

[0191] (12) Add 50 μL / well of stop solution to terminate the reaction. After the reaction is terminated, read the result on the analyzer within 15 minutes.

[0192] 6.3 mRNA expression results in CHO cells

[0193] The designed 41 mRNAs encoding bifunctional antibodies were expressed in CHO cells. Figure 1 , Figure 2 In the mRNA encoding 1194-NLA+12Z1-Fc ( Fig.10 ), the expression levels of sequence numbers 19 and 21 were the highest, reaching 800ng / mL; the expression levels of the remaining sequence numbers 1, 3, 4, 5, 6, 8, 9, 12, 13, 15, 16, 18, and 20 all exceeded 100ng / mL.

[0194] In the mRNA encoding Z15+12Z1-Fc ( Fig.11), the expression levels of sequence numbers 42 and 43 can reach 800 ng / mL, and the expression levels of the remaining sequence numbers 22, 27, 28, 33, 35, 36, 37, 38, and 39 all exceed 100 ng / mL.

[0195] Comparing the in vitro bifunctional antibody secretion amounts of sequence numbers 22, 42, and 43, relative to sequence number 22, adding a polypeptide mRNA sequence (i.e., the regulatory motif CAACCGCGGUUCGCGGCCGCU (SEQ ID NO.103)) to the mRNA sequence encoding the bifunctional antibody can significantly increase the secretion amount of the bifunctional antibody.

[0196] Example 7. In vitro mixed lymphocyte reaction of mRNA encoding bifunctional antibodies

[0197] 7.1 Preparation of mDC cells

[0198] Donor mDCs were prepared according to CN107574149B.

[0199] 7.2 CD4+ T cell sorting

[0200] CD4+ T cells were isolated from peripheral blood mononuclear cells of another donor using a human CD4+ T cell isolation kit (Milteniy Biotech, Bergisch Gladbach, Germany).

[0201] 7.3 Mixed Lymphocyte Reaction

[0202] 1) Activate 2*10 5 The DC / well culture medium was discarded, washed once with DPBS, and then 300 μL of serum-free Opti-MEM culture medium was added to each well.

[0203] 2) LNP encapsulated mRNA incubation: prepare 200 μL of Opti-MEM and LNP encapsulated mRNA mixed solution, and the final mRNA content of LNP added to each well is 0-2000 ng. The LNP encapsulation steps are as follows: Referring to PCT / CN2022 / 118198, the ionizable lipid compound (taking the following compound Target-1 as an example) is dissolved in ethanol with DOPE, cholesterol and DSPE-PEG2000 (all purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) at a molar ratio of 35:16:46.5:2.5 to prepare an ethanol lipid solution, and dilute the mRNA encoding the bifunctional antibody in 20 mM citrate buffer (pH = 6.1) to obtain an mRNA aqueous solution. By using a microfluidic device (Maianna (Shanghai) Instrument Technology Co., Ltd., model: INano TML) The ethanol lipid solution and the mRNA aqueous solution were mixed in a volume ratio of 1:3, and liposomes were prepared with a weight ratio of total lipid to mRNA of about 10:1. The solution was replaced by ultrafiltration, the ethanol was removed and the volume was fixed with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain an LNP preparation encapsulating mRNA with ionizable lipid / DOPE / cholesterol / DSPE-PEG2000 (35 / 16 / 46.5 / 2.5 mol%). The size and polydispersity index PDI of the lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in a 173° backscattering detection mode. The results showed that the parameters were as follows: particle size 90.5, PDI 0.133, encapsulation efficiency 95.21%, cell viability 95.0%, positive rate 90.2%, and fluorescence intensity 1.00.

[0204]

[0205] 3) Add 200 μL of liposome mixed solution dropwise along the top of each well. Shake the well plate crosswise. Place in a 37°C, 5% CO2 incubator for culture.

[0206] 4) After 4 hours of culture, add 8*10 5 CD4+T cells were co-cultured. On day 5, the culture supernatant was collected and the secretion of IL2 was detected using Human IL2 ELISA Kit (Sino-Qiao) according to the kit protocol.

[0207] 7.4 Experimental Results

[0208] The results of in vitro mixed lymphocyte reaction showed that ( Fig.12 ), DC cells transfected with mRNA, with the increase of mRNA dose, the effect of stimulating T cells to secrete cytokine IL-2 is gradually enhanced, and the stimulating effect of sequence number 22 is better than that of sequence number 26.

[0209] Example 8. Verification of expression of mRNA encoding bifunctional antibody in plasma level of C57BL / 6 mice

[0210] 8.1 Expression of bifunctional antibodies and mRNA encoding corresponding antibodies in C57BL / 6 mice

[0211] Thirty-six C57BL / 6 mice were divided into 9 groups and injected into the tail vein. The grouping is shown in Table 2. LNP encapsulation was as described in Example 7.

[0212] Table 8 Experimental groups

[0213]

[0214] Blood was collected from the eye sockets of all mice before administration. At 6h, 24h, D2, D3, D4, D5, D6, D7, D9, D11, D13, D15, D17, D20, D22, D24, and D26 after administration, with a blood volume of 0.1mL. Serum was separated from the blood samples and the blood samples were post-processed according to the standard. The antibody content in the serum samples was detected by ELISA, and the detection method was the same as 2.2.

[0215] 4.2 Experimental Results

[0216] From the expression level ( Fig.13 ), Z15-12Z1-0-2 5mg / kg=Z15-12Z1-0 5mg / kg>LNP Z15-12Z1-0-2mRNA 1mg / kg>LNP Z15-12Z1-0mRNA 1mg / kg>Z15-12Z1-0-2 0.5mg / kg=Z15-12Z1-0 0.5mg / kg>LNP Z15-12Z1-0-2mRNA 0.2mg / kg>LNP Z15-12Z1-0mRNA 0.2mg / kg. The results showed that the expression level of the antibody was proportional to the amount of nucleic acid, and the expression level of sequence number 43 was better than that of sequence number 22 as a whole, among which LNP Z15-12-Z1-0-2mRNA 1mg / kg≈3 times that of LNP Z15-12-Z1-0mRNA 1mg / kg.

[0217] From the perspective of sustainability ( Fig.13 ), the protein expression persistence of LNP encapsulated Z15+12Z1-FC_0 and Z15+12Z1-FC_0-2 mRNA was greater than that of Z15-12Z1-Fc and Z15-12Z1-Fc-0-2 proteins, indicating that LNP encapsulated mRNA can reduce the frequency of administration.

[0218] Example 9. Expression verification of saRNA encoding bifunctional antibodies

[0219] 9.1 Expression verification of saRNA encoding bifunctional antibodies in CHO cells

[0220] Referring to steps 6.1 and 6.2 in Example 6, the synthesized saRNA sequence was verified for cell expression.

[0221] 9.2 Experimental Results

[0222] From the expression results of CHO cells ( Fig.14 ), the dual antibody secretion of sequence number 44 can last for about 1 week, and the expression level of sequence number 45 is higher and lasts longer, and the dual antibody secretion can be maintained for about 3 weeks.

Claims

1. A pharmaceutical preparation containing a polynucleotide, characterized in that: The pharmaceutical preparation contains a polynucleotide encoding a bifunctional antibody that binds to CTLA4 and PD-1 and a delivery system, wherein the bifunctional antibody contains: a first functional region targeting PD-1, and a second functional region targeting CTLA-4, wherein the first functional region is an anti-PD-1 heavy chain antibody or an antigen-binding fragment thereof, and the second functional region is an anti-CTLA-4 heavy chain antibody or an antigen-binding fragment thereof, and the complementary determining regions of the anti-PD-1 heavy chain antibody include P-CDR1, P-CDR2 and P-CDR3, wherein P-CDR1 includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO: 1 or 4, P-CDR2 includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO: 2 or 5, and P-CDR3 includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO: 3 or 4. NO:3 or 6 has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical, and the complementary determining regions of the anti-CTLA-4 heavy chain antibody include C-CDR1, C-CDR2 and C-CDR3, wherein C-CDR1 includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO:7, C-CDR2 includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO:8, and C-CDR3 includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO:

9. The sequence shown in NO:9 has a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity.

2. The pharmaceutical preparation according to claim 1, characterized in that The anti-PD-1 heavy chain antibody has a sequence as shown in SEQ ID NO: 10, 11, 38 or 39, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, and wherein each CDR sequence maintains the same sequence as SEQ ID NO: 10, 11, 38 or 39, and / or The anti-CTLA-4 heavy chain antibody has the sequence shown in SEQ ID NO:12, or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, and wherein each CDR sequence maintains the same sequence as SEQ ID NO:

12.

3. The pharmaceutical preparation according to any one of claims 1 to 2, characterized in that The first functional region and the second functional region are fused via a linker, Preferably, the linker is (GGSGG)p or (G4S)mGn, where m, n and P are each independently a positive integer of 1-10. More preferably, the linker has a sequence shown in any one of SEQ ID NOs: 13-17.

4. The pharmaceutical preparation according to any one of claims 1 to 3, characterized in that The bifunctional antibody further contains: an Fc region; preferably, the Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4.

5. The pharmaceutical preparation according to claim 4, characterized in that The Fc region further comprises mutations that reduce the affinity constant of the bispecific antibody to FcγRIIIa and / or C1q.

6. The pharmaceutical preparation according to any one of claims 4 to 5, characterized in that The Fc region is an Fc region of IgG1, and according to the EU numbering system, the Fc region has one or more mutations selected from the following: L234A, L235A, G237A; or the Fc region is an Fc region of IgG4, and according to the EU numbering system, the Fc region has one or more mutations selected from the following: S228P, E233P, F234V, L235A, D254A, L309V, R409K.

7. The pharmaceutical preparation according to any one of claims 1 to 6, characterized in that The polynucleotide is any one of mRNA and saRNA.

8. The pharmaceutical preparation according to any one of claims 1 to 7, characterized in that The delivery system of the drug preparation comprises any one of lipids, liposomes, cationic liposomes, lipid nanoparticles (LNP), polymeric compounds, peptides, proteins, cells, nanoparticle mimics, nanotubes or conjugates, preferably liposomes, cationic liposomes, lipid nanoparticles.

9. The pharmaceutical preparation according to claim 8, characterized in that The lipid nanoparticles contain ionizable lipids, helper lipid molecules, cholesterol or cholesterol derivatives, and / or polymer-conjugated lipid molecules.

10. The pharmaceutical preparation according to claim 9, characterized in that The ionizable lipid is selected from any one of the following, In each formula, a, b, c, d, e and f are each independently an integer from 0 to 20, preferably an integer from 8 to 16, preferably, a, b, c, d, e and f are the same integer from 0 to 20, preferably the same integer from 8 to 16, more preferably the same integer from 9 to 15; Or DOTMA, ALC-0315, SM-102, DLin-MC3-DMA, DLin-KC2-DMA, Lipid L319, Lipid 5, DOTAP, DODMA, Lipid A9, Lipid C24, 93-O17S, DBAMEA-O61B, DLin-MC2-MPZ.

11. The pharmaceutical preparation according to claim 10, characterized in that The ionizable lipid compound is selected from any one of the following, 12. The pharmaceutical preparation according to claim 9, characterized in that The auxiliary lipid molecule is selected from any one of DOPE, POPC, EPC, DMPC, DPPC, DSPC, DAPC, DBPC, DLPC, DOPC, sphingomyelin, ceramide, DOPG, DPPG, POPE, and DOPE-mal; preferably DOPE or DSPC; The polymer-conjugated lipid molecule is selected from any one of PEG2000-DMG, PEG2000-DSPE, PEG2000-DPG, PEG2000-DSG, PEG5000-DMG, PEG5000-DPG, PEG5000-DSG, PEG-cDMA, PEG-C-DOMG, polyethylene glycol (PEG)-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, and PEG-ceramide (Cer), preferably PEG2000-DSPE or PEG2000-DMG.

13. The pharmaceutical preparation according to any one of claims 9 to 12, characterized in that In the liposome nanoparticles, the molar ratio of the ionizable lipid compound, the auxiliary lipid molecule, cholesterol or cholesterol derivative, and the polymer-conjugated lipid molecule is 60-5:60-5:50-5:10-1, preferably 40-10:30-10:50-20:8-1.

14. Use of the pharmaceutical preparation according to any one of claims 1 to 13 in the preparation of a drug for preventing and / or treating cancer, Preferably, the cancer is a PD-1 and / or CTLA-4 related cancer, More preferably, the cancer is selected from the group consisting of melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, etc., kidney tumors, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer and ovarian cancer.

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