WO2024098940A1 - Bispecific antibody and use thereof - Google Patents

Abstract

Provided is a bispecific antibody, comprising a first domain specifically binding to an interleukin-6 receptor (IL-6R), and a second domain specifically binding to a programmed cell death receptor-ligand 1 (PD-L1). Further provided is a method for the bispecific antibody to treat or prevent diseases related to IL-6R and PD-L1.

Description

Bispecific antibodies and their applications Technical Field

The present invention relates to bispecific antibodies targeting IL-6R and PD-L1 and applications thereof.

Background technique

Human programmed cell death receptor-1 (PD-1) is a type I transmembrane glycoprotein with 288 amino acids, which is expressed on activated T lymphocytes, B cells and myeloid cells. It plays an important role in the immune escape mechanism of tumors and is a known major immune checkpoint. It binds to its ligands PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death-Ligand 2) to inhibit the activity of T lymphocytes and related cellular immune responses in vivo. The expression of PD-L2 is relatively limited, mainly on macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies have shown that the interaction between PD-1 and PD-L1 is necessary to maintain the balance of the immune system in the body, and is also the main reason for PD-L1-positive tumor cells to avoid immune surveillance. New studies have found that high PD-L1 protein expression has been detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, ovarian cancer, cervical cancer, glioma, bladder cancer, esophageal cancer, oral squamous cell carcinoma, urethral epithelial carcinoma, pancreatic cancer, and head and neck tumors. By blocking the negative regulation of cancer cells on the PD-1/PD-L1 signaling pathway and activating the immune system, it can promote T cell-related tumor-specific cellular immune activity and help the immune system to eliminate tumor cells. Therefore, PD-L1 has become a popular target for the development of tumor immunotherapy drugs.

Interleukin-6 (IL-6) is a 26 kDa glycoprotein, a pleiotropic cytokine produced by various cell types such as T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells and some tumor cells. IL-6 is membrane-bound or binds to the soluble IL-6 receptor (IL-6R), and this complex binds to the signal transduction protein gp130, thereby activating the JAK-STAT3 pathway and ras-mediated MAP kinase signaling. IL-6 plays an important role in immune regulation, hematopoiesis, inflammation and tumor formation, and is highly expressed in the peripheral blood and tumor tissues of tumor patients. Diseases treated include: arthritis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, Castleman's disease, ankylosing spondylitis, solid tumors (renal cell carcinoma), prostate cancer and bladder cancer, pancreatic cancer, nervous system cancer and B-cell malignancies, etc. Tocilizumab (TCZ) is the first humanized monoclonal antibody targeting interleukin-6 receptor (IL-6R) and has been marketed both at home and abroad.

Among the many therapies currently under preclinical and clinical research, there are many targets that work synergistically with PD-1/PD-L1, but there is no drug that specifically binds to both PD-L1 and IL-6R targets at the same time.

Summary of the invention

In order to solve one of the technical problems existing in the prior art, the present invention provides a bispecific antibody, which can better maintain the activity of each monoclonal antibody, can specifically bind to two targets, IL-6R and PD-L1, and has similar or even better biological activity than the monoclonal antibody.

One aspect of the present invention provides a bispecific antibody, comprising: a first domain that specifically binds to interleukin-6 receptor (IL-6R), and a second domain that specifically binds to programmed cell death receptor-ligand 1 (PD-L1).

In some embodiments, the first domain is an antibody or a functional fragment thereof that specifically binds to IL-6R. In some embodiments, the first domain may include an immunoglobulin (Ig) domain of an IL-6R antibody.

In some embodiments, the second domain is an antibody or a functional fragment thereof that specifically binds to PD-L1. In some embodiments, the first domain may include an immunoglobulin (Ig) domain of a PD-L1 antibody.

In some embodiments, the first domain comprises an antibody Fab fragment, Fab' fragment, F(ab') ₂ fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment, or light chain variable region (VL) fragment that specifically binds to IL-6R.

In some embodiments, the second domain comprises an antibody Fab fragment, Fab' fragment, F(ab') ₂ fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment, or light chain variable region (VL) fragment that specifically binds to PD-L1.

In some embodiments, the first domain and the second domain are directly connected or connected through a linker.

In some embodiments, the linker has an amino acid sequence as shown in the general formula (G _n S) _m , where n and m are integers of 1-10, respectively; more preferably, n is an integer of 1-4, m is an integer of 1-3, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in the general formula (G _n S) _m . In some embodiments of the present invention, the linker comprises at least 6 amino acids.

In some embodiments, the bispecific antibody comprises a first domain and a second domain connected in any manner. In some embodiments, the bispecific antibody comprises a first domain-a second domain from the N-terminus to the C-terminus.

In some embodiments, the first domain includes: a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:1; and a light chain having an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:2.

In some embodiments, one or more of the amino acids 236, 237, 239, 332, and 333 corresponding to SEQ ID NO:1 are substituted.

In some embodiments, the amino acid at position 236 corresponding to SEQ ID NO: 1 is not leucine, and/or the amino acid at position 237 is not leucine, and/or the amino acid at position 239 is not glycine, and/or the amino acid at position 332 is not alanine, and/or Or the amino acid at position 333 is not proline.

In some embodiments, the amino acid at position 236 corresponding to SEQ ID NO:1 is substituted with alanine, and/or the amino acid at position 237 is substituted with glutamic acid, and/or the amino acid at position 239 is substituted with alanine, and/or the amino acid at position 332 is substituted with serine, and/or the amino acid at position 333 is substituted with serine.

In some embodiments, the first domain comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:4.

In some embodiments, the second domain comprises an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:5.

In some embodiments, the bispecific antibody comprises the amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO:6. In some embodiments, the bispecific antibody comprises the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO:8.

In some embodiments, the bispecific antibody also includes an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

In the present invention, the monoclonal antibody (206mab, which has the amino acid sequence shown in SEQ ID NO: 1) against interleukin-6 receptor (IL-6R) is based on the original drug tocilizumab (TCZ), and 5 mutations are made in the variable region, namely Phe51, Met57 and Ile103 on the heavy chain; Gln89 and Arg93 on the light chain, which increase the affinity with IL-6R by hundreds of times and the biological activity by about 30 times. 206mab is subjected to AEASS mutation (L236A, L237E, G239A, A332S, P333S, which has the amino acid sequence shown in SEQ ID NO: 4), which eliminates the Fc-mediated ADCC effect on immune cell killing to ensure the safety of clinical application.

Another aspect of the present invention provides an isolated nucleic acid molecule encoding the bispecific antibody of the present invention.

In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO:7, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the nucleotide sequence as set forth in SEQ ID NO:7. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO:9, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the nucleotide sequence as set forth in SEQ ID NO:9.

In some embodiments, the isolated nucleic acid molecule also includes a nucleotide sequence as shown in SEQ ID NO:3, or a nucleotide sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:3.

In some embodiments, the bispecific antibodies of the present invention form dimers. In some embodiments, the bispecific antibodies of the present invention form homodimers. In some embodiments, the bispecific antibodies of the present invention form heterodimers.

Another aspect of the present invention provides a nucleic acid delivery vector comprising the isolated nucleic acid molecule of the present invention. In some embodiments, the nucleic acid delivery vector comprises a nucleic acid delivery vector derived from adenovirus, adeno-associated virus, lentivirus or other acceptable nucleic acid delivery vectors.

Another aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention.

Another aspect of the present invention provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises the bispecific antibody of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the isolated nucleic acid molecule of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the nucleic acid delivery vector of the present invention and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is in the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained release agents or sustained release implants. In some embodiments, the pharmaceutical composition can be formulated into an injectable formulation. In some embodiments, the formulation is suitable for intravitreal injection, subcutaneous, intradermal, intramuscular, intravenous, intrathecal or intraspinal administration.

Another aspect of the present invention provides a medicine box, the medicine box includes a pharmaceutical composition of the present invention, and the pharmaceutical composition is packaged in a container. In some embodiments of the present invention, the container is a glass ampoule, a glass bottle, a plastic ampoule, a plastic bottle, a plastic bag or a prefilled syringe. In some embodiments, the present invention relates to a pharmaceutical unit dosage form suitable for parenteral administration to humans, the pharmaceutical unit dosage form is included in a pharmaceutical composition as described herein in a suitable container. In some embodiments, the suitable container is a prefilled syringe. In some embodiments, the prefilled syringe includes an injection needle.

Another aspect of the present invention provides a method for treating or preventing a disease associated with IL-6 and/or PD-1. The method comprises administering to a subject a therapeutically effective amount of the bispecific antibody of the present invention, the isolated nucleic acid molecule of the present invention, or the nucleic acid delivery vector of the present invention.

In some embodiments, the disease is selected from an immune system disease and a cancer-related disease. In some embodiments, the disease is selected from arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, systemic juvenile giant cell arteritis, giant cell arteritis, psoriasis, systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, Castleman's disease, ankylosing spondylitis, systemic sclerosis-related interstitial lung disease (SSc-ILD), COVID-19-related cytokine storm (CRS), severe or life-threatening cytokine storm (CRS) caused by CAR-T cells, acute myeloid leukemia, non-small cell lung cancer, liver cancer solid tumors (renal cell carcinoma), breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, ovarian cancer, cervical cancer, glioma, bladder cancer, metastatic esophageal squamous cell carcinoma, esophageal cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, head and neck tumors, prostate cancer, bladder cancer, pancreatic cancer, nervous system cancer, B cell malignancies and aging.

The present invention provides a bispecific antibody that can specifically bind to IL-6R and PD-L1. By electroporating a plasmid carrying an anti-IL-6R mab and an anti-PD-L1 scFv fusion gene into CHO cells, a stable and highly efficient expression cell line is obtained. The monoclonal cell line of the present invention can secrete and express a bispecific antibody against IL-6R and PD-L1. The bispecific antibody can block both the IL-6/IL-6R signaling pathway and the PD-1/PD-L1 signaling pathway, and can be used to treat renal cancer, glioma, and prostate cancer. Tumors, bladder cancer, pancreatic cancer, head and neck squamous cell carcinoma, acute myeloid leukemia, pancreatic cancer, non-small cell lung cancer, melanoma, liver cancer and other diseases. The bispecific antibody of the present invention comprises a specific antibody against IL-6R, which can improve the specificity of PD-L1 antibody and achieve a dual treatment effect.

definition

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a bispecific antibody or a bispecific binding molecule) and its binding partner (e.g., an antigen). Binding affinity can generally be expressed in terms of a dissociation constant ( _KD ), which is the ratio of the _dissociation rate ( _kd ) to the association rate ( _ka ), i.e., _KD = _kd / _ka . Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

As used herein, the term "specific recognition" or "specific binding" means that the antibody has recognition and binding selectivity for a ligand or receptor, and can be distinguished from unwanted or non-specific binding. In one embodiment, the degree of binding of the bispecific antibody of the present invention to an unrelated protein is less than about 10% of the degree of binding to IL-6R and PD-L1, for example, as measured by SPR. In certain embodiments, the bispecific antibody provided by the present invention binds to IL-6R and PD-L1 with a _KD of ^10-7 M or less, for example, ^10-10 M to ^10-11 M.

The term "substitution" as used herein for amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence by another different "replacement" amino acid residue. The term "insertion" as used herein for amino acids refers to the incorporation of at least one additional amino acid into an amino acid sequence. Although inserts typically consist of the insertion of 1 or 2 amino acid residues, larger "peptide inserts" can also be prepared, for example, inserting about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein for amino acids refers to the removal of at least one amino acid residue from an amino acid sequence.

The bispecific antibodies or fragments thereof disclosed herein may comprise conservative amino acid substitutions at one or more amino acid residues, for example at essential or non-essential amino acid residues. A "conservative amino acid substitution" is a replacement of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, In some embodiments, the bispecific antibodies or linkers are preferably replaced with another amino acid residue from the same side chain family. In some embodiments, the amino acid segments can be replaced with segments that are structurally similar and have different order and/or composition of side chain family members. Alternatively, in some embodiments, mutations can be randomly introduced along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into the bispecific antibodies of the invention and screened for the ability of these polypeptides to bind to the desired target.

The term "antibody" as used herein includes intact antibodies and any antigen-binding fragments (i.e., "antigen-binding portions," "antigen-binding polypeptides," or "immunobinders"), or single chains thereof. "Antibody" is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain includes a heavy chain variable (VH) region and a heavy chain constant region (CH). Each light chain includes a light chain variable (VL) region and a light chain constant region (CL). The VH region and the VL region each contain three regions with highly variable amino acid composition and arrangement order, called hypervariable regions or complementarity determining regions (CDR), CDR1, CDR2, and CDR3. The amino acid composition and arrangement order of the regions in the VH region and the VL region other than the CDR region are relatively conservative, called framework regions (FR). VH or VL each has four framework regions, represented by FR1, FR2, FR3, and FR4, respectively.

There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, and these major classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The CL lengths of different types (κ or λ) of immunoglobulins are basically the same, but the CH lengths of different classes of immunoglobulins are different, for example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The hinge region is located between CH1 and CH2, is rich in proline, and is easy to stretch and bend, thereby changing the distance between the antigen binding sites, which is conducive to the antibody binding to antigen epitopes located at different positions. The hinge region is easily hydrolyzed by papain, pepsin, etc., producing different hydrolysis fragments. Papain hydrolyzes Ig at the N-terminus of the two heavy chains connected by disulfide bonds in the hinge region, and can split Ig into two identical Fab segments and one Fc segment. The Fab segment is an antigen binding fragment (Fab), which consists of a complete light chain and the VH and CH1 domains of the heavy chain.

The term "homodimer" as used herein refers to a bispecific antibody of the invention, comprising two identical bispecific antibodies of the invention. The term "heterodimer" as used herein refers to a bispecific antibody of the invention, comprising two different bispecific antibodies of the invention.

The term "individual" or "subject" as used herein refers to mammals, including but not limited to humans and non-human mammals, for example, mammals include but are not limited to domesticated animals (such as cows, horses, dogs, sheep, goats, cats and dogs), primates (such as humans and monkeys) and rodents (such as rabbits, mice and rats).

Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

The term "connector" as used herein refers to a (peptide) linker of natural and/or synthetic origin, consisting of linear amino acids. Each domain in the bispecific antibody of the present invention can be connected by a connector, wherein each connector is fused to at least two polypeptides or domains and/or connected in other ways (e.g., via a peptide bond). In some embodiments, the amino acid sequences of all connectors present in the bispecific antibody of the present invention are identical. In other embodiments, the amino acid sequences of at least two connectors present in the bispecific antibody of the present invention are different. The connector should have a length suitable for connecting two or more monomer domains in this way, and the connector can ensure that the different domains connected to it are correctly folded and appropriately presented, thereby exerting the function of its biological activity. In different embodiments, the connector has a flexible conformation. Suitable flexible connectors include, for example, having glycine, glutamine and/or serine residues. In some embodiments, the amino acid residues in the connector can be arranged in small repeating units of up to 5 amino acids.

"Percent (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid sequence, after aligning the sequences and (as required) introducing gaps to obtain maximum percentage sequence identity, but without considering any conservative substitutions as part of the sequence identity. To determine the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, such as using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical preparation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "treat" as used herein refers to alleviating and/or ameliorating a disorder and/or a disease or symptom associated therewith, as well as preventing the deterioration of the symptoms of a disorder. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or alleviating symptoms, alleviating or improving prognosis. However, it should be understood that treating a disease or symptom does not require completely eliminating the disease or symptoms associated therewith.

As used herein, the term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive effect.

Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a schematic diagram of the structure of the bispecific antibody constructed in the present invention.

Figure 2 shows the identification results of the bispecific antibody PLS280 (206mab-PD-L1scFv) of the present invention, wherein 2A is the SDS-PAGE result, R is the reducing SDS-PAGE result, NR is the non-reducing SDS-PAGE result, and M is a marker; 2B is the SEC-HPLC spectrum of the PLS280 (206mab-PD-L1scFv) expression protein.

FIG3 shows the results of the activity of PLS280 (206mab-PD-L1scFv) of the present invention in blocking IL6/IL-6R on cells.

FIG. 4 shows the results of the activity of PLS280 (206mab-PD-L1scFv) of the present invention in blocking PD1/PD-L1 on cells.

FIG. 5 shows the SDS-PAGE electrophoresis gel images of the high-expression cell lines 9#17C2, 11#22D5, and 12#31G3 of the present invention, wherein 5A is the result of non-reducing SDS-PAGE, and 5B is the result of reducing SDS-PAGE.

FIG6 shows the SEC-HPLC detection of the repeatedly frozen-thawed samples of the protein expressed by the high-expressing cell line 9#17C2 of the present invention.

FIG. 7 shows the SDS-PAGE electrophoresis gel images of the protein expressed by the high-expressing cell line 9#17C2 of the present invention after repeated freezing and thawing, wherein 7A is the non-reducing SDS-PAGE result and 7B is the reducing SDS-PAGE result.

FIG8 shows the SEC-HPLC detection of the 40° C. accelerated sample of the protein expressed by the high-expressing cell line 9#17C2 of the present invention.

FIG9 shows the SDS-PAGE electrophoresis gel image of the protein expressed by the high-expressing cell line 9#17C2 of the present invention at 40° C., wherein 9A is the non-reducing SDS-PAGE result and 9B is the reducing SDS-PAGE result.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts disclosed herein.

The present invention constructs an anti-IL-6R×PD-L1 bispecific antibody by connecting the scFv (VL-linker-VH) of the anti-human PD-L1 monoclonal antibody in series to the C-terminus of the heavy chain of the anti-human IL-6R monoclonal antibody, and the structure is shown in Figure 1. Wherein, the general formula of linker is (G _n S) _m , n and m are integers of 1-10, preferably n is an integer of 1-4, and m is an integer of 1-3. The scFv region sequence of the anti-human PD-L1 monoclonal antibody is from Atezolizumab (obtained from the IMGT website).

The present invention first constructs the PLS280 (206mab-H-PD-L1scFv) fusion gene vector, and co-transfects HEK293 with 206mab-L for transient expression, obtains the bifunctional fusion protein through conventional purification, and after the obtained protein is correctly identified by electrophoresis, the purity, biological activity and affinity of the target protein obtained by transient transfection are detected. The results show that the bispecific antibody has good purity, and the biological activity and affinity of blocking IL6/IL-6R and PD1/PD-L1 are similar to or not lower than those of monoclonal antibodies. On this basis, five amino acid mutations (L236A, L237E, G239A, A332S, P333S) were performed on 206mab to remove the Fc-mediated ADCC effect on immune cell killing to ensure the safety of clinical application. 206mab(mut) and Anti PD-L1scFv were connected with linker ( _GnS ) _m to construct plasmid PLS301 (206mab(mut)-H-PD-L1scFv) for stable transfection of CHO cells. The plasmid was electroporated into CHO cells to obtain a CHO monoclonal cell line that stably and efficiently expresses human recombinant proteins. The stability and developability of the antibodies expressed by the screened monoclonal cell line were analyzed.

Example 1 Construction of expression plasmid

In this embodiment, the structure of the anti-IL-6R×PD-L1 bispecific antibody is shown in FIG1 , wherein the IL-6R antibody is 206mab (whose amino acid sequence and nucleotide sequence are shown below) and its mutant 206mab-mut (whose amino acid sequence and nucleotide sequence are shown below), 206mab is a genetically modified interleukin-6 receptor monoclonal antibody drug tocilizumab (TCZ). The drug was made by making five mutations in the variable region based on Tocilizumab: Phe51, Met57 and Ile103 on the heavy chain, Gln89 and Arg93 on the light chain, which increased the affinity of 206mab to IL-6R by hundreds of times and the biological activity by about 30 times. 206mab was further mutated with five amino acids (L236A, L237E, G239A, A332S, P333S) to obtain its mutant 206mab-mut, which removed the Fc-mediated ADCC effect on immune cell killing to ensure the safety of clinical application. The scFv region sequence of the anti-human PD-L1 monoclonal antibody is from Atezolizumab (obtained from the IMGT website, and its amino acid sequence and nucleotide sequence are shown below). A linker is provided between the IL-6R antibody and the anti-PD-L1 scFv, and the linker sequence is shown in the corresponding sequence underlined.

In this example, the scFv (VL-linker-VH, the specific sequence is shown below) of the anti-human PD-L1 monoclonal antibody was connected in series to the C-terminus of the heavy chain of the anti-human IL-6R monoclonal antibody to construct the PLS280 (206mab-H-PD-L1scFv, the specific sequence is shown below) and PLS301 (206mab (mut) -H-PD-L1scFv, the specific sequence is shown below) fusion gene vectors, and the bispecific antibody was obtained by co-transfection with 206mab-L for transient expression and conventional purification, which not only retained the activity of 206mab in blocking IL6/IL-6R, but also increased the activity of Anti-PD-L1scFv in blocking PD1/PD-L1.

The sequences involved in this example are shown in Table 1.

Table 1 Sequence Listing Related to Example 1

Example 2 Transient Expression and Protein Purification

The day before transfection, use KOP293 to prepare 50ml of HEK293 cells with a density of 1.2×10 ⁶ /ml, place it in a 250ml Erlenmeyer flask, and culture it at 135rpm, 37℃, 5% CO ₂ for 20-25h. The cell density reaches about 2×10 ⁶ /ml. The cells are in the logarithmic growth phase and the viability is above 95% before they can be used for transfection experiments. Add 50ug of recombinant plasmid to Opti-MEM with a total volume of 2ml, mix gently, add 250ug of PEI to Opti-MEM with a total volume of 2ml, and incubate at room temperature for 5min. Add 2ml of PEI dilution to the corresponding plasmid dilution, mix well, and incubate at room temperature for 20min. Gently rotate and shake the Erlenmeyer flask containing HEK293 cells, slowly drip the PEI-DNA mixture, and then culture the transfected cells in a shaker at 135rpm, 37℃, 5% CO _2. After 24h of transfection, 50×KT-Feed 1ml, 500×VPA 100ul were added, and after 5 days of transfection, the cell culture medium was collected by centrifugation at 3000rpm for 15min. After the cell expression supernatant was centrifuged at high speed, impurities were removed by filtration, and the obtained supernatant was subjected to affinity chromatography using HiTrap MabSelect SuRe pre-packed column. After the column was washed with pure water and balanced with PB buffer, the supernatant was loaded onto the chromatography column for binding. After the loading was completed, it was washed with PB buffer to the baseline, and then the protein was eluted with 0.1M citrate eluent. The eluted protein was neutralized with 1M Tris-HCl, appropriately concentrated, and loaded onto Superdex200 balanced with PBS for further purification. After the collected protein was concentrated to a certain concentration, SDS-PAGE electrophoresis and SEC-HPLC were performed. The results are shown in 2A and 2B of Figure 2. The results showed that the purified PLS280 protein had a high purity, and the SEC-HPLC purity was 98.61%.

Example 3 Biological Activity Assay

(1) Determination of biological activity of 206mab

The biological activity of 206mab was detected by 293-IL6Res cell/reporter gene method. Rinse the cells with preheated sterile PBS for 3-5s, remove the PBS, add trypsin for digestion, and add basal medium to terminate digestion when the cells fall off. Centrifuge and discard the supernatant, resuspend and count, adjust the cell density to 5×10 ⁵ cell/ml, and add 80μl/well to the corresponding wells. Dilute IL6 to 50ng/ml with basal medium, add 10μl/well to the sample well and positive control well, and add 10μl/well basal medium to the negative well. Dilute the test sample to 200μg/ml with basal medium, and dilute it into 8 concentrations in a 5-fold gradient. Add 10μl/well to the corresponding position wells of the 96-well plate, set two replicates for each concentration, and add 10μl/well basal medium to the positive and negative control groups. The cell plate was placed in a 37°C, 5% CO ₂ cell culture incubator for 22h. The melted and mixed One Lite detection reagent was added to the above 96-well plate at 100 μl/well, and the mixture was shaken and mixed for 3 minutes in an oscillator, and then allowed to stand for 3 to 5 minutes. The chemiluminescence value (RLU) was read using a multifunctional microplate reader. The data was processed using a four-parameter fitting curve, and the IC50 value of the test sample was calculated using the logarithm of the concentration of the standard or test sample as the horizontal axis and the RLU value as the vertical axis. The results are shown in Figure 3. The IC50 value of 206mab was 0.08212, and the IC50 value of PLS280 was 0.05973. After 206mab was connected to PD-L1scFv to form a bispecific antibody, the antibody activity of 206mab was not affected.

(2) Determination of biological activity of PD-L1 scFv

The biological activity of PD-L1scFv was detected by PD-L1/PD-1Luci cell line/reporter gene method. After counting huPD-L1-CD3L-CHO cells, the density was adjusted to 4*10 ⁵ cells/ml with complete medium and added to 96-well black transparent bottom cell culture plates at 50μL/well. Then count huPD-1-NF-AT-jurkat cells, adjust the density to 2*10 ⁶ cells/ml with complete medium, and add the cells to 96-well black transparent bottom cell culture plates at 50μL/well. Dilute the drug to 2μM with stress-free Jurkat complete medium, dilute 4-fold to 10 concentrations, add the cells to 96-well black transparent bottom cell culture plates at 50μL/well, and supplement 50μl of stress-free Jurkat complete medium for negative control. Place the 96-well black transparent bottom cell culture plate in a cell culture incubator at 37°C and 5% CO ₂ for 6h. The 96-well cell culture plate was taken out of the incubator and allowed to return to room temperature. The one-glo enzyme reaction substrate was thawed in the dark, and then 50 μL/well of the enzyme reaction substrate was added to the cell culture plate. The plate was incubated in the dark for 15 min, and the chemiluminescence value (RLU) was read using a multifunctional microplate reader. The data were processed using a four-parameter fitting curve, with the logarithm of the standard or test sample concentration as the horizontal axis and the RLU value as the vertical axis. The IC50 values of the test products were calculated using the coordinates. The results are shown in Figure 4. The IC50 value of Anti PD-L1Mab (Atezolizumab) is 0.3955, and the IC50 value of PLS280 is 0.5801, indicating that the biological activity of PD-L1scFv itself is basically not affected after it is connected to 206mab (mut).

Example 4 Affinity Determination

This example uses bio-layer interferometry (BLI) to determine affinity. First, PLS280 and Atezolizumab were diluted to 20nM and adsorbed onto the ProA probe at a speed of 400rpm/min. PD-L1 was diluted from 400nM to 5 concentration gradients. The program was set so that the diluted PD-L1 was bound to the ProA probes attached with PLS280 and Atezolizumab within a fixed time. After the binding was completed, the probe of the complex was transferred to the Q buffer without the analyte to dissociate the bound analyte. The data was fitted using the built-in software of the gator instrument to obtain the KD value. The results are shown in Table 2.

Table 2 Affinity of antibodies for PD-L1

Example 5 Screening and identification of stable cell lines

One day before transfection, adjust the density of CHO-K1 cells to 0.5×10 ⁶ cells/mL. On the day of transfection, prepare linearized, high-concentration endotoxin-free plasmids, measure the density and viability of CHO-K1 cells, and ensure that the cell viability is greater than 97%. After washing CHO-K1 cells twice with CD CHO medium, take 700μL of cell suspension, add 40μg of plasmid, mix well, transfer to a 4mm electrode cup, and put it into the electroporator. Set the electroporation parameters to 300V, 1000μF, electroporate once, transfer the electroporated cell suspension to preheated fresh CD CHO medium, and incubate at 37℃ for 20min. The incubated cell suspension is evenly inoculated in a 96-well plate. After 24h of transfection, pressurize and add CD CHO medium containing methionine iminosulfone (MSX). The final screening pressure is 25-50μM MSX, 5% CO ₂ , and static culture at 37℃. After the monoclones in the 96-well plate grow to a suitable size, start selecting monoclones, transfer all clones to a new 96-well plate, and culture at 5% CO ₂ and 37°C. After the cells in the wells are full, take the supernatant in the well plate for reduction electrophoresis to detect the expression of the bispecific antibody, select the cell line with the highest expression level for limited dilution screening of monoclonal cell lines, inoculate 96-well plates at 0.3 cells/well, and screen out three high-expressing cell lines 9#17C2, 11#22D5, and 12#31G3. The expression levels were measured, and the results are shown in Table 3; the three high-expressing cell lines were cultured in a 25mL volume of shake flasks, and the supernatants were purified using Mabselect sure and Superdex200. The purified protein was identified by reducing and non-reducing SDS-PAGE electrophoresis (the results are shown in Figure 5). The cell line 9#17C2 with good stability and high expression level was selected for drugability analysis.

Table 3 Protein expression determination of cell lines

Example 6 Drugability Analysis

The drugability analysis of the protein expressed and purified by the screened 9#17C2 cell line was performed. The protein was divided into 6 tubes, 1 ml/tube. One of them was not treated and used as a zero-point control. The other 2 tubes were taken and repeatedly frozen and thawed 3 and 5 times, respectively, and SEC-HPLC was tested. The results are shown in Table 4 and Figure 6; reduced SDS-PAGE (R) and non-reduced SDS-PAGE (NR) were tested, and the results are shown in Figure 7. The data showed that the purity of the protein was consistent before and after freezing and thawing, and it was relatively stable. Another 3 tubes were taken and accelerated in a 40°C water bath for 1 week, 2 weeks and 4 weeks, and SEC-HPLC was tested. The results are shown in Table 5 and Figure 8; SDS-PAGE electrophoresis, the results are shown in Figure 9; the biological activity of 206mab was tested, the results are shown in Table 6; the biological activity of PD-L1scFv was tested, and the results are shown in Table 7. The results showed that after the protein was placed at 40°C, there was no significant change in purity and biological activity, and the protein had good drugability.

Table 4 SEC-HPLC test results of freeze-thaw samples

Table 5 SEC-HPLC test results of 40℃ accelerated samples

Table 6 206mab biological activity test results of samples accelerated at 40℃

Table 7 PD-L1scFv biological activity test results of samples accelerated at 40°C

Example 7 Pharmacodynamic evaluation of PLS280 (206mab-PDL1scFv) bispecific antibody in acute myeloid leukemia (AML) mouse model

Human peripheral blood PBMCs were injected into NVSG severe combined immunodeficient mice (8-week-old mice) through the tail vein to form humanized mice with immune system. After 2 weeks, humanized mice were selected (humanized mice were considered to be successfully modeled when hCD45 ⁺ cells exceeded 10% as measured by flow cytometry). 6-7× ¹⁰⁶ HL-60 cells were injected into each tail vein, and the tumor volume was measured twice a week. When the tumor volume reached 100 mm ³ , group administration was performed. 48 mice that met the conditions were randomly divided into 6 groups (n=8): negative control group, 206mab monoclonal antibody group, PDL1scFv monoclonal antibody group, PLS280 (206mab-PDL1scFv) bispecific antibody low, medium and high dose groups. The group administration date was set as D1, D8, and D15. The administration method was tail vein injection.

The rats were observed for physical signs every day, mainly for discomfort, hair erection, weight loss and other related symptoms. The tumor volume was measured twice a week for tumor burden analysis. The measurements were continued for 3 weeks. The survival time and survival rate between groups were recorded. The data were expressed as mean ± standard error (Mean ± SEM) and plotted using GraphpadPrism 9 and Excel software, and statistically analyzed using one-way analysis of variance.

Tumor volume (V) calculation formula: V = 1/2 × L _long × L _short ²

Relative volume (RTV) = _VT / _V0

Tumor inhibition rate (%) = ( _CRTV - _TRTV )/ _CRTV (%)

V0 and VT are the tumor volumes at the beginning and end of the experiment, respectively. C _RTV and T _RTV are the relative tumor volumes of the blank control group (Blank) and the experimental group at the end of the experiment, respectively.

The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims (16)

1. A bispecific antibody comprises: a first domain that specifically binds to interleukin-6 receptor (IL-6R), and a second domain that specifically binds to programmed cell death receptor-ligand 1 (PD-L1).
2. The bispecific antibody according to claim 1, characterized in that the first domain is an antibody or a functional fragment thereof that specifically binds to IL-6R, and the second domain is an antibody or a functional fragment thereof that specifically binds to PD-L1.
3. The bispecific antibody according to claim 1 or 2, characterized in that the first domain comprises an antibody Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment or light chain variable region (VL) fragment that specifically binds to IL-6R.
4. The bispecific antibody according to any one of claims 1 to 3, characterized in that the second domain comprises an antibody Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment or light chain variable region (VL) fragment that specifically binds to PD-L1.
5. The bispecific antibody according to any one of claims 1 to 4, characterized in that the first domain and the second domain are directly connected or connected through a linker,

   Preferably, the linker has an amino acid sequence as shown in the general formula (GnS)m, where n and m are integers of 1-10 respectively; more preferably, n is an integer of 1-4, m is an integer of 1-3, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in the general formula (GnS)m.
6. The bispecific antibody according to any one of claims 1 to 5, characterized in that the first domain comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:1; and a light chain having an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:2.
7. The bispecific antibody according to claim 6, characterized in that one or more of the amino acids 236, 237, 239, 332, and 333 corresponding to SEQ ID NO: 1 are substituted;

   Preferably, the amino acid at position 236 corresponding to SEQ ID NO: 1 is substituted with alanine, and/or the amino acid at position 237 is substituted with glutamic acid, and/or the amino acid at position 239 is substituted with alanine, and/or the amino acid at position 332 is substituted with serine, and/or the amino acid at position 333 is substituted with serine;

   Preferably, the first domain comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:4.
8. The bispecific antibody according to any one of claims 1 to 7, characterized in that the second domain comprises an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least about 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO:5.
9. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 8.
10. A nucleic acid delivery vector comprising the isolated nucleic acid molecule according to claim 9, preferably, the nucleic acid delivery vector comprises a nucleic acid delivery vector derived from adenovirus, adeno-associated virus, lentivirus or other acceptable nucleic acid delivery vectors.
11. A host cell comprising the isolated nucleic acid molecule of claim 10.
12. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 8, the isolated nucleic acid molecule according to claim 9, or the nucleic acid delivery vector according to claim 10, and a pharmaceutically acceptable carrier.
13. The pharmaceutical composition according to claim 12, characterized in that the pharmaceutical composition is in the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained-release agents or sustained-release implants.
14. A medicine kit comprising the pharmaceutical composition according to claim 12 or 13, wherein the pharmaceutical composition is packaged in a container, and the container is preferably a glass ampoule, a glass bottle, a plastic ampoule, a plastic bottle, a plastic bag or a prefilled syringe.
15. A method for treating or preventing a disease associated with IL-6 and/or PD-1, comprising administering to a subject a therapeutically effective amount of the bispecific antibody according to any one of claims 1 to 8, the isolated nucleic acid molecule according to claim 9, or the nucleic acid delivery vector according to claim 10.
16. The method according to claim 15, characterized in that the disease is selected from immune system diseases and cancer-related diseases, preferably, the disease is selected from arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, systemic juvenile giant cell arteritis, giant cell arteritis, psoriasis, systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, Castleman's disease, ankylosing spondylitis, systemic sclerosis-related interstitial lung disease (SSc-ILD), COVID-19-related related cytokine storm (CRS), severe or life-threatening cytokine storm (CRS) caused by CAR-T cells, acute myeloid leukemia, non-small cell lung cancer, liver cancer solid tumors (renal cell carcinoma), breast cancer, lung cancer, gastric cancer, colorectal cancer, kidney cancer, melanoma, ovarian cancer, cervical cancer, glioma, bladder cancer, metastatic esophageal squamous cell carcinoma, esophageal cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, pancreatic cancer, nervous system cancer, B-cell malignancies and aging.