Novel cyclic anti-CD38 bispecific antibodies and uses thereof

Abstract

The invention provides a CD38 bispecific antibody with a novel ring structure and an Fc fusion type structure of the CD38 bispecific antibody. CD38 + tumor cells can be effectively targeted, T cells are combined and activated at the same time, and the T cells are mediated to kill leukemia cells in a targeted mode by connecting the T cells and the tumor cells. The BsAb is applied clinically and can be used for immunotherapy of tumors such as AML and autoimmune diseases.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to novel cyclic anti-CD38 bispecific antibodies and uses thereof. Background Art

[0002] In recent years, immunotherapy has made breakthroughs in the treatment of tumors and autoimmune diseases. Chimeric antigen receptor T cell (CAR-T) and bispecific antibody (BsAb) therapies targeting CD19 have been successfully marketed and used in clinical treatment, and are recommended as first-line treatments for B-ALL. However, the progress of immunotherapy in AML lags far behind that of B-ALL. The main reason is that ideal therapeutic targets for AML have not yet been found. Classic targets such as CD33, CD123, and CLL1 all have severe hematopoietic system toxicity, which limits the corresponding immunotherapy from the laboratory to the clinic. Therefore, there is an urgent need to explore new immunotherapy targets for AML.

[0003] CD38 is a potential immunotherapy target for AML. Currently, CD38 is generally considered an immunotherapy target for multiple myeloma (MM). However, it has been found that CD38 is also widely expressed on the surface of tumor cells in AML patients. Therefore, CD38 can be used as an immunotherapy target for a variety of hematologic malignancies.

[0004] Traditional BsAbs, such as belintuzumab, which targets CD19, are composed of a CD19-targeting scFv and a CD3-targeting scFv tandemly linked end-to-end, forming a linear molecular structure. Extensive research is underway on BsAbs with novel structural structures, hoping to achieve superior efficacy compared to traditional ones. However, no novel and highly effective anti-human CD38 bispecific antibody has yet emerged. Summary of the Invention

[0005] Technical issues solved:

[0006] The first aspect of the present disclosure is to address the shortcomings of the above-mentioned prior art and provide a novel cyclic anti-CD38 bispecific antibody and its use.

[0007] Specifically, the present disclosure provides a novel cyclic anti-CD38 bispecific antibody, which utilizes, for example, genetic engineering and molecular cloning techniques to construct an expression vector, expresses the fusion protein through a eukaryotic expression system, and provides a method for efficiently purifying the protein. The inventors utilize tumor-associated antigens (TAA) on the surface of blood tumor cells as immunotherapy targets, bind BsAb to the surface of blood tumor cells, utilize CD3 surface antigens as targeting antigens for human T cells, and achieve bispecific targeting of human T cells and tumor cells by binding to the CD3 binding portion of BsAb, thereby bringing T cells closer to tumor cells expressing specific TAAs, and activating T cells at the same time, achieving specific killing of tumor cells by T cells. At the same time, the present disclosure also provides an Fc-fused BsAb, which increases the molecular weight of BsAb and enhances the stability and function of BsAb by fusing a mutant immunoglobulin Fc segment to BsAb. Through the above means, the problems existing in the prior art are solved.

[0008] Technical solution:

[0009] A cyclic anti-CD38 bispecific antibody that specifically binds to human CD38 and human CD3 molecules, comprising:

[0010] a) variable regions CD38VH and CD38VL of the antigen-binding portion that specifically bind to human CD38;

[0011] b) variable regions CD3VH and CD3VL of the antigen-binding portion that specifically bind to human CD3; and

[0012] c) LoopLinker,

[0013] The amino acid sequence of the cyclic anti-CD38 bispecific antibody is arranged in the order of VL-LoopLinker-VH, and the amino acid sequence of the LoopLinker is as shown in SEQ ID No: 1 or a sequence having more than 80% identity thereto and having the same or substantially the same biological function.

[0014] In the present disclosure, the above-mentioned identity of more than 80% refers to a sequence that has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the target sequence.

[0015] In some embodiments, the antigen-binding portion is Fab, Fab', F(ab')2, Fd, FCL, dAb or scFv. In a more specific embodiment, the antigen-binding portion is a single-chain antibody (scFv).

[0016] In a specific embodiment, the amino acid sequences of CDRH1-3 of the above-mentioned CD38VH are shown as SEQ ID No: 2-4, respectively, and the amino acid sequences of CDRL1-3 of the above-mentioned CD38VL are shown as SEQ ID No: 5-7, respectively; the amino acid sequences of CDRH1-3 of the above-mentioned CD3VH are shown as SEQ ID No: 8-10, respectively, and the amino acid sequences of CDRL1-3 of the above-mentioned CD3VL are shown as SEQ ID No: 11-13, respectively.

[0017] In a specific embodiment, the amino acid sequence of the above-mentioned CD38VH is shown as SEQ ID No: 14, or a sequence having more than 80% identity thereto and the same or substantially the same biological function, the amino acid sequence of the above-mentioned CD38VL is shown as SEQ ID No: 15, or a sequence having more than 80% identity thereto and the same or substantially the same biological function; the amino acid sequence of the above-mentioned CD3VH is shown as SEQ ID No: 16, or a sequence having more than 80% identity thereto and the same or substantially the same biological function, the amino acid sequence of the above-mentioned CD3VL is shown as SEQ ID No: 17, or a sequence having more than 80% identity thereto and the same or substantially the same biological function.

[0018] To better achieve the objectives of the present disclosure, in some embodiments, the cyclic anti-CD38 bispecific antibody further comprises: d) an Fc region of a human immunoglobulin. In some specific embodiments, the human immunoglobulin is IgA, IgM, IgE, or IgG. In a more specific embodiment, the human immunoglobulin is IgG1.

[0019] To better achieve the purpose of the present disclosure, in one embodiment, the inventors modified the Fc segment of the immunoglobulin. The amino acid sequence of the Fc segment of the human immunoglobulin is shown in SEQ ID No: 18.

[0020] In some embodiments, the Fc segment of the human immunoglobulin is operably connected to the N-terminus of the VL-LoopLinker-VH structure through a hinge, and the hinge forms an immunoglobulin-like dimer structure through a disulfide bond.

[0021] In a specific embodiment, the amino acid sequence of the hinge is as shown in SEQ ID No: 19 or a sequence having more than 80% identity thereto and having the same or substantially the same biological function.

[0022] In the VL-LoopLinker-VH sequence described in the present disclosure, "VL" includes the CD3VL and CD38VL of the bispecific antibody, and "VH" includes the CD38VH and CD3VH of the bispecific antibody. In some embodiments, the VL-LoopLinker-VH sequence is:

[0023] 1)CD3VL-CD38VL-LoopLinker-CD38VH-CD3VH;

[0024] 2)CD38VL-CD3VL-LoopLinker-CD38VH-CD3VH;

[0025] 3) CD3VL-CD38VL-LoopLinker-CD3VH-CD38VH; or

[0026] 4)CD38VL-CD3VL-LoopLinker-CD3VH-CD38VH.

[0027] Preferably, in some embodiments, the order of the above VL-LoopLinker-VH is: 1) CD3VL-CD38VL-LoopLinker-CD38VH-CD3VH.

[0028] In some embodiments, a linker 1 (Linker 1) is operably connected between the CD3VL and the CD38VL, and a linker 2 (Linker 2) is operably connected between the CD3VH and the CD38VH. In some specific embodiments, the linker 1 or linker 2 is a flexible connecting peptide. In some more specific embodiments, the flexible connecting peptide is selected from (GGGGS) n , G x S x Repeating unit, one of GGG, GGSG or GSGG amino acid sequences.

[0029] In some embodiments, any peptide chain can be inserted into a suitable position between the aforementioned linkers as a spacer, and the peptide chain can be an oligopeptide or a polypeptide.

[0030] To enable protein expression or detection, in some embodiments, the cyclic anti-CD38 bispecific antibody further comprises a tag or signal peptide. In the present disclosure, such tags or signal peptides are well known in the art. In some specific embodiments, the tag is selected from at least one of a fluorescent marker, a radiolabel, or an enzyme marker.

[0031] In some embodiments, the cyclic anti-CD38 bispecific antibody is modified, and the modifications are one or more of cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycosylated, glycosylated, polysialylated, phosphorylated, adenylylated, or PEGylated. In the present disclosure, the methods for the above modifications and the effects of the modifications are well known in the art.

[0032] In one specific embodiment, the structure of the cyclic anti-CD38 bispecific antibody is HisTag-CD3VL-G4S-CD38VL-LoopLinker-CD38VH-G4S-CD3VH-StrepTagII (labeled as 38-3-loop-BsAb). In another specific embodiment, the structure of the cyclic anti-CD38 bispecific antibody is HisTag-CD3VL-G4S-CD38VL-LoopLinker-CD38VH-G4S-CD3VH-Hinge-CH2-CH3 (labeled as 38-3-loopFc-BsAb). The various portions of the cyclic anti-CD38 bispecific antibody shown in the primary protein structure are all connected in series. More specifically, the amino acid sequence of the cyclic anti-CD38 bispecific antibody is as shown in SEQ ID Nos: 20-21, or a sequence having 80% or greater identity thereto and having the same or substantially the same biological function.

[0033] The second aspect of the present disclosure provides an isolated nucleic acid molecule encoding the aforementioned cyclic anti-CD38 bispecific antibody.

[0034] In a specific embodiment, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No: 22 or a sequence having more than 80% identity thereto and having the same or substantially the same biological function.

[0035] The third aspect of the present disclosure provides a vector comprising the above-mentioned nucleic acid molecule.

[0036] The fourth aspect of the present disclosure is to provide a cell comprising the above-mentioned cyclic anti-CD38 bispecific antibody, the above-mentioned nucleic acid molecule or the above-mentioned vector. In some specific embodiments, the above-mentioned cell is a eukaryotic cell. In a specific embodiment, the above-mentioned eukaryotic cell is an ExpiCHO-S cell. When using the ExpiCHO-S cell expression system, it is necessary to introduce KOZAK and Igκ leader signal peptides into the expression vector. The nucleotide sequence of the signal peptide is shown in SEQ ID No: 22.

[0037] The fifth aspect of the present disclosure provides a pharmaceutical composition comprising the aforementioned cyclic anti-CD38 bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned cell.

[0038] The fifth aspect of the present disclosure provides use of the aforementioned cyclic anti-CD38 bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, or the aforementioned pharmaceutical composition in the preparation of a drug for treating tumors or autoimmune diseases.

[0039] In some embodiments, the tumor is a hematologic tumor. In some specific embodiments, the hematologic tumor is acute myeloid leukemia, lymphoma, multiple myeloma, or systemic light chain amyloidosis. In some embodiments, the autoimmune disease is immune thrombocytopenia, lupus erythematosus, or rheumatoid arthritis.

[0040] Beneficial effects:

[0041] The novel ring-structured CD38 BsAb and the structure fused with Fc provided by the present disclosure can effectively target CD38 + Tumor cells, while simultaneously binding and activating T cells, mediate T cell-targeted killing of leukemia cells by connecting T cells and tumor cells. The above-mentioned BsAbs can be applied clinically for immunotherapy of tumors such as AML and autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 For two BsAbs in the disclosed embodiments (wherein, Figure 1 A is 38-3-loop-BsAb and Figure 1 B is a schematic diagram of the expression plasmid of 38-3-loop Fc-BsAb, and the backbone plasmid is the pcDNA 3.4 expression vector;

[0043] Figure 2Figure 2 is a schematic diagram of the structures of two BsAbs in the disclosed embodiments, wherein the structure of 38-3-loop-BsAb is composed of HisTag-CD3VL-G4S-CD38VL-LoopLinker-CD38VH-G4S-CD3VH-StrepTagII in series, and the structure of 38-3-loopFc-BsAb is composed of HisTag-CD3VL-G4S-CD38VL-LoopLinker-CD38VH-G4S-CD3VH-Hinge-CH2-CH3 in series. The HisTag tag protein is used for the quantification of the two BsAbs, and StrepTagII is used for the purification of 38-3-loop-BsAb. 38-3-loop-FcBsAb is purified using an IgG antibody.

[0044] Figure 3 The structure prediction diagrams of two BsAbs in the embodiments of the present disclosure;

[0045] Figure 4 Figure 2 is a diagram of the purification process of two BsAbs in the embodiments of the present disclosure. The two antibodies were purified using a Cytiva AKTA purifier. The 38-3-loop-BsAb was purified using a Cytiva StrepTrap HP purification column, and the 38-3-loopFc-BsAb was purified using a Cytiva HiTrap Protein G HP purification column.

[0046] Figure 5 Western Blot (left) and Coomassie blue staining (right) images of two BsAbs in the disclosed embodiments, wherein the Western Blot shows the molecular weight of the two BsAbs, and the Coomassie blue staining shows the purity of the antibodies after purification;

[0047] Figure 6 For the two BsAbs and CD38 in the embodiment of this disclosure + Molm13 and CD3 + Figure 3: Specific binding assay results for Jurkat cell target antigens, using commercial flow cytometry antibodies PE-anti-Human-CD38 and PE-anti-Human-CD3 for competitive binding.

[0048] Figure 7 The results of the two BsAb-activated naive T cell proliferation and expansion experiments in the disclosed embodiments are shown in the figure, wherein the left and middle figures are the CFSE staining results, and the right figure is the proliferation counting result;

[0049] Figure 8Figure 2 shows the killing effect of two BsAbs on three AML tumor cell lines in the examples disclosed herein. Normal donor T cells were mixed with tumor cells at an effector-target ratio of 2:1, and different concentrations of BsAbs were added for co-culture killing experiments. Left: CD38 + Molm13, middle:CD38 + U937, right: CD38 - K562;

[0050] Figure 9 The two BsAb-mediated T cell responses to CD38 in the disclosed embodiments are shown in FIG. + Molm13 and CD38 + Detection of cytokine (IL2, TNF-a, IFN-γ, IL6) release during the killing effect of U937, where upper: Molm13, lower: U937.

[0051] Sequence description.

[0052] DETAILED DESCRIPTION Detailed description of the invention:

[0054] The present disclosure provides a novel cyclic anti-CD38 bispecific antibody and its uses. Those skilled in the art can refer to the disclosure herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention, and relevant persons can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0055] In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The terms "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example" etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0056] In this disclosure, when a range of values ​​is provided, it is understood that the endpoints are included in the range and that each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the stated range and any smaller range between the specified values ​​are encompassed unless the context clearly dictates otherwise.

[0057] In this disclosure, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0058] Throughout this disclosure, references to "one embodiment," "an example," "some embodiments," "specific embodiments," "related embodiments," "an example," "some examples," "additional embodiments," or "further embodiments," "further implementations," or "another embodiment," "other examples" mean that at least one feature or characteristic description is included in connection with an embodiment. Thus, references to these phrases in various places throughout this disclosure are not necessarily referring to the same embodiment. Furthermore, particular features may be combined in any suitable manner in one or more embodiments.

[0059] In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. For definitions of common terms in molecular biology, see Lewin's Genes, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. For definitions of common terms in biochemistry, see Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. For definitions of common terms in cell biology, see Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. For definitions of common terms in genetics, see Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0060] Unless otherwise specified, the laboratory techniques herein utilize conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard texts such as Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0061] definition:

[0062] The term "bispecific antibody" in the present disclosure refers to an antibody molecule or a functional fragment thereof having two different antigen binding sites, wherein the two antigen binding sites specifically bind to a first target antigen and a second target antigen, respectively, wherein the first target antigen and the second target antigen are different antigens, and the two antigen binding sites are present in the same antibody molecule structure, so that the antibody molecule can simultaneously bind to the first target antigen and the second target antigen. In the present disclosure, the bispecific antibody comprises a functional fragment (or antigen binding portion) of an antibody molecule, including but not limited to Fab, Fab', F(ab')2, Fd, dAb, a complementary determining region fragment, a single-chain antibody (scFv), a humanized antibody, a chimeric antibody or a diabody. It can form a protein-protein complex by the binding of an antigen and an antibody, the binding of a receptor ligand, etc.

[0063] The term "identity" or "sequence identity" as used herein refers to the exact same nucleotide or amino acid residues at the same position in two sequences. It is a fundamental and important concept in bioinformatics, commonly used to compare the similarity between two nucleic acid or protein sequences. Sequence identity is typically expressed as a percentage, reflecting the degree of sequence identity.

[0064] In some embodiments of the present disclosure, "a sequence having more than 80% identity and the same or substantially the same biological function" generally means that the actual sequence and the sequence described in the present disclosure may be at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical. The different parts may be due to substitution / insertion / deletion mutations introduced artificially or non-artificially. The artificially introduced mutations may be based on specific purposes, such as facilitating protein expression, obtaining better affinity and / or dissociation properties, etc. However, the introduction of these different parts is not sufficient to change the biological function of the original protein represented by the amino acid sequence, such as the binding ability between antigen and antibody, gene expression, etc.

[0065] The term "isolated" as used herein refers to a substance or entity that has been separated from its natural environment or the environment in which it existed prior to isolation and from other components. The separation can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Isolated substances may have varying levels of purity relative to their pre-isolation counterparts.

[0066] The term "vector" (or "vehicle") in this disclosure refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell through transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site.

[0067] Fc region of the novel cyclic anti-CD38 bispecific antibody:

[0068] Traditional BsAbs, such as belintuzumab, which targets CD19, are composed of a CD19-targeting scFv and a CD3-targeting scFv tandemly linked end-to-end. Their molecular weight is approximately 55 kDa, and their small size results in a short half-life in patients. Therefore, in clinical practice, belintuzumab requires continuous infusion to maintain plasma concentrations of the antibody to achieve its therapeutic effect, which presents a challenge for its clinical application. By fusing the immunoglobulin Fc region into BsAbs, the molecular weight of the BsAb can be increased, thereby addressing the short in vivo half-life of classic BsAbs. The native immunoglobulin Fc region requires modification to reduce the affinity of Fc-fused BsAbs for Fcγ receptors (FcγRs) in vivo. This prevents the nonspecific inflammatory response caused by the Fc portion and the phagocytic clearance of the BsAb by the mononuclear phagocyte macrophage system during in vivo application.

[0069] The term "Fc segment" (Fragment crystallizable region) in this disclosure refers to the C-terminal constant region fragment of an immunoglobulin (Ig) heavy chain, which is composed of the CH2 domain, CH3 domain, and the C-terminal portion of the hinge region of two or more heavy chains covalently linked by interchain disulfide bonds. Its structural characteristics are: (1) the spatial configuration in natural antibodies is a homodimer (IgG / IgA / IgD) or a multimeric complex (IgM / IgE); (2) the sequence position starts after the last cysteine ​​residue in the hinge region of the heavy chain (approximately amino acid position 226) and extends to the end of the C-terminus; (3) unlike Fab / F(ab')2, it does not contain an antigen binding site (i.e., it does not include the variable region VL / VH and the constant region CL / CH1).

[0070] In some embodiments, any suitable immunoglobulin Fc segment can be recombined with a bispecific antibody molecule according to the description in the prior art. The Fc segment can be derived from a human immunoglobulin such as IgA, IgM, IgE, or IgG. In other embodiments, the fusion CD38 BsAb is a BsAb in which the Fc segment of a human immunoglobulin IgG is fused to the BsAb. The human immunoglobulin IgG can be any suitable immunoglobulin subtype such as IgG1 or IgG4. In one embodiment, the Fc-fused CD38 BsAb is a BsAb in which the human IgG1 Fc segment is fused to the binding portion of the BsAb that binds to the CD3 antigen. In a more specific embodiment, the human IgG1 Fc segment is mutated to reduce its affinity for FcγRs, and the LALA mutation (L234A, L235A) is introduced into its CH2 domain.

[0071] Preparation of bispecific antibodies:

[0072] In some embodiments, the preparation method of the novel cyclic anti-CD38 bispecific antibody expression vector can be based on the amino acid sequence of the above-mentioned domain, and the nucleotide sequence encoding the amino acids of the above-mentioned domain can be obtained by known techniques such as chemical synthesis or molecular cloning. Generally, the codons encoding the amino acids of the above-mentioned domain can be optimized to optimize their expression in the host cell to obtain the fusion protein with the best function. In some embodiments, the two BsAbs in the present disclosure are prepared using a eukaryotic expression system (ExpiCHO-S cells) to obtain the fusion protein with the best function. The cells are selected from CHO cell lines and different strains thereof, such as FreeStyle CHO-S, ExpiHO-S, CHOK1SVGS-KO, CHODXB11, etc., other eukaryotic system expression system cells or prokaryotic cell expression systems. The above-mentioned nucleotide and amino acid information can be obtained by searching known literature or databases such as NCBI.

[0073] In some embodiments, the above-mentioned expression vector can be a linear vector or a circular vector. It can be a non-viral vector such as a plasmid, a viral vector, or a vector utilizing a transposon. For example, a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors. The vector can contain regulatory sequences such as a promoter and a terminator, as well as marker sequences such as a drug resistance gene and a reporter gene. In one embodiment, the above-mentioned vector comprises a KOZAK sequence, an Igκ leader signal peptide sequence, and the core structure of the above-mentioned two BsAbs.

[0074] In some embodiments, the basic structure of the vector is derived from the pcDNA3.4 expression vector, into which the gene sequences of the core structures of the two BsAbs described above are inserted via Nhe1 and Age1 digestion. In some specific embodiments, both BsAb vectors disclosed herein are verified to be correct by Sanger sequencing. In some embodiments, the two BsAb vectors disclosed herein are transformed with E. coli JM109 competent cells, and the expression plasmids are extracted using endotoxin-free extraction technology and used to transfect ExpiCHO-S cells to produce the two BsAbs described above.

[0075] In some embodiments, BsAbs can be prepared by expressing in host cells and secreting from the host cells using appropriate vectors. For example, mammalian cells or bacterial cells can be used. After expression and secretion of the BsAb, the BsAb can be isolated and purified using methods known in the art. In more specific embodiments, 38-3-loop-BsAbs are purified using Cytiva StrepTrap HP purification columns, and 38-3-loop Fc-BsAbs are purified using Cytiva HiTrap Protein G HP purification columns.

[0076] Codon optimization:

[0077] Codon optimization is an advanced technology that effectively enhances protein expression in vivo by improving the translation efficiency of target genes. In vivo, due to codon degeneracy—that is, multiple codons can encode the same amino acid—multiple different mRNA sequences may exist for a given amino acid sequence. However, different organisms or cells have their own biases in the selection of these synonymous codons, which is known as codon bias. Therefore, when a heterologous gene is expressed in a host cell, its codon usage may not match the host cell's optimal codon usage frequency, affecting protein expression levels. Codon optimization can select synonymous codons that better match the host cell's bias, thereby improving protein translation efficiency.

[0078] There are many methods that can be used to optimize codons. For example, the steps of some methods are as follows: (1) Analyze the codon usage frequency of the host cell: First, you need to understand the codon bias of the host cell, that is, which codons are used more frequently in the host cell. This can be obtained by consulting relevant literature or databases. (2) Design an optimization plan: Design an optimization plan based on the codon usage frequency of the host cell. This includes selecting which synonymous codons to replace the codons in the heterologous gene, and how to adjust the GC content. (3) Implement optimization: Use techniques such as gene synthesis or site-directed mutagenesis to apply the optimization plan to the heterologous gene. (4) Verify the optimization effect: Verify experimentally whether the expression level of the optimized gene in the host cell is improved. This can be evaluated by measuring indicators such as protein concentration and enzyme activity.

[0079] Example:

[0080] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0081] Example 1: Construction of BsAb expression vector

[0082] Based on the sequence, the 38-3-loop-BsAb core structure "KOZAK-Igκleader-HisTag-CD3VL-G4S-CD38VL-LoopLinker-CD38VH-G4S-CD3VH-StrepTagII" and the 38-3-loopFc-BsAb core structure "KOZAK-Igκleader-HisTag-CD3VL-G4S-CD38VL-LoopLinker-CD38VH-G4S-CD3VH-Hinge-CH2-CH3" were obtained by chemical synthesis and cloned into the pcDNA 3.4 expression vector by molecular cloning technology. The results are shown in the figure. Figure 1 A, B, Figure 2 The AlphaFold2 tool was used to predict the structures of the two antibodies. The results are shown in Figure 3 .

[0083] The molecular cloning steps are as follows:

[0084] 1. Primer design and introduction of enzyme cutting sites

[0085] 1) Primers for amplifying the 38-3-loop-BsAb core structure and introducing restriction enzyme sites

[0086] Nhe1-KOZAK-F: 5'-CTAGCTAGCGCCACCATGGAGAC-3'

[0087] StrepTagII-Age1-R:5'-CGCACCGGTTCACTTCTCGAACT-3'

[0088] 2) Primers for amplifying the 38-3-loop Fc-BsAb core structure and introducing restriction enzyme sites

[0089] Nhe1-KOZAK-F: 5'-CTAGCTAGCGCCACCATGGAGAC-3'

[0090] CH3-Age1-R: 5'-CGCACCGGTTCACTTGCCGGGAG-3'

[0091] 2. PCR amplification

[0092] 50L reaction system (mix the reaction solutions in an ice bath)

[0093]

[0094] Reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 61°C for 1 min, extension at 72°C for 2 min; 35 cycles: final extension at 72°C for 10 min.

[0095] After 1% agarose gel electrophoresis, the gel was cut and then recovered (for example, refer to the instruction manual of TaKaRa MiniBEST AgaroseGel DNA Extraction Kit Ver.4.0).

[0096] 3. Ligate the pcDNA3.4 expression vector and 38-3-BsAb core fragment after enzyme digestion:

[0097] 50L enzyme digestion system

[0098]

[0099] After the above system is mixed, enzyme digestion is carried out in a metal bath at 37°C for 2-4 hours, and the expression vector and target fragment are recovered for ligation.

[0100] 20L connection system

[0101]

[0102] Use the Vector Fragment Ligation Calculator tool on the NEB website to calculate x

[0103] After the above system was mixed, the cells were connected at room temperature for 30 minutes and then the plasmid transformation was performed.

[0104] 4. Transform the pcDNA3.4-38-3-BsAb expression vector plasmid and select the correct expression vector verified by Sanger sequencing for transfection into ExpiCHO-S.

[0105] Example 2: Preparation of BsAb

[0106] 38-3-loop-BsAb and 38-3-loop-Fc-BsAb were produced using a eukaryotic expression system (ExpiCHO-S). The BsAb-enriched supernatant was collected and centrifuged at 20,000g for 30 minutes. The supernatant was then stored at -80°C until further use or for immediate purification.

[0107] Example 3: Purification of BsAb

[0108] In this example, two BsAbs were purified using a Cytiva AKTA purifier, 38-3-loop-BsAb was purified using a Cytiva StrepTrap HP purification column, and 38-3-loop Fc-BsAb was purified using a HiTrap Protein G HP purification column. The results are shown in Figure 4 After protein purification, the protein was concentrated using a protein ultrafiltration membrane and stored in a -80°C freezer.

[0109] Example 4: Characterization and Functional Detection of BsAb

[0110] In this example, the molecular weights of the two BsAbs were verified by Western Blot. Figure 5 (Left). Coomassie staining was used to verify the purity of the purified antibody. Figure 5 (right).

[0111] In this example, two BsAbs were quantified using the His Tag ELISA assay.

[0112] The specific binding ability of the two BsAbs to the target antigens CD38 and CD3 in this example was tested by competitive binding experiments with commercial flow cytometry antibodies PE-anti-Human-CD38 and PE-anti-Human-CD3. The results are shown in Figure 6 .

[0113] The specific embodiments are as follows:

[0114] a) Take four tubes of 1×10 5 CD38 +Molm13 was added with PBS (negative control and positive control groups), 0.1 nM 38-3-loop-BsAb, and 38-3-loopFc-BsAb (experimental groups), respectively, and incubated at 4 °C for 2 h;

[0115] b) For the positive control group and experimental group, commercial flow cytometry antibody PE-anti-Human-CD38 was added for labeling and incubated at 4°C for 30 minutes;

[0116] c) detecting the degree of fluorescence intensity reduction using flow cytometry;

[0117] d) Using the same method to detect BsAb and CD3 antigen (CD3 + Jurkat cells) specific binding capacity.

[0118] The ability of the two BsAbs to activate the proliferation of primary T cells in this example was tested by CFSE staining proliferation assay and absolute T cell count. Figure 7 .

[0119] The specific embodiments are as follows:

[0120] a) Isolating human T cells.

[0121] b) CFSE staining proliferation assay method is as follows: human naive T cells were stained with CFSE. 1×10 6 Human naive T cells were added with PBS (control), 0.1nM 38-3-loop-BsAb and 38-3-loopFc-BsAb (experimental group), respectively. After culturing in a 37°C 5% CO2 incubator for 5 days, the degree of CFSE dye fluorescence intensity reduction was detected by flow cytometry ( Figure 7 left and center).

[0122] c) Absolute T cell count detection example is as follows: In a 24-well plate, 1×10 6 Initial human T cells were added with PBS (control), 0.1 nM 38-3-loop-BsAb, and 38-3-loopFc-BsAb (experimental group), respectively. The cells were counted every 2 days using a counting plate to maintain a T cell density of no more than 2 × 10 6 / ml, for 10 days ( Figure 7 on the right).

[0123] Example 5: BsAb anti-leukemia function detection

[0124] The anti-leukemia effects of the two BsAbs in this example were tested by a killing experiment in which human T cells were co-cultured with leukemia tumor cells (experimental group: CD38+Molm13 and CD38+U937; control group: CD38-K562). Figure 8 .

[0125] The specific embodiments are as follows:

[0126] 1. Take a 96-well plate and add T cells and tumor cells into the wells at a 2:1 effector-target ratio;

[0127] 2. Add PBS / different concentrations of 38-3-loop-BsAb and 38-3-loopFc-BsAb, respectively, and culture in a 37°C 5% CO2 incubator for 72 hours.

[0128] 3. Detect the killing function of anti-T cells against leukemia cells: label flow cytometry antibodies, use PE-anti-Human-CD33 to label tumor cells, use APC-anti-Human-CD5 to label T cells, and detect the proportion of residual tumor cells.

[0129] Example 6: Detection of Cytokine Release by BsAb-Mediated Anti-leukemia Function

[0130] The supernatant of the co-culture killing experiment in Example 5 was collected and the following cytokines were detected: IL2, TNF-a, IFN-γ, IL6. Figure 9 .

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A cyclic anti-CD38 bispecific antibody that specifically binds to human CD38 and human CD3 molecules, characterized in that: It includes: a) variable regions CD38VH and CD38VL of the antigen-binding portion that specifically bind to human CD38; b) variable regions CD3VH and CD3VL of the antigen-binding portion that specifically bind to human CD3; and c) LoopLinker, The amino acid sequence of the cyclic anti-CD38 bispecific antibody is arranged in the order of VL-LoopLinker-VH, and the amino acid sequence of the LoopLinker is as shown in SEQ ID No: 1 or a sequence having more than 80% identity thereto and having the same or substantially the same biological function.

2. The cyclic anti-CD38 bispecific antibody according to claim 1, wherein The antigen binding portion is Fab, Fab', F(ab')2, Fd, FCL, dAb or scFv; Preferably, the antigen binding portion is a scFv.

3. The cyclic anti-CD38 bispecific antibody according to claim 1 or 2, characterized in that The amino acid sequences of CDRH1-3 of the CD38VH are shown in SEQ ID Nos: 2-4, respectively, and the amino acid sequences of CDRL1-3 of the CD38VL are shown in SEQ ID Nos: 5-7, respectively; The amino acid sequences of CDRH1-3 of the CD3VH are shown in SEQ ID Nos: 8-10, respectively, and the amino acid sequences of CDRL1-3 of the CD3VL are shown in SEQ ID Nos: 11-13, respectively.

4. The cyclic anti-CD38 bispecific antibody according to claim 1 or 2, characterized in that The amino acid sequence of the CD38VH is as shown in SEQ ID No: 14, or a sequence having 80% or more identity thereto and having the same or substantially the same biological function; the amino acid sequence of the CD38VL is as shown in SEQ ID No: 15, or a sequence having 80% or more identity thereto and having the same or substantially the same biological function; The amino acid sequence of the CD3VH is shown as SEQ ID No: 16 or a sequence having more than 80% identity thereto and the same or substantially the same biological function, and the amino acid sequence of the CD3VL is shown as SEQ ID No: 17 or a sequence having more than 80% identity thereto and the same or substantially the same biological function.

5. The cyclic anti-CD38 bispecific antibody according to claim 1, wherein The cyclic anti-CD38 bispecific antibody further comprises: d) Fc region of human immunoglobulin; Preferably, the human immunoglobulin is IgA, IgM, IgE or IgG; More preferably, the human immunoglobulin is IgG1.

6. The cyclic anti-CD38 bispecific antibody according to claim 5, characterized in that The amino acid sequence of the Fc segment of the human immunoglobulin is shown in SEQ ID No:

18.

7. The cyclic anti-CD38 bispecific antibody according to claim 5, characterized in that The Fc segment of the human immunoglobulin is operably connected to the N-terminus of the VL-LoopLinker-VH structure through a hinge, and the hinge forms an immunoglobulin-like dimer structure through a disulfide bond.

8. The cyclic anti-CD38 bispecific antibody according to claim 7, characterized in that The amino acid sequence of the hinge is shown in SEQ ID No: 19 or a sequence having more than 80% identity thereto and having the same or substantially the same biological function.

9. The cyclic anti-CD38 bispecific antibody according to claim 1, wherein The order of the VL-LoopLinker-VH is: 1)CD3VL-CD38VL-LoopLinker-CD38VH-CD3VH; 2)CD38VL-CD3VL-LoopLinker-CD38VH-CD3VH; 3) CD3VL-CD38VL-LoopLinker-CD3VH-CD38VH; or 4)CD38VL-CD3VL-LoopLinker-CD3VH-CD38VH.

10. The cyclic anti-CD38 bispecific antibody according to claim 9, characterized in that Linker 1 (Linker 1) is operably connected between the CD3VL and the CD38VL, and Linker 2 (Linker 2) is operably connected between the CD3VH and the CD38VH.

11. The cyclic anti-CD38 bispecific antibody according to claim 10, characterized in that The connector 1 or connector 2 is a flexible connecting peptide; Preferably, the flexible connecting peptide is selected from (GGGGS) n , G x S x Repeating unit, one of GGG, GGSG or GSGG amino acid sequences.

12. The cyclic anti-CD38 bispecific antibody according to claim 1, wherein The cyclic anti-CD38 bispecific antibody further comprises a tag or a signal peptide; Preferably, the label is selected from at least one of a fluorescent label, a radioactive label or an enzyme label.

13. The cyclic anti-CD38 bispecific antibody according to claim 1, wherein The cyclic anti-CD38 bispecific antibody is modified, and the modification is one or more of cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycosylated, glycosylated, polysialylated, phosphorylated, adenylylated, or PEGylated.

14. The cyclic anti-CD38 bispecific antibody according to claim 1, wherein The amino acid sequence of the cyclic anti-CD38 bispecific antibody is shown in SEQ ID No: 20-21 or a sequence having more than 80% identity thereto and having the same or substantially the same biological function.

15. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the cyclic anti-CD38 bispecific antibody according to any one of claims 1 to 14.

16. The isolated nucleic acid molecule according to claim 15, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No: 22 or a sequence having more than 80% identity thereto and having the same or substantially the same biological function.

17. A carrier, characterized in that The vector comprises the nucleic acid molecule as claimed in claim 15 or 16.

18. A cell, characterized in that The cell comprises the cyclic anti-CD38 bispecific antibody according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15 or 16, or the vector according to claim 17; Preferably, the cell is a eukaryotic cell.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cyclic anti-CD38 bispecific antibody according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15 or 16, the vector according to claim 17, or the cell according to claim 18.

20. Use of the cyclic anti-CD38 bispecific antibody according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15 or 16, the vector according to claim 17, the cell according to claim 18, or the pharmaceutical composition according to claim 19 in the preparation of a medicament for treating tumors or autoimmune diseases; Preferably, the tumor is a hematological tumor; More preferably, the hematological tumor is acute myeloid leukemia, lymphoma, multiple myeloma, or systemic light chain amyloidosis; Preferably, the autoimmune disease is immune thrombocytopenia, lupus erythematosus, or rheumatoid arthritis.

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